(Ecological Studies 235) Oana Teodora Moldovan, Ľubomír Kováč, Stuart Halse - Cave Ecology-Springer International Publishing (2018)

Ecological Studies 235
Oana Teodora Moldovan

L’ubomír Kováč
Stuart Halse Editors
Cave
Ecology
Ecological Studies
Analysis and Synthesis
Volume 235
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Oana Teodora Moldovan • Ľubomír Kováč •
Stuart Halse
Editors
Cave Ecology
Editors
Oana Teodora Moldovan Ľubomír Kováč
Emil Racovitza Institute of Speleology, Faculty of Science
Romanian Academy P. J. Šafárik University
Cluj Napoca, Romania Košice, Slovakia
Stuart Halse
Bennelongia Environmental Consultants
Jolimont, Western Australia, Australia
ISSN 0070-8356 ISSN 2196-971X (electronic)

Ecological Studies
Analysis and Synthesis
ISBN 978-3-319-98850-4 ISBN 978-3-319-98852-8 (eBook)
https://doi.org/10.1007/978-3-319-98852-8
Library of Congress Control Number: 2018962260
© Springer Nature Switzerland AG 2018

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Contents
1 Preamble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Oana Teodora Moldovan, Ľubomír Kováč, and Stuart Halse
Part I Into Karst, Caves and Cave Habitats

2 Physiography of the Caves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Stein-Erik Lauritzen
3 Where Cave Animals Live . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Francis G. Howarth and Oana Teodora Moldovan
Part II There is Rich and Diverse Life in Caves

4 The Ecological Classification of Cave Animals and Their
Adaptations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
5 The Microbial Diversity of Caves . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Olivia S. Hershey and Hazel A. Barton
6 Phototrophs in Caves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Janez Mulec
7 Diversity of Terrestrial Invertebrates in Subterranean Habitats . . . 107
Louis Deharveng and Anne Bedos
8 An Overview on the Aquatic Cave Fauna . . . . . . . . . . . . . . . . . . . . 173
9 Subterranean Biodiversity Patterns from Global to Regional
Scales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Maja Zagmajster, Florian Malard, David Eme, and David C. Culver
v
vi Contents
10 Historical and Ecological Factors Determining Cave Diversity . . . . 229

Ignacio Ribera, Alexandra Cieslak, Arnaud Faille, and Javier Fresneda
Part III About Communities, Populations and Food

11 Cave Communities and Species Interactions . . . . . . . . . . . . . . . . . . 255
Stefano Mammola and Marco Isaia
12 Structure and Genetics of Cave Populations . . . . . . . . . . . . . . . . . . 269
Peter Trontelj
13 Caves as Oligotrophic Ecosystems . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Ľubomír Kováč
14 Food Webs in Caves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Michael P. Venarsky and Brock M. Huntsman
Part IV Particular Cave Ecosystems

15 Ice Caves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Ľubomír Kováč
16 Researches in Sulphide-Based Ecosystems . . . . . . . . . . . . . . . . . . . . 351
Alexandra Hillebrand-Voiculescu
17 Researches in Lava Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Pedro Oromí
18 Research in Anchialine Caves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Thomas M. Iliffe and Fernando Alvarez
19 Volcanic Anchialine Habitats of Lanzarote . . . . . . . . . . . . . . . . . . . 399
Alejandro Martínez and Brett C. Gonzalez
20 Research in Calcretes and Other Deep Subterranean Habitats
Outside Caves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
Stuart Halse
21 Subterranean Biodiversity in Ferruginous Landscapes . . . . . . . . . . 435
Rodrigo Lopes Ferreira, Marcus Paulo Alves de Oliveira,
and Marconi Souza Silva
Part V Conservation and Protection of Cave Habitats and Cave Fauna

22 Conservation of Cave Fauna, with an Emphasis on Europe
and the Americas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Matthew L. Niemiller, Steven J. Taylor, and Maria Elina Bichuette
23 Conservation and Impact Assessment of Subterranean Fauna
in Australia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
Stuart Halse
Contents vii
Part VI Cave Ecology for the Twenty-First Century

24 Where Angels Fear to Tread: Developments in Cave Ecology . . . . . 497
William F. Humphreys
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
Chapter 1
Preamble
Oana Teodora Moldovan, Ľubomír Kováč, and Stuart Halse
Wherefore each of you, when his turn comes, must go down to

the general underground abode, and get the habit of seeing in
the dark. When you have acquired the habit, you will see ten
thousand times better than the inhabitants of the cave, and
you will know what the several images are, and what they
represent, because you have seen the beautiful and just and
good in their truth.
Plato, Republica
1.1 On Caves, Cave Ecology, and Cave Inhabitants
Caves are seen as natural openings large enough to allow entrance of humans. Caves
are formed in many types of rock and by many processes, although the largest and
most common caves are those formed in limestone, dolomite, and solidified lava.
Nevertheless, when considering caves as living spaces, the size of the cave is often
less important than the size of the cave inhabitants. Most cave organisms are a few
millimeters or even less in size, and they can colonize any void of larger size than
this, especially where there is no light and environmental conditions are relatively
constant throughout the year. “The subterranean environment would be as insignifi-
cant by the small space it occupies on Earth, as by the low number of beings
O. T. Moldovan (*)
Emil Racovitza Institute of Speleology, Romanian Academy, Cluj Napoca, Romania
Ľ. Kováč
Faculty of Science, P. J. Šafárik University, Košice, Slovakia
e-mail: [email protected]
S. Halse
Bennelongia Environmental Consultants, Jolimont, Western Australia, Australia
© Springer Nature Switzerland AG 2018 1

O. T. Moldovan et al. (eds.), Cave Ecology, Ecological Studies 235,
https://doi.org/10.1007/978-3-319-98852-8_1
2 O. T. Moldovan et al.
inhabiting here; it would only be a kind of nature’s oddity. Now, there is no falser
idea!” (translated from French) was a statement by Racovitza in 1907, and he added
further that subterranean fauna consists of a heterogeneous mixture of very different
forms in origin, hereditary aptitudes, degree of organization, and time of immigra-
tion into caves.
We use the term ‘cave’ for any space or void that fulfills the environmental
requirements of living organisms for their survival. The anthropocentric view of a
cave size has been reconsidered in the last three decades, and the syntagm cave
ecology was replaced by subterranean ecology, not always in a justified way.
Ecology is the science that studies the interactions between organisms and their
relationship with the environment, and cave ecology studies the connections of cave
organisms with their cave environment, at multiple levels. The abiotic environment
is represented by the cave habitats, and the organisms are those typical troglobionts
or stygobionts, which live permanently in caves. The focus of the book is less on
troglophiles/stygophiles and guanobionts although all these categories are men-
tioned when appropriate.
1.2 Cave Ecology as the Center of Biospeleological

Researches
It is difficult to think about the beginning of cave ecology without referring to the
“Essai sur les problèmes biospéologiques” (Essay on biospeleological problems),
which was published by Racovitza in 1907 and ignored for a long time simply
because it was written in French. It is the manifesto of biospeleology, marking the
birth of this science, where the author defines the terms to be used, the habitats, and
the adaptation of cave inhabitants and outlines the ecological research to be done.
As knowledge of the fauna and habitats increased, details of the life histories of
various species were published by different authors (Delamare-Deboutteville 1960;
Vandel 1964; Ginet and Decou 1977), and almost one century later, the encyclo-
pedias (Juberthie and Decu 1994, 1998, 2001; Gunn 2004; Culver and White 2005;
White and Culver 2012) and books (Culver 1982; Camacho 1992; Wilkens et al.
2000; Romero 2009; Culver and Pipan 2009, 2014) brought together information on
the subterranean world, including its ecology.
1.3 What Can Bring a Multi-author Book on Cave

Ecology?
We want this volume to regenerate interest in the study of caves and their living
organisms by highlighting that caves are spaces of any size as long as they support
life. “Traditional” caves are still the places to which we have access, where
1 Preamble 3
organisms can be studied in situ and where communities can best be observed and
sampled. In caves we find the specimens used for laboratory experiments and
many other different studies in astrobiology, human health, paleoclimatology,
geomicrobiology, etc. The smaller caves, often referred to as micro- and
mesocaverns, may occur as a network of spaces around traditional caves or below
surface streams. The vast majority of these spaces, however, occur in groundwater or
weathering parts of the vadose zone elsewhere in the landscape, and the study of
these systems is in its infancy.
Cave ecology, as with all other areas of biological study, has numerous hypo-
theses and theories regarding cave inhabitants and the processes that sustain them. A
book with multi-authored opinions provides a more comprehensive overview of the
subject. Although it is the duty of the authors to provide an objective and embracing
view on what they write as scientists, the diversity of authors and their different
opinions enrich our understanding of cave ecology and its importance, as well as
increasing the range of topics covered and the perspectives on subterranean life.
This is a book about the state of the art and future directions in biospeleological
research addressed to students, teachers, and specialists who need simple definitions,
clear explanations of existing knowledge and theories, and informed opinions about
future directions for research in a single book. We do not pretend to have extensively
covered all the possible aspects and topics of cave ecology. Foremost in our aims
was to thrust the cave and the cave organisms into the spotlight in the hope that this
will lead to greater conservation and protection of this wonderful environment that is
under the increasing pressure from human activities.
Acknowledgment We would like to express our special thanks to Francis G. Howarth for the
reading of the book and useful suggestions and comments. OTM acknowledges the financial
support from the Romanian Academy and the grant of the Romanian Ministry of Research and
Innovation, CNCS—UEFISCDI, project number PN-III-P4-ID-PCCF-2016-0016, within
PNCDI III.
References
Camacho AI (ed) (1992) The natural history of biospeleology. Monografías del Museo Nacional de
Ciencias Naturales. CSIC, Madrid
Culver DC (1982) Cave life. Evolution and ecology. Harvard University Press, Harvard
Culver DC, Pipan T (2009) The biology of caves and other subterranean habitats. Oxford University
Press, Oxford
Culver DC, Pipan T (2014) Shallow subterranean habitats: ecology, evolution, and conservation.
Oxford University Press, Oxford
Culver DC, White WB (eds) (2005) Encyclopedia of caves, 1st edn. Academic, Amsterdam
Delamare-Deboutteville C (1960) Biologie des eaux souterraines littorales et continentales.
Hermann, Paris
Ginet R, Decou V (1977) Initiation à la biologie et à l’écologie souterraines. Jean-Pierre Delarge,
Paris
Gunn J (ed) (2004) Encyclopedia of caves and karst science. Fitzroy Dearborn, New York
4 O. T. Moldovan et al.
Juberthie C, Decu V (eds) (1994) Encyclopaedia biospeologica, Tome I. Société de Biospéologie,

Moulis (France), Bucarest (Romania)
Juberthie C, Decu V (eds) (1998) Encyclopaedia biospeologica, Tome II. Société de Biospéologie,
Juberthie C, Decu V (eds) (2001) Encyclopaedia biospeologica, Tome III. Société de Biospéologie,
Racovitza EG (1907) Essai sur les problèmes biospéologiques. Biospeologica 1. Archives de
Zoologie Expérimentale and Générale 4:371–488
Romero A (2009) Cave biology: life in darkness. Cambridge University Press, Cambridge
Vandel A (1964) Biospéologie. La Biologie des Animaux Cavernicoles. Gauthier-Villars, Paris
White WB, Culver DC (eds) (2012) Encyclopedia of caves, 2nd edn. Academic, Amsterdam
Wilkens H, Culver DC, Humphreys WF (eds) (2000) Subterranean ecosystems, ecosystems of the
world 30. Elsevier, Amsterdam
Part I
Into Karst, Caves and Cave Habitats
Chapter 2
Physiography of the Caves
Stein-Erik Lauritzen
2.1 Introduction
This chapter deals with the physiography (formation, morphology and internal
environment) of underground habitats (see also Chap. 3), generally known as
“caves”, and smaller interstitial spaces that can be occupied by organisms
(e.g. Poulson and White 1969). Caves are, by definition, natural underground
voids (White and Culver 2012), and passable caves (that are accessible to humans)
are just fragments of the hydrogeological network (Fig. 2.1). Caves exist in a variety
of geological materials, but they share many common characteristics with respect to
environmental factors: total darkness except in entrance zones, relatively constant
temperature and humidity and a compartmental geometry over a wide range of
dimensions.
The most commonly known “cave” types are karst caves, which are formed by
chemical dissolution of the host rock. These are so-called “true” karst caves
(Fig. 2.1). Other “pseudokarst” caves are lava tubes and various fissure and talus
caves that are formed in rocks that do not dissolve fast enough in water to make them
“karstic”. They are thus formed by processes other than chemical dissolution alone.
Caves may connect into vast, interconnected systems of complex architecture but
can also consist of physically isolated units, to be likened with islands, and can
therefore host endemic biota.
S.-E. Lauritzen (*)

Department of Earth Science, University of Bergen, Bergen, Norway

https://doi.org/10.1007/978-3-319-98852-8_2
8 S.-E. Lauritzen
Doline
Epikarst
Vadose
zone
Intermittent
spring
Epiphreatic Permanant
zone karst
spring
Water table River
Phreatic
zone Conduit flow
Conduit flow
Porous and
Deep fissure flow
pheratic
zone
Fig. 2.1 Conceptual cross section of a karst system
2.2 Cave Patterns
Depending on bedrock structure, and on hydrologic regimes, solutional caves

develop into several distinct architectures or patterns (Fig. 2.2). A basic difference
of pattern exists between caves developed along bedding planes and along fracture
networks. This difference is depicted by the top and bottom rows in Fig. 2.2.
Dendritic caves are the basic pattern formed on stream passages that converge as
tributaries, similar to surface rivers (Fig. 2.2a and e). Mazes or labyrinths consist of a
network of interconnected passages that have evolved simultaneously. They are
generally interpreted as created by periodic flooding that pumps into and drains all
available fractures with water, thus enlarging all fractures at the same time. An
alternative mechanism is diffuse recharge through a porous caprock or an underlying
porous formation, thus feeding every fracture with aggressive (corrosive) water. This
is particularly effective in situations where two waterbodies of different origin (and
chemistry) meet. Their different chemical composition may enhance the rate of
dissolution as a result of their mixing (mixing corrosion, Bögli 1964). The water
flow can be quite slow. Spongework and ramiform patterns are characteristics of
so-called hypogenic caves, where ascending water moves through porous rocks and
the voids are created by release of chemical aggressiveness due to mixing. In
particular, this may happen when saturated fresh and haline waters meet, forming
2 Physiography of the Caves 9
Fig. 2.2 Various plan-view patterns or architectures of solutional caves. Upper row shows caves
formed along bedding planes or in porous rock; lower row depicts joint- or fracture-controlled
caves. (a) and (e) dendritic or branchwork caves; (b) and (f) anastomotic and network mazes; (c)
spongework cave; (d) ramiform cave; (g) linear, joint-controlled cave. Adapted from Palmer (2007)
halocline caves, and where hydrogen sulphide (as gas or dissolved in water) encoun-
ters oxygenated waters and produces sulphuric acid, which boosts the dissolution
rate (ramiform caves).
2.3 Size Distribution of Cave Voids
Apart from the absence of light and, as a consequence, photosynthesis, the cave
habitat can be characterized by the size distribution of individual voids. A “cave”
may be defined in two ways, either anthropomorphic or hydraulic. In the first case, a
cave is defined as a natural underground space that is passable by man, i.e. it is at
least 30–60 cm diameter; in the second case, a (karst) cave is a continuous under-
ground space that allows turbulent water flow, i.e. >10 mm diameter. From a
biological point of view, caves are voids large enough to host cave-dwelling
organisms. The distribution of elements (voids) with diameter δ for proper caves,
sensu Curl (1986), follows a power or fractal law:
nδ ¼ aδ1D
10 S.-E. Lauritzen
Cave Void Distribution

6
4
log number of voids
3
–1
–2
–3
–4
–3 –2 –1 0 1 2
log void diameter (m)
Fig. 2.3 Size distribution of cave elements (voids) relative to a (spherical) unit, 1 m in diameter
(the red dot at 0,0). Assuming that this particular cave obeys a fractal dimension, D 2.73, i.e. the
Menger sponge, the size distribution of smaller voids would follow the graph. Associated with this
1 m diameter void, we may expect that there exists about 3000 and 150,000 voids greater than 1 cm
and 1 mm, respectively. This model illustrates the vast habitat available in a cave where only a few
passages are in fact passable to man
where n is the number of elements of diameter δ, a is a scaling constant and D is the

so-called fractal dimension. In practice, there are a lot smaller elements than there are
large ones. Empirically, many dissolutional caves have D 2.73, which means that
the cave is partially space filling, and with a dimension similar to the so-called
Menger sponge (Curl 1986). In consequence, if a person enters a cave with a
diameter of 1 m (e.g. Fig. 2.1), there may be another 105 voids in the surroundings
with a diameter down to about 1 mm (Fig. 2.3). The cave habitat is therefore vastly
larger than a human can reach, and it can accommodate space for small organisms.
The entrance of a cave is an intersection between the land surface and an
underground void. The formation of cave entrances is a stochastic process (Curl
1958, 1960); surface erosion processes may open or close entrances over time. The
longer the cave exists as a system, the more likely it is to have one or more entrances.
The likelihood is obviously greater for shallow caves than for systems deeply
beneath the land surface. In consequence, most caves in a region have no known
entrance and are therefore only indirectly accessible, e.g. through springs, boreholes
or construction works. This effect is not restricted to karst caves but applies to
pseudokarst voids as well.
Fig. 2.4 The carbonate

karst cycle
Box 2.1
Karst caves result from groundwater dissolving a rock mass consisting of
relatively easily dissolvable minerals: calcite, dolomite, gypsum and rock salt.
Carbonate karst is by far the most common type globally, followed by
evaporate (gypsum) karst (Ford and Williams 2007). For carbonates, the
process is an acid–base reaction, involving carbonic acid or other organic or
inorganic acids (Fig. 2.4).
The process from left to right creates caves (i.e. speleogenesis) and con-
sumes CO2, whilst the reverse process, from right to left, where H+ or CO2 is
removed from the system, actually fills the cave void—with speleothems.
From this, we realize that karstification is a CO2 sequestration process that,
in fact, is of global importance.
2.4 Karst Caves
2.4.1 Speleogenesis
Speleogenesis occurs when water penetrates joints, fissures or bedding plane part-
ings. There is a considerable latency (103–105 years), named “inception” or “break-
through time”, during which the downstream aperture of the proto-cave voids stays
below 10 mm (White 1977; Dreybrodt et al. 2005). This is because a relatively rapid,
first-order dissolution process consumes most of the aggressiveness over the first few
metres of flow along a tight fracture. After that, dissolution is extremely slow along
the remaining distance of the fracture, attenuated by a factor of 104–105. The key
issue is that the process approaches thermodynamic equilibrium very slowly, so that
the water emerging at the downstream end still has a minute dissolution potential and
consequently widens the downstream aperture (“bottleneck”) at a correspondingly
slow rate. When the downstream aperture has attained a diameter of 10–20 mm,
highly aggressive water emerges, and the aperture expands rapidly. This change in
process regime is named “chemical breakthrough”. At the same time, the conduit has
attained a dimension that permits the onset of turbulent flow, which greatly speeds
up the dissolution process. For a 200 m long fracture or bedding plane parting, the
nominal breakthrough time is of the order of 30,000 years. From this moment, the
conduit may grow to metre size in a few thousand years (Fig. 2.5). After this, the
12 S.-E. Lauritzen
20–50 m
the passage has grown
passage diameter
too large and collapses
Surface
erosion
3m removes
passage is drained the cave
and is transformed to
vadose key-hole
passage
1 cm
time
inception birth growth stagnation ‹‹death››
Fig. 2.5 The life cycle of a dissolutional (karst) cave passage; diameter as a function of time. Time
of growth from being a tight fracture (0.1 mm) to a 1 cm diameter proto-conduit may take 104–105
years, depending on flowpath length. After that, growth is very rapid and may attain 1 mm year1.
A diameter of >ca 3 m is often too large for natural recharge to keep it filled, and it turns into a
vadose passage. If situated deeply below the water table, the passage may continue to grow until it
becomes mechanically unstable and collapses. The ultimate speleothanatosis is when the surface
erosion destroys the cave. Compiled from White (1988) and Dreybrodt et al. (2005)
conduit may become too large for most surface streams to keep it completely full,
and it enters the vadose zone.
Alternatively, if the conduit is situated deep beneath the water table, it may
continue to grow until its diameter exceeds the mechanical strength of the host
rock, resulting in collapse. Such collapses may evolve through boulder chokes and
breccia pipes and eventually reach the surface. Collapses are high-porosity zones
which are not easily accessed by man. The cave’s life cycle ends when the surface
erosion destroys it (speleothanatosis, Šušteršič 1999) (Fig. 2.5). Caves are therefore
the most resistant elements of a landscape, and they do not disappear before the
surrounding rock is gone.
2.4.2 Hydromorphic Zones in Karst
Epikarst defines the interface between soil cover and the endokarst. It is generally a
10 m thick zone of intensely fractured and dissolved rock. With numerous,
interconnected fissures and narrow channels, the epikarst is more porous than the
bulk rock beneath it. Where there is scanty or no soil cover, the epikarst is exposed as
a barren surface with karren and grikes (lapiés).
The epikarst comprises a separate, perched aquifer that is poorly connected to the
vadose and phreatic zones deeper in the endokarst massif. The epikarst aquifer is
recharged through every fissure and hole, and water is transported horizontally
towards zones of denser and deeper fractures where water is conveyed vertically
into the vadose zone of the endokarst. These sites of capture are often situated
beneath dolines (closed depressions), which are also formed by this process through
a positive feedback loop (Williams 1985). The epikarst contain both air-filled and
water-filled voids.
The vadose zone occupies the upper endokarst in the vertical space between the
epikarst and the water table. The vadose zone is partially air-filled, and water
movement is mostly vertical as a result of gravity flow. There is a wide range of
types of water movement in the endokarst, including waterfalls and low-gradient
streams, trickles, seepage and slowly moving water films. Higher flow rates are
linked to inlets with correspondingly large catchments: streamsinks and dolines feed
streams and trickles, whilst fissures and percolation paths give rise to drips or film
movements. Vadose water is more or less directly connected to the surface environ-
ment and is an important nutrition vector for the cave habitats (Culver and Pipan
2009). Depending on climatic zone, cave geometry and ventilation, the vadose zone
may be quite wet or completely dry, as revealed by effervescence of moisture-
sensitive secondary minerals (Hill and Forti 1997; White 1997). The vadose zone
is the main locus for speleothem deposition, where carbonate speleothems are
precipitated by degassing CO2 (Fig. 2.4).
Water levels in caves may be viewed as a simple water table or, more correctly, a
piezometric level, below which all available voids are completely water-filled. There
is no continuous water table in karst; the water surface is only observed in passages
(syphons) which may be compared to a complex system of interconnected U-tubes,
with a piezometric level applying across them. Due to dynamic (venturi) effects, the
water table in karst does not always slope in a simple manner, and it will also vary
with recharge and local passage flow rate. This zone of variable water filling is
named the epiphreatic or floodwater zone (Fig. 2.1). Due to the pumping effect of
the fluctuating water table, aggressive water is replenished periodically, so that
dissolution may be more intense here than elsewhere in the phreatic zone.
The phreatic zone is the zone of permanent water saturation, below the lowest
water level in the epiphreatic, and thus there are no airspaces (Fig. 2.1). The phreatic
zone in karst is distinguished from groundwater in, for example, gravel by having
much larger spaces (conduits) and that water movement in well-developed tubes
may be turbulent. In general, phreatic water circulation is slow, and residence times
may be 102–103 years (Ford and Williams 2007). Deep circulation may go down to
about 300 m.
The deep phreatic zone designates the deepest limits of karstification, where
water movement is very slow in curved paths—downward at first, then rising
again towards the spring outlets at the boundary of the karst body. If the limestone
extends deep enough, there may be a relatively stagnant zone known as the
nothephreatic zone (Grimes 1999).
Even deeper-seated karst may form from ascending, thermal water, forming
hypogenic caves (Klimchouk 2007). The source of this water is connate
14 S.-E. Lauritzen
Fig. 2.6 Flow chart

depicting water recharge precipitation
and discharge in a karst evapotranspiration
system. The meteoric water
circulation penetrates
through the epikarst, the inlet boundary
vadose zone and phreatic
zones before discharging at
the karst boundary zones. In
the hypogenic case, deep- stream infiltration/ percolation
seated, thermal groundwater sinks
epikarst
enters the karst soil bare karst
dolines fissures
vadose flow vadose flow

in micro in fissures
caves
(thermal) hypogenic inlet boundary

cave stream
phreatic
phreatic cave phreatic
storage storage
in cave in
sediments deep fissures
phreatic
water
outlet boundary (springs)
(i.e. trapped within sedimentary rocks) or juvenile (of magmatic origin). Being
formed by ascending water, hypogenic caves display a characteristic, bulbous or
labyrinthic morphology (Ford and Williams 2007). It appears that most caves may
have a little recognized but significant, hypogenic prehistory, so that later
karstification tends to inherit older flowpaths and develop them further. Hypogenic
cave architectures are shown in Fig. 2.2c and d.
The cycle of meteoric water through a karst system is depicted in Fig. 2.6.
Infiltration from surface precipitation follows three main vectors, infiltration through
soil and through open fissures in bare karst or through discrete streamsinks. The
further movement through the karst is distributed among voids of a wide range of
dimensions (see Fig. 2.3), basically classified as epikarst fissures and micro-caves,
vadose fissures and shafts, sediment interstices and larger cave passages. The
discharge boundary of karst waters often coincidences with the lithological bound-
ary of the karst, and discharge occurs through discrete springs feeding surface
drainage and diffuse seepage into neighbouring sediments or adjacent waterbodies.
Deep-seated karst has another input boundary of hypogene, thermal waters that may
reach the surface through the phreatic zone (Fig. 2.6). Thus, in terms of energy and
nutrition transport into the subterranean ecosystem, there are two principally differ-
ent vectors: the meteoric, surface-derived pathway that conveys organic matter
connected to photosynthesis and the hypogenic, thermal pathway that is connected
to chemoautotrophic biosynthesis.
2.5 The Physics and Chemistry of Caves
In principle, one should expect the cave to pick up the geothermal gradient of the
surrounding rock mass, so that ambient temperature would increase monotonically
with depth. This is rarely the case, as ground heat is intercepted by flowing
groundwater and air currents (Badino 2005). Long or deep caves may show interior
thermal gradients that reflect geothermal influence (e.g. Ford et al. 1976). The
distribution of temperature and humidity in the epikarst and vadose zone is driven
by air and water movement.
Box 2.2
If the cave air is warmer than the surface air, e.g. a winter scenario, the density
of the cave air is less than surface air; there is a positive pressure into the lower
entrance, forcing air upwards and out of the higher-level entrance. The lower
entrance is the locus of lowest temperatures and potential for perennial ice
formation. In summer, the situation is opposite, and the air current is reversed
(Wigley and Brown 1976).
2.5.1 Cave Ventilation
Cave ventilation may have several origins. Air movement in multi-entrance caves is
commonly caused by seasonal, thermal differences between the cave and the surface,
the so-called chimney effect (Box 2.2. Fig. 2.7). The chimney effect is driven by
difference in air density between the cave and the surface. (Air density depends on
temperature and moisture content, so that the virtual temperature is the crucial
parameter.) The density difference results in a pressure difference between the inside
and outside of the cave at its entrances.
Air velocities depend on difference in elevation between entrances, temperature
contrasts and entrance diameter but can exceed 10 m s1. In extreme cases, this
ventilation effect (also named Balch ventilation) may result in the formation of
16 S.-E. Lauritzen
Fig. 2.7 The principle of the chimney effect of cave ventilation in a simple cave model.
Figure modified after Wigley and Brown (1976)
perennial ice behind the lower entrance, i.e. a “dynamic ice cave”. Such ice caves are
in fact pockets of local permafrost and occur in locations with mean annual temper-
atures far above zero degrees (Luetscher and Jeannin 2004; Perşoiu 2018; see also
Chap. 15). Single-entrance shafts may trap cold, dense air during winter, whilst the
summer condition will cap the opening with warm, dry air, preserving the low
temperature at the bottom. This effect may also sustain perennial snow or ice and
is the “static ice cave” type of Luetscher and Jeannin (2004). Also, deep dolines or
other karst depressions particularly when connected to caves that have other
entrances at higher elevation may function as cold traps and have inverted temper-
ature gradients. Such caves host particular fauna and microbiota (Iepure 2018;
Purcarea 2018; see also Chap. 15).
Atmospheric pressure variations may also control ventilation in single-entrance
caves. Depending on the total volume of the cave system, the diameter of the
entrance and the range and speed of external pressure change, quite strong winds
may occur (Deike III 1960). The phenomenon is called “cave breathing”, as exha-
lation and inhalation often alternate with the pressure change (Wigley 1967).
Running water may entrain air currents through streamsinks into vadose passages
and cause return flow along other parts of the cave or along the ceiling of the same
passage.
2.5.2 Attenuation of Surface Temperature
Surface diurnal and seasonal temperature variations are attenuated in the cave
environment, but they rarely attain complete constancy (Cigna 2002), although an
asymptotic mean can be identified. The deep interior of well-ventilated caves attain
the surface annual mean temperature, and the lower opening of multi-entrance caves
may develop a lower temperature regime than the annual mean surface (Wigley and
Brown 1971, 1976).
Neglecting latent heat effects, it can be shown that the amplitude is attenuated
exponentially along the cave passage from the entrance. Exponential damping is
characterized with a relaxation length, L0, over which the amplitude is attenuated by
1/e. L0 is a function of the passage diameter, d (in metres), and air velocity, v (m/s)
(Wigley and Brown 1976):
L0 ¼ 100 d 1:2 v0:2 ,
which means that L0 is much more sensitive to the diameter of the void than to the air
velocity through it. Temperature attenuation approaches an asymptotic value (Ta)
which, for a well-ventilated cave system, is close to the mean annual surface
temperature, although local effects, like ground heat flux, may cause deviation
from it. When introducing latent heat and humidity, the attenuation deviates signif-
icantly from the Wigley and Brown (1976) equation, although the concept of L0 is
still applicable. The attenuation depends on the relation between the external tem-
perature (Te), the asymptotic deep-cave temperature (Ta) and the dewpoint temper-
ature (Td). From this, three principally different situations may be identified
(Fig. 2.8).
In the summer situation, Te and Td are both greater than Ta, and condensation at
shallow depths may in some cases lead to a slight temperature rise (Fig. 2.8a). In
winter, Te and Td are both smaller than Ta, and the air is simultaneously warmed by
direct contact with the walls and cooled by evaporation of wall moisture so that a
cold zone forms next to the entrance where the temperature is lower than both Te and
Ta (Wigley and Brown 1976). In a similar way, humidity in caves depends on
ventilation and on the humidity of the external air. The model (Fig. 2.8b) suggests
that during summer, a well-ventilated cave never reaches saturation, although local
effects, like annexes and dead-end passages may override the model expectation.
During the transition regime, saturation is reached within one or two relaxation
lengths; in winter this distance is much shorter.
2.5.3 The Chemistry of the Cave Environment
Due to the carbon dioxide equilibria, the chemical environment in carbonate karst
water is buffered to pH 7–9, depending on the total hardness and hydrological history,
e.g. Figs. 2.4 and 2.5. Total hardness ([Ca2+] + [Mg2+]) depends on climate and
respiration in the environment (Atkinson 1977) and ranges from a few mMol L1 in
arctic and alpine environments to several hundred mMol L1 in a humic tropic
environment (Ford and Williams 2007). Similarly, the CO2 content of cave air
depends on respiration and ventilation and may vary from atmospheric values
18 S.-E. Lauritzen
Fig. 2.8 Season-dependent attenuation curves for temperature (a) and humidity (b) amplitudes
through a well-ventilated cave system. Figure modified after Wigley and Brown (1971). See text for
further discussion
(~400 ppm) to >104 ppm or more in closed chambers. Respiration-derived CO2

levels are independent of whether the cave is karstic or not.
In the case of noncarbonate host rocks (silicate, gypsum, halite), or where acces-
sory minerals that may oxidize and form acids are present (e.g. sulphides and skarn
minerals), the pH of the dry and aquatic cave environment may have extreme values.
For instance, the oxidation of pyrite produces sulphuric acid, as revealed by minerals
like jarosite (pH 1–2) and copiapite (pH < 1) (White 1997). Oxidation of base-metal
sulphides may release metallic ions of high toxicity (e.g. Zn2+, Pb2+, Hg2+), whose
concentration gradients may provide conditions where there is selection for metal
tolerance. Similarly, oxidation of hydrogen sulphide as expelled as gas or solute from
petroleum reservoirs or from sulphate reduction may produce quite low pH in

condensation and drip water (Macalady et al. 2007).
Soils in karst caves are often, but not always, basic and provide excellent
conservation for bones, which will otherwise dissolve in acidic soils
(e.g. Shahack-Gross et al. 2004).
2.6 Non-karstic Caves
Lava tubes are remnants of the internal drainage conduits of lava under a hardened
crust (see also Chap. 17). Lava tubes occur most commonly in low viscosity,
“pahoehoe lava”, but are also found in the coarser, “aa lava”. Lava tubes form
when the surface crust of a lava flow freeze, but liquid lava still flows underneath.
Lava tubes may form in two ways, by crusting over an active lava channel and, more
commonly, by liquid lava continuing to flow under a hardened crust. Hydrostatic
pressure from liquid lava may inflate the soft roof and enlarge the conduit (“infla-
tion”). When the upstream supply of lava ceases, the conduit may empty and leave
the roofed cave. Diameters can extend to >6 m, and many are branched. Lava tubes
can attain great lengths and complexities; the longest on Hawaii is some 65 km long
and 1000 m deep. However, the tubes are generally quite shallow; some 1–20 m
below the land surface, sloping lava flows may however give them “depth”. Lava
tubes are of the same age as the lava flow, and most are quite young, as the thin roof
makes them fragile and new lava flows often erode into them. At a temperature of
700–1200 C in flowing lava, the tubes are sterile and pristine at the moment of
formation.
Due to tensile fracturing during cooling and partings between individual lava
flows, basalts host transmissive aquifers. Collapse of cave roofs and occasional
stream capture emphasize the pseudokarstic appearance of lava flows. Due to
elevation differences between entrances, many lava tubes are well ventilated and
may host perennial ice.
Silicate Karst Caves Complex solutional cave systems, containing branchwork, anas-
tomotic and network patterns, exist in quartzites and quartz-sandstone rocks, of which the
most famous examples are those of the tepuis in Venezuela and in western Brazil. Various
modes of genesis have been attributed to such cave types, which display morphology that
is strikingly similar to carbonate karst caves of solutional origin. Quartz is slightly soluble
in water (ca 4 mg L1), amorphous silica cement is more soluble, and the dissolution rate
of quartz in water at moderate pH is in the range of 1012 mMol cm2 s1 (Ford and
Williams 2007). For comparison, the corresponding rates for calcite and dolomite are in
the order of 107 and 1011 mMol cm2 s1. Although slower, but comparable to
dolomite dissolution rates, given sufficient time, quartzite karst caves can form by simple
dissolution. However, it is sufficient to dissolve only the silica cement in quartzite
sandstones in order to create a cavity, as the loose grains can then be evacuated through
incipient fissures (arenization, e.g. Auler 2012) or simply by erosion through zones of
20 S.-E. Lauritzen
lateritization or incomplete cementation (Aubrecht et al. 2011). The latter views have
been challenged by Sauro et al. (2013). Deep weathering of feldspar- and mica-rich rocks,
like granite, gneiss and arkosic sandstones, will produce clay mineral residues that are
easily removed and so can also produce complex cave systems, as with quartzite.
Fissure and Talus Caves Tectonic stresses, due to faulting or unloading, may result
in open fractures and voids in any kind of rock. Along escarpments, tension joints
are likely to develop into fissure caves. Likewise, large talus deposits may host
complex underground cavities. For instance, the so-called Bodagrottorna in Sweden
are 2.6 km long over four levels and cover an area of 4 ha (Sjöberg 1986). In steep
fjord walls of Western Norway, deep talus caves contain ground ice and are efficient
aquifers. Weathered granite may host complex cavities of biospeleological impor-
tance, e.g. Sallstedt et al. (2014) and Bauer et al. (2015). There are also caves formed
by piping (resulted from removal of particles by channelled flow of groundwater; e.g.
Halliday (2007)).
References
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of groundwater hardness in limestones. J Hydrol 35:111–123
Aubrecht R, Lánczos T, Gregor M et al (2011) Sandstone caves on Venezuelan tepuis: return to
pseudokarst? Geomorphology 132:351–365
Auler AS (2012) Quartzite caves of South America. In: White WB, Culver DC (eds) Encyclopedia
of caves, 2nd edn. Academic, Amsterdam, pp 635–639
Badino G (2005) Underground drainage systems and geothermal flux. Acta Carsol 34:277–316
Bauer H, Exel T, Oberender P et al (2015) Die Gobholo-Höhle in Swasiland: Expedition in eine der
längsten Granithöhlen der Welt. Die Höhlen 66:27–42
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Press, Oxford
Curl RL (1958) A statistical theory of cave entrance evolution. Natl Speleol Soc Bull 20:9–21
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the vicinity of the Columbia Icefield, Alberta and British Columbia, Canada. J Glaciol
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of the Australian karsts. In: Proceedings of the 13th Australasian Conference on Cave and Karst
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Perşoiu A (2018) Ice caves climate. In: Perşoiu A, Lauritzen SE (eds) Ice caves. Elsevier,
Amsterdam, pp 21–32
Poulson TL, White WB (1969) The cave environment. Science 165:971–981
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Sallstedt T, Ivarsson M, Lundberg J et al (2014) Speleothem and biofilm formation in a granite/
dolerite cave, Northern Sweden. Int J Speleol 43:305–313
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197:190–196
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remains in cave sites. J Archaeol Sci 31:1259–1272
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geology. J. S. Tolson and F. L. Doyle, Int Assoc Hydrogeol, pp 503–517
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nisms for chemical reactions in karst terrains. Environ Geol 30:46–58
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caves, 2nd edn. Academic, Amsterdam, pp 103–107
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Z Geomorphol 29:463–482
Chapter 3
Where Cave Animals Live
3.1 Introduction
Subterranean habitats support discrete ecosystems composed of communities that

often include species highly specialized to live only underground. The physical
environment is rigidly constrained by the geological and environmental setting,
and because it is surrounded and buffered by layers of rock, it can often be defined
with great precision. These enclosed habitats represent rigorous, high-stress envi-
ronments for most surface organisms, and they are difficult for humans to access and
study. Nevertheless, because the environment is discrete, rigorous, and easily
defined, accessible subterranean habitats provide exemplary systems in which to
conduct biological studies (Culver 1982; Howarth 1983). Although they interact and
share many attributes (Fig. 3.1), the terrestrial and aquatic habitats are described
separately in this chapter.
Communities of specialized cave-adapted species are known from unglaciated
areas in the temperate zones to the humid tropics. In fact, they have been found
wherever suitable voids have been searched; however, many regions have yet to be
adequately surveyed. Biodiversity of troglobites is highest at mid-latitudes in the
north temperate zone (Culver et al. 2006), but this pattern may be an artifact of
incomplete sampling in tropical caves or other regions.
Only a small subset of the local surface taxa have successfully colonized subter-
ranean habitats in each region. The ancestors appear to have been preadapted for
cave life; that is, they already preferred to live in damp, dark habitats on the surface.
In temperate regions, many cave species were considered relictual since they have
survived severe climatic changes during glacial episodes, while their surface
F. G. Howarth
Bernice P. Bishop Museum, Honolulu, HI, USA
O. T. Moldovan (*)

https://doi.org/10.1007/978-3-319-98852-8_3
24
Fig. 3.1 Diversity of cave terrestrial and aquatic habitats and microhabitats. Original drawing by Oana T Moldovan
F. G. Howarth and O. T. Moldovan
24
3 Where Cave Animals Live 25
relatives became locally extinct (Barr 1968; Barr and Holsinger 1985). Recent
advances in molecular phylogenies (see Chap. 12) contradict this view, and active
colonization of subterranean habitats is advocated.
Box 3.1
In caves, habitats are of different sizes, but the degree of habitat interconnec-
tivity (the so-called permeability of rocks) makes possible the dispersal of cave
fauna throughout large and small voids in a karst massif. The common features
of cave (and other subterranean) habitats are constant darkness; relative
constancy of temperature and high relative humidity; food resources that are
primarily allochthonous and scattered; often limited potential for subterranean
dispersal, as this ability is contingent on the presence of suitable
interconnected voids; and absent or weakened environmental cues used by
surface species for dispersing, locating food, and reproduction in the three-
dimensional dark maze.
There is a continuum between caves and the other subterranean habitats

(Table 3.1) with barriers represented by different types of rocks, water, or sediments
that limit animals migration. Despite this continuum, the diversity of cave habitats is
reduced compared to the surface, with an increase of the physical inertia with depth
according to Gibert et al. (1994). Nevertheless, slight differences between different
parts of the cave habitats are discernible, and these define the microhabitats
(Table 3.1; Fig. 3.2). Spatial diversity of microhabitats within a three-dimensional
profile of a karstic massif can be much higher than supposed because environmental
factors, intrinsic population processes, and disturbances can shape spatial biodiver-
sity on very small scales. They also provide spatial and temporal protection from
disturbance and advantages in biotic interactions (Keppel et al. 2011). The use of
different microhabitats is one mechanism that makes possible the coexistence of
species with the same environmental requirements and using the same resources
(defined as niches), especially in caves where living conditions and food are patchily
Table 3.1 Examples of Microhabitats Caves MSS

terrestrial subterranean
Damp mud, clay, and other sediments √ √
microhabitats where cave-
adapted species are often Under and near rocks and debris √ √
found. MSS ¼ mesocavernous Decomposing organic material √ √
shallow substratum; see also Guano deposits √
explanation is the text Wet flowstone √
and Fig. 3.2
Damp walls √
Hygropetric habitat √
Biomineral oozes √
Plant roots √ √
Water surface √
26 F. G. Howarth and O. T. Moldovan
Fig. 3.2 Examples of microhabitats in caves: (a) biomineral ooze with larval trails of the crane fly,
Dicranomyia jacobus, on a wall in a Hawaiian lava tube; (b) tree roots (probably Ficus sp.) in the
stagnant air zone of Bayliss Cave, Queensland, Australia; (c) Hadesia sp. (Coleoptera) feeding on the
film of water flowing on calcite and defined as the hygropetric habitat. Photos by © Francis G
Howarth (a, b) and © Jana Bedek (c)
dispersed. Cave microhabitats were defined in studies on cave invertebrates and

vertebrates based on environmental characteristics for both terrestrial and aquatic
representatives, for example, substrate and the available space (Christiansen 1961,
1965; Trontelj et al. 2012); physicochemical characteristics of the water (Moldovan
et al. 2012; Brancelj 2015); water depth and flow regime (Culver 1970; Moldovan
et al. 2012; Jourdan et al. 2014); concentration of H2S in water (Sârbu et al.
1996; Jourdan et al. 2014); abundance of food, such as carbon and nutrients (Jourdan
et al. 2014); presence of biomineral oozes (Barton and Northrup 2007); and tree
roots (Howarth et al. 2007); and distribution of predators (Jourdan et al. 2014).
Microhabitats in caves can be temporary due to changing microclimatic features,
such as drying of pools or of wet surfaces. Some species can be found in different
microhabitats, while others are microhabitat specialists. The best examples are given
by microhabitat partitioning in the caves with the highest diversity in cave fauna.
One of these caves is Vjetrenica Pećina in Bosnia and Herzegovina with more than
60 cave species (Culver and Sket 2000), where some of the species can be found in a
single place of no more than few square meters (OTM, personal observation).
3.2 Terrestrial Cave Habitats
3.2.1 Abiotic Factors Characteristic of Cave Communities
Substrate Subterranean habitats occur in a broad range of sizes from tiny voids to
exceptionally large caverns and from shallow to great depth beneath the surface.
They also are found in many different rock and substrate types, but solution caves in
limestone and volcanic caves in basaltic lava are the best known. Subterranean
habitats can be divided into three size classes based on the environment and
communities supported. These are microcaverns (generally <5 mm in width),
mesocaverns (~5–500 mm in width), and macrocaverns (i.e., caves >50 cm in
width) (Howarth 1983).
Microcaverns rarely support terrestrial species because these tiny spaces quickly fill
or become blocked by debris. However, flowing water can keep such spaces open
and interconnected as well as transport food resources.
Mesocaverns are intermediate-sized spaces, large enough to remain open and
serve as dispersal corridors for cavernicolous animals yet small enough to restrict
airflow and gas exchange. Food resources can also be limiting especially in smaller
passages. Recent studies using new sampling methods have demonstrated that
mesocaverns provide a major part of the habitat as well as dispersal routes between
caves for many cave-adapted species (Howarth 1993; López and Oromí 2010).
Mesocavernous networks are especially important in young basaltic lava flows,
unconsolidated river deposits, and talus slopes. In addition, they can occur in almost
any rock type in which erosional or depositional processes create and maintain
interconnected air-filled voids. Based on faunal characteristics and environment,
mesocavernous shallow substratum (MSS) is considered distinct from deeper
mesocavernous profound substratum (MPS) voids (Juberthie 1983). If the MSS
merges with deeper caves, these shallow habitats can harbor a subset of the cave
fauna (Uéno 1987) as well as many unique species (Derkarabetian et al. 2010; Olmi
et al. 2014; Jiménez-Valverde et al. 2015).
Finally, macrocaverns include the accessible cave passages familiar to most
people. They commonly occur in soluble rock types (such as limestone, dolomite,
gypsum) and in basaltic lava flows, but they can also be created in other rock types
by a variety of geologic processes (see also Chaps. 17, 19, and 21). The fauna of
limestone caves is best known partly because these are the most accessible and long-
lasting stable caves. In the past few decades, lava tubes have been recognized as also
harboring highly diverse cave ecosystems (Ashmole et al. 1992; Peck and Finston
1993; Howarth 1996). Accessible cave passages also often harbor large colonies of
vertebrates (e.g., birds and bats) that introduce large quantities of food resources into
caves (Price et al. 2004; Holland et al. 2009; Ladle et al. 2012).
Darkness The single overarching characteristic of subterranean environments is the
absence of light. Total darkness affects nearly all aspects of how organisms adapt
and respond to environmental signals. Cues used by surface species (such as sight,
orientation, air motion, odor plumes, sound) are absent or act inappropriately in
caves and mesocaverns. Special behavioral and physiological characteristics must be
important for animals to avoid or escape harmful circumstances, locate food, and
reproduce in the dark three-dimensional mazes.
Atmosphere The atmospheric variables affecting cave ecosystems include temper-
ature, relative humidity and potential evaporation and condensation rates, diurnal
and seasonal periodicity, air movement, and gas concentrations (see also Chap. 2).
These factors vary according to the size, shape, and location of the passage within
the cave as well as by biogenic activity and climatic events. Fortunately, these
factors can be understood by considering caves as zonal habitats, with each zone
defined by these variables.
3.2.2 Environmental Zones
Box 3.2
Terrestrial subterranean habitats are strongly zoned, with five environmental
zones recognized, each with different communities of organisms and defined
on the basis of its physical environment, especially the amount of light,
moisture, air flow, gas concentration, and evaporative power of the air.
These are the entrance, twilight, transition, deep, and stagnant air zones
(Fig. 3.3 and Table 3.2) (Howarth 1993). Since potential evaporation rate
increases nearly exponentially with temperature, the boundaries between cave
zones are often more pronounced in tropical caves. Also, the boundaries
between zones are often highly dynamic within given caves, and animals
living in one zone may make brief forays into neighboring zones for food
and dispersal or even by accident, making static descriptions and predictions
on animal distributions tenuous. Nevertheless, the zonation provides a useful
classification scheme to understand cave ecology.
The entrance zone (euphotic) includes the area with sufficient light for vascular plant
life. It is the zone of mixing of surface and underground communities, and since the
more mesic environment can produce more food resources, this zone often supports
a larger number of species of plants and animals than is found in nearby surface or
cave habitats. The twilight zone, as the name implies, is the area with reduced light
between the limit of vascular plants and the region of total darkness. It is alternately
wet and dry, with a high and variable potential evaporation rate. Species diversity is
low and mostly composed of waifs from neighboring zones, surface animals seeking
shelter, scavengers, and predators. Beyond the twilight zone, there is often a
transition zone, which is characterized by total darkness and a variable abiotic
environment, especially changing humidity levels, airflow, and potential evaporation
Entrance-zone Twilight-zone Transition-zone Deep-zone Stagnant-zone
Stagnant-air-zone
Fig. 3.3 Stylized profile view of a cave showing the five environmental zones. Scale for length
greatly condensed relative to height. Figure modified after Howarth (1993), with permission
Table 3.2 Abiotic and biotic parameters of each of the five zones found in caves
Dark
Zones Entrance Twilight Transition Deep Stagnant air
Light Sunshine/ Shade to edge Total Total darkness Total darkness
vascular of darkness darkness
plants
Temperature Ambient Reduced Nearly stable Stable Stable
surface variation
Humidity Variable/ Reduced var- Reduced var- Saturated Saturated/
desiccating iation/ iation/ condensing
desiccating desiccating
CO2 (vol- 0.04% 0.04–0.5% 0.04–1% 0.1–3% 3–>6%
ume %)
Fauna Surface Trogloxenes, Trogloxenes, Troglobites, Predominately
troglophiles, troglophiles troglophiles, troglobites
waifs trogloxenes
rate. The variability is produced by diurnal and seasonal climatic cycles as well as by
local weather events on the surface. The habitat is generally dry, and species
diversity is generally low and composed of trogloxenes, scavengers, predators, and
waifs. However, locally large colonies of trogloxenes, such as bats or cave crickets
(Lavoie et al. 2007), can support a high diversity of guanophiles and guanobionts.
Twilight and transition zones are sometimes recognized as disphotic zone.
The deep cave zone (aphotic) lies beyond the transition zone where light is also
completely absent; the air remains still and saturated with water vapor, the substrate
remains moist, and potential evaporation rate is negligible, at least for relatively long
periods of time. There is usually a constriction in the passage that creates a barrier to
air exchange and marks the boundary between the transition and deep cave zones.
Caves that have multiple entrances or do not have such an obstruction may never
develop a deep cave zone. The most stable deep cave zone environments often occur
in dead-end upper-level passages since water vapor is roughly 0.625 as dense as air
and thus can become trapped in such passages. This is the zone in which cave-
adapted animals typically are found. Many troglophiles and trogloxenes, including
both invertebrates and vertebrates, also inhabit this zone.
The stagnant air zone occurs where air exchange is restricted even further; the
atmosphere periodically stagnates and gas concentrations, particularly carbon diox-
ide, become stressful. There is increasing evidence that this is the primary habitat of
troglobitic species (Howarth and Stone 1990). This zone is believed to be the
principal zone present in mesocavernous cracks and voids (Howarth 1993).
Behavioral, ecological, and environmental evidence all indicate that many of the
specialized cave animals live primarily within these medium-sized voids and enter
cave-sized passages only where the stagnant conditions are approximated. Thus,
important habitats may extend significantly beyond accessible cave passages. Caves
allow researchers to enter only the accessible fringes of subterranean habitats to
conduct studies on the underground community.
3.3 Aquatic Cave Habitats
Part of the precipitation that falls as rain or snow seeps underground and percolates
downward through soil and rocks until it reaches the water table or saturated zone
(Fig. 3.4). Subterranean water is called groundwater, which can fill voids completely
or only partially as it sinks to the water table (see also Chap. 2). The water-filled
voids comprise the groundwater habitats, which occur at different scales (Danielopol
1989), from the largest, the macrohabitat (known as aquifers), that can cover from
1 to more than 100 square kilometers in area; through mesohabitats, intermediate in
size and covering few square meters up to less than one square meter in area; to
microhabitats that are small in sizes that can be measured in square centimeters.
Macrohabitats are more heterogeneous and represent the principle living space of
most cave-living aquatic animals. Mesohabitats have relatively uniform physico-
chemical features and represent part of the species’ living space that is used during
different periods of the year or their life cycle. Microhabitats define the habitat of an
animal at a certain moment in time. Within microcaverns, liquid water movement is
controlled largely by capillary forces; however, flowing water under sufficient
pressure can keep such spaces open and interconnected as well as transport food
resources, which can provide suitable microhabitats for tiny aquatic species. Con-
solidated and unconsolidated rocks provide distinct microhabitats for different
species assemblages (Gibert et al. 1994; Malard et al. 2009). In consolidated rocks
(limestone, dolomite, granite, basalt, and sandstone), the voids can be from big
(especially in limestone and dolomites) to small in size, different from unconsoli-
dated rocks (gravel to sand) where voids are generally small but can be increased by
bioturbation (Datry et al. 2003).
Cave Surface Hypo- Perched Water Anchialine Littoral

stream Pools Spring stream thelminorheic aquifers table lake interstital
Hyporheic
zone
Phreatic
lake
Submarine
Confined aquifers lake
Legend
Limestone Clay Halocline
Gravel
Freshwater Sea water Rocks & debris
Fig. 3.4 The relationship between aquatic cave habitats and other subterranean aquatic habitats in
karstifiable limestone
3.3.1 The Aquatic Living Space
Groundwater habitats in unconsolidated sediments and in various rocks (Table 3.3)

represent one of the most extensive ecosystems on our planet (Gibert 2001). The
degree of connectivity between cave habitats and other types of groundwater habitats
depends of the rock permeability for animal migration and for nutrients, organic
carbon, and dissolved oxygen input (Cornu et al. 2013). Therefore, groundwater
habitats form a domain that extends from the superficial hypothelminorheic to the
interstitial of the marine littoral (Fig. 3.4). Groundwater animals (i.e., stygobionts;
see also Chap. 4) were considered to have much larger distribution areas than
troglobionts. However, more recent molecular analysis has brought a different
view on the distribution of groundwater species to less than 200 km in linear extent
(Trontelj et al. 2007; see also Chap. 9).
Box 3.3
Carbonate dissolution in karstic systems results in highly heterogeneous rock
permeability. Rock permeability is important because it defines the living
space of cave animals, including stygobionts. In karst massifs two major
zones are defined according to the hydrologic behavior: the saturated and the
unsaturated zones (Fig. 3.1). The unsaturated zone has two main parts, the
epikarst and the vadose zone, the last containing also most of the caves
accessible to humans.
Table 3.3 Examples of aquatic cave habitats/microhabitats and connected groundwater habitats
where stygobionts were found
Cave and microcave aquatic habitats/microhabitats Other groundwater habitats
Dripping water Hypotelminorheic
Small pools (gours)/puddles Springs
Big pools/lakes Wells
Streams Hyporheic zone ¼ freshwater interstitial
Riffles Littoral interstitial
Rocks in water
Sediments next to water bodies
Underwater/marine caves
Calcretes (see Chap. 18)
The epikarst is a highly weathered zone (see Box 3.3) where water can be stored
for long periods with a leaky capillary barrier at its base (Mangin 1975) that can
reach a depth of 0.5–2 m on average (Bonacci 1987). The epikarst is characterized by
slow percolation of air and water into narrow fissures and water storage. In the
vadose zone, water percolates from the surface to the water table by rapid drainage
through connected pipes (Ford and Williams 2007). The saturated zone is often
sandwiched between the unsaturated zone and an impermeable level. The saturated
karst is also known as the “flooded karst” (or phreatic zone) and represents the main
water storage of the karst aquifer (see also Chap. 2). Aquifers are rock formations
that can store, transmit, and yield economically significant amounts of water (Ford
and Williams 2007). Karst aquifers like those of other rocks are of three types:
unconfined, confined, and perched. The unconfined aquifers, known as water table
aquifers, extend to the surface and are more vulnerable to climatic and human influ-
ences. Confined aquifers are isolated from the surface by overlying and underlying
impervious geologic formations, but they can also be limited by thick layers of
un-karstified rocks (Ford and Williams 2007). Perched aquifers are suspended
above the water table, within the vadose zone, and they are of small volumes,
vulnerable to climate conditions, and therefore highly variable in time. Natural and
artificial wells are the windows to the confined aquifers, and springs are the outlets of
the karst aquifers. Both wells and springs represent accessible habitats for research
and fauna observations.
Aquatic cave habitats in karstic rocks were classified and defined into four
categories by Leruth (1939). Here, we redefine these categories and add a fifth one
(see also Fig. 3.1):
1. Highly dynamic flowing waters (sinking streams): these can be permanent or
temporary cave rivers with high flow that bring gravel from the surface or rework
the rocks inside the cave, shaping the walls and the riverbed by forming smooth
surfaces and few hiding places for aquatics. These waters provide few habitats
suitable for cave animals, although they can bring important amount of organic
matter and debris from the surface. The fast-flowing water and the lack of
constancy hamper the establishment of aquatic cave microhabitats, and they are
usually dominated by surface aquatic invertebrates and even vertebrates (sala-
manders, fishes, and frogs; Ginet and Decou 1977).
2. Slow-flowing waters and lakes are characterized by the fine sediments deposited
on the bottom. Slow or no flow and the presence of sediments of different sizes
are the prerogatives for forming microhabitats for aquatic fauna and even terres-
trial fauna on the water surface. The diversity of this type of habitat can be very
high, from free-swimming large stygobionts, such as crustaceans and fishes, to
minute animals, which rest in hidden places, such as under rocks or in sediments.
Slow-flowing waters are the most stable aquatic cave habitats because with their
high volume, they are less prone to complete drying. This type of habitat can be
fed by surface water, groundwater, or films of water flowing through the
vadose zone.
3. Gours or pools formed on flowstone are of different volumes and are present in
most limestone caves. They are formed by the dripping water and, thus, can be
permanently or temporarily filled with water; also, they can be filled during
flooding periods if a cave river flows nearby. These pools can have clay and/or
organic matter brought by water from the karstic voids above. However, such
gours can have important communities that belong both to aquatic and terrestrial
fauna: amphipods and harpacticoids hiding in the sediments, swimming
cyclopoids, and collembolans and acari on the surface of the water. They can
form a microcosm where dripping water brings new prey or predators and food
from the vadose zone and sometimes even an unexpected terrestrial visitor such
as beetles or pseudoscorpions can hide under the calcite and sediments near the
water.
4. Small pools on clay or mud can be dynamic aquatic habitats because they can
form only during periods of very intense percolation or by the flooding rivers.
During the periods with water, they can contain crustaceans from the vadose zone
(Brancelj 2015).
5. Dripping or percolation water brings inside caves both epigean fauna from the
surface and hypogean fauna from the unsaturated zone above. Dripping water is
an extremely rich domain that can contain hundreds of individuals of different
species, especially crustaceans, and its richness was for the first time mentioned
by Rouch (1968) and Delay (1969), followed by many other researchers in the
last decade. The ways taken by the water from the surface to the collection point
inside caves can be very different, even for drips few meters apart. Water crosses
different limestone layers, fractures, and voids of different sizes; it can remain
longer periods in the unsaturated zone, from minutes to days and even years
depending on the voids size and their interconnectivity.
Uniformity and relative stability characterize cave waters that are not in direct
contact to the surface waters (Camacho 1992). However, differences between
microhabitats exist and are sometimes measurable inside a single cave where one
can find different species if it collects different dripping points or pools (Moldovan
et al. 2012; Brancelj 2015; Fig. 3.5).
Fig. 3.5 Distribution and abundance of species in seven dripping stations of the Ciur Izbuc Cave
(Romania); species are in abundance order in each station. Modified from Moldovan et al. (2012)
3.3.2 Groundwater Habitat Features
Box 3.4
Aquatic subterranean habitats potentially include all the voids with water and
conditions of survival and reproduction. What best defines the groundwater
habitats, beside the features common with terrestrial subterranean habitats, are
usually the relatively constant chemical composition, low concentration of
organic matter, complex relationship with surface water (Schmidt and Hahn
2012), flow rate, permeability, and void size (Cornu et al. 2013). The con-
stancy of physicochemical features seems to be more important for the sur-
vival and range extension of groundwater fauna than the seasonality and
availability of habitats (Zagmajster et al. 2014).
Darkness is absolute in cave waters, as in all subterranean habitats. Water temper-

ature is relatively stable in deep habitats and very similar to air temperature inside
the cave. The slight difference is no more than 1 C lower (Camacho 1992).
Dripping water can have more variable temperatures that are in the order of few
degrees in the deep zones (e.g., ~ 5 C maximum in Meleg et al. 2012). Flow rate,
velocity, and water level in aquatic habitats can be variable if in direct contact to the
surface waters. High flow rates bring organic matter underground, but it destabilizes
the constancy of cave habitats, which in turn may promote the short-term coloniza-
tion of epigean species. Slow flow limits the input of nutrients inside caves that
become even scarcer with depth. Water pH is neutral or slightly acidic in limestone/
dolomite caves due to the presence of carbonic acid buffered by carbonates. Oxygen
content is generally low mainly because primary producers, the main water oxygen-
ators, are absent in cave waters. Cave waters with highly dynamic flows can have
more oxygen produced by physical processes. Hardness of the water is given by the
calcium ions that also give the karstic water the beautiful turquoise color. Water
mineralization depends on five independent variables (Bakalowicz 1974):
karstification degree, aquifer confinement, sediments cover, surface washaway and
its loss, and bioclimatic conditions on the surface.
Acknowledgements OTM acknowledges the financial support from the Romanian Academy and
the grant of the Romanian Ministry of Research and Innovation, CNCS—UEFISCDI, project
number PN-III-P4-ID-PCCF-2016-0016, within PNCDI III.
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Part II
There is Rich and Diverse Life in Caves
Chapter 4
The Ecological Classification of Cave
Animals and Their Adaptations
4.1 History of Classification Schemes
During the earliest studies of cave biology, researchers soon recognized that animal
species living permanently in caves often shared a suite of similar morphological
features, even when only distantly related (Racovitza 1907; Vandel 1964). The most
notable features are the loss or reduction of structures normally considered necessary
for life, such as eyes, bodily color, and robustness. To better understand cave
ecology, early workers attempted to classify cave animals by correlating the different
degrees of morphological change with the observed levels of their association with
caves. However, the high variability in morphology displayed by cavernicoles, lack
of ecological data for most species, and the subjective nature inherent in determining
their status in caves make ecological classification of some species problematic.
One of the earliest attempts to classify cave animals was presented by the Danish
biologist Schiødte (1849). He proposed four levels or categories distinguished by the
amount of light and the nature of the walls in the preferred habitat: (1) shadow
animals (Skygge-Dyr), (2) twilight animals (Tusmorke-Dyr), (3) obscure area ani-
mals (Hule-Dyr), and (4) obscure area with flowstone animals (Drypsteenshule-
Dyr).
Later, Schiner (1854) visiting several caves in southeastern Europe recognized
three categories of cave animals based on their ethology: (1) occasional hosts,
animals encountered in caves but also living in surface habitats; (2) troglophiles,
animals inhabiting regions where daylight still penetrates but which can be rarely
encountered on the surface; and (3) troglobionts, animals living exclusively in caves
F. G. Howarth
Bernice P. Bishop Museum, Honolulu, Hawaii, USA
O. T. Moldovan (*)

https://doi.org/10.1007/978-3-319-98852-8_4
and which are only encountered in epigean habitats following exceptional events
such as floods.
Joseph (1882) was a German biologist who proposed three categories
corresponding to topography: (1) inhabitants of cave entrances, i.e., well-lit regions
with variable temperature; (2) inhabitants of middle areas, i.e., where the noon sun
produces a kind of twilight; and (3) inhabitants of deep areas, with complete
darkness and constant temperature.
These early attempts were reviewed by Racovitza in 1907 who subsequently
provided more precise definitions of each category. Racovitza’s system proved
useful in understanding the evolutionary ecology of cavernicoles and his scheme,
which is called the Schiner-Racovitza classification, became the basis for all subse-
quent classifications (see Box 4.1).
Box 4.1
Racovitza’s original definitions are as follows:
1. Trogloxenes are lost or occasional visitors that do not live or reproduce in
caves. They rarely display any special adaptive features for life in caves,
and moreover, they usually stay near cave entrances.
2. Troglophiles are able to live and reproduce in subterranean environments
but are also able to live in surface habitats. In caves, they generally prefer
areas near entrances. They may have reduced eyes and other adaptations to
life in darkness.
3. Troglobionts exclusively inhabit subterranean environments and usually
are found only in the deepest areas of caves. They are highly modified
morphologically for underground life. Racovitza believed troglobionts were
among the oldest cave animals.
Unfortunately, placing cavernicoles in a classification scheme is often subjective

until their true association to caves is better known. Given the variability and
differing degrees of adaptation to caves and other subterranean habitats, it is often
not possible to determine the status of all species based on morphology or the
environment where they are found. Many species found in caves also live in soil
or cryptic habitats on the surface and their occurrence in caves may be accidental or
facultative. In cases where the status is uncertain, the general terms epigean and
hypogean are often used. Sket (2008) and Trajano and Carvalho (2017), among other
authors, discussed the difficulties and pitfalls in the application of the Schiner-
Racovitza classification. Trajano and Carvalho (2017) identified difficulties in the
“separation of subterranean organisms from accidentals, use of troglomorphisms to
infer the troglobitic status, distinction between troglophiles and trogloxenes, detec-
tion of obligatory trogloxenes.” Sket, retrieving a classification proposed by Ruffo
(1957), proposed to split the troglophiles in two categories, the subtroglophiles
(species inclined to perpetually or temporarily inhabit a subterranean habitat but
intimately associated with epigean habitats for some biological functions like
4 The Ecological Classification of Cave Animals and Their Adaptations 43
Fig. 4.1 Habitus of the epigean Catops fuliginosus (left, Photo by © Udo Schmidt) compared to
cave species at different degrees of adaptation to life in caves: Ptomaphagus hirtus, Speonomus
hydrophilus, Closania winkleri, and Leptodirus hochenwartii (Photos by © Pawel Jaloszynski)
feeding, seasonally or during the life history) and the eutroglophiles (epigean species
able to maintain a permanent subterranean population). Further discussions on
classifications are also presented in Chap. 7.
Christiansen (1962, 2012) coined troglomorphy as a general term for adaptive
traits commonly associated with cave-adapted species. An animal displaying at least
some troglomorphies and also known only from caves can be consid-
ered troglobiont/stygobiont (Fig. 4.1). The term troglomorphic is roughly equivalent
to “le facies morphologique cavernicole” (¼ the cavernicole morphological appear-
ance) used by Vandel (1964). How and why troglomorphies have occurred has long
intrigued both evolutionary biologists and laymen.
Preadaptation was originally proposed by Cuénot in 1911. As used here, the term
refers to a set of characters that evolved within a population, which allowed the
population to survive in a specific environment and which subsequently facilitated
the colonization of a novel habitat with a similar environment. For example, many
cave species have relatives living in cryptic dark and moist habitats on the surface,
such as in soil, under rocks, among moss, etc.
4.2 Overview of Common Features of Cave Inhabitants
The relationship of cavernicoles to subterranean environments ranges from being

casual visitors to highly specialized animals that are restricted to life underground.
Consequently, the traits displayed by cavernicoles are frequently related to their
Table 4.1 Common troglomorphic features for cave representatives. Modified after Langecker
(2000) and Christiansen (2012)
Features Troglobionts Stygobionts
Morphological features
Pigmentation loss All groups All groups
Loss or reduction of eyes/ocelli and eye All groups All groups
structures
Increased body size/body elongation Arachnids, Coleop- Some crustaceans, fishes
tera, Collembola,
etc
Elongation of appendages All groups Some crustaceans
Elongation/increased number/different All groups Cave Asellus aquaticus and
spatial distribution of sensory receptors some other crustaceans, fishes
Cuticle thinning/scale reduction All groups All groups
Physiological features
Low metabolism rate All groups All groups
Fasting/lipid storage All groups All groups
Dietary changes All groups All groups
Loss of water balance mechanisms All groups –
Tolerance to high CO2/low O2 All groups All groups
Behavioral features
Loss of circadian rhythm All groups All groups
Changes in mating behavior All groups All groups
Special features
Life cycle reduction All groups All groups
Egg number reduction All groups All groups
Egg volume increase All groups All groups
degree of association with caves (Howarth 1983; Trontelj et al. 2012). These traits
range from no discernible adaptation among waifs and casual visitors in caves to the
often-bizarre modifications displayed by obligate cave species. The morphological,
physiological, and behavioral modifications displayed by obligate subterranean
animals are convergent and arose independently in different taxa (see also
Table 4.1 and Fig. 10.6). Convergent morphologies of cave (subterranean) animals
are highly predictable (Derkarabetian et al. 2010), although explanations of their
function and fitness are rarely provided (Hüppop 2000; Jeffery 2009). In caves, there
is one major selective environmental feature whose extreme value gives predictabil-
ity and convergent traits, and this is the lack of light (Culver et al. 2010;
Derkarabetian et al. 2010).
4.3 Adaptations to Caves by Terrestrial Animals
4.3.1 Morphological Adaptations
Box 4.2
Loss of sight, bodily color, and fragile appearance are the classic hallmarks
displayed by cave-restricted species and initially were considered examples of
enigmatic regressive evolution (see also Chap. 10). Additional reductions of
morphological features include degeneration of wings and flight among
insects and thinning of the cuticle of arthropods. These traits are often con-
vergent, that is, evolved independently in different taxa inhabiting caves
(Figs. 4.2 and 4.3).
Although reductive troglomorphies are often the most conspicuous features of

obligate cavernicoles, there also are many “positive” changes. Examples include
an increase in size and number of sense organs (receptors); elongation of the body,
legs, antennae, and other appendages (Figs. 4.1 and 4.3); and specialization of foot
structures in Collembola and other terrestrial arthropods. The reduction of the
pronotum displayed by the most evolved cave Leptodirini and Trechini beetles is a
feature probably linked to the mechanics of locomotion associated with elongated
legs. The increase of the antennal surface is not by widening but by lengthening of
the antenna (Fig. 4.3a). This lengthening allows for an increase in the total number of
receptors, especially those responsible of mechanical reception (Moldovan et al.
2004). In springtails, the tarsal specializations allow the animals to walk on water
and climb the meniscus and thus escape from pools of water (Christiansen 1965).
Fig. 4.2 Hawaiian Lycosidae spiders comparing epigean, Lycosa hawaiiensis (left), and two cave
species: Lycosa howarthi with vestigial eyes (center) and Adelocosa anops with no eyes (right)
(Photos by © William P Mull)
Fig. 4.3 Examples of morphological adaptations in cave beetles (Leiodidae) compared to their
troglophilic relatives: (a) antennal length in troglophilic Ptomaphagus elabra (left) and cave
P. troglomexicanus (right); (b) sternal exocrine gland in endogean Bathysciola meridionalis (top)
and cave B. delayi (bottom) [Figures from Peck (1973; a) and Moldovan (1998; b)]
Changes in pretarsal structure also occur in some cave planthoppers (Howarth 1981;
Hoch and Howarth 1989). In this group, the arolium (a flexible pad at the apex of the
tarsus) is vestigial, whereas the claws are greatly elongated and adapted for walking
on barren wet rock instead of walking on vegetation (Fennah 1973). In beetles,
reduction of the sternal exocrine gland, which probably produces pheromones, has
been documented in cave representatives of Bathysciola (Fig. 4.3b; Moldovan and
Juberthie 1994). More about the specific adaptations in some groups can be found in
Chaps. 7 and 8.
4.3.2 Physiological Adaptations
Dietary Changes Troglobionts often broaden the types of food eaten compared to
their surface relatives; e.g., many species become omnivores, and some predators
add carrion to their diets. In addition, many troglobionts are able to consume
prodigious amounts of food and then are able to survive long periods without eating.
In caves, food resources are often scattered and difficult to find and exploit; there-
fore, cavernicoles cannot afford to be selective and adapt to take advantage of a
broad range of resources discovered.
The Leptodirini group of cave beetles are mainly detritivorous or saprophagous.
Differences in their mouthparts are obvious among genera and even among cave
species that display the same level of adaptation to life in caves. The main modifi-
cations concern the shape and structure of the different parts, as well as their length
and both position and number of hairs, bristles, and setae (Moldovan et al. 2004).
Some species show modifications of the mouthparts associated with their dietary
niche, being adapted to a semiaquatic way of life such as found in hygropetric
habitats on cave walls (Fig. 4.4). The adaptation to an aquatic niche has modified the
mandibles, which acquired a spoonlike form to bring water near the mouth, the
lacinia, which has taken on the role of stirring, and the galea, which can filter organic
particles with its modified fine and very dense hairs. Other terrestrial groups, such as
Diplopoda and Collembola, may also be adapted to take food from water with
modified mouthparts, especially structures evolving toward a spoonlike form
(Deharveng and Christian 1984; Enghoff 1985a, b; Adis et al. 1997; Enghoff et al.
1997).
Low Metabolism Rate Troglobionts often exhibit low levels of activity and low
mobility even during active periods. This is usually correlated with a low metabolic
rate (Hüppop 1985), but in some studies comparing closely related cave and surface
species, this relationship is unclear. In Caconemobius rock crickets in Hawaii, the
surface species has a high metabolic rate when active at night but rests in a torpid
state during daylight, whereas its cave-adapted relative is continuously active at a
Fig. 4.4 Frontal view of the mouth of Hadesia vasiceki (left) and ventral view of the head of
Tismanella chappuisi (right), both cave beetles (Leiodidae Leptodirini): A antenna, g galea, lb
labium, lp labial palp, M mandible, me mentum, mp maxillary palp [From Moldovan et al. (2004)]
low metabolic rate, as would be expected. However, averaged over 24-h periods and
corrected for weight, the two morphs have a similar overall rate of oxygen con-
sumption (Ahearn and Howarth 1982).
The reduced metabolic rate, as well as conspicuous loss of normally critically
important traits such as eyes and pigments, has been considered evidence that
troglobionts evolved to exploit food-poor environments (Hüppop 2005). This
hypothesis appears corroborated by the apparent barren rocky substrate usually
present in deep cave passages. However, energy conservation is a universal trait
shared by nearly all organisms in adapting to their environment, and therefore, such
adaptations may not indicate a food-poor environment. In fact, other evidence
suggests that food-rich environments may also foster reductions in morphological
and physiological traits. For example, among polymorphic arthropods living in
resource-rich habitats, the greatest reduction in morphological features often is
correlated with the presence of the largest amount of stable food resources, and
food scarcity triggers a switch to individuals with greater dispersal ability (Steffan
1973; Roff 1986). Also, the degree of cave adaptation found among troglobionts is
not closely linked to the amount of energy sources available, nor does it indicate the
age or time since the colonization of the cave system. Studies in Hawaiian (Hoch and
Howarth 1999) and Australian caves (Hoch and Howarth 1989) demonstrated that
the degree of cave adaptation is strongly correlated with the cave environment and
extent of geographic area available rather than with age of the cave or amount of
available food. The relative abundance of food resources, however, appears to be
correlated with the numbers of individuals and species that can share the habitat.
Fasting for long periods is possible due to the capacity of large body for fat
storing, a depressed metabolism, during which they mainly subsist on lipid stores, a
prolonged state of glycogen- and protein-sparing, and low energetic requirements
(Hervant and Renault 2002). This ability to store fat is found in all life stages, i.e.,
eggs, juveniles, and adults (Fig. 4.5).
Water Balance Deep cave environments, where troglobionts live, remain at or near
100% relative humidity, and occasionally the air becomes supersaturated. Saturated
atmosphere is stressful for most epigean organisms, and cavernicoles have altered
their water balance mechanisms to cope. Since saturated air is above the equilibrium
humidity of bodily fluids, troglobionts must deal with excess water rather than
desiccation. In doing so, they have lost many attributes (such as reduction and
change in composition of cuticular hydrocarbons and of thinning of the cuticle)
that conserve water and have become highly sensitive to desiccation (Howarth 1980;
Ahearn and Howarth 1982; Hadley et al. 1981).
CO2 Tolerance Decomposition of organic material can increase the concentration
of carbon dioxide to stressful levels in underground environments. Most inverte-
brates can tolerate brief exposure to high CO2 concentrations. Organisms living in
soil and shallow mesocaverns may be able to migrate to less stressful conditions, but
species living in deeper caves, especially those in deep mesocaverns, would be
unlikely to find refuge habitats since the dark, three-dimensional maze provides
Fig. 4.5 Electron microscope details of fat stored in the form of glycogen vesicles in the posterior
abdomen of cave beetle Drimeotus bokori (male; 11,400): g ¼ glycogen, m ¼ mitochondria
(Photo by © Oana T Moldovan)
few cues for escape. Humid bad air passages have been found to harbor highly
diverse communities of troglobionts (Howarth and Stone 1990).
4.3.3 Behavioral Adaptations
The perpetually dark, complex three-dimensional mazes in caves and mesocaverns

(see also Chap. 3) present special problems for organisms attempting to find food,
mates, and safe havens. Many important cues for life (e.g., light\dark cycles, air
currents, temperature changes, odor dispersion, and acoustics) are absent or provide
inappropriate signals in subterranean habitats. Thus, behavior must play a big role in
adaptations to live permanently underground. For example, an animal following a
scent plume, whether a pheromone or food odor, would spend most of its effort
walking away from the source since the animal must walk around or over numerous
irregularly shaped obstacles along the path. The main special behaviors exhibited by
troglobites are described below.
Loss of Circadian Rhythm Troglobites have lost the ability to entrain their activity
to daily cycles but instead exhibit a continuous level of activity, albeit movement is
often infrequent and irregular. The loss of daily rhythm results from the relaxed
selection pressure to maintain it, whereas the irregular activity allows the animal to
better exploit resources in the cave. Some cavernicoles living in shallow
mesocaverns or airy caves have reduced eyes and can entrain to light/dark daily
cycles.
Random Walks Troglobites move slowly in a random path, following a fractal

pattern (Moldovan and Paredes Bartolome 1998/1999). This behavior minimizes
energy consumption while maximizing the chance of finding food or mates. It also
minimizes the chance of falling victim to dangers, e.g., many troglobites actively
avoid entering pitfall traps (Kuštor and Novak 1980). Numerous pits and crevices in
cave passages often act as natural pitfalls, and selection to avoid these has been
harsh. Similarly, cave crickets and other jumping arthropods have greatly reduced
their ability and propensity to jump since this behavior is hazardous in total darkness.
Many, if not most, volant cave insects characteristically fly forward slowly with legs
extended to contact the substrate first (Howarth 1983).
Mating Behavior Finding mates and successfully reproducing in caves has been
little studied but is likely an important factor in the isolation and evolution of
troglobites. Visual cues are the primary means used to locate and recognize suitable
mates among many epigean animals, but of course this cue would be ineffective in
total darkness.
Some troglobites have elaborate pheromone glands (exocrine sternal gland and
isolated exocrine glands in cave beetles; Fig. 4.3), which indicate that pheromones
are important for sexual communication in some species (Cazals and Juberthie-
Jupeau 1983; Juberthie-Jupeau and Cazals 1984; Moldovan and Juberthie 1994).
Similarly, cuticular hydrocarbons, which are molecules with reduced or no volatility,
would function over short distances for intraspecific communication and mate
recognition (Moldovan 2003). Each species has a specific mixture of cuticular
hydrocarbons that is influenced by phylogenetical constraints and external factors,
and they might promote speciation in a changing climate much faster than genetic
isolation alone (Moldovan 2003).
Except for sonar used by cave-roosting bats and birds, airborne sound commu-
nication is exceptionally rare among cavernicoles, which is probably a result of the
poor acoustical properties within cave passages. However, substrate-borne sound
may be widely used at least for short range communication. This has been studied in
troglobitic cixiid planthoppers (Hoch and Howarth 1993; Hoch and Wessel 2005;
Wessel et al. 2013; see also Chap. 7). Epigean planthoppers duet on the host plant;
receptive females remain stationary, while males fly to potential host plants and sing.
When a receptive female hears the appropriate song, she responds and then the two
alternately sing until the male locates the female. In contrast, receptive female cave
cixiids initiate calling. They remain stationary on the host root and periodically call.
Their species-specific songs are transmitted along plant roots. Males wander among
patches of host plant roots and listen. When they hear a female song, they answer.
Upon hearing the appropriate male song, the receptive female responds, and the two
sexes then sing alternately until the male locates the female (Hoch and Howarth
1993).
4.3.4 Special Adaptations
Behavior May Precede Morphology Christiansen (1965) reported a stepwise

sequence in the evolution of the ability to climb a meniscus and thus escape from
pools of water in cave Collembola. Christiansen found that initially a small change in
behavior, which improved the animal’s ability to walk on water, was followed by
morphological change, which further improved the ability and which set the stage for
a subsequent behavioral change. The fully formed and functional troglomorphic foot
required several sequential behavioral and morphological steps.
Life History Adaptations to life in cave habitats often involve convergent changes in
life history traits. Troglobionts exhibit K-adapted traits, such as producing a few
large eggs (i.e., low fecundity; Vandel 1964; Poulson and White 1969). In addition,
the number of immature stages is reduced, and lifespan of the adult stage is extended.
For example, epigean wolf spiders (Lycosidae) produce more than 300 eggs per
clutch and the young ride on the female for a month or more until ready to disperse
(Fig. 4.6a). The small-eyed cave species (Lycosa howarthi) has about 40 spiderlings
per clutch, which stay with their mother for about 2 weeks (Fig. 4.6b). The no-eyed
wolf spider (Adelocosa anops) has about 25 spiderlings, which are able to leave the
mother in a few days (Fig. 4.6c) (Howarth and Mull 1992).
Increased lifespan also makes possible a longer investment in reproduction that
compensates in some groups the low number of offspring. Cave beetles show the
modification of life cycles at different levels of adaptation to life in caves
(Deleurance-Glaçon 1963; Deleurance and Deleurance 1964). Embryonic and larval
phases are longer in troglobionts compared to endogean relatives (Fig. 4.7). An
increase in the quantity of vitellus allows the development of the embryo for a longer
period. A larger egg ensures larger juveniles that have higher fitness; e.g., larger
young can cope better with the restrictive environment, have a higher resistance to
starvation, and exploit a broader range of food resources (Roff 1992; Hüppop 2000;
Wilhelm and Schindler 2000). Unless triggered by seasonal or regular pulsed input
of food resources, the timing of reproduction by cavernicoles is generally not
Fig. 4.6 Hawaiian Lycosidae comparing fecundity with each female carrying young. Epigean wolf
spider, Lycosa hawaiiensis (left; photo by © William P Mull), and two cave species, small-eyed
wolf spider, Lycosa howarthi (center), and no-eyed wolf spider, Adelocosa anops (right) (Photos by
© Francis G Howarth)
Fig. 4.7 The development cycle of cave Leptodirini (Coleoptera, Leiodidae): normal cycle (a) of
epigeans and intermediary (b) and contracted (c) cycles of cave beetles [Modified after Deleurance-
Glaçon (1963)]
synchronized, and all life stages are often present simultaneously throughout the
year.
Neoteny or Pedomorphosis Many of the troglomorphic changes commonly found
in cavernicoles may have evolved by neoteny (the retention of juvenile characters
into adulthood) (Culver 1982; Howarth 1993). Matsuda (1982), studying amphib-
ians and terrestrial amphipods, proposed that, in these groups, adaptive shifts to new
habitats are facilitated by stress-induced neoteny. Neoteny can arise rapidly as it
would require changes in only a few regulatory genes. Natural selection may favor
neoteny as an energy-saving mechanism, e.g., reduction of eyes and other traits by
changes in allometric development (Matsuda 1982). Neoteny may be positively
selected in cavernicolous species whose immature stages already live underground,
e.g., cixiid planthoppers. Cixiid nymphs feed on plant roots and characteristically
display many troglomorphies. In addition, the nymphs feed on xylem sap, which is
very dilute; thus, they have well-developed physiological and morphological adap-
tations to deal with excess water. Adults of epigean species metamorphose into
typical surface planthoppers and emerge from their underground lairs to disperse and
reproduce. Adults of the cave species have retained many features found in the
nymphal stage and remain underground (Howarth 1993).
Pseudo-physogastry The swelling of the posterior part of the more cave-adapted
Leptodirini (Coleoptera) body is due to a false physogastry caused by the enlarge-
ment of the elytra volume, while the abdomen remains normal (Fig. 4.1). One
possible explanation is that a spherical chamber formed by elytra can regulate the
hydric equilibrium of the body since cave animals living in saturated air humidity are
very sensitive to desiccation.
4.4 Adaptations to Caves by Aquatic Animals
4.4.1 Morphological Adaptations of Stygobionts
Pigmentation The lack of pigmentation (also called albinism) occurs not only in
stygobionts but also in some stygophiles that spend most of their life inside caves.
Some of the aquatic cave vertebrates are pigmented when young but then lose bodily
color as they age. For example, Proteus anguinus salamanders have black chro-
matophores as larvae but lose these and maintain a pink coloration of the body
during their entire life. The pink color is not due to pigments but to blood circulating
under a thin and depigmented skin (Durand 1983). Fish belonging to Typhlichthys
and Amblyopsis lose their grayish color during growth. Aquatic crustaceans may be
completely transparent (Troglocaris) or white and opaque, their integument being
impregnated by calcium salts (e.g., Proasellus, Niphargus, Cambarus). Some
aquatic invertebrates can also recover color when exposed to light, and some
populations are colored by pigments that are ingested together with the food, such
as carotenoids (Ginet and Decou 1977).
In cave populations of Asellus aquaticus, albinism (Fig. 4.8 and Chap. 8) can be
achieved in two different genetic ways involving three different genes within the
same population (Protas et al. 2011), unlike the cave fish Astyanax mexicanus where
the same gene is involved in the evolution of the albinism in three independently
evolved cave populations (Protas et al. 2006). Albinism might make stygobionts
more vulnerable to ultraviolet light with deleterious effects (Langecker 2000), and
direct light exposure is deadly or stressful for cave planarians (Merker and Gilbert
1932), ostracods (Maguire 1960), and for some amphipods, i.e., Niphargus virei
(Ginet 1960) and N. stygius (Simčič and Brancelj 2007).
Vision (Eyes or Ocelli in General) In completely dark caves, selection will strongly
favor individuals with a reduced visual system to reduce overall energy expenditure
(Moran et al. 2015), although some species retain remnants of these organs
(Fig. 4.9). Accordingly, the nerve centers are also reduced, and the reduction is
believed to reflect the degree of adaptation to caves or to the ancientness of
Fig. 4.8 The isopod Asellus aquaticus lives in surface waters (pigmented populations – upper left)
and inside caves (depigmented or translucent populations – center and right) (Photo by © Boris
Sket)
adaptation to cave life; some crustaceans have eye structures as embryos or young.
All blind cave vertebrates exhibit initial eye development followed by secondary
degradation during later development stages (Durand 1976; Jeffery 2009; Jeffery
and Strickler 2010). Proteus has normally pigmented eyes when they hatch, but
these become reduced and the structure altered during development. The two black
spots at the side of the head eventually disappear completely (Durand 1971), and this
is an ontogenetic regression rather than a phylogenetic regression, which occurs in
some fishes and crustaceans.
Eye loss and eye reduction have different genetic bases within populations of
Asellus aquaticus and in different populations of Astyanax mexicanus (Wilkens and
Strecker 2003; Jeffery 2005; Borowsky 2008; Protas et al. 2011). Nevertheless,
many cave species that were considered blind prove to be microphthalmic and
respond to light stimuli in a more buffered way than their stygophilic relatives living
in springs (Langecker 2000; Borowsky 2013; Soares and Niemiller 2013).
Other Senses It was assumed that other senses must compensate for the reduction or
complete lack of sight, e.g., the sensorial compensation proposed by Poulson (1963)
for amblyopsid fishes. The lateral line, olfaction, taste, and touch and electro-sense
are hypertrophied in cave species (Langecker 2000) together with drastic modifica-
tion of the brain (Fig. 4.10). Proteus anguinus, with its flattened and enlarged head,
can carry a larger number of sensory receptors (Schlegel et al. 2006) and use
Fig. 4.9 The normal eye in surface Astyanax mexicanus (a) is reduced in cave populations (b), and
lack of eyes is compensated by the neuromasts (not shown here) [Photos from McGaugh et al.
(2014), with permission]
Fig. 4.10 Adaptation of the brain in amblyopsid fishes to life in caves involves the reduction of the
optic lobes and the volumetric increase of both the cerebellum and otoliths from the epi-
gean Chologaster cornuta (a) to the cave adapted Forbesichthys agassizii (b) and Amblyopsis
rosae (c) [Modified after Culver (1982)]
chemical clues (Uiblein et al. 1992) as well as mechanoreceptors and

electroreceptors (Schlegel and Bulog 1997). The cave Astyanax have larger jaws
and more taste buds that together with a modified brain induce changes in behavior
(Yamamoto et al. 2009). The lateral line of fishes consists of a canal and superficial
neuromasts; the superficial neuromasts in cave Astyanax mexicanus are several times
more numerous than in surface fish (Schemmel 1967; Teyke 1990; Jeffery et al.
2000). Cave fishes neuromasts are also larger and contain more sensory hairs than
those of surface fish. The superficial neuromasts cupulae (hair cells covered by a
gelatinous case) are almost eight times longer in cave fishes compared to surface
fishes (Teyke 1990), which enhance detection and reaction to vibrating stimuli in
cave fishes (Yoshizawa et al. 2010). In populations of Asellus aquaticus, the
aesthetascs acting as chemoreceptors are longer, and setal patterns change in cave
populations compared to surface relatives (Turk et al. 1996; Prevorčnik et al. 2004).
In the cave shrimp Troglocaris, lengthening of the rostrum and significant enlarge-
ment of the postorbital carapace represent defense mechanisms against predation by
Proteus. This adaptation has been observed in places where these two taxa co-occur
(Jugovic et al. 2010).
Some species show reduction of senses in addition to vision, e.g., the lack of
statocysts in stygobiontic Bathynellacea and the scarcity of aesthetascs in
stygobiontic isopods (Camacho 1992), although in-depth studies are still scarce
and incomplete for most groups. The reduction of the optic lobe is correlated with
enlarged deutocerebral olfactory centers in malacostracans (Crustacea), which
implies an important role of the olfactory sense in this group (Stegner et al. 2015).
On the other hand, within Peracarida (Crustacea), the missing optic sense is not
correlated with an emphasized mechanosense.
The modification of the pineal organ is variable in different cave fishes, ranging
from disorganization to complete reduction, as in Typhlichthys subterraneus
(Langecker 2000). The pineal photoreceptors in fishes have the combined roles of
photoreception and as neuroendocrine cells for the synthetization and secretion of
the melatonin hormone under the control of light.
Body Size The void sizes of groundwater microhabitats strongly select the size of
the inhabitants resulting in giants or dwarfs of the cave world (Ginet and Decou
1977). In some Harpacticoida (Crustacea), that live in small and narrow spaces, the
body size, length and number of the swimming legs, and setae number are reduced
[e.g., in Parastenocaris, Speocyclops, and Morariopsis; Brancelj (2004)]. In some
genera, body size is highly variable even within species inhabiting the same cave
(Fig. 4.11).
4.4.2 Physiological Adaptations of Stygobionts
Metabolism The reduced metabolic rate and energy-saving behaviors of

stygobionts have been experimentally demonstrated in cave fishes and crustaceans
(Hervant et al. 1997, 1998); it is as an adaptation to food and oxygen stress in cave
habitats (Hervant and Renault 2002) or a response to energy economy (Hüppop
2005). In other species there is apparently no difference in the metabolic rates of
cave species and surface relatives, as in Astyanax mexicanus, a species that is not
experiencing food limitation (Culver 1982).
Fig. 4.11 Ecomorphs of Niphargus showing that the elongation of the appendages and the shape of
the body depend on the occupied microhabitat [Modified after Trontelj et al. (2012)]
Oxygen Consumption The first observations of low oxygen consumption in cave

inhabitants were made by Eigenmann in 1909 through observing the opercula
movement of Amblyopsis spelaeus. Deep cave waters, especially in the phreatic
zone, are often hypoxic (1 mg O2/L), but hypoxia can be also found few meters
below the water table (Malard and Hervant 2012). The response of the crusta-
ceans Niphargus and Stenasellus to hypoxia is a rapid reduction of locomotory
and respiratory activities, which allows longer survival. Recovery after periods of
low or no oxygen is also rapid, and the lactate resulting from the anaerobic
metabolism is reused, unlike surface Gammarus and Asellus crustacean relatives
which oxidize the lactate (Malard and Hervant 2012).
Fasting Stygobionts can store more fat than their epigean spring relatives (Hüppop
2000) and makes fasting for long periods a characteristic of stygobionts. Niphargus
can survive for more than a year (Gibert and Mathieu 1980; Mathieu and Gibert
1980), Stenasellus virei for more than 200 days (Hervant et al. 1997), Troglocaris up
to 2 years (Vogt and Štrus 1999), and Proteus up to 96 months of starvation without
showing any signs of disease (Vandel 1964; Hervant et al. 2001).
4.4.3 Behavioral Adaptations of Stygobionts
Circadian/Seasonal Rhythm Experiments on the cave Niphargus virei and the

epigean Gammarus pulex demonstrated that the cave species moved one-tenth as
much as the epigean species and that Niphargus had fewer periods of activity during
daylight than Gammarus (Ginet 1960; Camacho 1992). Cave fishes and crustaceans
have their own internal rhythms not linked with changes at the surface (Poulson and
Jegla 1969); Proteus is another cave representative that does not display a circadian
rhythm (Schatz et al. 1977). Weak activity patterns are connected to low oxygen
consumption in Orconectes pellucidus (Jegla and Poulson 1968), but these crayfish
still kept a rhythm of molting and reproduction in response to changes in both water
temperature and volume (Jegla and Poulson 1970).
The existence of periodicities in the life of aquatic cave animals might be inherited
from their epigean ancestors (Trajano and Menna-Barreto 1995) and subsequently
evolve in the absence of light inside caves as well as to the scarcity of food sources,
at least during some periods of the year. Photosensitivity or photokinesis (defined as
sensitivity to light) experiments showed that cave fishes and crustaceans react to a
certain intensity of light by an avoiding reflex and by hiding, which proved to be a
conserved ability to detect light of the pineal eye in cave fishes (Yoshizawa and
Jeffery 2008). Surface amphipod species showed a weak photophobic, photoneutral,
or in one case, photophilic response, whereas all cave species showed a strong
photophobic response (Fišer et al. 2015). On a regional scale, photophobia could
explain limited dispersal and a high degree of endemism observed among subterra-
nean species (Fišer et al. 2015) that cannot use surface waters for dispersal.
Agonistic Behaviors Such behaviors include the reduction in aggressive and sub-
missive reactions observed in cave fishes. The highly cave-adapted Amblyopsis
rosae and Typhlichthys subterraneus engage in much shorter, simpler, and less
intense agonistic bouts, most frequently being the tail-beating (Bechler 1983). The
explanations for reduced agonistic behavior are the reduced selection pressures,
decreased metabolic rate, and increased longevity that all confer the advantage of
energy conservation (Parzefall 1992). In contrast, aggressive behavior linked to
territoriality was observed at Proteus anguinus for short periods of time (Parzefall
1976).
Active search for cryptic microhabitats in cave waters (under rocks, in clay, and
within small voids in the calcite formations) was observed in Proteus, which
otherwise rest most of the time by “hiding” under rocks; and they preferred places
formerly used by other individuals, which had deposited a substance at the resting
place (Parzefall et al. 1980).
Schooling Behavior Fish schooling and aggregation behaviors are social and group
activities exhibited by fishes for foraging, reproduction, and defense from predators.
Fish in schools can also obtain a hydrodynamic advantage, thus reducing the cost of
locomotion. A reduction of this behavior has been observed in cave fishes and is
believed to be an adaptation to the lack of visual orientation or to a change in
stabilizing selection for schooling (Parzefall 1992). Tests on different generations on
Astyanax fasciatus led to the conclusion that the reduction of this behavior is
genetically based (Kowalko et al. 2013).
Feeding Behavior The studied cave fishes search for food by holding their body at
an angle of 55 to the bottom substrate (Schemmel 1980); they also use a much
larger area of dense taste buds on the ventral side of the head, compared to epigean
species that have such buds only at the tip of the mouth (Wilkens 1992). Some
stygobionts occasionally may use resources outside of caves during the night, i.e.,
Proteus has been observed feeding overnight outside caves (Sket 2008), and
Niphargus timavi stomach content was full of ephemeropteran larvae that live
outside caves (Fišer et al. 2010).
Predation The lack of predators may account for the observed reduction of defen-
sive behaviors in some cave fishes, although cannibalism by bigger individuals is
present in Astyanax (Parzefall 1983). The decapods Orconectes and Procambarus
have a more intense reaction to the presence of predators than their epigean relatives
if water movements accompany their presence, but not in the presence of chemical
cues. In the presence of chemical cues, epigean species responded quicker than cave
species (Ginet and Decou 1977).
4.4.4 Special Adaptations
Life Cycles The adult stage is exceptionally long in cave species, being up to
35 years for Niphargus (Turquin and Barthelemy 1985), more than 2 years for
harpacticoids (Ginet and Decou 1977), and reaching dozens of years for the cave
crayfish Orconectes australis australis (Cooper and Cooper 1976). Proteus females
become mature at an average age of 15.6 years and reproduce every 12.5 years for a
lifespan of 68.5 to 102 years (three times higher when compared to its most closely
related species; Voituran et al. 2011). The timing and rhythm of reproduction is easy
to check for many aquatics as ovigerous females are easily identified and censused.
For example, the cave isopod Stenasellus reproduces once every two years in good
conditions, but with low food supply and the interval between reproductions can be
much longer (Fig. 4.12). Nearly every phase of the development cycle is much
longer in cave animals than in related surface animals, as was demonstrated for
amphipods and harpacticoids (Ginet and Decou 1977; Rouch 1968). In contrast, the
life cycle of epigean invertebrates is normally completed within a single year or few
months during one year.
Number and Size of Eggs The number of eggs produced by stygobionts is often
one-tenth of their epigean relatives. There are also cases in which females carry a
single egg, as in Microparasellidae (Coineau 1971). Niphargus females lay more
voluminous eggs compared to their surface relatives (Fišer et al. 2012) but invest less
in a single brood, similarly to cave fishes from different genera (Poulson 1963).
Nevertheless, investment in egg quality and high survival rate in Niphargus is not
correlated with low number of eggs but to an increase in the body size of adults,
which can lay bigger eggs (Fišer et al. 2010); the increased size of the coxal plates in
Niphargus females provides better aeration of the large eggs (Fišer et al. 2012).
Coevolution of traits, such as egg size, aeration capacity, and number of eggs,
minimizes the overall costs and contributes to lifetime fitness (Fišer et al. 2012). A
female of Amblyopsis spelaea produces 40–60 large, heavily yolked eggs (Poulson
1963) and the cave Poecilia mexicana, with smaller females than their surface
relatives, produces larger offspring (Riesch et al. 2011). For Proteus, the clutch
comprises about 35 eggs of which less than 50% hatch (Voituran et al. 2011).
Reduced sleep is a novel trait aquired by stygobionts, with genes underlying the
behavioral regression independent from those responsible for eye and pigment
Fig. 4.12 The lifespan of the stygobiont Stenasellus (a), black ¼ time the female carries the
embryos, light gray ¼ juvenile period, dark gray ¼ adult period, and of cave fishes at different
levels of adaptation to life in caves (b), from the epigean Chologaster to the more cave adapted
Forbesichthys and Amblyopsis, black ¼ time to hatching, light gray ¼ time from hatching to first
reproduction, white ¼ reproductive lifespan [Modified after Ginet and Decou (1977; a) and Poulson
(1963; b)]
regression. In the same time, sleep regression evolved convergent in different,

isolated cave populations (Duboué et al. 2011). Cave Astyanax mexicanus have
drastically reduced sleep periods, and both daytime and nighttime sleep are reduced,
compared to their surface relatives (Soares et al. 2004).
Resistance to high concentration of sulfides in the water by Niphargus has been
associated with the resistance to low oxygen, or with traits involved in detoxifica-
tion, such as symbiosis with Thiothrix bacteria (Dattagupta et al. 2009). Adaptive
behaviors that improve oxygen uptake were proposed for Niphargus and a popula-
tion of Poecilia mexicana from Cueva de Villa Luz (Mexico), respectively (Plath
et al. 2007; Tobler et al. 2011, 2014).
Box 4.3
A comparison between cave-adapted Asellus from freshwater in Slovenia and
sulfuric waters in Romania shows that there is a parallel evolution of eye loss,
depigmentation, and elongation of some appendages. Water chemistry, nutri-
ent availability, and other factors like ancestral genetic variability and popu-
lation genetic structure are not hampering the convergent adaptation to caves
(Konec et al. 2015).
Thermal Tolerance Experiments on three cave Proasellus isopods show different

response for species that live in a habitat with less than 1 C yearly temperature
amplitude. Two of the species were sensitive to changes of less than 2 C, while the
third species tolerated a variation of 11 C, which shows that thermal physiology is
not solely shaped by the temperature in the natural environment for all groundwater
species (Mermillod-Blondin et al. 2013).
Salinity Tolerance Salinity tolerance is characteristic for the complex anchialine
systems where cave fauna is separated by salinity-stratified water layers (Iliffe and
Kornicker 2009; see also Chap. 17). Remipeds are restricted to the marine compo-
nent of the systems, and some atyid shrimps can be found in both marine and less
saline layers. The effect of salinity tolerance on habitat connectivity and
dispersibility of species living in these complex systems remains to be tested
(Moritsch et al. 2014).
Neoteny or pedomorphosis is striking for some of the cave salamanders. Eurycea
spelaea, Gyrinophilus subterraneus, and Proteus anguinus are neotenic, keeping the
gills outside the body and an aquatic way of life even as adults.
Ethophysiological Adaptations Cave animals display strong stereotropism (a reflex
in which the contact with a rigid surface is the orienting factor) and other
ethophysiological strategies (Ginet and Decou 1977; Holsinger 1988). For example,
Niphargus adapt to dry periods by digging a water-filled hole in wet clay that allows
survival of individuals even when water dries out (Ginet and Decou 1977).
Acknowledgments We are grateful to Jana Bedek, Pawel Jaloszynski, William P Mull, Udo
Schmidt, and Boris Sket for the permission to use their photos and to Ľubomir Kováč and Stuart
Halse for their corrections and suggestions. OTM acknowledges the financial support from the
Romanian Academy and the grant of the Romanian Ministry of Research and Innovation, CNCS—
UEFISCDI, project number PN-III-P4-ID-PCCF-2016-0016, within PNCDI III.
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Chapter 5
The Microbial Diversity of Caves
Olivia S. Hershey and Hazel A. Barton
5.1 Introduction
In attempting to describe microbial diversity, Baas-Becking famously stated that “Every-

thing is everywhere, but the environment selects” (Baas-Becking 1934). His hypothesis
was simple—the small size of microorganisms allows their broad environmental distri-
bution, with the appropriate conditions selecting species growth to dominate niche space
(Baas-Becking 1934). Given the many types of caves, and hence a myriad of environ-
mental conditions, it is difficult to describe a broadly relevant microbial diversity;
varying cave conditions provide opportunities to select a multitude of adaptations and
hence community diversity.
Despite this drawback, caves do represent an important environment for studying
microbial ecology, chiefly due to community adaptations to the resource limitation of the
subsurface; the lack of photosynthetic activity in caves requires microorganisms to rely
on either the heterotrophic breakdown of scant allochthonous organic carbon or auto-
trophic growth using in situ redox-active compounds (Peck 1986; Northup et al. 2003;
Carmichael et al. 2013; Desai et al. 2013; Parker et al. 2013; Jones and Macalady 2016).
While some caves, including sulfidic systems, are dominated by chemolithotrophic
primary production, such cave systems are relatively rare (and their microbial diversity
is reviewed in Chap. 15). In more common epigenic caves, nutrients entering from
surface-derived ecosystems are limited, with allochthonous organic carbon delivered by
vadose-zone groundwater generally measured below 0.5 mg/L (Barton 2015). This puts
cave environments firmly in the oligotrophic (<2.0 mg/L) spectrum of energetic
systems; for simplicity, we will to refer to such systems as oligotrophic caves (Engel
et al. 2010; Harmon et al. 2013; Barton 2015).
O. S. Hershey · H. A. Barton (*)

Department of Biology, University of Akron, Akron, OH, USA
e-mail: [email protected]; [email protected]

https://doi.org/10.1007/978-3-319-98852-8_5
70 O. S. Hershey and H. A. Barton
Fig. 5.1 A passage within Poor Farm Cave, West Virginia, USA. Based on the age of fossil
skeletons found within cave sediments, it is unlikely that the environmental conditions within this
passage have significantly changed in the last 400,000 years (Grady et al. 2000). Energy for
microbial growth likely comes from allochthonous organic carbon entering via cracks or fissures
in the bedrock, or the presence of redox-active compounds [Mn(II), Fe(II), NO3, or NH3].
Another unique feature of caves, and one commonly overlooked by microbiolo-

gists not used to working in such systems, is the static nature of the environment; once
cave forming processes have ended, the environmental conditions of a particular
niche might not change for thousands (to potentially millions) of years (Fig. 5.1;
Klimchouk et al. 2000; Palmer 2007). This is in stark contrast to surface ecosystems,
where plant species, foraging animals, anthropogenic impacts, and even the weather
can have a profound influence on microbial community structure in short (daily,
seasonal) time frames (Barton 2015; Palmer 2007). The absence of these dynamic
processes in caves means that the selective pressures driving diversity are dependent
on the variables that are present and strongly influenced by the geologic setting and
geochemical (environmental, local and regional) nature of the cave (Ortiz et al. 2013).
This review will not try to provide a synthesis of the ~400 papers published on cave
microbiology (Fig. 5.2). Instead, it will focus on the microbiology of oligotrophic caves
in limestone (carbonate; CaCO3) settings. We will also not produce a comprehensive list
of the microbial species found in caves, as this has recently been reviewed elsewhere
(Vanderwolf et al. 2013; Tomczyk-Żak and Zielenkiewicz 2016). Instead, we will
attempt to outline how the research history has led to our current understanding of
microbial ecology in caves, the potential for a core microbiome, and the common
ecological themes that might drive microbial diversity in caves.
5 The Microbial Diversity of Caves 71
5.2 Microbial Diversity in Caves Prior to 1996
Our understanding of the microbial ecology of caves is influenced by the technology

of the time (Engel 2015). As microbiology was built upon the ability to culture
microorganisms within the laboratory, early cave researchers used the same cultiva-
tion techniques as soil scientists and (somewhat unsurprisingly) found that caves
were a weak reflection of the microbiology of surface soils (Hess 1900; Scott 1909;
Høeg 1946; Caumartin 1963). The interpretation of microbial activity in caves was
therefore limited, and it seemed to be of little interest to the scientific community, with
less than 40 papers published prior to 1997 (Fig. 5.2; Engel 2015). Yet these papers
defined our understanding of cave microbiology, suggesting that caves were essen-
tially lifeless due to an absence of photosynthetic input, or simply home to transient
microbial species introduced by the activity of animals or humans (Caumartin 1963).
When endemic cave microorganisms were putatively identified, it was through
unusual metabolisms that were (incorrectly) thought to distinguish them from soil
species, such as iron oxidation (Caumartin 1963).
The primary limitation of cultivation-based approaches is that the vast majority
(>99%) of environmental microorganisms cannot be cultured; as in other microbial
environments, the ability to accurately describe microbial diversity within caves
required cultivation-independent techniques (Amann et al. 1996). Some early
non-cultivation approaches did support the idea that microbiology in caves was
more complex than originally thought: Fliermans and Schmidt (1977) used anti-
bodies to identify non-culturable Nitrobacter in Mammoth Cave sediments; the
microscopic techniques of Cunningham et al. (1995) demonstrated a rich structural
diversity from samples deep within Lechuguilla Cave; and Gonzalez et al. (1999)
demonstrated a rich diversity of actinobacteria in Spanish caves using fatty acid
methyl ester (FAME) analyses (Fliermans and Schmidt 1977; Cunningham et al.
1995; Gonzalez et al. 1999). Nonetheless, it wasn’t until the use of molecular
phylogenetics in the 1990s that the potential diversity of microorganisms in cave
environments emerged (Fliermans and Schmidt 1977; Cunningham et al. 1995; Pace
1997; Gonzalez et al. 1999; Barton 2006; Barton and Northup 2007; Engel 2010;
Lee et al. 2012).
5.3 Microbial Diversity in Caves: The Molecular Era

(1997–2012)
First introduced in the 1980s as a revolutionary way of identifying microorganisms

in the environment, Pace et al. used the 16S small ribosomal subunit rRNA gene
sequence as a genetic marker (phylotype) to distinguish previously uncultured
species (Stahl et al. 1984; Pace et al. 1986). It was also Pace (himself an avid
cave explorer) who facilitated the first molecular analysis of a microbial cave
community (Fig. 5.2). These investigators used molecular phylogenetic approaches
to examine the filamentous biofilms of a sulfidic stream within Sulfur River Cave,
72
Fig. 5.2 Impact of major events in cave microbiology on research in the field, as measured by publications in peer-reviewed journals (abstracts and conference
proceedings are not included in cumulative totals). The number of publications per year is indicated, cumulative total of publications, as well as the impact of the
WNS epidemic on cave microbiology publications. Redox Breakthroughs Conference ¼ Breakthroughs in Karst Geomicrobiology and Redox Geochemistry
Conference (see Sasowsky and Palmer 1994). WNS ¼ White-Nose Syndrome. 2009 ICS ¼ International Congress of Speleology Special Section: Microbiology
and Geomicrobiology of Cave and Karst Environments. The “*” symbol indicates the publication of a special issue on Cave Microbiology by the
Geomicrobiology Journal. Inset: histogram of publication rates by the primary journals in the field, including International Journal of Speleology (IntJS),
O. S. Hershey and H. A. Barton
Journal of Cave and Karst Studies (JCKS), and Geomicrobiology Journal (GeoJ); blue triangles correspond to the number of cave microbiologists who serve as
associate editors
Kentucky (Angert et al. 1998). This study revealed the surprising dominance of the
Epsilonproteobacteria, which were previously seen only in deep, oceanic hydro-
thermal systems; it was also the first clue to the important influence that members
of this phylum have within sulfidic cave environments (Campbell et al. 2006).
Most importantly, the study also demonstrated that microbial cave communities
could be remarkably distinct from their surface counterparts (Angert et al. 1998).
Through the 1990s molecular phylogenetics was the most powerful tool to study
microorganisms within the environment (Pace 1997). But the tool remained limited to
labs with both the molecular expertise and computing resources necessary to translate
genetic difference into the robust phylogenies necessary to identify uncultivated
microorganisms, with only two labs carrying out such analyses in caves (Vlasceanu
et al. 1997; Angert et al. 1998). The ability to analyze cave communities was further
complicated by the low biomass of these environments (routinely <106 cells/g), along
with a complex geochemistry, both of which interfered with the ability to obtain
sufficient DNA for analysis (Barton et al. 2006). This restricted early analyses to sites
with enough biomass to overcome DNA extraction limitations, such as those found in
the chemolithotrophic ecosystems of sulfidic caves, as examined by Pace (Sarbu et al.
1996; Angert et al. 1998; Engel et al. 2004a).
A lot changed over the next 15 years: new techniques improved the ability to
extract DNA from the environment (Barton et al. 2006; Tan and Yiap 2009); new
programs made phylogenetic analysis more accessible (Posada 2003; Wilgenbusch
and Swofford 2003; Edgar 2004; Kumar et al. 2006; Pruesse et al. 2007); sequencing
technologies reduced costs (Shendure et al. 2004); and high-impact journal articles
demonstrated the important contributions that the study of cave microorganisms could
provide (Cunningham et al. 1995; Engel et al. 2004b). A number of events further
raised the profile of cave microbiology, beginning with the 1994 Breakthroughs in
Karst Geomicrobiology and Redox Geochemistry Conference (59 conference pro-
ceedings), a special issue of the Geomicrobiology Journal in 2001 (10 journal articles),
and a special session on the Microbiology and Geomicrobiology of Cave and Karst
Environments at the 2009 International Congress of Speleology (32 conference
papers). The cumulative impact of these changes was a fivefold increase in the number
of journal articles on cave microbiology between 1997 and 2012 (from 43 to 256;
Fig. 5.2).
Traditional molecular phylogenetic approaches involve PCR amplification of 16S
rRNA gene sequences from environmental DNA, followed by cloning or denaturing
gradient gel electrophoresis (DGGE) to generate libraries of representative 16S rRNA
sequences (Pace 1997). These libraries can range in size from a few dozen to a few
hundred cloned sequences. Nonetheless, given the tens of thousands to potentially
millions of microbial cells in every sample, such “clone” libraries tend to identify the
species/phylotypes within an environment that make the greatest contribution to total
environmental DNA and/or 16S rRNA copy number (Fig. 5.4; DeSantis et al. 2007;
de Araujo and Schneider 2008; Kembel et al. 2012). While this does allow a snapshot
of the most successful microorganisms within an environment, it is also a limitation of
the technique, potentially missing a large number of organisms that play important
roles in ecosystem function (Fig. 5.4).
Box 5.1
This dramatic rise in microbial research also opened our eyes to the potential
diversity of microorganisms in cave environments. Rather than supporting the
idea that caves were dominated by a few specialized species adapted to nutrient
limitation, caves appeared to be home to a diverse assemblage of species from
multiple phyla, including the Alpha-, Beta-, Gamma-, and Deltaproteobacteria,
Chloroflexi, Planctomycetales, Bacteroidetes, Acidobacteria, and Actinobacteria,
with small but significant contributions from members of the Nitrospirae,
Gemmatimonadetes, and Verrucomicrobia (Fig. 5.3; Northup et al. 2003; Barton
et al. 2004; Chelius and Moore 2004; Barton et al. 2007; Zhou et al. 2007; Cuezva
et al. 2012; Lee et al. 2012; Porca et al. 2012; Rusznyak et al. 2012; Ivanova et al.
2013; Barton 2015). These data also demonstrated the potentially significant
contribution of the archaea to subsurface communities (Northup et al. 2003;
Chelius and Moore 2004; Shabarova and Pernthaler 2010).
60
Cave Biome Soil Biome

Percent Representation
40
(38)
(49)
(77)
20
(29)
(13)
(23)
0
Acidobacteria
Actinobacteria
Acidobacteria
Actinobacteria
Alphaproteobacteria
Betaproteobacteria
Deltaproteobacteria
Gammaproteobacteria
Verrucomicrobia
Alphaproteobacteria
Betaproteobacteria
Deltaproteobacteria
Gammaproteobacteria
Verrucomicrobia
Nitrospirae
Nitrospirae
Planctomycetales
Bacteroidetes
Firmicutes
Gemmatimonadetes
Planctomycetales
Bacteroidetes
Firmicutes
Gemmatimonadetes
Chloroflexi
Chloroflexi
Phylum
Fig. 5.3 Box plot comparison of soil microbial communities to those found in caves. The represen-
tation of major phyla of pooled soil and cave samples is shown. The soil biome was obtained from the
collated data of Chu et al. (2010). The cave biome was collated from the data of Northup et al. (2003),
Barton et al. (2004, 2007), Chelius and Moore (2004), Zhou et al. (2007), Cuezva et al. (2012), Porca
et al. (2012), Rusznyak et al. (2012), and Ivanova et al. (2013). Only datasets including at least
100 cloned 16S rRNA phylotypes are included. The boundaries for the first and third quartile are
shown, with the centerline representing the mean and whiskers representing the max/min values
(outlier values for the cave biome data are shown in parentheses)
5.4 Microbial Diversity in Caves: The Genomics Era

(2013–Present)
Among the many impacts of the Human Genome Project, the most powerful was the
development of optically based sequencing methods—collectively referred to as “next-
generation sequencing” (NGS) technologies (Ansorge 2009; Lander 2011). The dra-
matic increase in the number of bases that these technologies could sequence (>15
billion bases in as little as 4 h) combined with their significant cost reductions
revolutionized the ability to sequence DNA (Snyder et al. 2009; Forde and O’Toole
2013). Sogin et al. (2006) were the first to use NGS to identify environmental 16S
rRNA; rather than restricting the identification of phylotypes within a community to a
few hundred cloned 16S rRNA genes, NGS allowed Sogin and colleagues to sequence
120,000 PCR products directly. The results were transformative and demonstrated that
microbial ecosystems contained thousands of previously unidentified phylotypes
(Sogin et al. 2006). Sogin et al. referred to this extensive collection of previously
unidentified microorganisms as the “rare biosphere”—organisms of sufficiently low
number that they cannot be identified without deep-sequencing NGS approaches
(Fig. 5.4).
Due to limited access to NGS and the advanced statistical methods needed to
distinguish unique DNA sequences against a background of inherent PCR error, this
technology was also initially limited to a few specialized labs (Sogin et al. 2006). But as
Fig. 5.4 Idealized microbial diversity in cave environments and representative portions screened
via different molecular techniques. Some species are able to rapidly utilize the available nutrient and
energy sources, providing a competitive advantage that allows them to become dominant in the
ecosystem (similar to r-type selection). These dominant species are most often identified in shallow-
coverage analyses, such as clone libraries and DGGE. The rare biosphere contains a combination of
numerically low, slower-growing, poorly adapted, or even viable but non-growing species (similar
to K-type selection). The rare biosphere can usually only be identified using deep-sequencing
approaches, such as Illumina sequencing. NGS, next-generation sequencing
researchers developed more efficient mathematical algorithms to reduce the necessary

computational power, and techniques in bioinformatics simplified the analysis of large
NGS data sets, this technology rapidly became available to other researchers (Kuczynski
et al. 2012; Caporaso et al. 2010; Stamatakis 2014). The first to apply these applications
in cave environments were Ortiz et al. (2013) who used 454-pyrosequencing to examine
~400,000 PCR products from Kartchner Caverns, USA (Ortiz et al. 2013). Along with
the 13 phyla already identified in caves by cloning approaches, Ortiz et al. demonstrated
the presence of an additional 8 described and 12 candidate phyla, suggesting that caves
also contained rare biosphere microorganisms. In a significant step forward, these
researchers also used NGS to compare microbial communities in the cave with those
in surface soils directly above. These data demonstrated that only 16% of the sequences
were shared between the surface and the cave, confirming the uniqueness of microbial
cave ecosystems (Ortiz et al. 2013).
In the years since this study, there have been no other published 16S rRNA NGS
sequence datasets from oligotrophic caves; however, a number of studies have
submitted sequence data to public databases, such as the NCBI sequence read archive
(SRA) (Fig. 5.5; Leinonen et al. 2010). By processing these datasets, it is possible to
expand the work of Ortiz et al. across multiple cave systems with broadly distributed
geographical locations, including North America and Asia (Fig. 5.5). The results
confirm the robustness of the 13 dominant phyla already identified, along with another
14 phyla consistently represented in these populations (above a 0.1% threshold); these
include the Armatimonadetes (OP10), Chlorobi, Cyanobacteria, Elusimicrobia, Spi-
rochetes, and the candidate phyla BRC1, GN04, NC10, OP3 (Ca. Omnitrophica), TM6
(Ca. Dependentiae), WS1, and WS3 (Ca. Latescibacteria). Together these data support
the existence of a cave rare biosphere (Fig. 5.5). The cave NGS datasets also contain a
40
Clone Libraries Next-generation Sequencing

20
Rare biosphere
0
Alphaproteobacteria
Betaproteobacteria
Deltaproteobacteria
Gammaproteobacteria
Acidobacteria
Actinobacteria
Verrucomicrobia
Alphaproteobacteria
Betaproteobacteria
Deltaproteobacteria
Gammaproteobacteria
Acidobacteria
Actinobacteria
Verrucomicrobia
Armatimonadetes
BRC1
Chlorobi
Cyanobacteria
SBR1093
Spirochaetes
WS3
Unclassified
Nitrospirae
Nitrospirae
TM6
Planctomycetales
Bacteroidetes
Firmicutes
Gemmatimonadetes
Planctomycetales
Bacteroidetes
Firmicutes
Gemmatimonadetes
CN04
NC10
OP3
WS1
Chloroflexi
Chloroflexi
Elusimicrobia
Phylum
Fig. 5.5 Boxplot comparison of microbial cave community structure analyzed by 16S rRNA gene
clone libraries (from Fig. 5.3) or Illumina (next-generation) sequencing. The datasets SRR1686967,
SRR1686970, SRR1686976, SRR1693633, SRR1703816, SRR1703817, SRR1703818, SRR1703819,
SRR1703820, and SRR1703821, obtained from the NCBI sequence read archive (SRA), were used.
The datasets, which contained between 12,901 and 585,434 unfiltered sequence tags, were processed in
QIIME (Caporaso et al. 2010) to assign taxonomic identity. The boundaries for the first and third
quartile are shown with the centerline representing the mean and whiskers representing the max/min
values
significant proportion of sequences that cannot be classified using the taxon reference
databases, Greengenes and SILVA (Fig. 5.5; McDonald et al. 2012; Quast et al. 2013).
These sequences, which cannot be easily placed within the current taxonomic frame-
work, are known as microbial dark matter and represent the currently unexplored
diversity of microbial populations (Rinke et al. 2013). Such sequences tend to have a
higher representation in caves than other habitats, suggesting that the true diversity of
caves requires further description (Sogin et al. 2006; Rinke et al. 2013).
While targeted PCR amplification makes it possible to rapidly screen the 16S
rRNA sequences in the environment, it is also susceptible to significant technical
issues, including primer and amplification biases that preferentially select certain
DNA sequences for amplification (Chandler et al. 1997; Polz and Cavanaugh 1998;
DeSantis et al. 2007; Kembel et al. 2012). Overcoming these limitations requires
bypassing the PCR amplification step entirely and sequencing the sum of the genetic
information in the environment (Miller et al. 2011). This process requires randomly
fragmenting DNA into sizes appropriate for NGS sequencing (35–300 bp), either by
mechanical means or using transposons (Adey et al. 2010). These fragments are then
sequenced, and the overlapping ends are computationally reassembled back into a
full-length DNA contig, ranging in size from a few hundred to millions of bases—a
technique referred to as “shotgun sequencing” due to the randomness of the initial
DNA fragmentation (Sanger et al. 1977; Adey et al. 2010). Prior to the advent of
NGS, shotgun methods were not possible using environmental DNA as the com-
plexity of the samples reduced the likelihood of obtaining sufficient coverage for
assembly (Venter et al. 2004). But NGS dramatically increased sequence coverage,
making it possible to examine all the genes in an environment rather than just one—a
technique called metagenomics. Such metagenomic approaches allow the interac-
tions that support microbial ecosystem dynamics to be identified through the func-
tional gene composition of the community (Handelsman 2004; Tyson et al. 2004;
Venter et al. 2004).
Carrying out metagenomic approaches in oligotrophic caves continues to be prob-
lematic, primarily due to the significant amounts of DNA that are needed to create
shotgun libraries, from a minimum of a few hundred nanograms to multiple micrograms,
depending on the method (Thomas et al. 2012). Despite these limitations, in 2014 Ortiz
et al. were able to carry out metagenomic analyses of the microbial communities within
Kartchner Caverns. Their data identified over 365,000 gene fragments from the micro-
bial populations found on speleothems and walls within the cave and demonstrated that
the enrichment of genes involved carbohydrate metabolism and CO2 fixation. The
enrichment of these genes suggested that both heterotrophic and autotrophic metabolic
activity were important in community growth and subsistence, along with potentially
novel mechanisms of nutrient cycling, especially in regard to nitrogen.
To date, the technical limitations of low biomass have prevented other researchers
from publishing metagenomic studies from oligotrophic caves. Nonetheless, alter-
nate approaches still allow researchers to use NGS to explore evolutionary adapta-
tions, including genomic sequencing of cultured isolates (Lee 2008; Land et al. 2009;
Bhullar et al. 2012; Barton et al. 2013; Saw et al. 2013; Gan et al. 2014; Jiao et al.
2015). Land et al. (2009) were the first to sequence the genome of a bacterial species
isolated from a cave: Beutenbergia cavernae. Their data demonstrated the significant
contribution of carbohydrate catabolism and nutrient cycling genes to the genotype of
this organism, mirroring the results of Ortiz et al., despite the large geographic
distance between the two cave sites (China versus Arizona, USA) (Land et al.
2009). A culture study by Bhullar et al. (2012) on antimicrobial resistance phenotypes
in Lechuguilla Cave (USA) sequenced the genomes of a number of isolates, revealing
the presence of a novel antibiotic resistance pathway and suggesting the in situ
evolution of antibiotic resistance. Finally, a comparative study between cave and
soil strains of Pseudomonas fluorescens determined that cave isolates had adapted to
living within a mineral (rather than soil) environment, demonstrating genomic traits
that could be considered evidence of endemism, including horizontal gene transfer
events, increased scavenging efficiency using twitching motility, and an increased
ability to cycle nutrients, particularly nitrogen (Barton et al. 2013). Thus, while
culturable organisms within caves do represent a small minority (<1%), they are
still able to provide important clues to microbial adaptation within caves (Land et al.
2009; Bhullar et al. 2012; Barton et al. 2013).
5.5 Ecological Themes in Cave Microbial Communities
Box 5.2
Microbial communities in oligotrophic caves are dominated by the Alphapro-
teobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacte-
ria, Chloroflexi, Planctomycetales, Bacteroidetes, Firmicutes, Acidobacteria,
Actinobacteria, Nitrospirae, Gemmatimonadetes, and Verrucomicrobia
(Fig. 5.3). This distribution appears to be robust across a broad geographic
range, indicating that the key drivers of cave community structure are consis-
tent and that this diversity represents a core cave microbiome.
More than 80 years after Baas-Becking developed his theory of “everything is

everywhere. . .” the identification of a rare biosphere appears to provide the neces-
sary empirical support—a cosmopolitan distribution of microorganisms that can
proliferate or “bloom” under the appropriate conditions (Lynch and Neufeld
2015). If all environments contain such functionally diverse populations, then the
primary drivers of population dynamics and structure are simply those factors that
favor one microorganism over another (Lynch and Neufeld 2015).
Yet these same 13 phyla (see Box 5.2) are also dominant in soils, which have
remarkably different conditions of light, productivity, disturbance, and pH (Fig. 5.6).
The simplest explanation of such similarity is that soil microorganisms seed caves. The
seeding hypothesis for cave community structure would certainly provide an expla-
nation of how cave environments are populated: the solvent action of surface (mete-
oric) water creates the cave, while also carrying microbial species into this newly
60
Cave Biome Soil Biome
(pH 7–8)
40
20
0
Acidobacteria
Actinobacteria
Acidobacteria
Actinobacteria
Alphaproteobacteria
Betaproteobacteria
Deltaproteobacteria
Gammaproteobacteria
Verrucomicrobia
Alphaproteobacteria
Betaproteobacteria
Deltaproteobacteria
Gammaproteobacteria
Verrucomicrobia
Nitrospirae
Nitrospirae
Planctomycetales
Bacteroidetes
Firmicutes
Gemmatimonadetes
Planctomycetales
Bacteroidetes
Firmicutes
Gemmatimonadetes
Chloroflexi
Chloroflexi
Phylum
Fig. 5.6 Boxplot comparison of soil microbial communities under alkaline conditions to those
found in caves. The cave biome includes the data shown in Fig. 5.3. The soil biome data was
obtained from the studies of Lauber et al. (2009), Zhou et al. (2007), Ganzert et al. (2014), and
Zhalnina et al. (2015). The boundaries for the first and third quartile are shown, with the centerline
representing the mean and whiskers representing the max/min values (outlier values for the cave
biome data are shown in parentheses)
forming habitat. Recent studies support this idea, including the identification of
surface/epikarst-derived microorganisms in stalactite drip water (based on their covari-
ance with seasonal events) and the scant (<0.1%) population of cyanobacteria
observed in cave samples via deep sequencing (Fig. 5.5) (Gerič et al. 2004; Harmon
et al. 2013; Yun et al. 2015). Nonetheless, several pieces of evidence suggest that the
long-term impact of surface species on cave community structure may be diminished
over time, including (1) the finding by Ortiz et al. that less than 16% of the microor-
ganisms found in caves share taxonomic identity with soil species, (2) the significant
evolutionary adaptations observed in the genomes of indigenous microorganisms, and
(3) a study by Johnston et al., which demonstrated that human commensal species
introduced into a cave habitat are quickly lost (Land et al. 2009; Barton and Barton
2012; Johnston et al. 2012; Ortiz et al. 2013). Thus, while soil may seed these
environments, unique selective pressures within caves “sort” microbial species into
the observed cave populations (Fig. 5.3).
5.6 An Incomplete View
This review has focused on the contributions of bacteria to cave microbial community
structure as most studies likewise focus on the members of this domain (Peck 1986;
Vlasceanu et al. 1997; Schabereiter-Gurtner et al. 2002; Laiz et al. 2003; Barton et al.
2004; Engel et al. 2004b; Ikner et al. 2007; Zhou et al. 2007; Banks et al. 2010; Iker
et al. 2010; Bhullar et al. 2012; Cuezva et al. 2012; Ortiz et al. 2013). Nonetheless,
focusing on the bacteria provides an incomplete view of microbial populations, which
commonly include contributions from the archaea and microscopic eukarya—in
caves, primarily in the form of filamentous fungi (Pace 1997; Barton and Northup
2007; Vanderwolf et al. 2013). While some studies have incorporated the archaea into
their analyses and others have focused exclusively on mycology, none have attempted
to determine community structure and metabolic relationships across all three domains
of life (Woese and Fox 1977, Northup et al. 2003; Tetu et al. 2013; Vanderwolf et al.
2013; Barton et al. 2014; Ortiz et al. 2014). Given the significant contributions that
members of these domains can make in other microbial ecosystems, it is reasonable to
assume that the archaea and fungi help shape community metabolic interactions and
diversity in caves.
5.6.1 Archaea
The first demonstration of archaea in caves was by Northup et al. (2003), who
identified members of the Thaumarchaeota (at the time still phylogenetically
grouped within the Crenarchaeota) in the ferromanganese deposits of Lechuguilla
Cave, USA. Other studies supported the presence of archaea in caves, including the
significance of the Thaumarchaeota across multiple cave habitats (Fig. 5.7; Chelius
and Moore 2004; Barton et al. 2007; Barton et al. 2014; Ortiz et al. 2014). In all
cases, the numerical abundance of the archaea remains small (<2%), although these
studies are based on sequence-only approaches, rather than direct cell counts
(Fig. 5.7). In our work, which incorporated direct cell counts using archaeal-specific
fluorescent in situ hybridization, we have observed a strong correlation between the
availability of nitrogen and presence of archaea; when nitrogen levels are at their
lowest (ng/L), the contribution of the Thaumarchaeota to total population size can
exceed 15% (Johnston and Barton unpublished data), indicating that the contribution
of archaea to population structure covaries with resource limitation. The small size of
Thaumarchaeota (up to 100-fold smaller than their bacterial counterparts), their
slow growth rate, and innate resistance to severe energetic stress could certainly
make the archaea more competitive under extreme nutrient limitation and explain
why they make a larger contribution to microbial community structure in some cave
environments (Könneke et al. 2005; Valentine 2007; Brochier-Armanet et al. 2008;
Brochier-Armanet et al. 2012).
Fig. 5.7 Boxplot comparison of the relative distribution of three major Archaea phyla,
Thaumarchaeota, Crenarchaeota, and Euryarchaeota in cave environments as determined by
Illumina sequencing. The datasets used were SRR1686967, SRR1686970, SRR1686976,
SRR1693633, SRR1703816, SRR1703817, SRR1703818, SRR1703819, SRR1703820, and
SRR1703821, obtained from the NCBI sequence read archive (SRA) and processed in QIIME
(Caporaso et al. 2010) as described in Fig. 5.5. The boundaries for the first and third quartile are
shown with the centerline representing the mean and whiskers representing the max/min values. Inset:
A histogram of the relative distribution of all archaeal 16S rRNA sequences identified from caves
Despite this, the dominance of Thaumarchaeota in archaeal populations should

be difficult to reconcile with the low levels of nitrogen found in caves (Barton 2015);
the Thaumarchaeota play an important role in nitrification, where they utilize the
oxidation of mineralized nitrogen (NH3/NH4+) to generate energy for autotrophic
growth (Brochier-Armanet et al. 2008). In order to compete with bacterial species for
available NH3, the Thaumarchaeota express high affinity (nM) transport mecha-
nisms, making them better adapted to the low-nitrogen level found in caves, and
providing them an alternate growth strategy in an environment where competition
for other resources is likely to be high (Martens-Habbena et al. 2009). Given the
increased dominance of nitrogen cycling and recycling metabolic pathways
observed in bacterial populations in caves, the almost exclusive identification of
Thaumarchaeota in archaeal populations may suggest that nitrogen is one of the
most overlooked drivers of microbial community structure in caves (Fig. 5.7; Ortiz
et al. 2013; Tetu et al. 2013).
5.6.2 Fungi
One aspect of diversity that has generally lagged behind our understanding of cave
microbiology is the role of fungi. While the human pathogen Histoplasma capsulatum
was found in guano from cave hibernacula in 1957, very few papers had examined the
mycology of caves beyond this organism (Ajello et al. 1960; Hasenclever et al. 1967;
McMurray and Russel 1982; Sterflinger 2000; Burford et al. 2003); however, in 2009
this rapidly changed, when a fungal agent was found to be responsible for the
devastating White-Nose Syndrome (WNS) epidemic in bats (Fig. 5.2; Reynolds and
Barton 2014b). This disease was first identified in the winter of 2006–2007, when a
cave in New York State, USA, contained a number of dead and dying bats. Every one
of the sick bats appeared to have an unusual, white-powdery substance on their
muzzles and wing membranes (Frick et al. 2010). This powdery substance was
subsequently identified as the conidia (asexual spores) of a previously undescribed
fungal pathogen, Pseudogymnoascus (known as Geomyces) destructans (Pd) (Gargas
et al. 2009; Frick et al. 2010; Minnis and Lindner 2013). Since this initial outbreak,
WNS has spread to infect bats across 38 US states and 5 Canadian provinces with
mortality rates approaching 71%, making it one of the most devastating wildlife
diseases of North America in modern history (Boyles et al. 2011; Reynolds and
Barton 2014b). The WNS fungus (Pd) originated in Europe, where it likely emerged
from a Pseudogymnoascus sp. endemic to cave environments (Peuchmaille et al.
2011; Warnecke et al. 2012; Reynolds and Barton 2014a; Reynolds et al. 2015,
2016). The identification of such an important mycosis demonstrated a significant
lack of our understanding of cave mycology, and there has been a surge in studies
attempting to determine how the Geomyces/Pseudogymnoascus fit into the ecology of
cave systems (Fig. 5.2). By attempting to produce a broader ecosystem prospective,
such studies have dramatically increased our understanding of the diversity of fungi in
caves (Fig. 5.1; Vanderwolf et al. 2013).
The most commonly identified fungal species in caves are members of the phyla
Ascomycota (~70%), Basidiomycota (20%), and Zygomycota (~7%) (Vanderwolf
et al. 2013); however, the relevance of these findings should be viewed with caution,
as these studies utilize cultivation-dependent techniques, which are susceptible to the
same sample bias that once plagued cave bacteriology (Anderson and Cairney 2004;
Tedersoo et al. 2014). The dominance of the phylum Ascomycota, which contains
filamentous (mold) species such as Aspergillus and Penicillium, would suggest an
environmental advantage for members of these genera; however, these fast-growing
species readily utilize the nutrients found in media, often outcompeting other species
during cultivation. One cave study based on molecular techniques suggested that the
Ascomycota and Basidiomycota were almost equal in abundance (52 and 48%,
respectively), although the investigators could not rule out contamination for dom-
inance of the observed Basidiomycota (Connell and Staudigel 2013). Other investi-
gators have suggested that fungal spores found in cave locations have been
introduced by human or animal activity, reiterating the hypothesis that microbial
populations in caves do not represent endemic species, but environmental
Fig. 5.8 Scanning electron microscopy images of pristine surfaces from Lechuguilla Cave, USA.
Clearly visible on calcite mineral surface are fungal conidia (a), fungal hyphae (b), and etch marks
left behind by fungal growth (c; arrows), demonstrating how the fungi modify mineral surfaces
within the cave. Such etching of calcite by fungal species has been demonstrated before (e.g.,
Burford et al. 2003). Scalebars ¼ 10 μm
contaminants (Shapiro and Pringle 2009, Vanderwolf et al. 2013). The use of
molecular techniques to identify the true fungal ecology of caves therefore remains
a high priority (Tedersoo et al. 2014).
Whatever the true diversity of fungi in caves, it is likely that they play a significant
role in ecosystem processes. Fungi have been described from pristine cave environ-
ments, ruling out the idea that they are introduced contaminants, while there is
evidence that they play an important role in altering mineral chemistry (Fig. 5.8;
Cunningham et al. 1995). In other geologic settings, fungi are known to be important
weathering agents, whether mechanically sugaring or chemically dissolving the sur-
face in an attempt to access nutrients, or concentrating important nutrients and trace
metal ions, which can be readily utilized by other microorganisms for growth
(Sterflinger 2000; Burford et al. 2003). The growth of these filamentous fungi across
mineral surfaces (Fig. 5.8) also generates microfabrics that support the growth of other
microbial species (Burford et al. 2003). Together these diagenetic processes change
the mineral matrix to form other deposits, such as calcite, goethite, halloysite, and
montmorillonite, all of which have been detected in caves (Polyak and Güven 2000,
1996). Within surface soils, fungi play a most dominant role in breaking down
macromolecular structures, and it is likely that their dominance in caves is greatest
where particulate detritus (such as sticks and leaves) is brought in through flooding or
direct anthropogenic impact (Jurado et al. 2010; Schneider et al. 2012). The effective
ability of fungi to breakdown these recalcitrant carbon sources could subsequently
provide a pool of nutrients for the growth of other microorganisms (Barton 2015).
5.7 Toward a Better Understanding of Microbial Cave

Diversity
There has been a rapid increase in the pace of research in cave microbiology (Fig. 5.2).
While just a handful of labs in North America, Europe, and Australia published on the
topic in the 1990s, a tally of current publications reveals over 47 separate research
groups, including numerous labs in Asia and the emergence of the discipline in South
America. Such increased contributions have started to impact the broader microbiolog-
ical sciences, where interest is growing in the ability of cave environments to provide
important clues into the emergence of infectious mycoses, novel antibiotics, and the
evolution of antibiotic resistance (Bhullar et al. 2012; Fisher et al. 2012; Derewacz et al.
2013, 2014).
Despite this increased interest, there remain several barriers that need to be over-
come by new researchers wanting to enter the field. These barriers include the difficulty
in accessing and carrying out research in the challenging environment of caves and the
technical limitations of working with low biomass samples. Cave access limitations can
be overcome by reaching out to local cavers and speleologists, who often have the best
information on appropriate caves for access and can even help identify microbial
habitats for research; however, working with low biomass samples remains challeng-
ing, particularly in regard to preventing contamination, DNA extraction, and low DNA
template levels (Barton et al. 2006). Such limitations can be overcome by using targeted
cultivation approaches that take into account potential bias or circumvented by access
to technologies that make it possible to work with low biomass samples (Summons
et al. 2014). Currently, advances in DNA extraction and analysis make it possible to
extract and work with nanogram to picogram- levels of DNA from geochemically
complex environmental samples, while a new Nextera protocol only requires 1 ng/μL
of template DNA to prepare Illumina libraries for metagenomic sequencing (Pel et al.
2009; Grunenwald et al. 2010; Rinke et al. 2013). Low biomass limitations may be
overcome in the future by emergent technologies, such as new NGS approaches;
PacBio (sequencing individual DNA fragments >40,000 bp in length) or Nanopore
(sequencing single DNA molecules up to millions of bases in length) sequencing could
be combined with single-cell whole-genome sequencing (SCWGS) (Branton et al.
2008; Rinke et al. 2013; Rhoads and Au 2015). While these methods have not yet
been used in caves, such technologies could revolutionize the way low biomass
environments are examined.
Over the next decade, it is likely that cave research will adopt many of the technology
trends currently advancing the field of environmental microbiology, including the use of
metabolomics (studying whole community metabolic products), metatranscriptomics
(studying changes in whole community transcription), and metaproteomics (studying
whole community protein expression patterns). Such data could help us understand the
relative contributions of heterotrophy, autotrophy, mutualism, and competition to com-
munity energetics, or the unique role geochemistry plays on microbial community
structure, with a goal of integrating the domain-level contributions of bacteria, archaea,
and fungi that make it possible for microbial communities to subsist in such nutrient-
limited habitats (Barton et al. 2007; Banks et al. 2010; Engel 2010; Lee et al. 2012;
Barton 2015). Such studies may identify the fundamental ecological principles and
adaptations that drive community dynamics and diversity and provide a more satisfying
answer as to what constitutes a cave microbiome.
Acknowledgements The authors would like to thank Dr. Raina Maier for access to sequencing
data, Drs. Soumya Ghosh and Naowarat Cheeptham in compiling the list of international research
groups, and Dr. Max Wisshak for the SEM images used in Fig. 5.8.
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Chapter 6
Phototrophs in Caves
Janez Mulec
6.1 Introduction
Cave passages that open to the surface experience some level of direct or indirect
illumination by sunlight. Natural light is available not only at the cave entrance but
also deeper inwards, and its amount depends upon entrance orientation, type of cave
(horizontal cave/shaft) and the surrounding relief around the cave entrance which
affects the pattern of light scattering and shading. Under suitable conditions, indirect
light reflected from water, ice or snow can penetrate deeper into underground
passages. In this illuminated zone, extending to the zone of complete darkness,
phototrophic organisms can utilize light as an energy source (Dobat 1998).
Vegetation commonly masks cave openings, but in some cases, its presence is
indicative of a cave, for example, where humidity from underground reaches the
surface in arid areas. With gradual light decrease, the zonation of phototrophs
reduces sequentially from phanerophytes, pteridophytes and bryophytes to algae
and cyanobacteria. Microscopic phototrophs including lichens and, especially, algae
and cyanobacteria colonize rocky and sedimentary surfaces at entrances. Colourful
vertical streaks named also ink streaks or tintenstriche (originally described as
Tintenstriche in German) are clearly visible on exposed limestone and dolomite
surfaces dominated by cyanobacteria (Luttge 1997). Their colour is intensified when
seeps become active. Cyanobacteria in particular live not only on the surface
(epilithic) but also beneath the surface (endolithic) and in crevices within stony
substrata as euendoliths (Komárek and Anagnostidis 2000). Differently coloured
patinas of biofilm on walls, ranging between greenish, bluish and brownish, span
J. Mulec (*)
Karst Research Institute, Research Centre of the Slovenian Academy of Sciences and Arts,
Postojna, Slovenia
UNESCO Chair on Karst Education, University of Nova Gorica, Vipava, Slovenia

https://doi.org/10.1007/978-3-319-98852-8_6
92 J. Mulec
from the cave mouth deep into the twilight zone. Phototrophs submerged in
entrances of water cave colonize rocky surfaces as a part of periphyton. Researchers
have to improvise many times during sampling in caves. Once a proper sample is
collected, it is generally subjected to well-established laboratory methodologies
from plant and microbial ecology and lately more and more from molecular biology.
Light is a prerequisite for growth of phototrophs (Figs. 6.1a and b), but commonly
in cave entrances, there is no clear zonation of vegetation (Mulec et al. 2008). In the
Frasassi Caves (Italy), the cyanobacterium Phormidium sp. exhibited a light com-
pensation point at 13 μmol photons/m2/s and captured an average photosynthetic
photon flux density (PPFD) only between 6 and 10 μmol photons/m2/s (Giordano
et al. 2000). The quantity of energy (PPFD) available for photosynthesis is photo-
synthetically active radiation (PAR) between 400 and 700 nm. The quantity of
photons available for photosynthesis does not correspond to illuminance sensed by
the human eye, which is expressed in luminous flux (lux). In extreme habitats, the
light compensation point of bryophytes is similar to that of algae (Glime 2007).
Some algae and cyanobacteria can readily switch photoautotrophic metabolism to
override the lack or insufficient quanta of light by adopting photoheterotrophic or
entirely heterotrophic metabolic pathways (Droop 1974; Lloyd 1974; Pentecost and
Whitton 2012).
In some caves, phototrophic organisms are actively involved in lithogenesis
(Figs. 6.1a and c). These formations commonly include stromatolitic stalagmites,
stalactitic tufas (Taboroši 2006) and biocalcification associated with the mosses
Eucladium verticillatum and Didymodon tophaceus termed eucladioliths and
didimodontoliths, respectively (Dalby 1966).
In show caves equipped with electric lighting, there are areas around lamps that
are colonized by lampenflora (Fig. 6.1d). Lampenflora also referred to as lamp flora
is a result of light eutrophication and is composed predominantly of algae,
cyanobacteria, commonly also with bryophytes and ferns (Mulec 2012). Because
the community of lampenflora is responsible for the deterioration of substrata, this
issue is particularly urgent in artificially lit historic underground sites (Asencio and
Aboal 2001; Saiz-Jimenez 2010; Zammit et al. 2011; Albertano 2012; Zucconi et al.
2012).
6 Phototrophs in Caves 93
Fig. 6.1 Phototrophs in caves: (a) schematic representation of natural flora in a cave entrance
(1 phanerophytes, 2 bryophytes and ferns, 3 biocalcification on a moss under a seepage, 4 a
tuffaceous stalactite oriented towards incoming sunlight, 5 a stromatolitic stalagmite under a
seepage, 6 surfaces colonized predominantly by cyanobacteria) and lampenflora (7 bryophytes,
8 a speleothem predominantly colonized by algae); (b) cave entrance colonized by phototrophs,
Osojca Cave, Slovenia; (c) stromatolitic stalagmites, Škocjan Caves, Slovenia; (d) lampenflora,
preferential colonization of sediment surfaces, Postojna Cave, Slovenia
94 J. Mulec
6.2 Zonation of Phototrophic Communities
Box 6.1
The pattern of decrease in light and increase in humidity towards the cave
interior, accompanied by zonation of vegetation (Fig. 6.1b), gives four distinct
regions of the phototrophs distribution (Dobat 1970, 1998). The outermost
region, the “access zone”, can be rather shady but is always well illuminated,
and some soil is present; phanerophytes predominate over pteridophytes and
bryophytes. The “entrance zone” has less soil, receives indirect sunlight and is
distinguished by a more hygrophilous and ombrophilous vegetation, which
still includes phanerophytes, ferns and bryophytes. Etiolated growth of plants
is also activated and is expressed as bigger or sparser leaves, weak stems,
elongated internodes or even chlorosis (Dalby 1966; Burgess 1985; Dobat
1998; Whippo and Hangarter 2006). Cytological changes, such as alteration of
chloroplasts, grana and thylakoids, were observed in mosses cultivated in
complete darkness inside a cave (Rajczy 1978–1979). In the “transition
zone”, which receives weak, indirect light, there are phanerophytes, but fairly
abundant communities of ferns and bryophytes also occur. Non-colonized
patches between these organisms clearly show a greenish patina of algae and
cyanobacteria. Finally, the “deep twilight zone” ends with the absence of
surface colonization of algae and/or cyanobacteria. As the distance down the
light gradient increases, there is a corresponding decrease in cyanobacterial
diversity. There are also reports of a lowering of the proportion of coccoid
vs. filamentous cyanobacteria in the deep zone in some caves (Vinogradova
et al. 1998), whereas in some other caves, there are reports of predominance of
Oscillatoriales over Chroococcales (Lamprinou et al. 2012a).
There are also changes in the phototrophic community related to eukaryotic algae.
For example, towards the inner part of Sefunim Cave in Israel, the diversity of
diatoms was greater than that of Chlorophyta, and Xanthophyta gradually
disappeared from the community (Vinogradova et al. 2009). Additionally, an ideal
clear zonation related to species diversity and their abundance in cave entrances is
not always expressed for bryophytes (Pentecost and Zhaohui 2001), nor for algae
and cyanobacteria (Mulec et al. 2008; Lamprinou et al. 2012a). In show caves,
lampenflora can colonize wide surfaces, up to or locally exceeding a few square
metres around an individual lamp. Distinct light-related zonation of different groups
of phototrophs is not common among lampenflora (Mulec et al. 2008).
Light and climatic conditions differ between horizontal caves and shaft systems.
Stronger climatic gradients are generally better expressed in shafts, with a more
clearly delimited zonation of vegetation (Rosselló and Ginés 1980; Fiol 1995) giving
favourable conditions for some uncommon bryophytes (Pericàs et al. 2009). This is
particularly important in hot places such as the Mediterranean region, where the
summer is extremely dry and the shaft microclimate offers a refuge to species such as
Asplenium scolopendrium which is most common in shaded places, moist soil and
damp crevices (Ginés and Ginés 1992). Interestingly, in this respect, approximately
25% of the Balearic (Spain) bryoflora is reported from karst caves and shafts
(Rosselló and Pericàs 2011). Furthermore, because of microclimatic peculiarities
and passage morphology at cave entrances, such as in the Škocjan Caves (Slovenia),
glacial floral relicts (e.g. Primula auricula, Saxifraga incrustata, Viola biflora) and
thermophilic taxa (e.g. Adiantum capillus-veneris, Asparagus acutifolius) can be
found at the same location (Martinčič 1973). It seems that some cave entrances in
SW China act as a refuge for diverse angiosperms (flowering plants) due to defor-
estation of the surrounding areas in the past, which caused drastic changes in the
microclimate and landscape (Monro et al. 2018).
6.3 Ecology and Relevance of Cave–Associated

Phototrophs
Phototrophs fix carbon dioxide into biomass (Berg 2011) which becomes available
higher in the food web (Briand and Cohen 1987). In situ fixed biomass in cave
entrances and the eventual fate of its organic carbon in a cave ecosystem have not yet
been evaluated, though probably its significance is not negligible. Prokaryotes, as
well as (micro)eukaryotes, enter into interactions with phototrophs in these habitats,
where there is the potential to discover new organisms, such as the amoebae
Allovahlkampfia spelaea, first described from an illuminated stromatolitic stalagmite
(Walochnik and Mulec 2009). Independent of photosynthetic reactions, there are
also cave ecosystems based upon in situ microbial chemolithoautotrophy (Sarbu
et al. 1996; Porter et al. 2009; Por et al. 2013).
6.3.1 Diversity
At least 278 unique cyanobacteria species have been documented from caves in the
temperate zone (Hauer et al. 2015). Various authors have pointed out the relative
dominance of cyanobacteria in the community of cave entrances (e.g. Golubić 1967;
Vinogradova et al. 1995; Asencio and Aboal 2000; Selvi and Altuner 2007; Uzunov
et al. 2007; Mulec et al. 2008; Czerwik-Marcinkowska and Mrozińska 2011; Mazina
and Maximov 2011; Lamprinou et al. 2012a; Popović et al. 2015). The most
commonly documented cyanobacteria were from the genera: Aphanocapsa,
Aphanothece, Chroococcus, Gloeocapsa (Fig. 6.2), Lyngbya, Phormidium and
Scytonema. The evidence suggests that caves may be diversity hotspots for some
cyanobacteria, especially Hapalosiphonaceae and Symphyonemataceae, and some
species representatives are believed to be obligatorily cavernicoles. This could be
96 J. Mulec
Fig. 6.2 Gloeocapsa sp., a

common cyanobacterium in
cave entrances
attributed to the fact that in comparison to subaerial habitats, where cyanobacteria

are not exposed to high UV irradiation, desiccation and major temperature oscilla-
tions, the only stressor in cave entrances is low PPFD (Hauer et al. 2015). Addi-
tionally, in cave, micro-niches with higher PPFD values, large filaments of algae and
cyanobacteria can physically protect any underlying small phototrophs (Golubić
1967).
Mosses, liverworts and ferns in cave entrances are common species that also
occur in adjacent areas (Buckallew 2003; Moseley et al. 2013). Flowering plants are
restricted to the outermost parts of cave entrances (Dobat 1998). Several bryophytes
are commonly found in cave entrances, for example: Anomodon viviculosus,
Conocephalum conicum, Ctenidium molluscum, Encalypta streptocarpa,
Homalothecium sericeum, Plagiochila asplenioides and Porella platyphylla. Taxa
such as Fissidens cristatus, F. minutulus, Leiocolea muelleri, Mnium stellare,
Pedinophyllum interruptum and Thamnobryum alopecurum have a larger ecological
tolerance for the dim light conditions in caves. The most commonly reported
representative of pteridophytes in cave entrances is Asplenium trichomanes (Dobat
1998). Some caves provide a habitat for endangered and protected species, for
example, the fern Polystichum aculeatum in Nagi Vizes Cave, Hungary (Buczkó
and Rajczy 1989). Fern prothalli and moss protonema are commonly encountered
deeper inside cave entrances (Rajczy et al. 1986).
In lampenflora, the percentage of cyanobacteria is generally smaller, commonly
with essentially equal proportions of cyanobacteria, Chlorophyta and Chrysophyta.
Chrysophyta are represented mostly by diatoms (Mulec 2015). A typical represen-
tative of lampenflora is an ubiquitous, fast-reproducing aerophyte terrestrial alga
(Chlorophyta or Chrysophyta) or cyanobacterium that is adapted to low PPFD
values (Mulec 2015). In lampenflora, mosses Fissidens taxifolius and Amblystegium
serpens are frequently identified (Glime 2007) among other moss protonema and
fern prothalli.
Cave habitats are sources of species new to science; some recent examples are the
cyanobacteria, Iphinoe spelaeobios and Loriellopsis cavernicola (Lamprinou et al.
2011), Toxopsis calypsus (Lamprinou et al. 2012b) and Culatella subterranea
(Zammit et al. 2012) and the diatoms: Cholnokyella aerophila (Taylor and Lange-
Bertalot 2013), Nupela troglophila (Falasco et al. 2015), Mayamaea cavernicola and
Sellaphora barae (Van De Vijver and Cox 2013). Algal diversity in submerged
marine caves is, as yet, understudied (Gerovasileiou et al. 2015); also there is only
limited information about psychrophilic algal diversity in ice caves (Kol 1964;
Hillebrand-Voiculescu et al. 2014), fumaroles and lava tubes (Goff et al. 1979;
Van De Vijver and Cox 2013). Molecular methods provide opportunities to extend
understanding of the total diversity of subaerial and underground communities
(Pentecost and Whitton 2012).
6.3.2 Environmental Conditions and Estimates

for Colonization
Box 6.2
There are many environmental parameters crucial for the development of
aerophytic communities in cave entrances: light, type of substrata
(e.g. limestone, lava, ice, soil, sediment) and their characteristics
(e.g. porosity, inclination), water and nutrient availability, concentration of
carbon dioxide and biological factors such as the availability of inoculum
(cells, spores and thalli), ecological interactions and organic debris including
excrements (Mulec 2015). For colonization by higher plants, other important
considerations are the cave entrance morphology (e.g. horizontal cave or shaft)
and its geographic position (altitude and orientation), the characteristics of the
surrounding area (such as whether it is open, shrub-covered or forested) and
the past and ongoing activities of humans and animals (Fiol 1995).
The inoculum of phototrophs from adjacent areas reaches cave entrances either with
air currents, normal water flow, floods of sinking rivers and seeps through crevices in
the rock mass or by the intervention of animals and humans (Mulec 2015). Typical
planktonic species are sometimes found in cave phototrophic communities, for
example, a nonmotile coenobial green alga Pediastrum boryanum was identified
as a part of lampenflora and in a community from the cave entrance. Their presence
indicates not only the efficient transport but also the existence of a suitable niche in
water droplets in aerophytic habitats (Mulec et al. 2008). Alongside light levels and
the depth of the cave entrance, hydrodynamics is the most important factor in the
establishment of phytobenthos within submerged caves (Alongi et al. 2012). In
Vlychada Cave, Greece, the distribution of cyanobacteria in a water gallery was
influenced predominantly by concentration of carbon dioxide, in a dry part of the
98 J. Mulec
cave by relative humidity and PPFD, and at the cave entrance by temperature
(Lamprinou et al. 2014). In Arubota’im Cave, Israel, low light, water deficit and
peculiarities of the halite bedrock were responsible for extremely low cyanobacterial
diversity (Vinogradova et al. 2011). In the Alpine show Cave Bossea, in Italy, the
presence of seeping water and the distance from the cave entrance had significant
impacts on the growth of lampenflora, but tourist presence did not (Piano et al.
2015).
Concentration of photosynthetic pigments per unit of surface is a measure of
colonization and organic biomass of phototrophic biofilms. In the illuminated cave
entrance of Božana Cave, Serbia, the concentration of chlorophyll a was up to
7.07 μg/cm2 (Popović et al. 2015), whereas in Škocjan Cave, Slovenia, the maxi-
mum concentration was 1.71 μg/cm2. Using a pulse amplitude modulated fluorim-
eter on lampenflora in Cave Bossea, the chlorophyll a concentration for
cyanobacteria was up to 5.90 μg/cm2, 6.14 μg/cm2 for diatoms and 4.32 μg/cm2
for green algae (Piano et al. 2015). These are higher values compared to the
maximum concentration of total lampenflora chlorophyll a (2.45 μg/cm2) in Pekel
pri Zalogu Cave, Slovenia, where pigment extraction procedures were used (Mulec
et al. 2008). Furthermore, in Cave Bossea, illuminance (lux) was the major factor
that correlated positively with the presence and productivity of these main groups of
phototrophs (Piano et al. 2015).
6.3.3 Adaptations of Phototrophs for Light–Deprived Cave

Environments
Cyanobacteria can live in caves at minuscule PPFD values, ranging as low as

0.0008–0.06 μmol photons/m2/s (Martínez and Asencio 2010). Changes of the
ultrastructure of photosynthetic membrane systems (generally they become densely
packed) in light-deprived cave environments have been documented for several
genera and organisms: Chlorella (Cox 1977), Chroococcus (Cox 1977), Gloeocapsa
(Cox et al. 1981), Geitleria calcarea (Couté 1982), Chroococcidiopsis,
Cyanosarcina, Leptolyngbya, Phormidium and Pseudocapsa (Asencio and Aboal
2004). The authors suggested that the loss of a raphe system in Diprora can also be
attributed to the cave environment (Kociolek et al. 2013).
Box 6.3
Physiological changes, such as increased biosynthesis of photosynthetic pig-
ments (Mulec et al. 2008) and chromatic adaptation, for example, in
Phormidium valderianum (Pentecost and Whitton 2012), were recognized
among the isolates from some caves. Algae and cyanobacteria in caves can
change their cell morphology, and this makes microscopic identification
(continued)
Box 6.3 (continued)

problematical; thus, the use of cultures is essential. Various morphological
modifications have been observed, for example, among the cyanobacteria:
sheath pigments, cell walls and envelopes and formation of resting or dormant
cells (Komárek and Anagnostidis 2000; Komárek and Anagnostidis 2005;
Komárek 2013).
One of the well-known cytological adaptations of bryophytes to low levels of

light is the example of the moss Schistostega pennata. It commonly grows in cave
entrances in the northern temperate zone and, due to its characteristic light reflection,
it is known as “dragon’s gold”. The lens-shaped cells of the moss protonema are
curved to focus the light, and chloroplasts orient themselves so that they are always
located at the most intensely illuminated point at the inner wall of the cell (Glime
2007). In the southern hemisphere, the moss Mittenia plumula exhibits similar
protonema characteristics (Dobat 1998).
6.3.4 Lampenflora: A Problem for Show Caves
The term lampenflora is applied to the phenomenon of the proliferation of mainly

phototrophic organisms in newly formed ecological niches close to artificial lights in
caves (Fig. 6.1d). Vascular plants are rarely present at these locations, but if they do
occur, they are normally found as germinating shoots (Mulec 2012). Additionally,
mass tourism and artificially induced air circulation can enhance the expansion of
lampenflora (Mulec 2015). Surfaces with lampenflora are likely to develop more
quickly in show caves with an active underground stream. For example, two show
caves in Slovenia with comparable visitor numbers and lighting regimes (annual
illumination of >60 h/sector and average PPFD of 28.0 μmol photons/m2/s in
Kostanjeviška Cave and > 80 h/sector and average PPFD of 30.3 μmol photons/m
2
/s in Županova Cave) differ in terms of their lampenflora colonization. In Županova
Cave, 58.2% of lamps are colonized by algae and 7.7% by bryophytes, whereas in
Kostanjeviška Cave, with an active underground river causing occasional floods,
85.5% of lamps are colonized by algae and 43.6% by bryophytes. Bryophytes, as
well as algae and cyanobacteria, can proliferate at low PPFD values. For example,
Eucladium verticillatum was identified at PPFD ranging from 1.4 to 530.0 μmol
photons/m2/s (Mulec and Kubešová 2010).
The effects of lampenflora may differ between cave ecosystems. Surfaces covered
by phototrophic lampenflora that are a source of organic carbon can become the
physical and nutritional base for niche ecosystems populated by higher trophic levels
of heterotrophs. Abundant lampenflora biomass might impact the ecology of fauna
and their interactions. The potentially irreversible deterioration effect of lampenflora
biofilm on underlying substrata is especially serious in caves with painted rock art
100 J. Mulec
(Albertano and Urzi 1999; Saiz-Jimenez 2010; Albertano 2012; del Rosal et al.
2014).
Different physical methods have been employed in attempts to control
lampenflora: cleaning of colonized surfaces with brushes and water jets, shortening
the lighting period, reducing the light intensity, partially altering the emission spectra
of lamps and the use of UV lamps (Mulec and Kosi 2009). UVC-emitting lamps have
a proven deleterious effect on a mixture of Chlorophyta from lampenflora (Borderie
et al. 2011). UVC irradiation causes thymine-thymine dimers leading to mutations
(Sinha and Häder 2002) and thus also has a deleterious effect upon other cave biota in
a range of irradiation. Various chemicals have been applied in attempts to kill
lampenflora, for example: Atrazine (6-Chloro-N-ethyl-N0 -(1methylethyl)-1,3,5-tri-
azine-2,4-diamine), bromine compounds, calcium hypochlorite, cupric ammoniac
compounds, Diuron (N-3, 4-dichlorophenyl-N0 -dimethyl urea), formalin, hydrogen
peroxide, quaternary ammonium derivatives, Simazine (6-Chloro-N,N0 -diethyl-
1,3,5-triazine-2,4-diamine) and sodium hypochlorite (Mulec and Kosi 2009; Bastian
et al. 2010). Sodium hypochlorite and hydrogen peroxide are nowadays the most
widely used chemicals (Hebelka 2014). At the moment, a buffered (pH 7.0–7.5)
solution of hydrogen peroxide (15–20%) seems to be the most environmentally
friendly method for the remediation of insensitive calcite surfaces affected by
lampenflora (Mulec 2014).
Some progress towards the long-term preservation of delicate, historically impor-
tant, surfaces that are artificially lit might be achieved by installation of a tuneable
LED lighting system. This would have the capability to modulate the spectral
distribution to minimize damage (such as colour fading) by the effective irradiance,
would partially inhibit biodeterioration and would improve the apparent colour
reproduction of the illuminated objects (de Luna et al. 2015). The use of lamps
with altered emission spectra is only partially successful in inhibiting biodeteriora-
tion because some phototrophs can readily and rapidly modify synthesis of (acces-
sory) photosynthetic pigments (Roldán et al. 2006; Mulec 2014). Alongside regular
removal of lampenflora, in order to reduce lampenflora growth and expansion, the
lighting regime should also be restricted; illumination of speleothems and artefacts
should be as brief as possible, with the minimum PPFD (Mulec 2014).
6.4 Phototrophs Impact Cave (Micro)Morphology
Biota participates actively in changes to the cave environment as a

biogeomorphological factor that involves litholysis (rock weathering, erosion) and
lithogenesis (mineral formation). Light availability undoubtedly enhances biologi-
cally mediated erosion in marine caves via the formation of notches (Coombes et al.
2015). Biodeterioration of colonized illuminated surfaces in caves with rock-art
paintings is an especially urgent problem (Urzi et al. 2010). Mineral deposits such
as gypsum, halite and calcite related to biochemical activities contribute to biodete-
rioration of artworks in underground archaeological sites (Zammit et al. 2011). In
some caves, especially those close to the surface, plant roots can penetrate deep and
impact upon cave formations. On the other hand, lithogenesis attributed to
phototrophs in caves results in the development of some particularly obvious and
peculiar formations (e.g. Fig. 6.1c).
Few biologically influenced speleothems are known in caves (Taboroši 2006),
some of them particularly related to phototrophs, calcified plants
(e.g. eucladioliths—Eucladium verticillatum, didimodontoliths—Didymodon
tophaceus), generally irregularly shaped stalactitic tufas (in literature also termed
tuffaceous stalactites or calcareous tufa stalactites) and stromatolitic stalagmites
(Fig. 6.1c), referred also as crayback stalagmites (Dalby 1966; Cox et al. 1989a).
Calcite deposited on E. verticillatum as eucladioliths appears first on leaves, as
isolated crystals, then increases gradually and coalesces until the whole plant is
embedded in calcareous sheaths (Dalby 1966). All such biogenic structures are
oriented towards the incoming sunlight. The term microbialite was applied to a
formation containing the cyanobacterium Phormidium laysanense and hanging from
a cave ceiling on Tikehau atoll, French Polynesia (Sprachta et al. 2001). These
biolithogenic structures are normally soft, fragile and commonly porous. Until they
are not completely lithified, they can physically protect organisms against desicca-
tion and excessive light quanta (Mulec et al. 2007). Only a few cyanobacteria that
actively precipitate carbonates are documented in caves, for example, Geitleria
calcarea (Couté 1989), Scytonema julianum (Couté and Bury 1988), Loriella
osteophila (Hernández-Mariné et al. 1999) and Herpyzonema pulverulentum
(Hernández-Mariné and Canals 1994).
Laminae of stromatolitic stalagmites resemble those of stromatolites formed in
shallow lakes and seas, but they differ in their mode of formation. The proposed
model based on observations at Wombeyan and Jenolan caves (New South Wales,
Australia) includes an active seep within the illuminated zone at the cave entrance.
Wind helps to shape the size of the formation, but most importantly, fixation of
carbon dioxide by cyanobacterial biofilm results in an increase of pH during
photosynthesis and consequently in biocalcification. During drier periods, grains
of aeolian sediment are lithified together by microbial exopolysaccharides on sta-
lagmites, creating typically laminar strata. Low molecular weight fatty acids, amino
acids and n-alkanes derived from cyanobacteria were identified within the structures
(Cox et al. 1989b; James et al. 1994). Recent observations revealed a variety of
crayback morphologies that reflect different local conditions, e.g. wind velocity, drip
level, and activity of phototrophs (Dodge-Wan et al. 2012). Formation of unique
phosphatic stromatolites of hydroxyapatite (Deer Cave, Sarawak, Malaysia) requires
photosynthetic activity of cyanobacteria and a low pH (2.4) of guano drainage water
that dissolves bedrock (Lundberg and McFarlane 2011). It has been suggested that
microbiota also has an important role in the formation of silica stromatolites in a
volcanic cave in the Azores, Portugal (Brunet and Revuelta 2014). Nevertheless, in
some caves, there are also old and inactive stromatolites from past geological
periods, which can be used to aid reconstruction of environmental conditions during
their development (Lisker et al. 2009).
102 J. Mulec
The importance of phototrophs as a potential food source for the cave fauna is
part of Chap. 14.
6.5 Conclusions
Biodiversity and the genetic pool within naturally illuminated parts of caves are still
understudied, especially in lava tubes, ice caves, submerged caves and cenotes,
i.e. open water pools of groundwater. Particularly, for certain higher plants, cave
entrances represent life at an extreme as well as refuge against extinction. Exactly
what, and how much, phototrophs do for the cave ecosystem in conjunction with
other biota remain open questions. There is considerable potential for biotechnolog-
ical applications among cave phototrophs, for example, some cyanobacteria from
caves have displayed antibacterial activity against some human pathogens
(Lamprinou et al. 2015). Phototrophs play an active role in weathering processes
that are especially undesirable in show caves and caves with rock-art paintings.
Regular removal of lampenflora in such places is recommended for bioremediation
of insensitive surfaces. The lighting regime in underground places should be
severely restricted. In addition, in some caves, phototrophs can be the main driver
in the formation of specialized types of speleothems.
Acknowledgements The author acknowledges the financial support from the Slovenian Research
Agency (research core funding No. P6-0119), and Angel Ginés, Tina Eleršek and Andreea Oarga-
Mulec for their valuable comments on an earlier version of the manuscript, and David Lowe for
comments and language editing assistance.
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Chapter 7
Diversity of Terrestrial Invertebrates
in Subterranean Habitats
Louis Deharveng and Anne Bedos
7.1 Introduction
The most comprehensive contribution to the knowledge of world subterranean

biodiversity is the monumental Encyclopaedia Biospeologica in three volumes
coordinated by Juberthie and Decu (1994, 1998, 2001). Since then, a very large
number of subterranean taxa have been described, while the understanding of
diversification processes has considerably progressed (see also Chap. 1). The diver-
sity of terrestrial subterranean fauna has been summarized more recently in dedi-
cated chapters of several books, generally limited to large taxa or large regions
(Culver and Pipan 2009, 2014; Romero 2009; White and Culver 2012). In this
chapter, we shall give a broader overview at the world scale of the patterns of
morphological diversity among terrestrial subterranean invertebrates of the world,
in their taxonomic and adaptive aspects.
Subterranean habitats encompass all kinds of voids in mesoporous below-ground
substrate, including caves, cracks, MSS, upper hypogean voids, and various kinds of
screes (see Chap. 3). Interstitial habitats, where voids are less than a few mm in their
larger size, are traditionally dealt with soil habitats and will not be considered here
(Deharveng and Bedos 2012; Culver and Pipan 2014).
Cave invertebrates are classically categorized as troglobites, troglophiles, and
trogloxenes (but see below for definitions). We shall focus here on troglobites.
Troglophiles will be touched on for species of uncertain ecological status, or when
they belong to lineages where both lifestyles are represented. Amphibious species
and free stages of parasites when only known from caves will be considered as well.
L. Deharveng (*) · A. Bedos

Institut de Systématique, Evolution, Biodiversité, ISYEB - UMR 7205 - CNRS, MNHN,
UPMC, EPHE, Museum national d’Histoire naturelle, Sorbonne Universités, Paris, France

https://doi.org/10.1007/978-3-319-98852-8_7
108 L. Deharveng and A. Bedos
Table 7.1 World troglobitic Hexapoda by order. Source of the total number of species; Zhang:
values extracted from Zhang (2011) (ed.); text: values extracted from various sources, see text; total:
total number of species in the world. Tb: number of troglobites in the world, see text; Orthoptera:
rough estimate including troglophiles
Group Source Total Tb %
Grylloblattodea Text 32 7 21.8750
Diplura Text 984 131 13.3130
Zygentoma Text 568 45 7.9225
Collembola Text 8128 500 6.1516
Orthoptera Text 27,267 250 0.9169
Coleoptera Zhang 386,500 2500 0.6468
Blattodea Zhang 7314 40 0.5469
Psocoptera Text 10,880 30 0.2757
Dermaptera Zhang 1978 2 0.1011
Hemiptera Zhang 103,590 85 0.0821
Diptera Zhang 155,477 22 0.0142
Lepidoptera Zhang 157,338 2 0.0013
Hymenoptera Zhang 116,881 1 0.0009
Zoraptera Zhang 37 0 0
Rhaphidioptera Zhang 254 0 0
Embioptera Zhang 463 0 0
Megaloptera Zhang 354 0 0
Mecoptera Zhang 757 0 0
Protura Zhang 804 0 0
Strepsiptera Zhang 609 0 0
Ephemeroptera Zhang 3240 0 0
Mantodea Zhang 2400 0 0
Microcoryphia Zhang 513 0 0
Neuroptera Zhang 5868 0 0
Odonata Zhang 5899 0 0
Phasmatodea Zhang 3014 0 0
Phthiraptera Zhang 5102 0 0
Plecoptera Zhang 3743 0 0
Siphonaptera Zhang 2075 0 0
Thysanoptera Zhang 5864 0 0
Trichoptera Zhang 14,391 0 0
7.1.1 Statistics, Patterns, Sampling
Globally, terrestrial subterranean diversity represents only a small fraction of surface

diversity (Gibert and Deharveng 2002). Except rare root-feeders, animals linked to
green plants, which constitute the largest part of biodiversity on earth, are absent
underground. Other invertebrate groups have colonized the subterranean environ-
ment with unequal success (Table 7.1), for reasons that remain largely unknown.
7 Diversity of Terrestrial Invertebrates in Subterranean Habitats 109
Fig. 7.1 Cumulated number of taxa along years. (a) Genera of Chinese troglobitic Trechinae (data
from Tian et al. 2016); (b) species of Pyrenean cave Trechinae (data from Faille et al. 2010b); (c)
species of French Pyrenean cave Collembola (data from Thibaud 2017); (d) species of Southeast
Asian cave Diplopoda, genus Glyphiulus. (Data from Golovatch et al. 2007a, b, 2011a, b, 2012;
Likhitrakarn et al. 2017)
Sampling efforts continue to be considerably uneven, and the underlying diversity

patterns generated by biogeographical history are emerging only slowly
(Christiansen 2012). Hard data on cave species number are scattered in the literature,
uneven and incomplete, and the statistics given here are indicative.
It is clear that several zoological groups or regions of Japan, the USA, or Europe,
thoroughly investigated for more than a century, are progressively running dry of
discoveries for many groups. For instance, of the 47 species and subspecies of the
large beetle genus Anophthalmus that occur in the Dinarides, only three have been
described after 2000, against 9 from 1910 to 1920; of the 36 current species and
subspecies of the mostly Romanian beetle genus Drimeotus, none has been
described after 2000, against 11 between 1910 and 1920 (Hlaváč et al. 2017).
Some taxa escape however from this trend, even in the best documented hotspots
like the beetle genus Typhloreicheia: 35 of its 50 Sardinian species, some of them
cave restricted, have been described after 2000 (Magrini et al. 2013). Nevertheless,
globally, the contribution of such regions to global cave biodiversity is decreasing
while that of newly surveyed areas is growing rapidly (Fig. 7.1).
The fast pace of hypogean species discoveries that Australia has experienced for
several years is for instance not likely to slow down with the total number of
subterranean invertebrate species estimated to be 10 times their current number
(Guzik et al. 2011). In Russia, the number of described troglobites doubled since
2001 (Turbanov et al. 2016), with an emerging hotspot in Caucasus. A similar
progress occurred in parts of the Iberian Peninsula (Reboleira et al. 2011; Sendra
et al. 2011). It is nevertheless in the tropics and subtropics that the progress for now
two decades has been the fastest, particularly in China, Southeast Asia, and Brazil
(Fig. 7.1). Recent discoveries have reinforced the demise of the paradigm of a very
low diversity of terrestrial non-guanobitic fauna in these regions, first questioned by
Howarth (1972) based on the fauna of Hawaiian lava tubes: troglobites, some clearly
troglomorphic, have been discovered in Cuba (Silva Taboada 1974), Jamaica (Peck
1975), New Guinea and Borneo (Chapman 1976, 1980), Central America (Reddell
1981), Sulawesi and Thailand (Deharveng and Leclerc 1986), and many other
regions. The lower richness of South America underlined by Deharveng and
Bedos (2012) does not hold anymore with the discovery of a rich troglobitic fauna
in Brazil (Souza-Silva and Ferreira 2016; Trajano et al. 2016). The knowledge of
southern China and Southeast Asia cave diversity has experienced a similar growth
(Deharveng et al. 2009b; Golovatch 2015; Tian et al. 2016). Both Brazil and China
have now emerged as world-class diversity hotspots for subterranean fauna, at the
level of the traditional hotspots of temperate regions (Culver et al. 2006).
In spite of these advances, large blanks subsist on the map. Particularly puzzling
is the paucity of Africa in non-guanobitic troglobites, probably a result of insufficient
sampling and rarity of favorable habitats. A second gap concerns high altitude
tropical caves, where limited available information suggests a high richness in
troglomorphic species (Chapman 1976 and Smith 1980 for New Guinea; Reddell
1981 for Guatemala and Mexico).
7.1.2 Original Features of Terrestrial Subterranean Diversity
Aside from the low and uneven species richness evoked above, terrestrial subterra-
nean biodiversity departs considerably from that of other ecosystems by a higher
proportion of narrow endemics, a higher contribution of radiations to diversity, a
much higher frequency of relictual taxa, and a very original taxonomic composition.
The high representation of short-range endemics in subterranean compared to
surface habitats and the much smaller size, on average, of their distribution range are
documented in many publications (Gibert and Deharveng 2002; Harvey 2002).
A number of speciose lineages contribute substantially to terrestrial subterranean
diversity, as illustrated later on for various groups, like Aphaenops beetles in
Pyrenean or Telema spiders in Southern China (see also Fig. 7.2).
The high contribution of relicts to overall diversity is one of the most exciting
features of cave fauna. With major climate changes having generated species
extinction much more rapidly in surface than in subterranean habitats, many
Fig. 7.2 Contribution of the dominant genus to the total richness in troglobites of different
suprageneric taxa and regions. (a) Coleoptera of the USA (248 species, Pseudanophthalmus the
richest with 157 species); (b) Araneae of the USA (86 species, Cicurina the richest with 47 species);
(c) Trechini of China (104 species, Dongodytes the richest with 12 species); (d) Leptodirini of the
world (935 species, Bathysciola the richest with 95 species); richest genus in light grey, pooled
richness of other genera in dark grey. a and b from Culver et al. (2000), c from Tian et al. (2016) and
d from Perreau (2000, and personal communication)
subterranean species are today geographically disconnected from their closest rela-
tives and considered as relicts (Peck 1990; Humphreys 2000).
Four kinds of relicts can be recognized in terrestrial cave fauna: (1) cold climate
relicts, generated by the glacial reflux after Pleistocene glaciations, are surprisingly
rare; (2) tropical climate relicts, witnesses of warmer or more humid climates, are
frequent. The cave fauna of Cape Range in Western Australia, under arid climate
today, is for instance clearly related to the fauna of rainforest of Eastern Australia,
3500 km apart (Humphreys 1993); (3) most often, disjunct distributions cannot be
explained in climatic terms, and such patterns are much more common in caves than
in any other habitat. Many remarkable examples are listed later in the text for each
taxon, like the spider Telema tenella (Wang et al. 2012) from Catalonia, or the beetle
Dalyat mirabilis (Ribera et al. 2005) from Spain; (4) phyletic relicts, i.e., taxa without
“close” relatives at world level (Humphreys 2000), are also numerous in caves, like
the monospecific and micro-endemic millipede genera Niphatrogleuma (Switzer-
land) and Marboreuma (Pyrenees).
7.1.3 Taxonomic Composition
The prime characteristic of subterranean diversity is to be severely truncated, in the

absence of green plants, which constitute the largest component of surface terrestrial
diversity on earth (Gibert and Deharveng 2002). Second, invertebrate groups that
colonized underground habitats do not contribute to cave diversity in proportion to
their diversity in surface habitats, as clearly illustrated for Hexapoda in Table 7.1:
troglobites are in particular absent or rare in all large groups of insects except
Coleoptera. Non-insects Arthropods (arachnids, millipedes, terrestrial isopods, and
springtails) often linked to soil have a higher proportion of cave-related species.
However, several groups such as mites, Symphyla, Pauropoda, Protura, many
Collembolan families, a large diversity of Coleoptera, though largely dominating
in deep soil communities at the interface with subterranean environments, have
almost no troglobitic representatives. The interface between subterranean and soil
habitats is perhaps a severe filter rather than a major evolutionary pathway for cave
colonization.
7.1.4 Temperate Versus Tropical Diversity
Terrestrial diversity is significantly higher in tropics than that in temperate regions.

This is not the case for cave diversity (Deharveng and Bedos 2012), though recent
discoveries and the integration of guanobites in statistics may shake this view.
Tropical and temperate terrestrial subterranean diversity differs as follows
(Deharveng and Bedos 2000):
A—among troglobites and troglophiles not related to guano: (1) higher diversity of
arachnids except mites; (2) lower diversity of springtails; (3) lower diversity and
lower levels of troglomorphy in beetles; and (4) higher diversity of several other
cave groups, like crickets, cockroaches, millipedes, and woodlice.
B—among guanobites and guanophiles: (5) much higher richness at all taxonomic
levels.
Trajano and Bichuette (2010) retrieved several of these features in Brazil, but
rightly stressed that (points 1 and 4 above) are for a part correlated to the character-
istics of local epigean faunas as source of colonizers rather than to that of subterranean
ecological factors per se. In subtropical regions, as well as at high altitude in the
tropics, diversity patterns exhibit a mix of tropical and temperate subterranean
elements, such as local diversification of troglobitic beetles and lower diversity of
higher Arachnid taxa and guano species.
7.1.5 Lifestyles and Life Forms
Lifestyles Clearly delimited ecological categories are useful for describing and
understanding the diversity, origin, and dynamics of cave communities. Subterranean
animals are most often classified according to the strength of their link to subterranean
environment, under the classical terms of troglobites, troglophiles, and trogloxenes.
We understand here ecological categories as defined below, as a trade-off between
practicality, scientific relevance, etymological coherence, and commonness of use.
The terminology used by Trajano and Carvalho (2017) is given in brackets when
relevant: (1) troglobite: a species only reported from cave and (2) troglophile: a
species reported from inside and outside caves. The subdivision of troglophiles into
eu- and subtroglophiles proposed by Pavan (1944) and endorsed by Sket (2008) is
retained when possible, as meaningful information: eutroglophile: a species with
permanent populations inside and outside caves (¼troglophile); subtroglophile: a
species linked to cave for part of its life cycle (seasonal or circadian) (¼trogloxene);
(3) trogloxene: a species not linked to caves, whatever its morphology (¼accidental).
Some species, of major importance in the tropics, seem to be more dependent on the
presence of guano than on cave habitat itself. Following the rationale of “affinity to
the environment,” Decu (1986) and Gnaspini and Trajano (2000) consider that
species should be characterized along two separate gradients, i.e., their dependence
on cave habitat and their dependence on guano habitat, in order to meaningfully
characterize species ecology. For instance, “guanobites” may be “troglophilic” or
“troglobitic.” Applied to invertebrate fauna, this might greatly change the current
statistics on terrestrial subterranean diversity. For Orthoptera, Desutter-Grandcolas
(1998) uses an even more elaborated system (see below).
Assignation of a species to an ecological category faces in many cases with lack
of biological information (Giachino et al. 2011; Trajano and Carvalho 2017). The
use of morphological traits as indication of troglobitic lifestyle is discussed below.
Life Forms, Facies, Troglomorphy Life forms are combinations of morphological
traits that are only or most frequently encountered in species of unrelated groups
living in similar environments. They are assumed to result from convergent evolu-
tion, acquired under a same set of selective pressures (Trontelj et al. 2012). Their
description and interpretation in terms of convergence and divergence, as well as of
regressive and progressive evolution, is a major focus of current investigation in
subterranean biology (Culver and Pipan 2015, see also Chap. 4).
The concept of facies or “life form” (morphotypes of Golovatch and Kime 2009)
is actually widely used in ecology. Gisin (1943) characterized different life forms in
Collembola that were characteristic of various habitat types. A similar system has
been devised for Diplopoda (Golovatch and Kime 2009). This categorization was
greatly improved by Christiansen (1964), who redefined the terminology on a purely
morphological basis and introduced the term troglomorphic. Life forms and life-
styles are considered separately in this conception, allowing a double characteriza-
tion, ecological and morphological, of a species.
Based on the screening of all cave terrestrial groups of invertebrates, we propose

here a system of life forms characterized as follows (Table 7.2): (1) hypogeomorphic:
regressed eyes, pigment, and wings, without significant modification of body size,
appendage length and claw thickness compared to surface relatives;
(2) troglomorphic: regressed eyes, pigment, and wings + larger size, more slender
body, longer appendages, thinner claws; (3) euedaphomorphic: regressed eyes,
pigment, and wings + smaller size, more compact body, shorter appendages, thicker
claw (Coiffait 1958); (4) epigeomorphic: no regressive evolution, no particular
modification of body size and appendage length.
Troglomorphy as defined above applies more easily to arthropods than to other
invertebrates. Troglomorphic and euedaphomorphic life forms are well recognizable
in these groups, being at the opposite ends of gradients of appendage and body size
(Pipan and Culver 2017). All intermediates exist nevertheless with hypogeomorphic
life forms.
Life forms can be used as proxy for assigning species to ecological or functional
categories, as far as they have been sufficiently documented in the group. However,
such ecological assignation on purely morphological grounds can be misleading as
mismatch between life forms and habitat is not exceptional. Culver and Pipan (2009,
2015) point out that the springtail Acherontides eleonorae is troglomorphic by its
claw elongation but ecologically a guanobite and, based on such mismatch,
questioned the use of the term troglomorphy. In fact, guanobites with more or less
reduced eyes and pigment compared to their epigean relatives are not exceptional in
Diplopoda and Collembola (see later). Nevertheless, the concept of troglomorphy
remains statistically robust across a large array of taxa and remains an integrative and
descriptive tool to approach evolutionary issues related to convergence and diver-
gence hypotheses.
Troglomorphy illustrates striking convergent morphological evolutions that
occurs in many unrelated living groups (see also Chap. 4). However, several of
these traits are also observed separately in epigean life forms, from completely
different habitats (Christiansen 2012 and Table 7.2). Thus, eye, pigment, and wing
regression are shared with interstitial species and are not sufficient to qualify as
troglomorphic. They need for that to be combined with appendage or claw elongation
in the definition adopted here.
As stated in Chap. 4 and by Christiansen (2012), morphological changes associated
with cave life may affect various traits: body size, body slenderness, eyes, pigment,
appendage length, foot complex, and wing development. Other more taxon-specific
traits are recognized by taxonomists as cave related, like cuticle thinning,
pseudophysogastry in beetles, or development of specialized sensory organs in
Palpigradi (Condé 1998), Rhagidiidae mites (Zacharda et al. 2011), and Diplura
(Bareth and Pagès 1994), increase in number and sensorial organs in Collembola.
Some of these characters may be extremely modified in a few cave species that
are qualified as highly troglomorphic. Several examples are given later
on. Appendage elongation is particularly spectacular in this respect, in a large variety
of groups (Ricinulei, Collembola, Zygentoma, Diplura, Carabidae, and Leiodidae).
Such exaggerated traits raise evolutionary and functional questions beyond the scope
of this chapter. The last point to be stressed is that, in all diversified terrestrial groups,
Table 7.2 Terminology and definition of terrestrial life forms of Arthropods with most common trends in morphologic traits, and preferential habitats. Derived
from Gisin (1943) and Christiansen (1964). 0 very reduced or absent; 1 reduced or short; 2 intermediate or present; 3 large, long, slender, or well developed. Foot
complex is too group dependent to be considered here. In brackets, organs which concern only some groups
(Antennae), legs, Lifestyle best
Life form Size Eyes Pigment (pedipalps) (Furca) Claw (Wings) Preferential habitats match
Atmobiomorphic 3 3 3 3 3 2 3 Vegetation in open space Epigean
Hemiedaphomorphic 2 2 2 2 1 or 2 2 1 or 2 Superficial soil Hemiedaphic,
guanobitic
Hygrophilomorphic 2 3 3 2 3 3 3 Wet habitats Epigean
Troglomorphic 3 0 0 3 3 3 1 Caves, (MSS) Troglobitic
Hypogeomorphic 2 0 or 0 or 1 2 2 2 1 or 2 Caves, MSS, soils Troglobitic,
1 guanobitic
7 Diversity of Terrestrial Invertebrates in Subterranean Habitats
Euedaphomorphic 1 0 0 1 0 or 1 1 0 Deep soil, interstitial habitats, Euedaphic,

MSS, caves troglobitic
115
regressive and troglomorphic characters do not evolve at the same pace within a
lineage. It is this decoupling that generates the observed diversity in the expression
of troglomorphy.
7.2 Non-arthropod Invertebrates
7.2.1 Annelida: Hirudinea (Leeches, Fig. 7.3a)
Hirudinea (more than 680 species distributed worldwide, Sket and Trontelj 2008)
mostly live in freshwaters, but are also very common in terrestrial habitats in South
Asia and Australasia. Troglobitic species of the family Haemadipsidae are known
from Southeast Asian caves (Chapman 1976; Deharveng et al. 2007), but only two
have been described: Leiobdella jawarerensis from Papua New Guinea and
Haemadipsa cavatuses from China. Both have reduced skin pigmentation, unlike
surface leeches, but retain pigmented eye patches. They may be found in rather large
populations on cave walls and have been observed sucking the blood of bats.
7.2.2 Gastropoda (Snails, Fig. 7.3b)
Gastropoda include 80,000 species (Bouchet et al. 2005). Among them, only
80 were considered troglobitic or troglophilic by Bernasconi (2004). They belong
to Pulmonata, characterized by the absence of operculum and a portion of the mantle
cavity modified for respiration, as adaptations to terrestrial life.
Cave species are distributed in 12 families (Culver 2012), but cave-restricted ones
are probably few. They include carnivorous species like the troglobitic slug
Troglolestes sokolovi from Caucasus and several troglophilic Oxychilus. Most
detritivorous troglobites were described from the Dinarides, but some species are
also known from other Mediterranean regions like the Pyreneo-cantabric range. The
genus Zospeum (Ellobiidae) is the richest in species, all of minute size, with a disjunct
distribution Eastern and Dinaric Alps—Pyreneo-cantabric range. Other microsnails
were recently described from Eastern Asia, some less than one mm long, hence
among the smallest snails on earth (Páll-Gergely et al. 2015). Several of them (e.g.,
Koreozospeum nodongense from Korea) are only known from caves (Weigand et al.
2013; Jochum et al. 2015). The Subulinidae so frequent on the guano of tropical caves
are considered eutroglophiles.
Eye and pigment reduction are retrieved in all troglobitic species, and partly in
cave Subulinidae. Elongation of tentacles is also obvious in some species, and the
shell is often thinner than that of surface species (Vandel 1964).
Fig. 7.3 Cave non-arthropods and Arachnida; not at the same scale. (a) Haemadipsa sp. (Hirudinea;
Sarawak); (b) Zospeum bellesi (Gastropoda Ellobiidae; Pyrenees); (c) Peripatopsis alba
(Onychophora; South Africa); Opiliones (d to g): (d) Shearogovea mexasca (Cyphophthalmi;
Mexico), (e) Taracus silvestrii (Ischyropsalidae; USA), (f) Speleonychia sengeri (Travuniidae;
USA), (g) Trojanella serbica (Travuniidae; Serbia); (h) Alacran tartarus (Scorpions; Mexico); (i)
Troglobisium racovitzai (Pseudoscorpiones; Spain); (j) Siamacarus sp. (Opilioacari; Vietnam); (k)
Rhagidia (Deharvengiella) paralleloseta (Rhagidiidae; France); Araneae (l to n): (l) Anapistula
ataecina (Symphytognathidae; Portugal), (m) Amauropelma matakecil (Ctenidae; Indonesia), (n)
Trogloraptor marchingtoni (Trogloraptoridae; USA); Amblypygi: (o) Whip spider eating a cricket
7.2.3 Nematoda
The phylum Nematoda includes 24,773 extant species (Hodda 2011) of which
295 have been reported underground (Du Preez et al. 2017). The two species
considered as troglobites (Mylonchulus cavensis and Stenonchulus troglodytes
both from Europe) were recently retrieved in soil.
7.2.4 Onychophora (velvet worms, Fig. 7.3c)
The phylum Onychophora has 180 extant species and 49 genera (Mayer and Oliveira
2011). All are terrestrial ambush predators and show a number of highly peculiar
morphological and biological features. They are distributed in two families, each
with one troglobite: Peripatopsidae in the southern hemisphere with Peripatopsis
alba from South Africa and Peripatidae in the tropics with Speleoperipatus spelaeus
from Jamaica. Both are devoid of eyes and pigment contrary to all surface species
except one, Tasmanipatus anophthalmus from Tasmania, completely white and
blind.
7.3 Arachnida
Arachnida encompasses 112,201 extant species (Zhang 2011) in 16 orders, all

represented in subterranean habitats except Solifugae. They comprise parasites,
guanobites, troglophiles, and troglobites, but in very uneven proportions. In spite
of their huge diversity, mites (Parasitiformes + Acariformes, formerly Acari) are
very poorly represented in oligotrophic cave habitats, being only diversified in guano
or as parasites. On the contrary, spiders and Opiliones, rare in guano, have success-
fully colonized subterranean oligotrophic habitats.
Fig. 7.3 (continued) (Amblypigi; Indonesia), (p) Typopeltis cf. magnificus (Thelyphonida; Laos);
(q) Hubbardiidae (Schizomida; Vietnam). Photos by © A Cressler (a), © O Gargominy (b), © G
Giribet (c), © G Giribet (2011) (d), Shear and Warfel (2016) (e), © M Hedin (f), I Karaman in Kury
et al. (2014) (g), P Sprouse in NSF (2017) website (h), © A Meseguer (i), © M Lukić (j, k), Cardoso
and Scharff (2009) (l), Miller and Rahmadi (2012) (m), Griswold et al. (2012) (n), © C Rahmadi
(o), © L Deharveng and A Bedos (p, q)
7.3.1 Opiliones (Fig. 7.3d–g)
Statistics and taxonomic hierarchy for this order follow Kury (2017) unless specified.
Opiliones contain more than 6650 extant species in ca. 1670 genera and four
monophyletic suborders: Cyphophthalmi, Eupnoi, Dyspnoi, and Laniatores
(Fernández et al. 2017). Most are predators of small arthropods. Troglobites and
troglophiles are numerous, but very few are guano dwellers. Eupnoi has
subtroglophiles not dealt with here, but no troglobites. The other suborders include
troglophiles and troglobites, some of them strongly troglomorphic. In 1994, Rambla
and Juberthie estimated that Opiliones included 82 troglobites and 33 troglophiles, of
which 50 belonged to Gonyleptoidea and 17 to Triaenonychidae. These numbers
have since enormously increased, though detailed statistics are not available.
Cyphophthalmi suborder (147 species, six families) is patchily distributed world-
wide. Its species live in soil and leaf litter and a few are recorded from caves. They
are homogeneous in habitus, small, uniformly and often darkly pigmented, oval,
compact, and relatively short-legged. Many species have been described during the
last two decades, with considerable progress in the knowledge of their phylogeny
and biogeography (Giribet et al. 2016). Stylocellidae family of Southeast Asia
includes seven troglobites, three of which are eyeless species of Fangensis (Clouse
2012). Among the 61 species of Petallidae, of Gondwanan distribution, only
Purcellia argasiformis from South Africa is troglobitic, but slightly modified for
cave life. Troglosiro aelleni, endemic to New Caledonia, is the only cave represen-
tative of this genus of 13 species. It is eyeless, but darkly pigmented and without
appendage elongation. The Afro-American Neogoveidae include the Brazilian
troglobite Canga renatae, eyeless but short-legged. The relictual troglobites
Marwe coarctata from Kenya and Shearogovea mexasca from Mexico combine
anophthalmy, pale coloration, and elongated appendages. Sironidae family (52 spe-
cies, eight genera), distributed in the northern Mediterranean region with isolated
spots in Japan and the USA, is the richest in troglobites, all members of the large
Northeastern Mediterranean genus Cyphophthalmus, and representing more than
half of its 32 species (Karaman 2009). Anophthalmy and depigmentation are shared
by all species of the genus; appendage elongation, including chelicerae, is significant
in some species like C. paradoxus.
Eye reduction and depigmentation in troglobitic Cyphophthalmi are associated
with subterranean life in some lineages (Stylocellidae), but phyletic characters in
others (Sironidae). Appendage elongation is at most moderate in troglobitic species.
The suborder Dyspnoi (ca. 390 species) is mostly Holarctic in distribution. Tens
of species have been reported from caves as troglobites or troglophiles (Schönhofer
2013). In southern Europe, half of the ca. 23 species of the genus Ischyropsalis are
cave related, and several like Ischyropsalis pyrenaea from Pyrenees are weakly
modified troglobites (Schönhofer et al. 2015). Eye reduction, depigmentation, and
appendage elongation are common in other genera, with more than 10 eyeless
species, including seven of the Caucasian genus Nemaspela.
Laniatores suborder (ca. 4248 species, 29 families) is extremely diverse morpho-

logically and includes many eutroglophiles or troglobites (but no guano species).
Since the 1990s, its taxonomy has progressed at a fast pace, through taxonomic
rearrangements (Kury 2015), phylogenetic studies (Sharma and Giribet 2011), and
new taxa descriptions. The richest continent in cave Laniatores is America. They are
considerably less diversified in Africa, the Palaearctic, and tropical Asia. The eight
superfamilies recognized by Kury (2017) include cave-obligate species.
About 13 troglobites, mostly African, are known in Assamioidea superfamily
(535 species). Three of them are eyeless, whereas anophthalmy is reported in at least
ten non-cave species of the group (Starega 1992). Two species of the superfamily are
highly troglomorphic: Maiorerus randoi from Canary Islands and Jarmilana pecki
from Belize, the only American species of the African family Pyramidopidae.
Gonyleptoidea superfamily (2039 species), mostly American, contributes largely
to the overall diversity of troglobitic Laniatores. Several families are concerned, with
Stygnopsidae the richest. Of its more than 30 troglobites, about one-third are blind.
A few species have significantly elongated appendages associated with anophthalmy
and depigmentation, like Giupponia chagasi from Brazil or Mictlana inops from
Mexico.
Phalangodoidea superfamily (132 species) is as rich as Gonyleptoidea in
troglobites (>30), but they represent a much higher proportion of the total richness.
Most European Phalangodoidea are subterranean, i.e., about ten species including
two rare, eyeless, highly troglomorphic ones in relictual monospecific genera of
Balkans: Paralola buresi and Lola insularis. North America is much richer than
Europe, with a remarkable hotspot in California where about 50 species and four
endemic genera are known (Derkarabetian et al. 2010), including non-cave and cave
species at various levels of eye reduction and appendage elongation. Among them,
Banksula comprises ten species, nine of which are troglobites and blind or quasi-
blind. The most troglomorphic species of northern America, like Texella mulaiki,
have very long appendages.
Only 4–5 troglobites are known among Epedanoidea superfamily, mostly diver-
sified in the Indo-Malayan and Australasian regions (ca. 410 species). None is
troglomorphic, though Sungsotia uenoi from Vietnam exhibits some eye and pigment
reduction. Samooidea superfamily (215 species) is a small group of Laniatores with a
few troglobites, mostly diversified in tropical America. A single species, Stygnomma
fiskei, is eyeless and has elongated appendages. More than half of the 78 species of
Travunioidea superfamily are cave dwellers, linked to oligotrophic habitats. They are
concentrated in two regions: the USA and Mediterranean Europe. The USA has a
dozen of weakly cave-adapted troglobites, all oculated, in two genera (Erebomaster
and Sclerobunus), and three troglomorphic eyeless species in two genera
(Speleonychia, monospecific, and Speleomaster, bispecific). In Europe, more than
20 cave species are known, all in Travuniidae, including about 15 troglobites in
mono- or oligospecific relictual genera scattered across northern Mediterranean
regions. They are highly troglomorphic in facies, like Trojanella serbica from Serbia.
Adaptive traits include reduction or absence of eyes (about ten species are eyeless),
very pale coloration, and elongated appendages.
Triaenonychoidea superfamily (ca. 490 species), temperate Gondwanan in dis-

tribution, is poorly diversified in caves with only four troglobites. Two of them are
eyeless and troglomorphic: Picunchenops spelaeus from Patagonia and Lomanella
troglodytes from Tasmania. Zalmoxoidea superfamily (272 species), widely distrib-
uted in tropical regions, have troglobites in America, and one in Australia. Reduced
eyes or anophthalmy are reported in several of them, but also in some soil species.
The cave-restricted Relictopiolus galadriel from Brazil has moderately elongated
appendages and minute size (Pérez-González et al. 2017).
Troglomorphy among Opiliones is mostly expressed through the classical trends
of eye reduction, depigmentation, and appendage elongation, while body size may
increase or decrease. Eye reduction up to disappearance is also reported in non-cave
species of Assamioidea in Africa (Kauri 1985) or Zalmoxoidea in Brazil (Pinto-da-
Rocha and Kury 2003), associated with soil or termite nests.
7.3.2 Scorpiones (Fig. 7.3h)
Scorpiones (1947 species, 18 families, Prendini 2011a) are all predators. Lourenço
(1994) mentioned 13 troglobitic species in four families, of which 11 are from
Mexico. Sixteen years later, Prendini et al. (2010) considered as unequivocally
troglobitic 23 species, in 16 genera and 10 families. Today, we know about
40 cave-restricted species in 25 genera and 12 families and a few eutroglophilic ones.
More than half of the troglobitic scorpions have eyes and pigment reduced or
absent, often associated with appendage elongation. Anophthalmy is however
retrieved in non-cave species, like Chaerilus telnovi from Halmahera. Cave scorpions
are patchily distributed. They are absent in temperate regions, except the relictual
genera Belisarius in Catalonia, almost blind but troglophilic, and Akrav in Israel,
troglobitic and blind. Most cave scorpions are distributed in two spots: Mexico and
central Indochina. In Mexico, troglobites (ca. eight species in four genera, all eyeless)
belong to Typhlochactidae. Alacran species are rather large (6–7 cm) and highly
troglomorphic, and two of them are the only known amphibious scorpions
(Santibáñez-López et al. 2014). Troglobites of the three other genera, like
Typhlochactas reddelli, are often minute (less than 2.5 cm). In Cuba, the guanobitic-
troglobitic Alayotityius delacruzi is not morphologically modified. Central Indochina
between 15 N and 20 N is the second spot of diversity, recently discovered, with five
cave species in the small relictual family Pseudochactidae (Lourenço and Pham 2010),
including three blind Vietbocap. In the tropics south of 15 N, scorpions are highly
diverse in surface habitats, but rare underground. The only blind cave species in this
area is Chaerilus sabinae from Sulawesi. No troglobite is known from Africa, only a
few, weakly modified, from Madagascar (Lourenço and Goodman 2008), South
America (Gallão and Bichuette 2016), and tropical Asia. At least, Australia has a
single troglobite, Aops oncodactylus, which is small, blind, and depigmented.
7.3.3 Pseudoscorpiones (Fig. 7.3i)
Pseudoscorpiones (3533 extant species, 26 families in 2012, Harvey 2013) are

small-size predators of arthropods, frequent in soil, mosses, under barks, and in
caves, where most of their 26 families are represented. Cave-dwelling species can be
allocated to two ecological categories: guano species and oligotrophic habitat
species. Guano species, mostly Chernetidae and Cheliferidae, are moderately diver-
sified, not troglomorphic and often widely distributed through phoretic behavior.
Troglobites of oligotrophic habitats are narrow endemics and much more diverse,
though often extremely rare. Most have reduced or no eyes, and elongated append-
ages, to an extreme degree in some Ideoroncidae and Bochicidae. On the other hand,
several eyeless troglobites are euedaphomorphic in facies.
Troglobitic species are especially numerous among Chthoniidae and Neobisiidae.
Edward and Harvey (2008) list 49 species of blind cave-dwelling Chthoniidae in the
world, most of them troglobitic members of Lagynnochthonius and Tyrannochthonius,
diversified in various regions like the USA and Spain (Zaragoza 2017), Australia, or
the Dinarides (Ozimec 2004). The family Neobisiidae exhibits a similar richness in the
latter region, with many cave endemics.
Relictual Pseudoscorpiones are frequent in caves. The Iberian Peninsula is of
particular interest, with two highly troglomorphic relicts, Titanobochica magna and
Troglobisium racovitzai, only representatives in Europe of the American family
Bochicidae (Reboleira et al. 2010), and Arcanobisium comasi, the only species of the
subfamily Arcanobisiinae within Syarinidae. In Southeast Asia, the subfamily
Cybellinae was recently defined for two non-troglomorphic cave species of
Feaellidae, a family mostly diversified in Africa (Judson 2017).
7.3.4 Palpigradi
Palpigradi (82 species, two families, Prendini 2011b) are small (less than 2.2 mm),
delicate, white and eyeless, and live in soils and caves of tropical, Mediterranean,
and more rarely temperate regions (Condé 1996). In the northern part of their
distribution range, they are only found in caves. In the tropics, they are more frequent
in soils. They seem to prey on small microarthropods like Collembola (Condé 1996).
Most cave species are members of the cosmopolitan genus Eukoenenia. Europe has
more than 25 described troglobitic species (Condé 1998), while the two richest
tropical countries, i.e., Brazil and Thailand, have seven and five species of
Eukoenenia (Souza and Ferreira 2016).
Troglomorphic characters observed in cave species include larger size and longer
appendages, and an increase in the number of blades in the lateral organ of the
prosoma (Condé 1998), of unknown function. These characters change at different
pace during evolution. Thus, E. thais from Thailand which has long appendages and
the highest number of blades in the lateral organ is a small-size species (about 1 mm).
7.3.5 Ricinulei
Ricinulei (58 species, a single family, Prendini 2011c) are predators of nematodes
and small arthropods. They are well diversified in tropical America, less in Africa,
and absent in Asia. Cave species, restricted to tropical America, are usually
guanobitic, blind but pigmented like surface species, and of moderate size (10 mm
at most). A wide gradient of appendage elongation is known in Pseudocellus, which
contains the most troglomorphic species, Pseudocellus krejcae.
7.3.6 Acari (Mites) (Fig. 7.3j, k)
Though no more recognized as monophyletic, mites are very abundant and highly
diversified in energy-rich habitats of caves, while very few have adapted to energy-
poor habitats. Troglomorphic species are exceptional, limited to a few Opilioacari
and Rhagidiidae. This low representation of mites in cave oligotrophic habitats
illustrates that soil has not been a highway for the colonization of these habitats by
this invertebrate group. Two superorders and four orders contain troglobitic species:
Parasitiformes with the orders Opilioacarida and Gamasida, and Acariformes with
the orders Sarcoptiformes and Trombidiformes.
Opilioacarida order includes ca. 35 species in a single family (Beaulieu et al.
2011), that live in soil or under tree barks mainly in the tropics. Four species in two
genera are known as cave dwellers. Two are Opilioacarus from Cuban caves; they
have eyes, pigment, and non-elongated appendages. Two are Siamacarus from
southern Thailand caves where they live in large populations on guano; they have
delicate and long legs and are almost devoid of pigment, though eyes are still
present. This genus is present in Vietnam with undescribed troglobitic species
(Deharveng et al. 2009a). Gamasida, the largest order of Parasitiformes (11,424
species, 109 families; Beaulieu et al. 2011), are distributed worldwide. A large
number of cave species are permanent or temporary ectoparasites of bats, but most
are free living. Parasitidae and several related families are active predators in high-
energy habitats. Many Gamasida are only known from caves. Thus, 60% of the
50 subterranean species of Uropodidae are cave restricted, with 12 of them in the
genus Uroobovella alone, but we ignore in most cases to which extent these
non-modified species are dependent on subterranean environment. Many species
of Gamasida disperse by phoresy, which may explain their frequently wide distri-
bution range.
Among Sarcoptiformes the suborder Oribatida contains 16197 extant species in
249 families (Schatz et al. 2011). Its species, all decomposers, are the most diver-
sified and abundant mites in soil. They are represented in caves as troglophiles or
guano dwellers, but cave-restricted species are surprisingly rare: some species of
Schwiebea (Acaridida) from humid hypogean habitats, devoid of adaptations to
subterranean life, and a few European Belbidae, such as Metabelbella
phalangioides—pale and long-legged. Trombidiformes (25821 extant species,

151 families; Zhang et al. 2011) is one of the most diverse order of mites. Its larger
subdivision, Prostigmata (more than 20,000 species), includes a few poorly known
cave species (Palacios-Vargas et al. 1998) in the families Leeuwenhoekiidae,
Trombiculidae, and Trombidiidae, frequent on or close to guano accumulations in
tropical caves, and bat or invertebrate parasites at larval stage. They are large,
unpigmented, eyeless, and long-legged, i.e., of typical troglomorphic facies. The
family Proterorhagidiidae has a single relictual troglobite, Proterorhagia oztotloica
from Mexico. Rhagidiidae is the only mite family that has colonized cave oligotro-
phic habitats. Its species are hunting predators frequent in soils worldwide, all blind
and unpigmented (Zacharda 1980). Troglobitic Rhagidiidae are often linked to cold
caves in the Holarctic. They may exhibit substantial appendage elongation. Peculiar
to the family, the rhagidial organ, a group of solenidia of unknown function, shows
significant elongation or multiplication in most cave-obligate species.
7.3.7 Araneae (spiders, Fig. 7.3l–n)
In 2017, extant Araneae encompassed almost 47,000 species in ca. 4060 genera and
112 families (World Spider Catalog 2017). Spiders have colonized most terrestrial
habitats and are a major faunistic element in caves and subterranean voids. While
they are second to mites in soil layers and guano, spiders are the main predators in
parietal and often in oligotrophic habitats. Two syntheses on cave spiders diversity
and biology were recently published (Mammola et al. 2017; Mammola and Isaia
2017). At the world level, these authors provide an estimate of 1000 cave-related
species, in at least 48 families.
Northern temperate regions are the richest in cave spiders, but increasing sam-
pling in the tropics may change the story. No less than 40 eyeless cave spiders are
already described from tropical regions (Mammola and Isaia 2017), and we know
that a same cave may harbor several strongly modified troglobitic species (Bloom
et al. 2014; Deharveng and Bedos 2000). Sparassidae (Heteropoda and related
genera), prominent predators of the giant arthropod communities of Southeast
Asia, have diversified into a huge number of mostly epigean species worldwide
(Jäger 2014), with several cave species, some of them with eye reduction up to
anophthalmy (Sinopoda scurion from Laos).
In Europe, 486 cave-related species are known, of which 194 are considered
troglobites (Mammola et al. 2017). This modest number of troglobites compared to
the huge number of troglophiles is related to the large contingent of darkness-loving
species that exist all over the world. Among troglobites, the families Linyphiidae and
Dysderidae are dominant, like in other temperate regions, in contrast to many
tropical and southern hemisphere regions, where other families contribute more to
biodiversity.
Like the cited Sinopoda, most genera of troglobitic spiders have epigean and
troglophilic counterparts (Ribera 2004). Regional radiations involving surface and
cave species contribute for a large part to spider diversity, like that of the genus
Dysdera in Canary Islands. In southern China, the recently disclosed radiation of the
genus Telema encompasses dozens of species, probably all troglobitic, some eyed
and some eyeless (Lin and Li 2010; Wang and Li 2010). The radiation of Cicurina
(Cicurella) in Texas includes 80 surface and troglobitic species with extensive
gradients of eye regression, from full-eyed to eyeless species (Paquin and Dupérré
2009). Conversely, relictual species that have no close relatives in surface fauna are
surprisingly rare compared to other cave groups. The case of Telema tenella is the
best documented: the species limited to a few caves of Catalonia (Spain) is thousands
km away from other species of other Telemidae in Africa, America, or Asia. A
second remarkable species, Anapistula ataecina from Portugal, is the only member
of the family Symphytognathidae in Europe. A last remarkable relict is Trogloraptor
marchingtoni recently discovered in caves of Oregon (USA), for which a special
family (Trogloraptoridae) has been erected.
Morphological diversity within cave spiders is illustrated among various features
by their size range (Mammola and Isaia 2017): from 0.4 mm (Anapistula ataecina
from Portugal, one of the smallest spiders in the world) to 30 cm of legspan
(Heteropoda maxima from Laos). The troglomorphic “syndrome” is a classical
combination of eye reduction, depigmentation, size increase, and appendage elon-
gation, but is less obvious in spiders than in other arthropods for the last two
characters. Tiny species, particularly in the families Oonopidae and Tetrablemmidae,
frequently encountered in tropical caves, have a euedaphomorphic facies, and may
prove to be actually soil dwellers, except Bacillemma leclerci, a troglobite from
Thailand, which combines very small size and very long legs.
7.3.8 Amblypygi (Whip Spiders, Fig. 7.3o, p)
Amblypygi comprise ca. 190 extant species grouped into five families (Seiter and
Wolf 2014) and two unequal suborders: Paleoamblypygi and Euamblypygi. Statis-
tics on the different families are drawn from Prendini (2011d). The group is
widespread in the tropics. All species are predators using a “sit-and-wait” hunting
strategy. Till recently, only two or three cave species of Amblypygi were considered
troglobitic (Weygoldt 2000). A number of new species have been described since,
mostly in the genus Charinus in Brazil, several troglobitic with reduced eyes and
pigment (Giupponi and Miranda 2016).
Paleoamblypygi suborder contains a single extant species of small size
(Paracharon caecus), which lives in termite nests in Guinea-Bissau and is completely
blind (Weygoldt 2000). Almost half of Euamblypygi species are troglophiles and few
are troglobites. Most troglobites belong to Phrynidae and Charinidae. Phrynidae
(70 species, four genera) are often of large size and are all American except Phrynus
exsul from an Indonesian cave. Phrynidae include troglobites from Mexico and Cuba
with eyes variously reduced, like Paraphrynus reddelli, an eyeless species of Mexico.
Charinidae (ca. 90 species, three genera) are small-size circumtropical Amblypygi,
including at least 30 short-range troglobites or troglophiles, mostly from Southeast

Asia and mostly in the genera Charinus and Sarax. Cave species tend to be
depigmented and their eyes variously reduced, up to anophthalmy for instance in
Charinus caribensis from Jamaica or C. stygochthobius from Socotra Island.
Charontidae (14 species, two genera) are limited to Southeast Asia, Australasia, and
the Pacific. Some large species are components of the giant arthropod cave fauna of
Southeast Asia. None exhibits clear morphological adaptations to cave life.
The only certain cave-adaptive characters of cave Amblypygi are the reduction of
eyes and pigment. Both traits are however also observed in some non-cave species.
The long appendages are similar in cave and non-cave species. Adaptation of
Amblypygi to cave life is therefore weakly expressed in their morphology.
7.3.9 Thelyphonida (Giant Whip Scorpions, Fig. 7.3p)
Thelyphonida (110 species, one family; Prendini 2011e) are large-size tropical
Arachnida. No cave dwellers were reported by Juberthie and Decu (1994, 1998).
The recently described Typopeltis magnificus is however, in spite of its dark
coloration, a regular inhabitant of caves in southern Laos, so far unnoticed outside
caves.
7.3.10 Schizomida (Microwhip Scorpions, Fig. 7.3q)
Schizomida (ca. 260 species, two families; Prendini 2011f) are fast-moving preda-
tors of moderate size (5–10 mm), living in soils and in caves, preferentially in high-
energy habitats. Protoschizomidae, restricted to Mexico and Texas, has 16 species in
two genera, all troglobitic. Hubbardiidae, widely distributed in tropical and subtrop-
ical regions up to Japan and the USA, include the remaining species, with troglobites
or troglophiles. Several show weak troglomorphic traits such as loss of eyes or
relatively elongated appendages (Cokendolpher and Reddell 1992).
7.4 Myriapoda
Of the four classes of Myriapoda, one has no cave species (Pauropoda) and another a
few uncertain troglobites (Symphyla; Juberthie-Jupeau 1994).
7.4.1 Chilopoda (Centipedes, Fig. 7.4a)
Chilopoda (ca. 3110 extant species, 24 families; Minelli 2011) is divided into 5 orders.
Scutigeromorpha and Scolopendromorpha are mostly diversified under warm cli-
mates, Lithobiomorpha and Craterostigmomorpha in temperate regions, while
Geophilomorpha are present worldwide. All Chilopoda are carnivorous and play a
major role in soil and cave communities. Cave species are known in six families:
Scutigeridae (Scutigeromorpha), Lithobiidae (Lithobiomorpha), Cryptopidae,
Scolopendridae and Scolopocryptopidae (Scolopendromorpha), and Geophilidae
(Geophilomorpha). Scutigeridae and Scolopendridae include troglophiles, but no
troglobites. An estimate of 55 troglobitic Chilopoda was given by Negrea and Minelli
(1994). This number has increased to at least 70 species today.
Lithobiidae (1000 species, 43 genera, Minelli 2011) are mostly, though not only,
diversified in the western Palaearctic. Bonato et al. (2016) list 519 species for the
genus Lithobius alone, which is the richest in troglobites and troglophiles. In France,
16 of its 62 species are troglobites (Geoffroy and Iorio 2009). Eye reduction and
appendage elongation are frequent among cave species, where elongation of anten-
nae and legs may be extreme, like in L. matulicii (Dinarides) or L. lorioli (Cantabric
range). Other troglomorphic species are known in the eastern Mediterranean genus
Eupolybothrus, like E. cavernicolus from the Dinarides.
Cryptopidae and Scolopocryptopidae have a few troglobites in Europe, Canary
Islands, and Australia, and more in tropical and subtropical America with ca. five taxa
of Cryptops (Azara and Ferreira 2014) and ca. eight of Scolopocryptopidae (Chagas
and Bichuette 2015). Strongly elongated antennae and legs are known in
Scolopocryptops troglocaudatus and several Newportia. Only two troglobitic
Geophilidae with significant antennal elongation exist in Europe: Geophilus perseph-
ones from Pyrenees and Geophilus hadesi from Croatia (Stoev et al. 2015).
7.4.2 Diplopoda (Millipedes, Fig. 7.4b–i)
Diplopoda is a mega-diverse group of mostly saprophagous soil species (7753

species, 147 families, Shear 2011). They are present in all regions of the world,
with a large number of cave-related species in 11 of the 16 orders of the class,
taxonomically arranged as follows (Shear 2011): subclass Penicillata (Polyxenida),
subclass Chilognatha (infraclass Pentazonia (Glomeridesmida, Glomerida),
infraclass Helminthomorpha (Siphonophorida, Julida, Spirostreptida, Spirobolida,
Callipodida, Chordeumatida, Stemmiulida, Polydesmida)).
Polyxenida order (86 extant species, four families, Shear 2011) is distributed
worldwide. Its species are abundant in dry soils and under barks, with ca. three
troglobites: Lophoproctus pagesi from Balearic Islands and two recently described
species from Christmas Island (Lophoturus speophilus and L. humphreysi). A weak
elongation of appendages is noted in L. pagesi and L. speophilus.
Fig. 7.4 Cave Myriapoda, Isopoda, Collembola, Diplura, Zygentoma; not at same scale. (a)
Eupolybothrus cavernicolus (Chilopoda; Croatia); Diplopoda (b to i): (b) Illacme tobini
(Siphonorhinidae; USA), (c) Glyphiulus sp. (Cambalopsidae; China) feeding on a dead bat, (d)
Pseudonannolene lundi (Pseudonannolenidae; Brazil), (e) Biokovella mauriesi (Biokovellidae;
Glomeridesmida order (31 species, two families, Shear 2011) is locally frequent
in tropical forest of Asia and America. Unidentified species are cited from various
caves of Southeast Asia (Shelley and Golovatch 2011), and two troglobitic
Glomeridesmus species have been described from Mexico and Brazil.
Glomerida order (pill millipedes; 212 species, two families, Shear 2011) is widely
distributed in East Asia and the Euro-Mediterranean region, with a few representatives
in central and northern America (Shelley and Golovatch 2011). In America, only the
genus Glomeroides is present underground, with six microphthalmic troglobites from
Mexico (Palacios-Vargas et al. 2015). The Euro-Mediterranean region is much richer,
especially its western part, with several oligospecific genera including cave species of
euedaphomorphic facies, such as Doderia, Geoglomeris, and Spelaeoglomeris
(Mauriès 1994). Four troglobites are reported from southern Spain, northern Africa,
and Canary Islands in the large, ordinarily epigean genus Glomeris. Hyleoglomeris
known till recently by a few cave species from southeastern Europe and Southeast
Asia, unpigmented and blind or microphthalmic, is in fact diversified, with more than
20 troglobitic species described since 2006 in these regions (Golovatch et al. 2012).
Cave Glomerida are depigmented, their eyes are reduced or absent, but their append-
ages not elongated.
Siphonophorida order (ca. 70 species, 14 genera, Shear 2011) includes three
subterranean species: Yucatanium sabachana from Mexico (Palacios-Vargas et al.
2015) and two species of the relictual Californian genus Illacme: I. plenipes, an MSS
inhabitant and the leggiest animal of the world with up to 750 legs, and I. tobini
known by a single specimen from cave. They are phyletically eyeless, but not
troglomorphic.
Julida order (ca. 740 extant species, 163 genera, Shear 2011) is mostly distributed
in the Holarctic and Oriental regions. Since the review of Mauriès (1994), a number
of cave species and genera have been described in the families Julidae, more
diversified in eastern Mediterranean regions, and Blaniulidae, more diversified in
western Mediterranean regions. In Julidae, the genus Typhloiulus has ca. 30 cave
species (WoRMS 2017), scattered in the Balkans and surroundings, eutroglophilic or
troglobitic (Mauriès 1994). Cave species are known in several other genera, like
⁄
Fig. 7.4 (continued) Croatia), (f) Dobrodesmus mirabilis (Dobrodesmidae; Brazil), (g and h)
Pacidesmus spp. (China), non-troglomorphic (above) and troglomorphic (below), (i)
Eutrichodesmus astrisimilis (Vietnam); Crustacea (j to m): (j) Erebusa calobates (Brachyura; Laos),
(k) Trogloniscus trilobatus (Oniscidea; China), (l) Iuiuniscus iuiuensis (Oniscidea; Brazil), (m)
Xangoniscus odara (Oniscidea; Brazil); Collembola (n to t): (n) Yuukianura judithae on bat guano
(Vanuatu), (o) Verhoeffiella longicornis (Bosnia-Herzegovina), (p) Sinella longiantenna (China), (q)
Lepidonella lecongkieti (Vietnam), (r) Oncopodura sp. (Spain), (s) Tritomurus veles (Croatia), (t)
Galeriella liciniana (Bosnia-Herzegovina); (u) Gollumjapyx smeagol (Diplura; Spain); (v)
Texoreddellia sp. (Zygentoma; USA). Photos by Stoev et al. (2013) (a), Marek et al. (2016) (b), Latella
and Hu (2008) (c), Iniesta and Ferreira (2015) (d), Antić et al. (2016) (e), Shear et al. (2016) (f), © L.
Deharveng and A. Bedos (g, h, j, k, p, q, r), D VandenSpiegel in Golovatch et al. (2016) (i), Souza et al.
(2015) (l), Campos-Filho et al. (2016) (m), © B Lips (n), © M. Lukić (o, s, t), JM Azkarraga in Sendra
et al. (2006) (u), Espinasa et al. (2016) (v)
eyeless Dolichoiulus in Canary Islands (Enghoff 2012). The six species of the
Pyreneo-Cantabric genus Baskoiulus are troglobites, as well as the two species of
the Catalan genus Paratyphloiulus. Several monospecific genera have also
troglobitic species, like Mammamia from Italy. Blaniulidae have more than
50 cave-dwelling species, particularly diversified in southwestern Europe (Antić
et al. 2015). Of the 11 species of Blaniulus, six are troglobitic (Kime and Enghoff
2017), and others are known in a number of oligo- or monospecific genera of western
(e.g., Euzkadiulus, Iberoiulus, Tarracoblaniulus) and eastern Mediterranean regions
(Cibiniulus, Thassoblaniulus). Nemasomatidae includes some remarkable
troglobites, like Antrokoreana gracilipes from Korea, and two species of the
genus Thalassisobates (continental Spain and Canary Islands) closely related to a
littoral species. Some Mongoliulidae from the Palaearctic Far East seem to be
troglobitic as well (Mikhaljova 1997).
All Julida are cylindrical and devoid of well-marked integument ornamentation
with a homogeneous habitus, accentuated in cave species by convergent regressive
evolution of eyes and pigment and absence of appendage elongation or shortening. A
few species like Vascoblaniulus cabidochei (Pyrenees) and Typhloiulus spp. (Balkans)
have modified mouthparts suggesting life in hygropetric habitats.
The tropical order Spirostreptida is subdivided into Cambalidea (242 species, five
families) and Spirostreptidea (772 species, five families, Shear 2011). Cambalidea
includes a high proportion of cave-related species, while Spirostreptidea has only
two troglobites from Yucatan Peninsula caves. Aside from a few eutroglophiles
known in and outside caves, many Spirostreptida, listed as troglophiles due to lack of
troglomorphic traits, are probably cave restricted, as most were so far not found
outside caves. Cambalopsidae (noodle millipedes), widespread in continental
Southeast Asia and southern China, are the main contributor to cave species richness
of Cambalidea. Two of its genera are highly diversified underground: Glyphiulus
and Plusioglyphiulus. Of their 81 species, 57 have been described since 2007 (see for
instance Golovatch et al. 2011a, b). Most live in eutrophic cave habitats, reaching
impressive density on guano. They exhibit various degrees of pigment and eye
reduction up to anophthalmy in several Glyphiulus species, without clear appendage
elongation. Endemism, usually low among guano species, is very high in cave
Cambalopsidae. Cambalidae includes several eyeless troglobitic species, distributed
in Central America and southern USA (Cambala, Mexicambala, Jarmilka). They are
replaced in South America by Pseudonannolenidae which includes several recently
described troglobites, all members of the large genus Pseudonannolene. They prefer
high-energy habitats (debris or guano), are depigmented, have reduced eyes, but
their size is smaller than that of their epigean relatives (Iniesta and Ferreira 2015).
The pantropical order Spirobolida (506 species, 13 families, Shear 2011) has at
least two troglobites: the troglomorphic guanobite Reddellobus troglobius in Mexico
and Speleostrophus nesiotes, depigmented and eyeless, in Australia.
Callipodida order (ca. 140 species, seven families, Shear 2011) has a patchy
distribution in northern hemisphere. Its species, very large—up to 10 cm long, live
preferentially in rocky habitats including caves. About 30% are troglobites or
troglophiles (Stoev et al. 2008). However, they are not or are only weakly
troglomorphic (depigmentation and eyes sometimes reduced). Their appendages are

elongated, like those of their epigean relatives. The main genera which contain
troglobites are Tetracion (USA), Sinocallipus and Paracortina (continental South-
east Asia), Acanthopetalum, Apfelbeckia, Balkanopetalum with six troglobites, and
Callipus (Europe, especially Balkans).
Chordeumatida order (ca. 900 species, 50 families and four suborders, Shear 2011)
are absent in Africa and South America, and patchily distributed elsewhere, with a
peak of richness in Europe (Shelley and Golovatch 2011). About 40% of its 50 families
include troglobitic or troglophilic species, usually linked to oligotrophic habitats. This
high taxonomic diversity is paralleled by a wide morphological range of adaptive
traits, with gradients of pigment and eye reduction till complete loss within several
genera and families. Cave Chordeumatida are particularly diversified in the Pyreneo-
cantabric range, Dinarides, and Eastern United States (Culver and Shear 2012), with
many oligospecific genera. Caucasus has recently emerged as a new spot of diversity,
with 11 troglobitic Anthroleucosomatidae species described at once, several clearly
troglomorphic for appendage length (Antić and Makarov 2016). Surprisingly, cave
Chordeumatida are only moderately diversified in southern China, a hotspot for cave
Diplopoda (Golovatch 2015) and are absent in Macaronesia.
Several species of Chordeumatida are restricted to cold habitats (Golovatch and
Kime 2009). Brolemanneuma gayi and Biokovella spp. live for instance in cold
caves of Alps and Croatia. Two other species are remarkable relicts of uncertain
phyletic position: Niphatrogleuma wildbergeri from northwestern Alps and
Marboreuma brouquissei from high altitude shafts in Pyrenees. All these cryophilic
troglobites are markedly troglomorphic.
Stemmiulida (ca. 150 species, a single family; Mauriès et al. 2010) is an order
distributed in tropical regions of Africa and America, with a few occurrences in
tropical Asia and New Guinea (Shelley and Golovatch 2011). Three species have
been recently described as cave restricted. Two, one from Brazil, and one from
Malukku associated with bat guano belong to the widespread genus Stemmiulus
(Mauriès and Golovatch 2006). They are eyed and pigmented. A third one from
Vietnam is the only species of the relictual genus Eostemmiulus and the only blind
species of the order.
With 3374 species in 28 families (Shear 2011), Polydesmida is the richest order
of Diplopoda. Distributed worldwide, it has subterranean representatives in all
temperate and tropical regions except Sub-Saharan Africa. No species seems to be
guano dependent, but troglobites and troglophiles are plenty. All four suborders and
at least 16 of the 28 families of Polydesmida have cave species. Troglophilic
Polydesmida, including tramps, are numerous in cave ecosystems. Many are conge-
neric with troglobitic species within large genera, like Polydesmus (ca. 250 species,
including several weakly modified troglobites in Europe). The genus
Eutrichodesmus of East and Southeast Asia is constituted of ca. 50 microendemics
of which 44 are troglobitic. Such a mix of troglobitic and non-troglobitic species is
also found in smaller genera. On the other hand, relictual oligospecific genera, like
Caucasodesmus from Crimea or Cantabrodesmus lorioli (Chelodesmidae) from
Cantabric range, are rare. The most remarkable is Dobrodesmus mirabilis recently
discovered in a Brazilian cave, and sole species of the family Dobrodesmidae.
Polydesmida are phyletically blind but show large gradients in other cave-related
traits, from tiny, short-appendaged Nevadesmus from the USA to large, long-
appendaged troglomorphic Pacidesmus from China. Most troglobites range between
these extremes. Depigmentation is very common, and appendages are elongated in
troglomorphic species of various families, like Balkanodesmus biokovensis from
Croatia (Trichopolydesmidae), Selminosoma chapmani from New Guinea
(Paradoxosomatidae), or Nepalella grandoides from China (Megalotylidae).
Troglomorphy and regressive morphological evolutions in Diplopoda were
recently the object of a detailed analysis based on several Chinese examples (Liu
et al. 2017). Like most arthropods, cave Diplopoda frequently show eye and pigment
reduction, elongation of legs and antennae, and body slenderness (Golovatch and
Kime 2009). Body size increase is frequent, but the reverse is also reported in some
lineages (Enghoff 1992).
Interesting features of cave Diplopoda diversity can be summarized as follows:
(1) a very important contribution to global subterranean diversity; (2) a diversifica-
tion in all subterranean environment, from guano to the most oligotrophic habitats;
(3) an exceptionally large number of relicts, including phyletic relicts, especially in
Blaniulidae and Chordeumatida; (4) a hotspot of diversity and troglomorphy in
southern China caves (Deharveng et al. 2009b); (5) an overwhelming importance
of Cambalidea, especially Cambalopsidae and Pseudonannolenidae, in many caves
with guano of Asia and to a lesser extent of the Neotropics; and (6) a frequent
occurrence of regressive features among guanobitic Cambalopsidae, undermining
the paradigm of the morphological stasis of guano-dependent invertebrates (Culver
and Pipan 2009).
7.5 Crustacea Malacostracea
7.5.1 Decapoda: Brachyura (Crabs, Fig. 7.4j)
Crabs are usually omnivorous and aquatic Crustacea, mostly diversified in seawater,
with more than 7000 species (Ng and Davie 2017). A strong minority is constituted of
nonmarine species (1476 species, 238 genera, Yeo et al. 2008), rich in cave dwellers,
mostly freshwater species. Cave terrestrial species, mainly known from Southeast
Asia, belong to the families Chasmocarcinidae, Gecarcinidae, Gecarcinucidae,
Potamidae, and Sesarmidae, with probably less than 20 species. The status of troglo-
or stygobite is inappropriate for some of them, which are amphibious, like the
Sesarmidae Karstarma balicum from Bali. The genus Karstarma is the richest in
troglobites, all narrow endemics. Surprisingly, the highly troglomorphic Trogloplax
joliveti (Chasmocarcinidae) has a large distribution, being present in caves 200 km
apart in New Guinea (Guinot 1994).
Morphological modifications of troglobitic crabs are eye and pigment reduction

and leg elongation. These trends are moderately expressed in most species; eye loss
in particular is exceptional. The species Erebusa calobates from Laos, one of the
most modified with its elongated legs and pale bluish coloration, still has small eyes.
Highly troglomorphic facies combining anophthalmy, pigment loss, and very long
legs are known in several freshwater crabs (Guinot 1994), but limited among
terrestrial species to Trogloplax joliveti from New Guinea.
7.5.2 Amphipoda: Talitridae
The only troglobitic Amphipods are three Talitridae, a small family of ca. 320 species
(WoRMS 2017): the microphthalmic species Palmorchestia hypogaea from Canary
Islands, and two eyeless species, Spelaeorchestia koloana from Hawaii and
Minamitalitrus zoltani from Japan (White et al. 2013). These species have elongated
appendages and can be qualified to be troglomorphic.
7.5.3 Isopoda: Oniscidea (Woodlice; Fig. 7.4k–m)
Oniscidea, the most successful group of terrestrial Crustacea, included 3710 species
in 37 families distributed worldwide in 2014 (Sfenthourakis and Taiti 2015). Many
are troglophilic, and about 10% are troglobitic. Half of the families include
troglobites, with Trichoniscidae the richest (about 70% of all known troglobitic
species, Taiti 2004). The number of subterranean Oniscidea was estimated at 270 by
Argano (1994) and at 300 by Taiti (2004). They are probably more than 350 today,
significant discoveries having been recently made in caves of Brazil (Campos-Filho
et al. 2017), Socotra (Taiti and Checcucci 2009), Greece (Alexiou and Sfenthourakis
2013), and Portugal (Reboleira et al. 2015).
Eutroglophiles and guanophiles are well represented among cave Oniscidea.
Guano species, sometimes largely distributed, lack troglomorphic traits, but some,
like several tropical Trichorhina, have reduced eyes and pigment. Many troglobitic
Trichoniscidae, not attracted by guano, have a euedaphomorphic facies, i.e., short
appendages, eyes reduced or absent, no pigment, and small size. They are well
represented in Western Europe in the genus Trichoniscoides.
Troglomorphic or hypogeomorphic facies are common as well in meso- and
oligotrophic habitats. Among them, Trichoniscidae and to a lesser degree
Armadillidiidae dominate cave assemblages in temperate regions, usually replaced
by Styloniscidae, Philosciidae, and Armadillidae in the tropics. In Crimea, however,
troglobitic Ligiidae are more diverse than Trichoniscidae (Gongalsky and Taiti
2014). Most of these species (e.g., Scotoniscus in Pyrenees) are eyeless and
depigmented, but appendage elongation is always moderate.
Several troglobitic Oniscidea are secondarily adapted to life in freshwater. From

1994, their number has more than doubled. They belong to the families
Trichoniscidae, Styloniscidae, Philosciidae, and Scyphacidae. Recently described
are Macedonethes stankoi from the Balkans, Haloniscus spp. from Australia (Taiti
and Humphreys 2001), two species of Trogloniscus from China (Taiti and Xue
2012), and several species of Xangoniscus, and Iuiuniscus iuiuensis from Brazil
(Souza et al. 2015). Adaptation to freshwater is apparently not associated with
particular morphology, but these amphibious species are often clearly
troglomorphic.
Several genera of cave Oniscidea have given local radiations. Oritoniscus of
southwestern Europe radiated in the Pyrenees, where about 20 troglobitic, soil, and
epigean species are known, from minute and blind euedaphomorphic cave-restricted
species to eyed and well-pigmented epigeomorphic ones. Within Alpioniscus
(ca. 30 species), a probably nonadaptive radiation involves more than 25 species
in the Balkans, all eyeless troglobites.
Oligospecific or monospecific genera are numerous in Oniscidea, and a few of
them are considered phyletics relicts, like the amphibious Cantabroniscus primitivus
from northwestern Spain, or Thaumatoniscellus spp. from southeastern Europe. At a
finer scale, disjunct distributions and local relicts are common. Within Alpioniscus
for instance, one species (Alpioniscus (Illyrionethes) escolai) is known from Cata-
lonia, while its congeners are distributed from Alps and Sardinia to Greece.
7.6 Collembola (Springtails; Fig. 7.4n–t)
Collembola (8128 species in 30 families; Janssens and Christiansen 2011) are

divided into four orders: Poduromorpha (3154 species), Entomobryomorpha (3753
species), Symphypleona (1188 species), and Neelipleona (33 species). Further
taxonomic statistics in the text are drawn from Bellinger et al. (2017). Collembola
are terrestrial arthropods, rarely neustonic, abundant, and diversified worldwide
from tree canopy to soil and caves. Most are decomposers, feeding on microorgan-
isms or vegetal debris. More than 400 troglobitic species are known from subterra-
nean habitats, where they are preys for various predators. Eutroglophilic species are
also well represented. Many species are linked to guano, where they are second to
mites numerically (Deharveng and Bedos 2012). In terrestrial oligotrophic cave
habitats, Collembola usually dominate invertebrate communities in abundance:
Entomobryidae and Paronellidae in tropical caves, Hypogastruridae, Onychiuridae,
and Entomobryidae in temperate caves.
Cave species exist in four of the nine families of Poduromorpha order:
Gulgastruridae, Hypogastruridae, Onychiuridae, and Neanuridae. Gulgastruridae,
phyletically isolated, has a single species, Gulgastrura reticulosa, only known
from cave entrances in Korea (Lee and Thibaud 1998). Hypogastruridae and
Onychiuridae (ca. 690 and 660 species, respectively) have successfully colonized
the subterranean environment, mostly in temperate climate of the northern
hemisphere. Troglobitic Hypogastruridae of oligotrophic habitats are often charac-

terized by slender (Taurogastrura in Crimea, Typhlogastrura and Bonetogastrura in
the Holarctic) or extremely slender claw (the relictual Ongulogastrura longisensilla
from Pyrenees), but elongation of appendages is moderate. Guanobitic
Hypogastruridae have usually reduced eyes and pigment and sometimes elongated
claw like several Pseudacherontides.
Guano-related Onychiuridae are few, but species of meso- or oligotrophic habi-
tats are plenty. They mostly belong to the speciose genera Deuteraphorura and
Onychiurus. At least four relictual monospecific genera are also cave restricted,
including Ongulonychiurus colpus from Spain which has the most slender claw
among Poduromorpha and Absolonia gigantea from Slovenia of unusually large
size.
Neanuridae (ca. 1480 species) differ from other Collembola by strongly modified,
often sucking mouthparts. Cave species are known in three subfamilies. Frieseinae
contain a single relictual, non-cave-adapted troglobite, Gisinea delhezi, from Belgium.
Pseudachorutinae include ca. 10 troglobitic Anurida in the Coreo-Japanese region and
one in Caucasus, blind, white, and of large size like some of their epigean congeners,
but without morphological modifications. Troglobitic Neanurinae are few: Yuukianura
judithae—a guanobite from Vanuatu, several Coecoloba—Japan, C. plumleyi—New
Guinea, the only Neanuridae with elongated claws, and two eyeless and unpigmented
Deutonura in France (D. anophthalma) and in Austria (D. mirabilis).
Entomobryomorpha order is constituted by nine extant families (Deharveng
2004). Six of them include cave species. Isotomidae (ca. 1380 species) have many
guanophiles in the tropics, one cosmopolitan troglophile (Folsomia candida) and
three troglobites restricted to oligotrophic habitats: Gnathofolsomia palpata from
Austria, an isolated relict, and two Isotomiella species from France. All have
moderately elongated claws. Entomobryidae (ca. 1850 species) have a few
troglophiles, tropical guanophiles, and many troglobites. Cave-restricted species
are members of the large widespread genera Pseudosinella, Coecobrya, and Sinella,
which all have epigean, edaphic, and cave species. Less speciose genera, more
restricted geographically, are entirely constituted of troglobites, like Verhoeffiella
in the Balkans. Subterranean radiations are known for all the cited genera, in various
regions of the world (Pyrenees for Pseudosinella, southern China for Coecobrya and
Sinella, Dinarides for Verhoeffiella). Within each of these genera, several species
exhibit spectacular troglomorphic characters, i.e., extremely long antennae (like
Verhoeffiella longicornis from the Dinarides, Pseudosinella christianseni from the
USA, Sinella longiantenna from China, Coecobrya polychaeta from Thailand),
others very elongated claw (like Pseudosinella cabidochei from Pyrenees, Sinella
longiungula from China). The highly troglomorphic Bessoniella procera, sole
species of the subfamily Bessoniellinae, is a phyletic relict restricted to cold Pyre-
nean caves.
Paronellidae (ca. 400 species), mostly diversified in tropical regions, include large
colorful species living on the vegetation, and a strong minority of soil and cave
species. This last component, widespread in tropical and Mediterranean regions, is
constituted of guanobites and troglobites of the genera Cyphoderopsis, Lepidonella,
Troglopedetes, and Trogolaphysa. They are the most diversified Collembola in

many tropical caves but are absent in large areas like southern Sunda Islands. In
Mediterranean regions, Troglopedetes species have been described from caves and
are considered as climatic relicts. Troglomorphic species with eyes and pigmentation
reduced or absent, elongated appendages, and thin claws are frequent, like
Troglopedetes multispinosus in Thailand or Trogolaphysa jacobyi in Belize. In
many cases however, especially among guano dwellers, appendages are not elon-
gated (Jantarit et al. 2013).
Cyphoderidae (ca. 140 species) are all phyletically blind and unpigmented. Most
are myrmecophilous or termitophilous, but Cyphoderus and Troglobius have
troglobitic-guanobitic species in tropical caves. None exhibits appendage or claw
elongation.
Tomoceridae (ca. 170 species), diversified in northern and southern temperate
regions with an extension into Southeast Asia, include cave species in Holarctic
regions not affected by Quaternary glaciations. They are especially numerous in
North America and eastern Asia (Yosii 1967). They include troglophiles, many
troglobites, but no guanophiles. Some troglobitic species do not exhibit differences
with their outside relatives in eyes, pigment, or appendage length, like Plutomurus
unidentatus, widespread in Western Europe. Many show various regressions of eyes
and pigment (Yu and Deharveng 2015). Several eyeless species are known, like
Tritomurus scutellatus (Slovenia, Croatia), Lethemurus missus (USA), Plutomurus
ehimensis (Japan), or Tomocerus caecus (China). None of them exhibit significant
appendage elongation, and claws are moderately slender. Two relictual troglobites
only, Tritomurus falcifer (Pyrenees) and T. veles (Croatia), combine anophthalmy
and very elongated claw (Lukić et al. 2010).
Oncopoduridae has 49 species in three genera, of which 35 are cave restricted,
mostly in oligotrophic habitats. Described troglobites are Holarctic, but undescribed
species are reported from tropical and Australian caves. All are phyletically blind,
and some like O. lebretoni (Pyrenees) are troglomorphic with long claw and
elongated antennae.
Cave Symphypleona belong to Arrhopalitidae and Sminthuridae. More than
80 species are known today, living mostly in oligotrophic or mesotrophic habitats,
a number that increased to about two-thirds since Thibaud and Deharveng (1994).
Arrhopalitidae, with the genera Arrhopalites and Pygmarrhopalites, are the richest
in troglobites. Especially diversified in temperate regions of northern hemisphere
(Christiansen and Bellinger 1996; Vargovitsh 2009), they are present in South
America (Zeppelini 2006) and Southeast Asia (Nayrolles 1990), but absent in Africa.
In tropical and southern hemisphere caves, Arrhopalitidae are usually replaced by
Sminthuridae: Adelphoderia in Australia, Troglospinotheca in Argentina, and
Pararrhopalites in the tropics, all poorly known taxonomically. All cave
Symphypleona are microphthalmic or blind, weakly or not pigmented, but their
appendages and claws are moderately or not elongated. Long antennae and very
slender claws are however known in recently described troglobites from Caucasus
like Arrhopalites macronyx, but they reach their highest expression in Galeriella
liciniana from Dinaric caves, which has extremely elongated antennae and legs.
Neelipleona order, with its single family Neelidae, includes six genera and
47 species, distributed worldwide, of which 15 are troglobites. The genus
Spinaethorax has a highly disjunct distribution with two species in Mexico and
one in Vietnam (Schneider and Deharveng 2017). Several troglobites belong to
Megalothorax, the smallest cave Collembola (often less than 0.5 mm long). All
Neelidae are phyletically blind and devoid of cuticular pigment, but cave species
show larger size, more slender claw, and hypertrophy of some antennal sensillar
organs (Kováč and Papáč 2010). Appendage elongation is significant in Neelus
lackovici (Croatia) and N. klisurensis (Kosovo).
The high taxonomic diversity of troglobitic Collembola is associated with a high
diversity in troglomorphic and regressive traits. Troglomorphy is expressed by larger
body size, reduced eyes and pigment, elongated appendages and claws, and modi-
fications more specific to Collembola: shorter, thinner, and pointed tenent hairs on
tibiotarsi, reduction and basal shift of inner teeth on claw, hypertrophy or multipli-
cation of antennal sensilla (Christiansen 2012). Each trait evolves at its own pace
within lineages. Anophthalmy without appendage elongation is for instance frequent
among cave and soil species, while some cave species are oculated, weakly
pigmented, but have elongated appendages (e.g., Pseudosinella theodoridesi from
Pyrenees). Extreme elongation of appendages is not paralleled by extreme elonga-
tion of claws. These differences in evolutionary changes result in complex patterns
of troglomorphy among Collembola.
7.7 Diplura (Fig. 7.4u)
Sendra (2015) reports 984 species of Diplura in ten families and two orders,
Dicellurata (484 species, short sclerotized cerci) and Rhabdura (500 species, seg-
mented filiform cerci). Most Dicellurata are predators in deep soil layers. Rhabdura
are decomposers or predators from upper soil layers. Six Dicellurata are troglobitic
against more than 125 Rhabdura. Since the last review of subterranean Diplura
(Bareth and Pagès 1994), a single new cave Dicellurata has been described, against
about 35 cave Rhabdura.
Troglobitic Dicellurata are members of the family Japygidae. Gollumjapyx
smeagol from Spanish Catalonia, the most recently described, is remarkable by its
slender forebody and elongated legs (Sendra et al. 2006). Other troglobitic species
do not exhibit troglomorphic features.
Subterranean Rhabdura belong to Campodeidae, represented underground by
many eutroglophilic and troglobitic species. MSS habitats may host various species,
but only Campodea alluvialis, a weakly adapted species from Spain, is considered
restricted to this habitat (Sendra et al. 2017b). Troglobitic campodeids are mostly
known from mesotrophic or oligotrophic cave habitats. They are much less diverse
under tropical (ca. 10 species, Lepidocampinae and Campodeinae) than temperate
climate (more than 115 species, Campodeinae and Plusiocampinae). Diversity is
concentrated in the northern Mediterranean region with about 70% of the troglobites,
and in the southern Nearctic region with about 13%. Elsewhere, troglobitic
Rhabdura are rare.
Blindness and lack of cuticular pigment are phyletic traits shared by all Diplura.
Troglomorphy is expressed in other traits: increase in body size, slenderness,
elongation of appendages, increase in number and/or size of antennal and cercal
articles, and modifications of claw structure. An original troglomorphic “syndrome”
is the increase of sensorial receptors, especially in the cupuliform organ of antennae
(Sendra et al. 2017a). These different trends are variously combined. In
Oncinocampa for instance, minute euedaphomorphic troglobites with short append-
age like O. trajanoae from Brazil (less than 2 mm, with cerci less than 1/3 of body
length) contrast sharply with highly troglomorphic species like O. asonensis from
Spain (5–7.4 mm long, with cerci three times longer than body and very slender
claws). Extreme appendage elongation is encountered in other European species,
such as Paratachycampa hispanica (Spanish Catalonia) and Plusiocampa christiani
(Serbia). Similarly modified species are also known in tropical Lepidocampinae
(e.g., Lepidocampa beltrani from La Réunion).
7.8 Zygentoma (Fig. 7.4v)
Zygentoma included 568 extant species, all decomposers, and two families in 2014
(Gaju-Ricart et al. 2015). Sixteen troglobites were recognized by Mendès (1994),
and 45 are known at present. Most belong to Nicoletiidae, except two
Protrinemuridae from Greece and Thailand (Mendes 2002). Nicoletiidae is
subdivided into four subfamilies: Atelurinae (a single species from Philippines),
Coletiinae, Subcoletiinae, and Cubacubaninae. Coletiinae have about 12 troglobites
in the Mediterranean basin, and three outliers in South Africa (Mendès 1994), Brazil
(Mendès and Ferreira 2002), and Thailand (Mendes 2002). Subcoletiinae have three
troglobites in Southeast Asia, one in New Guinea and one in Australia, where several
euedaphomorphic species are known from deep bores. Cubacubaninae, more diverse
underground than the other subfamilies, have 25 troglobites distributed from Central
America to southern USA.
Ecologically, cave Zygentoma live preferentially in oligotrophic habitats, except
two guano-troglobitic species of Cuba, of which one, Cubacubana decui, has
elongated appendages. Anophthalmy and depigmentation are phyletic traits shared
by all Zygentoma, but body size and appendage length increase in troglobites, like
Coletinia capolongoi (Spain) and Squamatinia algharbica (Portugal), which are
among the largest species of the group in the Palearctic. Appendage elongation is
remarkable in several unrelated lineages, like Speleonycta anachoretes (USA),
Lepidospora (Brinckina) makapaan (South Africa), or Coletinia longissima (Tur-
key). Antennae and caudal appendages are three times longer than the body in
Cubacubana negreai (Cuba) and two times longer in Anelpistina levidensis (Guate-
mala), while they do not usually exceed body length in non-cave species of
Anelpistina.
7.9 Insecta Except Coleoptera
7.9.1 Orthoptera (Fig. 7.5a–c)
Unless specified, statistics on Orthoptera are drawn from Cigliano et al. (2017). The
taxonomic arrangement of the order is provisional (Chintauan-Marquier et al. 2016).
Orthoptera encompass 27,267 extant species in 43 families distributed in two sub-
orders of similar specific richness, Ensifera and Caelifera. All cave species are
members of Ensifera, which are omnivorous or scavengers, and none of Caelifera,
which are phytophagous. They are known in half of Ensifera families, representing
hundreds of troglobitic and troglophilic species. Many are nonpermanent cave
dwellers, i.e., obscuricole and “cavicolous” sensu Desutter-Grandcolas (1993,
1998), which forage at night for feeding outside cave. Some are cave restricted.
Crickets are the most important primary consumers in many subterranean food webs,
as stressed for caves of North America (Lavoie et al. 2007) or Southeast Asia
(Deharveng and Bedos 2000). Most belong to the large families Gryllidae,
Phalangopsidae, Trigonidiidae, and Rhaphidophoridae. A noticeable exception is
the two cave Anostostomatidae from Venezuela (Derka et al. 2013), which are the
only aquatic or semi-aquatic cave Orthoptera.
Gryllidae is rich in species, but few have colonized caves. The troglobite
Anurogryllus typhlos from Galapagos has reduced eyes, elongated legs, and no
epigean congener in the island. Conversely, Thaumatogryllus cavicola, a reduced-
eye species from Hawaii, has relatives in epigean habitats. Several weakly modified
Petaloptilini (moved to Phalangopsidae; Chintauan-Marquier et al. 2016), such as
Petaloptila pyrenaea, have been described from northern Mediterranean caves.
Phalangopsidae are mainly tropical, with extensions in Mediterranean regions and
Asia (if Gryllomorphini and Petaloptilini are included). They comprise numerous cave
crickets, which are more often cavicolous than strictly troglobitic (Desutter-Grandcolas
1998). The African genus Phaeophilacris is probably the most diversified, with at least
30 of its 74 species troglobitic or troglophilic-cavicolous and narrowly distributed. The
monospecific genus Socotracris from Socotra (Yemen) is troglobitic, but fully eyed. In
tropical Asia, Phalangopsidae number five or six genera that include both cave and
epigean species. They are usually absent when Rhaphidophoridae are dominant. None
of them exhibits well-marked troglomorphic traits, apart from reduced eyes in some
cases, like Arachnomimus microphthalmus from Malaysia. Australasia has very few
troglobitic Phalangopsidae and all are epigeomorphic in facies, like Endacusta irrorata.
Neotropics are the richest region in epigean and cave Phalangopsidae. Desutter-
Grandcolas (1998) lists 40 cave species, a number that has significantly increased
with recently described Brazilian species (Souza-Dias et al. 2014). Many are
troglobitic or subtroglophilic species, with wings, pigmentation, and eyes variously
reduced (Longuripes in Mexico, Eidmanacris and Endecous in Brazil). Paracophus
caecus from Mexico is probably the most troglomorphic species, eyeless, wingless,
depigmented, and with elongated appendages.
Fig. 7.5 Cave Insecta including part of Coleoptera; not at same scale. Orthoptera (a to c): (a)
Cophonemobius faustini (Trigonidiidae; Vanuatu), (b) Thaumatogryllus sp. (Gryllidae; Hawaii), (c)
Diestrammena sp. (Rhaphidophoridae; China) eating a dead Diplopoda; Blattodea (d and f): (d)
Helmablatta louisrothi (Nocticolidae; Vietnam), (f) Miroblatta baai (Blaberidae; Indonesia);
Hemiptera (e, g, h): (e) Reduviidae larva (Vietnam), (g) Phasmatocoris labyrinthicus (Emesinae;
USA), (h) Kinnapotiguara troglobia (Kinnaridae; Brazil); (i) Troglocladius hajdi (Diptera
Chironomidae; Croatia); Coleoptera Carabidae (j to q): (j) Duvalius lucidus (Trechinae; Croatia),
(k) Rhadine exilis (Platyninae; USA), (l) Coarazuphium tapiaguassu (Dryptinae; Brazil), (m) Subilsia
senenti (Trechinae, Morocco), (n) Goedetrechus damperi (Trechinae; Tasmania), (o) Duvalius iblis
Distributed worldwide, Trigonidiidae include two subfamilies, Nemobiinae and

Trigonidiinae. Troglobites are only known in the former, scattered in Spain, India,
Australia, and Pacific islands. Most have an epigeomorphic facies, without marked eye
reduction, like Nemobius interstitialis (Spanish MSS), Speonemobius decoloratus
(Assam), several Caconemobius (Hawaii), or Cophonemobius faustini (associated
with guano in Vanuatu). In contrast, Ngamarlanguia luisae, the only truly troglobitic
cricket of Australia, is eyeless, pigmentless, and wingless (Humphreys 2012).
Rhaphidophoridae include the largest number of cave Ensifera. It is widespread in
the Nearctic, Central America, Europe, southern and eastern Asia, and Gondwanan
lands. Half of its ca. 300 species are cave dwellers (Sbordoni and Cobolli 2004). Of its
nine extant subfamilies, six include cave species: Aemodogryllinae, Ceuthophilinae,
Dolichopodinae, Macropathinae, Rhaphidophorinae, and Troglophilinae, each
restricted to a well-delimited region.
Aemodogryllinae, from tropical Asia, eastern Asia, and Western Europe with a
small patch in North America, uncover a large array of life forms from epigeomorphic
to troglomorphic. They include the only Rhaphidophoridae strongly modified for cave
life, i.e., eyeless, unpigmented, and with elongated appendages: Eutachycines cassani
(Laos), Eutachycines caecus (Assam), and Diestrammena (Gymnaeta) omninocaeca
(Southern China).
Ceuthophilinae are limited to America north of Mexico. Many species in the
genera Ceuthophilus, Hadenoecus, and Euhadenoecus are found in caves, often in
large populations. All are troglophiles except possibly Ceuthophilus longipes which
has reduced pigmentation. Interestingly, a blind species exists in the subfamily,
Typhloceuthophilus floridanus, but it lives in rodent burrows, not in caves.
Dolichopodainae comprise 53 species in a single genus, Dolichopoda. One-third
of them have been described after 2001, and half of them from Greece. They are the
dominant crickets in caves of the northern Mediterranean region but are absent in
Iberia south and east of Catalonia and represented by a single species in the
Dinarides. When ecologically studied, these troglobites usually turn out to be
subtroglophiles, being active outside caves at night.
Macropathinae have a typical Gondwanan distribution with a large number of
cave species (some troglobitic) in Gondwanan lands (Di Russo and Sbordoni 1998).
These species do not exhibit strong morphological modifications related to cave life.
Rhaphidophorinae are distributed across tropical Asia, Australasia, and eastern
Asia. Its cave species are large in size (4 cm for Rhaphidophora oophaga from
Sarawak) and the trophic basis of the giant arthropods community of Southeast
Asian caves (Deharveng and Bedos 2000). They may live in very dense populations
⁄
Fig. 7.5 (continued) (Trechinae; Algeria), (p) Velebitaphaenops giganteus (Trechinae; Croatia),
(q) Giraffaphaenops clarkei (Trechinae, China). Photos/drawings by Desutter-Grandcolas (2009)
(a), Howarth et al. (2007) (b), © MY Tian (c), © L Deharveng and A Bedos (d, e, f), Pape (2013)
(g), Hoch and Ferreira (2013) (h), J Bedek in Andersen et al. (2016) (i), Jalžić et al. (2013) (j),
Krejca and Weckerly (2008) (k), Pellegrini and Ferreira (2011) (l), Español (1967) (m), PM
Giachino in Eberhard and Giachino (2011) (n), Peyerimhoff (1910) (o), Casale et al. (2012) (p),
Deuve (2002) (q)
on walls and ground, from the entrance to the deepest recesses of caves as soon as
guano or food resources are plenty. Some are suspected to forage outside at night for
feeding (Chopard 1969), but this is unlikely to occur for deep-cave populations.
None of them exhibits modifications related to cave life (Di Russo and Rampini
2017).
Troglophilinae, with its single genus Troglophilus (21 species, all troglophiles or
at least obscuricoles), are the dominant crickets in Eastern Mediterranean regions.
The classical regressive traits, i.e., reduction of wings, eyes, and pigment in cave
compared to non-cave species, can be found in all Orthoptera lineages (Chopard
1969; Di Russo and Sbordoni 1998; Desutter-Grandcolas 1999), but less strongly
marked than in other cave insects. Anophthalmy is rare. Size increase, change in leg
tip structure, and appendage elongation are not observed or tenuous. The strikingly
elongated appendages are largely a phyletic character for most cave species. Leg
elongation has been reported to occur within groups of closely related species, but it
needs to be confirmed (Leroy 1967; Di Russo and Sbordoni 1998; Desutter-
Grandcolas 1999). In this context, evolutionary reversion from troglobitic to epigean
life, as suggested by Desutter-Grandcolas (1993) from phylogenetic analyses of
Neotropical Phalangopsidae, seems plausible. The importance of initial exaptations
for subsequent cave adaptation has been stressed by the same author who showed
that straminicolous-cavicolous Phalangopsidae are more prone to colonize subterra-
nean habitats than other species.
Some interesting characteristics of cave Orthoptera diversity may be summarized
as follows: (1) a large number of subtroglophiles that use caves as daily refuge
(Desutter-Grandcolas 1993); (2) a large contribution of radiations to overall biodi-
versity, like in several other Arthropod groups. These radiations involve in various
proportions cave and non-cave species, as illustrated by European Dolichopoda or
Southeast Asian Diestrammena; (3) a low proportion of relicts compared to several
Arthropod groups; and (4) moderate levels of troglomorphy compared to most
Arthropod groups.
7.9.2 Grylloblattodea
Grylloblattodea are large predatory insects, restricted to the northern Pacific region,
and have 32 extant species (Wipfler et al. 2014) living in mountain forest litter, some
near the snow, except seven that are cave dwellers. These troglobites have reduced
eyes and larger size than their epigean relatives, like the two blind species of
Namkungia from Korea.
7.9.3 Blattodea (Cockroaches, Fig. 7.5d, f)
In 2011, extant Blattodea (not including Isoptera) contained 4779 species, 9 families
(Beccaloni 2014). Cockroaches are almost absent in caves of temperate regions, but
well represented in tropical caves, with many troglophiles and guanophiles (often
tramp species of large size), and about 40 troglobitic species, all saprophagous. The
troglobitic Blaberidae Miroblatta baai, endemic to a Kalimantan cave, is one of the
largest cockroaches (up to 6 cm long). It is dark pigmented, eyed, and long-legged
but moves slowly, with the body elevated up over the substrate (Bell et al. 2007).
Another rather large troglobite is Neostylopyga jambusanensis (Blattidae) from
Borneo, which has reduced eyes and pigmentation, but no appendage elongation.
All other troglobites are smaller in size. They belong to Ectobiidae (ca. 18 species,
on a total of ca. 2840; Beccaloni 2014), particularly diversified in Canary Islands and
Australia, and Nocticolidae (ca. 22 species, on a total of 34; Vidlička et al. 2017)
particularly diversified in Southeast Asia and Africa. They may be encountered near
guano (the Nocticolidae Helmablatta louisrothi from southern Vietnam), but live
preferentially in oligotrophic habitats with stagnant air (Stone 1988). Troglobites of
both families have more or less reduced eyes, many being highly troglomorphic,
eyeless, unpigmented, wingless, and with very long appendages, like Nocticola
flabella (Australia). Several have lost the specialized adhesive structures of the tip
of the legs usually present in epigean species, a possible adaptation to cave life (Bell
et al. 2007).
7.9.4 Dermaptera (Earwigs)
Extant Dermaptera contain 1978 species (Zhang 2011), present worldwide, though
more diversified in the tropics. Two families (ca. 16 species) are parasitic on or
linked to mammals, including bats. All others are free-living omnivores, more rarely
predators. Very few are strictly associated with caves, and none has been described
since the synthesis of Brindle and Oromi (1994). There are only two troglobites with
well-marked troglomorphic traits, Anataelia troglobia (Canary Islands) and
Anisolabis howarthi (Hawaii). Their eyes are much reduced, their appendages
elongated, and they are depigmented and wingless. Three other Dermaptera species
from deep soil layers are blind and wingless, but of much smaller size and with
shorter appendages (Brindle and Oromi 1994).
7.9.5 Psocoptera
Johnson and Smith (2017) report 10,880 extant species of Psocoptera in 64 families.
In Badonnel and Lienhard (1994), 12 species were listed as associated with caves at
world level. About 40 species are known today, distributed in two suborders:
Trogiomorpha and Troctomorpha. Cave Psocoptera live preferentially in dry habi-
tats, often on guano. The most common are a few widespread troglophilic species,
like Prionoglaris stygia, a quasi-troglobite of Europe. Northern America also has a
single troglobite, Speleketor flocki. Tropical regions are richer in troglobitic species,
most of them recently described: three in Malaysia (Moseley et al. 2012), six or
seven in Africa (Lienhard and Ashmole 2011), and more than 20 in the Neotropics,
mainly in the genus Psyllipsocus in Brazil (Lienhard and Ferreira 2015). All belong
to the suborder Trogiomorpha and are weakly or not modified in relation to cave life,
except for three Afro-Atlantic Troctomorpha, which include the only eyeless cave
Psocoptera known so far (Sphaeropsocopsis myrtleae from St. Helena Island).
7.9.6 Hemiptera: Heteroptera (Fig. 7.5e, g)
Schuh and Slater (1995) estimated that Heteroptera comprised 38,000 species world-
wide. They are highly diversified in all terrestrial and freshwater habitats on earth and
feed in sucking a large range of living plants or animals. A small proportion of them
are found in subterranean habitats, as guanophiles (Leptopodidae, Lygaeidae, and
Reduviidae), bat ectoparasites (Cimicidae), or troglophiles and troglobites of parietal
associations (Reduviidae Emesinae) (Maldonado Capriles 1994). Emesinae have
44 cave species (24 cave-restricted) on a total of 900 (Pape 2013). All are immediately
recognized by their very long appendages, but this character is lineage dependent and
not an adaptation to cave life. All species have well-developed eyes and most are
pigmented, with the exception of three troglobites: Collartida anophthalma and
C. tanausu from Canary Islands, and Nesidiolestes ana from Hawaii. Other
Reduviidae, poorly represented underground, are mostly troglophilic-guanophilic
species, with eyes, pigment, and normal appendages. Southeast Asian caves also
host large, unpigmented, reduced-eyes Reduviidae larvae (possibly Oncocephalus
sp., Cai pers. comm.), frequently found wandering on or around guano.
7.9.7 Hemiptera: Fulgoromorpha (Fig. 7.5h)
About 7000 species of Fulgoromorpha are known on earth. Four families include
cave species: Cixiidae, Meenoplidae, Kinnaridae, and Delphacidae. All are
rhizophagous and most are troglobites. Since the last review of Hoch (1994) who
listed ca. 40 cave species, at least 20 additional ones have been described.
Fulgoromorpha have given local radiations with nice series of eye and wing regres-
sion, without significant appendage elongation, in Australia (e.g., Solonaima),
Canary Islands (e.g., Cixius), and Hawaii (e.g., Oliarius). Several species are eyeless
with well-developed wings. Recently, species with regressed eyes, pigment, or
wings have also been described from temperate regions (Italy, Croatia), tropical Asia
(Sulawesi, Vietnam), and South America (Brazil).
7.9.8 Hymenoptera (Ants)
Hymenoptera is the fourth largest order of insects (116,881 species; Zhang 2011).
Various troglophilic ants are regularly encountered in guano of tropical cave (Decu
et al. 1998a). Leptogenys khammouanensis, collected in oligotrophic habitats of a
Laotian cave, is possibly the only troglobitic ant.
7.9.9 Lepidoptera (Butterflies)
The 157,338 species of Lepidoptera (Zhang 2011) include very few cave species.
Only two of them are troglobitic: the reduced-eyes Tineidae, Tinea microphthalma
(Philippines; Robinson 1980), and the Erebidae Schrankia howarthi (Hawaii;
Howarth and Hoch 2012), which presents several regressive traits: flightlessness,
reduced pigmentation, and reduced eye size.
7.9.10 Diptera (Flies, Fig. 7.5i)
Diptera (155,477 species, Zhang 2011) are poorly diversified underground with
about 100 species of which 22 are troglobites after Matile (1994). Aside from
parasites of bats, Diptera include many guanobites or guanophiles associated with
guano in tropical caves, as well as a number of non-guano-dependent troglophiles.
Among non-hematophagous Diptera, the emblematic Mormotomyia hirsuta from
Kenya, a remarkable relict of aberrant morphology, is actually not troglobitic
contrary to literature claims (Copeland et al. 2011). Clisa australe from Australia
and Spelobia tenebrarum, the only troglobitic Diptera of northern America, are cave
restricted. This last species has reduced eyes, like its congeners living in mammal
burrows. Gymnomus troglodytes from the Dinarides is the only troglobitic
Heleomyzidae. Among Sciaridae, several troglobitic or troglophilic species, like
Allopnyxia patrizii from Italy, have a minute size, regressed eyes and pigment, and
shortened appendages, i.e., euedaphomorphic attributes. Six troglobitic species of
the large genus Crumomyia (Sphaeroceridae) have reduced eyes, elongated antennal
arista and legs, weak sclerotization, pale pigmentation of body, and sometimes
reduced wings (Roháček and Papp 2000). The last discovered troglobite is a
Chironomidae collected from a deep pit of Croatia, Troglocladius hajdi. It combines
pale color, strongly reduced eyes, and very long legs, but its antennae are short and
its wings well developed.
Hematophagous Diptera include many troglophilic and some troglobitic species

of Psychodidae and Culicidae. Psychodidae, abundant in tropical caves, are well
studied as vectors of Leishmaniasis. Several species of Phlebotomus and Chinius
from Africa, Asia, and Australia are considered troglobites (Matile 1994). However,
most have been found outside caves when searched for, and should be considered as
troglophilic (Carvalho et al. 2013). The only species firmly established as a
troglobite, Deanemyia maruaga from Brazil, is parthenogenetic, nonvector, and
can complete its full life cycle in cave. A few tropical Culicidae seem be troglobitic
in a way similar to Psychodidae (hematophagous-guanophagous).
All troglobitic Diptera are not or weakly modified for cave life.
7.10 Insecta: Coleoptera (Beetles)
With 386,500 species in 29,500 genera and 176 families (Slipinski et al. 2011),
Coleoptera is the most species-rich order of living organisms. Beetles are present in
all terrestrial and freshwater habitats and in all climatic zones. They are common in
terrestrial subterranean habitats worldwide, as troglophiles, troglobites, or associated
with guano. Most species are terrestrial, estimated at 1927 by Decu and Juberthie
(1998), and probably outnumbering today 2500 species. Understanding of cave
Coleoptera diversity and evolution is hampered the lack of data on the larval instars
of troglobitic species, which seem to have developed adaptive traits different from
those of adults.
Three families, Carabidae, Staphylinidae (including Pselaphinae and Scydmaeninae),
and Leiodidae count for 98% of the diversity of troglobitic and troglophilic beetles, with
1180, 110, and 599 species (Decu and Juberthie 1998), for total species richness of
40,000, 56,000, and 3700 species, respectively (Slipinski et al. 2011). This taxonomic
unbalance is retrieved at lower taxonomic levels. It is also striking at the geographical
scale, with lineages developing large subterranean radiations locally and being almost
absent from caves in other regions.
Guanobites and guanophiles, largely neglected taxonomically, represent a signif-
icant component of subterranean Coleoptera diversity. Thus, 28 cave beetles asso-
ciated with guano from 10 families are listed by Peck et al. (1998) for Cuba, and
82 species from 19 families by Moulds (2004) for Australia. The diversity of guano
beetles is much higher than that of non-guanobite troglobitic beetles at single-cave
scale in the tropics, but not in temperate regions.
7.10.1 Adephaga: Carabidae (Groundbeetles; Figs. 7.5j–q

and 7.6a–d)
All terrestrial cave species of Adephaga belong to Carabidae. We follow here

Anichtchenko (2017) for the taxonomic hierarchy of the family. Casale et al. (1998)
published a major synthesis of cave Carabidae diversity and mentioned 14 subfamilies
(of a total of ca. 33) having representatives underground: Anthiinae, Brachininae,
Dryptinae, Harpalinae, Lebiinae, Nebriinae, Paussinae, Platyninae,
Promecognathinae, Psydrinae, Pterostichinae, Scaritinae, Siagoninae, and Trechinae.
Trechinae comprise 90% of all described species of cave Carabidae (Decu and
Juberthie 1998). Of their six tribes, three contain troglobites (Trechini, Bembidiini,
and Zolini). Distributed worldwide, they are however mostly diversified in temperate
climates. Half of their more than 2000 species are cave dwellers (Casale et al. 1998).
The basic outlines of the taxonomy of the subfamily were set up by Jeannel
(1926–1928). Since the last review of Casale et al. (1998), numerous new taxa have
been described, and molecular phylogenies of several major lineages of the subfam-
ily have been published (Faille et al. 2010a, 2011, 2013), bringing new insight on
their origin and evolution.
Of the four subtribes of Trechini, only Trechodina and Trechina include cave
species. Trechodina have a few troglobites of euedaphomorphic facies, limited to
Macaronesia and eastern Spain. Three anophthalmic ones are Canarian endemics in
the genera Canarobius and Spelaeovulcania. The others are microphthalmic and
belong to the European genus Thalassophilus.
Trechina includes all other cave Trechini. More than any other cave group, this
subtribe illustrates the major features of the diversity patterns known among cave
fauna: unbalanced distribution, large evolutionary radiations, high number of relicts,
and a broad range of morphologies from euedaphomorphy to extreme troglomorphy.
Cave-restricted Trechini inhabit almost exclusively caves of temperate and sub-
tropical climates. They are absent north of 47 N and south of 45 S, probably impacted
by Quaternary glaciations. They are also extremely rare in tropical caves. Though
present in the southern hemisphere, they are more diversified in the Holarctic. At fine
grain, pattern of taxonomic richness is complex and uneven, characterized by intense
but scattered local radiations (Casale et al. 1998). Recent phylogenetic analyses (e.g.,
Faille et al. 2010b) combined with a fast increase in taxa discoveries and species
distribution have allowed robust hypotheses on divergence and biogeographical
history of various lineages.
Ecologically, most cave Trechini are troglobites of oligotrophic habitats. Their
absence in guano parallels their rarity in tropical regions. Surprisingly, troglophilic
species also are few.
Till recently, Southern Europe was known as the major diversification area for
cave Trechinae on earth (Casale et al. 1998). Its species richness is mostly a result of
local evolutionary radiations, reflected in the occurrence of 10 genera having more
than 15 troglobites each. On the other hand, the large number of phyletically isolated
and oligospecific genera recognized by taxonomists (of which more than
Fig. 7.6 Cave Coleoptera, Carabidae and Staphylinidae; scales as specified. Carabidae (a to d): (a)
Xuedytes bellus (Trechinae; China), (b) Morimotoidius zhushandong (Platyninae; China), (c)
Zariquieya boumortensis (Pterostichinae; Spain), (d) Dalyat mirabilis (Promecognathinae;
Spain); Leiodidae (e to m): (e) Ptomaphagus trezzii (Guatemala), (f) Pholeuon (P.) knirschi
(Romania), (g) Nafarroa sorogainensis (Spain), (h) Graciliella ozimeci (Croatia), (i) Leptodirus
hochenwartii croaticus (Croatia), (j) Speoplanes giganteus biokovensis (Croatia), (k) Remyella
20 monospecific genera) illustrates a high phyletic richness. Cave Trechini diversity

is maximal on the biodiversity ridge between 42 and 46 of latitude (Culver et al.
2006), more particularly in Pyrenees and the Dinarides. Pyrenees is home of the
Aphaenops lineage, with 87 mostly troglobitic species in two genera, Aphaenops and
Geotrechus (Queinnec 2014). This lineage has no clearly relatives with Trechus
surface lineage or European Trechinae genera (Faille et al. 2010a, b). All species are
blind and depigmented, and most are troglomorphic regarding body slenderness and
appendage elongation.
The Duvalius lineage spreads from northeastern Spain to China (Moravec et al.
2003), but is absent in Pyrenees. It is richer in species than the Aphaenops lineage
but spread across a much larger area. It ranges from epigean winged and eyed species
(life forms that do not exist in the Aphaenops lineage) to highly troglomorphic ones,
like Duvalius (Trechopsis) iblis from Algeria or Alpine Trichaphaenops. The genus
Anophthalmus, closely allied to Duvalius, represents a third important radiation
diversified across southeastern Europe.
Casale et al. (1998) recorded nine cave genera of Trechini in southern China. This
number has since increased impressively. The region is today the richest on earth for
troglobitic Trechini, with more than 130 species in ca. 45 endemic genera (Tian et al.
2016, 2017). Like for the Pyrenean radiation, species of the Chinese radiation(s) are
all troglobites, blind, wingless, and unpigmented. They exhibit an extremely large
range of morphologies, reflected in the small number of species by genus, many
being monospecific and the richest, Dongodytes, having only 12 species. Their
maximal diversification area stretches from 23 N to 30 N, more southerly than the
Japanese, European, or American hotspots. Outside this hotspot, the single
troglobitic Trechini known in 1998 for the region was the reduced-eyes monospe-
cific genus Trechiamiotes from Thailand. Recent discovery of blind and highly
troglomorphic species in lowlands caves, south of the southern China hotspot, has
been a surprise: three species of Lanxangaphaenops in Vietnam and Laos, and the
monospecific genera Tonkinaphaenops in Vietnam, Laosaphaenops in Laos, and
Birmaphaenops in Burma. The origin of this large radiation is unclear, no species
having been related so far to eyed Trechinae. Most species are troglomorphic.
Several, in the genera Giraffaphaenops, Dongodytes, and Xuedytes are the most
modified troglobitic ground beetles known so far in the world due to extreme
elongation of appendages.
⁄
Fig. 7.6 (continued) scaphoides scaphoides (Serbia), (l) Radziella styx (Croatia), (m) Baronniesia
delioti (France); Staphylinidae (n to s): (n) Domene (D.) lencinai (Paederinae; Spain), (o) Euconnus
(Tetramelus) longipedes (Scydmaeninae; Croatia), (p) E. (T.) tronqueti (France), (q) Metopiellus
painensis (Pselaphinae; Brazil), (r) Pseudophanias spinitarsis (Pselaphinae; Nepal), (s)
Zopherobatrus tianmingyii (Pselaphinae; China). Photos by Tian et al. (2017) (a), Pang and Tian
(2014) (b), Faille et al. (2011) (c), © A Anitchenko (d), Perreau (2009) (e), Hlaváč et al. (2017) (f, i,
j, k, l), Fresneda and Dupré (2010) (g), Njunjić et al. (2016) (h), Fresneda et al. (2009) (m), Vives
(2010) (n), Hlaváč and Jalžić (2009) (o), Orousset (2014b) (p), Asenjo et al. (2017) (q), Yin et al.
(2015a) (r), Yin and Li (2015) (s)
Though close to Southern China, the Japanese hotspot of cave Trechinae biodi-
versity is located more northerly, centered on the island of Shikoku at about 33 N
(Uéno 2001). It hosts speciose lineages, especially in the Trechoblemus and
Trechiama groups. Contrary to the radiations of southern China, the richest lineages
of cave Trechinae in Japan comprise edaphic and troglobitic species: Rakantrechus
and Yamautidius with c.a. 20 species each, Kurasawatrechus with more than
30 species, and Trechiama with more than 130 species (Moravec et al. 2003).
Though many species lack eyes and pigment, a few only are highly troglomorphic.
In North America, the highest diversity of cave Trechini occurs at 35–40 N
(Culver et al. 2006). There, 145 species of the genus Pseudanophthalmus (Barr
2004) contribute for almost 85% to the total richness of cave Carabidae of the USA
(Culver et al. 2000). Most species are obligate cave dwellers; a few are edaphic.
Their eyes are absent or very reduced and their appendages only moderately
elongated, though some species like Nelsonites jonesi are more strongly
troglomorphic. Further south subterranean Trechini are confined to high elevation
caves and have more elongated appendages: several Mexaphaenops, and four
species of oligospecific genera: Mayaphaenops, Chiapadytes, Guatemalatrechus,
and Mamesdytes.
Cave Trechini are much less diversified in the southern than in the northern
hemisphere. They include troglomorphic species of uncertain affinities in
New Zealand, which combine vestigial eyes and rather long appendages (Townsend
2010), like Erebotrechus infernus or Scototrechus orcinus. In Tasmania, they are
also frequent, with local radiations of the genera Goedetrechus and Tasmanotrechus,
almost entirely constituted of troglobites which exhibit mild degrees of eye reduction
and moderate appendage elongation (Eberhard and Giachino 2011).
Zolini include six troglobites of the genera Idacarabus and Pterocyrtus in
Tasmania, all oculated and weakly or not modified for cave life (Eberhard and
Giachino 2011). Bembidiini have cave species in five of their six subtribes, most
of them troglophilic of euedaphomorphic habitus, i.e., small, blind, and with short
appendages. Anillina, particularly in the northern Mediterranean region (Hlaváč
et al. 2017), include a large number of such species, living in soil or MSS, and a
few troglobites that exhibit weak troglomorphic facies (slenderness, appendages
slightly elongated), like Microtyphlus alegrei from Spain or Hygranillus kuscheli
from New Zealand. Six genera and 17 species of Anillina have been recently
discovered from deep bores in Western Australia.
The subtribe Bembidiina has troglophilic species, but probably no troglobites.
Some have regressed eyes, but none has significantly elongated appendages.
Horologionina is a monospecific subtribe, established for a subterranean species of
atypical morphology from the USA. The four troglobitic species of the enigmatic
Balkanic subtribe Lovriciina also show, aside from anophthalmy, moderate
troglomorphic traits, like Paralovricia beroi (Hlaváč et al. 2017). Among troglophilic
Tachyina, especially in the tropics, a few linked to soil or caves are blind and
euedaphomorphic in facies (like some Lymnastis of Canary Islands; Machado 1992).
Of the three tribes of Dryptinae, only Zuphiini are present in caves with
ca. 15 species in seven genera, besides a number of epigean and edaphic forms.
Ecologically, most cave Zuphiini are troglobites and none is guanophile. Three of
them are relicts of outstanding interest. The monospecific genus Ildobates from
Catalonia, blind and highly troglomorphic, is the only cave representative of
Zuphiini in Europe (Ortuño et al. 2004; Ribera et al. 2006). The two Australian
species from the Nullarbor karst Speothalpius grayi and Speozuphium poulteri)
belong to monospecific genera of uncertain affinities and are by far the most
troglomorphic Carabidae of Australia. Other cave species of the tribe have shorter
appendages, but remain slender, unpigmented, and blind or microphthalmic: eight
troglobitic Coarazuphium in Brazil and one Parazuphium in Canary Islands. Several
species with a similar morphology have also been collected in deep soil in Morocco
and in deep bores in Australia.
Though one of the richest subfamily of Carabidae, Harpalinae has only a few cave
species, restricted to Australia and New Zealand. Australian Notospeophonus, prob-
ably guanophilic, are fully oculated and devoid of cave-adapted traits (Moore 1964).
New Zealand species of Syllectus and Pholeodytes live in oligotrophic cave habitats,
have reduced eyes, and have a typical aphaenopsian habitus (Larochelle and
Larivière 2005).
Paussinae include highly modified species living as commensals of ants, and free-
living species, mostly Ozaenini, with several tropical troglobites. Itamus cavicola
from New Ireland is a guanobitic troglobite. The genus Eustra, sporadically distrib-
uted in soil and caves of Southeast Asia, includes several troglobites living of
oligotrophic habitats, unpigmented and with eyes reduced or absent. They are
morphologically euedaphomorphic, except two species from northern Thailand
with relatively elongated appendages (Deuve 2001).
Platyninae are subdivided into three tribes, of which two include cave species:
Platynini and Sphodrini. Platynini have colonized subterranean habitats, to the exclu-
sion of guano, in various regions on earth. Troglobites are numerous. Most belong to
radiations including epigean species: Rhadine in North America (ca. 50 species;
Gómez et al. 2016), Jujiroa in eastern Asia, Gastragonum in Australasia,
Mexisphodrus in Mexico, or Blackburnia in Hawaii. They are moderately modified
morphologically, though eyes and pigmentation are often reduced. Some species
however, like Rhadine exilis, have a typical troglomorphic facies, with vestigial
eyes, depigmentation, very slender forebody, and long appendages. Aside from
these speciose genera, a few mono- or oligospecific genera contain highly
troglomorphic species like Speocolpodes franiai from Guatemala or Speagonum
mirabile from New Guinea, both at high altitude. In eastern Asia, some eyed and
pigmented species exhibit surprisingly long appendages, like Morimotoidius from
China. The only cave Platyninae of Europe is Galiciotyphlotes weberi from Spain,
considered as a relict (Ortuño and Gilgado 2010). Many of the ca. 700 species of
Sphodrini are linked to subterranean habitats. Since the revision of Casale (1988) a lot
of new species have been described, but the main lineages, the global patterns of
distribution, and the troglomorphic gradients have only changed a little. The group
seems to be absent from caves in the tropics and in the southern hemisphere. Within
Sphodrini, the Laemostenus lineage is the richest and most widespread, distributed in
West Palearctic and Central Asia. Its remarkable radiation of ca. 200 species of
relatively uniform habitus includes epigean, guanobites, troglophiles-guanophiles, and

troglobites, often of large size compared to other cave Carabids. Troglomorphic traits
are not or weakly marked in species of the subgenera Actenipus and Pristonychus,
usually troglophilic or guano dwellers, but troglobites of the subgenus Antisphodrus,
like L. (A.) navaricus from Pyrenees, are clearly modified (eye and pigment reduced,
appendages elongated). No species is eyeless. Aside from Laemostenus, a few
oligospecific genera of the Palaearctic (Canary Islands, Morocco, Turkey, Japan)
comprise also weakly modified troglobites. In America, Sphodrini are represented
by a single relictual troglobite of Mexico, Miquihuana rhadiniformis, which is also the
most modified of the tribe with an unusual combination of depigmentation, vestigial
eyes, long and slender forebody, but moderate elongation of antennae.
Several lineages of Pterostichinae have colonized caves (Casale et al. 1998). Of
the eight tribes of Pterostichinae, two contain troglobites: Abacetini and
Pterostichini. Abacetini include two weakly modified troglobitic species of Indone-
sia: Mateuellus troglobioticus, from Sulawesi and Metabacetus willi, from Java.
Pterostichini, distributed in Canary Islands, the Pyreneo-Iberian region, and Eastern
Europe, have subterranean species in three subtribes (Molopina, Euchroina, and
Pterostichina). Molopina includes rare relictual troglobites, not attracted by guano, in
four mono- or bispecific genera (Faille et al. 2011), all blind, but without appendages
elongation: Speomolops sardous in Sardinia, Henrotius jordai in Balearic Islands,
Oscadytes rovirai, Zariquieya boumortensis, and Z. troglodytes in Catalonia and
eastern Pyrenees. The subtribe Euchroina is present underground only in Canary
Islands, with three species of the genus Orthomus from caves or MSS (Machado
1992). Their eyes and pigmentation are reduced, but their appendages are not
significantly elongated. The subtribe Pterostichina, rich in epigean species, has
several representatives underground. In France, Stomis benoiti from Massif Central
is oculated, pigmented, and with normal appendages, but cave restricted. Several
species of Pterostichus (subgenera Rambousekiella, Troglorites, Lianoe, and
Tinautius) are scattered in Europe. They are blind or microphthalmic and have
relatively elongated appendages, but a rather stout body. The genus Speluncarius
is the richest, with 27 species in the eastern Mediterranean region, of which about
one-third are troglobites of hypogeomorphic facies.
Scaritinae are represented underground by several species of the tribe Clivinini,
subtribes Reicheina and Clivinina. Within Reicheina, the radiation of Typhloreicheia
in Sardinia includes a majority of soil species and several cave species of
euedaphomorphic facies (Magrini et al. 2013). Eight anophthalmous Clivinini
have been described from southern Europe, Mexico, Papua New Guinea, and
China (Tian 2013). Other reduced-eye species and a few troglophiles mentioned in
Casale et al. (1998) are known in the tribe. All these species have the same strong
burrowing forelegs as their epigean relatives and an overall euedaphomorphic facies.
A single species, Italodytes stammeri, is clearly troglomorphic, with much less
robust forelegs and slender forebody.
Other cave species, interesting in various respects, are scattered in several sub-
families of Carabidae. Of outstanding interest, Dalyat mirabilis, unique species of
the subfamily Promecognathinae in Europe, is a large-size relictual beetle of
southern Spain, related to South African and North American taxa (Mateu and
Bellès 2003). Brachininae include a few endogean species, and Brachynillus
varendorffi from Tanzania, the only troglobitic Carabidae of Sub-Saharan Africa,
long considered as a relict. Nebriinae at least include hygrophilic-cryophilic species
in Holarctic region, of which the troglobitic Nebria nudicollis from Algeria, with
reduced eyes and pigment, and slightly elongate appendages.
7.10.2 Polyphaga
Polyphaga are present in caves with ca. 30 families and a very large number of species
(Decu et al. 1998b). Most troglobites and guanobites are distributed in 10 families
according to the following taxonomic hierarchy: superfamily Hydrophiloidea
(Histeridae), Staphylinoidea (Ptiliidae, Leiodidae, Staphylinidae), Scarabaeoidea
(Trogidae, Scarabaeidae), Derodontoidea (Jacobsoniidae), Tenebrionoidea (Aderidae,
Tenebrionidae) and Curculionoidea (Curculionidae); several other families include
troglophiles or guano species of uncertain ecological status (Elateridae and
Cantharidae among Elateroidea, Salpingidae and Zopheridae among Tenebrionidae).
Histeridae (4300 species, 350 genera, Slipinski et al. 2011) have various feeding
habits from carnivory to saprophagy. They include, aside from soil and inquiline
species, several guanophiles and troglophiles, and about 15 troglobites. These last
species, distributed in the Mediterranean region, Africa, and Central America
(Vomero 1998; Lackner 2013), small in size and wingless have reduced or absent
eyes, with sometimes weak appendage elongation.
Ptiliidae (650 species, 80 genera, Slipinski et al. 2011) are saprophagous soil
dwellers, present worldwide. They may be numerically important in the guano of
tropical caves. Two species are listed as troglobitic by Decu et al. (1998b). One of
them (Malkinella cavatica from South Africa) is clearly guanobitic, eyeless,
pigmentless, and wingless, but its appendages are not elongated, traits that are
retrieved in several epigean Ptiliidae.
Leiodidae (Fig. 7.6e-m). Statistics for Leiodidae taxa are drawn from Perreau
(2000, updated). Leiodidae is a moderately diverse family among beetles, with
ca. 4000 species worldwide in 450 genera. Giachino et al. (1998) published a
reference synthesis at world level on its underground diversity. The rough lines of
its phyletic structure are well documented (Fresneda et al. 2011). Leiodidae encom-
passes a higher proportion of cave-restricted species than any other large beetle
family and is only second to Carabidae in number of troglobites. Since 1998, our
taxonomic knowledge of the family has considerably progressed. The last available
estimate gave 599 species of cave Leiodidae (Decu and Juberthie 1998). In the
Balkans alone, 369 species are currently recorded, about the same number as
Carabidae (368 species, Hlaváč et al. 2017). All cave Leiodidae are scavengers,
and most are linked to oligotrophic habitats.
Leiodidae comprises six subfamilies of unequal species richness. Camiarinae and

Coloninae have no cave representatives. Platypsyllinae group a few pholeophilous
species including troglophilic Leptinus spp. which are blind and wingless.
Catopocerinae group five genera and ca. 50 species of which four are cave
dwellers. Three of them belong to the genera Catopocerus and Pinodytes, which
are blind and wingless, but without significant appendage elongation (Fresneda et al.
2011). The fourth troglobite is Glacicavicola bathyscioides from western USA, a
blind, troglomorphic, relictual endemic of leptodiroid facies (see below for expla-
nation). Leiodinae have more than 1700 species, but less than 10 cave species, of
which none is markedly modified for cave life. Regressive evolution of wings and
eyes is not exceptional among non-cave species of the subfamily. Thus, 32 of the
ca. 825 species of Agathidium have reduced eyes or are eyeless (Švec 2014), but
none inhabits cave.
Cholevinae (ca. 2500 species in seven tribes) have a large number of cave species,
mostly in Leptodirini and Ptomaphagini. Sciaphyini from Eastern Asia (one genus,
three species) are blind, but not cave dwellers. Anemadini, with ca. 100 species
distributed worldwide except in Africa and South America, are poorly diversified in
subterranean habitats. Its cave species considered as troglobites (Speonemadus
escalerae from Spain for instance) have wings, eyes, and pigment, but two
anophthalmic species of the tribe (Anemadus kabaki and A. imurai) have been recently
described from soil or MSS-like habitat in China. Ptomaphagini (ca. 360 species)
contain a large proportion of troglobitic species of bathyscioid facies in two main
areas: Southeast Asia and northern and central America. Southeast Asia caves host a
few Ptomaphaginus and several Ptomaphaminus. They are attracted by decaying
organic matter, but rarely guanobites. They are not or hardly troglomorphic, having
eyes and short appendages, but wings are sometimes reduced. In America, the genus
Ptomaphagus (ca. 140 species) includes soil and cave species, with more than
35 troglobites. Morphological gradients from fully eyed, fully winged, small size,
and short appendages to eyeless, wingless, large size, and moderately long appendages
species have been documented in the genus Ptomaphagus (Peck 1986). Marked
troglomorphic cave species, like Ptomaphagus (Adelops) trezzii from Guatemala,
are rare. Surprisingly, the only indubitably blind species of the genus,
P. troglodytes, is a troglobite from Spain (Fresneda et al. 2011).
Leptodirini (ca. 950 species, 235 genera) is the richest tribe of Cholevinae. It is
mostly distributed in Europe and western Asia, with only three genera in eastern
Asia and one genus in the Nearctic. Its species are eyeless and depigmented, with
very rare exceptions (Jeannel 1924; Fresneda et al. 2011). About three-fourths of
them are troglobites, living in oligotrophic habitats of cave and in the MSS, the
remaining ones living in soil. Eutroglophiles are not rare, but guanobites are
infrequent.
Balkans is the richest spot of Leptodirini, followed by the Pyreneo-Cantabric range
and Alps (Perreau 2000). The whole tribe constitutes a remarkable radiation of the
northern Mediterranean region, including local radiations of several clades like
Antroherpon in the Dinarides (Njunjić et al. 2016), Quaestus and Speonomus in the
Pyreneo-Cantabric range (Cieslak et al. 2014), and Drimeotus in Romania (Moldovan
2000). On the other hand, half of the Leptodirini genera are monospecific, reflecting a
relictual status (like Speophyes in France) or rapid rates of morphological evolution.
Most species of the tribe are narrow endemics, with more than one-third known from a
single site (Moldovan 2008). Life forms of Leptodirini, extremely diverse, have been
allocated to four facies defined by Jeannel (1943) and illustrated by Laneyrie (1967):
bathyscioid, pholeuonoid, leptodiroid, and scaphoid, characterized by different com-
bination of slenderness, appendage elongation, and body outlines. The bathyscioid
facies, considered as primitive, characterizes most non-Leptodirini Leiodidae, and
among Leptodirini, many soil and litter species, as well as troglobites like
Pholeuonella from the Dinarides, Quaestus from Spain, Sophrochaeta from Romania,
or Speonomus from Pyrenees. The pholeuonoid facies, characterized by longer
appendages, more slender body, wider head, and thorax more elongated and sinuous,
is retrieved in many “normal” troglobites of various phyletic lines, like Pholeuon from
Romania, Antrocharis from Pyrenees, or Speoplanes from Croatia. The spectacular
leptodiroid and scaphoid facies are limited to Dinaric species of the subtribes
Leptodirina and Antroherponina. Antennae are much longer than the body, legs
very long, and head and thorax narrow and sometimes elongated like in the most
troglomorphic Trechinae. Abdomen is distinctly swollen in the leptodiroid facies,
versus narrow in the scaphoid facies. These life forms are known among Leptodirina
(like Leptodirus hochenwartii, Albanodirus trezzii, Nonveilleriella ognjenovici) and
Antroherponina (like various species of Antroherpon, Leptomeson, and Graciliella).
Species of hygropetric environment often show intermediate facies between
pholeuonoid and leptodiroid, associated with strongly modified mouthparts (Sket
2004; Polak et al. 2016). They are known in Italy (Cansiliella) and the Balkans
(Croatodirus, Hadesia, Kircheria, Nauticiella, Radziella, Tartariella, and
Velebitodromus).
Staphylinidae (Fig. 7.6n-s). With more than 55,000 species, Staphylinidae is the
largest family of the whole animal kingdom. It comprises a low number of subter-
ranean species, including guanobites, very few troglophiles, and more than
220 troglobites (Hlaváč et al. 2006; Yin et al. 2015a).
Guanophilic Staphylinidae are common in northern temperate regions (Bordoni
and Oromi 1998) but rarer in the tropics and do not exhibit modifications related to
cave life, with the exception of the large-size Scydmaeninae Clidicus gracilipes from
Sumatra.
Non-guano troglobitic Staphylinidae, much more diverse, are known in seven
subfamilies: Omaliinae, Tachyporinae, Paederinae, Aleocharinae, Pselaphinae,
Scydmaeninae and Scaphidiinae. Among them, several troglobites are clearly
troglomorphic, i.e., blind, depigmented, long-legged, and large-sized. Cave
Omaliinae include two troglobites (Uenohadesina styx from South Korea and
Lesteva (Lestevina) sbordonii from southern Italy), while a single one is known
among Tachyporinae (Ischnosoma spelaeum from Spain) after Hlaváč et al. (2006).
None of these species show troglomorphic features.
Paederinae and Aleocharinae contain 28 and 15 troglobitic species (Hlaváč et al.
2006; Perreau and Faille 2012), as well as several eutroglophiles. They include cave
species nested in large genera, with local spots of underground diversification in
Canary Islands (Domene, Medon, Micranops for Paederinae, Alevonata for

Aleocharinae), Northwestern Spain (Domene for Paederinae), and perhaps Japan
(Lathrobium for Paederinae). Remaining species belong to less speciose genera,
some relictual, in Galapagos (Pinostygus galapagoensis), India (Typhlomalota
glenniei), and Spain (Cantabrodytes vivesi). All show eye and pigment reduction,
body slenderness, and appendage elongation, with several highly troglomorphic
species like Domene vulcanica from Canary Islands or Anopsapterus bordati from
Sahara.
The ca. 8600 species of Pselaphinae (Poggi et al. 1998) are predators of smaller
arthropods and live mostly in soils (Fig. 7.6q–s). About 170 cave species have been
described (Yin et al. 2015a) mostly from northern temperate regions. Africa has a
single genus (Speobaxyris) with a few eyed and weakly modified species (Poggi
et al. 1998), and a single species, slightly troglomorphic, was recently described
from Brazil (Metopiellus painensis). The world hotspot of cave Pselaphinae diver-
sity is the Balkans. The number of cave taxa in this region increased at a fast pace
during recent years, from 25 species in 11 genera in 1998 to ca. 64 species in
ca. 18 genera in 2017 (Hlaváč et al. 2017). Among recently described troglobites,
some show euedaphomorphic or atypical facies, but two species of the monospecific
genera Pauperobythus and Velebythus are highly troglomorphic.
In other Euro-Mediterranean regions, cave Pselaphinae number more than 25 spe-
cies in ca. 10 genera, almost all in the tribe Bythinini. Most species have reduced
eyes and weakly elongated appendages Besuchet 1974) A few are eyeless and
strongly troglomorphic, such as Bathybythus bleyi (Pyrenees). Since 1998, many
species have been described from East Asia (China and northern Indochina), which
emerges today as a novel spot of diversity for cave Pselaphinae. Batrisini dominate
this fauna, with for instance 13 species of Tribasodites in Southern China,
microphthalmic and more or less troglomorphic (Yin et al. 2015b), or the Chinese
genus Zopherobatrus, which is blind and has very elongated appendages. North
America is rich in troglobitic Pselaphinae. Barr and Holsinger (1985) recognized
three components in its fauna, a pattern still valid today: (a) troglobites lacking
pronounced troglomorphic features, e.g., about 20 troglobitic Batrisodes on the
ca. 85 North-American species of this genus; (b) a few troglomorphic troglobites,
eyeless, and with often slightly elongated appendages, e.g., the two monospecific
genera Speleobama and Texamaurops, and cave species of the genus Arianops, a
radiation of 42 species from southeastern USA, all blind and wingless, either edaphic
or troglobitic (Carlton 2008); and (c) minute eyeless euedaphomorphic species of the
genus Speleochus (Carlton 2012).
Life forms of cave Pselaphinae vary from euedaphomorphic to highly
troglomorphic. Regressive characters are, as usual in beetles, eye reduction, pigment
reduction, and loss of wings. However, eyes are often retained in troglobites, and
appendages elongation is limited: antennae as long as body are only known in the
phyletically isolated species Decumarellus sarbui from Romania.
Two supertribes, Scymaenitae and Mastigitae, include 98 and 2% of the species
of Scydmaeninae (Orousset 2014a, b). Mastigitae have one dubiously guanobitic
species from a Sumatran cave, Clidicus gracilipes, with large eyes and wings but
elongated appendages. Five troglobitic species, eyeless and wingless, are known.
Scydmaenus (Heteroeustemmus) aelleni from New Caledonia was already listed in
Decu et al. (1998b). Four species have been described since, three from Croatia
[Euconnus (Tetramelus) bazgoviensis, E. (T.) longipedes, and Scydmoraphes
speluncarius], and one from French Pyrenees [E. (T.) tronqueti]. S. speluncarius
has a facies of soil Scydmaeninae, but the three others, especially E. (T.) longipedes,
have more slender body and longer appendage than their epigean relatives. Two
species of Scaphidiinae, only known from caves in Madagascar (Toxidium cavicola)
and Thailand (Bironium troglophilum), do not exhibit any character linked to cave
life (Löbl and Faille 2017).
Trogidae and Scarabaeidae species linked to cave guano are known from many
tropical regions (Decu et al. 1998b). They do not show characters associated with
cave life, except for the Scarabaeidae Mozartius uenoi from a Japanese cave, which
is wingless and has regressed eyes.
Jacobsoniidae (22 species, three genera; Hava 2016) includes the minute
guanobites Derolathrus cavernicolus and D. troglophilus, both fully oculated,
respectively in the USA and Fidji.
Aderidae (ca. 900 species worldwide, Slipinski et al. 2011) are sometimes in large
numbers on guano piles or organic debris in caves of Africa (Zarcosia) and tropical
Asia (Aderus and Euglenes), with up to five species in a single cave (Moseley et al.
2012). None of them is blind or troglomorphic. Tenebrionidae (ca. 20,000 species
worldwide, Slipinski et al. 2011) usually live in dry and warm habitats, but some
colonized caves where they often dominate guano assemblages in biomass. Most are
eutroglophiles, winged, fully eyed, dark pigmented, and with short appendages, like
their epigean congeners. A few are dark pigmented but with reduced eyes and
moderately elongated appendages, like some cave-restricted North American
Eleodes (Caverneleodes).
Curculionidae (ca. 51,000 species, 4600 genera worldwide, Slipinski et al. 2011) are
mostly phytophagous, those found in caves being apparently root feeders. Osella and
Zuppa (1998) list 34 troglobitic or troglophilic species. Since this review, most generic
assignations have changed, and at least 35 additional species have been described
(Morrone and Hlaváč 2017). They are so far limited to the Euro-Mediterranean region,
Canary Islands, and North America, though undescribed species are reported from the
tropics or China. Within Europe, 50% of the species are described from Italy (Morrone
and Hlaváč 2017) and few from the Balkanic or Pyreneo-Cantabric hotspots. The vast
majority of cave Curculionids belong to the subfamily Entiminae (54 species), with the
large genera Baldorhynchus and Otiorhynchus, almost entirely European. Molytinae
have eight species from Canary Islands and North America, and the subfamilies
Brachycerinae and Cossininae eight European species (Morrone and Hlaváč 2017).
Morphologically, these cave root feeders tend to have a hypogeomorphic or
euedaphomorphic facies, i.e., eyes reduced or absent, no pigmentation, relatively
small size, and appendages not elongated.
Acknowledgements We thanks Oana Moldovan for her tireless support during the redaction of
this chapter, and colleagues who helped reviewing parts of the chapter: Brachyura (Peter Ng),
Coleoptera (Arnaud Faille, Jean Orousset, Michel Perreau, Mingyi Tian), Collembola (Marko
Lukić), Diplopoda (Sergei Golovatch), Gastropoda (Olivier Gargominy), Heteroptera (Wanzhi
Cai), Oniscidea (Jana Bedek, Stefano Taiti), Opiliones (Adriano Kury), Orthoptera (Laure
Desutter-Grandcolas), Pseudoscorpions, Acari and Palpigradi (Mark Judson). This contribution
would have not been possible without all those who kindly gave us the permission to use their
photos and illustrations.
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Chapter 8
An Overview on the Aquatic Cave Fauna
8.1 Introduction
The community of organisms that live in groundwater, which is the obligate subter-
ranean water community, was defined as stygon by Husmann (1966, 1967) who gave
the name stygobiology to the science that studies groundwater life. Earlier, in 1925,
Thienemann proposed a terminology for groundwater animals that paralleled the
ecological classification of terrestrial cave fauna (see also Chaps. 1 and 4):
(1) stygoxenes—occasionally found in groundwater, (2) stygophiles—live both in
groundwater and in epigean habitats; (3) stygobionts—live only in groundwater.
The first discovered and described cave inhabitants were aquatic animals.
In 1436, in Yunnan (China) a local doctor mentioned a cave fish, the
stygophile Sinocyclocheilus grahami described centuries later (1904) by C. Tate
Regan (Ma and Zhao 2012). In 1540, also in China, a stygobiont fish was mentioned
by the local governor of Guangxi province and described as Sinocyclocheilus
hyalinus in 1944 by Chen and Yang (Ma and Zhao 2012). Two centuries later, in
1768, the baby dragon—Proteus anguinus—was described by Laurenti from a small
karstic source in Carniola (parts of the present-day Slovenia). This dragon was for a
long time thought to be the first described cave species in the world.
In 1986, Botosaneanu stated that more than 7000 groundwater species occur
worldwide, a number that was soon recognized to be an underestimate (Gibert and
Culver 2005), and there has been a steady upward trend each year in the number of
species known. Even without the description of new taxa the discovery of cryptic
species, due to improvements in molecular techniques, is steadily increasing the
number of known and described species (Trontelj et al. 2009; Fišer et al. 2018).
Groundwater species richness is higher in Europe (~2000 species) compared with
the other continents, ~561 species in Asia, ~500 species in North America, ~335
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174 O. T. Moldovan
species in Africa (Gibert and Culver 2005), and ~500 in Australia (Stuart Halse,
personal communication), although this can be the result of a more concentrated
effort of identification of new groundwater species in the Old Continent until
relatively recently. Europe was the center of biospeleological researches for almost
a century and the first researches on groundwater fauna were concentrated mostly on
cave aquatic habitats, especially cave rivers and pools, and on wells and springs
outside the caves (see also Chap. 3). It was only later that the hyporheic zone was
described (Orghidan 1959; Motaș 1963) and studied more intensively than caves
(reviews in Danielopol and Rouch 1991; Rouch 1992; Boulton et al. 2003; Di
Lorenzo et al. 2013). The deeper aquatic zones, such as the phreatic zone (see
Chap. 3) known to harbor specific fauna (Marmonier et al. 1993; Stoch et al.
2009), have been less studied due to limitations such as difficulties of access,
boundaries assignment, and spatial heterogeneity (Larned 2012).
8.2 Groundwater Unicellular Organisms (see Table 8.1)
8.2.1 Protista: Protists
Protists are a group of unicellular eukaryotic organisms ranked as a separate king-

dom. More than 180 species have been described from caves, of which ~20% occur
in guano, decomposing organic matter, and humid clay (Golemansky and Bonnet
1994). They are also abundant in show caves, in pools and also around lampenflora
(Varga 1959, 1963). Most of the studied groundwater protists are from interstitial
habitats and display some adaptations such as small, fine, and transparent shells of
Foraminifera that inhabit deep groundwater of the Kara-Kum desert (Brodsky 1928,
1929; Nikoljuk 1948, 1968 cited by Golemansky and Bonnet 1994). Protists are
mostly introduced in caves by waters that come from the surface. The species found
in deserts groundwater are sometimes remnants of ancient seas that covered the
respective areas (Delamare-Debouteville 1960). In an unpublished study of Sheila
Seale (cited by Lavoie 2015), protists were found in all aquatic pools and streams
sampled in Mammoth Cave (USA) but not in drip water. González-López et al.
(2013) also found protists on stalagmites in different caves around the world but all
were edaphic species. Protists have also been found in the microbial mats of Movile
Cave (Romania) together with bacteria and fungi (see also Chap. 16). Epibiontic and
endoparasitic species of protists were found on and in cave animals and some are
specific for their stygobiont hosts, such as Lagenophrys monolistraeand
Spelaeophrya troglocaridis (Golemansky and Bonnet 1994).
Table 8.1 The main groundwater organismal groups of this chapter (in italics) and their simplified systematic position
Kingdom Phylum Subphylum Class Subclass Superorder Order Suborder
Protista Foraminifera
Animalia Porifera
Cnidaria Hydrozoa
Platyhelminthes Tricladida
Temnocephalida
Rotifera
Kinorhyncha
Nematoda
Nemertea
Gastrotricha
Tardigrada
8 An Overview on the Aquatic Cave Fauna
Annelida Aphanoneura
Clitellata Oligochaeta
Hirudinea
Polychaeta
Mollusca Gastropoda
Bivalvia
Arthropoda Crustacea Branchiopoda Cladocera
Remipedia
Maxillopoda Copepoda Calanoida
Cyclopoida
Harpacticoida
Gelyelloida
Ostracoda
Malacostraca Eucarida Decapoda
Peracarida Isopoda
175
(continued)
Table 8.1 (continued)
176
Kingdom Phylum Subphylum Class Subclass Superorder Order Suborder

Amphipoda
Spelaeogriphacea
Theormosbaenacea
Mysidacea
Bochusacea (Mictacea)
Syncarida Anaspidacea
Bathynellacea
Chelicerata Arachnida Acari Trombidiformes Prostigmata-Hydrachnidia
Hexapoda Insecta Coleoptera
Chordata Vertebrata Pisces
Amphibia Urodela
O. T. Moldovan
8 An Overview on the Aquatic Cave Fauna 177
8.3 Groundwater Multicellular Animal Groups (see

Table 8.1)
8.3.1 Porifera: Sponges
Sponges, multicellular organisms with a body full of pores and channels, are poorly
represented in caves or other subterranean environments, and the few that inhabit
groundwaters are considered to be stygoxenes. The only true stygobiont sponge
found to date occurs in Croatia, Eunapius subterraneus, with the following adaptive
characteristics: gemmule reduction and reduced thickness of both skeleton and body
consistence (Sket and Velikonja 1984, 1986; Fig. 8.1a).
There are sponges that inhabit marine caves, because of their constant conditions,
without particular adaptations to life in caves (Vacelet 1990; Manconi et al. 2013).
8.3.2 Hydrozoa: Hydrozoans
The only known cave hydrozoan is Velkovrhia enigmatica described by Matjašič and
Sket in 1971 from caves of the Dinaric karst. Other hydrozoans found in caves of
Mexico, the USA, the Czech Republic, Austria, Australia, and Dinarides belong to
the genus Hydra and are not adapted to subterranean life (Zagmajster et al. 2011).
8.3.3 Platyhelminthes: Flat Worms
The order Tricladida order contains stygobiont planarians that are depigmented, lack
eyes or have reduced eyes and have slower biological processes (ontogenesis,
respiration, and metabolism) than their epigean relatives (Fig. 8.1b). One of the
peculiar characteristics of cave planarians is that they have a higher number of
chromosomes (Gourbault 1968, 1970). Their origin can be either freshwater or
marine waters, and half of the described freshwater species are hypogean. Among
stygobionts the genus Dendrocoelum contains ~57 species inhabiting groundwaters
in the Palearctic, including the special ecosystem of the Movile Cave (Stocchino
et al. 2017; see also Chap. 16). Stygobiont planarians have also been described from
the Americas and Australia. For example, Hausera hauseri is a typical stygobiont,
unpigmented and eyeless, presumably an oceanic relict found in Brazil (Leal-
Zanchet et al. 2014), while the genus Girardia has diversified in caves of Brazil
and Mexico (Souza et al. 2016).
Most planarians, however, are considered stygophiles because the lack of eyes
and depigmentation are the group characteristics and no other adaptations to ground-
water were observed. There are studies indicating that the presence of planarians in
groundwater could be a good indicator of organic pollution (Holsinger 1966;
Eberhard 1990).
178 O. T. Moldovan
Fig. 8.1 Stygobiont invertebrates from caves and calcrete aquifers; (a) Eunapius subterraneus
(Porifera), (b) Dendrocoelum sp. (Tricladida), (c) Zospeum tholussum (Gastropoda), (d) Congeria
sp. (Bivalvia), (e) Pristina pastoral (Oligochaeta), (f) Croatobranchus mestrovi (Hirudinea), (g)
Marifugia cavatica (Polychaeta), (h) Arenurus sp. (Hydrachnidia). Note that the relative size of the
The order Temnocephalida contains smaller parasitic species that live on cave
crustaceans, with the exception of Bubalocerus pretneri, which is a predator. All
subterranean described Temnocephalida lack eyes and most of the genera have been
found in caves of the Dinaric range—Stygodyticola, Scutariella, Subtelsonia, and
Troglocaridicola—, other than a single unidentified species from Papua New
Guinea (Matjašič 1994).
8.3.4 Rotifera: Rotifers
Many rotifers have been observed in cave waters but none seem to be groundwater
adapted, with the exception of some interstitial species, found outside caves
(Pourriot 1994). Nevertheless, the presence and diversity of Rotifera in caves and
other groundwater might be underestimated because most of the sampling has been
done with larger mesh of planktonic nets unable to catch these extremely tiny
organisms. A study on fauna in dripping water in caves in Brazil found rotifers to
be more diverse than Copepoda as usually found in such habitats in Europe (Simões
et al. 2013).
8.3.5 Kinorhyncha: Mud Dragons
Species of kinorhynch found in marine caves in the Mediterranean regions of Europe

and Australia belong to Echinoderes (Sørensen et al. 2000), and those in Japan
belong to Ryuguderes (Yamasaki 2016).
8.3.6 Nematoda: Roundworms
Nematodes colonized different environments and do not have pigments or eyes, which
makes it difficult to distinguish stygobionts from epigean or stygophile species. There
are a few species that were described from continental and marine caves that are
considered to be cave adapted. They belong to genera such as Desmocolex,
Cylindrolaimus, Plectus, Halalaimus, and Thalassoalaimus (Delamare-Deboutteville
1960). In Movile Cave, five species live in the microbial mats: Chronogaster troglo-
dytes—endemic to this cave—, Panagrolaimus sp., Protorhabditis sp., Udonchus
tenuicaudatus, and probably a species of Monhystrella (Riess et al. 1999). Their
⁄
Fig. 8.1 (continued) animals is not respected. Photos by © Marko Lukić (a), © Jaroslav Stankovič
(b), © Jana Bedek (c, d, f, g), © Jane McRae (e, h)
180 O. T. Moldovan
population is controlled by the abundant copepod Eucyclops subterraneus scythicus,

revealing a trophic linkage between nematodes and copepods (Muschiol 2009).
8.3.7 Nemertea: Ribbon Worms
There is no evidence of subterranean adaptations in marine species, while some

freshwater cave species are depigmented and lack eyes. Eyeless species of Prostoma
have been described from cave waters in Europe (Botosaneanu 1998), as have
species of Potamonemertes from gravel riverbeds in New Zealand (Moore and
Gibson 1972).
8.3.8 Gastrotricha: Hairybacks
A single species can be considered as stygobiontic, Marinellina flagelata, a gastro-

trich that lives in the hyporheic zone. This interstitial species has reduced size,
reduced number of eggs, is depigmented, and lack eyes (Kisielewski 1998). Few
gastrotrichs have also been recorded in continental cave waters, such as Chaetonotus
antrumus from a cave in Montenegro (Kolicka et al. 2017). The only comprehensive
study of the gastrotrich fauna in a cave habitat was carried out in an Italian sea cave
by Todaro et al. (2006).
8.3.9 Tardigrada: Water Bears
Only interstitial stygobiont species are known from this group; there are no occur-
rences in continental caves. They are depigmented and lack eyes. Tardigrades in
marine caves are more frequent although there is no information on their adaptation
to the environment. However, Jørgensen et al. (2014) recently found dozens of
species in marine caves, with nine from Australia; Fujimoto and Yamasaki (2017)
described a new genus and species collected from sandy beaches of Ryukyu
Archipelago, Japan.
8.3.10 Annelida: Segmented Worms
Representatives of the class Aphanoneura and subclass Oligochaeta (Clitellata

class) occur frequently in continental and marine caves. Some terrestrial taxa also
live in cave waters, such as Fridericia and Allolobophora (Dumnicka and Juberthie
1994). Cave Annelida do not show any adaptations to subterranean life, because they
already lack eyes and appendices (Fig. 8.1e), except for the smaller size and shorter
hairs of some Naididae, or longer hairs of some Enchytraeidae (Dumnicka 1986;
Dumnicka and Wojtan 1989). However, in a recent study Gonzalez et al. (2018)
have shown that Aphroditiformia suborder show a significant elongation of sensory
parapodial cirri (bundles of cilia), while lacking eyes and pigmentation (eyes could
be also lost during colonization of deep-sea habitats).
In caves, the oligochetes prefer the sediments at the bottom of the water bodies,
leaving only their posterior parts free, above the sediments, for respiration
(Dumnicka and Juberthie 1994).
Some families, such as Potamodrilidae (Aphanoneura) and Dorydrilidae
(Oligochaeta), live exclusively in groundwater habitats. Other representatives of
Aelosomatidae (Aphanoneura) have been described from caves in Europe
(Rheomorpha neiswestnovae, Aeolosoma gineti) and Cuba (Aeolosoma cubana),
although their typical habitats are interstitial waters. Among oligochetes,
Dorydrilidae has one groundwater genus (Dorydrilus), Haplotaxidae has three
genera known only from caves, Lumbriculidae has seven genera with cave taxa,
and Tubificidae has 14 genera of which five are known only from groundwater.
Enchytraeidae, although known as soil inhabitants, has subterranean species that live
exclusively in water, while stygobiont species of Naididae have been described only
from the Antilles (Dumnicka and Juberthie 1994); groundwater appears to be an
important habitat for Phreodrilidae in northern Australia (Pinder 2008).
Hirudinea subclass has few cave-adapted leeches around the world, and their
adaptations to life in caves include depigmentation and lack or reduction of eyes.
The slight widening of the oral sucker in Dina absoloni was proposed as another
cave-specific adaptation by Sket (1986). In the family Haemopidae, Haemopis caeca
is the only known stygobiont and was found in Movile Cave (see also Chap. 16).
Most of the aquatic cave leeches belong to Erpobdellidae in the Northern Hemi-
sphere; within this family the stygobionts belong to Dina, Trocheta, and
Croatobranchus which occur in the Dinaric karst, the last with an unique mouth
morphology (Sket et al. 2001) (Fig. 8.1f). Erpobdella borisi is a possible new
stygobiont found in Iran (Cichocka et al. 2015).
Polychaeta class are marine species with few representatives living in anchialine
caves (see also Chap. 18) or in freshwater cave habitats. There are two well-known
polychaetes from freshwater cave habitats: Troglochaetus beranecki and Marifugia
cavatica. The minute and transparent T. beranecki is widely distributed in Europe,
and a related species was found in Japan and attributed to Speochaetes (although
never described according to Uéno 1957; from Juberthie and Decu 1998).
M. cavatica is depigmented and eyeless, lives in a calcified tube, and probably
colonized cave waters during Pliocene or Pleistocene from freshwater lakes in the
Dinaric karst region (Sket 1997; Mihevc et al. 2001; Fig. 8.1g). Twelve of the
17 described Namanereis species inhabiting subterranean habitats were mentioned
from Australia, Yemen, Canary Islands, and Mexico (Williams 2004; Glasby et al.
2014; Conde-Vela 2017).
182 O. T. Moldovan
8.3.11 Mollusca: Mollusks
Among several classes of mollusks, some are marine and others only fossils; only
two have subterranean representatives: Gastropoda and Bivalvia. Cave adaptations
are reflected in features such as a thin and fragile shell, white-depigmented body, fine
and semi-transparent tegument, and more or less reduced eyes. The small size of the
groundwater species can also be an adaptation (Ginet and Decou 1977).
Gastropoda (snails). Unlike epigean snails, the groundwater species belong to a
group that can breathe under water with the aid of gills positioned in front of their
internal cavity (Ginet and Decou 1977). Altogether, 350 stygobiont gastropods have
been described around the world, with 97% of the taxa belonging to Hydrobiidae
(Culver 2012), of which 168 species inhabit caves in the Balkan Peninsula (Sket
et al. 2004; Fig. 8.1c). Most of the described species were found in the western
Palearctic.
Bivalvia (clams). The cave clams belong to the genera Congeria and Pisidium.
Only one species was assigned to Congeria until 2013, when C. kusceri was split
and two other species added: C. jalzici and C. mulaomerovici (Bilandžija et al.
2013). They all inhabit groundwater of the Dinaric region (Fig. 8.1d). Four species
of Pisidium are known to inhabit caves of Caucasus, Turkey, and Japan (Bole and
Velkovrh 1986). However, the number of described species underestimates true
species richness since various cave populations of a species have different shell
shape and organization of the hinge teeth, which are usually specific characters, so
that each cave is likely to have at least one endemic species (Chertoprud et al. 2016).
8.4 Arthropoda (see Table 8.1)
8.4.1 Crustacea
Crustaceans are the most diverse stygobiont group. Crustaceans live in marine and
freshwater environments and their adaptations to groundwater include reduction of
eggs number (less than 10 compared to dozens at epigean species), reduction of the
reproductive rhythm, longer development, longer life, depigmentation, eye reduc-
tion, and reduction of the swimming legs and hairs (see also Chap. 4).
There are only a few stygobiont Cladocera (water fleas) worldwide, and their
adaptations to subterranean life include valve depigmentation and eye regression.
To date, five stygobiontic chydorid species belonging to Alona and Brancelia are
known (Negrea 1994; Van Damme and Sinev 2011), while other cladoceran species
belonging to Macrothrix, Neothrix, Moina, and Leptodora require further research.
Stygobiont Copepoda belong to the orders Platycopioida, Calanoida,
Misophrioida, Cyclopoida, Harpacticoida, and Gelyelloida, although the number
of stygobiont species is not evenly distributed among and within these orders
(Galassi 2001; Fig. 8.2). Stygobiont species are particularly common within
Fig. 8.2 Number of species and subspecies of different orders of cave Copepoda (modified after
Galassi 2001)
Harpacticoida, Cyclopoida, and Calanoida. The Cyclopoida and the Harpacticoida

have almost 900 species and subspecies (Galassi 2001) distributed worldwide.
Most of the stygobiont Harpacticoida (Fig. 8.3e) belong to the families
Ameiridae, Canthocamptidae, and Parastenocaridae, with Praeleptomesochra,
Pseudoleptomesochrella, Parapseudoleptomesochra, Nitocrellopsis, Stygonitocrella,
Nitocrella, Antrocamptus, Spelaeocamptus, Gulcamptus, Stygepactophanes,
Ceuthonectes, Parastenocaris, Forficatocaris, Paraforficatocaris, and Potamocaris
being exclusively subterranean genera. Chappuisidae has only two exclusively subter-
ranean species (Rouch 1994). The exclusively subterranean Cyclopoida (Fig. 8.3g)
belong to genera Speocyclops, Graeteriella, Allocyclops, and Kieferiella,
with Speocyclops as the most specious genus. The 11 known freshwater stygobiont
Calanoida belong to Diaptomidae (Bowman 1986; Shu et al. 2017). Freshwater
calanoids are distributed from Mexico to Europe and to the Far East, including
Australia (Shu et al. 2017), although marine and anchialine cave Calanoida are much
more diversified (see also Chap. 18).
184 O. T. Moldovan
Fig. 8.3 Stygobiont crustaceans from caves and calcrete aquifers; (a) Microcerberidae (Isopoda),
(b) Gomphodella yandi (Ostracoda), (c) Mangkurtu sp. (Spelaegriphacea), (d) Halosbaena tulki
(Thermosbaenacea), (e) Phyllopodopsyllus wellsi (Harpacticoida), (f) Stygiocaris stylifera
Stygobiont orders Platycopioida and Misophrioida are found only in marine and
anchialine caves (see also Chap. 18). The order Gelyelloida is represented by two
species that live in groundwater in France and Switzerland and by another species
living in the interstitial sediments of a stream in the USA (Galassi 2001).
The class Remipedia was first described from an anchialine cave (Yager 1981).
They lack eyes and are depigmented and have been found only in marine cave
systems in the Caribbean Sea, Australia, Canary Islands, Mexico, and Dominican
Republic (Koenemann et al. 2018; see also Chaps. 18 and 19). There are 29 described
species (Koenemann et al. 2018) and most speciose genera are Speleonectes,
Cryptocorynetes, and Morlockia.
According to Danielopol and Hartmann (1985), 310 species and subspecies of
the class Ostracoda (seed shrimps) were known from hypogean habitats in 1986, of
which only 50 were reported from karst and caves habitats (Fig. 8.3b). Most of
subterranean ostracods belong to the order Podocopida and some of the genera have
only stygobiont species (i.e., Mixtacandona, Caribecandona, Danielocandona,
Phreatocandona). Since 1986, dozens of species have been described from fresh-
water and marine cave habitats. The most notable is the discovery of more than
80 stygobiont ostracods in the Pilbara region of Australia (see also Chap. 20), most
of them belonging to endemic genera (e.g., Deminutiocandona, Pilbaracandona,
Areacandona; Karanovic 2007). Other interesting discoveries have enlarged not
only the number of cave ostracods and their biogeographic distribution but also
the knowledge on the origin and history of the group; for example, the discovery of a
Frambocythere species in a South Korean cave, a genus considered extinct in
Eocene (Smith et al. 2017). Subterranean ostracods are mostly blind and
depigmented, their size is reduced, and chaetotaxy simplified, while some of the
chemosensory aesthetascs are enlarged (Martens 2004).
Groundwater Mysidacea (opossum shrimps) are known from coastal caves,
phreatic waters, and anchialine habitats (see also Chap. 18) mostly in the Caribbean
region, Mexico, and Mediterranean basin, with only a few species described from
south-east Africa and southern India. Stygobiont species belong to Spelaeomysis,
Stygiomysis, Antromysis, Troglomysis, Heteromysoides, Burrimysis, and
Palaumysis. They are blind and depigmented and have a reduced carapace; their
sensory setae also show some differences from their epigean relatives in terms of
their fine morphology (Crouau 1978, 1980).
Three stygobiont species of Bochusacea (formerly Mictacea) have been described
from marine caves of the Caribbean Sea: Mictocaris halope and two species of
Thetispelecaris (Bowman and Iliffe 1985; Ohtsuka et al. 2002).
Eyeless and unpigmented spelaeogriphaceans and thermosbaenaceans are
represented by few species from subterranean waters. The Spelaeogriphacea are
found only in continental waters, in limestone or sandstone caves, or in calcrete
⁄
Fig. 8.3 (continued) (Decapoda), (g) Thermocyclops decipiens (Cyclopoida). Note that the relative
size of the animals is not respected. Photos by © Jane McRae
186 O. T. Moldovan
aquifers where they are represented by three genera, Mangkurtu (Fig. 8.3c),
Potiicoara, and Spelaeogriphus (Jaume 2008). Thermosbaenacea are marine crus-
taceans and most taxa occur in the anchialine environment. Only 18 species of
Thermosbaenacea have been recorded in limnic or brackish waters, in caves, or in
interstitial habitats (Jaume 2008), and they belong to Thermosbaena, Tethysbaena,
Limnosbaena, Halosbaena (Fig. 8.3d), Theosbaena, and Tulumella (Boutin 1998).
Subterranean thermosbaenaceans have a larger distribution, matching precisely the
area covered by the ancient Tethys Sea (Jaume 2008).
One of the most diverse orders of crustaceans, Isopoda, is found in all subterra-
nean marine and freshwater habitats (see also Chap. 7 for a review of terrestrial
isopods). The order contains small individuals that live in interstitial habitats and
bigger individuals that occur in larger groundwater bodies. They lack eyes, are
depigmented, have relatively longer antennae than their epigean relatives and
sometimes very elongated uropods, have slower development and produce fewer
eggs, —even one single bigger egg that is richer in vitellus as in Microparasellidae
(Coineau 1998). With one exception all suborders have also colonized groundwater
habitats. Few species belong to the most primitive suborder of isopods, the
Phreatoicidea (Coineau 1998; Knott and Halse 1999; Wilson 2008). The rest of
groundwater representatives belong to Asellota [i.e., Caecidotea, Proasellus,
Synasellus, Stenasellus, Microcerberus (Fig. 8.3a), Microcharon, etc.], Cymothoida
(i.e., Cyathura, Stygocyathura, Thyphlocirolana, Faucheria), Sphaeromatidea (i.e.,
Monolistra), and Calabozoida (from Coineau 1998). In the world, the most diverse
regions in aquatic isopods are Caribbean and Mediterranean. Asellus aquaticus is a
special example of a largely distributed isopod inhabiting various aquatic habitats in
Europe, with only two subspecies restricted to caves, one in Movile Cave and
another in the Dinaric karst caves. The importance of this species is that it shows
how surface populations diverge after subterranean colonization and that only about
one-third of all changed traits can be considered as troglomorphisms (Konec et al.
2015).
Amphipoda is another diverse order of crustaceans characterized by the lack of
both eyes and body pigmentation and a diversity of body and appendage sizes
depending on the groundwater habitat it occupies. They also have longer life and
lay larger and fewer eggs. More than half of the known species (~750 species) occur
in caves (Holsinger 1994; Hobbs 2004). Groundwater amphipods belong to 32 fam-
ilies; among the most speciose genera are Niphargus, Stygobromus, Metaniphargus,
Bogidiella, Pseudoniphargus, Ingolfiella, Gammarus, Salentinella, Metacrangonyx,
Paramelita, etc. Two broad regions are especially rich in amphipods, namely, a
region comprising eastern and southern North America and West Indies and the
Mediterranean region of Europe. Niphargus species are used in biospeleology as
models for phylogeographical and evolutionary studies (see a recent paper by Delić
et al. 2017).
The most evolved crustacean order, the Decapoda, have numerous cave repre-
sentatives in the infraorders of Brachyura (crabs), Caridea (shrimps), Astacidea
(crayfishes), and Anomura. The cave decapods have thin tegument, fine and long
pereiopods and antennae with increased number of aesthetascs and are depigmented,
except for some anchialine shrimps that are orange, red, or pink in color. The visual
organs are reduced or animals are completely blind with a slow metabolism
and increased longevity (Cooper and Cooper 1978; Hobbs 1998).
More than 30 species of crabs with marine or freshwater origins have
been described from caves. Sesarmoides, Trogloplax, and Cancrocaeca are the
main cave genera with a marine origin, while the freshwater crabs belong to
Cerberusa, Isolapotamon, Phaibulamon, Stygothelphusa, Adeleana, Thelphusula,
Phricothelphusa, Sendleria, Holthuisana, Rouxana, Typhlopseudothelphusa,
Neostrengeria, Chaceus, and Rodriguezia. The cave crabs are distributed in Indo-
nesia, Papua-New Guinea-New Britain, Thailand (the species of marine origin),
Jamaica, Mexico, Guatemala, Belize, and South America (the species of freshwater
origin) (from Guinot 1994).
Cave shrimps belong to different families and some genera are especially
speciose: Procaris, Caridina, Parisia, Troglocaris, Typhlatya, Macrobrachium,
Troglomexicanus, and Typhlocaris (Fig. 8.3f). They are distributed in North Amer-
ica, West Indies, Australia, and the Mediterranean regions (Hobbs 1998; Page et al.
2008).
Freshwater crayfishes are considered successful in colonizing caves, with the
Cambaridae as the best represented family. Cambarus, Orconectes, and
Procambarus are the most diversified genera distributed in North America and
north of Mexico (Hobbs 1998).
Syncarida superorder have subterranean representatives in both its orders,
Anaspidacea and Bathynellacea. Anaspidacea has families restricted to caves or
interstitial habitats, like Psammaspididae and Stygocarididae, that lack eyes and
have reduced appendages (Coineau 1998). They were identified in Tasmania,
Australia, and South America. Bathynellacea have exclusively groundwater repre-
sentatives distributed on all continents. They lack eyes and statocysts and have
reduced appendages. The order has more than 95 species, most of them belonging
to Bathynella with more than 50 species and subspecies (Camacho and Valdecasas
2008). Hexabathynella has a cosmopolitan distribution being as rich in species as
Iberobathynella with a limited distribution to the Iberian Peninsula (Coineau and
Camacho 2004; Camacho 2003). Most genera have small geographic distribution
(Camacho and Valdecasas 2008).
8.4.2 Hydrachnidia: Water Mites
Water mites are known only from phreatic and hyporheic zone waters (Fig. 8.1h).
Specializations to these habitats include eye reduction or lack of eyes, depigmenta-
tion and thinner body cuticle, smaller and elongated body, shorter legs than their
epigean relatives, and reduction of the number of eggs (Teschner 1963). Only one of
the 10 suprafamilies of this subclass has no subterranean representatives. The most
frequent genera in phreatic waters are Stygotrombidium, Cerberotrombidium,
Wandesia, Tartarothyas, Bandakia, Torrenticola, Neomamersa, Kawamuracarus,
Atractides, Frontipodopsis, Aturus, Stygomomonia, etc. (Schwarz et al. 1998).
188 O. T. Moldovan
Fig. 8.4 Stygobiont beetles

from a single calcrete
aquifer in the Australian
desert (Dytiscidae:
Hydroporini), from left to
right: Paroster
macrosturtensis,
P. mesosturtensis,
P. microsturtensis. Photo by
© Chris Watts
8.4.3 Insecta, Coleoptera aquatica: Aquatic Beetles
The first stygobiont beetle (Siettitia balsetensis) was discovered in 1904, in France,
by Abeille de Perrin and until the end of the twentieth century only 16 more species
and one subspecies were reported from Japan, China, Thailand, Indonesia,
and Ecuador (Spangler and Decu 1998). They belong to Dytiscidae, Elmidae,
Hydrophilidae, and Noteridae families. The number of stygobiont species increased
dramatically with the study of the calcrete aquifers in Western Australia (see also
Chap. 20) where approximately 100 dytiscid species with regressed or absent eyes
have been found within 45 separate calcrete bodies (Leys et al. 2003; Watts and
Humphreys 2009; Watts et al. 2016).
Along with the typical adaptations of subterranean inhabitants, stygobiont beetles
have unique adaptations that are not found in their epigean water beetle relatives,
such as the change of pupation habitat from terrestrial to the bottom of the subter-
ranean waters (Uéno 1957). Smaller size was also considered as one of the general
adaptations of stygobiont beetles until the discovery of calcrete Dytiscidae of very
different sizes (Fig. 8.4).
8.5 Chordata
8.5.1 Pisces: Fishes
Fish are the most studied group of cave inhabitants, especially in relation to
processes of adaptations. All stygobiont fishes belong to the Teleostei and have a
small and slender body. More than 80 taxa were described but new species are added
each year. Cave fishes belong to the orders Characiformes, Cypriniformes,
Siluriformes, Gymnotiformes, Percopsiformes, Ophidiiformes, Cyprinodontiformes,

Synbranchiformes, and Perciformes. The richest in species are Cypriniformes and
Siluriformes. The most diversified genera are Sinocyclocheilus, Triplophysa,
Rhamdia, Trichomycterus, Ancistrus, and Lucifuga. Cave fishes are most common
in tropical and subtropical caves, but a few have also been described from temperate
caves (Weber et al. 1998; Romero 2001; Trajano et al. 2010). The most recent
discovery was the first cave fish in Europe, found in the cave labyrinth called the
Danube-Aach System, in southern Germany (Behrmann-Godel et al. 2017).
Adaptations of cave fish include the ones common for all stygobiont
animals—depigmentation and reduction or lack of eyes, as well as some typical
adaptations for the group: reduction of the pineal organ in some species, enhanced
olfaction, taste and touch senses, and the development of the lateral line (see also
Chap. 4).
8.5.2 Amphibia
Only the order of Urodela (salamanders) has cave representatives. The most
advanced adaptations include the development of the lateral line, slender legs, a
flat and wide head, and neoteny. Eurycea, Typhlotriton, Haideotriton, and
Gyrinophilus are known from North America; the species of these genera are
depigmented and have reduced eyes. The only European cave salamander is Proteus
anguinus which reaches sexual maturity at 11–14 years for the males and 15–18
years for the females, while hatching takes place more than 100 days after oviposi-
tion (Juberthie et al. 1996; Durand 1998). A first black Proteus was found almost
three centuries after the description of this species by Von Valvasor in 1689, and
described as a subspecies endemic to a small area in Slovenia (Sket and Arntzen
1994).
Acknowledgements I am grateful to Ľubomir Kováč and Stuart Halse for the useful suggestions
and comments that improved the quality of the manuscript. I am also indebted to Jana Bedek, Marko
Lukić, Jane McRae, and Jaroslav Stankovič for allowing me to use their photos in this chapter. OTM
acknowledges the financial support from the Romanian Academy and the grant of the Romanian
Ministry of Research and Innovation, CNCS—UEFISCDI, project number PN-III-P4-ID-PCCF-
2016-0016, within PNCDI III.
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Chapter 9
Subterranean Biodiversity Patterns
from Global to Regional Scales
Maja Zagmajster, Florian Malard, David Eme, and David C. Culver
9.1 Introduction
Interest in the geographic pattern of species richness became a central theme in

macroecology in the late 1980s (Wilson 1988). The quantitative study of biodiver-
sity was made possible by (1) the development of spreadsheets and databases that
could store and manipulate large amounts of data, (2) GPS devices and detailed
digital maps that allowed accurate and rapid georeferencing, and (3) mapping
software that made display and analyses of georeferenced occurrence data straight-
forward. In the light of rapid environmental changes due to human impacts, which
may bring species close or even to extinction, the need to map biodiversity patterns
and improve the understanding of the mechanisms underlying the patterns became
obvious.
Caves and other subterranean habitats offer many challenges and opportunities in
tackling general biodiversity questions, but large-scale biodiversity studies started
M. Zagmajster (*)
Biotechnical Faculty, Department of Biology, Subterranean Biology Lab, University of
Ljubljana, Ljubljana, Slovenia
F. Malard
University of Lyon 1, ENTPE, CNRS, Univ. de Lyon, UMR5023 LEHNA, Villeurbanne,
France
D. Eme
New Zealand Institute for Advanced study, Institute of Natural and Mathematical Sciences,
Massey University, Auckland, New Zealand
D. C. Culver
Department of Environmental Science, American University, Washington, DC, USA

https://doi.org/10.1007/978-3-319-98852-8_9
196 M. Zagmajster et al.
1200
Number of species 1000
800
600
400
200
0
1800 1850 1900 1950 2000
Year
Fig. 9.1 Cumulative number of aquatic crustacean species described, by year, for surface species
(thin line) and subterranean species (thick line) in Europe (Modified from Stoch and Galassi (2010))
much later below ground than in other habitats for several reasons. First, discovery
and description of subterranean species lagged behind the description of surface
species, at least by several decades (Fig. 9.1; Stoch and Galassi 2010). Second, until
relatively recently, the number of stygobiotic and troglobiotic species seemed so low
that quantification was unnecessary, as can be seen in Fig. 9.1. In past decades this
changed significantly. There are some taxonomic groups that are even richest in the
subterranean realm; for example, in Europe, the number of stygobiotic crustacean
species exceeds the number of surface-dwelling crustacean species (Stoch and
Galassi 2010: 1174 vs. 1111 species, respectively). There are more troglobiotic
palpigrades (Arachnida) than surface ones in temperate regions (Giribet et al.
2014). The most speciose genus of Amphipoda, Niphargus, has more than 400 spe-
cies, all but a handful of which are strictly subterranean (Fišer et al. 2008; Horton
et al. 2017). Third, because many species have very small ranges, local species
richness is low compared to regional species richness (Gibert and Deharveng 2002),
which in turn means large amounts of data are required for large-scale analysis.
In past few decades, there has been a substantial progress in mapping biodiversity
patterns of subterranean taxa, as presented in this chapter. But there is one aspect that
becomes obvious in such an overview—there is a large bias toward analyses of
groundwater taxa, compared to terrestrial subterranean taxa, especially on global and
continental scales.
9 Subterranean Biodiversity Patterns from Global to Regional Scales 197
9.2 Challenges in Describing and Explaining Subterranean

Biodiversity Patterns
Biodiversity studies foremost depend on the quality of species distribution data,

which are inevitably incomplete. At any point in time, data can be improved, more
records collected, and additional species discovered, so that “perfectly complete
datasets” can never be obtained and waiting to get them may lead to biodiversity loss
prior to discovery. Even incomplete datasets can give valuable information for
scientific studies, as well as the development of conservation strategies, if interpreted
and evaluated properly. This is especially true of subterranean biodiversity studies,
which deal with habitats that are difficult to access, species that are difficult to
sample and, even when collected, mostly difficult to identify (Fišer and Zagmajster
2009; Delić et al. 2017). Consequently, studies of subterranean biodiversity may be
more challenging. But if the characteristics of such environments are taken into
account, and advantage is taken of the data resources and analytical capacity
currently available, robust conclusions are possible. An overview of potential pitfalls
in comparative studies of subterranean biodiversity was prepared by Culver et al.
(2012), and we refer to this work at several places. In the following paragraphs, we
describe challenges related to documenting biodiversity patterns, which are followed
by challenges in explaining these patterns.
9.2.1 Spatial Scale and Availability of Data
One of the main challenges is to improve accuracy and availability of data. It was
only relatively recently that databases on subterranean species distributions devel-
oped from species lists per country/region to sets of species occurrences with
accurate spatial coordinates defined at the level of sampling sites (Culver et al.
2001; Zagmajster et al. 2008, 2014; Deharveng et al. 2009; Christman et al. 2016).
While species lists per country/region can be used for inferences at global or
continental scales, spatially accurate species occurrences enable studies at regional
and local scales as well as application of dedicated analytical tools for the study of
spatial patterns (Christman and Zagmajster 2012). Databases with georeferenced
records on subterranean species distributions have been developed, but are not freely
available. Metadata on some databases can be found online, with instructions on
how to retrieve the data (e.g., CKmap2000 2003; BioFresh Portal 2014). In rare
cases, publicly available databases can also be downloaded (e.g., for Bathynellacea
in Iberian Peninsula, Camacho et al. 2014). Finally, occurrences of species at the
country level are directly available, for example, in Fauna Europaea database
(de Jong et al. 2014).
9.2.2 Not All Species Are Troglobionts
An issue that is somewhat specific to subterranean habitats is that not all species are
exclusively subterranean, as some live underground only during some parts of their
life cycle or can occur there accidentally (see Chap. 3 for discussion on classification
of subterranean fauna). Comparative studies of distributional patterns of subterra-
nean biodiversity should be done with species or combination of species that have
the same level of dependency on subterranean habitats. This is because troglobiotic
and non-troglobiotic species differ in their responses to different environmental
factors. This does not imply that only troglobionts should be considered in analyzing
subterranean biodiversity patterns. Indeed, it could be useful to include data on
troglophiles, but such data are very rarely gathered in databases. The same is the case
with guanobionts, species that are specialized to live in bat guano but do not exhibit
troglomorphies—they can present a substantial amount of species in guano-rich
tropical caves (Deharveng and Bedos 2012).
9.2.3 The Omnipresence of Undescribed Species
A recurrent problem in subterranean research is that a lot of specimens remain

determined to the level of “undescribed species” due to the lack of taxonomists.
Yet, not all undescribed species become new valid species when checked by taxon-
omists—some of them turn to be already described species. This issue was referred to
by Culver et al. (2012) as the fallacy of “equality of the described and undescribed
species,” with recommendation that only formally described species should be used
for regional comparisons. This may represent a problem for some regions, for
example Brazil, where many new recent discoveries yielded many specimens attrib-
uted to undescribed species based on visual morphological differences (Trajano and
Bichuette 2010; Souza and Ferreira 2016). But, compared to morphological estima-
tion, molecular methods may be better in distinguishing different “units” that could
be considered as separate species (Guzik et al. 2011). Indeed, molecular approaches
can facilitate the discovery of species diversity, and molecular data offer valuable
distinctive characters which can be used for species diagnosis (Delić et al. 2017).
9.2.4 Dealing with Sampling Difficulties
Even though the problem of sampling incompleteness is common to biodiversity

studies in general (Beck and Schwanghart 2010), it is more apparent in caves and
other subterranean habitats. Indeed, accessing subterranean habitats is challenging
and may require special physical skills from collectors (vertical caving, cave diving)
or specialized equipment to reach the animals where humans cannot physically
enter. As abundance of species in caves is generally low (Culver and Pipan 2009),
repeated visits or trapping techniques are needed to collect most of the present
animals. Repeated visits with comparable sampling effort can be used to estimate
species detectability—this is the probability that the species will be sampled when
present (Tyre et al. 2003). However, taking this into account in multispecies
subterranean studies is fairly uncommon due to the difficulty of evaluating the
detectability of each species.
Even working with incomplete datasets can lead to sound and firm conclusions, if
differences in sampling are considered properly. On large scales, robustness of the
pattern can be evaluated by comparing the patterns at different snapshots in time
(Culver et al. 2004a; Bregović and Zagmajster 2016). Species accumulation curves
enable comparisons of regions considering their potential for additional species—the
steeper the curve, the more species are still expected to be found (Gotelli and Colwell
2001). Species richness estimators, which predict missing species on the basis of rare
species (Colwell and Coddington 1994), have been widely used in subterranean
comparative studies (e.g., Culver et al. 2006; Zagmajster et al. 2008; Deharveng
et al. 2009). Using the ratio of observed species richness to estimated species
richness has been proposed as a direct measure of sampling/completeness (Soberón
et al. 2007). This approach was applied to large-scale subterranean biodiversity
studies, to get the subsample of “well-sampled” areas (Zagmajster et al. 2014). A
different approach to identify such areas was proposed by Zagmajster et al. (2010),
who used the number of single-cave species per quadrat as indicators of sampling
completeness when quadrats of similar species richness were compared.
Some approaches used in subterranean studies can lead to biased conclusions.
One is the use of best expert opinion rather than sampling results to estimate the
expected number of species, which was referred to by Culver et al. (2012) as “misuse
of expert opinion.” Guzik et al. (2011) treated the expected numbers of subterranean
species richness in Western Australia, given by taxonomic experts, as a number for
comparisons with other regions of the world, where no such expert estimates had
been made. Another false approach is presuming one should not expect new species
in areas where high numbers of described species already occur. Treating various
regions differently in this respect was termed by Culver et al. (2012) as fallacy of
“provincialism.” The point is that new species are being discovered not only in
species-poor but also in species-rich areas. This has been statistically confirmed in
the Dinaric karst in the Balkans, where additional species are not to be expected only
in species-poor but also within species-rich areas (Zagmajster et al. 2010). On the
other hand, this does not imply that eventually all regions of the same size will yield
the same number of subterranean species (termed fallacy of “isotropy” by Culver
et al. 2012). This means that number of species per certain area cannot be simply
extrapolated to other areas of the same size.
9.2.5 Area Matters
In comparative studies, surface areas that are used to report number of species should
be taken into account—this is termed as fallacy of “scale invariance” by Culver et al.
(2012). Clearly, species richness tends to increase with increasing area, also known
as the species-area relationship (Rosenzweig 1995). Thus, only species richness of
similar or comparable areas should be compared. If this is not possible, corrections
due to differences in area should be included. For example, Stoch and Galassi (2010)
used adjusted formula controlling for area to investigate groundwater crustacean
biodiversity patterns in Europe.
9.2.6 Spatial Autocorrelation
There are also challenges in studies that attempt to explain the patterns of species
richness. First is the consideration of spatial phenomena termed spatial autocorrelation
(Cressie 1993), referring to the observations that spatially closer areas are more related
than the ones further apart. Spatial autocorrelation, which is the result of dispersal
(or the lack of it), should be considered when inferences in explaining the mechanisms
behind the observed patterns are sought. Different approaches to considering spatial
autocorrelation exist and have been employed in subterranean biodiversity studies,
including Moran’s I (Christman et al. 2005; Zagmajster et al. 2008), conditional
autoregressive models (Christman and Culver 2001), simultaneous autoregressive
models (Zagmajster et al. 2014; Eme et al. 2015), generalized least squares models
(Eme et al. 2017), and spatial eigenvectors (Bregović and Zagmajster 2016).
9.2.7 Selection of Relevant Variables
Selection of variables used to explain subterranean biodiversity patterns should be

done with consideration of their potential effect on subterranean communities. For
example, in cave/subterranean habitats, daily and even yearly fluctuations of tem-
perature are attenuated (e.g., Freeze and Cherry 1979; Šebela and Turk 2011), so
such variables are not expected to have direct impact on subterranean communities.
In species richness studies, number of caves and cave length were initially used as a
measure of habitat availability (Christman and Culver 2001; Culver et al. 2001,
2004a, b), but recent publications of the distribution map of groundwater habitats in
Europe (Cornu et al. 2013) and the karst map database in the United States (Weary
and Doctor 2014) enabled the development of additional variables (Zagmajster et al.
2014; Eme et al. 2015, 2017; Christman et al. 2016). Bearing in mind the recent
increase in availability of different environmental variables for statistical modeling,
it is important to select those variables that are related to the central question and
scale of analyses (Zagmajster et al. 2014; Eme et al. 2015, 2017; Bregović and
Zagmajster 2016; Mammola and Leroy 2017).
9.2.8 The Importance of Spatial Scale and Spatial

Non-stationarity
The importance of different explanatory variables may differ according to the scale
of analysis (Field et al. 2009), as well as to the region under study (Eme et al. 2015).
The latter, termed spatial non-stationarity, acknowledges that the relationship
between response variable (biodiversity metrics) and environmental factors may
vary across space. Geographic weighted regression models that take into account
spatial non-stationarity (Brunsdon et al. 1996) were used by Eme et al. (2015) to
reveal geographic variation in the relative contribution of environmental factors to
species richness of groundwater crustaceans in Europe.
9.3 Global Patterns of Species Richness: The Tropics Are

Not Hotspots
Owing to the lack of comparable data on cave and other subterranean species from
most countries, little is known on global patterns of subterranean biodiversity. Attempts
to collect all known data on subterranean species of the world started already in the first
half of twentieth century. Species lists of Wolf (1934–1938) and numerous heteroge-
neous country lists of taxa were included in publications within Encyclopaedia
Biospeologica (Juberthie and Decu 1994, 1998, 2001). The per country distributions
of subterranean fishes indicate that their diversity is highest between 16 and 25 N
(Culver and Pipan 2009). This case is specific to rather low number of cave fishes on a
world scale (Proudlove 2010) and may reflect also some other specificities of the group.
A few global overviews of species richness of other subterranean taxa [Amphipoda, by
Holsinger (1993) and Nematoda, by Du Preez et al. (2017)] are also interesting but of
little use to evaluate global subterranean biodiversity patterns.
9.3.1 Global Distribution of Biodiversity Hotspots
In 2000, Culver and Sket published a list of caves and wells of the world where at
least 20 troglobionts, both terrestrial and aquatic, had been recorded. Their list and
the map they produced was the first global overview of the distribution of hotspots of
species richness per site. Since then, the number of such sites quickly doubled
(Culver and Pipan 2009; Souza and Ferreira 2016). Culver and Pipan (2013) set a
more stringent criterion to define the hotspots in cave biodiversity and listed the sites
with at least 25 species, where aquatic and terrestrial troglobionts were treated
separately. The list became much shorter, including only nine aquatic and seven
terrestrial sites on the globe (Fig. 9.2). These two lists show that the distribution of
hotspots is not uniform around the globe and that areas with largest aggregations of
hotspots do not occur in the tropical belt. Only one site in Australia is situated at the
edge of the tropical belt in the Southern Hemisphere, while all the others are located
in temperate latitudes. Deharveng and Bedos (2012) brought forward the potential
for increased species richness in the tropics, if guano-specialized species
(guanobionts) were included in the lists.
9.3.2 Global Patterns of Groundwater Crustacean Species

Richness
Mapping sites containing at least 25 troglobionts is only a rough approximation of

the global patterns in subterranean biodiversity. Additionally, there are resources
available that enable mapping of biodiversity of at least some subterranean taxa at
global scale.
We tentatively mapped the global distribution of groundwater crustaceans
(Fig. 9.2), using first, data from Botosaneanu (1986) and second, data from various
sources published over the last two decades (Africa: Kayo et al. 2012; Asia: Brancelj
et al. 2013; Australia: Guzik et al. 2011; Halse et al. 2014; Europe: Zagmajster et al.
2014; North America: Peck 1994; Mejia-Ortiz et al. 2013; Niemiller and Culver,
unpublished; Southern America: Pérez-González and Yager 2001; Trajano and
Bichuette 2010; Rios-Escalante et al. 2016). Even though species richness data
were not available for some countries, the maps provide valuable insights into the
global pattern of groundwater crustacean species richness. The number of species per
country is rising in time, supporting the need for further worldwide species invento-
ries. The updated map of groundwater crustacean species does not indicate any rise in
species richness near equator or in tropical regions. Highest numbers of species can
be found at temperate latitudes, especially in the Palearctic and Nearctic regions.
9.4 Striking Features of Subterranean Biodiversity Patterns

at Continental Scales
The first initiatives in mapping continental subterranean biodiversity patterns started in

North America, covering contiguous states of the United States (Culver et al. 1999,
2000). An important impetus in promoting the mapping of subterranean biodiversity on
larger scales was presented by a thematic workshop on Mapping Subterranean Biodi-
versity (Culver et al. 2001). The first large-scale quantitative study was the PASCALIS
Fig. 9.2 Different presentations of global patterns of subterranean biodiversity: (a) distribution of
caves with at least 25 troglobionts and/or stygobionts (data from Culver and Pipan 2013); (b)
distribution of groundwater crustacean species per km2, based on data from Botosaneanu (1986); (c)
distribution of groundwater crustacean species per km2, based on data from publications in the last
two decades (see text for the list). Species richness per country (SRc) in (b) and (c) is corrected for
the area effect using the following formula SRc ¼ SRobs/A0.15 where SRobs is the raw species count
for each country and A is the country area (Stoch and Galassi 2010). Class delimitation in (b) is done
according to the highest value, this is >85%, >60%, >40%, >20% and less than 20% of the richest
area. In (c) class delimitation follows limits from (b). Grey colour in (b) and (c) refers to
the countries with no data
project (Protocols for the ASsessment and Conservation of Aquatic LIfe in the Subsur-
face), a western-central European-wide study (Gibert et al. 2004). Recently, studies
reporting on high subterranean biodiversity areas have been reported from Brazil
(Trajano et al. 2016) and Western Australia (Guzik et al. 2011; Halse et al. 2014), but
they are of limited use to evaluate continental patterns. Most of what we know about
spatial patterns of subterranean species richness at continental scale is from studies
conducted in Europe, which are almost exclusively limited to groundwater habitats.
9.4.1 Scale Dependence of Species Richness Patterns
Not surprisingly, patterns depend on the spatial grain used to map species richness.
Coarse-grain studies revealed a northward monotonic decline in the number of
species in Europe (Fig. 9.3). This latitudinal gradient of aquatic species richness
was first documented by Hof et al. (2008) for the groundwater fauna, based on
934 groundwater species taken from Limnofauna Europaea (Illies 1978). Stoch and
Galassi (2010) documented the pattern of groundwater crustacean species richness in
Europe using data from Fauna Europaea (de Jong et al. 2014). Their analysis
included 1174 groundwater crustaceans. They calculated species richness for each
country, corrected for area, and clustered the countries into three latitudinal bands.
For each band, they calculated the number of species per area. The pattern of
decreasing species richness with increasing latitude became visible (Fig. 9.3a). In
Fig. 9.3b, we used a more recent dataset of 1570 groundwater crustacean species in
Europe (Zagmajster et al. 2014), calculated number of species per each bioregion
(sensu Illies 1978), and corrected for area of mainland within each region (species
Fig. 9.3 Coarse-grain patterns of species richness for the groundwater crustaceans in Europe. (a)
Latitudinal bands of species richness, joining countries with similar groundwater crustacean species
richness. The legend presents species richness per area [spp/km2] within each band (Stoch and
Galassi 2010). (b) Groundwater crustacean species richness from the European groundwater
crustacean dataset (Zagmajster et al. 2014), calculated per each biogeographical region (sensu Illies
1978). Species richness was corrected for the area of mainland within each region [spp/km2].
Dashed lines denoted region borders, and pale gray denoted country borders for reference
richness [SRc] is corrected for the area effect using the formula SRc ¼ SRobs/A0.15
where SRobs is the species count and A is the area of the region; as in Stoch and
Galassi 2010). Presented in three classes according to species richness, the latitudi-
nal decrease in species richness is again visible (Fig. 9.3b).
In contrast to coarse-grain studies of continental biodiversity patterns, once a finer
spatial grain was used, a ridge of high terrestrial and aquatic species richness at
latitudes ranging from c. 42 to 46 N became visible (Culver et al. 2006; Zagmajster
et al. 2014). Culver et al. (2006) assembled georeferenced records of terrestrial cave
species for three European regions and four north-American regions ranging in size
from 2000 to 6300 km2. By mapping the number of species for a total of 23 regions,
Culver et al. (2006) hypothesized that the pattern for Europe was one of a ridge of
high species richness (Fig. 9.4). In North America, the ridge of high species richness
was reduced to a single region (northeast Alabama), later expanded to south central
Tennessee (Niemiller and Zigler 2013).
Zagmajster et al. (2014) assembled in the European groundwater crustacean
dataset (EGCD) a total of 21,700 database records collectively representing 1570
species and subspecies of Crustacea. To analyze the spatial patterns of species
richness at a European scale, the authors used bands of 0.9 latitude, with cells in
the bands having an area of 10,000 km2 (Fig. 9.5). The average species richness per
cells within 0.9 latitudinal band exhibited a hump-shaped pattern: species richness
for groundwater crustaceans peaked at roughly the same latitudes (c. 42–46 N;
Fig. 9.5) as richness of terrestrial cave species (Fig. 9.4). Zagmajster et al. (2014)
showed that the hump-shaped pattern of groundwater crustacean species richness
was robust to sampling bias.
9.4.2 Range Size Follows a Rapoport Rule in the Palearctic
The species richness patterns are inherently related to the patterns of range size that
were explored in groundwater crustaceans (Zagmajster et al. 2014). The “Rapoport
rule” was first coined by Stevens (1989) to describe the pattern of increasing species
range size at higher latitudes observed among a number of plants and animals.
Zagmajster et al. (2014) measured the range size of groundwater crustaceans by
calculating the maximum linear extent (MLE) of each species—defined as the
straight-line distance between the two most distant known localities of a
species. Then, they computed and mapped the median MLE of species contained
in 10,000 km2 cells across Europe (Fig. 9.6). The groundwater crustacean ranges
were found to exhibit a marked Rapoport rule, as the cell average of median MLE
per 0.9 latitudinal band increased markedly with latitude above a latitudinal thresh-
old of 43 N (Fig. 9.6).
Fig. 9.4 Map of species richness patterns of European terrestrial cave species. The open triangles
are areas with few if any terrestrial cave species, the gray triangles are areas with <50 species, and
the gray circle is Ardèche with <50 species in 5000 km2 of area. The black circles are the diversity
hotspots in Slovenia and Ariège (France). Black triangles are other possible diversity hotspots. The
boundary of the Pleistocene ice sheet is shown as solid line. A pair of dashed lines indicates the
hypothesized position of the high diversity ridge (From Culver et al. (2006))
9.4.3 A High Spatial Turnover in Species Composition Over

Short Distances
Subterranean fauna typically consist of narrow range species, which affect patterns
of change in species composition (i.e., beta diversity). Culver et al. (2000) provided
data on species composition of stygobionts, troglobionts, and single-county
endemics for the contiguous counties of the United States, based on 927 species.
Endemism was high and 54% of the species were known from a single county. High
endemism of troglobionts was reported for calcrete bodies of Western Australia,
with very little common species also among closely positioned calcrete bodies (e.g.,
only 8 and 20% shared species among three calcrete bodies; Humphreys et al. 2009).
Of a total of 313 groundwater species collected in six regions of Central Europe as
part of the PASCALIS project, only 1, 3, 4, and 27 species were shared by 6, 4,
3, and 2 regions, respectively (Dole-Olivier et al. 2009).
Zagmajster et al. (2014) quantified the latitudinal pattern in the intraregional
turnover component of beta diversity of groundwater crustaceans across Europe
using large squares of equal area (Fig. 9.7a). The turnover component of beta diversity
represents differences in species composition caused by species replacements as
Fig. 9.5 Map of species richness patterns of European groundwater crustacean species. (a) Species
richness of 10,000 km2 cells. (b) Relationship between the cell average of species richness per 0.9
latitudinal band and latitude. Black horizontal bars and boxes show the median and interquartile
range, respectively, for latitudinal bands. The maximum length of each whisker is up to 1.5 times
the interquartile range, and open circles represent outliers. The thick red line represents the fit of
generalized additive model to the averages of latitudinal bands (From Zagmajster et al. (2014))
Fig. 9.6 Map of range size patterns of European groundwater crustacean species. (a) Median
maximum linear extent (MLE) of species contained in 10,000 km2 cells. (b) Relationship between
the cell average of median maximum linear extent per 0.9 latitudinal band and latitude. Black
horizontal bars and boxes show the median and interquartile range, respectively, for latitudinal
bands. The maximum length of each whisker is up to 1.5 times the interquartile range, and open
circles represent outliers. The thick red line represents the fit of generalized additive model to the
averages of latitudinal bands (From Zagmajster et al. (2014))
Fig. 9.7 Spatial variation of intraregional species turnover of groundwater crustaceans in Europe.
(a) Map of species turnover expressed as a proportion of beta diversity (Jaccard index of dissim-
ilarity). Species turnover was calculated by resampling 11 cells of 10,000 km2 in each 250,000 km2
square 1000 times. (b) Plot of intraregional species turnover versus latitude. White squares and
whiskers show means and standard deviations, respectively (From Zagmajster et al. (2014))
opposed to the nestedness-resultant component of beta diversity, which represents

differences in species composition caused by species losses or gains (Baselga 2012).
Zagmajster et al. (2014) showed that the mean intraregional beta diversity was as high
as 0.95 0.019 (n ¼ 19 squares), and it was almost entirely due to species replace-
ment (mean species turnover component ¼ 94.6% 4) (Fig. 9.7a). Moreover, the
proportion of beta diversity explained by species replacement was found to decrease
with increasing latitude (Fig. 9.7b).
9.5 Subterranean Biodiversity Patterns at Regional Scales
There are some general objectives that can be pursued based on existing studies of
regional patterns in subterranean biodiversity. The first is to determine the overall
number of species in a region. It is undisputable that there are not enough human and
financial resources to sample in a reasonable time frame any large region to properly
delineate the distribution range of all species. Therefore, different approaches have
been employed to estimate the size of regional species pools from known local
communities, and they have been extensively used in subterranean studies. The next
interesting objective is to explore the differences and similarities between different
regions—and the contribution of regional diversity to overall species richness.
Within regions, spatial patterns of species richness are not uniform—aggregations
of species in certain parts can be recognized.
9.5.1 Regional Species Accumulation Curves Rarely Reach

Saturation
Over the last decade, considerable effort has been made to identify the most efficient
strategy for assessing the size of the regional species pool (Dole-Olivier et al. 2009).
An efficient strategy must provide an unbiased estimate of the number of species
present in a region while minimizing the sampling effort. Subterranean communities
are characterized by an overrepresentation of rare species, which makes it difficult to
obtain an unbiased estimate of the regional species pool. Extensive sampling carried
out as part of the project PASCALIS in six European regions of less than 10,000 km2
in size showed that the proportion of rare groundwater species (i.e., species occurring
in less than 3% of the 192 sites sampled in each region) varied from 40 to 77%
depending on the region (Dole-Olivier et al. 2009). Terrestrial subterranean commu-
nities also support large proportions of rare species. For example, single-site species
represented 45% of all terrestrial troglobionts in the Eastern United States (Christman
et al. 2005); almost 70% of all terrestrial troglobionts in Tennessee are known from
five or fewer caves (Niemiller and Zigler 2013). It is therefore not surprising that
despite extensive sampling of subterranean fauna carried out in a number of regions in
Europe and North America, species accumulation curves began to level off but never
reached an asymptote (Fig. 9.8) (Culver et al. 2006; Castellarini et al. 2007;
Zagmajster et al. 2008; Dole-Olivier et al. 2009, 2015; Stoch and Galassi 2010).
There are various reasons why species accumulation curves are unlikely to
saturate at the regional level. When the same data on distribution of subterranean
beetles in the Dinaric region in the Balkans were associated with grids of different
sizes, species accumulation curves started to level off only for the grids of the
smallest size, 5 5 km (Zagmajster et al. 2008). The size of the unit at which
species accumulation curves can reach an asymptote depends on the dispersal
capacity of organism studied. Generally, stygobiotic species have higher dispersal
capacity than troglobionts (Lamoreux 2004). In Tennessee, species accumulation
curves for terrestrial troglobionts were rising, while the curves for stygobionts
reached an asymptote (Niemiller and Zigler 2013). Dole-Olivier et al. (2009) showed
that the efficiency of sampling design for estimating the number of species in any
region could also be improved by accounting for different sources of environmental
heterogeneity such as catchment boundaries, aquifer types (i.e., karst versus porous
aquifers), and habitat types within aquifers. However, it is not possible to provide a
universal sampling design that is efficient in all regions because the most relevant
sources of heterogeneity to be considered are region-specific.
Fig. 9.8 Regional species accumulation curves for (a) groundwater species in six European regions
(from Dole-Olivier et al. 2009), using sites as units; (b) terrestrial cave species in seven regions in
Europe and the United States (from Culver et al. 2006), using 100 km2 hexagons as units; and (c)
subterranean beetles in Dinarides in the Balkans, using grid cells of different sizes (from Zagmajster
et al. 2008)
a b
100
b regions
b catchments
Total species richness (%)
80 b aquifers 100
Total species richness (%)

a aquifers
80
60
60
40
40
20 20
0
0
β regions β caves α caves
a
ra
ia
l
ta
ni
illo
br
Ju
To
lo
ta
ss
al
an
ou
W
C
R
Fig. 9.9 Additive partitioning of groundwater crustacean diversity in (a) four European regions
(from Malard et al. 2009) and (b) the eastern Alpine area, Italy (from Stoch and Galassi 2010);
α ¼ alpha diversity, β ¼ beta diversity
9.5.2 The Disproportionally High Contribution of Regional

Diversity to Overall Richness
Total diversity at any spatial scale can be additively partitioned into a within-sample
component (alpha diversity, α) and a between-sample component (beta diversity, β)
(Lande 1996). Additive partitioning can be extended across multiple hierarchical
spatial levels to determine the contribution of each level to total richness (Crist et al.
2003). Additive partitioning was applied by Malard et al. (2009) to analyze hierar-
chical patterns of groundwater crustacean diversity in four European regions and by
Stoch and Galassi (2010) to analyze the hierarchical patterns of groundwater crus-
taceans in the eastern Alpine area, Italy. In both datasets, the contribution of each
spatial level to total species richness increased as its size increased (Fig. 9.9):
βregions > βcatchments > βaquifers > αaquifers, for the four European regions and
βregions > βcaves > αcaves for the eastern Alpine area. However, beta diversity between
regions made by far the highest contribution to total richness in both datasets (i.e.,
71.8% for the four European regions and 75.2% for the eastern Alpine area). The
disproportionally high contribution of beta diversity between regions to total rich-
ness implies that differences in community composition between similar habitats in
different regions are typically more pronounced than between different habitats (i.e.,
karst and porous habitats) of the same region. From a conservation perspective, it
implies that a high proportion of groundwater species in Europe can be protected by
focusing conservation efforts on a few species-rich aquifers distributed in distinct
regions (Michel et al. 2009).
Fig. 9.10 Species richness of subterranean beetles in the Western Balkans, using grid cells of
20 20 km (From Bregović and Zagmajster (2016))
9.5.3 Hotspots Within Hotspots
When species are mapped onto a grid within regions, the pattern is not uniform—there
are hotspots of species richness, as well as areas with low species richness. Even
within generally high diverse region, areas of low species diversity can be recog-
nized—as well as peaks in subterranean biodiversity (Fig. 9.10).
However, the number of species accounted for by the hotspots within any region
represents a small proportion of the regional species pool. In the updated dataset on
subterranean beetles of the Western Balkans, the highest number of species per
20 20 km cell reached only 5% of all species known in the study region (Bregović
and Zagmajster 2016). The contribution of the species richness within hotspots in
Tennessee was somewhat higher, 15–20% to the overall species richness of
troglobionts (Niemiller and Zigler 2013).
Factors Energy Heterogeneity History
Evolutionary processes Speciation Extinction Dispersal
Biodiversity patterns Species

richness
Fig. 9.11 Relationships between environmental factors, evolutionary processes, and resulting
patterns of species richness
9.6 Explaining Patterns of Subterranean Biodiversity
A major goal in subterranean ecology is to understand the determinants of species

richness at a range of spatial scales (herein, from global to regional scales). The
recent availability of different environmental variables has increased immensely in
recent decade, enabling complex modeling using statistical tools.
9.6.1 Environmental Factors: Energy, Spatial Heterogeneity,

and History
The number of species in a region ultimately depends on three evolutionary pro-

cesses—speciation, extinction, and dispersal (Wiens 2011; Fig. 9.11). Yet, a multi-
tude of environmental factors act upon each of these three evolutionary processes.
For the sake of clarity, environmental factors have been grouped into three broad
classes of environmental factors: energy, spatial heterogeneity, and history (Field
et al. 2009; Tisseuil et al. 2013; Fig. 9.11). This broad classification echoes the main
hypotheses that were proposed in the macroecological literature to explain spatial
variation of species richness at global to regional scales.
The energy hypothesis proposes that climate controls species richness by acting
directly on the physiology of organisms (e.g., via temperature) or by acting indirectly
on the amount of food resources available to them (e.g., via temperature and water
availability) (Brown et al. 2004; Evans et al. 2005). Under the heading “energy”
(Fig. 9.11), actual evapotranspiration (AET), temperature, and precipitation are often
used in studies of surface ecosystems as surrogates of productive energy, ambient

energy, and water availability, respectively (Evans et al. 2005).
Heterogeneity across space or time is associated with a number of hypotheses
(Brown 1995; Bonn et al. 2004; Ohlemüller et al. 2008) which propose that
environmental heterogeneity can either increase speciation through increased spe-
cialization, decrease dispersal by restricting the movement of individuals across
space, or decrease extinction by promoting local survival. For subterranean taxa,
number of caves, surface area of aquifers, surface area of karstic aquifers, and the
karst connectivity index were used as surrogates for area (Christman and Culver
2001; Zagmajster et al. 2014; Bregović and Zagmajster 2016). Subterranean habitat
diversity was calculated by Eme et al. (2015) at the scale of Europe using the areal
proportion of 12 groundwater habitat types as defined in the groundwater habitat
map of Europe (Cornu et al. 2013).
History refers to the long-lasting effect of past climatic and paleogeographic
events on present-day pattern of species richness. A popular hypothesis states that
species extinction has been more severe in regions that experienced large Quaternary
climatic oscillations (Dynesius and Jansson 2000; Leprieur et al. 2011). However,
the effect of history is intrinsically linked to dispersal constraints. In species showing
low dispersal ability, the effect of extinction on present-day species richness is less
likely to be compensated by subsequent dispersal phases (Foulquier et al. 2008; Hof
et al. 2008). Under the heading “history” (Fig. 9.11), a commonly used surrogate of
historical climatic variability is temperature anomaly, defined as the difference in
mean annual temperature between the present and last glacial maximum. In regional
studies of groundwater species richness, the distance from historical sea margins
(e.g., late Cretaceous) is also used as a factor because present-day species richness
may also reflect past colonization events of groundwater by marine species ancestors
(Boutin and Coineau 2000; Culver et al. 2003). Speleogenesis, the process in which
a cave was formed, was used as an explanatory variable in study of species richness
of caves in the Iberian Peninsula (Jiménez-Valverde et al. 2017).
9.6.2 Multi-causality and Spatial Non-stationarity
The concept of multi-causality acknowledges that geographic variation in species

richness is more likely shaped by multiple factors (Fig. 9.12). Eme et al. (2015)
showed that productive energy, spatial heterogeneity, and historical climate vari-
ability were equally important in explaining geographic variation of groundwater
crustacean species richness in Europe when the importance of each factor was tested
separately (i.e., single-hypothesis testing). However, when the three factors were
analyzed simultaneously (i.e., multiple-hypothesis testing), historical climate vari-
ability was only marginally important because its influence became hidden in the
variation shared with productive energy and spatial heterogeneity (Eme et al. 2015).
The concept of spatial non-stationarity acknowledges that the relative contribu-
tion of factors may vary among regions (Fig. 9.12). Geographical variation in the
Fig. 9.12 Biodiversity patterns are shaped by multiple environmental factors (i.e., multi-causality),
merged here into three broad classes of factors, but the relative influences of factors on biodiversity
patterns vary among regions (i.e., spatial non-stationarity). The three boxes in the panel indicate that
concepts of multi-causality and spatial non-stationarity can be applied at different scales
relative contribution of factors can be analyzed using local regressions models

(Gouveia et al. 2013; Eme et al. 2015). And last, multi-causality and spatial
non-stationarity in the determinants of species richness can be analyzed across a
series of nested spatial scales.
9.6.3 The Importance of Historical Climate Variability
Historical climate variability has long been viewed by subterranean biogeographers

as a primary factor driving both the pattern of species richness and the pattern of
species range in the Palearctic (Hof et al. 2008; Stoch and Galassi 2010). Indeed,
until the 2000s, the disproportionate extinction of narrow range species at higher
latitudes during cold Pleistocene climate was put forward to explain both the pattern
of decreasing species richness and the pattern of increasing range size with increas-
ing latitude. Recent data and analyses by Culver et al. (2006), Zagmajster et al.
(2014), and Eme et al. (2015) provided a broader perspective of subterranean
biodiversity patterns. Although the latitudinal increase in range size north of the
ridge of high species richness undoubtedly reflects the long-lasting effect of cold
Pleistocene climate (Zagmajster et al. 2014), historical climate variability alone is a
poor predictor of geographic variation in species richness (Eme et al. 2015, 2017). In
Northern Europe, the long-lasting effect of cold Pleistocene climates adds up to the
a LC (0.4%) b
PE
9% HH LC
6% LC:PE
LC:HH 28.2% 28.8%
PE:HH Unexplained
16%
48% SC (0.2%)
5.7%
Unexplained
HH
LC:SC 36.7%
21%
SC:HH
LC:SC:HH
LC:PE:HH LC:HH
Fig. 9.13 Multi-causality in the determinants of species richness (a, data from Eme et al. 2015) and
range size (b, data from Zagmajster et al. 2014) of groundwater crustaceans in Europe. Pie charts
show the proportion of variance attributed to different environmental factors in the explanation of
geographic variation of species richness and range size of groundwater crustaceans in Europe; LC
long-term climate variability, PE productive energy, HH habitat heterogeneity, SC short-term
climate variability. Colons denote shared variance between factors
effects of reduced spatial heterogeneity and lower productive energy to limit the
number of species. In Southwestern Europe, the increasing aridity since the last
glacial maximum potentially adds up to the lower productive energy to limit the
number of species. Species richness peaks in regions of mid-latitude where the
combined beneficial effects of a high productive energy and high habitat heteroge-
neity have not been counteracted by cold or arid historical events.
A study by Zagmajster et al. (2014) testing the contribution of the three broad
classes of environmental factors to the range size pattern of European groundwater
crustaceans found that historical climate variability, more especially the long-term
variability of temperature rather than precipitation, had the highest independent
contribution (i.e., 28.8%) to geographic variation in range size (Fig. 9.13). Historical
climate variability also shared a substantial amount of variance with habitat hetero-
geneity (i.e., 36.7%), which by itself explained a relatively small proportion of
variation in range size (i.e., 5.7%; Fig. 9.13). It has proven very difficult to tease
apart the relative importance of temperature seasonality and the amplitude of long-
term climate oscillations in shaping patterns of range size in other taxa, because the
two factors covary in most environments (Veter et al. 2013). The study by
Zagmajster et al. (2014) has shown that groundwater organisms offer very useful
systems to resolve this issue because they lack strong thermal seasonality. Ground-
water and its fauna is one of the systems where historical climate can be decoupled
from present seasonality and its importance in forming large-scale range size
patterns can be evaluated.
9.6.4 Productive Energy as a Driver of Species Richness

at Large Scales
Low food supply has long been regarded as a major factor constraining subterranean
life (Gibert and Deharveng 2002). The role of productive energy in controlling
subterranean species richness has long been restricted to studies conducted at local
spatial scales (Strayer et al. 1997; Datry et al. 2005; Foulquier et al. 2011). Culver
et al. (2006) were the first to suggest that continental patterns of subterranean species
richness might be driven by geographic variation in productive energy. They
hypothesized that the mid-latitude ridge of high species richness for the terrestrial
cave fauna in Europe and the United States (see Fig. 9.4) might correspond to
regions where density of caves (habitat availability) was high and where productivity
remained high over recent geological time. The productivity hypothesis was corrob-
orated by Eme et al. (2015) for the groundwater crustacean fauna of Europe (see
Fig. 9.5). They found that energy, herein AET, explained spatial variation in species
richness better than spatial heterogeneity and history (Fig. 9.13).
Considering the inferred importance of productive energy at continental scale, the
reason why tropical regions of high productive energy contain fewer groundwater
crustacean species remains unclear. One hypothesis may be that a high food supply
to groundwater may restrict the acquisition of troglobiotic traits and hence the
diversification of obligate groundwater lineages arising from the colonization of
groundwater by surface ancestors. On the other hand, cave fish tend to show the
opposite pattern to groundwater invertebrate fauna, as they seem to be most diverse
in tropical regions (Culver and Pipan 2009). As there are many other past and present
factors that can influence diversification processes, this question remains open.
9.6.5 Explanations Vary Among Regions
Spatial non-stationarity in the importance of environmental factors was shown in the

aforementioned continental study of Eme et al. (2015). These authors studied the
contribution of several environmental factors to spatial variation in species richness at
regional scale (regional meaning 10,000 km2 squares), and regional differences in the
relative contributions of factors became apparent. Notable differences in factors
explaining species distributions among regions in Europe also differed (Gibert et al.
2009). In a study of factors that drive terrestrial cave biodiversity pattern in Dinarides
in the Western Balkans, productive energy had very little effect on the observed
species richness pattern of cave beetles within the region (Bregović and Zagmajster
2016) (Fig. 9.14). The highest proportion of variance was explained by habitat
heterogeneity. Even though Dinaric region compared to other parts of the continent
is the area with high surface productivity (Zagmajster et al. 2014; Eme et al. 2015),
low variation in productive energy within the region contributed little to explaining
the biodiversity pattern within the region (Bregović and Zagmajster 2016). In a study
Fig. 9.14 Variance PE HH

partitioning in nonspatial
model of subterranean
beetles in the Balkans; PE
productive energy, HH
habitat heterogeneity, HCS 0.095 –0.058 0.489
historical climate stability
(From Bregović and
Zagmajster (2016))
–0.046
0.130 0.186
0.204
HCS
in Portugal, comparing individual caves, the presence of organic material in the cave
and the origin of the cave were the most important variables in explaining terrestrial
species richness (troglobionts and troglophiles combined) among caves (Jiménez-
Valverde et al. 2017).
9.7 Subterranean Macroecology in the Molecular Era
Molecular methods have become a widely used tool also in subterranean biology, in
studies of evolution and distribution of various taxa (see also Chap. 13). Due to high
levels of convergence in many morphological characters, molecular approaches have
turned out to be crucial in revealing hidden (cryptic) biodiversity. Detection of
species-specific molecular sequences represents the base for studies of species
distribution via environmental DNA approaches that are starting to be used in
subterranean studies. Incorporating phylogenetic information represents a promising
but not yet used approach in studies of evolutionary processes shaping subterranean
biodiversity patterns.
9.7.1 Cryptic Species Do Not Challenge Biodiversity

Determinants
Recent developments in molecular species delineation methods have enabled highly

distinct genetic lineages to be identified within morphologically similar taxa. Those
lineages represent putative species commonly described as “cryptic species”

(Bickford et al. 2007). A review by Trontelj et al. (2009), based on 14 groundwater
species, revealed that small distribution ranges among 51 cryptic species were the
rule, while large range sizes above 200 km were extremely rare. However, a few
phylogeographic studies also revealed that cryptic species have similar distribution
range sizes as their corresponding morphologically described species (Lefébure et al.
2006; Eme et al. 2013, 2014).
Eme et al. (2017) conducted one of the first studies to test the effect of different
species delimitation methods on the richness and range size patterns using data for
groundwater crustaceans in Europe (Niphargidae and Aselloidea). Despite doubling
the number of putative species when shifting from morphological to molecular
methods, the spatial patterns of species richness were qualitatively similar among
delimitation methods. All delimitation methods supported a ridge of high species
richness between 42 and 46 N with a smaller peak between 49 and 51 N
(Fig. 9.15). The number of species delimited by the different molecular methods
increased linearly with the number of morphologically described species along the
latitudinal gradient. These results supported the controversial view that cryptic
species might be evenly distributed across large geographic regions (Pfenninger
and Schwenk 2007; Trontelj and Fišer 2009; Vodă et al. 2015). For range size
pattern, the shift from morphologically to molecularly delimited species led to a
tenfold decrease in the average range size of species. However, the increase in
median range size per cell with increasing latitude (i.e., the Rapoport rule) was
typically more pronounced for molecularly delimited species (Fig. 9.15). The pres-
ence of widely distributed species at northern latitudes confirmed the differential
fragmentation of species ranges between Southern and Northern Europe (Fig. 9.15).
Furthermore, they supported the view that large-scale dispersal is an important driver
of species range size in Northern Europe (Lefébure et al. 2006; Eme et al. 2013).
Interestingly, Eme et al. (2017) found that the relative proportions of variation in
species richness explained by productive energy, historical climate, and spatial
heterogeneity were very stable regardless of the species delimitation methods
used, which is in line with the conclusions of Eme et al. (2015). For range size
pattern, historical climate variability, more especially temperature anomaly,
remained by far the most important predictor (as in Zagmajster et al. 2014).
Although these results suggest that the presence of cryptic species may be evenly
distributed along large geographical regions and environmental gradients, they do
not provide any evidence that the mechanisms driving cryptic diversity are invariant
across space and environmental gradients. The lack of morphological distinguish-
ability between species may be due to recent divergence, niche conservatism, or
morphological convergence (Trontelj et al. 2012; Meleg et al. 2013). These three
mechanisms may be structured along space or environmental gradients in such a way
that they compensate each other to produce the homogeneous distribution of cryptic
species as described by Eme et al. (2017).
Fig. 9.15 Relationships between species richness per latitudinal band and latitude for the morpho-
logically (a) and molecularly delimited (b) species and relationships between median range size
(maximum linear extent) per latitudinal band and latitude for both sets of morphologically (c) and
molecularly delimited (d) species. In all panels, black horizontal bars, red dots, and boxes show the
median, average, and interquartile range, respectively, for 0.9 latitudinal bands. The maximum
length of each whisker is up to 1.5 times the interquartile range. Continuous red lines represent the
fit of a generalized additive model to the averages of latitudinal bands, and dashed red lines show
95% confidence intervals (From Eme et al. (2017))
9.7.2 Environmental DNA Can Speed Up Species Inventory
Detecting environmental DNA (eDNA) is a noninvasive sampling procedure that

relies on the presence of DNA in the environment to detect species’ presence (Ficetola
et al. 2008; Bohmann et al. 2014). It has started to be successfully applied also to
detecting subterranean species. The pioneering use of eDNA for subterranean species
detection was the work of Gorički et al. (2017) on the olm Proteus anguinus, distributed
in caves of Dinaric karst in Southeastern Europe. The eDNA approach proved to be
successful in detection of the presence of two stygobiotic amphipod species

(Stygobromus tenuis potomacus, S. hayi) in shallow subsurface habitats in the vicinity
of Washington, DC (USA; Niemiller et al. 2017). The potential of eDNA analyses in
biodiversity sampling in subterranean habitats is enormous because of the rarity of
many subterranean species and the difficulty in accessing subterranean habitats.
9.7.3 Making Use of Phylogeny to Foster Our Understanding

of Biodiversity Patterns
In continental-scale studies of subterranean biodiversity patterns, the inferences

about the relative role of evolutionary processes remain extremely limited, which
is mostly due to considering all species as equivalent evolutionary and functional
entities. Phylogenetic diversity metrics which integrate the evolutionary relation-
ships among species hold much promise to disentangle the relative influence of
different evolutionary processes (Tucker et al. 2017). Geographic variation of alpha
phylogenetic diversity can provide insight into the relative contribution of speciation
and extinction (see Davies et al. 2007 for further details). Species-rich areas associ-
ated with a low phylogenetic diversity (closely related species) reflect a process of
recent and rapid speciation in situ. These areas are commonly referred to as “cradles
of diversity” (Jablonski et al. 2006). For a given species richness, an increase in
phylogenetic diversity (species with long independent evolutionary histories) sug-
gests a decrease in the extinction rate occurring in regions often qualified as
“museums” because they preserve diversity through time (Jablonski et al. 2006). It
is not yet clear which of the two hypotheses better support the ridge of high species
richness in groundwater crustaceans (Zagmajster et al. 2014; Eme et al. 2015, 2017).
Phylogenetic diversity metrics can also be used to tease apart the role of dispersal
in shaping biodiversity patterns. Some regions—which we refer to as “Noah’s
Arks”—can be characterized by high species richness and high phylogenetic diver-
sity, both because they preserve lineages through time and because they receive
phylogenetically distant migrants from neighboring regions. Recent decomposition
of phylobetadiversity indices offered the possibility to tease apart the effects of
spatial turnover and gradient of phylogenetic diversity caused by differences in
species richness among communities (Baselga 2010, 2012; Leprieur et al. 2012).
For a given species richness, the spatial turnover of phylogenetic diversity is
expected to be higher in a museum than in a Noah’s Ark. Phylobetadiversity indices
may thus be used to test whether the ridge of high subterranean species richness
might also have served as refugia during glacial periods and/or as a source of
colonizers during interglacials.
Developing a phylogenetic framework of subterranean communities, which com-
bines the use of both alpha and beta phylodiversity metrics, can provide new insights
into the links between environmental factors, evolutionary processes, and biodiver-
sity patterns (see Fig. 9.12). The development of georeferenced molecular datasets
over large spatial scale as proposed by Eme et al. (2017) and the ever-increasing
availability of robust phylogenetic trees for a number of subterranean taxa offer an
exciting perspective to apply this macro-phylogenetic framework.
9.8 Conclusions
Sampling difficulties, numerical rarity, and high levels of endemism make the
analysis of geographic patterns of subterranean species especially difficult. Never-
theless, there are now georeferenced datasets with thousands of individual records
from hundreds of sites that make it possible to describe and begin to explain global,
continental, and regional patterns. For all countries and all regions, there remain
many undescribed and undiscovered species, and this makes comparisons difficult.
But, even incomplete datasets can be used for analyses, if critically evaluated and
properly compared. Different statistical procedures can be applied to take into
consideration differences in sampling effort as well as spatial properties of the
different datasets being compared.
There are some general conclusions that can be briefly summarized at each spatial
scale. At the global scale, there is a consistent pattern of highest species richness in
north temperate regions of Europe and North America, as well as Australia. At the
continental scale, the pattern profoundly depends on the size of the grain used for
analysis. Using a coarse grain, like countries or large biogeographical regions,
groundwater diversity in Europe declines with increasing latitude, but a ridge of
high species richness becomes apparent between 42 and 46 N when using a finer
grain. The contribution of beta diversity to total diversity increases with spatial scale,
but beta diversity between regions disproportionally contributes to total diversity.
Patterns within regions are not uniform, but hotspots represent only a small propor-
tion of the regional species pool.
A number of environmental factors, lumped into energy, spatial heterogeneity,
and history, affect species richness. While productivity, spatial heterogeneity, and
severity of the Pleistocene climate are all important, productivity is the most
important at least in explaining the European subterranean ridge of species richness.
However, the spatial non-stationarity of explanations really predominates because
the contribution of different factors appears to vary substantially among regions.
Although severity of the Pleistocene climate alone is a poor predictor of subterranean
species richness pattern at the whole of Europe scale, it exerts a major control on the
pattern on increasing range size with increasing latitude in Europe (i.e., the Rapoport
rule). Once again, scale of analysis is important. For example, the importance of
productivity in shaping patterns of species richness disappears in smaller areas such
as the Balkans. The inclusion of cryptic species appears not to change overall
geographic patterns or their explanations. It does of course greatly increase the
number of species. A more thoroughly phylogenetic approach is likely to produce
greater clarity with regard to the geographic patterns of subterranean biodiversity as
well as for their explanations.
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Chapter 10
Historical and Ecological Factors
Determining Cave Diversity
Ignacio Ribera, Alexandra Cieslak, Arnaud Faille, and Javier Fresneda
10.1 Introductory Background
In this chapter, we do not aim to review the historical views on the origin and
evolution of cave fauna, of which there are several excellent accounts (see,
e.g. Bellés 1987; Culver et al. 1995; Romero 2009; Culver and Pipan 2014), but to
try to understand the origin of some persistent ideas that have traditionally shaped
the study of the subterranean fauna and its diversity and that still have a recognisable
influence. We will mostly refer to terrestrial fauna and mostly to the groups with
which we are most familiar through our own work (Coleoptera Leiodidae and
Carabidae), which are also the ones with the highest diversity in the subterranean
environment. For the evolution of the stygobiontic fauna, see, e.g. Marmonier et al.
(1993), Culver et al. (1995), Danielopol et al. (2000), Lefébure et al. (2006) or
Trontelj et al. (2009).
The origins of most of the current views on the evolution of the subterranean
fauna can be traced back to Emil Racovitza and René Jeannel (e.g. Racovitza 1907;
Jeannel 1926, 1943), which were the first to document extensively and systemati-
cally the diverse fauna of the European caves. They were strongly influenced by the
earlier work of North American biospeleologists (e.g. Packard 1888), but they
reframed their ideas according to the evolutionary views prevalent in the first
decades of the twentieth century. The work of R. Jeannel and other biospeleologists
I. Ribera (*) · A. Cieslak · A. Faille

Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Barcelona, Spain
J. Fresneda
Ca de Massa, Lleida, Spain
Museu de Ciències Naturals (Zoologia), Barcelona, Spain

https://doi.org/10.1007/978-3-319-98852-8_10
230 I. Ribera et al.
had by then firmly established some generalities about the subterranean fauna that
demanded an evolutionary explanation, which could be summarised as follows:
1. Species living in the deep subterranean environment tend to share similar mor-
phological characters, such as reduction or lack of eyes, wings and pigment,
elongated appendages, and in many cases extremely similar body shapes (Jeannel
1943; Vandel 1964).
2. These characteristics (the troglomorphic phenotype, Christiansen 1962, 2012; see
Chap. 4) evolved independently from epigean ancestors in multiple zoological
groups, showing a high degree of convergence (Fig. 10.1).
3. Some zoological groups are more prone than others to colonise the subterranean
environment. In the Northern Hemisphere, the terrestrial fauna in caves is dom-
inated by a few groups, mostly among insects and spiders. Two lineages show a
clear dominance in terms of number of species with troglomorphic characters,
both of them among the Coleoptera: tribe Trechini within the Carabidae (ground
beetles) and, in Europe and the Middle East up to the Caspian Sea, tribe
Leptodirini within the Leiodidae (Jeannel 1911, 1928).
4. Most species with troglomorphic characters have very restricted distributions.
They are often known from a single cave or a group of nearby caves and are likely
to be restricted to their surrounding subterranean system (e.g. Jeannel 1911, 1928;
Barr 1968; Assmann et al. 2010). This is in sharp contrast to many epigean
species of the same groups that often have wider distributions.
The usually restricted ranges of subterranean species would imply that, once
underground, they do not tend to move, at least not outside the area directly
accessible through subterranean fissures or cavities. This means that “islands” of
subterranean habitat, isolated from other such islands by substratum not suitable for
subterranean species, would have an independent subterranean fauna. The difficulty
with this scenario is that in some cases, subterranean species in different units
(“islands”) of subterranean habitat, and thus of supposed independent origin, can
have extremely similar morphologies, forming an apparently well-defined lineage
with very close phylogenetic relationships among species and without any easily
identifiable epigean relative. In these cases, an independent origin would require first
a parallel evolution of their morphology and second the extinction of all epigean
relatives. It is well established that there were many independent colonisations of the
subterranean environment in different groups of organisms, and many of them had
also unquestionably converged to a similar troglomorphic phenotype (e.g. Racovitza
1907; Vandel 1964). As an extension of these two generally accepted facts, it was
assumed that the independent evolution of troglomorphy occurred not only in
distantly related lineages but in virtually all subterranean species. In addition to
this, the increasing knowledge on the history of the effect of the glaciations in
Europe and North America provided a convenient driving force to explain the
extinction of the epigean relatives: with the onset of a glaciation, all species that
took no refuge in caves would have been extirpated from the surface (Peyerimhoff
1906; Jeannel 1943). The joint extinction of all surface ancestors would account for
the lack of easily identifiable close relatives of many troglomorphic species, and
10 Historical and Ecological Factors Determining Cave Diversity 231
Fig. 10.1 Convergence in body shape in species typical of the deep subterranean environment. (a)
Species of suborder Adephaga, from left to right Graptodytes eremitus (Dytiscidae) from the High
Atlas of Morocco, Aphaenops pluto (Carabidae, Trechinae) from the French Pyrenees and
Oscadytes rovirai (Carabidae, Pterostichinae) from the Spanish Pyrenees; (b) species of suborder
Polyphaga, from left to right Laccobius fresnedai (Hydrophilidae) from the Western Mediterranean,
Anthroherpon charon (Leiodidae, Cholevinae, Leptodirini) from Bosnia-Herzegovina and
Hydraena exasperata (Hydraenidae) from the Iberian Peninsula. Note the extreme similarity
between the two middle species despite belonging to different suborders; this similarity cannot be
solely due to the fact that they are both subterranean, as the three represented species of Adephaga
share the subterranean habits (G. eremitus is a stygobiont species, and O. rovirai is found in MSS,
Ribera & Faille 2010; Fresneda et al. 1997). Scale bars, 1 mm; Photos by © Javier Fresneda
given that each colonisation was independent, there was no need to explain how
subterranean species diversify or disperse.
Under this view, troglomorphic species would have originated from ancestors
living in the soil or forest litter, a dark, cold and humid environment in some aspects
similar to that of a cave (see also Chap. 3). These ancestors may already have been
adapted for the subterranean life (in fact, under some criteria the deep layers of litter
or the upper layers of soil may be considered a shallow subterranean environment,
see below), as they are frequently wingless, partially unpigmented and sometimes
even blind. Once underground, species would go through the fixed pathways of
morphological modification due to the effect of the environment and phyletic
evolution. The different morphologies we observe would thus be due to the different
stages in which the species are in this path from epigean to extreme troglomorphy,
which in turn depends on the time elapsed since the colonisation (a morphological
clock; e.g. Poulson 1963; Wilkens 1987). Extremely modified species would be the
most ancient colonisers, including the only survivors of lineages of which all
epigean relatives are long extinct, the “antequaternaires” (i.e. before the Quaternary)
of Racovitza (1907) or the “living fossils” of Darwin (1859, p. 107) or Jeannel
(1943). On the contrary, those with still incomplete modifications (e.g. partly
pigmented or microphthalmic) would be the most recent newcomers.
On a more theoretical ground, to fundament this view of the origin of the
subterranean species, R. Jeannel argued that regressive troglomorphic characters
are not adaptive and appear as a result of the lack of use of the affected organs in the
caves. This view can be traced at least to Lamarck (1809, p. 242), mentioning the
Proteus anguinus, together with the mole and other vertebrates of subterranean
habits, as examples of the reduction or loss of eyes through disuse. In Darwin
(1859, p.138), there is also mention to the loss by disuse, “By the time that an
animal had reached, after numberless generations, the deepest recesses, disuse will
on this view have more or less perfectly obliterated its eyes”. However, Darwin and
others also argued that some characters commonly present in troglomorphic species
were the result of natural selection (e.g. elongation of appendages, increased sensi-
tivity of some sensorial organs). Thus, the second part of the sentence quoted above
reads: “and natural selection will often have effected other changes, such as an
increase in the length of the antenna or palpi, as a compensation for blindness”.
Contrary to Darwin’s view, the role of natural selection was minimised, or plainly
rejected, by early biospeleologists (e.g. Packard 1888), but most prominently by
R. Jeannel and most of the biospeleologists of the early twentieth century. Instead,
they argued that there was a genetic tendency of lineages to follow fixed evolution-
ary paths, going through different phases (origin, expansion, diversification and
senescence, Jeannel 1950; see also Vandel 1964) until the last stage, which is
characterised by a lack of variation causing their ultimate extinction. The number
of these orthogenetic paths is also limited, leading to further convergence onto
certain morphological “syndromes”.
Jeannel developed multiple hypotheses on the geographical and temporal origin
of different subterranean lineages, based on their morphology, their current distri-
bution and the geological knowledge of the time (he was one of the first biogeog-
raphers to consider the then highly unorthodox theories of Wegener on continental
drift; e.g. Jeannel 1942, 1959). There were no fossils or other evidence to support
many of his biogeographic hypotheses, but still many of them have proved to be
surprisingly accurate and have been corroborated by recent studies using molecular
data and an improved knowledge of the geological history of the areas involved
(e.g. Faille et al. 2013; Andújar et al. 2016).
To understand the evolutionary hypotheses developed by R. Jeannel,

E. Racovitza and other early biospeleologists, it is necessary to interpret them in
the context in which they were first formulated. In the early decades of the twentieth
century, there was a general acceptance of the evolution of the species, but the
mechanisms driving this evolution were still highly controversial. Darwinian natural
selection was out of favour, even among some of the founders of modern genetics
and evolutionary theory, such as T.H. Morgan, which were—at least in their earlier
works—strong advocates of the mutationist theories of De Vries and followers,
dismissing the role of adaptation and natural selection in evolution (Bowler 1983).
The observation that eyeless and wingless flies could suddenly appear in a “glass
milk bottle” (the type used for the laboratory cultures of Drosophila) in one
generation led Morgan to deny the role of natural selection in the origination of
new phenotypes (and specially of characters considered to be regressive), which
needed not to have any apparent adaptive value to become established in a popula-
tion (Morgan 1919). Even more relevant for shaping the views of E. Racovitza and
R. Jeannel was the lack of experimental evidence against the inheritance of acquired
characters, which was assumed by default to be one of the prevalent mechanisms of
inheritance even by Darwin himself (Bowler 1983).
It was also known that in some species, subterranean populations showing typical
troglomorphic characters could at least partially reverse to an epigean phenotype,
and in particular to recover pigmentation, when exposed to light for long periods
(e.g. in Proteus, Dubois 1892). Individuals of epigean species in a subterranean
environment could also develop troglomorphic characters, as was known for some
cave fishes (e.g. Packard 1888; see Mazza et al. 2014 for a recent example). The
assumption was then that these modifications could become fixed and be transmitted
to the descendants. Although not in this context, it is noteworthy that the evolution-
ary role of phenotypic plasticity as originator of morphological diversity has been
recently vindicated (e.g. Gómez-Mestre and Jovani 2013).
The definitive ostracism of the neo-Lamarckism did not arrive until the discredit
of Paul Kammerer’s experiments with midwife toads (which are still subject of
controversy, e.g. Vargas 2009). But even then—and until the end of his life (see,
e.g. Jeannel 1950)—R. Jeannel argued that troglomorphic characters were the direct
result of a neo-Lamarckian inheritance. He acknowledged the lack of experimental
evidence but considered that laboratory experiments were maintained for a time too
short to observe any appreciable effect, which should only be visible at much larger
temporal scales (Jeannel 1950) (something that, incidentally, sounds as a rather
reasonable objection). The problem was that the lack of experimental evidence
was accompanied by a lack of any plausible molecular mechanism, which led to
the complete rejection of neo-Lamarckism in the standard view established after the
evolutionary synthesis of the neo-Darwinism.
In the second half of the twentieth century, the evolutionary views of some
European biospeleologists became increasingly marginalised, but perhaps due to
the enormous importance of the taxonomic and biogeographic publications of
R. Jeannel, his unorthodox evolutionary views did not affect the recognition or the
relevance of his work. In any case, and despite the influence of the early
biospeleologists, most studies on the diversity and ecology of cave faunas done in
the last decades are in perfect agreement with contemporary evolutionary ideas,
especially in North America (among others the works of T. C. Barr, K. Christiansen,
D. C. Culver or T. L. Poulson; see References below and Culver and Pipan 2015 for
a review). The discovery of troglomorphic species in tropical caves (Howarth 1972)
introduced a full new field of research and helped to shape the different views on
how cave colonisation could have proceeded. As seen above, the prevalent view at
the time involved strong climatic fluctuations as a driver of colonisation, something
that clearly did not apply to tropical faunas. However, the two proposed modes of
colonisation (Howarth 1972; Peck and Finston 1993; Hampe and Jump 2011) can be
seen as extremes of a continuum in which the different factors (gene flow between
epigean ancestors and subterranean species, ecological differences between the
ancestral and subterranean habitat) merge and combine in each particular circum-
stance. But these differences refer only to the process of colonisation, not to the
subsequent evolution of subterranean lineages, for which there was hardly any solid
evidence available.
In the last years, there have been huge advances in the understanding of the origin
of troglomorphic characters, thanks mostly to the research conducted on some
species with epigean and subterranean populations (e.g. Culver et al. 1995), and in
particular the cave fish Astyanax mexicanus. This is a species with multiple
populations, some of them living in caves and with typical troglomorphic modifica-
tions: fishes living in caves are unpigmented and blind, with different sensorial
capabilities and with some other ecological and physiological modifications (see,
e.g. Jeffery 2008, 2009 or Protas and Jeffery 2012 for reviews). There are different
cave populations with an independent origin showing these modifications, which has
allowed to investigate convergence in some characters (Protas et al. 2006; Bilandzija
et al. 2012; Kowalko et al. 2013).
The focus on species with epigean and cave populations allows the direct
comparison of modified and unmodified phenotypes and the identification of the
precise genetic and physiological origin of troglomorphic characters. It is, however,
necessarily limited to fast-evolving characters (those first evolving after the coloni-
sation) and is silent on the long-term evolution and diversification of the subterra-
nean species and lineages.
10.2 The Crucial Dilemma: No Dispersal but Multiple

Origins, or Single Origin but Geographic Dispersion
and Diversification
Despite the growing understanding of the origin of the morphological characters

typical of the subterranean fauna (Culver and Pipan 2015), until recently there was
still little evidence on the long-term evolution of subterranean species. The dilemma
framed by the early biospeleologists was still unsolved: either subterranean species
cannot disperse, they have independent origins, and their similarity is due to
generalised convergent evolution or there is a single origin of the subterranean
adaptations at least in some cases, with the implication that they can disperse over
non-subterranean habitats and diversify to generate species-rich and widely distrib-
uted lineages with only subterranean species. To distinguish between the two
possibilities, it would be necessary to know the detailed phylogeny of the lineages
in which subterranean species occur and to have a time estimate of their evolution. In
most cases this was not possible. There is little—if any—fossil data that could be of
any use, and the extensive occurrence of phenotypic convergence makes difficult the
estimation of phylogenetic relationships based on morphological data only (Wiens
et al. 2003; Fresneda et al. 2007).
The increasingly widespread use of molecular data in the last years allowed
addressing both issues for a number of subterranean lineages. Molecular data
provide a set of characters to reconstruct robust evolutionary relationships between
species (phylogenies), which are in principle neutral and thus not affected by the
strong convergence that preclude the use of morphological characters to reconstruct
relationships between subterranean species. The use of molecular clock techniques
allows establishing a broad temporal framework for the reconstructed phylogenies,
even with the absence of fossil data and despite unavoidable uncertainties (Kumar
2005; Ho and Duchêne 2014).
Molecular data have been used to find the phylogenetic position of isolated and
highly modified cave species, without apparent relatives in the geographic area in
which they are found. These species are sometimes referred to as “living fossils” (see
Sect. 10.1), although this term may be more appropriate to species (or genera) that
have been first described as fossils to be subsequently discovered alive and well,
such as the coelacanths (genus Latimeria) or the Mallorca midwife toad (Alytes
muletensis; Alcover and Mayol 1980) (also referred as “Lazarus taxa”). In the case of
subterranean species, there is usually no fossil record, and the term “living fossils”
refers to the fact that they are presumably relicts, the only survivors of an ancient
fauna that became extinct on the surface and of which only some isolated examples
remain, thanks to the protection offered by the caves and the surrounding subterra-
nean environment (Assmann et al. 2010). Some highly modified subterranean
species do seem to be highly isolated phylogenetically, with the only known distant
relatives living in remote geographical areas. This is the case, for example, of Dalyat
mirabilis, the only Palaearctic Promecognathinae (Coleoptera Carabidae), which
otherwise have some species living in the forests of western North America and
South Africa (although with a Cretaceous fossil in Central Africa; Ribera et al.
2005), or Ildobates neboti, a Zuphiini (also a Carabidae), which closest relatives are
unknown (Ribera et al. 2006). Other cases of likely isolated highly troglomorphic
species (or a small group of closely related taxa) within Trechini are the genera
Paraphaenops in the Iberian System, Sardaphaenops in Sardinia or Jeannelius in
the Caucasus (Faille et al. 2013; see Assmann et al. 2010 for examples in other
groups). However, it should be stressed that the actual age and origin of the
colonisation of the subterranean environment of these species are unknown, so it
may well be that it happened only recently and that the role of the subterranean
environment for the survival (or extinction) of their respective lineages was abso-
lutely irrelevant.
Of more interest for the understanding of the evolution of the subterranean fauna
are the studies of the phylogenetic relationships and the diversification of large
clades with abundant subterranean species. There have been a number of works on
stygobiontic groups, most of them Crustaceans (e.g. Zakšek et al. 2007; Fišer et al.
2008; Bauzà-Ribot et al. 2012; Botello et al. 2012; Meleg et al. 2013; Esmaeili-
Rineh et al. 2015) but also diving beetles (Dytiscidae, e.g. Leijs et al. 2012).
Terrestrial groups include mostly spiders (e.g. Arnedo et al. 2007; Mammola et al.
2015) and beetles, which, as already noted, will be treated in more detail and are the
main subject of this chapter.
10.3 Once or Many? Single Versus Multiple Origin

of the Subterranean Colonisation
When trying to answer this question, there is a consideration to be made which,

despite its formal appearance, is of crucial importance. Imagine we have a group of
species which are presumably each other closest relatives and of which some are
subterranean and some are epigean, and we want to test if there are single or multiple
origins of the subterranean way of life. When our initial hypothesis—i.e. the null
hypothesis that has to be falsified—is that there is a single origin of the subterranean
species, to falsify, it we only need to demonstrate that one of the epigean species is
the closest relative of any of the subterranean ones, as this would require at least two
origins and possibly more. On the contrary, when the null hypothesis is that all
subterranean species have colonised the subterranean environment independently
but that their epigean closest relatives are all extinct, there is no simple observation
that allows us to reject it and accept the alternative hypothesis of a single origin.
Even when all subterranean species are found to be monophyletic and phylogenet-
ically nested within the epigean ones, there is always the possibility that the epigean
relatives that should have appeared interspersed among them had become extinct.
Only additional evidence could make this hypothesis increasingly unlikely, but not
falsify it unquestionably.
In this sense, it may be considered that, when hold to the extreme, the hypothesis
of multiple origins with the corresponding extinction of epigean relatives is unfal-
sifiable. Fortunately, in practice this rarely happens, as in some cases, the additional
evidence is strong enough to make the multiple origins extremely unlikely.
Box 10.1
We assume that the morphological modifications leading to troglomorphism
are in general irreversible, in particular the loss of eyes, wings or pigmentation.
Note that we refer to lineages with subterranean species with fixed
troglomorphic characters, without polymorphisms or known phenotypic plas-
ticity. It is known that in some species it is possible to recover pigmentation
(e.g. the already noted example of Proteus) and, at least under laboratory
conditions, it is even possible to partially recover some functionality of the
eyes (Porter and Crandall 2003; Niven 2008). But, to our knowledge, there is
no evidence of the reversal of troglomorphic characters implying the recovery
of completely lost complex structures. There have been some claims of
recovery of wings in otherwise wingless lineages (Whiting et al. 2003), but
these are controversial (e.g. Stone and French 2003; Collin and Miglietta
2008; Goldberg and Igic 2008) and in any case do not refer to subterranean
species, in which there is always an association of multiple modified charac-
ters. We also refer to morphological modifications, not to changes in the
habitat or the way of life: it is known that within subterranean lineages,
some species may become secondarily epigean, but to our knowledge, this
never implies the reversal of their troglomorphic characters.
Starting with the simplest case and assuming the irreversibility of the
troglomorphic characters (see Box 10.1), there is now strong evidence of the
multiple independent origin of the subterranean way of life within different lineages
of Coleoptera and especially in Trechini. This was the case of the genus Duvalius
(Faille et al. 2013), the species of the Trechus fulvus group (Faille et al. 2014) or the
Cantabrian species of Trechus (Faille et al. 2012; Fresneda et al. 2015). In all these
examples, subterranean species with different degrees of morphological modifica-
tion (or in some cases small clades of closely related species) are sisters to epigean
species, demonstrating their independent colonisation of the subterranean environ-
ment. Trechus fulvus is a good model to study the process of colonisation of the
subterranean environment, as it is widely distributed in Western Europe and North
Africa, with many isolated populations under different ecological conditions,
although always requiring high humidity. The existence of multiple replicates of
isolated populations leads to morphological variability, with some populations
having only apterous individuals with more or less reduced eyes, whereas other
populations include only macropterous individuals (Jeannel 1920). The evolution of
the Cantabrian lineage of Trechus within the Iberian Peninsula seems to follow a
similar pattern, with T. escalerae, T. valenzuelai and a clade including some species
of Apoduvalius having independently developed some troglomorphic characters
(Faille et al. 2013; Fresneda et al. 2015).
Contrary to the examples above, there is evidence for the single origin of the
colonisation of the subterranean environment—and of the origin of the associated
modifications—in a number of diverse lineages both within Trechini and
Leptodirini. Within Trechini, the largest and most unexpected (so far) clade of
subterranean species with troglomorphic characters is the Pyrenean lineage (Faille
et al. 2010), with all 87 species of the 2 genera Aphaenops (including subgenus
Hydraphaenops) and Geotrechus, the latter previously considered to belong to a
distinct lineage (Jeannel 1919). In addition to a strong support of the monophyly of
the Pyrenean subterranean lineage including most of the species of those genera in
all phylogenetic analyses (Faille et al. 2010, 2011, 2013), there is also considerable
additional evidence confirming the single origin of the colonisation of the subterra-
nean environment and the relatively simple evolution of the troglomorphic charac-
ters. Thus, the topologies observed might suggest a gradation in the specialisation of
the species inside the karstic massif, associated with morphological and ecological
changes and modification of the life cycle (Faille and Pluot-Sigwalt 2015, Faille
et al. manuscript in preparation; Fig. 10.2). There are other instances in various
geographic areas in which an unexpected clade of subterranean species of Trechini
turned out to be monophyletic, such as the Dinaro-Alpine hypogean clade gathering
all the highly troglomorphic cave Trechine of the Eastern Alps and Dinarids (Faille
et al. 2013).
Within Leptodirini there is also strong evidence to support the single origin of the
subterranean colonisation, and the troglomorphic characters, of many of the geo-
graphically localised radiations (Ribera et al. 2010; Fresneda et al. 2011; Cieslak
et al. 2014). In addition to the strong support of the monophyly of the exclusively
subterranean lineages in the molecular phylogenies, there is additional support from
morphological and physiological characters. Thus, in the Pyrenean lineage (the
Speonomus clade, Ribera et al. 2010), including 141 described species, the evolution
of the life cycle from the ancestral 3 instars to a reduced larval development with
only 2 instars and finally a larvae that does not mould, is not active and does not feed
follows a simple path with a single transition from 3 to 2 instars at the end of the
Oligocene, and from 2 to 1 in the early Miocene (Fig. 10.2). To explain the
monophyly of all known species with one instar cycle from the Pyrenees, we
could always argue that from an ancestor with three instars, there were multiple
independent origins of the modifications of the life cycle to produce species with one
instar, and what we observe is just the result of the extinction of all the species living
in the surface that would be interspersed within the clade. However, in addition to
the extinction of all epigean species, this would require that, depending on their
phylogenetic placement, subterranean species independently (but in coordination)
either did not modify their cycle, evolved to a two-instar cycle or to a one-instar
cycle. This would require the widespread occurrence of orthogenesis in its most
extreme version. The evolution of the life cycle followed similar simple patterns in
other geographic areas, although data are scarcer than for the Pyrenees and the
patterns were less firmly established (Cieslak et al. 2014).
Speonomus pyreneus gr
A Speonomus zophosinus gr
B
Paraspeonomus
Speonomus carrerei
Stygiophyes+ increased diversification?
Lagariella 1 ovariole
Naspunius
Salgadoia
o Cerbaphaenops
increased
diversification Trapezodirus
o Troglocharinus
Hydraphaenops ehlersi
Speonomites
1 instar Pallaresiella 2 ovarioles

Trocharanis
o o
Geotrechus gallicus
Paratroglophyes
Antrocharis
1 instar?
o
Gesciella 2 ovarioles?
Geotrechus orpheus
o
Troglophyes increased diversification?
Perriniella 1 ovariole
Ceretophyes o Aphaenops s.str.
Phacomorphus+
Bathysciella
2 instars Baronniesia
Bellesia Trechus ovatus
o Machaeroscelis
5 ovarioles
Speonomus speluncarum gr
Parvospeonomus Trechus partim

3 instars
Pseudospeonomus
Bathysciola partim
Typhlotrechus
Josettekia
Euryspeonomus beruetei
Nafarroa
Euryspeonomus partim
Speocharidius
Aranzadiella
Bathysciola partim
Ma
30.0 25.0 20.0 15.0 10.0 5.0 0.0

Oligocene Miocene Pliocene Pleisto.
Fig. 10.2 Schematic phylogeny of the Pyrenean clades of (a) Leptodirini and (b) Trechini,
showing the reconstructed transitions from the ancestral life cycle (3 instar larvae in Leptodirini,
5 ovarioles in Trechini) to an intermediate cycle (2 instars in Leptodirini, 2 ovarioles in Trechini)
and a highly modified cycle (1 instar Leptodirini, 1 ovariole in Trechini), in which the larvae are not
active and do not feed. In the Leptodirini this transition was estimated to have occurred only once,
associated with an increased diversification rate, but in Trechini it likely occurred twice, probably
also associated with shifts in diversification rates. In bold, clades for which actual data of the life
cycle is known. Modified from Cieslak et al. (2014) and Faille and Pluot-Sigwalt (2015)
10.4 Relation Between Age and Troglomorphism
The single origin of the colonisation of the subterranean environment in large,

diverse lineages questions the relationship between the age of a species and its
degree of troglomorphism, i.e. the extent of the morphological and physiological
modifications from the ancestral phenotype. When the assumption is that virtually all
subterranean species had an independent origin from epigean ancestors, it follows
naturally that the more modified the species, the more ancient the colonisation of the
subterranean environment (e.g. Jeannel 1950; Poulson 1963). However, in a mono-
phyletic lineage in which all species share a single ancestor with subterranean habits
(i.e. a single colonisation of the subterranean environment), the relationship between
the degree of troglomorphism and time underground is meaningless, as for all
species of the lineage this time would be the same. The age of the extant species
may differ, but this is irrelevant, as the important factor is how much a species differs
from its direct ancestor, that is, the rates of phenotypic evolution. These rates may
depend on the environment or way of life of a species and their ancestors, and thus
the relationship of interest is not between phenotype and age but between phenotype
and habitat or ecology. Old species may be less troglomorphic than younger ones if
they have slower rates of phenotypic evolution but also if the ancestor of the younger
one was already a troglomorphic species or if they have a different ecology.
There may still be continuous trends in the evolution of troglomorphism, but not
in individual species (or closely related species) but in whole lineages. Thus, as seen
above (Fig. 10.2), among Pyrenean Leptodirini, there was a single evolutionary path
from forest litter species with three-instar larvae to subterranean species with a
two-instar cycle and finally species with a highly modified, one-instar cycle (Cieslak
et al. 2014). Among Pyrenean subterranean Trechini, there were two independent
developments of the same type of contracted life cycle, with two evolutionary paths
starting with endogean species of the genus Geotrechus (Faille and Pluot-Sigwalt
2015; Fig. 10.2). But extant species of the unmodified lineages can be as old, or
older, than any of the highly troglomorphic ones. In fact, in these cases it can be
argued that the older phenotypes are the less troglomorphic ones, which have
remained closer to the ancestral type, while the more derived—and thus more
recent—phenotypes are those of the highly troglomorphic species. The importance
of considering the evolution of a lineage and not only of the individual species is also
clear in the case of recently diverged species which still are very similar to each other
(and likely to their most recent common ancestor). Thus, the highly modified and
isolated genus Sardaphaenops has two extant species (and one subspecies) of very
recent origin, as are many of the species of the highly diverse and not very modified
genus Duvalius (Casale 2004; Faille et al. 2013). In both cases, what is of interest is
not the age and morphology of the extant species, which in both cases are of recent
divergence and likely very similar to their direct ancestors, but those of the whole
lineage.
10.5 Dispersal and Diversification
It is generally accepted that subterranean species can disperse to some degree

through the network of fissures and cavities within a karst massif (e.g. Barr and
Holsinger 1985), and there have been attempts to quantify the movement of subter-
ranean species through mark-recapture methods (Cabidoche 1966; Delay 1975;
Fejér and Moldovan 2013) or to estimate gene flow (e.g. Crouau-Roy 1989). The
generally accepted single origin of groups of closely related species in close geo-
graphical proximity also implied the possibility of some dispersal with subsequent
isolation. What is usually questioned is the possibility of range expansions at large
geographical scales, implying displacements over areas without subterranean
environment.
However, if we accept that lineages that occupy large geographic areas—such as

the Pyrenees—are monophyletic and have colonised the subterranean environment
only once, it necessarily follows that species with subterranean habits and some
degree of troglomorphism were able to expand their ranges and colonised new
isolated subterranean units by epigean dispersal.
Some examples are species of Anophthalmus (Faille et al. 2013), different
populations of Aphaenops cerberus (Faille et al. 2015) or, at a larger geographic
scale, the genus Troglocharinus. This genus belongs to the Pyrenean lineage of
Leptodirini with a highly modified 1-instar life cycle (Cieslak et al. 2014). It was
estimated to have an origin in the late Miocene, derived from a lineage in which all
species have troglomorphic characters and estimated to have colonised the subter-
ranean environment in the late Oligocene (Cieslak et al. 2014). Troglocharinus has
some 20 species, mostly distributed in the pre-Pyrenees, but there is a monophyletic
group of species living in the coastal mountain ranges of Barcelona and Tarragona
(Rizzo et al. 2013). The linear distance between the closest localities of the Pyrenean
and coastal clades is not exceedingly large (some 60 km), but they are separated by
Miocene sediments of the upper Ebro basin without any trace of subterranean
environment (Fig. 10.3). The only possibility is thus that the ancestor of the coastal
lineage expanded its range from the Pyrenees, necessarily crossing the upper reach
of several rivers and some non-karstified areas. During the estimated time for this
range expansion, the end of the Miocene-early Pliocene (Rizzo et al. 2013), tem-
peratures were decreasing, but precipitation remained high—higher than today—and
seasonality was low, as the Mediterranean climate, with its high seasonality, would
not become established until ca. 2.3 Ma (Suc 1984). It seems likely that most of
Catalonia was at that time covered by a dense forest, with a high humidity and no
temperature extremes. Physiological experiments have demonstrated that coastal
species of Troglocharinus can tolerate temperatures up to 20 C for long periods
of time, provided that humidity is maintained close to saturation (Rizzo et al. 2015).
There are some records of the presence of specimens of Troglocharinus—and some
other highly troglomorphic species—closer to the entrance of caves or directly
outside (Rizzo et al. 2013), and the possibility of finding troglomorphic species
typical of the deep subterranean environment close to the surface, under stones or in
the MSS has been always recognised. For example, Packard (1888), quoting Bedel,
states that “It appears that not all the species [of Leptodirini] live in caves, but occur
in the open air under large stones, moss, vegetable detritus, or at the entrance to
caves”. A particularly interesting case are species that at lower altitudes occur
exclusively in caves but at high elevation can be found under stones or in shallow
environments (among others, species of Duvalius, Trechus or some Leptodirini;
Jeannel 1943; Hernando et al. 1999). More recently, Pipan and Culver (2012) and
Culver and Pipan (2014, 2015) have comprehensively documented the regular
presence of troglomorphic species in the shallow subterranean habitats (SSHs),
questioning the relationship between troglomorphy and the deep subterranean envi-
ronment (see Fig. 10.4 for some examples with terrestrial fauna).
Although still insufficient, the available data on the physiological limits of
troglomorphic species point to humidity as a more stringent limiting factor than
Fig. 10.3 Distribution of the species of the genus Troglocharinus. In red, species occupying the
ancestral range of the lineage, in the Spanish Pyrenees. In blue, species in the Catalonian coastal
ranges (see Rizzo et al. 2013 for details). The shortest distance between them, ca. 60 km, crosses
Miocene sedimentary deposits of the upper Ebro valley
temperature (Rizzo et al. 2015; Sánchez-Fernández et al. 2016). The importance of

humidity as a limiting factor for subterranean species has been stressed by many
authors (e.g. Peyerimhoff 1906; Racovitza 1907; Jeannel 1943, see also Chap. 3). It is
likely that during climatic periods with low seasonality, without extreme temperatures,
species could move through microenvironments maintaining a high humidity, such as
the deep layers of forest litter. In the northern temperate regions, this may have been
the case during some periods of the late Miocene or the early-middle Pliocene, with
temperatures similar or slightly above the present but higher precipitations (Jiménez-
Moreno et al. 2010). The Pleistocene glaciations, and, in the Mediterranean area, the
onset of the strong seasonal variation typical of the Mediterranean climate, ended these
favourable climatic window, reducing the possibility of range movements on the
surface and confining subterranean species to their current areas of distribution
(Rizzo et al. 2013). Even if occasionally subterranean species can disperse across
non-subterranean medium, in general their geographic ranges are extremely reduced,
suggesting that most species have a very restricted mobility most of the time. One of
the most robust macroevolutionary patterns is the strong negative correlation between
range size and extinction probability (Ribera 2008), so for old lineages of subterranean
species to persist, either they have somewhat escaped extinction (becoming highly
Fig. 10.4 Some circumstances in which troglomorphic species can be found close to the surface.
(a) Doline in Sardín, Asturias, NW Spain. Some dolines are likely to act as “gates” to the deep
subterranean environment, providing a direct connection to the surface; (b) below stones like the
one in the image in the lowest part of the doline, there were troglomorphic species which have also
been associated with caves: Apoduvalius franzi (Carabidae), Cantabrodytes vivesi and Domene
sp. (Staphylinidae); (c) MSS in the Spanish Pyrenees (Barruera), only partially covered by soil. In a
pitfall trap placed below the stone marked with the arrow, Stygiophyes aldomai allomorphus was
found; (d) doline in Collau de la Plana del Turbón (Spanish Pyrenees), type locality of Trapezodirus
orobios orobios. The last two are highly derived troglomorphic species (Cieslak et al. 2014), the
first also found in caves (Fresneda and Salgado 2016); photos by © Ignacio Ribera (a and b) and
© Javier Fresneda (c and d)
isolated due to the extinction of most of their close relatives, Assmann et al. 2010) or
have to compensate a high extinction probability with an also high speciation rate.
Without fossil data the only evidence to estimate evolutionary turnover are phyloge-
nies of extant species, from which it is notoriously difficult to estimate speciation and
extinction rates (Rabosky 2015). But what can be estimated is the net diversification
rate, i.e. the result of speciation minus extinction, and it is also possible to identify
significant changes in this diversification rate and associate them to particular nodes in
the phylogeny (Rabosky and Huang 2016). There is still few data on diversification
rates for subterranean lineages, but for some groups, there is good evidence of an
increased diversification in highly troglomorphic species associated with changes in
the life cycle. This is evidenced in Leptodirini (Cieslak et al. 2014) and suspected in
Trechini (Faille and Pluot-Sigwalt 2015).
10.6 General Overview and Concluding Remarks
In the last years, different lines of research have converged in a renewed view of the
biology and evolution of species living in subterranean environments and on the
subterranean environment itself. The discovery of highly troglomorphic species in
lava tubes was the first challenge to the established paradigm on the origin of
subterranean species but, in the last years’ comprehensive studies of what were
considered marginal habitats, has further faded the limits of the subterranean envi-
ronment. Thus, the SSHs of Culver and Pipan (2014) include a wide range of
habitats for which the limits between them, and with more “traditional” subterranean
environments, are impossible to draw precisely. Similarly, among more terrestrial
environments, the limits of the MSS as originally understood (Juberthie et al. 1980)
have been challenged by the recent discovery of highly troglomorphic species in
areas and habitats in where their presence was not suspected (Giachino and Vailati
2010; Fig. 10.4). Although without highly troglomorphic species, other shallow
environments have been recognised as subterranean due to their physical conditions
and some peculiarities of the fauna (e.g. Ortuño et al. 2013). There is a basic
dichotomy when defining and limiting the subterranean environment, which is
whether using the characters of the species or the physical conditions of the habitat.
There does not seem to be a one-to-one correspondence, and as seen above, there are
many examples of subterranean environments without troglomorphic species and
troglomorphic species in habitats that do not have the typical characteristics of the
subterranean environment. These discordances are of particular interest to under-
stand the evolution of the subterranean fauna (Culver and Pipan 2014).
Despite the progress made in recent years in the study of the subterranean species
and their adaptations, as seen here there are still a number of questions that remain
largely unanswered. Without claiming to be comprehensive, in what follows we list
some of them, delineating some potential areas of research.
Why Troglomorphic Species Are More Frequent in Some Lineages? As already
noted, the number of species with troglomorphic features is highly unevenly dis-
tributed through the terrestrial invertebrates, suggesting that some of them have a
stronger propensity to colonise the subterranean environment. In some cases, there
are obvious reasons for what species cannot successfully colonise the underground,
such as for species strongly depending on green plants (e.g. leaf beetles) or with a
predominantly aerial adult way of life (e.g. dragonflies, butterflies). There are some
known cases of troglomorphic species in otherwise almost entirely epigean groups,
such as some Diptera (Andersen et al. 2016), Hymenoptera (Juberthie and Decu
1998) or even Plecoptera (López-Ródriguez and Tierno de Figueroa 2012), but these
are anecdotic and of little quantitative importance.
It is commonly assumed that groups with multiple independent invasions of the
subterranean environment (e.g. spiders, pseudoscorpions, springtails, some families
of beetles) are somehow “preadapted” to it, mostly because species living in forest
litter or the upper soil layers show some typical troglomorphic characters
(e.g. reduced eyes, lack of pigment and wings), suggesting that these environments
may be stepping stones for the colonisation of the subterranean environment. The
abundance in the subterranean environment of trophic resources adequate for some
groups of forest litter species, such as fungi or decayed organic matter for
Collembola or Leptodirini, or the appropriate prey for Trechini may also be a key
factor facilitating their colonisation. However, there is little phylogenetic data to
support this hypothesis, and there are many other possible routes for the origin of
subterranean lineages. They may directly derive from a fully epigean ancestor, from
which forest litter and subterranean lineages may have derived independently, or
from endogean species (see Fig. 10.5 for some possibilities). It could also be that the
key characters facilitating a subterranean life are less apparent and may have
originally evolved for an unrelated reason, such as changes in sensory organs, type
of development or other physiological characters.
What Are the Factors Driving the Colonisation of the Subterranean Environment?
Or in other words, what pushed species to the underground? There are many
hypotheses broadly related to abiotic (e.g. climatic) or biotic factors (mostly eco-
logic, such as resource availability or to avoid competition), as emphasised by the
climatic relict or adaptive shift hypotheses, respectively (Howarth 1972; Peck and
Finston 1993). It can, however, be questioned that the colonisation of the subterra-
nean environment needs a particular explanation more than, say, the colonisation of
the aquatic environment, the forest canopy or a particular species of plant. It may
seem rather unattractive and inhospitable, but, like any other habitat, it has advan-
tages and disadvantages that have to be overcome by the species living in it. Why did
species colonise subterranean habitats in the first place is probably a question with
little biological sense, unless it is reframed to ask why some colonisations are more
successful than others, which is the same as to ask why some groups have more
troglomorphic species than others.
What Are the Selective Advantages, if Any, of the Troglomorphic Phenotype? There
is still little hard evidence as to what could be the advantage, if there is any, of some
common troglomorphic characters that have repeatedly evolved in different groups.
Prominently among them are some body shapes found among Coleoptera (the
morphotypes of R. Jeannel; see above and Fig. 10.1), in which potential adaptive
value is unknown. The same applies to some modification of the life cycle, such as
the reduction of larval instars and the loss of activity that has evolved multiple times
in different groups (Deleurance 1963; Cieslak et al. 2014; Faille and Pluot-Sigwalt
2015). It has been hypothesised that by concentrating all the foraging and the
resource acquisition in the more mobile stage (the adult), species can survive in
environments with a lower density of resources, as a less mobile larva would not be
able to cover all the surface needed to gather the resources needed for the develop-
ment into an adult. This would allow these species to colonise the deeper part of the
caves, or caves in which resources are scarcer, for example, at higher altitudes
(Cieslak et al. 2014).
There are, however, many characters showing some degree of convergence for
which virtually nothing is known, not even if they are what are usually considered
Fig. 10.5 Schematic representation of some among many possible modes of colonisation, range
expansion and diversification within the deep subterranean environment (DSE). (a) In alpine
conditions, epigean species may be forced to seek refuge in superficial subterranean environments
(MSS), colonising from there the DSE; (b) a subterranean species living in the DSE may reach other
geologically isolated DSE units through MSS or forest litter, leading to range expansion and
diversification; (c) subterranean species in different geological units may maintain gene flow
through soil or forest litter but be isolated from other subterranean species through other geological
features (e.g. rivers); (d) an endogean species, derived from an epigean one, may independently
colonise different geological units leading to subterranean species in each of them
“regressive” or “progressive” characters, as, for example, modification in the sensory

organs, including the setae (Fig. 10.6).
How Linked Are Troglomorphic Species to the Subterranean Environment? The
erosion of the limits between the epigean and subterranean environments questions
the factors that determine the distribution of the troglomorphic species. Physiolog-
ical experiments can be of great help, although it is difficult to discriminate between
different factors or recreate them in controlled conditions. As already noted, avail-
able data and recurrent observations suggest that humidity is the main limiting
factor. Temperature, at least during periods up to several days, seems to be of lesser
importance as far as it is maintained below ca. 20 C, at least for some Leptodirini
(Rizzo et al. 2015). There is, however, no data on the long-term resistance, and the
possibility of acclimation of troglomorphic species to environmental conditions.
Some of the work done in the Laboratoire Souterrain de Moulis (France) suggests
that there may be long-term (at the scale of generations) acclimation to high
Fig. 10.6 Convergence in the eye and chaetotaxy reduction in some Cholevinae. (a) Speonemadus
bolivari (Anemadini); (b) Leptodirus hochenwartii (Leptodirini); (c) Anemadus smetanai
(Anemadini); (d) Notidocharis calabrezi (Leptodirini). C and D have independently developed
reduced eyes and similar modifications of the setae of the head and antennae. Photos by © Ignacio
Ribera
temperatures, at least in some developmental parameters (Delay 1978). A better

knowledge of the physiological and ecological limits of troglomorphic species will
contribute to understand the range expansions that necessarily took place in some of
the lineages.
Why Some Colonisations of the Subterranean Environment Gave Origin to Diverse
Radiations and Others to Single Species? Some lineages of subterranean species
are very diverse, while others remain species poor. To inquiry the reasons for these
inequalities would require first to assess the general macroevolutionary pattern of
lineage diversification over a large group. Due to random factors in the processes of
speciation and extinction, some lineages are expected to be more diverse than others,
so that no evolutionary explanation may be required for differences in species
numbers unless there are significant deviations with respect to the assumed null
model of diversification. There is also the difficulty of precisely determining the
moment in which a possible change of diversification rates occurred (i.e. in which
node in the phylogeny the diversification rates changed, Vogler and Ribera 2003).
Some of the few data available suggest that shifts may be associated not with the
original colonisation of the subterranean environment but with changes occurred
when species had already evolved all typical troglomorphic characters (Cieslak et al.
2014; Faille and Pluot-Sigwalt 2015).
Acknowledgements We thank our friends and colleagues Carmelo Andújar, Charles Bourdeau,
Achille Casale, Jordi Comas, Carles Hernando, Valeria Rizzo and Enrique Valenzuela for their
collaboration and ideas on the origin and evolution of the subterranean fauna, and Achille Casale for
his comments to previous versions of the work. The SEM photographs of Fig. 10.6 were taken in the
Phyletisches Museum (Jena) with the support of Rolf G. Beutel and Hans Pohl.
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Part III
About Communities, Populations and Food
Chapter 11
Cave Communities and Species Interactions
Stefano Mammola and Marco Isaia
11.1 Cave Communities
In ecology, a community—or also a biocoenosis—is defined as an assemblage of

different species living in a particular space and time. Entering a cave and moving
toward the inner sections, the abundance and the diversity of organisms progres-
sively decrease, going from the highly populated and diverse fauna crawling in the
twilight zone to the energy-deprived and poorly populated habitats of the deeper
parts (see also Chap. 3). The explanation for the high abundance and diversity of
organisms dwelling at the cave entrance has to be generally referred to the fact that
the entrance is an input site of potential food materials (Poulson and White 1969; see
also Chap. 12). Moreover, an array of organisms exploits this sheltered habitat for a
variety of functions—trophic purposes, reproduction, etc.—thus increasing local
diversity. While some subterranean specialized elements from the superficial sub-
terranean habitat (SSH) fauna may be present (Novak et al. 2012), the communities
of the twilight zone are usually dominated by species exhibiting a poor degree of
adaptation to subterranean life. In contrast, true troglobionts dominate in the deep
subterranean domain, where strong selective pressures promote a highly specialized
community and, at the same time, limit diversity and abundance (Fig. 11.1).
It is, however, an open question whether species within a cave are interdependent
on each other for keeping balance and stability of the system—recalling Clement’s
(1916) holistic view—or whether the community is primarily defined by the envi-
ronmental conditions following the individualistic concept of Gleason (1926). While
both concepts may apply to cave communities in different circumstances, neither of
them has been demonstrated to apply entirely. In a third, controversial view pro-
posed by Hubbell (2001), differences between members of an ecological community
S. Mammola · M. Isaia (*)

Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy

https://doi.org/10.1007/978-3-319-98852-8_11
256 S. Mammola and M. Isaia
Fig. 11.1 The classic representation of cave communities. In the twilight zone, species exhibiting a
poor degree of subterranean adaptation dominate. In contrast, true troglobionts are preferentially
found in the deep zone, where high selective pressures promote highly specialized communities
and, at the same time, limit diversity and abundance (original illustration by Stefano Mammola)
of trophically similar species are “neutral” or irrelevant to their success. The neutral
view has been criticized because it requires all species to be equivalent and in
constant environmental equilibrium, which is a rare condition in nature, where
climatic and geographical conditions change too frequently for this to be attained
(Ricklefs 2006). The existence of semi-stable conditions would make cave ecosys-
tems ideal candidates for testing this theory, although no direct support for this
concept has ever been demonstrated in cave communities to date.
Support for the organismic view of the cave community is offered by authors who
postulate the existence of so-called species associations in caves, which form
discrete units. One of the first authors stressing this concept was Jeannel (1926),
who focused on spatial variations in the community from the cave floor, walls, and
ceilings. In particular, he noticed how the animal community thriving on wall
surfaces—that he termed parietal association—differed greatly from the one dwell-
ing on the cave floors. Pushing this concept further, Bourne (1976) described local
variations of parietal associations according to different wall morphologies, vertical
height, and distances from the cave floor. Recently, Moseley (2009) retrieved this
idea, identifying and defining different associations of species within the cave
microhabitats, such as the parietal association, the pool surface association, the
guano pile association, etc.
11 Cave Communities and Species Interactions 257
On the other hand, the occurrence of a transition between the communities of

the twilight and the dark zones, characterized by species commuting to the sur-
face—Poulson and White’s (1969) “middle zone”—supports an individualistic
rather than a holistic view of cave communities. Under this perspective, transitions
in cave communities are gradual and not easily defined, and the relations between
species are mostly the results of similar requirements and tolerances.
11.2 Diversity of Cave Communities
The number of species within a cave is likely to depend on the carrying capacity of
the system and the biogeographic context of the area under consideration. Further-
more, being strongly related to external inputs—as well as to the extent of the system
of rock fissures in which the cave develops (Culver and Pipan 2014; Mammola et al.
2016a)—estimations of species richness can be ambiguous. However, as a rule of
thumb, species richness in caves is orders of magnitude lower than in neighboring
epigean ecosystems. Caves have indeed low energy inputs and are subjected to
strong environmental filters, which pose a challenge to the adaptation of organisms
(see also Chaps. 3, 4, and 10).
Culver and Sket (2000) published a very influential contribution, in which they
documented species richness in 20 hot spots of subterranean biodiversity across the
world, hosting 20 or more stygobionts and troglobionts. Later, more sites were added
to this list (Culver and Pipan 2009), including a few subterranean hot spots in the
tropics (Souza-Silva and Ferreira 2016; Trajano et al. 2016). The high richness in
these caves—up to 84 troglobiont species in the case of the Postojna-Planina Cave in
Slovenia—possibly represents the maximum numbers of obligate species compris-
ing a hypogean community. Moreover, the number of species almost doubles when
considering troglophile and occasional species interacting with the deep hypogean
assemblage of obligate cave species. In many circumstances, occasional species
have indeed been reported far inside the cave entrance, testifying to the potential
exchanges between epigean and hypogean communities. An emblematic case is the
occurrence of troglophile flies Trichocera maculipennis (Diptera, Trichoceridae) in
the deepest sections (–2140 m) of the deepest abyss in the world (Krubera-Voronja
Cave, Western Caucasus) (Sendra and Reboleira 2012).
However, compared to epigean ecosystems, the average number of species in a
cave community is generally low, especially when considering intra-guild diversity.
For example, the coexistence of multiple troglobiont spiders in the same cave is rare
and often mediated by niche partitioning (Mammola and Isaia 2017 and unpublished
data). Typically, deep cave communities host a single troglobiont spider or rarely
two or three species. As far as we are aware, the maximum number of troglobiont
spiders documented from a single cave in the temperate zone is four (Balićeva špilja,
Croatia; Martina Pavlek, personal communication) and two in the case of the tropical
regions (Bloom et al. 2014).
Fig. 11.2 Members of cave communities belong to different trophic and functional groups. (a)
Phototrophic organisms may be present in the twilight zone or in areas artificially illuminated; (b)
occasional external elements such as winged insects can colonize the cave entrance and neighboring
areas; (c) fungi represent the base of the typical detritus-based food chain of the cave environment;
(d–f) detritivorous and microbivores, such as diplopods, collembolans, and isopods, generally
dominate the arthropod communities in caves; (g–i) predators such as chilopods, spiders, and
carabid beetles occupy the apical positions in the food chain. All photos by courtesy of © Francesco
Tomasinelli
11.3 Functional Composition of the Community
Because of the considerable hindrance of the low flux of energy in cave ecosystems
(see also Chaps. 13 and 14), caves generally host simplified communities, less diverse
and less functionally redundant than their epigean counterparts (Fig. 11.2). Deprived
of primary photosynthesizing producers, herbivores are therefore absent in subterra-
nean habitats, cave root-feeders being the exception (Howarth 1983). Accordingly,
subterranean communities are considered to be “disharmonic” (Gibert and
Deharveng 2002). A complication to this general pattern is provided by microbivores
organisms, small arthropods which are able to ingest bacteria or fungi—micro-
floras—but exclude detritus from their guts. However, the larger the animal, the
less able it is to distinguish between microflora as food and the plant or animal detritus
on which these are growing. In fact, the majority of the detritivorous animals involved
in the decomposition of dead organic matter are generalist consumers of both the
detritus itself and the associated microfloral populations.
Furthermore, there is evidence that in energy-deprived ecosystems such as most
of the cave/subterranean ones, obligate predators may decrease their disappearance
being due to lack of prey, as well as secondary evolutionary shifts toward polyphagy
(Gibert and Deharveng 2002). For example, in the troglobiont beetle Aphaenops, the
mouthparts are highly adapted for predation, to feed on dead plant matter and to
ingest earth to extract nutrients (Gers 1995).
It can be argued that the richness and the abundance of detritivores in a cave act as
regulating factor for the entire cave community through a bottom-up control mech-
anism—strongly dependent on external energy inputs—which, in turn, limit the
abundance and the diversity of predators. As in other detritus-based food chains,
the most important interspecific interactions are competition—among decomposers
and among predators—and predation. However, variations to these general patterns
are expected in relation to the type and nature of the trophic inputs. It is self-evident,
for instance, that guanobionts—organisms feeding on bat guano—will be present
only if there are bat colonies within the cave and that specific parasites and
parasitoids will be part of the community only in presence of their hosts.
Primary producers and true herbivores may be also locally present in cave
entrances. Phototrophic organisms generally occur at the entrance zone, where they
may support small groups of true herbivores. Primary producers exploiting different
sources of energy such as chemolithotrophic bacteria (e.g., Northup and Lavoie
2001)—especially ferrobacteria, sulphobacteria, and nitrificant bacteria—may be
also present in caves.
Another exception to the general pattern can be found in caves that are show
caves, where primary production dramatically increases because of the installation of
artificial lighting systems (Albertano 2012; see also Chap. 6).
11.4 Turnover in Space
Beta-diversity or species turnover is the extent of change in community composition

along a gradient (Whittaker 1960). At the regional scale, the species turnover is the
species loss/replacement from site to site—also referred to as nestedness. Most of the
studies focusing on species turnover in caves have focused on single taxonomic
groups (e.g., Malard et al. 2009; Stoch and Galassi 2010; Cardoso 2012; Zagmajster
et al. 2014) and not on subterranean communities as a whole. Considering that most
subterranean species exhibit restricted ranges, we may expect that even at a small
geographical scale, caves should display higher levels of beta-diversity in compar-
ison to most epigean ecosystems. In other words, it is theoretically expected that
communities in caves relatively close to each other should exhibit faunal differences.
The high level of endemism of most subterranean species seems to confirm this
hypothesis in most cases (Sharratt et al. 2000; Christman et al. 2005).
More throughout considerations can be drawn when considering the spatial
community turnover at the local scale—e.g., along a certain gradient within a single
cave. It is generally observed that in a typical cave, there is a gradient of subterranean
specialization of the biological community from the entrance zone toward the
deepest sectors (see, e.g., Tobin et al. 2013; Fig. 11.1)—or, more generally, from
shallow toward deep subterranean spaces (see Mammola et al. 2016a). Nevertheless,
this perspective does not take into account the connections that exist between the
habitats accessible to humans—the caves themselves—and the habitats that are not
directly accessible (e.g., the SSHs; Culver and Pipan 2014).
Accordingly, the existence of a linear turnover in the community with the
distance from the entrance has been recently questioned by Novak et al. (2012).
By studying cave communities in Slovenia, they observed and characterized two
distinct communities, one dwelling close to the surface (0–10 m depth) that is
characterized by non-troglobionts and a major group of troglobionts belonging to
the SSH fauna and another consisting of a minor group of troglobionts, dwelling in
the deeper parts of the caves. Such spatial separation in the community directly
implies sharp variations in the beta-diversity from the surface toward the subsurface
(i.e., a bimodal distribution), rather than a constant variation along the cave gradient
(i.e., a linear distribution). Although the generality of this pattern remains to be
proven, other authors have reached similar conclusions. For instance, in their studies
on ecotones, Prous and colleagues noticed how the animal community in the twilight
zone presents higher diversity in respect to the neighboring epigean and deep
hypogean habitats, with sharp decreases in both directions (Prous et al. 2004,
2015; see also Chap. 3). In particular, they interpreted the entrance zone as an
ecotonal area harboring an enriched community, capable of exploiting its unique
and intermediate characteristics (Fig. 11.3).
11.5 Turnover in Time
There is a very old paradigm in cave-based science that subterranean communities

are temporally stable. However, it is nowadays well acknowledged that subterranean
habitats are characterized by a constant flux of “invaders” (Culver and Pipan 2009)
and that there is a temporal variability in the distribution of invertebrates found in
caves (Delay 1969; Tobin et al. 2013). For instance, subterranean invertebrates move
actively in search of food in the larger chambers and move back to the more stable
fissures in response to physiological stress—e.g., variations in relative humidity.
Moreover, external species enter the subterranean habitat for a number of specific
purposes, e.g., day/night refuge and seasonal refuge. As a consequence, in a typical
cave community, a significant temporal turnover in the species composition is
observed, particularly near the surface (Bento et al. 2016).
Twilight zones are also frequently colonized by external elements—e.g., winged
epigean arthropods—which cause greater fluctuations in species composition over
the year. This is particularly true in the case of epigean species that are linked to cave
habitat in a certain period of their biological cycle. For instance, lepidopterans such
as Scoliopteryx libatrix (Erebidae) and Triphosa spp. (Geometridae) hibernate in
European caves in winter, whereas Vanessa spp. (Nymphalidae) enter caves in
summer, possibly to avoid extreme temperature (Mammola S., personal observa-
tions). Similarly, epigean harvestmen and crickets commonly overwinter in
Fig. 11.3 The epigean/hypogean ecotone: in a recent study on the community of a Brazilian
limestone cave (Lapa do Mosquito), Prous et al. (2015) sampled the animal biocoenosis from the
exterior of the cave toward the dark zone—up to 70 m inside. By this they identified a transition area
of approximately 20 m (10 m inside the cave and 10 m outside) acting as an epigean/hypogean
ecotone and hosting 55 species. Out of these species, 49 were shared with the epigean community,
37 with the hypogean community, and 14 species shared by the 3 communities. Modified from
Prous et al. (2015)
gregarious formations on cave walls and ceiling in the twilight zone, both in
temperate (Kozel et al. 2015; Lipovšek et al. 2016) and subtropical areas (Chelini
et al. 2011). Variations in the community may also be detected within day-night
cycles, especially with respect to nocturnal species taking shelter in caves during the
day. Classic examples in this sense are bats, but other organisms such as nocturnal
lepidopterans can display a similar behavior.
Although less documented, temporal turnover may also occur in communities
occupying the deeper sections of caves. Crouau-Roy et al. (1992) observed how the
abundance of the subterranean beetle Speonomus hydrophilus (Leiodidae,
Leptodirini) underwent a significant decline both in winter and summer, in relation
to temperature variations. They argued that this species was able to take shelter in the
network of fissures when the climatic conditions were not favorable. Chapman
(1985) observed a similar pattern in the Hawaiian Islands in certain highly vagile
specialized species, which were able to appear and disappear rapidly in the cave,
visiting the larger chamber from the adjacent labyrinth of voids and vice versa. In a
similar way, Mammola et al. (2015) documented the occurrence in troglophile
beetles of local seasonal migrations from the cave entrance toward the inner parts
of the cave in response to mean daily temperature and humidity drops. Such
evidence directly implies that, even in the most stable sections of caves, the
composition of the animal community cannot be considered stable, and the study
of cave communities should preferably incorporate a temporal perspective.
11.6 Ecological Niche and Interspecific Competition
The biological structure of a community is defined by its composition of species,

namely, their number and relative abundance. However, to be part of a community,
individuals of a certain species have to maximize their survival in the given
environmental conditions characterizing the habitat in which the community
develops. The probability of surviving in certain conditions is the consequence of
an array of physiological, ethological, and morphological adaptations, which from
one side may allow the survival of a certain species but from the other may preclude
survival of another.
We define the ecological niche of a species as the array of adaptations determin-
ing its success in terms of survival, growth, and reproduction in a certain habitat.
Therefore, along environmental gradients, species may exhibit distinctive bell-
shaped abundance patterns, which reflect their differential survival probability as
the environmental conditions change. In caves, for example, troglobionts exhibit an
array of characters, which make them more competitive—and more abundant—in
total absence of light and in energy-deprived habitats. Conversely, troglophiles tend
to be more abundant in the twilight zone, either because their adaptations are
efficiently exploited or because they are poorly suited to the darkest parts of the cave.
In one of the most influential contributions on subterranean biology, Poulson and
White (1969) argued that microclimate, habitat structure, and food availability are the
primary and secondary factors which determine subterranean biodiversity, rather than
competition- and niche-based processes. Remarkably, however, a few years later,
Poulson himself (1977) demonstrated the existence of spatial niche partitioning in
two linyphiid spider species—Phanetta subterranea and Anthrobia monmouthia—in
North American caves. He documented how the two species were able to exploit
distinct spatial niches, in order to avoid competition for food resources. Parallel
observations by Culver (1973, 1975) led to similar results, underlining that compe-
tition in caves was indeed more common and important than previously thought.
In line with these early observations, recent studies unraveled that niche-based
processes operating in cave/subterranean ecosystems are more the rule than the
exception (e.g., Fišer et al. 2012; Mammola et al. 2016b; Resende and Bichuette
2016). Accordingly, Culver and Pipan (2015) included competition among the three
most important factors of the selection pressures existing in the subterranean envi-
ronment. They argued that (p. 442): “[. . .] Interspecific competition, a divergent
selective force, is important in shaping morphology when competing species are
present.”
Commonly, ecological niches and species interactions are represented as normal
functions of the abundance a species—or its probability density—along a certain
Fig. 11.4 An example of species interaction in a cave: the abundance of a competitor affects the
distribution pattern of other species of the cave community. In this example, the abundance of the
spider Pimoa graphitica (Pimoidae) is highest in the vicinity of the entrance, where trophic
resources are more abundant (top panel). As the abundance of the competitor Meta menardi
(Tetragnathidae) increases (bottom panel), P. graphitica is forced to “step back,” rearranging its
niche deeper inside the cave, a suboptimal habitat in terms of trophic availability. Modified from
Mammola et al. (2016b)
gradient. For instance, in a study in caves in the Alps focusing on top predators in the
twilight zone, it has been shown that niche overlaps lead to strong species interac-
tions, which determine the rearrangement of the niche of the less competitive species
toward suboptimal habitats (Mammola et al. 2016b; Fig. 11.4).
More recently, thanks to the advances in the mathematical characterization of the
niche, Blonder et al. (2014) developed a simple method to calculate the n-dimen-
sional hypervolume (sensu Hutchinson 1957) of a certain species and to quantify its
reciprocal intersection (i.e., overlap) with other species sharing the same habitat. By
this means, it is possible to use field collected data to map the species distribution in
3 4
2
Distance from the
1
entrance
–2 –1 0 –1 0 1 2 3 4
4
3
2
Illuminance
1
–1 0
Troglophile spider
–1 0 1 2 3 4
4
3
Prey
2
1
0
–1
–3 –2 –1 0 1
Niche centroid
Visual representation Relative humidity

of the niche boundary Troglobiont spider
Fig. 11.5 The niche hypervolume of two cave predators. Graphical representation of the four-
dimensional niche hypervolume (sensu Blonder et al. 2014) of two spiders showing different
degrees of subterranean adaptation and coexisting within the same cave habitat. The combination
of multiple environmental factors determines the niche of the two species—all variables are
rescaled. For instance, in the central row, the first panel illustrates high partitioning in the niche
of the two species concerning the optimal sites for hunting. The troglophile species finds its prey in
most illuminated area of the cave (twilight zone), whereas the troglobiont hunts in the darker area.
Similarly, the right panel in the same row shows that the troglobiont species has a preference for a
narrow range of relative humidity, while the troglophiles is more tolerant. Data derived from
Mammola and Isaia (2016)
relation to environmental features of the cave habitat and to use these data to
represent the species niche in a mathematical space (e.g., see Fig. 11.5).
From a more morphologically oriented perspective, competitive exclusion can be
studied by analyzing variations in functional morphological traits in
multidimensional morphospace. By these means, it is possible to detect signatures
of character displacement and divergent evolution due to interspecific competition.
In recent times, using species pairs of subterranean amphipods of the genus
Niphargus coexisting within the same habitat, Fišer et al. (2012, 2015) demonstrated
that functional morphological diversity within subterranean communities was higher
than expectation, suggesting that extreme habitats maintain a high potential for
diversification via niche partitioning.
These examples testify the ecological potential of simplified ecosystems, such as
caves, as model systems for the advance of niche theory. However, despite their
potential, the use of subterranean organisms as models to study competition has

lagged behind the use of their epigean counterparts. This is perhaps surprising, given
that in complex epigean ecosystems, confounding effects may increase the com-
plexity in defining and quantifying the niche. These include higher species diversity,
multi-species interactions, and the intrinsic variability of the environmental condi-
tions. On the other hand, due to their intrinsic simplicity and the absence of most
confounding effects, caves offer unique natural laboratories. We are indeed con-
vinced that the idea of basing niche-based studies in cave/subterranean habitats has
strong potential in improve ecological theory.
Acknowledgments We are grateful to Wolfgang Nentwig for providing insightful comments on

an early version of the chapter.
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Biogeogr 23:1135–1145
Chapter 12
Structure and Genetics of Cave Populations
Peter Trontelj
12.1 Introduction
12.1.1 The Genetics of Cave Populations Reflects

Evolutionary History
Genetics of cave species can be and occasionally has been studied in its own right,
just for the sake of the genetic and genomic mechanisms behind their captivating
biology. But far more often, genetics in all its forms has served to support or reject
concepts and hypotheses within the current paradigm or some particular research
program of biospeleology. Over the past four decades, the predominant view has
been that rare and localized colonization events give rise to small, isolated subter-
ranean populations. These eventually become species of their own with hardly any
opportunity to disperse beyond the bounds of an aquifer or a fissured, permeable
geotectonic unit. The resulting biodiversity pattern is a patchwork of tiny ranges with
large gaps in-between, as well as high allopatry of closely related and morpho-
logically similar taxa (Sbordoni et al. 2000; Culver and Pipan 2014). Over the years,
evidence from numerous studies from around the globe has only consolidated this
model, despite occasional reports about larger ranges and subsurface dispersal
(reviewed below).
There is a major, albeit largely overlooked, conceptual problem with the current
model. While each single troglobiotic (used here for both troglobionts and
stygobionts, unless specified) species has a small range, groups of closely related
and morphologically very similar species can be distributed over much larger areas.
This could have happened either by independent cave invasion and convergent or
parallel evolution, or by a single troglomorphic ancestral species spreading over the
P. Trontelj (*)
Biotechnical Faculty, Department of Biology, University of Ljubljana, Ljubljana, Slovenia

https://doi.org/10.1007/978-3-319-98852-8_12
270 P. Trontelj
entire, vast range that subsequently became fragmented. The principle of Ockham’s
razor, or parsimony, advises us to be cautious when postulating copious repetitions
of a complex evolutionary transition. On the other hand, it is hard to imagine how a
specialized cave species might traverse belts of non-permeable substratum, be it
terrestrial or aquatic. In a few cases, empirical genetic evidence points toward long-
range subsurface dispersal (e.g., Lefébure et al. 2006; Zakšek et al. 2007; Eme et al.
2013), but the biological means remain speculative. The most solid explanation, still
far from being universally applicable, seems to be dispersal via the hyporheic
interstitial habitat along gravel-filled riverbeds (Ward and Palmer 1994), which
might occasionally work also for terrestrial species (Ortuño et al. 2013). In Lefébure
et al. (2006), we have asked ourselves “whether wide distributions should always be
bent to accommodate a nondispersal paradigm”, or perhaps some other hypotheses
should be given priority. More than 10 years later and in the light of overwhelming
genetic evidence, it has become clear that the non-dispersal is the winning paradigm,
as exemplified by the studies discussed in this chapter.
12.1.2 The Paradox of Large Subterranean Ranges
But how can the paradox of large ranges be resolved for all those species that do not
disperse via the interstitial? On their evolutionary transition from surface to subter-
ranean life, populations are believed to undergo four stages: (1) colonization, (2) a
“trial phase” when their success or failure is determined, (3) speciation, and (4) dis-
persal of the new subterranean population (Culver and Pipan 2014). Unfortunately,
this hypothesized linear chain of events will not help us find a solution. On the
contrary, it strengthens the paradox, because it permits only subterranean dispersal.
A sobering example comes from recent molecular phylogeographic work on the
widespread Cuban cave fish Lucifuga dentata. Hernández et al. (2016) have shown
that the species occurs in five disjunct clusters of populations in isolated karst areas
spanning nearly 400 km of western Cuba. The population clusters are genetically
completely isolated from each other, as inferred from mitochondrial DNA (mtDNA)
sequences. Genetic differences between them are not much larger than the diver-
gence within, implying that they still belong to the same species, and that whatever
events caused that structure must have taken place quite recently. Using approximate
Bayesian computation, Hernández and his co-workers estimated that the present
distribution was reached through gradual westward dispersal following a stepping-
stone model and starting from the eastern-most population cluster. Paradoxically, the
population clusters are separated by tens, sometimes hundreds of kilometers of
unsuitable substratum. So, how could L. dentata, as an obligate subterranean fish,
disperse so far westwards? The authors adhere to some unknown means of subter-
ranean dispersal, mentioning that, under certain hydrological conditions, barriers to
underground dispersal can be transiently overcome. Such an explanation is not far
from the “just-so” stories of dispersal from the pre-vicariance era of historical
12 Structure and Genetics of Cave Populations 271
C A A' B B'C C' D D'

A D
B
a) C'
A' D'
B'
S A' B' C' D'

S
b) D'
A'
B'
A' B' C' D'

C'
c) A' D'
B'
A' B' C' D'
d) A' D'
B'
Fig. 12.1 Hypotheses explaining wide and discontinuous ranges of subterranean populations. (a)
Structuring took place already among ancestral population at the surface, followed by multiple
independent and geographically separate (polytopic) invasions; (b) a single surface population gave
rise to a single invasion, and the new subterranean population subsequently dispersed or expanded
endogenously; (c) whether a group of allopatric subterranean populations evolved by polytopic
invasions, by subterranean dispersal, or by vicariant fragmentation of a once larger contiguous
subterranean range cannot be inferred by phylogeny alone, once the surface ancestor has gone
extinct; (d) occasional chance dispersal of troglobionts via the surface could partly homogenize
gene pools of disjunct cave populations
biogeography (Morrison 2014). It shows that subterranean biology has yet to search
for answers to some of its basic questions.
As a side note, the usual biogeographic alternative of vicariant fragmentation of a
once large and contiguous subterranean range faces exactly the same limitation as
any “just-so” story about dispersal. For subterranean vicariance to occur, a
troglobiont would have first to spread over a large area of subterranean habitat.
To illustrate another aspect of the problem, let us take the example of a widely
distributed but strongly structured subterranean species (Fig. 12.1). The linear model
of cave species evolution can be applied in two ways to explain this pattern. The first
272 P. Trontelj
is through a single colonization, followed by subterranean dispersal over a wider

area of subterranean habitat and, finally, vicariant fragmentation of the subterranean
range creating the well-known pattern of patchy, isolated subterranean populations.
The second one is to postulate a strongly structured surface species, each of its
population giving rise to a separate subterranean population in the same way as
postulated above but not followed by any substantial subterranean dispersal and
range fragmentation. The problem starts when the surface ancestor goes extinct,
which is the rule for cave species (Culver et al. 2009). A phylogeographic analysis of
the cave populations will not be able to discern between the two scenarios. The
limitations of molecular phylogenies in tests of classical biospeleological hypotheses
have been noticed and described before by Desutter-Grandcolas (1997) and Culver
et al. (2009). Researchers therefore focused on questions (see examples discussed
below) that can be addressed more easily and that do not require explicitly differ-
entiating between processes taking place at the surface or during colonization and
those taking place after the subterranean realm has been colonized. This essentially
means that despite the use of sophisticated phylogeographic tools, we are often still
not able to tell whether the most basic ecological and evolutionary events that shape
the characteristics of cave species are taking place at the surface or underneath it.
The mentioned paradox is mostly a feature of continental subterranean habitats,
both aquatic and terrestrial. The marine and coastal subterranean fauna from habitats
such as anchialine caves and the coastal interstitial is governed by different spatial
and temporal scales of dispersal and differentiation. It is dealt with in another chapter
of this book (see Chaps. 18 and 19).
12.1.3 Exogenous and Endogenous Processes Determine

the Structure of Subterranean Populations
Culver et al. (2009) pointed out that, in order to reconstruct these historical pro-
cesses, one needs to obtain additional information, like times estimated by molecular
clocks or data on paleoclimatic changes. The list can be expanded by data on genetic
signatures of demography and genetic diversity of populations, pointing to hypo-
thetical bottlenecks or founder events during colonization and population growth
during phases of range expansion but also to events that increase diversity such as
multiple invasions and secondary contact of subterranean populations. Further hints
might be obtained by studying biological traits related to dispersal and subterranean
lifestyle as well as comparative phylogenetic reconstruction of their evolution. The
third category of data in this non-exhaustive list contains past and current abiotic and
biotic factors enabling or obstructing dispersal above or below the surface, including
changes in the availability of subterranean habitat, and vicariant events causing
fragmentation of a wider, contiguous historical range.
Using these data, innumerous scenarios can be coined to formulate testable
hypotheses. Some of them have been tested (described below) but hardly ever in a
formal conceptual framework that would address the central problem in an explicit
and comparable way: “Which of the processes that shape subterranean biodiversity
take place at the surface and which within the subterranean realm?” And, as an
extension to this question: “How did large subterranean ranges form?”
In its simplest form, such a framework might imply that the studied processes and
events belong to either of two classes with respect to the ecological space in which
they take place. In an operational sense, it requires the formulation of hypotheses that
discriminate between these two classes:
1. Exogenous processes: they take place at the surface or during colonization and are
generally common (e.g., fragmentation of ancestral populations, cave invasions,
and the accompanying evolutionary changes).
2. Endogenous processes: they take place in the subterranean realm, after a new
troglobiotic population has been established. They are comparatively rare (e.g.,
subsurface dispersal, vicariant fragmentation of subterranean ranges, secondary
sympatry by two closely related troglobionts, evolving into new subterranean
niches).
The idea that evolutionary processes shaping subterranean populations act at both
levels, surface and subterranean, has been put forward already in the 1960s by
Thomas Barr and has been later explicitly elaborated by John Holsinger (reviewed
in Holsinger 2000). Although often cited and illustrated in biospeleological texts, the
“two-phase model”, as Holsinger called it, has not received much consideration in
empirical studies using genetic and phylogenetic approaches. Nevertheless, as will
be shown in this chapter, our ability or failure to identify the level at which certain
processes take place may have important consequences for our understanding of
various biospeleological problems as well as practical implications, for example for
the assessment of conservation priorities.
12.2 Genetic Diversification of Subterranean Populations
12.2.1 Brief Historical Account
In the early years of molecular population genetics, allozyme electrophoresis and the
allele frequency data derived from it were the preferred tools to study population
structure, diversity and differentiation. Studies of the 1960s to 1990s have shaped a
picture of strongly structured populations of troglobionts with low to very low
amount of gene flow between them (reviewed by Kane 1982; Sbordoni et al.
2000). Comparative analyses revealed much lower rates of gene flow taking place
over considerably shorter distances in cave species than in closely related surface
taxa. Interestingly, in populations that were examined over a range of geographical
distances, e.g., cave crickets, beetles, spiders and amphipods, only negligible gene
flow was detected whenever average distances between populations exceeded
20–40 km. That was one of the first empirical indications of a phenomenon that
274 P. Trontelj
would two decades later become popular under the expression “cryptic species”.
Population genetic research in caves has soon proven a valuable new tool in
biospeleology, and development of the discipline has gone hand in hand with the
advancement of genetic methodology ever since.
While allozyme data and the analytical methods applied to them enabled pro-
found insight into population genetic structure and diversity, they fell short when it
came to the detection of the temporal dynamics of population and lineage-level
processes. The measure used (Wright’s F-statistics) presumes an equilibrium situa-
tion where gene flow by migration counteracts drift and other factors that tend to
drive allele frequencies of different populations apart. But what if we knew that
migration between physically isolated subterranean populations is impossible or
nearly impossible? Then, the amount of gene flow, determined based on measured
allele frequencies, “was supposed to reflect the historical gene flow that occurred
between cave and epigean populations” (Sbordoni et al. 2000). This, of course, is a
conceptual oversimplification that obfuscates the temporal and spatial dimension of
evolution. We have accurately described the pattern but remain largely ignorant
about which of many possible scenarios of surface and subterranean processes
(Holsinger 2000) might have produced it.
With the start of routine use of DNA sequence methods in the 1990s, a temporal
treatment of genes and their genealogies became possible. DNA sequences from
individual specimens could be arranged to phylogenetic trees and networks. By
mapping the distribution of genealogical lineages, one could draw conclusions about
colonization and fragmentation history in an approach known as phylogeography
(Avise 2000). First attempts at resolving questions of colonization history and
isolation showed that simple cladistic approaches are too naïve to accommodate
the complexity of possible scenarios (Rivera et al. 2002). Rivera and his co-workers,
studying troglobiotic Hawaiian oniscidean isopods, concluded that “the known cave
species of Hawaiioscia evolved from a widespread ancestral surface species or a
group of closely related species through multiple, independent adaptive shifts on
each of the islands separately”. Their paper can be seen as the beginning of modern,
DNA-based molecular biospeleology. It tentatively announced that cave invasions
by the same surface species are frequent in both time and space, leaving a compli-
cated imprint on the molecular architecture of cave populations. The study showed
that the question of whether isolated cave populations represent independent species
is important not just from a taxonomic point but also for our understanding of the
evolution of subterranean biodiversity. Furthermore, it underscored the need for an
explicit temporal framework for the evolutionary dynamics of cave population. The
temporal component was soon addressed by other authors (Caccone and Sbordoni
2001; Leys et al. 2003; Ketmaier et al. 2003; Verovnik et al. 2004), and it is
presented in more detail in another chapter of this book (see Chap. 10).
12.2.2 Genetic Structure Results from Historical Processes

and Concurrent Brakes in Connectivity Between Cave
Populations
The term genetic structure is applied loosely, to denote any spatial or ecological
departure of complete genetic homogeneity. In most cases, genetic structure is
detected by comparing the genetic setup of several individuals from different
caves. When in a population distributed across several caves individuals from
different caves differ genetically more than individuals from the same cave this
population is said to be structured into smaller populations (also subpopulations or
demes). This is primarily done by measuring (counting) allele frequencies, be it in
the form of allozymes, microsatellites, or single-nucleotide polymorphisms (SNPs),
and by comparing DNA sequences and calculating average within- and between-
population genetic distances. Alternatively, geographic population structure can be
revealed by a phylogeographic approach, which is essentially a phylogenetic study
of DNA sequences sampled from many individuals from different caves.
Phylogeographically, the clearest case of population structure is obtained when
members of different populations constitute mutually exclusive monophyletic
groups and thus constitute separate species, at least under the phylogenetic species
concept. It is the most commonly reported form of genetic structure, often in
association with taxonomic proposals regarding cryptic or unrecognized species
(Fig. 12.2a). This, however, is not the type of genetic structure that is particularly
interesting for the ecology of cave populations.
Essentially, the ecologically challenging patterns of differentiation between cave
populations are produced in two ways or a combination thereof:
1. As an historical imprint of exogenous processes, determined by the genetics of
the surface ancestor and the way and number of times it invaded the caves
(Fig. 12.2c)
2. As a consequence of concurrent endogenous factors acting upon the connectivity
between caves, such as physical or other ecological barriers, limited biological
dispersal abilities of troglobionts, and their potential to disperse over the surface
(Fig. 12.2b)
Thus, we are looking at cases where cave populations are actually or at least
theoretically in contact with each other via ongoing or recent gene flow. Such cases
are remarkably rare. One reason for this is that, when researches look at subterranean
populations at ever smaller geographical scales, they keep discovering new cryptic
species instead of moderately structured populations (Zakšek et al. 2009). Other
reasons are discussed below.
276 P. Trontelj
Fig. 12.2 Different types of genetic structure of cave populations as revealed by DNA sequence
networks. (a) Mutually exclusive monophyletic clusters of sequences, indicating two separate
species; (b) several sequences shared between populations, differences mostly in terms of haplotype
frequencies; this network suggests ongoing gene flow between subterranean populations; (c) similar
or same sequences in two separate cave populations can stem from a shared surface ancestor or from
ongoing polytopic immigration from a widespread surface population. Most analytical methods
would indicate gene flow, although no migrants are being exchanged between caves. Note how
multiple invasion events, although low in numbers, can lead to high diversity by introducing highly
divergent sequences. Each circle represents a DNA sequence haplotype, the diameter reflecting its
gene pool frequency and the color the population of origin. The length of the path between circles is
proportional to the number of substitutional differences between haplotypes. The networks are
hypothetical but based on examples from studies discussed in this chapter
12.3 Population Structures in Different Ecological

and Evolutionary Settings
12.3.1 The Sluggish Interstitial Highway Produces Strong
Genetic Structuring and Isolation by Distance
An illustrative example is the small-scale genetic structuring of aquatic fauna in

calcrete aquifers in the arid zone of central Western Australia. Technically, calcretes
are not caves but shallow subterranean habitats formed in carbonate deposits in the
groundwater of paleodrainages (Humphreys 2008; see also Chap. 18). They are
completely isolated from each other for more than 5 million years (Leys et al. 2003),
each harboring its own assemblage of endemic species (e.g., diving beetles, amphi-
pods and parabathynellids) and therefore called a subterranean archipelago of
groundwater islands in the desert (Cooper et al. 2007). The distribution of genetic
diversity between calcretes is clearly a matter of exogenous processes taking place
millions of years ago and in no relation to genetic connectivity between them. Yet,
calcretes extend over tens and even hundreds of kilometers, which is huge for a
contiguous area of subterranean habitat (Trontelj et al. 2009). Therefore, single
calcretes are an ideal place to look for “internal” subterranean population structure.
Inaccessible to humans, they are sampled via boreholes. In two recent studies, dense
fields of orthogonally arranged boreholes were used to gain insight into the small-
scale genetic structure of selected aquatic troglobionts. First, at the 3.5 km2 large
borefield of the Sturt Meadows calcrete, three sympatric species of dytiscid diving
beetle were examined for genetic structure using mtDNA sequences (Guzik et al.
2009). In all three species, a remarkable diversity of mtDNA haplotypes was
discovered that was highest within single bores and showed an isolation-by-dis-
tance-like behavior at a distance of maximally 2.5 km in the larger two species. A
lack of population structure in the smallest species was interpreted of consequences
of its better dispersal capability across matrix with small pore size. A similar study in
the same area on chiltoniid amphipods using allozymes in addition to mtDNA
(Bradford et al. 2013) revealed the possibility of an east-west bipartition and a
weak (FST up to 0.14) structuring by some of the individual bores for the most
common species. With the second most common species showing isolation by
distance, the amphipods, just as diving beetles appeared to be slightly limited in
their dispersal through the subterranean matrix. Guzik et al. (2011) chose a larger
window to look at potential population structure of dytiscid diving beetles, an
amphipod, and an isopod in the Laverton Downs calcrete. They sampled the fauna
via a series of boreholes at three different points over a distance of 15 km and looked
at mtDNA sequences. Just as in Sturt Meadows calcrete, the majority of genetic
diversity was within bores, and some structuring was observed around groups of
boreholes, in part possibly because of the influence of a nearby salt lake that
restricted dispersal. Isolation by distance was detected for most of the species but
only at distances larger than 6.5 km. This second study confirmed that limited
dispersal and habitat heterogeneity shape the genetic structure within continuous
subterranean habitats at the scale of 5–10 km in a similar way as they do at a smaller
scale.
Strong genetic structuring at much shorter geographical distances (<50 m) was
reported for small crustaceans, the parabathynellid Bathynellaceans, in an alluvial
interstitial habitat in New South Wales, Australia (Asmyhr et al. 2014; Fig. 12.3).
The entire studied borefield was no longer than 300 m. Inferred migration rates
between more distant bores were below one percent, which, upon taking into
account an average sample size of about 20, can be translated to an FST value of
about 0.9 (very high). It is noteworthy that the mtDNA sequences of the sampled
278 P. Trontelj
Fig. 12.3 Fine-scale genetic structure of small-bodied crustaceans in interstitial groundwater.

Spatial autocorrelation serves as measure of the genetic similarity between pairs of individuals
within a specified distance range. Spatial autocorrelation coefficient ¼ 0 (gray line) corresponds to
the null model of random dispersal within a given distance. Only for boreholes within the range of
about 30 m are individuals more similar than expected by chance. At larger distances, migration in
the interstitial is slower than expected by chance (Based on microsatellite data of an unspecified
species of Parabathynellidae from the alluvial interstitial in New South Wales (Australia; Asmyhr
et al. 2014))
parabathynellids averaged at about 2% and did not indicate a deep taxonomic

structure, demonstrating the superiority of highly variable nuclear markers
(microsatellites) for population-level studies.
The work by Asmyhr et al. (2014) is not directly comparable with the previous
ones as the type of molecular markers and analytical methods (Bayesian assignment
tests) applied were different, but the basic message is the same. Both the alluvial
interstitial and the calcrete matrix are small-pore aquatic subterranean habitats, and
both show very low migration rates and slow dispersal despite a continuous distri-
bution in a continuous habitat. From the point of view of the interstitial highway
hypothesis (Ward and Palmer 1994), the “highway” may still validly describe the
riparian or hyporheic interstitial as continuous linear habitat, but certainly not the
speed at which its inhabitants disperse. With respect to the new model, the important
message is that the endogenous process of subterranean dispersal, while possible
along continuous habitats, is likely to be extremely slow compared to surface
dispersal and thus in a purely numerical sense is a less probable scenario. Further-
more, recently developed mathematical models suggest that when dispersal proceeds
at a very slow pace and the reach of single individuals is short compared to the entire
range, new species may evolve parapatrically without the need for ecological
discontinuities (Hoelzer et al. 2008), especially in linear habitats (De Aguiar et al.
2009). This possibility, so far not yet considered by biospeleologists, offers an
additional and elegant explanation to the all-pervading phenomenon of subterranean
cryptic species.
12.3.2 Cave Populations Tend to Have an “All-or-Nothing”

Structure
In caves and karstic (i.e., large pore) aquifers, a number of studies revealed two
possible patterns of genetic structuring of populations. One is a nearly panmictic or
admixed pattern; the other one is strong structuring according to local and regional
hydrological boundaries.
A typical situation is that found by Carlini et al. (2009), who sampled three caves
and several surface populations of the gammarid amphipod Gammarus minus along
a 50 km stretch of the Greenbrier River in West Virginia, USA. They sequenced
mitochondrial DNA and nuclear internal transcribed spacers (ITS1) from up to
20 individuals per sampling site. The three caves contained completely different
sequences, belonging to separate clades, and were obviously colonized at three
separate occasions from different ancestral populations. They are completely iso-
lated from each other. Conversely, within Organ Cave, at a distance of about 0.5 km,
no genetic structuring was detectable, meaning that the amphipods disperse and mate
without restriction inside the large cave.
On the other side of the Atlantic, another widespread and common aquatic
crustacean, the asellid isopod Asellus aquaticus, is known for its rich and complex
history of cave invasions. In the northwest Dinaric Karst, studies using mito-
chondrial and nuclear DNA sequences (Verovnik et al. 2004), randomly amplified
polymorphic DNA (Verovnik et al. 2003), and microsatellites (Konec et al. 2015)
revealed deep genetic differentiation between cave population from different drain-
age basins. A phylogeographic analysis based on mtDNA suggested that caves in
each basin have been invaded separately, which, besides the lack of hydrological
connections, explains the deep structure. In the central part of the Slovenian Dinaric
Karst, inside the Postojna-Planina cave system, a seemingly different pattern popped
up. Inside a single cave system, three separate cave populations coexist in a
parapatric manner (Verovnik et al. 2003, 2004). At a first glance, this looked like a
rare or possibly the first known case of endogenous, subterranean fragmentation
within a single cave. However, later microsatellite studies (Konec et al. 2015 and
unpublished data) suggested that the three populations are simply three inde-
pendently founded and evolved subterranean populations in secondary contact and
partially reproductively isolated. A tentative example of a vicariant range frag-
mentation within a subterranean species was nevertheless identified in the nearby
subterranean Reka river system, inhabited by the recently described close relative
Asellus kosswigi (Konec et al. 2016). In the uppermost part of the system, about
400 m higher than other known sites, a small population with different mitochondrial
haplotypes has been discovered. It is believed that such an altitudinal difference at a
short horizontal distance presents an insurmountable barrier for the aquatic isopods.
The area is tectonically very active, so today’s disjunct distribution could have
resulted from times when the piezometric levels where more even.
A similar system of a widespread surface population and numerous but localized
cave populations as in both aforementioned crustaceans is presented by the famous
280 P. Trontelj
Mexican characid cave fish, Astyanax mexicanus. All cave populations live within a
relatively small area (about 150 km by 40 km), the Sierra de El Abra region of east-
central Mexico. No other cave animal has been studied as thoroughly and by as many
researches as the Mexican cave fish. Three recent comprehensive studies, building
upon previous mtDNA phylogeographies, used microsatellite (Bradic et al. 2012;
Strecker et al. 2012) and genome-wide SNP data (Coghill et al. 2014) to unravel the
complex story of multiple invasions from multiple surface lineages to multiple cave
populations and occasionally the reverse. The three studies partly disagree about the
number of lineages that independently invaded the caves (from three in Strecker
et al. 2012 to five or more in Bradic et al. 2012) and on some details in timing of gene
flow and dispersal. Nonetheless, they all uncovered a common pattern. Some cave
populations exchange genes with surface population in both directions. Hence, these
cave populations are directly or indirectly connected by migration via the surface,
where there is hardly any genetic structuring (Bradic et al. 2012). Cave populations
or groups of cave populations are genetically either completely distinct from each
other (the El Abra, Guatemala, and Micos cave populations), or they show almost no
structure at all. In the central group of six El Abra caves, spanning about 30 km,
microsatellite-based FST values are mostly insignificant and rarely exceed 0.1
(Bradic et al. 2012). Strong structure (FST 0.3) arises only compared to the Pachón
Cave population that is 60 km away, but this might well stem from a separate
invasion or recent genetic exchange with different surface populations.
12.3.3 Dispersal of Evolutionarily Old and Large-Bodied

Cave Species Probably Proceeds via the Surface
Just like the crustaceans G. minus and Asellus, the cave fish A. mexicanus is not the
typical, evolutionary mature cave animal that has ceased exchanging genes with its
surface ancestor. The genetic structure of their cave populations is still determined
exogenously, by the genetics of their surface ancestors and the dynamics of coloni-
zation events rather than by intrinsic conditions of the cave environment. Large
ranges, beyond the boundaries of physical connectivity of the cave habitat, are
readily imaginable as consequence of multiple, spatially separate colonizations
followed by parallel or convergent troglomorphic evolution. Conversely, there is
no easy answer to the question how evolutionarily old cave species that have lost
contact to their surface relatives long ago reach and maintain their large distributions.
For example, the last surface ancestor of Niphargus, a diverse genus of European
cave and groundwater amphipods, was marine and lived in the Mesozoic. There
probably never was a surface proto-Niphargus to disperse over the continent and
colonize the karst areas one by one. It all had to be achieved by fully troglobiotic
animals.
The next interesting question therefore is whether such evolutionarily old cave
species undergo some active concurrent processes that maintain their populations
structured and yet not entirely separated from each other. In theory, these processes
are endogenous, because they must have originated from an already evolved
troglobiotic population and should be controlled by the conditions of the subterra-
nean environment only. Potential aquatic examples that have been extensively
studied for their mtDNA and nuclear gene variation include cave amphipods of the
genus Niphargus (Niphargidae) and atyid cave shrimps Troglocaris sp. Most of
these species as adult measure several centimeters in length and are thus bound to
large-pore habitats, usually karstic aquifers and caves. Nominal species like
Niphargus virei and N. rhenorhodanensis from western France, N. laticaudatus
from the Western Carpathians and T. anophthalmus from the Dinaric Karst all
have relatively large ranges, reaching up to several hundred kilometers (Lefébure
et al. 2006, 2007; Zakšek et al. 2009; Meleg et al. 2013). All these nominal species
harbor considerable cryptic diversity. At a closer look, even within some of the
cryptic species a further, hierarchically lower level of mutually exclusive clades
exists that contains further cryptic species. Such is the pattern within two of the
geographically largest clades of N. virei (Lefébure et al. 2006) and some of the
N. rhenorhodanensis clade, while in at least one of the N. rhenorhodanensis clades,
a geographically admixed DNA haplotype distribution suggested recent dispersal
(Lefébure et al. 2007). Similarly, one of the phylogeographic clades of
T. anophthalmus sensu lato extending across 300 km of hydrographically
fragmented karst terrain displayed a genetic pattern of recent dispersal, while other
large-range clades were genetically structured either in an isolation-by-distance-like
way or by allopatric fragmentation (Zakšek et al. 2009). Where sampling was dense
enough, e.g., in the W-Slo clade, good connectivity and dispersal within existing
hydrological connections were found, as a small number of common haplotypes
were shared among interconnected caves.
Thus, we are observing two types of genetic and phylogeographic patterns in
these evolutionarily old subterranean species. One is a hierarchically arranged
subclustering from large to smaller geographical areas. The other is the lack of
structure within small but occasionally also quite large areas. While the lack of
structure in small areas of contiguous habitat is expected, all distributions crossing
hydrological boundaries require some unknown means of dispersal. Vicariant frag-
mentation of a wide ancestral range, like the one of Niphargus virei sensu lato,
spanning over 800 km, is no solution to the puzzle, because it still requires wide-
range dispersal of a subterranean ancestor to start with. Therefore, most authors have
considered the possibility of some form of passive or active dispersal via surface
habitats. Some are very cautious about it, e.g., “it seems that its ancestor was more
ubiquitous than today, and that it was able to disperse through surface water”
(Lefébure et al. 2006). Others are more explicit: “Stygobitic crayfishes are severely
limited in dispersal abilities by both subterranean and surface barriers, except during
high water levels when they can migrate (or wash) out of caves” (Buhay and
Crandall 2005). Whoever is more correct, occasional occurrence of troglobionts
outside caves is a well-known phenomenon (Bressi et al. 1999; Graening et al.
2006), and it is only a matter of time when one of them will reach the next section of
subterranean habitat. As for young troglobionts independently invading multiple
282 P. Trontelj
caves, the key to understanding large subterranean ranges here, too, lies on the
surface. When we are interested in exclusively endogenous processes, we should
focus on smaller areas of presently or historically contiguous subterranean space.
Freshwater stygobionts with phylogenetic, ecological or geographic affinities to
marine environments often have large, disjunct ranges. A number of scenarios have
been proposed about how they might have dispersed across such large areas using
coastal or marine waters as medium (Notenboom 1991; Boutin and Coineau 2000).
The proposed scenarios consist of two major evolutionary transitions, one from
marine to freshwater and the other from pelagic or epibenthic lifestyle to a subter-
ranean one. While the order by which they occur may vary, they are usually
supposed to be irreversible, i.e., a population, once it has colonized the subterranean
environment and become stygobiotic, does not return to the sea. However, for
young, non-relictual but disjunct populations, this may be a valid alternative hypoth-
esis to consider. The five disjunct population clusters of the Cuban cave fish
Lucifuga dentata mentioned at the beginning of this chapter have evolved by
gradually “jumping” from one isolated karst area to the other (Hernández et al.
2016). All five areas are close to the current shoreline; thus past sea-level fluctuations
might have created occasional connections to the sea, enabling the fish to disperse
via a non-subterranean habitat. In that way, exogenous processes might shape the
population structure of widely distributed stygobionts with marine affinities. Here,
too, the real barriers to dispersal seem to lie between caves, not between the surface
and the cave.
12.3.4 Non-dispersal Has Different Meanings in Karstic

and in Interstitial Groundwater
A general problem with most of these studies is that they were not designed for
detecting fine-scale genetic structure within caves or cave systems. Other molecular
markers, preferably microsatellites or SNPs, and a much denser sampling would be
required for this purpose. But from what has been found so far, we can infer the
following. Evolutionary old, large-bodied cave species and troglobionts from deep
cave/subterranean habitats, both terrestrial and aquatic, are highly mobile within the
limits of their interconnected subterranean habitat. In single caves and cave systems,
even very large ones, genetic structure is weak or lacking. They have evolved
locomotory, sensory, and energetic mechanisms optimally adapted to moving
through the large-pore subterranean space. The proof of existence of such mecha-
nism was one of the great achievements of the American neo-Darwinian school of
subterranean biology (Poulson 1963; Christiansen 1965; Culver 1982; Culver et al.
1995).
On the other hand, hydrological divides, geomorphological brakes,
non-permeable bedrock, and other discontinuities of their habitat represent formi-
dable barriers that sharply shape the structure of populations and species. Therefore,
in aquatic organisms, the non-dispersal paradigm does not mean the same for karstic
and interstitial groundwaters. In the latter, low dispersal results from an intrinsic
combination of small body size and physical or ecological limitations of the small-
pore labyrinth structure of habitat, which in itself is not necessarily fragmented. In
the former, locomotion is probably just as fast as in similarly sized and built surface
organisms, but their dispersal is restricted extrinsically, by insurmountable ecolog-
ical barriers.
12.3.5 Terrestrial Troglobionts Are Less Studied but Seem

to Share Basic Patterns of Genetic Structuring
with Aquatic Ones
There is much less recent data available for the genetic population structure of
terrestrial troglobionts than for aquatic ones. A lot of work has been done in the
allozyme era on rhaphidophorid cave crickets of the genus Dolichopoda (Sbordoni
et al. 2000) and leiodid (Speonomus, e.g., Crouau-Roy 1989) and carabid
(Pseudanophthalmus, Darlingtonea, e.g., Kane et al. 1992) cave beetles.
Box 12.1
The results of these studies agree remarkably well with what has recently been
found for aquatic subterranean fauna using DNA sequences and other modern
molecular markers. For instance, the small Speonomus beetles living in caves
as well as in deep soil and MSS showed a pattern of genetic differentiation
among populations resulting from slow dispersal, just as in the interstitial. The
population structure of larger-bodied cave beetles responded to geological
features and rivers, indicating brakes in the connectivity of the subterranean
habitat and possibly barriers to surface dispersal. In the large Dolichopoda
cave crickets, surface dispersal played an essential role for the genetic struc-
turing of populations. Populations in areas with unfavorable surface habitat
were strongly structured, while those in areas with favorable surface habitat
were only weakly structured.
A small number of population-level phylogeographic studies on terrestrial

troglobionts using mitochondrial and sometimes also nuclear DNA sequences are
in agreement with the forgoing suggestions: cave populations form interconnected
clusters that are completely isolated from each other (all-or-nothing structure). The
current subterranean distribution is mostly believed to reflect the distribution and
structure of ancestral surface populations (Villacorta et al. 2008). A detailed
phylogeography of Caribbean Amblypygi of the genus Phrynus, which are not
exclusively cave bound, suggested that barriers to surface dispersal such as
284 P. Trontelj
geological substratum, unsuitable habitat, and climate are responsible for the island-
like structure of cave populations (Esposito et al. 2015).
Alternatively, disjunct distributions separated by extensive areas of non-karstic
habitat can be explained by surface movements of animals that are otherwise deemed
strict troglobionts during short periods of favorable climatic conditions (Rizzo et al.
2013). Both scenarios require hypothesizing exogenous processes to explain an
endogenous pattern. A noteworthy possible exception has been reported for cave
plant hoppers of the Oliarus polyphemus species complex (Wessel et al. 2013). The
authors suggest that the island-like population structure resulted from a combination
of very low random dispersal through the subterranean lava tube network of Hawai’i
island and very fast, non-ecological speciation. It is questionable, however, whether
this model is applicable to other cave species.
Two recent phylogeographic and molecular systematic studies on cave beetles
illustrate how difficult it can be to distinguish between dispersal and fragmentation,
past and present habitat connectivity, as well endogenous and exogenous processes.
Both studies are primarily systematic and not intended to disentangle population
structure. However, since they cover small areas (>100 km), densely packed with
even smaller allopatric ranges of closely related species, these can be used as proxy
for strongly structured populations. The first case study, conducted in the eastern
Pyrenees on the carabid genus Geotrechus, suggested that the strictly allopatric
distribution of seven closely related species resulted from a series of vicariant events
that, over a period of about 10 million years, had fragmented a once contiguous,
larger ancestral range (Faille et al. 2015). Since these beetles are quite capable of
dispersal, as they are found also in artificial caverns, occasional dispersal events
across geomorphological boundaries cannot be ruled out. Ancestral dispersal might
have proceeded during wetter and therefore more favorable conditions for surface
dispersal. The processes behind the current allopatric and highly fragmented struc-
ture were therefore probably exogenous or mixed rather than exclusively endoge-
nous. In a superficially very similar situation in the southern Dinaric Karst, the
cholevid genus Hadesia has evolved into five allopatric species during the past
3 million years (Polak et al. 2016). However, circumstantial evidence clearly points
toward a purely endogenous process ever since the ancestral Hadesia evolved. All
species of the genus share a highly specialized way of life, filter-feeding on cave
walls in films of flowing water. Their complexly elaborated mouthparts and feet are
identical in all five species and thus very unlikely to have evolved independently.
One cannot imagine these animals surviving and migrating in any other habitat but
wet caves. The most likely scenario thus foresees an endogenous evolutionary origin
of the ancestral Hadesia, followed by subterranean dispersal over the entire range of
the genus during the warm and wet mid-Pliocene, when cave development in the
Dinaric Karst was at its height. The possibility that there existed a huge, at least,
temporarily interconnected cave and crevice system is supported by the fact that
today this is one of the most highly karstified areas in the world (Milanović 2015).
During later period of cooler and drier climate, the range gradually became restricted
to isolated habitat fragments, where there was enough percolating water to sustain a
permanent cave hygropetric environment. The five known Hadesia species,
although descendant from a single endogenous ancestor, thus probably evolved

allopatrically in what may be one of very few documented cases of genuine
subterranean vicariance.
The above examples demonstrate that the inferential procedure to discern
between exogenous and endogenous evolutionary processes is not a straightforward
one. Adequate background knowledge on the biology of organisms is crucial for the
study of genetic connectivity (Pante et al. 2015). Explaining large ranges, structured
populations and high diversity of subterranean populations requires data on the
geological, hydrological and climatic history (Rizzo et al. 2013). Only then can we
incorporate our population genetic and phylogenetic data into hypotheses in a way
that makes biological sense. An essential part of these hypotheses is the origin and
evolutionary dynamics of genetic variability of cave populations, which is the
subject of the next section.
12.4 Genetic Diversity of Cave Populations
12.4.1 Conflicting Theoretical Predictions and a Paradox
Genetic diversity determines a population’s fitness and adaptive potential (Reed and
Frankham 2003; Allendorf and Luikart 2007). The question of how subterranean
populations obtain and maintain their genetic diversity is therefore central to the
understanding of the evolutionary success of subterranean life. Over the past four
decades, it has been the matter of some debate and paradigm shifts. Even nowadays,
researchers keep reporting genetically surprisingly diverse as well as unexpectedly
uniform populations, reflecting the uncertainties of theoretical predictions.
Before the first empirical data on genetic diversity of cave populations became
available in the form allozyme data, two contrasting views had formed (Fig. 12.4).
Barr (1968) suggested a model in which a small founder population becomes
isolated underground and, if successful, gradually recovers from the reduced diver-
sity by expanding into the unoccupied subterranean space. Conversely, Poulson and
White (1969) proposed that genetic variability of a cave population gradually
decreases because of the constant selective environment, isolation, small population
size and longer generation times as compared to the surface ancestor.
Soon thereafter, the results of several allozyme studies announced that a simple
and unequivocal solution is not to be expected. Some researchers found evidence in
support of the reduced genetic variability hypothesis (e.g., Avise and Selander 1972;
Laing et al. 1976; Swofford et al. 1980). Others detected high genetic diversity that
equaled or exceeded that of related surface populations (Turanchik and Kane 1979;
Sbordoni et al. 1980; Culver et al. 1995). These studies were not without drawbacks.
Only two of them—on Astyanax cave fish (Avise and Selander 1972) and the
amphipod Gammarus minus (Culver et al. 1995)—were able to compare surface
populations with their derived cave populations. All others had to use surface
populations of different species, with no guarantee that they truthfully represent
286 P. Trontelj
Fig. 12.4 Two contrasting models of the dynamics of genetic diversity of cave populations. Left:
low diversity after colonization results from a genetic bottleneck; if successful, the population
gradually adapts and expands into the new habitat, eventually restoring its genetic variability (Barr
1968). Right: the initial genetic variability of the surface ancestor decreases after colonization as
consequence of selection in an isolated, unvarying environment that is spatially limited and poor in
food (Poulson and White 1969)
the genetic diversity of the direct surface ancestor. Reliable age estimates for cave
populations are crucial for testing the two hypotheses but were mostly lacking.
Culver (1982) reformulated the hypotheses explaining genetic diversity of cave
populations to incorporate the neutralist-selectionist debate that was very popular
at that time. This is important because the DNA-based genetic markers that became
widely used in the late 1990s are mostly selectively neutral. The prediction for
neutral genetic variation is to be low in small populations and to reflect temporary
reduction in population size (low effective population size, Ne ¼ an idealized
measure of the average number of individuals of a population that contribute their
genes in equal shares to the gene pools of succeeding generations) that is believed to
have occurred during cave colonization. Culver (1982) commented that genetic
variability in cave populations shows no consistent pattern and that it is unlikely
that this pattern will become any simpler when new data become available. It turned
out he was right.
The one thing most authors seem to agree with is the existence of some form of
genetic bottleneck or founder event reducing the initial genetic diversity of a new
cave population. Paradoxically, such a restriction in population size and available
genetic variation for selection to act on is incompatible with the observed multitude
of highly successful, replicated, and ongoing cave invasions as well as the fast and
consistent evolutionary change taking place. This is a dilemma well known from
invasion biology: how do newly founded populations overcome the low genetic
diversity and the expected low evolutionary potential, typically associated with
extinction risk, to become established outside their native habitat (Roman and
Darling 2007)? The paradox has been largely resolved by discovering that successful
biological invasions tend to be driven by high propagule pressure (Simberloff 2009).

Propagules are the invading individuals, and high propagule pressure means that
these are invading in higher numbers and diversity than expected by a single chance
dispersal event. This can happen simply because the numbers of invading individ-
uals are high, through ongoing migration between the original and the new habitator
by augmentation through repeated invasions or by any combination thereof. Because
colonization of caves is nothing but a specific form of biological invasion, it is
reasonable to expect that the success of cave colonization is determined by propa-
gule pressure, too.
12.4.2 Exogenous and Endogenous Factors Affect Genetic

Variability in Different Ways
There are three ways by which genetic diversity becomes available to a cave/
subterranean population:
1. Directly from an ancestral surface population. Since no new mutations are
involved, this kind of genetic variability is called standing genetic variation. It
is more important in young populations than in old ones, and it probably plays a
major role in early stages of adaptation. It determines the potential of a population
to adapt. Standing genetic variation of a new cave population depends entirely on
exogenous factors: the genetic diversity of the ancestral population, random
sampling of alleles (drift) and propagule pressure. During the later course of its
subterranean existence, a cave population will inevitably loose some its initial
genetic variability due to drift. This is controlled by endogenous factors such as
effective population size, mating system, and demography of the cave population.
Several molecular studies (see next section) provide insight into the genetic
diversity of young cave populations and the demography of cave lineages.
2. By endogenous exchange with other cave populations. Separate cave populations
could, in theory, become interconnected when barriers to subterranean dispersal
between them break down. The resulting genetic admixture or gene flow would
increase the genetic diversity of the populations receiving migrants. Although this
is a pattern commonly encountered in surface species, no such case has been
documented for troglobionts so far. The handful of troglobiont populations that
are known to exchange genes by underground migration, e.g., Astyanax
mexicanus in Mexico (Bradic et al. 2012; Strecker et al. 2012) and Asellus
aquaticus in Romania and Slovenia (Verovnik et al. 2004; Konec et al. 2015),
are geographically close and relatively young populations with their surface
ancestor still present. For these populations it is difficult to disentangle exogenous
or mixed from purely endogenous processes.
3. By new mutations. This is a purely endogenous process. The accumulation of
new (neutral) mutations is expected to proceed at slightly lower rate when the
generation cycle becomes longer. There are suggestions that this might be
288 P. Trontelj
compensated by a higher mutation rate, for example, because of higher radon

concentrations in caves (Allegrucci et al. 2015). This subject remains speculative.
It is much clearer that the available subterranean habitat is usually much smaller
than surface habitats, as is the available energy. This, in combination with low
connectivity, is expected to lead to lower Ne on the long term than in surface
populations of taxonomically and functionally similar species. However, using
various methods for reconstructing demographic history, researchers have found
that populations of old troglobionts can be more dynamic and experience popu-
lation growth long after they have invaded the subterranean realm. This could
happen when ecological conditions are not as constant as usually assumed, for
example after a long-term rise of the groundwater table in calcrete aquifers of
Western Australia (Guzik et al. 2009; Bradford et al. 2013).
Of these three groups of processes that are generating genetic diversity of cave
populations, only the first—the exogenous one—has been sufficiently explored.
However, new molecular data seem to be more in support of the high propagule
pressure hypothesis of invasion biology than of the traditional genetic bottleneck
view of subterranean biology. This dilemma has rarely been addressed by
biospeleologists.
12.4.3 Exogenous Propagule Pressure Appears to Shape

the Genetics of Young Cave Populations More than
Bottlenecks
The most direct test of the genetic bottleneck hypothesis is to compare the gene pool
of recently established cave populations to that of the surface population from which
it has been derived. This kind of analysis is hampered by the scarcity of known cases
where troglobiotic populations exist alongside their direct surface relative.
The heterozygosity of Astyanax populations estimated on 26 microsatellite loci
was significantly lower in caves of the El Abra region (0.55 0.07) than in adjacent
surface populations (0.82 0.04); so was the allelic number corrected for sample
size, 2.54 0.26 in El Abra caves and 3.63 0.14 in surface populations. By
comparison of gene pools, Bradic et al. (2012) found out that the allelic contents of
cave populations are subsets of alleles of the surface stock. In agreement with the
genetic diversity, data were estimates of effective population size, which averaged
about 1000 in caves, and were about two to three times larger in surface populations.
However, error rates of these estimates were high and largely overlapping. It needs
to be mentioned that Ne is a tricky parameter to estimate, and estimates depend
heavily on the method and its underlying assumptions. Comparisons between
estimates based on different methods and markers can be misleading (Skrbinšek
et al. 2012). While differences in genetic diversity were mostly significant, their
magnitude is not what one would expect to be after a severe bottleneck. An analysis
of migration rates between surface and cave populations explains why this is so: all
Table 12.1 Genetic diversity indices of cave populations and co-occurring surface populations
with the relative extent of diversity in caves expressed in %
Observed
Nucleotide diversity (π) heterozygosity (HO)
Taxon Surface Cave % Surface Cave % Source
Astyanax 0.0011a 0.0002 18 0.76 0.53 70 Panaram and Borowsky
mexicanus (2005), Bradic et al.
(2012)
Poecilia mexicana – – 0.50 0.28 56 Plath et al. (2007)
Asellus aquaticus 0.0170b 0.0180 106 0.34 0.19 56 Konec et al. (2015)
Romania
Asellus aquaticus 0.0035b 0.0025 71 0.15 0.05 33 idem
Postojna-Planina
cave system,
Slovenia
Gammarus minus 0.0035b 0.0071 203 – – Carlini et al. (2009)
Georissa 0.0013c 0.0025 192 – – Schilthuizen et al.
filiasaulae/ (2012)
G. saulae
Presented are simplified average values; for original figures, standard errors, and population sizes,
the cited references should be consulted
a
π-values are derived from a genome-wide analysis of randomly amplified polymorphic DNA
(RAPD)
b
π-values are derived from mitochondrial COI gene sequences and their frequencies
c
π-values are derived from mitochondrial 16S rRNA gene sequences and their frequencies
cave populations exchange genes with surface populations, either directly or indi-
rectly via other cave populations. Altogether, recent studies agree that the current
genetic setup of Astyanax cave populations resulted not only from a number of
temporally distinct invasions but also from ongoing genetic exchange with surface
populations. One can safely say that subterranean Astyanax populations are under
strong exogenous influence. Nevertheless, the gene flow from the surface has to be
counterbalanced by strong natural selection to maintain the troglomorphic traits.
The cave molly (cave form of Poecilia mexicana) is less differentiated and
structured than the cave Astyanax, but the pattern of genetic diversity of the only
known troglobiotic population in southern Mexico is quite similar. The Cueva del
Azufre is a sulfidic cave, which means that colonizers are facing a double ecological
gradient—the permanent darkness of the cave combined with toxic hydrogen sulfide
(H2S). Measured by allele frequencies of ten microsatellite loci, the cave population
was significantly less diverse than the surface reference population form non-sulfidic
water (Table 12.1). No migration from the surface into the cave could be detected by
analysis of gene flow. Even though genetically isolated, the population showed no
convincing signs of a recent genetic bottleneck (Plath et al. 2007).
On the invertebrate side, Asellus aquaticus with several independent pairs of
ancestral surface and derived cave populations offers a powerful study system.
Konec et al. (2015) compared population pairs in Slovenia and Romania. Mitochon-
drial DNA diversity was not (Romania) or only slightly (Slovenia) lower in caves
290 P. Trontelj
than at the surface. Microsatellite diversity, measured at eight loci, was up to three
times lower in caves (Table 12.1). High mtDNA diversity of the Romanian cave
population is the consequence of at least two independent invasion events, delivering
two groups of highly divergent mitochondrial DNA haplotypes into the cave gene
pool (Konec et al. 2015). For the Slovenian cave population, the number of coloni-
zation events cannot be inferred from the haplotype network, but shared high-
frequency haplotypes speak against a founder bottleneck. Natural selection seems
to be uncoupled from mitochondrial and microsatellite genetic variability. Neither
did the added selection pressure of toxic H2S in the sulfidic Romanian cave restrict
genetic variation, nor did the smaller genetic variability of the Slovenian cave
population affect the evolutionary potential, as shown by the overall higher number
of changing morphological traits. The Asellus example highlights the importance of
choosing the correct populations for comparison. By matching, for example, the
Romanian surface and the Slovenian cave population one would get the impression
of a strongly reduced genetic diversity in caves. In reality, the Romanian cave
population is just as diverse as the surface population.
In Gammarus minus, two of the cave populations showed very low mtDNA
diversity, which is consistent with a genetic bottleneck. The others got colonized
twice from different sources, which is more consistent with the propagule pressure
hypothesis of cave invasion. Carlini et al. (2009) looked at codon usage bias as a
pointer to population size (small populations are expected to have low bias due to
drift) and found it was lower in cave than in surface populations. While this is
consistent with a low long-term population size, it is not necessarily a consequence
of a genetic bottleneck.
The only terrestrial example comes from a nonstandard cave species. The tropical
cave snail Georissa filiasaulae from Borneo lives in contact with its surface ancestor
G. saulae. It is thought to have evolved in a process of ecological speciation and has
experienced recent gene flow from the surface species (Schilthuizen et al. 2012). The
important point is that, although genetically distinct, it shows a similarly shaped
mtDNA haplotype network and more genetic variability than the surface species.
Apparently, one-way gene flow from the surface is genetically enriching the cave
population. The concepts of speciation with gene flow and ecological speciation are
compatible with the propagule pressure hypothesis but much less so with founder
effects and genetic bottlenecks.
12.5 Conclusions and Perspectives
The predominant, traditional view of the genetics of cave populations is that they are
strongly structured because of the patchiness of subterranean habitats and low
dispersal of cave species. When a group of populations or closely related species
is distributed over a larger area, they are usually explained as the result of multiple
separate invasions with subsequent extinction of the surface ancestor. Genetic
bottlenecks caused by small numbers of invading individuals additionally contribute

to genetic differences between populations.
There is a discrepancy between large and disjunct ranges of troglobionts and low
migration capability inferred by genetic analyses, somewhat similar to the mysteri-
ous occurrence of nonswimmers and non-fliers on remote oceanic islands. Also, the
notion of countless replicated successful invasions is incompatible with the genetic
bottleneck hypothesis because low genetic diversity means low evolutionary poten-
tial and high extinction risk. The past two decades of molecular population genetics
and phylogeography have provided solid solutions to these controversies but left
some points unanswered.
A dual approach that differentiates between exogenous and endogenous pro-
cesses can provide the necessary new perspective. Exogenous processes take place
at the surface or during the invasion phase. Although having a strong impact on cave
life, they can be studied and understood using a conventional ecological and
evolutionary framework. Endogenous processes, on the other hand, are governed
by the specific conditions of cave/subterranean environments. Cave life is shaped by
both, but it is essential to separate the two classes of processes, if we wish to find out
what is really special about caves. With this distinction in mind, the main conclu-
sions of this overview paper are the following:
1. Genetic similarity between allopatric cave populations is rarely the consequence
of contemporary gene flow but rather reflects shared ancestry. Such populations
can be considered as cryptic species under the phylogenetic species concept.
Whether these species evolve by exogenous or endogenous processes is one of
the key questions of subterranean biology.
2. The central question, whether a wide range is reached by dispersal via the surface
or subterranean migration, cannot be answered directly by molecular phylo-
genetic and phylogeographic analyses. Geological, biological and ecological
information needs to be combined with data points from molecular clocks and
demographic analyses in order to formulate testable hypotheses about genetic
connectivity of subterranean populations.
3. Evidence for endogenous vicariance—the breakup of a large, contiguous subter-
ranean range through the action of extrinsic forces—is exceedingly rare. This
might be in part because of epistemological reasons, in part because it requires a
large and interconnected subterranean population to begin with.
4. Evidence for contemporary gene flow between allopatric cave populations is
sparse. Most known cases of recent migration between non-neighboring
populations entail the possibility of dispersal via the surface, even if the species
is an obligate cave dweller. Superficial and small-pore subterranean habitats do
not seem to contribute much to the genetic connectivity between cave
populations. Genetic data suggest gene flow and dispersal within these habitats
are very low. The real barriers to dispersal lie between caves, not between the
surface and the cave.
5. Time since colonization is an essential parameter though difficult to measure for
older cave species. Young populations are those with their direct ancestors or
292 P. Trontelj
sister species still present at the surface. The gene pools of young cave
populations often show signs of multiple colonization events and/or recurring
gene flow from the surface. This is compatible with the high propagule pressure
hypothesis of successful biological invasions. Conversely, convincing molecular
evidence for the genetic bottleneck hypothesis of cave colonization is lacking.
6. The dynamics of genetic diversity of old cave populations is poorly understood.
Published studies suggest that it is shaped exogenously, by ecological influences
from the surface (e.g., long-term groundwater fluctuations), and not by endoge-
nous selective forces. These fluctuations might induce genetic bottlenecks in
already established troglobiotic/stygobiotic species and leave similar genetic
imprints as founder events. That the same genetic pattern can result from two
fundamentally different processes remains a challenge for future investigations.
Acknowledgments The ideas presented in this chapter have grown through intriguing discussions
with David Culver, Cene Fišer, Slavko Polak and numerous other colleagues, students and friends.
They are based on research supported by and conducted under the Slovenian Research Agency
Program P1–0184 and associated projects.
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Chapter 13
Caves as Oligotrophic Ecosystems
Ľubomír Kováč
13.1 Introduction
Box 13.1
Hypogean systems are usually relatively energy-limited (Poulson and Lavoie
2000). Most caves are oligotrophic with low amounts of available organic
matter and low energy density. The trophic structure of these caves is less
complex than that of the less common energy- and nutrient-rich eutrophic
caves, which are characterized by larger amounts of animal faeces (such as bat
guano) and plant debris transported into caves by floods and/or gravity.
Longer caves, where sections differing in amounts of organic material may
be recognized, are called poecilotrophic (Humphreys 2000a). Generally, food
scarcity along with darkness seems to be the main factors shaping the selective
regime in caves (Trontelj 2012).
Oligotrophic systems are environments with low amounts of trophic resources in the
form of organic substances. The limited trophic base of such systems leads to low
diversity and biomass and less developed biotic communities. Such systems occur in
the Arctic, Antarctic and high-mountain tundra where harsh conditions result in very
low primary production in both aquatic and terrestrial habitats. Similarly, oligotro-
phic systems are characteristic of subterranean environments that are relatively
isolated from the surface, thus having very limited organic input.
Chemolithoautotrophy by bacterial communities in oligotrophic systems is usually
negligible. Despite the low organic supply resulting in less developed and less
Ľ. Kováč (*)

https://doi.org/10.1007/978-3-319-98852-8_13
298 Ľ. Kováč
diverse biotic communities, oligotrophic cave systems have been in the focus of
attention from the beginning of cave explorations. Recently, this attention has even
increased as scientists study the composition of the biotic communities of oligotro-
phic caves, the peculiar adaptations of organisms to this extreme environment and
the functional roles of different species in these systems. The focus of this chapter is
primarily on cave passages that appear oligotrophic, while examples from food-rich
or exceptional caves are included for comparison.
Organization of the trophic structure of oligotrophic caves largely depends on
their connectivity and interactions with the epigean environment. Oligotrophic cave
systems represent relatively isolated subterranean spaces without extensive above-
ground connectivity. In most cases, there is a complicated structure with numerous
links between the hypogean environment and its surrounding epigean one (Dunne
et al. 2002; Romero 2009). The connectivity between epigean and hypogean envi-
ronments is relatively high in limestone massifs, where the vadose zone plays an
important role as an ecotone. Mostly vertical downwards movement of percolating
water is characteristic of this zone.
There are two main routes of organic carbon input to caves: (1) localized flow
through sinks and shafts, and (2) diffuse flow through soils, epikarst and the vadose
zone. Oligotrophic caves, and their characteristic low-energy biota, are mostly
associated with diffuse flow (Simon et al. 2007a). The food base for cave biota in
nutrient-poor caves is mostly represented by organic matter transported by percolat-
ing water. This includes dissolved organic carbon (DOC), represented by complex
molecules of fulvic and humic acids that are metabolized very slowly by
actinobacteria. It also includes leached particulate organic matter (POM) that is of
low quality and only slowly used (Poulson 2012). Transport of organic matter from
the surface is limited, with the quantity progressively reduced with depth below the
ground surface. The low amounts of organic matter in the remote and isolated parts
of caves create a kind of biotope suitable for troglobionts, which are adapted to cope
with low food resources (Deharveng and Bedos 2000, see also further text). In Organ
Cave (USA) and Postojnska jama (Slovenia), Simon et al. (2007a) found that most of
the organic carbon entering through the epikarst was DOC at concentrations aver-
aging around 1 mg/L. Overall, the amount of DOC in percolation water depends on
surface ecosystems and residence times of the water in soil, epikarst and the vadose
zone. The amount of DOC in percolation water may be strongly reduced in arid areas
such as Cape Range, Australia (Humphreys 2000b; Simon et al. 2007b). DOC and
POM are further processed within caves, and probably in all voids of the vadose
zone, with an important element for their transformation being microbial films on
rocks—“epilithion” (Culver et al. 2012). In addition, percolating water mediates
transport of living organisms into subterranean habitats, such as microorganisms,
aquatic meiofauna and terrestrial microarthropods (Culver and Pipan 2009; see
Chap. 14 for more information on this topic).
13 Caves as Oligotrophic Ecosystems 299
13.2 Food Variability and Communities of Biota
Food variability and predictability in caves are different for aquatic and terrestrial
habitats. Abiotic input of food by water is represented by plant detritus degraded
through the activity of surface decomposers and leaching by water, so bacterial and
fungal decomposers are also much reduced. On the other hand, irregular and heavy
floods can bring in unleached detritus that may be a food base for the cave
communities for 10–100 years. Biotic input of organic material from trogloxenes
moving into caves is more predictable, and, although it does not bring as much food,
the quality is high (Poulson 2012).
Aquatic species are limited by food that is diluted and dispersed widely, whereas
terrestrial species rely on food that remains concentrated in a local area. Intervals at
which detritus is renewed at the base of the aquatic food web can be of the order of
decades, so there is the selection for long life (Poulson 2012). According to Weber
(2000), food scarcity is stronger in the terrestrial than in the aquatic cave habitats. In
groundwater, low oxygen supply and food availability are overlapping aspects
determining the development of adaptive strategies and distribution patterns of
animals (Hervant and Malard 2012).
Generally, in deep subterranean systems food webs depend on diffuse and
continual input of very small amounts of dissolved organic matter in percolating
water. These cave sections are more oligotrophic and support a small number of species
that tend to be long-lived troglobionts which are adapted to cope with low food
resources (Deharveng and Bedos 2000; Poulson and Lavoie 2000). Specialized
troglobionts can persist in very low-energy environments, being documented even
from isolated spaces of the deepest vertical caves such as Lukina jama–Trojama
( 1421 m) in Croatia (Bedek et al. 2012; Andersen et al. 2016) and Krubera–Voronja
( 2140 m) in Abkhazia, Georgia (Sendra and Reboleira 2012).
Box 13.2
In subterranean environments, food input is not uniformly distributed in space
or time. Cave animals share numerous adaptations to the relative food scarcity
in their habitat. Hüppop (2012) distinguishes three aspects of food scarcity in
caves. General food scarcity holds for nearly all caves and occurs especially in
caves with limited food input. Periodic food supply characterizes caves that
are regularly flooded or caves that regularly receive food input from visiting
animals. During the wet season, food supply can be very high and even
abundant for some weeks or months. After exhaustion of these food reserves,
animals in such caves suffer food scarcity like animals in generally food-poor
caves. Some cave animals have to cope with patchy food scarcity. This means
that food is not necessarily limited but is difficult to find and exploit. Under
such conditions, cave organisms can be observed aggregated at patchy food
resources.
300 Ľ. Kováč
Oligotrophic caves occur in bedrock of all types, i.e. limestones, volcanic (lava
tubes) and ferruginous rocks (see Chaps. 17 and 21), and also sandstones. Recent
exploration in Venezuelan tepuis, table-top mountains in the Guiana Highlands of
South America, provided surprising discoveries of caves and cave systems in
quartzite rocks that were formerly assumed to be insoluble substrate. These caves,
mainly created by the water currents submerging from the upper plateau and flowing
through the tectonic faults, are very scarce in food. Orthoquartzite is characterized by
poor buffering capacity, when compared to carbonates, and limited nutrient avail-
ability. Barton et al. (2014) observed a significant microbial activity on exposed
surfaces within a quartzite cave on Roraima Tepui that appeared to be linked to a
stream flowing through the cave. Biospeleothems were described in several Vene-
zuelan tepui caves (Aubrecht et al. 2012), along with a few cave animals such as the
troglophilic orthopteran Hydrolutos breweri, numerous individuals of which were
observed in Cueva de Charles Brewer, one of the greatest quartzite cave systems in
Venezuela (Derka and Fedor 2010).
Oligotrophic conditions may be also associated with hypogenic caves (see also
Chap. 2). These caves are usually created by the ascending flow of reactive fluids,
characterized by deep-seated solutional aggressiveness in soluble lithologies.
Hypogenic caves may be also the result of the circulation of meteoric groundwater
flow in the uppermost zones of the earth crust. Hypogenic karst thus originates from
void-conduit systems occurring at depth and evolves without direct geological
linkage with the surface (Palmer 2011; Klimchouk et al. 2017). Hypogenic caves
developed by fluids with high concentrations of carbon dioxide, but without sulfidic
emanations, are very poor in trophic sources. Such caves are isolated deeper
underground and may represent larger networks of fissures and voids or great
chambers. Based on the first zoological observations carried out in the eastern
Iberian Peninsula by Sendra et al. (2014), it appears that this type of hypogenic
cave has a depauperate fauna, consisting of non-cave adapted species. This compo-
sition is the result of limited opportunity for faunal colonization during and after
genesis of the hypogenic habitats because of their isolation from the surface, i.e. the
lack of connection between voids and the surface during the entire history of this
type of cave.
13.3 Microorganisms in Oligotrophic Caves
Many caves appear to represent an essentially oxidized and nutrient-limited envi-

ronment. Consequently, any subaerial microorganisms in these systems likely sub-
sist by scavenging sparse nutrients (Barton et al. 2004, 2007; see also Chap. 5). Cave
microorganisms use varying metabolic pathways to obtain nutrients and gain energy.
In autotrophs, carbon for cellular growth originates by converting inorganic carbon
(CO2, HCO3 ) to organic carbon. In contrast, some microorganisms, including most
fungi, have heterotrophic metabolism and use organic compounds for most or all of
their carbon requirements. The physiological mechanisms for capturing chemical
energy are diverse, and the distinction between a chemosynthetic and a photosynthetic
organism is based on whether the initial source of energy is from inorganic chemicals
(litho) or light (photo). Microbes that gain energy through chemosynthesis and fix
inorganic carbon are chemolithoautotrophs. Some microbes are mixotrophs, in which
both chemolithoautotrophy and heterotrophy are expressed simultaneously. Microor-
ganisms that gain cellular energy from chemical transformations but use organic
carbon compounds for their carbon source are chemoorganotrophs (Engel 2012).
Cave microbiota include Bacteria, Actinobacteria, Archaea and Fungi and more
rarely some algae and Cyanobacteria. Bacteria often establish their colonies in caves
without evident sources of organic material (Northup and Lavoie 2004). Moist
surfaces of cave walls and speleothems are colonized by microorganisms in the
form of biofilm. In this way, microorganisms contribute significantly to both the
creation of speleothems and the erosion of limestone (Cuezva et al. 2009).
Subterranean microbial communities were originally considered to be a subset of
soil communities flushed underground by meteoric drip waters, surface streams and
air currents or carried by animals into caves. Because of oligotrophic conditions,
microbial biomass in caves was considered to be low and insufficient to impact most
geological or geochemical processes (Engel 2012). The studies from recent decades,
using modern methods for detecting microbial forms and revealing their metabolism
pathways, have brought much deeper insight into cave microbial communities in
oligotrophic caves and revealed more complex systems than previously thought.
Many of the cave microbes are genetically divergent from surface microbes and
adapted to the aphotic and oligotrophic cave environment. It was shown that in
nutrient-poor caves heterotrophic bacteria dominate the microbial community,
accompanied by a number of chemoautotrophs. Bacteria present under the oligotro-
phic environment of caves survive using complex metabolic pathways (Ortiz et al.
2014; Oliveira et al. 2017; De Mandal et al. 2017). Thus, despite being starved of
nutrients, caves contain surprisingly diverse microbial communities. Chapters 5 and
6 provide more details about the diversity of microorganisms in caves.
In addition to having values in their own right, microorganisms play a very
important role in subterranean ecosystems as a major food source for other, more
complex organisms. Microorganisms stand at the base of the subterranean food web
(see further text).
13.4 Faunal Communities in Oligotrophic Caves
In contrast to assemblages of guano and other energy-rich microhabitats (flood

debris, root mats) with rich communities of non-troglobiotic/stygobiotic species,
oligotrophic microhabitats are characterized by high proportions of the community
being troglobiotic or stygobiotic. In Southeast Asia, troglobionts are mainly found
foraging around scattered micropatches of food resources, mostly dispersed faeces of
swiftlets, bats or rhaphidophorid crickets, and never colonize guano accumulations.
Stygobionts are found in subterranean water flows and pools, in phreatic and
302 Ľ. Kováč
percolating waters and more rarely in surface streams which have been filtered
through fine boulders. They are usually absent in large tropical sink-resurgence
systems, where epigean fauna is often abundant (Deharveng 2004a). This is, how-
ever, not the case of some large temperate hydrological systems, such as Postojna-
Planina Cave System, which hosts 62 stygobionts (Sket 2012). The pattern of high
proportions of troglobionts and stygobionts being associated with oligotrophic
habitats is not universal, since a high proportion of troglobionts may sometimes be
associated with high amounts of organic debris. For example, root mats often
support diverse community of troglobionts, as is the case of Bayliss Cave, a lava
tube in North Queensland, Australia (Howarth and Stone 1990; see also Chap. 3).
The food supply in tropical and subtropical caves is richer than in temperate ones
because of the higher production of biomass in the tropical biomes (Poulson and
Lavoie 2000). As a consequence, selection pressure can be expected to be weaker,
the rate of evolution slower and the occurrence of troglobionts limited when
compared to caves with low energy input such as temperate ones (Hüppop 2012;
see also Chap. 4). However, as emphasized above, patterns are not universal and the
rarity of troglobionts in tropical caves may be only the result of sampling error; very
few tropical caves have been appropriately sampled.
In spite of the different nature and origin of the various sources of nutrients, the
composition of terrestrial troglobiotic communities is rather similar in all oligotro-
phic habitats for a given underground system, provided that humidity is high (see
also Chap. 3). The amount, rather than the nature, of the food supply seems to control
the presence of troglobionts and the composition of their assemblages. In most cases,
troglobiotic faunas have fewer species than guano communities. In tropical oligo-
trophic caves, larger predators, such as amblypygids and scorpions, are mostly
absent, probably because of the low density of potential prey (Deharveng and
Bedos 2000), although some exceptions are found among scorpions (see Chap. 7).
Most primary consumers are insects, while predators are arachnids, mainly small
spiders. Most of these species exhibit some degree of troglomorphy, particularly the
Collembola (Deharveng and Bedos 2000).
13.5 Morphological and Biological Adaptations of the Cave

Fauna
In caves, food scarcity acts as a selective force and requires evolutionary adaptations
(Deharveng 2004b; Trontelj 2012). Food scarcity in caves is associated with devel-
opment of morphological adaptations, so-called troglomorphisms (Christiansen
1965, 2012; see also Chap. 4) or more appropriately troglobiomorphisms (Juberthie
1984). The most obvious morphological modifications are longer legs and antennae
in arthropods and fins and barbels in fishes. Elongated appendages combined with
enlarged or multiplied sensory areas may improve or change food-finding ability and
foraging behaviour of animals in hypogean environments through better sensory
perception. Thus, cave animals can detect food faster and at a greater distance from
their bodies than their epigean relatives and spend less energy searching for food. As a
consequence, when foraging, they have abandoned shoaling and aggregation behav-
iour and adopted a continuous moving mode to cover a greater area. The benefits of
shoaling behaviour in terms of reduced predation risk are lacking in most cave fishes
(Timmermann et al. 2004; Hüppop 2012). In contrast to the situation in caves,
movement through an interstitial medium is presumably facilitated by shortened
appendages and elongated bodies that some troglobionts and stygobionts possess
(Hüppop 2012). Reduced scales in stygobiont fish and reduced cuticle in troglobionts
are other characteristic traits of cave animals. Weber (2000) argued that the body size
of stygobiotic fishes and salamanders, which are usually small in comparison to
epigean relatives, is an adaptation to the low food supply. However, the considerable
reduction in morphological features is often correlated with the presence of the large
amounts of stable food resources. And finally, food scarcity may trigger a switch to
individuals with greater dispersal ability (see Chap. 4 for more details).
Special morphological traits have evolved in the inhabitants of moist hygropetric
habitats (see also Chap. 3). Conspicuously, troglobiotic leptodirine beetles of the
genera Tartariella, Hadesia, Nauticiella, Radziella and Cansiliella occupy such
places in caves of the Eastern Alps and Balkan Peninsula. The hygropetric
leptodirines have enlarged and curved claws, and modified mouthparts, character-
ized by a marked setation and by knee-shaped mandibles. The modified mouthparts
are an adaptation for filtering organic particles from the percolating water. A similar
set of morphological traits occurs in semiaquatic Diplopoda and in some aquatic
crustacean genera of the families Asellidae and Niphargidae (Moldovan et al. 2004;
Sket 2004). Another set of morphological modifications for hygropetric habitats is
found in the troglobiotic collembolan Pygmarrhopalites aggtelekiensis, which is
endemic to the Western-Carpathian caves. It has significantly extended antennae and
legs with elongated claws that are adapted to move over the slippery surfaces of
sediments and speleothems covered with a percolating water film (Kováč et al.
2014). The same habitat is occupied by the strikingly troglomorphic collembolan
Disparrhopalites tergestinus, found in a single cave in the Eastern Alps (Italy) and
Disparrhopalites sp., which occurs in several caves in Croatia and Bosnia and
Herzegovina and has extremely long antennae.
An extreme case of adaptation to oligotrophic habitats is the chironomid dipteran
Troglocladius hajdi discovered in Lukina jama–Trojama Cave System in Croatia at
980 m depth. The combination of strongly reduced eyes and large, broad wings is
unique and might indicate that the species is able to fly or hover in the total darkness
of the cave (Andersen et al. 2016). In vertebrates, there are also extreme cases of
adaptations to low food in underground streams. These include the waterfall climbing
loach Cryptotora thamicola, a troglobiotic fish distributed in a few caves of northern
Thailand (Trajano et al. 2002; Fenolio 2016), and the climbing troglophilic catfish
Chaetostoma microps from Ecuador (Hoese et al. 2015). The loach lives out of water
for extended periods, using its well-developed pectoral and pelvic fins to crawl out of
the water to feed on bacteria growing on damp rocks; the caves where this species
lives often have elevated concentrations of carbon dioxide gas.
304 Ľ. Kováč
Besides a general food scarcity, many cave animals are faced with temporal
periodicity of food. The inputs of food in oligotrophic caves may be seasonal,
based on periodic flooding, or result from animals, such as bats, visiting the cave
sporadically. Hence, cave animals need the ability to survive long periods of
starvation, which may be the capacity to accumulate and store large energy reserves
as a way of coping with food scarcity (see also Chap. 4). Cave animals can enable
their young to survive periods of food shortage by producing eggs with more yolk
(Thibaud and Deharveng 1994; Hüppop 2012). This phenomenon has been studied
in many invertebrates, such as amphipods, decapods, collembolans and beetles, as
well as in salamanders and fishes.
A reduced energy demand is also highly adaptive in caves with limited food
sources, and many cave species exhibit striking energy economy (Hüppop 2012).
Poulson (1963) demonstrated a decreasing trend in the metabolic rate from the
epigean species of the Amblyopsidae family through troglophiles to gradually
more cave-adapted troglobionts. The body temperature of troglobionts, which are
exclusively ectotherms, is determined primarily by passive heat exchange with its
environment. Thus, troglobionts do not spend energy for thermoregulation. Owing
to mostly low metabolic rates, the troglobionts are successful in zones with limited
resource supplies (food, oxygen, water). Reduction of metabolic rate is associated
with prolonged lifetime and iteroparity (multiplied reproduction) (Hüppop 2012).
Another adaptation is that most cave animals exhibit feeding generalism, or
dietary shift, compared with surface relatives (Hüppop 2000, 2012; Gibert and
Deharveng 2002; Moldovan 2004). To cope with the general food scarcity in
caves, the life history of terrestrial and aquatic animals changes towards
K-selection and limited energy loss in the relatively predictable and stable habitats.
K-selected species have low or no population growth, a reproductive strategy based
on fewer but larger eggs, increased hatching time, prolonged larval stage, decreased
growth rate, delayed reproduction, iteroparity and increased longevity (Thibaud and
Deharveng 1994; Hüppop 2000, 2012). Other features enhancing energy economy
include late hatching, protection of eggs and embryos, viviparity and dormancy
during dry or cold periods (tardigrades, nematodes). In addition, some aquatic
organisms are able to regulate their relatively low oxygen consumption (Albuquer-
que and Coineau 2004). In amblyopsid fishes, Poulson (1963) observed that popu-
lation growth rate and density of amblyopsid fishes decrease with phylogenetic age
of the cave species, and the population structure shifts towards adults.
Food scarcity and stability of the environment also help explain the tendency for
paedomorphosis and neoteny (retention of larval characters) in some subterranean
vertebrates (Culver 1982). However, neoteny was also observed in a food-abundant
cave microhabitat, namely in tree root mats in Hawaii Island lava caves occupied by
cixiid planthopper Oliarus polyphemus that have evolved from an ancestor by
retaining the beneficial nymphal characters to adulthood (Howarth 1993). Besides
the morphological features that characterize this retardation of somatic development,
some anatomical changes have been observed in cave fish and amphibians, including
reduced muscles and ossification (Moldovan 2004).
Acknowledgments During writing this chapter, the author was supported from project of the
Slovak Development and Research Agency APVV-17-0477 and the Slovak Scientific Grant
Agency VEGA 1/0346/18. I am very indebted to Oana T. Moldovan, Stuart Halse and Francis
G. Howarth for the useful suggestions and comments that improved the quality of the manuscript.
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Chapter 14
Food Webs in Caves
Michael P. Venarsky and Brock M. Huntsman
14.1 Introduction
A fundamental goal of ecology is to understand the factors influencing spatial and

temporal patterns of biodiversity. A central tool in these efforts is the quantitative
and/or qualitative description of food webs (see Hall and Raffaelli 1991; Ings et al.
2009; Baiser et al. 2013). Food webs can be used to visualize how energy and
materials flow through ecosystems and are complex adaptive systems, meaning that
they represent the emergent properties governing community dynamics (Power and
Dietrich 2002; Shurin et al. 2006). Food web studies, for example, have identified
multiple mechanisms that influence population demographics and/or community
structure, including bottom-up versus top-down forces (Power 1992), donor-
controlled resource dynamics (Polis and Strong 1996), and the importance of both
grazer- and detritus-based energy pathways in supporting community productivity
(see Moore et al. 2004; Shurin et al. 2006). In this chapter, we explore cave food
webs to elucidate the potential mechanisms that structure these communities. Given
the fundamental differences in energy sources (see Box 14.1), we discuss detritus-
based and chemolithoautotrophically based cave ecosystems separately.
M. P. Venarsky (*)
Australian Rivers Institute, Griffith University, Nathan, QLD, Australia
United States Geological Survey, Fort Collins Science Center, Fort Collins, CO, USA
B. M. Huntsman
Division of Forestry and Natural Resources, West Virginia University, Morgantown, WV, USA

https://doi.org/10.1007/978-3-319-98852-8_14
310 M. P. Venarsky and B. M. Huntsman
Box 14.1
Two types of food webs can be found in cave ecosystems, those based on
detritus (dead animal or plant matter) and those based on chemolithoautotrophic
bacteria. While each of these food webs is ultimately supported by autotrophic
primary production, the pathway by which inorganic carbon (carbon dioxide or
bicarbonate) is transformed into organic compounds (prokaryotic and eukaryo-
tic biomass) differs. Detritus-based cave ecosystems are supported via classic
photosynthetic pathways, whereby plants and algae utilize solar light energy to
transform carbon dioxide into the primary producer biomass that supports cave
consumer productivity via detrital inputs. In contrast, chemolithoautotrophically
based cave ecosystems are supported by bacteria that acquire energy via the
oxidation of inorganic compounds, including sulfide (most common), methane,
ammonium, iron, and manganese. This energy is then used to transform carbon
dioxide or bicarbonate into the microbial biomass that supports cave consumer
productivity (for details see Sarbu 2001; Engel 2007; Madigan et al. 2010).
14.2 Detritus-Based Cave Ecosystems
Detritus-based cave ecosystems rely on detritus (dead animal or plant matter and
associated microbial decomposer assemblages) imported from surface environments
to support in situ biological productivity (Poulson and Lavoie 2001; see also Box
14.2). These types of cave ecosystems are donor-controlled, meaning that consumers
(bacteria to top predators) do not influence the rate of detritus input (Polis and Strong
1996). This contrasts with food webs in surface environments that are supported by
primary producers, where consumers at all trophic levels can either directly
(top-down control) or indirectly (trophic cascade) influence the quantity, quality,
and productivity of the primary producers (algae, macrophytes, and terrestrial plants;
Hairston et al. 1960; Fretwell 1977; Oksanen et al. 1981; Power 1992). While
detritus-based food webs are common in surface environments (forested headwater
streams and leaf-litter communities; Chen and Wise 1999; Wallace et al. 1999),
caves are unique by having relatively weak connections to their surface donor
habitats, which ultimately limit the detrital inputs that fuel cave productivity (see
Moore et al. 2004 for a review of detritus in ecosystems processes).
Box 14.2
When detrital inputs/availability is compared among detritus-based eco-
systems, caves appear to anchor the extreme low end of the detrital spectrum,
while surface ecosystems occupy the opposing high end (Venarsky et al.
2018). Thus, the structure and productivity of detritally based cave communi-
ties are strongly influenced by the factors that control the type, quantity, and
(continued)
14 Food Webs in Caves 311
Box 14.2 (continued)

quality of detritus inputs (see Moore et al. 2004 for a review of detritus in
ecosystem processes).
In this section we begin by discussing the input dynamics of dissolved and

particulate organic matter and then examine the role of microbial communities in
transferring these detrital resources to higher trophic levels. We end this section with
an examination of how detritus inputs influence various aspects of cave community
structure, including biodiversity, taxa abundance, and community productivity.
14.2.1 Dissolved Organic Matter
Dissolved organic matter (DOM) dominates detritus budgets in both cave and
surface ecosystems (Graening and Brown 2003; Simon et al. 2007; Tank et al.
2010). DOM originates in the soil horizons from the breakdown and leaching of
plant organic matter, exudates from plant roots, and the by-products of microbial
metabolism (Schiff et al. 1997; Birdwell and Engel 2010; Simon et al. 2010). The
concentration of DOM decreases as water percolates through the soil horizon and the
complex matrix of fissures within the vadose zone (Baker and Genty 1999; van
Beynen et al. 2000; Pabich et al. 2001; Tatár et al. 2004; Simon et al. 2010). Upon
reaching the phreatic zone, DOM concentrations decrease further with increasing
depth below the groundwater table (Pabich et al. 2001; Datry et al. 2005; Foulquier
et al. 2010). The composition of DOM also changes along transport pathways, with
concentrations of humic, fulvic, and large molecular weight compounds generally
decreasing from the soil horizons to the groundwater table (Baker and Genty 1999;
van Beynen et al. 2000; Simon et al. 2010). These changes to the DOM detritus pool
are due to both biotic and abiotic processes, including microbial consumption,
sorption to clay, and flocculation (see Schiff et al. 1997).
The complex hydrologic setting in karst systems (see Bakalowicz 2005) can
strongly influence DOM inputs to caves. Water flows relatively slowly through the
soil horizons and vadose zone, causing the DOM to be in contact with biologically
active surfaces (biofilms on soil particles and rock surfaces) for extended periods of
time. In contrast, the rapid transport of water through large conduits (large fissures
and sinking streams) limits the time that water is in contact with microbial biofilms,
which potentially limits DOM consumption (Simon et al. 2010). These differences in
transport pathways were suggested to influence DOM concentration and composi-
tion between water from drips (i.e., from soil horizon and vadose zone) and a cave
stream (i.e., from large conduits). Furthermore, DOM concentration and compo-
sition can differ among epikarst drips within the same cave, indicating that large
amounts of variability in DOM inputs are also found at much finer spatial scales
(Ban et al. 2008; Simon et al. 2010). Thus, the heterogeneity of water flow paths at
both small and large spatial scales influences DOM inputs.
DOM inputs are also influenced by local precipitation patterns. Several studies
have reported that high concentrations of DOM are flushed from soil horizons and
the vadose zone during storms (Baker and Genty 1999; van Beynen et al. 2002; Tatár
et al. 2004; Datry et al. 2005; Tissier et al. 2013; Foulquier et al. 2010). These
changes in DOM concentration caused by fluctuating hydrologic conditions are also
accompanied by shifts in DOM composition (Simon et al. 2010; Tissier et al. 2013).
DOM inputs to cave ecosystems are thus likely influenced by complex antecedent
hydrological conditions, including the length of time between and the intensity of
each precipitation event (see Baker and Genty 1999; van Beynen et al. 2002;
Tissier et al. 2013; Shabarova et al. 2014).
14.2.2 Particulate Organic Matter
Particulate organic matter (POM) comes in various forms, including plant material
(leaves, wood, fruit, seeds), guano, and animal carrion. Measures of quality [com-
monly reported as carbon/nitrogen/phosphorus (C/N/P)] ratios can differ greatly
among types of POM but follow the general pattern: plant-POM < guano < animal
carrion (see Sterner and Elser 2002; Cross et al. 2005 for consumer-resource C/N/P
comparisons). Importantly, quality can vary within each POM type. For example,
leaf litter is higher in quality than wood debris, and leaf quality can differ signifi-
cantly among and within plant species (Webster and Benfield 1986). Litter quality
also changes throughout the stages of decomposition, generally increasing in the
early stages of decomposition and then decreasing in quality during the later stages
(Webster and Benfield 1986). Similar to leaf litter, differences in diet can cause
guano quality to vary considerably among animal species, with bat diet (frugivorous,
hematophagous, and insectivorous) strongly influencing guano C/N/P ratios
(Shahack-Gross et al. 2004; Emerson and Roark 2007; Wurster et al. 2015). While
we are unaware of studies that have reported C/N/P ratios for cave cricket guano,
their herbivorous feeding habits suggest that their guano would be of lower quality
than bat guano (Lavoie et al. 2007).
In most cave ecosystems, POM is a small portion of the detrital budgets
(Graening and Brown 2003; Simon et al. 2007). However, the type and quantity of
POM inputs can vary substantially among caves within the same geographic region
(Simon and Benfield 2001; Huntsman et al. 2011a; Venarsky et al. 2012a). The
primary factor that appears to control POM inputs is the number and size of large
conduits (cave entrances and large fissures) in the vadose zone that connects caves to
surface environments. For example, large quantities of plant-POM can accumulate in
cave passages near entrances, pits, large fissures, and inflowing and sinking streams
(Simon and Benfield 2001; Souza-Silva et al. 2011, 2012; Venarsky et al. 2012a).
Cave pits can trap animals (rodents, snakes, frogs, deer, and turtles), while large cave
entrances provide access to bat roosting habitats where significant amounts of guano
can accumulate. Additionally, large root mats can form in caves as plant roots snake
through the maze of fissures in the vadose zone in search of water (Jasinska et al.
1996; Eberhard 2004; Howarth et al. 2007).
The strength of connection to surface environments also appears to dictate
temporal patterns in POM inputs. Souza-Silva et al. (2011, 2012) examined inputs
of animal- and plant-POM in neotropical caves that have strong connections to
surface environments (e.g., inflowing stream and multiple cave entrances), finding
that POM inputs were six times higher during the rainy season compared to the dry
season. Input rates of bat guano can also substantially fluctuate throughout the year
due to bat migrations. Iskali and Zhang (2015) reported that monthly guano depo-
sition rates ranged from 66 to 11,000 kg, with the highest deposition rates occurring
when the maternity bat colony was present (20 million individuals). In contrast,
several cave stream studies in the eastern United States found that plant-POM
standing stocks varied little throughout the year (Simon and Benfield 2001;
Venarsky et al. 2012a, 2018). While not a direct measure of input rate, these studies
suggest that plant-POM inputs were constant on an annual basis.
14.2.3 The Gatekeeper: The Role of Microbes in Detritus-

Based Cave Food Webs
Box 14.3
While caves receive a diverse array of detritus inputs, the DOM and plant-
POM that dominate most cave energy budgets do not directly support inverte-
brate or vertebrate consumer growth and reproduction. Instead, these
low-quality detrital resources are first converted into higher-quality microbial
biomass by bacteria and fungi (Cummins and Klug 1979; Simon et al. 2003).
Detritally based microbial communities support the entire food webs (small crusta-
ceans to vertebrate predators) in both cave and surface ecosystems (Hall and Meyer
1998; Simon et al. 2003, 2007; Graening and Brown 2003; Venarsky et al. 2018; see
also Box 14.3). Cave microbial communities are composed of two groups, bacteria
and fungi. Both bacteria and fungi contribute to the decomposition of plant-POM,
with fungi typically dominating microbial biomass in the early stages of leaf litter
decomposition, followed by bacterial dominance in the latter stages (Webster and
Benfield 1986; Simon and Benfield 2001). In contrast, bacteria appear to dominate
the uptake of DOM, transforming these resources into both bacterial biomass and a
porous polysaccharide matrix that is the structural backbone for biofilm development
(Hall and Meyer 1998; Simon et al. 2003; Madigan et al. 2010). The relative
importance of bacteria and fungi among cave habitats, ecosystems, or consumers
remains poorly understood because bacteria and fungi are generally lumped into a
single “microbial” trophic level. However, two lines of evidence suggest that cave
macroconsumers (i.e., large-bodied consumers such as insects or amphipods) likely

rely on bacteria more than fungi. First, the bacterial uptake of DOM can support the
entire cave food webs (Simon et al. 2003). Second, plant-POM is an ideal substrate
for the growth of fungi (Webster and Benfield 1986), but the low quantity of plant-
POM in most cave ecosystems likely minimizes the role of fungi in cave food webs.
However, the role of fungi could be disproportionately important compared to
bacteria as some aquatic surface species will selectively feed on fungi over bacteria
(Kostalos and Seymour 1976; Arsuffi and Suberkropp 1989). Thus, studies that
explore the relative roles of bacteria and fungi will provide a more nuanced under-
standing of the role of “microbes” in cave food web dynamics.
Some detritus inputs can be directly assimilated by macroconsumers and thus do
not require a microbial intermediate. Animal carrion is a high-quality resource that
may quickly attract high numbers of macroconsumers and is often used in both
aquatic and terrestrial bait traps for cave survey studies (Hunt and Millar 2001;
Huntsman et al. 2011b; Schneider et al. 2011). Several studies have reported that
animal guano can be directly consumed and assimilated by macroconsumers
(Salgado et al. 2014). The most interesting of these studies is Fenolio et al. (2006)
who reported that a population of cave salamanders was partially supported by the
direct consumption and assimilation of bat guano. However, microbial colonization
of guano can likely increase the quality of some guano types, such as herbivorous
cave crickets that likely produce low-quality guano (Lavoie et al. 2007).
14.2.4 Detritus Quantity
Detritus-based cave food webs are broadly similar to their surface counterparts (e.g.,
soil habitats and forested headwater streams) in that caves support three broad
trophic levels: detritus and associated microbes, primary consumers, and predators
(Gers 1998; Simon et al. 2003, 2008; Graening and Brown 2003; Souza-Silva et al.
2013). Classic bottom-up forces (i.e., limited detrital quantity) are thought to control
community structure and productivity within each trophic level (Poulson and Lavoie
2001; Hüppop 2001). This hypothesis is broadly supported by observational and
experimental studies. Bacterial productivity, for example, can be stimulated by
increased DOM availability (Cooney and Simon 2009; Chelius et al. 2009;
Foulquier et al. 2010, 2011a, b), and invertebrate and vertebrate consumer abun-
dance is positively correlated with organic matter availability (Humphreys 1991;
Datry et al. 2005; Fenolio et al. 2006, 2014; Huntsman et al. 2011a; Venarsky et al.
2014, 2018). Rapid increases in organic matter availability have also been linked to
broad shifts in community composition (Sinton 1984; Smith et al. 1986; Madsen
et al. 1991; Notenboom et al. 1994; Simon and Buikema 1997; Sket 1999, 2005;
Wood et al. 2002, 2008; Culver and Pipan 2009; Venarsky et al. 2018). However,
this appears to be a relatively simplistic view of the mechanisms that structure
detritus-based cave food webs. Rather, we suggest that cave communities are
structured through a complex array of bottom-up forces where detrital quantity
interacts with other factors, including the strength of surface connectivity, size of
detrital surpluses, spatial distribution of detrital resources, and consumer-resource
stoichiometry.
14.2.5 Surface Connectivity, Detritus Surpluses, and Spatial

Distribution of Detritus
The strength of surface connectivity appears to be one of the most important factors
that can interact with detritus quantity to structure cave communities. While surface
connectivity affects detritus input rates (see above), it also controls the migration rate
of surface species (sensu troglophile and trogloxene; Humphreys 2001) into cave
ecosystems. Surface and obligate cave species (sensu troglobite; Humphreys 2001)
have distinct biological traits that have been shaped by their respective evolutionary
histories. Obligate cave species have low metabolic rates, increased starvation
resistance, and K-selected life-history traits (long life span, slow growth rate,
reduced fecundity; see also Chap. 4), which are presumed adaptations to the
energy-limited cave environment (Hüppop 2001; Venarsky et al. 2012b; Craig
2013). In contrast, surface species are adapted to survive in energy-rich surface
environments and thus have more r-selected life-history traits, including fast growth
rates and high fecundities (Hüppop 2001). Surface species often dominate commu-
nity biomass and biodiversity in caves with little or no anthropogenic influences
(e.g., low pollution; Ferreira et al. 2000; Souza-Silva et al. 2011, 2012; Venarsky
et al. 2012a, 2018; Pellegrini and Ferreira 2013) and are thus important in several
ecosystem processes, including organic matter decomposition (Brussock et al. 1988;
Galas et al. 1996; Simon and Benfield 2001; Venarsky et al. 2012a), predator
productivity (Huntsman et al. 2011a; see also the Glossary for more explanations
on productivity), and food web energetics (Huntsman et al. 2011a; Venarsky et al.
2014, 2018). Given the proper environmental conditions (temperature and food
resources), many vertebrate (salamanders) and invertebrate (crayfish, amphipods,
isopods, insects, arachnids) surface species are capable of completing their life cycle
in caves. Furthermore, large portions of cave microbial communities have surface
origins (e.g., soil and streams; Chelius and Moore 2004; Griebler and Lueders 2009;
Engel 2010).
Two factors appear to allow obligate cave and surface species to coexist: the size of
the detritus pool and its spatial distribution. In detritus-based surface ecosystems (e.g.,
soils, forested headwater streams, and large rivers), detritus inputs generally exceed
consumer demand (Smock and Roeding 1986; Wallace et al. 1999; Hall et al. 2000,
2001; Stagliano and Whiles 2002; Cross et al. 2007), leaving a large pool of surplus
detritus that is either exported from (e.g., streams) or stored in (e.g., soils and
floodplains) the ecosystem. This is in contrast to some cave ecosystems, where nearly
all resources (detritus and detritivorous prey) are consumed (Venarsky et al. 2014).
The near-complete consumption of all detrital resources creates an energy-limited
environment, where little surplus of detritus is available to support additional cave

macroconsumer productivity. Thus, obligate cave species, with their adaptations to
energy limitation, have the competitive advantage in cave ecosystems where detrital
surpluses are limited. However, rapid increases in detrital availability cause a
corresponding increase in the detrital surplus pool which transfers the competitive
advantage to the surface species that are better adapted (higher growth rates and
fecundities) to exploit the newly available energy (Venarsky et al. 2014). This is the
likely mechanism that is responsible for the extirpation of obligate cave species
following organic pollution episodes (Sinton 1984; Smith et al. 1986; Madsen et al.
1991; Notenboom et al. 1994; Simon and Buikema 1997; Sket 1999, 2005; Wood
et al. 2002; Culver and Pipan 2009). Venarsky et al. (2018) recently provided
experimental support for this mechanism using a stream-reach scale litter amendment
experiment, which showed that surface species responded strongly to litter amend-
ment, while no response was seen in obligate cave species.
While the total amount of energy within caves influences the outcomes of
competitive interactions among obligate cave and surface species, the spatial distri-
bution of energy throughout a cave ecosystem likely dictates if obligate caves
species can persistently coexist with surface species. Cave ecosystems with large
bat colonies can contain large deposits of guano that support high abundances of
both guano specialist taxa (i.e., exclusively inhabit guano) and guano generalist taxa
(i.e., inhabit plant and animal detritus; Gnaspini and Trajano 2000) (Ferreira et al.
2000; Gnaspini and Trajano 2000; Iskali and Zhang 2015). However, measures of
species abundance and biodiversity decrease with the increasing distance from
guano piles (Pellegrini and Ferreira 2013), indicating the influence of guano on
cave community structure is spatially restricted. Similarly, the litter amendment and
control (i.e., no litter amendment) reaches in Venarsky et al. (2018) were separated
by <25 m, but the control reach contained ambient species densities, while the litter
amendment reach had significantly higher densities of surface species.
14.2.6 Nutrients and Detrital Quality
Our discussion has thus far approached the topic of detritus inputs and cave
community structure from a strictly energy- or organic carbon-based perspective.
However, the growth and reproduction of consumers in detritus-based ecosystems
are controlled by several elements (carbon, nitrogen, phosphorous, calcium, potas-
sium) and compounds (fatty acids, vitamins), of which the inadequate supply of one
or more can significantly limit consumer productivity (Sterner and Elser 2002;
Torres-Ruiz et al. 2007). Two elements, nitrogen and phosphorus, have received
considerable attention in the ecological literature, and their availability (forms
include nitrate, nitrite, ammonium, orthophosphate, animal and plant detritus) has
been directly linked to the productivity of bacteria, fungi, and invertebrate and
vertebrate consumers (Sterner and Elser 2002; Cross et al. 2003, 2006, 2007;
Johnson et al. 2006). To date, however, little attention has been given to the roles
of inorganic nutrients and/or detrital quality (C/N/P ratio) in cave community

dynamics.
In cave streams, dissolved inorganic nutrient concentrations can be relatively high
and thus likely do not limit microbial productivity (Simon and Benfield 2001).
Instead, DOM quality and/or lability appear to strongly influence microbial produc-
tivity in cave streams. Using microcosms, Cooney and Simon (2009) provided cave
bacteria communities either high (glucose and soil and leaf extracts) or low (tannic
acid) lability DOM, finding that bacterial productivity was only stimulated with the
highly labile forms of DOM. In contrast to aquatic cave environments, microbial
communities in terrestrial cave environments can potentially be co-limited by both
detrital quality and nutrients. A study by Chelius et al. (2009) reported that microbial
abundance increased with the separate additions of low (lint)- and high (nutrient-rich
rat guano)-quality sources of POM. The strongest response, however, was found in
combined lint-rat guano treatments which supplied large quantities of both carbon
and nutrients. Interestingly, the productivity in terrestrial cave environments can be
limited by factors other than carbon or nutrients (e.g., nitrogen or phosphorus).
Humphreys (1991) conducted litter addition experiments in an arid Australian
cave and found that macroconsumers only responded following the addition of
water to the litter, suggesting that water availability was the primary factor limiting
both microbial and total community productivity. Collectively, these studies suggest
that cave stream microbial communities are limited by detrital quantity and quality
rather than inorganic nutrient availability, while cave terrestrial microbial commu-
nities could potentially be limited by a multitude of interacting factors, including
carbon, nutrient (e.g., nitrogen or phosphorus), and water availability.
To date, only one study has explicitly examined the role of detrital quality in
structuring macroconsumer cave communities. Schneider et al. (2011) experimen-
tally manipulated detrital quality in cave pits by supplying food webs with either
high (rat carrion)- or low (leaf litter)-quality detritus. While species richness (i.e.,
troglophiles) did not differ among treatments, higher abundances of different species
were present on the high-quality rat carrion. The response in this study bears some
resemblance to the patterns in community structure observed in bat guano cave
communities (Ferreira et al. 2000; Gnaspini and Trajano 2000; Pellegrini and
Ferreira 2013; Iskali and Zhang 2015), suggesting that detrital quality plays an
important role in structuring cave communities in bat guano caves. However, the
results of these studies should be viewed within the context of surface connectivity,
which is strong in both cave pits and bat caves.
Another approach to viewing the role of detritus quality in cave community
dynamics is to examine consumer-resource stoichiometry (Sterner and Elser
2002). In detritus-based cave and surface ecosystems, macroconsumers (shredders
and collector-gatherers) usually have much lower C/P and C/N ratios than either the
plant-POM or epilithic biofilms that support their productivity (Cross et al. 2003;
Schneider et al. 2011). These large stoichiometric imbalances indicate that P and N
availability probably limits macroconsumer growth and reproduction. On short
timescales, consumers can cope with these large imbalances by increasing consump-
tion rates, selectively feeding on high-quality microbes, and dispersing to more
suitable foraging habitats or through trophic omnivory (see Schneider et al. 2011).
Prolonged exposure to these nutrient imbalances, however, can be a strong evolu-
tionary force that selects for life histories (more K-selected) and physiologies
(modified body C/N/P ratios; Elser et al. 2000; Fagan et al. 2002) that reduce the
organism’s demand for limiting nutrients. Thus, detrital quality is likely an important
evolutionary driver in both cave and surface ecosystems (Elser et al. 2000; Fagan
et al. 2002; Schneider et al. 2011; Craig 2013).
Adaptation to large stoichiometric imbalances appears to have occurred for some
detritivorous insects, which have C/P and N/P ratios that are 2–3 times higher (which
indicates lower nutrient demand) than other terrestrial or aquatic insects (Cross et al.
2003). The potentially lower quality of detritus resources in caves suggests that
obligate cave species would likely have even higher C/N/P ratios than their surface
counterparts (Schneider et al. 2011). Support for this hypothesis remains tentative.
Detritus quality is rarely quantified in cave ecosystems, with recent studies
suggesting that resource quality is similar among cave and surface ecosystems
(Schneider et al. 2011; Craig 2013). Using different approaches, both Schneider
et al. (2011) and Craig (2013) suggest that obligate cave species nutrient demand is
lower than surface species but that large stoichiometric imbalance between obligate
cave species and their food resources is a factor that likely limits the growth of
obligate cave species.
14.2.7 Beyond Detritus
The discussion above outlined how bottom-up forces structure cave communities.
However, several lines of evidence suggest that cave community structure is also
under the influence of top-down forces. Similar to surface ecosystems (Pace and
Cole 1994), microbial populations in both cave and groundwater environments can
be strongly affected by the feeding activities of heterotrophic nanoflagellates and
larger macroconsumers (Edler and Dodds 1996; Kinner et al. 1998; Sintes et al.
2004; Kinsey et al. 2007; Cooney and Simon 2009). Interestingly, Cooney and
Simon (2009) found that amphipods influence bacterial production on rock surfaces
but not in fine sediments, suggesting that substrate characteristics (e.g., fine sedi-
ments vs rock surfaces) dictate whether epilithic microbial biofilms are top-down
controlled (e.g., Foulquier et al. 2010). Venarsky et al. (2014) found that nearly all
detrital and prey resources needed to be consumed to support community produc-
tivity, which implies cave communities could simultaneously be under both bottom-
up and top-down control.
14.3 Chemolithoautotrophically Based Cave Ecosystems
Our understanding of population and community dynamics, in both cave and surface
ecosystems, has been built around the assumption that complex food webs can only
be supported via photosynthetically derived organic compounds. This notion, how-
ever, was overturned in the 1970s and 1980s with the discovery of diverse consumer
communities (grazers, filter feeders, and predators) surrounding geothermal deep-sea
vents and in sulfidic caves (Engel 2007). These complex food webs are supported by
chemolithoautotrophic primary production rather than classic photosynthetic pri-
mary production. Since their initial discovery, chemolithoautotrophically based
ecosystems have been documented in numerous habitats, including phreatic aquifers
and marine sediments (Engel 2007). In this section we begin with a general descrip-
tion of the factors influencing chemolithoautotrophic primary production in caves
and then explore what factors structure communities in these especially harsh
environments. We end with a discussion of cave food webs that are supported by
both detrital- and chemolithoautotrophic energy pathways.
14.3.1 Chemolithoautotrophic Primary Production
Rates of chemolithoautotrophic primary production reported within caves

(1.8–281 g C m 2 year 1; Porter et al. 2009) fall within the lower range of global
photosynthetic primary production estimates (<1 to ~10,000 g C m 2 year 1;
Cebrian and Lartigue 2004). Similar to how light availability limits photosynthetic
primary production, the availability of an energy source also limits
chemolithoautotrophic primary production. Porter et al. (2009) found that rates of
chemolithoautotrophic primary production in four sulfidic caves were positively
correlated with the concentration of hydrogen sulfide (energy source). Local redox
conditions also influence chemolithoautotrophic primary production by dictating the
amount of energy generated from the energy source (e.g., oxidation of inorganic
compounds; Pohlman 2011). The highest energetic gains are attained when oxygen
is present, with gains decreasing as other compounds are used as terminal electron
acceptors (e.g., nitrate or sulfate; Madigan et al. 2010).
Other factors that limit photosynthetic primary production, such as inorganic
carbon and nutrient (N and P) availability (Elser et al. 2000), do not appear to limit
chemolithoautotrophic primary production in caves. Inorganic carbon in chemo-
lithoautotrophic caves is abundant, being supplied via the dissolution of carbonate
bedrock, oxidation of methane, or respiration (Sarbu 2001; Engel 2010). Inorganic
nutrient availability in chemolithoautotrophic caves is not well documented, but high
nitrogen content of chemolithoautotrophic bacteria suggests that nitrogen is not
limiting productivity (Kinkle and Kane 2001; Sarbu 2001).
14.3.2 A Complex Story: The Role of Chemolithoautotrophic

Primary Production in Structuring Cave Communities
Similar to our previous discussion of detritus-based caves, energy availability is not

the only factor influencing community structure in chemolithoautotrophically based
cave ecosystems. Redox conditions for chemolithoautotrophic primary production
commonly include low oxygen and high hydrogen sulfide concentrations, which
produces a toxic environment for most eukaryotes (Tobler et al. 2006; Engel 2007;
Tobler 2008). Coping with these extreme conditions requires specific physiological
and behavioral adaptations. Some taxa are capable of excreting hydrogen sulfide and
have enlarged gills to allow more efficient extraction of oxygen from the low-oxygen
environment (Tobler 2008). The Mexican cave molly (Poecilia mexicana), for
example, uses a behavior called active surface respiration to occupy the air-water
interface of some sulfidic caves where concentrations of oxygen and hydrogen
sulfide are relatively low and high, respectively (Plath et al. 2007; see also
Chap. 4). Both terrestrial and aquatic taxa have also been observed exploiting
areas of chemolithoautotrophic primary production for only short time periods to
minimize exposure to toxic conditions (Sarbu et al. 1996; Dattagupta et al. 2009).
Interestingly, Dattagupta et al. (2009) described a symbiotic relationship between an
amphipod and an epibiotic chemolithoautotrophic bacterium (Thiothrix) where the
bacterium assists with detoxification. Undoubtedly, the harsh conditions in these
caves strongly dictate community structure by acting as a filtering mechanism on
regional species pools.
While most chemolithoautotrophically based cave ecosystems are considered
energy-rich and can support a highly diverse and abundant community, the commu-
nities in these caves could still be energy-limited. Behavioral strategies to cope with
the extremely harsh conditions in sulfidic caves are thought to be energetically
costly. Plath et al. (2007) reported that Mexican cave mollies in sulfidic caves
spend 73% of their time engaged in aquatic surface respiration, which leaves a
small amount of time for foraging activities. Further exacerbating this issue are the
increased energetic costs likely associated with the need to detoxify after hydrogen
sulfide exposure (Plath et al. 2007; Tobler 2008). The increased energetic demands
of living in this harsh environment are reflected in the physical condition of Mexican
cave mollies, with populations in sulfidic caves having lower rates of fecundity and
reduced body conditions compared with populations in non-sulfidic environments
(Tobler 2008; Riesch et al. 2010). Whether these communities are living at a
carrying capacity set by limited energy availability or the physiological demands
of living in a harsh environment is an exciting avenue of future research.
14.3.3 The Linked Conceptual Model:

Chemolithoautotrophically Based and Detritus-Based
Ecosystems
In previous sections we discussed chemolithoautotrophically based and detritally

based cave ecosystems separately. However, some cave and groundwater ecosys-
tems are supported by both chemolithoautotrophic and detrital energy pathways
(Pohlman et al. 1997; Sarbu 2001; Opsahl and Chanton 2006; Roach et al. 2011;
Pohlman 2011; Neisch et al. 2012; Hutchins 2013; Tobler et al. 2013). These types
of cave ecosystems are similar to many surface ecosystems in that they are simul-
taneously supported by both living autotrophic (photo- vs. chemoautotrophs) and
detrital energy pathways. However, the role of detritus appears to differ between
ecosystems. In surface ecosystems, photoautotrophic primary production can be
spatially and temporally variable, while the large detrital pool is comparably con-
stant and thus stabilizes food web dynamics (Moore et al. 2004). In contrast, detrital
inputs to cave ecosystems are generally low and can fluctuate substantially on both
spatial and temporal scales (see Sect. 14.2). Instead, chemolithoautotrophic produc-
tion can provide a large, continuous input of high-quality resources to the base of
cave food webs.
The presence of both detritus and chemolithoautotrophic energy pathways can
strongly influence cave community structure. The heterogeneous redox conditions
allow multiple microbial metabolic pathways to exist in sympatry, which increases
microbial biodiversity (Pohlman 2011; Porter et al. 2009). The Edwards Aquifer in
Texas (USA) offers an excellent example of how the presence of two energy
pathways can influence macroconsumer community structure. Macroconsumer bio-
diversity is highest in areas where both detritus inputs and chemolithoautotrophic
primary production support in situ productivity and lowest in areas where detrital
inputs are the sole energy source (Hutchins 2013). The higher biodiversity near areas
with both energy sources appears to be the result of trophic niche differentiation
(Hutchins et al. 2014), where the increased productivity at the base of the food web
creates more trophic niches for macroconsumers to exploit (Pianka 1966; Abrams
1995). High amounts of biodiversity have also been reported in several Mexican
sulfidic caves whose communities are supported by both detrital and
chemolithoautotrophic energy pathways (Engel 2007; Roach et al. 2011; Tobler
et al. 2013). The cave communities appear to be dominated by surface species,
indicating that the strength of surface connectivity plays a similar role in structuring
communities in these types of caves as it does in detritus-based cave ecosystems (see
Sect. 14.3.2).
14.4 The Donor Habitat: The Role of Surface Ecosystems

in Structuring Cave Food Webs
Rates of photosynthetic primary production in surface environments are controlled

by multiple factors, including floral community structure (see also Chap. 6), tem-
perature, precipitation, light, and nutrient availability (Begon et al. 2006). Given that
most cave ecosystems are partially supported by detritus inputs from surface eco-
systems, the factors that govern primary productivity in surface donor habitats can
influence cave community structure by modifying detritus inputs. Both local- and
regional-scale patterns in obligate cave species biodiversity have been qualitatively
linked to patterns in surface ecosystem productivity (see Culver and Sket 2000;
Culver et al. 2003; Christman et al. 2005). Culver et al. (2004) found that measures
of cave biodiversity were higher in caves at lower elevations and suggested that this
was the result of increased surface productivity, which was assumed to be negatively
correlated with elevation. Culver et al. (2006) observed that cave biodiversity in
Europe and North America peaked in areas that corresponded with warm and wet
regions which were assumed to have the highest rates of surface productivity (see
also Chap. 9). To date, however, we are not aware of studies that have quantitatively
linked cave productivity or biodiversity to rigorous estimates of primary or second-
ary (animal) production (g C m 2 year 1) in surface ecosystems. While this is a
difficult task, quantitatively linking cave ecosystem processes (food web and com-
munity structure and productivity) to both surface donor ecosystem productivity
(land use, vegetation type, local climate; Goldscheider 2012) and the physical
aspects of karst systems (soil type and depth, conduit size, depth, and distribution;
Williams 2008; Tuttle and Stevenson 1977) would allow the identification of “hot
spots” or “hot times” of cave productivity (sensu Simon 2008; Simon et al. 2008).
Furthermore, these studies would provide a potential link between cave ecosystem
processes and the most pressing problems of our time, global climate change and
biodiversity loss.
Acknowledgments Comments from Alex Huryn, Paul Cryan, Daniel Nelson, Michael Kendrick,
Stuart Halse, and Oana Moldovan greatly improved this book chapter.
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Part IV
Particular Cave Ecosystems
Chapter 15
Ice Caves
Ľubomír Kováč
15.1 Introduction
Ice caves and glacier caves contain perennial ice, representing unique types of
oligotrophic subterranean habitats characterised by low temperature as well as low
amounts of organic material. The broad topic of the ice caves is central in a recent
comprehensive monograph by Perşoiu and Lauritzen (2018) that is recommended
for further reading.
Glacier caves are formed in subpolar zones or high mountains by melting of the
ice of glaciers and may be categorised as glacier pseudokarst. The caves form along
the crevices in the ice and along its contact with bedrock, through the action of
invading water that is usually derived from ice melting on the surface of the glacier.
As glaciers melt, this water drains down into the glacier through fissures. This water
then moves along the base of the glacier and, because it is slightly warmer than the
freezing point of water, it gradually carves out long tunnels that open at the front of
the glacier forming the rivers that drain the glaciers. Glacier caves are ice tunnels
with floors of rock and walls and ceilings of ice. When the surface of the glacier is
below freezing, the tunnels drain and become open to exploration. When the glacier
is melting, the tunnels are often filled with water (White and Culver 2012). Glacier
caves tend to be ephemeral, but some form quite large integrated systems,
e.g. Paradise Ice Caves System in Washington State, USA, which extended for
over 19 km. However, by the 1990s, its glacier had melted completely (Halliday
2004).
Contrary to glacier caves, ice caves can occur in various climatic zones, with a
few occurrences in the hot arid climates, such as the Southwestern USA and central
Turkey (Perşoiu and Onac 2012). However, the global distribution of ice caves,
Ľ. Kováč (*)

https://doi.org/10.1007/978-3-319-98852-8_15
332 Ľ. Kováč
Fig. 15.1 Location of important ice caves in Europe. (1) Casteret Cave, Pyrenees (France);
(2) Grotte de la Glacière, Jura Mts (France); (3, 4) Eisriesenwelt and Dachstein-Rieseneishöhle,
Alps (Austria); (5, 6) Dobšinská and Silická ľadnica ice caves, Western Carpathians (Slovakia); (7, 8)
Gheţarul Focul Viu and Gheţarul de la Scărişoara, Apuseni Mts (Romania); (9) Kungurskaya Ice
Cave, Ural (Russia). Map source: http://www.gifex.com
containing perennial ice accumulations, is limited to the Northern Hemisphere, and

their occurrence follows a general trend of decreasing altitude with increasing
latitude. In Europe, ice caves are found generally at mid-altitudes, where tempera-
tures are below freezing for a long-enough period that (1) dripping water will freeze
to ice and (2) summer warming will be insufficient to completely melt the accumu-
lated ice (Perşoiu 2018). Two major types of ice are recognised in ice caves: firn and
congelation ice. Firn ice results from the regelation of snow accumulated at the base
of the cave entrance during the winter season. Congelation ice develops preferen-
tially during spring, when exterior snowmelt and precipitation result in the infiltra-
tion of water which refreezes in the cave (Luetscher and Jeannin 2004).
Many caves in European mountains at altitudes over 1500 m harbour snow and
ice deposits whole year in their entrance sections (Pyrenees, Alps, Carpathians,
Dinarides and others). Well-known sites, with high volumes of ice, are Dobšinská
Ice Cave in Slovakia, Gheţarul de la Scărişoara (see details for these two caves at Sect.
15.5) and Gheţarul Focul Viu in Romania, and Eisriesenwelt and Dachstein-
Rieseneishöhle in Austria (Fig. 15.1). Eisriesenwelt is a multiple-entrance dynamic
cave system with a classical bidirectional airflow pattern developed in the Upper
Triassic Dachstein Limestone in the Tennengebirge massif in Austria. The cave
consists of over 42 km of passages; the cave entrance is situated at 1641 m asl in a
steep rock wall. In the first 700 m from the entrance, the cave houses a perennial ice
body with an overall surface area of 10,000 m2 and estimated volume of about
33,000 m3. Inside the cave a clear daily cycle in air temperature was observed from
15 Ice Caves 333
late spring until autumn with a maximum temperature of 0.7 C in summer that is
independent of the weather outside the cave (Schöner et al. 2011). Analysis of an ice
core revealed the age of the glacier to be several thousand years (May et al. 2011). The
neighbouring massif of Dachstein hosts another show cave with ice, Dachstein-
Rieseneishöhle. It is a rare static type of ice cave that contains underground glaciers.
The ice is not more than 800 years old, as shown by 14C dates of organic debris from
the ice base (Silvestru 1999; Audra and Pavuza 2004). The Casteret Ice Cave in the
Pyrenees, France, occurs in 2640 m asl, being the highest ice cave in the world
(Fig. 15.1). It is a multiple-entrance dynamic cave, holding 220 m3 of ice along its
entire length. Another well-known ice cave in France, Grotte de la Glacière in the
French Jura Mountains, is located at only 525 m asl. Silická ľadnica Ice Cave in
Slovakia (Fig. 15.2a–c) is a static cave containing a glacier (213–340 m3) in its
entrance section at 470 m asl and is the lowest-lying perennial ice cave in the
temperate climatic zone (less than 50 north). Formation of ice in this abyss (light
hole) started between 400 and 0 years BC (Bella and Zelinka 2018). In Asia, the best-
known ice cave is the 4700 m long Kungurskaya Ice Cave in the Ural Mountains. This
multiple-entrance dynamic cave is carved in sulfidic sediments (gypsum rock) of the
Lower Permian at 700 m of elevation, with an estimated ice volume of 500 m3
(Mavlyudov and Kadebskaja 2018). Fuji Fuketsu in Japan is a lava tube with the
entrance opening at 1120 m of elevation, formed during a volcanic eruption in 864 BC.
The ice-covered section has a static climate with an ice volume of 3000 m3 (Silvestru
1999).
Ice caves may be found in different geological conditions, most often in karst
caves and lava tubes. Caves at high latitude or altitude are often under permafrost
conditions. As a consequence, the air temperature is below 0 C, which provides
favourable conditions for ice preservation in the cave. But permafrost conditions
restrict water inflow, and therefore only a limited amount of ice forms in these
conditions (Perşoiu and Onac 2012). Perennial ice accumulations in caves may occur
at medium altitudes where the presence of ice is restricted to vertical or downward
sloping spaces with only one entrance in which the dense, cold air sinks and remains
trapped (Racoviţǎ and Onac 2000). A peculiar combination of climatic, hydrologic
and cave morphology factors is required for the formation of ice in caves in
conditions that are otherwise not favourable for its formation. Cave cooling is a
prerequisite for ice genesis, which can be achieved by (1) trapping of cold air, the
most common mechanism, (2) unidirectional ventilation and (3) evaporative cooling
or (4) as a consequence of the cave’s geographic setting in the subpolar climatic
zone. Trapping of cold air is most common in caves with a single entrance and
descending passages, in which cold air sinks in winter, displacing the warm air,
which is pushed out through the same entrance or through other entrances. The direct
cooling under the influence of sinking cold air is accompanied by evaporative
cooling, induced by evaporation of moisture from the walls. In summer, the air
inside the cave is colder and thus heavier than the external air and is trapped inside
the cave. Cooling begins in early autumn and lasts until late spring. In summer, the
cave air temperature begins to rise, due to the heat conduction through the walls and
air column in the entrance shaft(s), and the warmer percolating water. Thus, the heat
334 Ľ. Kováč
Fig. 15.2 Silická ľadnica Ice Cave, Slovak Karst, Slovakia (a–c), a lowest-lying perennial ice cave
in the temperate climatic zone: (a) entrance ice decorations, (b) a glacier behind the cave entrance,
(c) entrance section of the cave with a collapse doline. Inhabitants of subalpine ice cave Ľadová
pivnica, Belianske Tatry Mts, Slovakia (d, e): (d) herald moth Scoliopteryx libatrix, (e)
ichneumonid parasitoid wasp Exephanes ischioxanthus. Photos by © Jaroslav Stankovič (a–c), ©
Ľubomir Kováč (d), © Zuzana Višňovská (e)
15 Ice Caves 335
delivered to the cave leads to ice melting and hence cooling of air, so that cave
atmosphere is maintained at 0 C and the cold air remains trapped inside the cave
(Perşoiu and Onac 2012). The ice is formed mainly from recrystallisation of snow,
from refreezing of percolation water or, less significantly, from deposition of cave-
air vapour (Luetscher and Jeannin 2004). If ice formation exceeds loss due to ice
melting and ice evaporation (sublimation), a layered ice body will be formed, similar
to mountain glaciers. Therefore, the term glacier is extended to perennial ice
accumulations in caves that represent large, stratified ice blocks with marked
seasonal mass fluctuations and active flow (Perşoiu and Onac 2012).
15.2 Ice Caves as Ecosystems
Studies on cave fauna have a long history, but biospeleological surveys on caves
with perennial ice are still unusual, and very little is known about the diversity and
activity of their biota (Iepure 2018; Purcarea 2018). According to Poulson (2005),
the food supply in glaciated caves is variable, and there are few or no cave-adapted
species. If they were present, most became extinct when glaciers covered the caves.
Current ice caves tend to be poor in terms of biodiversity (even considering species
not adapted to caves), essentially as a consequence of the high latitude and/or
altitude (Romero 2009). The small number of studies reflects the perception that
environmental conditions of ice caves are too harsh to sustain terrestrial and aquatic
populations, yet biological communities in ice caves consist of a variety of organ-
isms (Iepure 2018).
Among microorganisms commonly found in frozen environments, phototrophic
prokaryotes and eukaryotes from light-exposed environments play an important role
in carbon and nitrogen enrichment of the cave environment by photosynthetic
assimilation of atmospheric nitrogen and CO2, respectively (Hillebrand-Voiculescu
et al. 2014). The specific microclimate of ice caves, with their low air temperatures,
leads to low concentration levels of fungal spores in the air (Ogórek et al. 2017). In
general, perennial cave ice deposits host a lower microbial biomass than other cave
sediments (Purcarea 2018). A thorough review of microbiome diversity (Bacteria,
Archaea, Fungi and Algae) in ice caves is provided by Purcarea (2018).
Box 15.1
Ice caves at lower elevations have microclimate conditions that are distinctly
different from the surrounding surface and may serve as a significant refuge for
cryophilic fauna. Data from several ice caves in the Western Carpathians,
Slovakia, indicate that habitats in contact with ice deposits are inhabited by
sparse invertebrate communities compared with other, non-glaciated parts of
the caves. This is the case of Demänovská Cave System where, however, some
invertebrates occasionally prefer sites close to the ice. Several species are even
(continued)
336 Ľ. Kováč

active on the ice surface, including the troglophilic beetle Choleva glauca and
the ichneumon wasp Exephanes ischioxanthus (Fig. 15.1e, Kováč et al. 2014).
Animals most often enter ice caves to avoid unsuitable external conditions and
usually become inactive in a dormant stage during summer (aestivation) or winter
(hibernation), for example, hymenopterans (ichneumons; Fig. 15.2e). Moreover,
entering the ice caves probably helps animals to avoid predators. From the early
spring, various insects may be seen directly on ice in caves, such as the troglophilic
moth Scoliopteryx libatrix (Fig. 15.2d). It is not clear if they are attracted by the
surface of the ice itself, or by specific internal microclimatic conditions. Sometimes
insects are observed to copulate on ice, such as Choleva glauca in Demänovská Ice
Cave. This behaviour is known in some arthropods active on snow, such as stoneflies
(Plecoptera), scorpionflies (Mecoptera) and chironomid midges (Diptera) (Hågvar
2010).
Castleguard Cave, a cave system in the Rocky Mountains, Alberta, Canada,
extends underneath the Columbia Icefield glacier for total length of about 20 km
as a meteoric water dissolutional cave in limestone. The internal air temperature of
the most insulated parts is approx. 3 C. Two species of troglobiotic or fully adapted
crustaceans live in pools, where the water apparently never freezes. Whilst the
isopod Salmasellus steganothrix is also known elsewhere in the Canadian Rockies,
the amphipod Stygobromus canadensis has been found only in Castleguard. It is
possible that the cave served as a subglacial refuge for these stygobionts during the
last glaciation or even longer (Holsinger et al. 1983).
Terrestrial and aquatic invertebrates in ice caves are mainly represented by
arthropods (Iepure 2018), including beetles of the tribe Leptodirini (Leiodidae,
Cholevinae) and subfamily Trechinae (Carabidae). Examples of troglobionts that
populate ice caves at temperatures of 0–2 C are Isereus xambeui which is recorded
from the French Alps (Trou du Glaz cave near Grenoble), Arctaphaenops
angulipennis from the Austrian Alps (Dachstein Mts), Pholeuon knirschi from
Romania (Apuseni Mts), and Bathyscimorphus globosus and congeners of the
genus Astagobius from Dinaric Mountains (Vandel 1965; Decu and Juberthie
2004; Sket 2014). Figure 15.3 shows Ledena Pećina Ice Cave in Durmitor, Monte-
negro, as an example of a high-altitude ice cave inhabited by coleopterans.
Another example of beetles adapted to ice caves is the genus Pretneria, which
includes seven species spread over a karst area extending from north-eastern Italy to
western Slovenia. All species have been found in caves and superficial subterranean
habitats characterised by low air temperature (0–5 C), high relative humidity and
the presence of temporary or permanent deposits of snow and ice. P. ternovensis
occupies two caves, Ledenica pri Dolu, a 180 m long and 80 m deep subhorizontal
cave, as well as the 2.2 km long and 650 m deep Velika ledena jama v Paradani.
Entrances of both caves are situated on the karst plateau of Trnovski gozd, at
15 Ice Caves 337
1:500
–5 0 5 10 15 20m
Fig. 15.3 Ledena pećina Ice Cave in Durmitor, Montenegro, an example of a high-altitude ice cave
(ca. 2100 m asl): (a) firn ice slope at the cave entrance and ice fillings at the bottom, (b) detail of ice
fillings, (c) horizontal and vertical cross section of Ledena pećina (Barović et al. 2018), with
permission of Elsevier, (d) Neotrechus suturalis, a subterranean carabid beetle active on cave ice
in early summer. Photos (a, b, d) by © Ľubomir Kováč
altitudes of 1000–1100 m on the bottom of deep dolines, characterised by microcli-

mate inversion and deposits of snow that persist until late spring. The beetles are
concentrated in the twilight zones within a range of about 50 m from cave entrance.
The seasonal activity is intense from May to July, in association with a slight
338 Ľ. Kováč
increase of temperature (from 1 C to 4–5 C), when the deposits of snow and ice
melt and the relative humidity is close to saturation (78–84%; Bognolo 2016).
Westcott (1968) discovered a remarkable blind cholevid beetle Glacicavicola
bathyscioides in three separate lava tube ice caves in southern Idaho, USA. Based on
the degree of troglomorphy, this beetle is a troglobiont and a likely descendant of an
old lineage. All the individuals have been found either on ice, where they crawled
slowly, or floating in meltwater above the ice floor.
Diptera is another group of invertebrates in which some species are found to be
associated with ice in caves. Two new taxa of Sphaeroceridae were recently
described from the high-alpine Obstanser Eishöhle (2320 m asl), an ice-bearing
cave in East Tyrol, namely, the troglophilic subspecies Crumomyia cavernicola
oculea and the possibly troglobiont Crumomyia microps (Christian and Spötl
2010). The Buso del Valon cave is one of the few karstic caves of the Venetian
Prealps in Italy that has a permanent ice body, fed by seasonal snowfall entering
from the wide entrance. The cave opens with a shaft of about 30 m in diameter and
about 50 m deep, having a vertical trend with a depth of about 70 m. The period of
major activity of Chionea dipteran coincided with the period of major extent of the
snow cover. The cave serves as a seasonal refuge for nivicolous species of dipterans
during summer when the outside snow cover is absent. The constant temperature and
the presence of snow and ice in the cave, together with the presence of organic
material coming from the outside, make this cave an optimal environment for the
survival of Chionea (Avesani and Latella 2016).
In western North America, 11 species of ice crawlers of the genus Grylloblatta
(Grylloblattodea, Notoptera, Insecta) are known for their adaptation to cold condi-
tions. These cryophilic insects are found on north-facing talus slopes and snow
patches at high elevations (1500–3000 m asl), and in caves with permanent ice at low
elevations (300–1000 m asl) (Jarvis and Whiting 2006).
Collembola is another group frequently found near ice in caves. A cryptic
collembolan species has been discovered in large numbers in Silická ľadnica Ice
Cave, Slovakia (Fig. 15.2a–c). The collembolan aggregations occur regularly in the
vicinity of the floor ice with air temperature of approximately zero throughout the
year. The species is morphologically identical to Folsomia manolachei, which is
associated with thermophilous deciduous forests. In a laboratory experiment, the
cold-adapted cryptic Folsomia species survived significantly lower temperatures
than forest congeners from the same area (Raschmanová et al. 2017).
The examples of fauna associated with ice caves may be found also among aquatic
crustaceans. Kungurskaya Ice Cave in the Ural Mountains is occupied by the
stygobiotic amphipod Crangonyx chlebinkovi, endemic to groundwater habitats of
the Priuralye karst area, living under constant low water temperatures (0–5.5 C)
(Sidorov et al. 2012). The stygobiotic syncarid Bathynella glacialis is associated with
an interstitial habitat in a subterranean lake in Ledyanaya Ice Cave near Khabarovsk in
the Russian Far East (Birstein and Ljovuschkin 1967). Stygobromus allegheniensis is a
stygobiotic amphipod commonly found in caves of the Northeastern USA. Its several
populations occupy the unique tectonic ice caves in the Shawangunk Ridge in
New York, USA; these small caves represent simple faults and cracks in the bedrock,
15 Ice Caves 339
covered with ice until spring. During the warmer months, some of the ice caves have
small streams and several 2 m deep pools where the amphipods can be found in great
numbers. In the winter months, the ice caves freeze, and the floor and walls become
covered in solid ice. The amphipods may seek warmer waters within the cave but can
also survive being frozen in solid ice (Espinasa et al. 2015). This has been experi-
mentally documented in another aquatic subterranean amphipod, Niphargus
rhenorhodanensis. After inoculation at high sub-zero temperatures, cold-acclimated
specimens survived. The accumulation of cryoprotective molecules such as glycerol
and free amino acids may be the mechanism allowing the survival of this species when
it is cold-acclimated (Issartel et al. 2006; see Box 15.2).
Novak et al. (2014) found that the resistances to freezing had a decreasing
trend from trogloxenes over troglophiles to troglobionts since trogloxenes are not
adapted and troglophiles not completely adapted to thermally stable subterranean
environments. A weak resistance was preserved in nearly all troglobionts. The
authors found that troglobionts inhabiting the superficial subterranean habitats
withstand freezing to a limited degree but have increasing freezing tolerance in
winter. Troglobionts inhabiting deep subterranean or other thermally buffered sub-
terranean habitats had equal (limited) tendency to withstand freezing in both summer
and winter.
Box 15.2
Life conditions in ice caves are extreme for most living creatures, and coloni-
sation of ice caves by biota requires specific adaptations. A few cavernicoles
can develop and exist at temperatures near to freezing point. The adaptations to
low temperatures are well known in invertebrates inhabiting cold climatic
zones or high mountain regions.
The freeze tolerance strategy in cave invertebrates appears to have evolved

according to the habitats species occupy. For example, in two caves in the Venetian
Prealps (NE Italy), the leptodirine beetle Neobathyscia pasai, abundant close to the
entrances where the temperature is variable, was strongly freeze tolerant. On the
contrary the congener N. mancinii, confined to the deeper part of the caves where
the temperature is constant throughout the year, was only moderately freeze tolerant
(Lencioni et al. 2010).
Some insects are physiologically able to tolerate the formation of ice in their
extracellular body fluid. This freeze tolerance is widespread in some families of
Coleoptera, Diptera, Hymenoptera and Lepidoptera, especially in larvae and pupae.
However, most species are freeze susceptible and depend on supercooling to survive
lower temperatures. These adaptations are usually associated with the evacuation of
body water and gut content to reduce freezing of body liquids. In overwintering
insects, the degree of supercooling is increased by the accumulation of cryoprotec-
tive substances in the haemolymph, which are low molecular weight compounds,
such as polyhydric alcohols (e.g. glycerol) and sugars. Antifreeze proteins (thermal
hysteresis proteins) stabilise the supercooled state of insects and prevent the
340 Ľ. Kováč
inoculation of ice from outside through the cuticle and protect the gut fluids from
spontaneous nucleation (Sømme 1999; Willmer et al. 2005). We may expect that
these adaptations are less pronounced in arthropods dwelling ice caves because this
environment is protected against very low external temperatures. Generally, knowl-
edge of the phenological and behavioural adaptations of invertebrates inhabiting ice
caves is very limited. Studies of population dynamics of Pholeuon knirschi in
Scarişoara Ice Cave in Romania, conducted by G. Racoviţa, provide one of the
few examples of this kind of observations (see the further text).
15.3 Ice Caves and Palaeoclimate Reconstruction
The rather stable environments within caves have long been recognised to have the
potential to preserve past environmental information in various types of sediments
(speleothems, fluvial sediments, guano, bones, etc.). Perennial ice accumulations in
caves may also host a wide range of palaeoclimatic and palaeoenvironmental
proxies, of which the most important one is the stable isotopic composition of
water (ice). Many past climate studies have examined the oxygen and hydrogen
stable isotope variation in ice on the assumption that the isotopic composition of ice
mirrors that of meteoric water before freezing inside the cave. Thus, there is a strong
correlation between outside temperature and the oxygen isotopic composition in the
ice. By examining the ages of the ice at different depths with high-resolution stable
isotope analyses along the ice core gained by drilling the ice block, a detailed record
of climate changes in the cave’s region (mean annual temperature, source of
moisture, etc.) can be derived for the period since ice accumulation began (May
et al. 2011; Perşoiu and Onac 2012; Perşoiu 2018). However, obtaining a climatic
signal from cave drip water and associated ice is not a straightforward process
because of the complex climatic regime of a given cave, specifically due to a strong
influence of various ice mass-balance change-related processes on cave air temper-
ature (Perşoiu et al. 2011a).
Among the available techniques, short-lived radionuclides, such as tritium,
radiocaesium, americium and radiolead, are useful dating tools that provide an objec-
tive control in age-accumulation models based on cave ice core analyses (Kern 2018).
Fossil remains (e.g. pollen grains, plant fossils, charcoal) can also be excavated from
cave ice deposits and provide very useful numerical radiocarbon (14C) data (Holmlund
et al. 2005; Feurdean et al. 2011). Radiocarbon analysis of surface-derived ephemeral
plant and animal remains is currently the most potentially accurate dating approach for
the older cave ice deposits (Kern 2018). However, melting phenomena may cause
great limitations in the potential analysis of ice deposits, making it impossible to
calibrate the palaeoclimatic signals recorded (Kern and Perşoiu 2013).
The radiocarbon ages of cave ice blocks (glaciers) from Europe and North
America indicate that they are younger than 1000 years. Eisriesenwelt and
Scǎrişoara are notable exceptions with the age of their ice blocks estimated as
~4000 and 10,000 years, respectively (Perşoiu and Onac 2012).
15 Ice Caves 341
15.4 Ice Caves as Show Caves
Fascination with ice caves was the main reason for their opening to the public more
than 100 years ago. There are about 20 ice caves worldwide that function as show
caves (Oedl 2018) and require proper and sensitive conservation management.
Human activities associated with the construction of tourist pathways and cave
maintenance may directly threaten the whole ice cave ecosystem. Artificially created
ice corridors, tunnels and new cave openings may induce new air currents and
subsequently undesired decline in the amount of ice. Moreover, the ice surface is
polluted by dirty deposits that are transported inside the cave directly by the visitors
or as the natural dust, which stems from water entering the cave or being deposited
by drafts. Electric lighting can be a significant problem when the light bulbs emit
heat in close proximity to the ice formations creating small melting depressions.
Recently, LED lamps as a new technology have become the standard system for
lighting in show caves that considerably reduce heat emissions (Oedl 2018).
The high number of visitors may potentially increase the cave air temperature
adjacent to human traffic by up to half a degree centigrade for short periods of time.
However, in dynamic caves, there is a pronounced natural exchange of air which
means that even large numbers of visitors influence the temperature minimally. Even
in static ice caves with a much lower level of air exchange, such as Scărişoara Ice
Cave, the influence of visitors on the temperature is minimal. The temperature levels
inside the cave are influenced to a significantly greater extent by natural causes such
as warm rainwater. The internal cave climate is continuously monitored, and visitors
have access only during a limited part of the year, for example, for 6 summer months
in Eisriesenwelt and 4.5 months in Dobšinská Ice Cave, currently with 180,000 and
80,000 annual visitors, respectively (Oedl 2018; Bella and Zelinka 2018).
Detailed studies are lacking on the potential effect of the management of ice show
caves on local biological communities which are considered to be less sensitive to
disturbance because of extreme temperature conditions around freezing point. Thus,
it is thought that installation of biological materials, such as wooden pathways, does
not alter significantly the composition of ice cave biota (Oedl 2018).
15.5 Examples of Ice Caves Ecosystems
In the next section, three examples of complex biotic communities of ice caves are
provided: Gheţarul de la Scărişoara in Romania, Dobšinská Ice Cave in Slovakia and
fumarolic ice caves on Mt Erebus, Antarctica.
The cave Gheţarul de la Scărişoara (700 m long, 105 m deep) belongs to the
most intensively studied ice cave systems. It is situated at 1165 m of elevation in the
Apuseni Mountains, NW Romania, in Upper Jurassic limestone. At the bottom of the
circular shaft, 60 m in diameter and 48 m deep, the cave hosts 1–3 m thick layer of
perennial snow. Beyond the entrance, an ice block with a volume of more than
342 Ľ. Kováč
100,000 m3 forms the floor of the Great Reservation hall, making it one of the
world’s largest subterranean glaciers. The surface area of the top of the ice block
covers approximately 3000 m2, and its thickness exceeds 22 m. The ice block is
preserved due to unusual microclimate and permafrost conditions within the cave.
There is seasonal bidirectional ventilation in which the active phase is limited to the
winter season when cold air is trapped inside the cave. During summer the vertical
temperature gradient becomes steep in the shaft: the upper part has temperatures
close to the surrounding outside area, whilst the lower part is close to freezing.
Below this level the cold air masses fill the cave as a result of negligible air mixing
between the systems. Thus, the internal air temperature is governed by the winter
cold and the cooling effect of the ice block, and only to a minor extent influenced by
summer temperatures. The average annual mean temperature at the ice surface in the
Great Reservation is 0.9 C, which is almost 8 C lower than in the surroundings of
the cave. At the ice surface, temperatures may drop as low as 14.5 C during the
winter season; during summer the temperature is around 0 C, rarely exceeding
+0.5 C. The radiocarbon age of the block in the Little Reservation of Scărişoara is
~1200 cal year BP; the total age of ice mass in the cave was estimated around 10,500
years, based on radiocarbon dating of the containing debris (Perşoiu et al. 2017). The
accumulation of the perennial ice block must have started after the collapse of the
passage in the Great Reservation that was linking to the nearby cave, Pojarul Poliţei
Cave, a passage that allowed free circulation of air through the cave and thus
preventing its warming. A period of rapid and almost continuous melting of the
ice block was observed in the middle and late twentieth century (Silvestru 1999;
Holmlund et al. 2005; Perşoiu and Pazdur 2011; Perşoiu et al. 2011a, Perşoiu and
Onac 2012). Investigations of the behaviour of oxygen and hydrogen stable isotopes
(δ18O–δ2H) during the formation of ice by the freezing of water were carried out in
Scărişoara Ice Cave. A model for the reconstruction of the initial isotopic composi-
tion opens a new direction in palaeoclimatic research in ice caves (Perşoiu et al.
2011b). Derived from a precisely dated isotopic record (14C, δ18O, δ2H values) of
cave ice deposit in Scărişoara, Perșoiu et al. (2017) presented a reconstruction of late
autumn through early winter air temperature and moisture source changes in East-
Central Europe (ECE) for the Holocene. It was found that during this time period
winter temperature in ECE was mainly controlled by insolation changes.
Studies of pollen, charcoal and macrofossil recovered from Scǎrişoara ice pro-
vided valuable palaeoecological data (Fig. 15.4e–f). The collected data provide a
picture of past vegetation dynamics and composition at both the local and regional
scale, along with clear signals of human impact over the past 1000 years (Feurdean
et al. 2011).
Microorganisms belonging to Bacteria and Eukarya domains were found in ice
layers of different age (recent, 400, and 900 years). A small section of the ice block
surface (~10 m2) exposed to sunlight promotes the development of an abundant
population of phototrophic prokaryotes and eukaryotes living in the supraglacial
pond at the water-ice interface. The chemical composition and organic content of
both deeply buried (>10 m inside the ice block) and surface (supraglacial pond
water) parts of the ice block were analysed in relation to their age and organic
composition. The composition of cold-adapted ice embedded microbiota varied with
15 Ice Caves 343
Fig. 15.4 Dobšinská Ice Cave, Slovak Paradise, Slovakia (a–d): (a) tourist passage carved in a
compact ice block of the cave glacier, (b) upper part of the glacier with ice decorations. Photos (a, b)
by Ľubomir Kováč. (c, d) Seasonal patterns of air exchange in Dobšinská Ice Cave, schematic cross
section (Korzystka et al. 2011). With permission of the Slovak Caves Administration. Scărişoara Ice
Cave, Apuseni Mountains, Romania: (e, f) ice layers of an exposed ice wall in the Little Reserva-
tion. The red dot indicates the position of the pollen profile, whereas the numbers in red represent
the position and number of the samples used for the radiocarbon dating (from Feurdean et al. 2011).
With permission of Elsevier
age and organic content of the section (Hillebrand-Voiculescu et al. 2014; Iţcuş et al.
2016).
The cavernicolous leptodirine beetle Pholeuon knirschi glaciale is a subspecies
endemic to four caves in Bihor massif of the Apuseni Mountains, which have an
internal air temperature of 6–7 C. The species also frequently occupies warmer zone
of Scǎrişoara where temperature is usually 2–5 C throughout the year. A population
occupying the periglacial zone of the Little Reservation has unique dynamics since it
is periodically exposed to low winter temperatures (ca. –1 C) which lead to
emigration of the beetle during this season. In summer the population is
344 Ľ. Kováč
re-established when the temperature rises to ca. 0.6 C. This cave is thus a borderline
habitat in terms of the beetle’s cold tolerance. Mark-recapture techniques showed
that only ca. 1% of beetles were recaptured in a 5-month period, indicating that a
large population was present in the cave and interconnected fissures of the surround-
ing karst. The winter population decrease is associated with the migration of
individuals from cave to fissures and crevices in bedrock to avoid less suitable
microclimate. P. k. glaciale is considered to be a strictly troglobiotic animal. More
recent studies, carried out in mesovoid shallow substratum (MSS) in a neighbouring
area, found that this beetle may migrate closer to superficial layers of karst and also
non-karst bedrock. The summer population maximum in the cave is thus most
probably the result of seasonal immigration of individuals from different sectors of
the massif, including MSS (Racoviţǎ 1976, 1980, 1987, 2000).
Dobšinská Ice Cave (Dobšinská ľadová jaskyňa, Fig. 15.4a–d) is a famous tourist
cave located in the Slovak Paradise karst area, Slovakia, in an area of temperate
climate. The entrance of this static-dynamic ice cave is a collapsed doline at an
elevation of 969 m asl in a north-facing slope covered by a coniferous forest. It is the
isolated end of the Stratenská jaskyňa Cave System, a 23.6 km long multilevel cave
formed by sinking palaeo river in the Middle Triassic Steinalm and Wetterstein
limestones. The collapse separated the cave with 1483 m of passages and vertical
span of 112 m from the remainder of the system, probably in the Middle Quaternary.
The beginning of the ice filling development is estimated to ca. 250–140,000 years
ago. The main part of the cave consists of a large collapsed chamber containing
perennial ice masses, descending to a depth of 70 m. Most of its volume is filled with
a glacier with an ice volume of more than 110,132 m3, glaciated surface of 9772 m2
and a maximum depth of 26.5 m, making it the largest known compact ice monolith
in a cave in the world (Fig. 15.4a–b). The average air temperature in the glaciated
Great Hall is 2.7 to 3.9 C in February and around +0.2 C in August; in lower
parts of the cave, the air temperature is below freezing year-round (Bella and Zelinka
2018). The Dobšinská Ice Cave features a different winter and summer regime of air
circulation and thus is classified as a statodynamic cave. The colder air circulates
from the surface into the cave during the winter season and reverses during the
summer season (Korzystka et al. 2011; Fig. 15.4c–d). The ice monolith grows in the
upper parts and melts on the contact with the bedrock, a process that is in equilib-
rium. The ice is slowly moving in the direction of the floor at a rate of 2–4 cm/year.
Continuous replacement of the glacier is estimated to take around 1280 years. The
typical stratification of ice with distinct layers is formed in relation to the cyclic
freezing of cave seepage water over the years during winter season (Bella and
Zelinka 2018; Fig. 15.4a, f).
A comprehensive study of ice cores has been undertaken in Dobšinská Ice Cave
by Vrana et al. (2007) and Clausen et al. (2007). Six stages were specified in the
development and degradation of ice crystal sediment (Pflitsch et al. 2007). Diverse
colonies of microfungi were isolated from sediments and cave air (Nováková 2006).
It was observed that the arthropod diversity at glaciated sections is markedly lower
than in warmer deeper parts of the cave (air temperature between +0.8 C and
+3.5 C) as a consequence of the oligotrophic conditions and low air temperature
15 Ice Caves 345
limiting decomposition of organic matter and the development of microbial colonies,

which are direct food sources for microbivorous arthropods. The cave is occupied by
four troglobiontic collembolans, Protaphorura janosik, Deuteraphorura kratochvili,
Pygmarrhopalites aggtelekiensis and Megalothorax carpaticus, and by two crusta-
cean stygobionts, Bathynella natans and Elaphoidella sp. (Kováč et al. 2006). An
aggregation of the cryophilic collembolan Protaphorura janosik was observed in the
vicinity of the ice floor. The species is endemic to the Western Carpathians where its
isolated populations preferentially inhabit colder caves. According to the recent
molecular study (Parimuchová et al. 2017), populations of P. janosik diverged
during the Early Pleistocene between 1.3 million and 800,000 years ago.
The fumarolic ice caves on Mt Erebus (Antarctica) are a unique example of an
oligotrophic cave system, characterised by volcanic bedrock as a substrate. The Mt
Erebus ice caves are moist, cold habitats (on average ~0 C) at high altitude in one of
the most remote and oligotrophic environments on Earth. Subglacial fumaroles issue
air-dominated gasses with 80–100% humidity and up to 3% CO2; some contain also
CO and H2 but are essentially devoid of CH4 and H2S. Many of the caves are
completely dark and therefore unable to support photosynthesis. In these dark
oligotrophic volcanic ecosystems, the only possible sources of organic carbon are
from atmospheric deposition or ice algae that may grow on the surface of the ice
during summer and subsequently being introduced into the caves through burial
from above and melting from below. The microbial communities in these caves are
composed primarily of bacteria and fungi. Low phylum-level bacterial diversity
confirms a commonly found adaptation to extreme environmental conditions; the
energy driving CO2 fixation and supporting the ecosystem is likely derived from
reducing volcanic rocks and gas emissions. Thus, the fundamental support for
biological systems in fumarolic Antarctic ice caves is provided by
chemolithoautotrophy (Tebo et al. 2015). For more details on this phenomenon,
see Chap. 5.
15.6 Ice Caves and Global Change
Perennial ice masses can accumulate in caves far outside of the boundaries of the
permafrost climatic zone which makes them especially vulnerable to any warming
trend. It has been highlighted that radiocarbon dating showed relatively young age of
ice blocks of European and North American ice caves, being mostly younger than
1000 years. Caves hosting ice bodies have been reported to suffer significant loss of
ice mass worldwide over the past decades (Kern and Perşoiu 2013; Colucci et al.
2016). This widely observed process threatens cave ice deposits with tremendous
volume loss or ultimately total disappearance. It is suggested there is an urgent need
to investigate the probably unique palaeoenvironmental information stored in cave
ice deposits (Kern and Perşoiu 2013). Cave glaciers have a complex response to
external climate variations; therefore their future behaviour under changing climatic
conditions is more difficult to predict than for surface glaciers (Perşoiu et al. 2011a).
346 Ľ. Kováč
Warmer and more intense precipitation may be crucial in the future mass balance
evolution of permanent cave ice deposits. The close relationship between global and
local climate change and the evolution of the ice deposits has been highlighted in the
dynamic ice caves, especially in regard to the extreme weather events (Colucci et al.
2016). Mammola et al. (2018) pointed out the importance of subterranean organisms
as model species for ecological studies dealing with climatic changes. We may
expect that populations of the cryophilic fauna of ice caves, often representing
glacial relicts, will decline in ice cave systems, and species may even be threatened
with progressive melting of the cave ice due to climate warming.
Acknowledgements I am very grateful to Oana T. Moldovan and Stuart Halse for the very useful
suggestions and comments that improved the quality of the manuscript. I am indebted to Jaroslav
Stankovič (Slovakia) for the photographs of Silická ľadnica Ice Cave and Zuzana Višňovská
(Slovakia) for the photograph of a hymenopteran insect on ice. The contribution was partly
supported from the research grants APVV-17-0477 and VEGA 1/0346/18.
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Chapter 16
Researches in Sulphide-Based Ecosystems
Alexandra Hillebrand-Voiculescu
16.1 Introduction to Sulphide-Based Ecosystems
The concept of chemolithotrophy, as the ability of bacteria to obtain energy by the

oxidation of reduced inorganic compounds such as H2S, NH3 and Fe2+, which led
eventually to the entire concept of sulphur and nitrogen cycles in nature (Dworkin
2012) was discovered by Sergei Winogradsky (1887), at the end of the nineteenth
century, when microbiology was taking off as an independent science. The process
was initially considered insufficient to support ecosystem-level processes (Summers
Engel 2007), and life in the absence of light was thought to be impossible until the
1970s when the deep-sea hydrothermal vents and their associated biocenoses were
discovered. These vents showed that in extreme conditions, such as complete
darkness, low temperatures and extraordinary high pressure, where no one imagined
life to be possible, life not only exists but presents an astonishing abundance and
diversity. The dogma according to which life exists only in the presence of light was
abolished, but the sustaining processes were still not very well understood until
biologists, geologists and chemists put their efforts together to explain the richness
and great biodiversity in the vents. As a result, new C/N and energy sources as well
as pathways alternative to photosynthesis were found to be enough to sustain
ecosystems. One-carbon compounds, other than CO2, were shown to function as
source of C, while oxidation of reduced compounds such as NH4+ and H2S provides
almost all the energy requested for biomass formation. Still, the hydrothermal vent
ecosystems do receive some organic matter from the water column, plankton,
various types of detritus, dead animals, etc.
Chemolithotrophy was confirmed to exist also in habitats associated with sul-
phurous water, but again, these systems are also open, receiving organic matter from
A. Hillebrand-Voiculescu (*)
Emil Racoviţă Institute of Speleology & Group for Underwater and Speleological Exploration,
Bucharest, Romania

https://doi.org/10.1007/978-3-319-98852-8_16
352 A. Hillebrand-Voiculescu
the surroundings. In contrast, at the end of the 1980s, a secluded, peculiar

chemosynthesis-based ecosystem, isolated from any source of organic matter, was
discovered in Romania, inside a cave. This is Movile Cave, the first terrestrial
chemolithotrophy-based ecosystem discovered (Sarbu et al. 1996).
Caves are habitats characterised by partial or complete darkness, constant climate
(constant air/water temperature, relative humidity near saturation) as well as a
restricted input of nutrients (see also Chap. 3). Caves are classified based on various
criteria, one being the mechanism of their formation, the speleogenesis. Based on this,
caves formed by bedrock dissolution can be epigenic or hypogenic. The majority of
caves, known also as solution caves, are epigenic, being formed by the movement of
water from overlying or immediately adjacent recharge surfaces to springs in nearby
valleys (Palmer 1991; see also Chap. 2). Atmospheric CO2, as well as CO2 from soil
and dissolved in water, forms carbonic acid (CO2 + H2O ! H2CO3) that corrodes the
carbonate (CaCO3) bedrock (H2CO3 + CaCO3 ! 2HCO3 + Ca2+).
16.2 Hypogenic Caves
In contrast to the solution caves formed by meteoritic water dissolving the limestone
in karstic regions, caves of hypogenic origins are formed by fluids ascending through
various geological and tectonic settings at different depths (ranging from a few tens
of metres to several kilometres) by different dissolution mechanisms operating in
various lithologies (Klimchouk 2009). Klimchouk found that hypogene
speleogenesis is a complex hydrogeological process, with deeper groundwaters in
regional or intermediate flow systems interacting with shallower and more local
systems to support multiple dissolution mechanisms that differ in their physical and
chemical properties. Despite the variability of the speleogenesis mechanisms and of
the geological conditions, hypogenic caves can be recognised by specific meso-
morphological features indicating sluggish flow conditions (Klimchouk 2009).
Hypogenic caves differ depending on the origin and thus on the chemical
composition and temperature of the rising waters as well as on the nature of the
host rock. In the case of caves with inputs of gases such as hydrogen sulphide (H2S)
and/or methane (CH4), which could provide energy sources for microbial commu-
nities, H2SO4 corrosion and condensation corrosion promoted by HCO3– coexist and
reinforce each other (Kumaresan et al. 2014).
The deep-seated aquifers rich in H2S and/or CH4 are usually thermal as compared
to the average temperature of the regional groundwater and have a higher temper-
ature than the underground environment. This results in water condensation on the
substrate and sulphuric acid formation due to H2S exposure to atmospheric O2
(Eq. 16.1). The limestone (CaCO3) of the walls is then rapidly corroded by
H2SO4, while gypsum (CaSO4 2H2O) deposits form to cover the walls, and CO2
is released (Eq. 16.2). The CO2 further reacts with H2O vapours and the condensed
water with H2CO3production (Eq. 16.3), which further forms HCO3– that will again
corrode the walls (Eq. 16.4) (Kumaresan et al. 2014).
16 Researches in Sulphide-Based Ecosystems 353
H2 S þ 2O2 ! H2 SO4 ðsulphide oxidationÞ ð16:1Þ

H2 SO4 þ CaCO3 þ H2 O ! CaSO4 2H2 O þ CO2
" ðsulphuric acid driven corrosionÞ ð16:2Þ
CO2 þ H2 O ! H2 CO3 ðcarbonic acid formationÞ ð16:3Þ
H2 CO3 þ CaCO3 ! 2HCO3 þ Ca2þ ðcondensation corrosionÞ ð16:4Þ
16.2.1 Trophic Webs in Hypogenic Caves
Epigenic cave biocenoses are strongly dependent on photosynthesis-derived organic

matter. Subterranean trophic chains are typically short and based on detritivores. In
contrast, hypogenic caves usually have a high degree of isolation from the surface,
being confined between layers with low porosity. The energy in these systems is
mainly provided by the rising fluids and gases (Forti et al. 2002), with the input of
exogenous organic matter being insignificant. The energy sources for
chemolithotrophic bacteria are provided by dissolved reduced compounds, such as
H2S and CH4, in the ascending water.
There are hypogenic caves, such as Movile (Romania) and Ayyalon (Israel),
which host an astonishingly rich and diverse fauna despite their complete isolation
from the surface (see review in Summers Engel 2007). In these caves, the organic
matter required to support the entire trophic web is synthesised in situ, by
chemolithotrophic bacteria performing chemosynthesis (Sarbu et al. 1996; Levy
2007). The microbial communities form biofilms on the surface of the sulphurous
water and on the walls/rocks in the proximity of the water. Since the nutrients are
derived from the water, and because organic matter also forms here, owing to the
chemosynthetic bacteria, the cave species are clustered mainly near the water, with
the rest of the galleries being almost depleted of fauna. The trophic net is a
surprisingly complex one, with primary producers (chemolithotrophic sulphoxidant
and methylotrophic bacteria using the dissolved compounds in the water), primary
consumers (heterotrophic bacteria, fungi), secondary consumers (protozoa as well as
grazers feeding on the microbial pellicle and plankton), tertiary consumers and
quaternary consumers (or predators, feeding on grazers and on lower-level con-
sumers). In other less isolated, open caves, such as Cueva de Villa Luz (Mexico) and
Frasassi (Italy), in which sulphurous waters flow, fauna is more uniformly distrib-
uted among the galleries, but specific groups of animals tend to cluster depending on
the particular features of the cave habitats (Hose and Pisarowicz 1999). Such caves
present several diverse habitats, localised in specific galleries and/or chambers that
differ by the amount of light coming through the skylights and the entrance, the
presence/absence of sulphurous water, the air currents and the anthropogenic influ-
ences (tourism, visits by local people for various rituals, etc.).
Owing to their isolation as well as to the peculiar habitats they host, hypogenic
caves can be used as natural laboratories where biological researches of sulphide-
based ecosystems mainly cover topics such as biodiversity of both micro- (prokary-
otic and eukaryotic microorganisms) and macrobiota, energy and C/N sources, food
web/trophic net (more precisely trophic chains), the toxicity of the environment and
the detoxification mechanisms developed by the organisms to overcome this threat
as well as morphological adaptations of those troglo-/stygobitic organisms. Of
course, there has also always been a quest for new metabolic pathways, for new
potentially valuable microorganisms, able, for instance, to degrade various materials
or/and potentially valuable sources for enzymes useful in different biotechnologies.
Currently, more than 20 years after the ALH84001 meteorite brought what appeared
to be first evidence of primitive life on early Mars, when exo- and astrobiology
gravitates mainly around the concept of habitability, the sulphide-based ecosys-
tems—especially in caves—are providing valuable information on evolution of
life on Earth and in the Universe through insights about conditions apparently
incompatible with life but in which life actually thrives (Schirmack et al. 2015).
16.3 Examples of Sulphide-Based Caves

16.3.1 Movile Cave: The First Terrestrial Chemosynthesis-
Based Ecosystem Ever Discovered
Movile Cave is located in south-eastern Dobrogea, a region of Romania limited to

the east by the Black Sea and well known for its thermomineral sulphurous waters,
which have been a spa facility since antiquity. The artificial shaft drilled during
geological prospection works in 1986 became the entrance to the system of natural
passages partially flooded with hydrogen sulphide-rich thermal water that is Movile
Cave. The cave represents the access to a deep captive sulphurous aquifer located in
Barremian-Jurassic limestone, extending 15 km north and 50 km to the south of
Mangalia. Through a system of geological faults, the deep water ascends towards the
surface and mixes with the Sarmatian oxygenated waters. Movile Cave is part of a
broad underground maze of fissures and passages associated with the sulphurous
aquifer in Mangalia as is shown by the wide distribution of Movile Cave fauna in
hand-dug wells in nearby localities, sometimes at distances of tens of kilometres
from the cave (Sarbu et al. 1994b).
Movile Cave is developed at a depth of 21 m (Lascu et al. 1995), in oolitic- and
fossil-rich limestone of Sarmatian age (i.e. late Miocene, about 12.5 Ma) that
contains numerous mollusc fossils (Lascu 1989). Almost from the very entrance in
the vertical shaft that leads to the main gallery, the visitor is struck by a smell of
rotten eggs, which becomes ever stronger on the way to the end of the cave. The
underground passages form a network, with only about 200 m length accessible to
humans. In the deep part of the cave, the galleries become wider and higher, their
Fig. 16.1 Movile Cave main gallery; arrows indicate the cupolas of the ceiling. Photo by © Petr
Zajíček
heights ranging from 40 cm to 2.5 m; the walls are soft, easily penetrable by finger,
and the ceiling becomes cupola shaped (Fig. 16.1). This feature reflects severe
corrosion of the bedrock, as confirmed also by petrographic analysis. The difference
of about 0.5 C between the wall temperature (19–21 C) and the atmosphere, with
the latter being warmer (Sarbu and Lascu 1997), results in water condensation on the
cave walls/ceiling and formation of carbonic acid that accelerate the dissolution of
the surface of the limestone walls.
The superficial corroded layer of bedrock reaches a thickness of up to 8 cm and
consists of uncemented oolites (Horoi 1994). To this condensation corrosion process
is added the sulphuric acid corrosion, as previously described (see Eqs. 16.1 and
16.2), and as a consequence of sulphide dissolved in the water condensed on the
walls, gypsum crystals form and cover the limestone surfaces (Fig. 16.2).
The passages are distributed on two levels, merging in one chamber—the Lake
Room (Fig. 16.3). The upper level is very dry and cooler than the lower one that is
warmed by thermal sulphide-rich water. Due to this temperature regime, warm
vapours produced at the surface of the sulphidic lake rise along the passages into
the upper level. Here, the atmosphere is enriched in CO2 (up to 1.5%), resulted
mainly from methane oxidation and metabolic activity of the cave biota as demon-
strated by C isotopic analysis (Sarbu et al. 1996).
The lower, flooded level (approx. 40 m in length) is accessible from the Lake
Room. This water accumulation is actually not a true lake, since the water flows,
with a velocity of 5 L/s at the bottom (Sarbu 2000) and makes the flow barely
noticeable. The water has a milky aspect, due to white sulphur suspended particles
Fig. 16.2 Gypsum crystals on the walls of Movile Cave. Photo by © Alexandra Hillebrand-
Voiculescu
Fig. 16.3 The Lake Room in Movile Cave. Photo by © Cristian Lascu
(Fig. 16.4), has a temperature of 21–22 C, pH near neutral (7.2–7.4) and is rich in
H2S (0.2–0.3 mM), CH4 (0.02 mM) and NH4+ (0.2–0.3 mM). Dissolved oxygen is
measurable (9–16 μM) only in the first 5 cm from the surface; deeper than that, the
conditions are anoxic. The ceiling of the flooded galleries rises in several places
forming air pockets between the surface of the water, the walls and ceiling of the
Fig. 16.4 Microbial biofilm on the surface of the sulphurous water in AirBell II, Movile Cave.
Photo by © Alexandra Hillebrand-Voiculescu
gallery—the so-called air bells—with air unusually rich in CO2 (up to 2.5%) and
CH4 (1–2%) and poor in O2 (7–10%; Sarbu 2000). In this lower level of the cave,
almost no condensation occurs since the walls and ceiling are warmed by the thermal
water (Sarbu and Lascu 1997).
No seasonal variations in the chemical composition of the cave atmosphere were
registered, and no infiltrations from the surface occur since the fissures and cracks in
the limestone are sealed by clay and loess. The isolation from the surface was proven
by the lack of radioactive artificial nuclides 90Sr and 137Cs that are otherwise
abundant in the region since the nuclear accident at Chernobyl in 1986. Faecal
streptococci that should normally infiltrate underground as a result of the intense
grazing occurring on the surface are also absent from the cave environment (Sarbu
et al. 1994a). This surprising isolation raised questions about: (1) how a void in
limestone can be so secluded and (2) what is the energy base of the very rich and
diverse biocenosis, characterised by an abundance of predators in the trophic
structure, unusual for a cave ecosystem (Sarbu 2000).
At the time of Movile Cave discovery, its biocenosis was represented by 48 spe-
cies of invertebrates, of which, at that time, 31 were not described (Sarbu and Kane
1995). Later, even more new species endemic to the Movile Cave and/or the
sulphidic aquifer associated with Movile Cave were identified. The last identified
species was Dendrocoelum obstinatum by Stocchino et al. (2017). The species is
unpigmented and lacks eyes and is one of the top predators of the aquatic community
of the sulphidic groundwaters at Mangalia, along with the leech Haemopis caeca and
the heteropteran Nepa anophthalmia. In contrast to any other species that are
restricted either to sulphidic or non-sulphidic environments, D. obstinatum occurs
both in sulphidic and non-sulphidic wells, a fact that allows two scenarios of
subterranean colonisation. According to the first scenario, D. obstinatum used to
live exclusively in a sulphidic aquifer that in time became fragmented, thus being
presently found in areas that once were sulphidic but are not any more. The second
hypothesis proposes that cave colonisation occurred more recently, from
non-sulphidic areas. D. obstinatum is the first triclad species living in both sulphidic
and non-sulphidic conditions, as it is the first subterranean planarian species
recorded to be infested by a nematode (Stocchino et al. 2017).
Currently, there are 51 species of invertebrates from which 35 are endemic to
Movile Cave or the sulphidic aquifer associated (Sarbu et al. 2018). The fauna of the
cave is concentrated in close proximity to and within the sulphidic water (i.e. in the
Lake Room and the Air bells). The aquatic species living in the cave belong to the
phyla Platyhelminthes (flatworms), Nematoda (round worms), Rotifera, Annelida
(segmented worms), Mollusca (snails) and Arthropoda (ostracod, copepod, isopod,
amphipod crustaceans and insects) (Sarbu and Kane 1995). A peculiarity of the
aquatic organisms in Movile Cave is the ability to survive high concentrations of
H2S and very low concentration of O2/anoxic conditions (see also Chaps. 4 and 11).
Organisms exposed to H2S (Sarbu and Kane 1995) have developed several detox-
ification/resistance mechanisms such as an association with sulphur bacteria that
may be involved in the transformation of the toxic gas (Nepa anophthalmia),
lowered ventilation rate (Asellus aquaticus) and increased tolerance to H2S poison-
ing (Tobler et al. 2011; Greenway et al. 2014).
Referring to the terrestrial fauna, 30 new species of terrestrial invertebrates
belonging to the classes Arachnida, Crustacea, Myriapoda and Insecta appear to be
endemic for the Movile Cave- associated ecosystem (Sarbu et al. 2018; Sarbu and
Kane 1995). They have morphological adaptations to the dark environment that are
typical for obligate cave-dwelling organisms, such as lack of eyes, depigmentation
and elongated antennae and legs (e.g. Agraecina cristiani, Hahnia caeca, a new
species of Nesticus, Trachelipus troglobius).
While aquatic fauna has probably colonised the cave continuously through the
points of discharge of underground springs located along the Black Sea coast and on
the bottom of sulphide- rich lakes, two major events could have isolated cave
populations from their surface ancestors. These are the Messinian Salinity Crisis
(MSC) about 5.96–5.33 Mya (Sarbu and Kane 1995), the onset of the Pleistocene
and later glacial periods (Sarbu and Kane 1995; Falniowski et al. 2008). During the
MSC, the connection of the Mediterranean Sea with the Atlantic Ocean was closed,
and due to intense evaporation, the Mediterranean basin was reduced to hypersaline
lakes. The Black Sea level dropped about 3000 m compared with the present level,
and the climate in Dobrogea became extremely dry. Passages at depths of about
200 m were formed by underground waters and were later flooded by deep sulphide-
rich waters (Sarbu and Kane 1995). Data obtained using the molecular clock
technique indicate that the aquatic snail Heleobia dobrogica invaded the under-
ground voids associated with the thermal sulphurous water about 2.172 Mya ago,
when both temperature and precipitations decreased, initiating the Pleistocene in

Europe (Falniowski et al. 2008). Much later, the Würm glaciations in quaternary
(115,000–11,700 years ago), when the level of the Black Sea dropped with about
100–110 m (Panin and Strechie 2006), provided ideal conditions for new colonisa-
tion. Species such as Heteromurus nitidus and Cryptops anomalans are abundant in
the cave and are also found at the surface.
In the Air bells, but not in the Lake Room where the O2 concentration is close to
normal, the surface of the water is covered by a biofilm as thick as up to 2 cm, kept
afloat by CH4 bubbles, which sinks as soon as it is disturbed (Fig. 16.4). The biofilm
consists of bacteria, archaea and fungi and represents both habitat and food for
various protozoans and metazoans. The protozoan microbiota consists of flagellates
and ciliates. About 95% of total metazoans living in the microbial mat are nematodes
(Poikilolaimus sp., Monhystrella sp., Panagrolaimus c.f. thienemanni,
Udonchustenui caudatus and the endemic Chronogaster troglodytes), followed by
cyclopoid copepods (Eucyclops subterraneus scythicus, 3.5%) and ostracods
(1.1%). Harpacticoid copepods (Parapseudoleptomesochra italica), gammarids,
isopods, acarids, rotifers and gastropods occur in very low densities (Muschiol
et al. 2015).
When incubated with 14C-labelled bicarbonate, the microbial biofilm was shown
to incorporate radioactive carbon in its lipids (Sarbu et al. 1996). Ribulose-1,5-
bisphosphate carboxylase/oxygenase (RuBisCO, the key enzyme of the Calvin
cycle, responsible with CO2 fixation) was determined to be present and active both
in the microbial pellicle floating on the surface of the water and covering the walls of
the galleries and in lysates obtained from bacterial cultures isolated from water/
sediments samples (Sarbu et al. 1994a). These data demonstrated that Movile Cave
is a self-sustained ecosystem dependent on chemoautotrophically fixed carbon
(Kumaresan et al. 2014; Sarbu et al. 1996). At the time of this work, Movile Cave
ecosystem was the first terrestrial ecosystem known to be almost entirely based on in
situ production of organic matter through chemosynthesis. In 2006, another similar
terrestrial ecosystem (Ayyalon Cave) was discovered in Israel. A detailed overview
on the animal life in this hypogenic groundwater cave is given by Por et al. (2013).
A preliminary microbiological characterisation of the Movile ecosystem was
provided by Sarbu et al. (1994b) who, based on specific morphological and physi-
ological features, provided evidence of the presence of Thiobacillus and
Thiosphaera genera as well as of filamentous bacteria from the genus Beggiatoa.
Later, while the sulphur cycle in Movile Cave was investigated, it became apparent
that the main group of primary producers are the sulphur oxidisers, both aerobic and
facultatively anaerobic, and that these bacteria might account for the high concen-
tration of sulphide at the surface of the water (Rohwerder et al. 2003). Sulphate-
reducing bacteria were also shown to occur, the sulphur cycle being complete and
seemingly connected with the nitrogen cycle through denitrification linked to sul-
phur oxidation. Detection of sulphur-oxidising bacteria in cultures enriched with
thiosulphate and nitrate and incubated in anoxic condition, together with the pres-
ence of 16S rRNA gene sequences related to Denitratisoma denitrifiers in microbial
biofilm samples from the cave, supports the hypothesis that sulphate and nitrogen
cycles are linked. The use of NO3– by facultatively anaerobic sulphur oxidisers such
as Thiobacillus denitrificans, as an alternative electron acceptor for respiration,
might account for the depletion of nitrate from this environment, which is otherwise
very rich in ammonium (Rohwerder et al. 2003; Kumaresan et al. 2014). Microbial
N2 fixation might also occur in the cave, many of the bacteria like Beggiatoa and
Methylocystis being known N2 fixers; nevertheless, further work in the area is
needed because reduction of N2 to NH4+ is highly energy-consuming and generally
carried out during nitrogen-limited conditions (Kumaresan et al. 2014).
Along with sulphide oxidisers and sulphate-reducing bacteria, the second most
important primary producers are methano- and methylotrophic bacteria, using as
sole source of carbon and energy, CH4 and C1-compounds, respectively, such as
methanol and methylated amines resulted from the degradation of the microbial
biofilm, as sole source of carbon and energy (Rohwerder et al. 2003; Chen et al.
2009; Wischer et al. 2015). The first methanotrophs identified in Movile Cave were
retrieved from the water and the microbial pellicle and belong to Alpha-
(Methylocystis/Methylosinus) and Gammaproteobacteria (Methylomonas,
Methylococcus) (Hutchens et al. 2004). The methanol released by methanotrophs,
as a result of methane oxidation, is probably used by methylotrophs as source of
C. Among the obligate methylated amine users present in the microbial biofilm
Methylotenera mobilis is dominant (Kumaresan et al. 2014). Other abundant
methylotrophs are Methylobacterium extorquens and Methylovorus but also bacteria
that were not known to grow methylotrophically, such as Catellibacterium (98%
identity to Catellibacterium caeni), Cupriavidus (99% identity to Cupriavidus
necator), Porphyrobacter (99% identity to Porphyrobacter neustonensis) and
Altererythrobacter (99% identity to Altererythrobacter epoxidivorans) (Wischer
et al. 2015). For extensive description of bacterial metabolism of methylated amines
as well as for an overview on novel methylotrophs in Movile Cave refer to Wischer
et al. (2015).
Small-scale metagenomic sequencing of DNA from mat samples provided
approximately 960,000 sequences with a mean length of 360 bp, from which
36.8% corresponded to annotated proteins, 33.7% to unknown proteins and 1.9%
to ribosomal sequences. Of the annotated sequences, 96.5% were of bacterial origin,
1.8% eukaryotic, 1.3% archaeal and 0.2% were viral sequences. Phylum-level
phylogenetic classification revealed that 60% of the total annotated sequences
belong to Proteobacteria, alongside Bacteroidetes (12.1%) and Firmicutes (7.6%).
Nearly 3% of the total sequences retrieved were representative of cyanobacterial
sequences, which is not expected in a deep cave ecosystem (Kumaresan et al. 2014).
Data about microbiota from sediments, i.e. the microoxic and anoxic sites of
Movile, are still scarce. For the presence of methanogens in the sediment accounts,
amplicons of the mcrA gene from DNA extracted from sediment samples
(Kumaresan et al. 2014); to our knowledge, Methanobacterium movilense
(Schirmack et al. 2014) and Methanosarcina spelaei (Gazert et al. 2014) are the
only methanogens isolated so far from the anoxic sites of the cave.
Methanobacterium movilense is a hydrogenotrophic, secondary alcohol-utilising
methanogen isolated from sediments at the bottom of the cave water (Schirmack
et al. 2014), while Methanosarcina spelaei is a strictly anaerobic archaeon, growing

autotrophically on H2/CO2 and additionally using acetate, methanol,
monomethylamine, dimethylamine and trimethylamine (Gazert et al. 2014).
An inventory of the cultivable microscopic fungi has been recently published by
Nováková et al. (2018). Samples of dry material, including sediment and clay
scrapped from the walls/ceiling of the upper level galleries, isopod faeces and
invertebrate carcases (dead isopodes and spiders) were collected, while the wet
samples, taken from the lower level (Lake Room and AirBell II), included water,
microbial biofilm floating on the surface of the water and underwater sediment. Air
from both dry and flooded parts of the cave was assessed for airborne fungi by the
gravity settling method. To evaluate the degree of cave contamination with exoge-
nous fungi, the underground microfungal community was compared with the sur-
face, by collecting air and soil samples from the proximity of the cave entrance and
similarly processed. For Aspergillus and Trichoderma, the morphological, physio-
logical and chemical analyses were combined with molecular techniques. 90 and
28 fungi species were recovered from the dry part of the cave and from AirBell II,
respectively.
Sixty-seven species were found only in the dry part of the cave, while 20 were
found both in dry galleries and AirBell II. The most diverse genera were Penicillium,
Aspergillus and Cladospora. Undescribed species of Aspergillus sect. Candidi,
Talaromyces kendrickii and Talaromyces ruber are confined to AirBell II. The
presence of Aspergillus sp. sect. Candidi in the anoxic sediment might indicate its
involvement in denitrification processes. Aspergillus thesauricus, Cylindrocarpon
didymum, Penicillium griseofulvum and P. janthinellum were isolated only from
invertebrate cadavers while Spiniger meineckellus, Aspergillus sydowii, Cadophora
malorum and Paecilomyces sp. from isopod faeces only. Aphanocladium album,
Aspergillus creber, Cladosporium variabile, Lecanicillium psalliotae, and
Stachybotrys chartarum were only isolated from the dry part of the cave and not
from AirBell II nor from outside the cave. The new species Aspergillus movilensis
and A. europeus were isolated from outside the cave, isopod faeces and cave
sediment and from outside the cave, respectively (Hubka et al. 2015, 2016). Of
great abundance and variability in the cave is also the fungi Trichoderma. The
association with the chemoautotrophic bacteria and archaea present in the microbial
pellicles floating on the surface of the water might have ecological importance, in the
consumption of organic material produced chemoautotrophically and in dead cell
degradation (Nováková et al. 2018).
Classical microbiological and molecular biology methods, together with stable
isotope probes (SIP), have enabled a broad understanding of the ecosystem associ-
ated with the sulphurous aquifer present in South Dobrogea, to which Movile Cave
is only an access gate. Strategies combining classical methods, SIP and
metagenomic sequencing can provide genome information for various microbes,
especially that these are difficult to cultivate in the laboratory (Kumaresan et al.
2014). Active microbial communities in the cave ecosystem could be further
documented using metatranscriptomics and metaproteomics (Kumaresan et al.
2014). Ongoing as well as future research, focussing on the anoxic niches of the
cave, namely, the sediment and the anoxic water, may answer questions such as
whether there is any biogenic methane production or anaerobic methane oxidation.
Still to be investigated are the trophic interactions between various microbial
functional groups. Next-generation sequencing, combined with a variety of molec-
ular ecology techniques and a concerted effort to isolate novel organisms, will
improve our understanding of the functional diversity of the microbial communities
and allow us to study the contributions of different functional guilds in maintaining
this self-sustaining chemoautotrophic ecosystem (Kumaresan et al. 2014).
16.3.2 Cueva de Villa Luz (Mexico): A Cave Where

Speciation Occurs in Its Multiple Habitats
Another sulphidic cave in which complex research has been undertaken is Cueva de
Villa Luz (also called Cave of the Lighted House/Cueva de las Sardinas/Cueva de la
Sardina/Cueva del Azufre) located 2 km south of the village Tapijulapa, Municipio
de Tacotalpa, Tabasco, Mexico on the northern edge of the Chiapas Highlands
(Palacios-Vargas et al. 2015; Borgonie et al. 2010). La Cueva de Villa Luz
(Fig. 16.5) began to be scientifically studied only in the 1960s although its occur-
rence has been known for a very long time and it was used for various ceremonies
and rituals by the native Zoque (Soque) population (Tobler et al. 2008, 2011).
Decades before scientific studies began, unidentified specimens of a diverse fauna
in the cave were collected both alive and preserved (Gordon and Rosen 1962;
Palacios-Vargas et al. 2011). However, the first description of the cave as well as
detailed studies on the cave molly fish (Poecilia mexicana) alongside with a partial
inventory of the bats species living here was completed by the biologists Gordon and
Rosen (1962). Unfortunately, their work was put aside for over 20 years, until 1987,
when speleologists Jim Pisarowicz and Warren Netherton started to explore the
region of Teapa (Tabasco). This is when the systematic and scientifically research at
Cueva de Villa Luz really took off. A sketch of the entrance segment as well as the
geological profile of the area where the cave is located was presented by Gamboa
and Ku (1998), and the first accurate map and in-depth description were provided by
Hose and Pisarowicz (1999).
The cave is located in a region with very well-represented endokarst, developed
in lower cretaceous limestone and bounded to the south by a normal fault, a structure
that probably controls the cave inlet’s location (Hose et al. 2000). Cueva de Villa
Luz is situated at an altitude of 100 m asl in a valley (Arroyo del Solpho) in between
Amatán and Oxolatan streams, before they join to form the Tacotalpa river, about
100 km away from the Campeche Bay. The cave is surrounded by semi-tropical
jungle as a consequence of both high temperatures and heavy rains resulted from the
cooling of the moist winds coming from over the bay, uplifted by the Chiapas
Highlands (Sierra Madre de Chiapas; Gordon and Rosen 1962).
16
LEGEND
Breakdown Stream Mud or Silt
Slope
Block Flow
Snottites
~ Bedrock 32
Breakdown Floor Dip
Sand, Silt or Selenite Gypsum Skylight or
N
CUEVA DE VILLA LUZ Dirt Paste Entrance
Drop or
Tabasco, Mexico M Moonmilk
Stalagmite Ledge ? Lead Too
Windway and Stalactite Elemental Not surveyed Tight
Springs Sulfur
PLAIN
N VIEW Water Ceiling Drop
Flowstone
CASA DE LOS Springs
MURCELOGOS ? ?
E E'
? ? E
? F SAG
D' CCCC F' 32 PAS
D 38 ZING
~ BUZ ? MAIN
M ~~ 32 42
M SALON
SALON DE LOS
DE LOS 85 ? ENTRANCE
SULFUR SPRING ~~ XXX
~ ~ ~ ~
~ ~ ~ MURCELOGOS
MURCELOGOS
SNOT ~ ~ SOPRESA DE JAIME
~ ~ 32
~ ~ ~
~ 45 ~ ~
HEAVEN ~ ~ 32 4re Drop Water
~ 52 ~ XXX
~ XXX
M ~ ~ ~ ~ ~
~ ~ ~ ~
~ ~
~ ~ ~ CAT BOX
~ SALA RAGU
RAGU ~ ~
MAIN PASSAGE 37 ~ ~ ~ ~ ~
Fresh air
Fresh Air PASSAGE
PASSAGE
GRANDE ~ ~ 1 km
Profile View ~ section
Section ~~
M
~
~~ ~ ~~
~
~ Itctly Natural ?
TH E O
Researches in Sulphide-Based Ecosystems
B B' M 28 ~ ~ ~ ? ~
~ ~ ~ ~ ~
~ Passage Bridge ~ ~ ~
T HE
~ ~ ~ ~
~
R
YELLOW ROSES 37 ~ ~ M ~ ~ ~ ?
BU
~ ~ ~ ~
A'ROOM C' ZZ ~~
A IN G GE ~
P A SSA M ~
C The Zoo Passage Damas Primeras ~
I' ~
F G' ~~ ~
M
H' I
G H ~ ~
87 SCALE ~
0 10 20 30 ~ Resurgence
The Zoo BAT M
BAT U' Meter Area
~
Profile View ROOM
ROOM U
Joins Main
THE ZOO PASSAGE - Profile View Stress Passage
MAIN
0 ENTRANCE 0
10m SNOT SALA SALON DE LOS 10m

HEAVEN GRANDE MURCIELOGOS SOPRESA
Sopresa Resurgence
DE
de JAIME
jaime Area
20m 20m
30m 30m
MAIN PASSAGE - Profile View
Fig. 16.5 Map of Cueva de Villa Luz; with permission from Hose and Pisarowicz (1999)
363
Regarding its speleogenesis, Cueva de Villa Luz was formed mainly by the
slightly rise of thermal and sulphur-rich water from multiple springs through discrete
conduits or gravel deposits on the floor of the cave (Hose et al. 2000). These inlets
merge into a low-gradient, milky coloured stream of about 10–30 cm deep that flows
through the cave (Hose and Pisarowicz 1999) at a rate of 200–300 L/s (Hose et al.
2000). Along with the H2S-rich inlets (containing up to 300–500 mg/L H2S and no
measurable O2), there are also discharge inlets of oxygenated water (4.3 mg/L O2)
and no detectable H2S. The origin of the cave water is still under debate. The source
of the H2S-rich water might be the rich petroleum fields near Villahermosa, within
50 km of the cave, as indicated by the nature of dissolved solids in the cave water, as
well as El Chichón volcano (also about 50 km away from the cave) which has
sulphur-rich waters in its caldera (Hose et al. 2000).
The stream discharge occurs year round at a constant level, with flow discharges
measured between 290 L/s (January 1998, early in the dry season) and 210 L/s
(August 1999, early in the wet season); even after heavy rains, when the discharge in
the Río Xoxocotlan, east of Tapijulapa, rose from 186 to 538 m3/s (Hose et al. 2000)
changes in the stream discharge at the resurgence and within the cave were not
significant. The pH of the water is nearly neutral, with values of 7.2 and temperature
of 27.5–28.38 C—slightly thermal as compared with the regional groundwater
temperature of about 22–24 C (Hose et al. 2000).
The length of the explored galleries is of about 1.9 km (Borgonie et al. 2010),
most of them being flooded by the sulphurous stream. The heights of the passages
range from 25 cm to 15 m with several vertical tubes extending tens of metres
upward from the stream level, intersecting the surface to form skylight entrances
(Hose et al. 2000). The presence of these holes in the ceiling gave the name of the
cave Cueva de Villa Luz (Cave of the Lighted House) and together with the sulphidic
inlets determine various habitats, which differ drastically in terms of the presence/
absence of the light and the presence/absence of H2S. These factors can represent
sources for natural selection and shape the structure of fauna populations as well as
the trophic web in the cave.
The biocenosis of the cave is very diverse, represented by invertebrates and
vertebrates. The trophic chains benefit of three sources of energy, namely, the litter
that enters the cave through the skylights, the entrance or/and brought by animals,
the bats guano and the chemosynthetic bacteria forming the snottites (biofilms in the
shape of filamentous, gelatinous, elastic stalactites; Palacios-Vargas 2009). Among
the invertebrates, ubiquitously present in huge densities is the dipteran
Goeldichironomus fulvipilus, whose larvae are in their highest densities within the
bacterial mats, where they graze on the bacteria (Palacios-Vargas et al. 2015). Within
the snottites, live the nematode Mesorhabditis acidophila whose life cycle is tightly
linked to the acidity of this microbial structures (pH 0–3), probably to escape
predation by mites (Borgonie et al. 2010). The giant water bug Belostoma
(Heteroptera, Belostomatidae) occurs also in high numbers in the cave. Planarians
of the genera Dugesia, crabs Trichodactylus bidens (Trichodactylidae) and
Avotrichodactylus bidens, whip spiders Phrynus (Amblypygi) and different spiders
of the genera Tetragnatha (Tetragnathidae), Maymena (Mysmenidae), Eidmannella
(Nesticidae), Tinus sp. (Pisauridae), Hemirrhagus pernix (Theraphosidae) and a

Ctenidae representative (Palacios-Vargas et al. 2011; Horstkotte et al. 2010;
Palacios-Vargas 2009) are also present in the cave. The majority of acari live in
guano (Mesostigmata and Astigmata, 79% and 6% of the microarthropods, respec-
tively) or in litter. Associated with bacteria are only two species of Histiostomidae
(Astigmata) and Oppidae (Cryptostigmata), in low number of individuals (Palacios-
Vargas 2009).
From vertebrates, one species of fish was recorded in the cave, Poecilia
mexicana, family Poeciliidae, notorious for being intensively studied for their
pronounced ecomorphological and geographic variation (Tobler et al. 2006) and as
models for speciation process triggered by peculiar environmental conditions such as
H2S and/or rotenone (a well-known inhibitor of the mitochondrial complex I, NADH
coenzyme Q reductase; Palacios et al. 2013). As an adaptation to the high concen-
tration in the water of the extremely toxic H2S, cave mollies perform aquatic surface
respiration (ASR) to exploit the more oxygenated topmost layer of the water column
(Horskotte et al. 2010). The synbranchid eel Ophisternona enigmaticum was occa-
sionally reported from the cave (Tobler et al. 2007). The other vertebrates are the
bats from the families Mormoopidae (Mormoops megalophylla, Pteronotus davyi,
Pteronotus parnellii, Pteronotus personatus and Pteronotus gymnonotus),
Emballonuridae (possible Baliantopterix), Phyllostomidae (Carollia) and the vam-
pire Desmodus rotundus (subfamily Desmodontinae; Palacios-Vargas et al. 2011).
In contrast to Movile Cave, Cueva de Villa Luz has three sources of energy,
namely, the bat guano, the organic matter (leaves, soil, wood, detritus) that enters the
cave through the skylights and the microbial communities formed of chemoautotro-
phic bacteria (Palacios-Vargas 2009). A pellicle of condensed or infiltrated fresh
water covers the walls resulting in droplets in which hydrogen sulphide outgassed
from the sulphidic water dissolves. In the droplets, the H2S reacts with dissolved O2
forming sulphuric acid. The pH value of the droplets is around 1.4. Sulphur-
oxidising bacteria such as Acidithiobacillus thiooxidans facilitate this process
(Hose et al. 2000) and form white deposits, called snottites that drip sulphuric acid
at their extremities and grow quickly in length, up to a centimetre per day (Palacios-
Vargas et al. 2015). The biofilm covering the walls of the galleries, as well as the
snottites are dominated by the genus Acidithiobacillus (Jones et al. 2016) serving as
one of the food bases in the cave, directly eaten by very abundant larvae of midges
(Hose et al. 2000). While both prokaryotic microbiota and macrofauna of Cueva de
Villa Luz are well characterised, fungal and protists communities have been
neglected. To our knowledge, no study of these two components of the cave
ecosystem has been undertaken yet, so that Cueva de Villa Luz remains still a
very fertile subject for future investigation.
Acknowledgements I thank Dr. Şerban Sârbu and Cristian Lascu for wonderful explanations and
discussion on the underground ecosystems and to Vlad Voiculescu, Mihai Baciu and Dr. Virgil
Drăguşin for assisting me in my research of Movile Cave. I am most grateful to my collaborators,
Prof. Colin Murrell (UK), Dr. Deepak Kumaresan (UK) and Dr. Alena Nováková (CZ) for valuable
scientific discussions and great time spent together during sampling trips and throughout our
collaborations.
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Chapter 17
Researches in Lava Tubes
Pedro Oromí
17.1 Introduction
Volcanic terrain usually lacks limestone and other karstifiable rocks, but the forma-
tion of caves is frequent, though in a very different way from karstic processes. The
most common caves in volcanic terrain are lava tubes, which form only in smooth,
ropy basaltic flows (“pahoehoe”) very soon after eruption, while molten lava is still
flowing. Once a lava tube has cooled, it becomes habitable by a specialized fauna
only through a process of ecological succession, on a variable time span depending
on the local climate. Due to collapses, erosion and silting, lava tubes persist as
suitable habitats for much shorter periods than limestone caves, usually not exceed-
ing a few hundred thousand years (Howarth 1973).
The Canary archipelago in the eastern Atlantic is exclusively of volcanic origin
and includes seven main islands with a variety of ages (from 1 to 21 Ma), volcanic
rocks, type of eruptions and local climates. Tenerife is the largest, highest and most
diverse island, with abundant lava tubes and other subterranean environments which
harbour a rich troglobiont fauna (Oromí 2004). The highest density of lava tubes on
the island is found in the lavas emitted by the initial basaltic eruptions of Pico Viejo,
a stratovolcano in the central-western part of the island. Three caves located in these
lavas, Cueva del Viento-Sobrado, Cueva de Felipe Reventón and Cueva de los
Roques, have been the most attractive lava tubes for scientific studies in the Canary
Islands (Montoriol and de Mier 1974; Wood and Mills 1977; Martín and Oromí
1986; Oromí 1995; Arechavaleta et al. 1996, 1998, 1999) together with the
anchialine part of Corona lava tube on the island of Lanzarote (Wilkens et al.
2009; Martínez et al. 2016 and references therein).
P. Oromí (*)
University of La Laguna, Tenerife, Canary Islands, Spain

https://doi.org/10.1007/978-3-319-98852-8_17
370 P. Oromí
Fig. 17.1 Map of the northwest part of Tenerife. Dark spots: present outcrops of Pico Viejo
pahoehoe lavas where the studied lava tubes are located. Dashed lines: boundaries of Icod and
Orotava landslides. Modified after Carracedo et al. (2008)
17.2 Geological Aspects
Between 2.5 and 0.2 Ma, there were several cycles of volcanic activity in the centre
of Tenerife, with large increases in height and three or more major explosive events,
with the consequent steep sloping and instability. At least three huge landslides
occurred during the last million years, at 0.8 Ma, 0.6 Ma and 0.15 Ma, respectively
(Masson et al. 2002). The two older ones created the Güímar and La Orotava valleys,
but the third one, known as the Icod landslide, released the island of such weight that
volcanism was strongly reactivated, refilling the landslide scar and building up the
existing Teide stratovolcano (3714 m) and its twin Pico Viejo (3135 m). These two
volcanoes filled the upper part of the landslide scar and formed the northern limit of
the modern 16 9 km Las Cañadas caldera (Fig. 17.1).
The initial eruptions of Pico Viejo were exclusively basaltic, producing fluid
pahoehoe flows which readily form lava tube caves. Lava flows dated at
27,030 430 years ago extended towards the west and the north, reaching the
coast at both sides of the island, but a few short flows went southwards inside Las
Cañadas caldera at over 2000 m.a.s.l. (Carracedo et al. 2008). The northern branch
spreads over a large area and gave rise to many lava tubes, constituting the highest
cave concentration in the archipelago. However, later eruptions of Teide and Pico
17 Researches in Lava Tubes 371
Fig. 17.2 Cueva de Felipe Reventón has many interconnected labyrinthine passages, typical of
lava tubes originated on steep slopes. Photo by © Juan S. Socorro
Viejo produced extensive intermediate lavas and phonolites which covered most of
the northwest of the island (except the ancient Teno massif), leaving exposed on the
surface only some big kipukas (outcrops of the older pahoehoe lava surrounded by
later flows). The entrances to the most important lava tubes in Tenerife—Cueva del
Viento-Sobrado, Cueva de Felipe Reventón and Cueva de San Marcos—are in
outcrops of this kind in the vicinity of Icod de los Vinos. The more limited Pico
Viejo pahoehoe flows inside the caldera were hardly covered by newer lavas and
contain a highly interesting lava tube, Cueva de los Roques (see locations in
Fig. 17.1).
As Wood and Mills (1977) pointed out, Cueva del Viento is characterized by
unusually complex planimetric form (see Fig. 17.2), sinuosity of the main passages,
steep, multilevel long profile and great variety of passage forms. These features are
also found at Felipe Reventón and to a lesser extent at Cueva de los Roques. The
multilevel profiles were formed by lavas flowing over one another, and their
passages were sometimes connected by the lower tube capturing the lava flowing
through an upper, newly forming tube. Such captures, indicated by solidified lava
falls up to 12 m high, connect as many as three distinct levels in Cueva del Viento-
Sobrado (Fig. 17.3). Environmental parameters can differ between shallow and deep
passages, with significant ecological consequences.
The known extent of Cueva del Viento-Sobrado (see Table 17.1), the world’s fifth
longest lava tube (Gulden 2015), must be just a small part of its real dimensions,
given that its highest known passage is more than 10 km away from the eruptive vent
on Pico Viejo. Felipe Reventón is actually an independent cave from a strict
372 P. Oromí
Fig. 17.3 Cross section of interconnected galleries in Viento-Sobrado cave complex, showing the
three different levels formed by consecutive overlapping flows of the same eruption. Drawing by
© Ángel Vera
Table 17.1 Data on the most important lava tubes from Pico Viejo pahoehoe lavas. Temperatures
measured in galleries away from entrances
Temperature
Cave Linear extent (m) Altitude range (m) ( C) No. of entrances
San Marcos 2130 30–99 15.0–16.0 2
Viento-Sobrado 18,500 480–1040 13.8–15.2 6
Felipe 1845 595–734 14.5–15.5 1
Reventón
Los Roques 900 2160–2200 13.0–13.5 4
speleological point of view, but its projection on the map reveals its continuity at
some distance with one of Cueva del Viento-Sobrado main branches, now
interrupted by a lava siphon.
17.3 Ecological Features
Lava tube caves that have not been covered by later flows are usually shallow,
forming a few metres below the surface of a solidifying flow and running parallel to
the slope. While in limestone caves ecological succession advances downwards
creating new habitats ever deeper, in lava tubes the situation is reversed, and
succession progresses upwards as soil develops on the surface and vegetation
grows on it, and the cave environment becomes humid and stable (Howarth 1996).
Conditions in the deepest passages in the lowest levels of Cueva del Viento-Sobrado
differ from those in shallower ones, the former with scarcer food resources, more
Fig. 17.4 Roots hanging from the ceiling in one of the main passages of Cueva del Viento (Photo,
Juan S. Socorro). Upper left: an individual of the planthopper Tachycixius lavatubus on the roots.
Photo by © Pedro Oromí
constant temperature and humidity and higher CO2 concentrations. Conditions in

deep tubes are unsuitable for non-adapted animal species and lead to dominance of
troglobionts (Arechavaleta et al. 1994).
Young lava tubes are usually dry and are occupied by particular epigean species
shared with recent lavas and known as lavicoles; as ecological succession pro-
gresses, the caves mature, and this fauna is replaced by troglobionts (Ashmole
et al. 1992). Most caves from Pico Viejo lavas are in a mature stage due to the
time elapsed since their origin (appr. 27,000 years) combined with the mesic climate
prevailing on the island. Pahoehoe lavas from the Icod region are mostly covered by
soil, with a mixed pine forest on those at intermediate altitudes (Cueva del Viento-
Sobrado and Cueva de Felipe Reventón). Stable temperatures (see Table 17.1) and
high humidity prevail throughout the year in these caves, suiting them for cave-
adapted fauna. The pahoehoe flows containing Cueva de los Roques are in an earlier
stage of succession due to the drier climate at this altitude; here there is generally
little soil on the surface and only scattered shrub vegetation. Nevertheless, environ-
mental stability in Cueva de los Roques is adequate in galleries where drying air
currents are absent (Martín and Oromí 1986).
The shallow situation of lava tubes and the abundant cracks in the lava allow plant
roots to reach these caverns, dangling from the ceiling and walls (Fig. 17.4). This is
an essential resource for sapsucking and rhizophagous insects, which are usually
absent or very rare in limestone caves from geographically comparable areas. These
species are significant contributors to the cave community and its trophic network,
and are probably the most abundant cave-dwelling animals. The profuse network of
cracks and small collapses also facilitate entry by many trogloxene animals, which
374 P. Oromí
form an important resource for predatory species in many parts of the cave, far from
any large entrance. The abundant epigean flies captured in traps demonstrate the easy
connection with the outside and may account for the striking abundance and variety
of troglobiotic web spiders, which could not survive feeding only on other
troglobiont arthropods.
Relative humidity is usually high and ceiling drip very frequent, but water is
rarely present on the cave floor due to the abundance of cracks connected with a
network of voids in the host rock. Thus, no significant water ponds are present, and
no stygobiont species have ever been found because the water table is much deeper
than the caves. The absence of water streams within the caves is a major cause of the
poverty of passages at deeper levels, where scarce food supplies arrive with perco-
lating water and only a few troglobionts can survive.
Environmental features in Viento-Sobrado and Felipe Reventón caves are very
similar since they are in the same lava flow, mainly at the same altitude and at an
identical stage of maturity, sharing most of the troglobiont species. From the
ecological and the speleogenic points of view, they should be considered as a single
cave, even though they lack connections for humans. Accumulations of soil in some
passages provide the only habitat better represented in Felipe Reventón and may
explain the occurrence of some species never found in Cueva del Viento-Sobrado.
Cueva de los Roques is within Teide National Park; its entrances are fenced and
visits highly restricted, so it is in a very good state of conservation. However, Viento-
Sobrado and Felipe Reventón caves are in an inhabited area, with houses on the
surface, so they are threatened to some extent. The lower part of Viento (Piquetes) is
absolutely polluted by raw sewage, the central part (Breveritas) is reasonably well
preserved but endangered, while the upper part (Sobrado) is in a natural area under
pine forest and is owned by the local government, gated and in very good condition
(Oromí 1995). A 150 m long stretch of Sobrado is dedicated to public visits.
17.4 Animal Communities
The diversity of terrestrial troglobionts in the three lava tubes is really remarkable,
competing with the richest caves in the world (see Table 17.2) and being the highest
among volcanic caves (Culver and Pipan 2013). The communities in these cavities
are by far the most varied in cave-adapted animals within the Canary archipelago
(see Table 17.3), the only one on a comparable level being the community in the
anchialine Cueva de la Corona on Lanzarote, which only harbours stygobiont
species (Martínez et al. 2016).
Due to the shallowness of these Tenerife caves, which facilitates the input of
accidental trogloxenes, the total number of arthropod species found in them is
roughly double the number of troglobionts (see Table 17.4, bottom), with the
exception of Cueva de los Roques which is in a dry subalpine zone with a poorer
epigean fauna. The deepest levels of Cueva del Viento-Sobrado are usually free of
these trogloxenes.
Table 17.2 Caves with more than 25 troglobionts (modified from Culver and Pipan 2013). Cueva
del Viento and Cueva del Sobrado, actually the same cave system, harbour a total of 36 species
between them
Cave Country No. of species Cave type
Postojna-Planina Cave Slovenia 38 Karst cave
Cueva de Felipe Reventón Canary Islands 37 Lava tube
Vjetrenica Bosnia and Herzegovina 30 Karst cave
Movile Cave Romania 29 Chemoautotrophic
Cueva del Viento Canary Islands 28 Lava tube
Cueva del Sobrado Canary Islands 28 Lava tube
Mammoth Cave USA 26 Karst cave
Table 17.3 Number of cave-adapted species (troglobionts and stygobionts) so far known in the top
ten richest lava tubes and one artificial mine (a) on the Canary Islands
Cave Island Habitat No. of species
Felipe Reventón Tenerife Terrestrial 37
Viento-Sobrado Tenerife Terrestrial 36
La Corona Lanzarote Anchialine 33
Los Roques Tenerife Terrestrial 24
Labrada Tenerife Terrestrial 19
Palmeros La Palma Terrestrial 19
Grande de Chío Tenerife Terrestrial 16
Bucio Tenerife Terrestrial 15
Salto de Tigalate La Palma Terrestrial 15
Los Llanetesa Gran Canaria Terrestrial/freshwater 11
Viento-Sobrado and Felipe Reventón have a total of 42 species if considered as a single cave.
Local troglophiles are usually widespread species, some clearly introduced like
the millipedes Blaniulus guttulatus and Choneiulus subterraneus and some probably
native like the spider Meta bourneti, the psocid Psyllipsocus ramburii and also the
moth Schrankia costaestrigalis, whose caterpillars are active root eaters. None of the
mentioned troglophiles except the psocid are present in Cueva de los Roques,
probably due to the colder climate at this altitude.
The cave-dwelling communities of Cueva del Viento-Sobrado and Cueva de
Felipe Reventón are very alike and probably constitute a single unit for the animals
because of their proximity and underground connection through mesocaverns (see
Chap. 3). No less than 42 troglobiont species have been found in them together (see
Table 17.4), which represents 60% of the total species known from the central,
young part of Tenerife at low and intermediate altitudes. Most of these troglobionts
have spread out along the island through different subterranean environments
(mesocaverns, MSS, etc.), occupying most of the island except the old, caveless
Anaga Peninsula in the NE, which has remained isolated in terms of subterranean
dispersal (Oromí and Martín 1992). The troglobiont community from Cueva de los
Roques includes six species only found at over 1800 m.a.s.l. on the island; some of
Table 17.4 Troglobiont arthropods occurring in the studied caves of Pico Viejo lavas
376
Lava tube
Fauna group Family Species Trophic spectrum Viento Sobrado Felipe Reventón Los Roques
Pseudoscorpiones Chthoniidae Paraliochthonius setiger Predator x
Paraliochthonius superstes Predator x x
Paraliochthonius tenebrarum Predator x x
Lagynochthonius curvidigitatus Predator E
Syarinidae Microcreagrina subterranea Predator x x x
Araneae Dysderidae Dysdera ambulotenta Predator x x x
Dysdera esquiveli Predator x x
Dysdera labradaensis Predator x x x
Dysdera sibyllina Predator x x
Dysdera unguimmanis Predator x x
Dysdera gollumi Predator E
Dysdera chioensis Predator x
Pholcidae Spermophorides reventoni Predator x x
Pholcus roquensis Predator E
Liocranidae Agraecina canariensis Predator x x
Linyphiidae Metopobactrus cavernicola Predator x x
Troglohyphantes oromii Predator x x x
Walckenaeria cavernicola Predator x x
Nesticidae Canarionesticus quadridentatus Predator x x
Diplopoda Glomeridae Glomeris speobia Detritivore x x x
Iulidae Dolichoiulus labradae Detritivore x
Dolichoiulus ypsilon Detritivore x x x
Chilopoda Cryptopidae Cryptops vulcanicus Predator x
Lithobiidae Lithobius speleovulcanus Predator x x x
Isopoda Porcellionidae Porcellio martini Detritivore x x
P. Oromí
Armadillidae Venezillo tenerifensis Detritivore x x x

Trichoniscidae Trichoniscus bassoti Detritivore x x
17
Collembola Paronellidae Troglopedetes cavernicola Detritivore x x

Troglopedetes vandeli Detritivore x x
Blattaria Blattellidae Loboptera subterranea Omnivore x x
Loboptera troglobia Omnivore x x
Loboptera sp. Omnivore E
Hemiptera Cixiidae Tachycixius lavatubus Rhizophagous x x x
Coleoptera Carabidae Lymnastis subovatus Predator x x x
Lymnastis thoracicus Predator x x
Spelaeovulcania canariensis Predator x x
Researches in Lava Tubes
Canarobius oromii Predator x

Gietopus martini Predator x x x
Wolltinerfia tenerifae Predator x x x
Histeridae Aeletes oromii Predator E
Staphylinidae Ocypus n. sp. Predator E
Domene alticola Predator x x x
Domene vulcanica Predator x x x
Micranops bifossicapitatus Predator x
Micranops spelaeus Predator E
Micranops mlejneki Predator x
Alevonota oromii Predator x
Alevonota outereloi Predator x
Alevonota canariensis Predator x
Curculionidae Oromia hephaestos Rhizophagous x x
Troglobionts 50 36 37 24
Total no. of species 86 77 38
In bold: taxa with no congeneric epigean species on the archipelago. (x) presence; (E) exclusive to one cave. Total species: non-adapted + troglobionts.
377
378 P. Oromí
them are exclusive to this cave (see Table 17.4 and Arechavaleta et al. 1998). Some
of these high-altitude species like Dysdera gollumi, Alevonota canariensis,
Micranops mlejneki and Ocypus sp. are vicariants to other congeneric troglobionts
occurring at lower levels.
It is remarkable the high diversity of predators (37) occurring in these caves in
relation to the total number of troglobiont species (50). Particularly interesting is the
presence of 4–5 species per cave of Dysdera spiders (Fig. 17.5), a genus with nine
troglobiont species in Tenerife, all but two with different epigean sister species
(Arnedo et al. 2007). This implies that at least eight independent colonizations of the
underground have taken place, with eight different evolutionary events towards
troglomorphism. Only the ninth species derived from an already troglobiont sister
taxon. Multispecies lava tubes are generally inhabited by large-, medium- and small-
sized Dysdera species, as happens with D. ambulotenta, D. chioensis and D. gollumi
in Cueva de los Roques (Arnedo et al. 2007). Agraecina canariensis is also a
wandering spider, while the remaining six spiders found in these caves are
web-building species.
Beetles are the most varied order, the majority being predatory species. The
ground beetles are all eyeless but not highly evolved with respect to other
troglomorphic characters, as is usual with other cave-adapted Canary species of
this family. However, most of the nine rove beetles listed in Table 17.4 are highly
troglomorphic (see Fig. 17.6), a common feature of the 24 troglobiont rove beetles
from the archipelago, which together with those from Morocco form the top world’s
hot spot for this group (Hlaváč et al. 2006).
Omnivore species are represented by three Loboptera cockroaches, completely
troglomorphic like three other species of the genus occurring in central Tenerife,
Fig. 17.5 Dysdera unguimmanis, the most troglomorphic among nine cave-adapted species of the
genus occurring in Tenerife. Photo by © Pedro Oromí
Fig. 17.6 The rove beetle Domene vulcanica is one of the most troglomorphic arthropods
occurring in Tenerife, with a marked “aphaenopsid” appearance. Photo by © Pedro Oromí
while in the MSS of the older Anaga peninsula, there are two-eyed ambimorph
species (Oromí and Martín 1992). Three permanent cave-dwelling insects feed on
living roots inside these lava tubes: the caterpillar of the troglophilic moth Schrankia
costaestrigalis, the scarce eyeless weevil Oromia hephaestos and the very abundant
planthopper Tachycixius lavatubus. The latter is frequent in most lava tubes on the
island, showing subtle morphologic but marked bioacoustic differences across its
range, which suggests the existence of at least two allopatric species on the island
(Hoch and Asche 1993). Detritivore species are represented by Venezillo tenerifensis
woodlice and Glomeris speobia pill millipedes, both occurring in most Tenerife
caves, and two true millipedes of the diverse genus Dolichoiulus, which has given
rise to a swarm of 53 endemic species on the archipelago, including 7 troglobionts
(Enghoff 2012 and references therein). Two troglomorphic springtails of the genus
Troglopedetes have been recorded for these caves (da Gama and Ferreira 2000), but
their identity should be checked since it is difficult to accept that continental
troglobionts have colonized oceanic islands.
Concerning their possible evolutionary origin, 18 out of 50 troglobionts occurring
in these caves (Table 17.4, in bold) have no epigean close relatives and can be
considered as relicts. The Canaries have never been glaciated, but other climatic
changes like droughts could have promoted isolation underground (Rando et al.
1993). The classic Climatic Relict Hypothesis could explain the origin of these
troglobionts although not necessarily promoted by severe cooling. The remaining
32 taxa belong to genera also including epigean species living in the archipelago,
often on Tenerife and even on the surface near the caves. The Adaptive Shift
Hypothesis fits many Canarian troglobionts which live in sympatry even with their
epigean sister species (Contreras-Díaz et al. 2007).
380 P. Oromí
Acknowledgements I am indebted to Juan S. Socorro for his pictures of the caves and for
comments on geology, to Antonio J. Pérez for his collaboration studying Los Roques cave fauna, to
Ángel Vera for an original drawing, to Heriberto López for his arrangements on some figures and to
Philip Ashmole for revising the text and the English version. Most of the information included
herein is due to the task of all GIET members along many years surveying lava tubes and studying
their fauna.
References
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Cueva del Viento (Tenerife, Canary Islands). In: Oromí P (ed) Proceedings 7th international
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Libros de la Frontera, Sant Cugat del Vallés, pp 11–14
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volcánicas del Parque Nacional del Teide (Tenerife): Los Roques, Cuevas Negras y Chavao.
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Chapter 18
Research in Anchialine Caves
Thomas M. Iliffe and Fernando Alvarez
18.1 What Are Anchialine Caves?
Prompted by his discovery of a variety of unusual shrimps from coastal pools in

diverse tropical locations, Dutch carcinologist Lipke Holthuis (1973) coined the
term anchialine for habitats “with no surface connection with the sea, containing salt
or brackish water, which fluctuate(s) with the tides.” Finding similar pools in the
interior of otherwise dry caves and associated networks of extensive submerged
passageways accessible only to specialized cave divers prompted Stock et al. (1986)
to refine the definition of anchialine as “bodies of haline waters, usually with a
restricted exposure to open air, always with more or less extensive subterranean
connections to the sea, and showing noticeable marine as well as terrestrial influ-
ences.” Most recently, Bishop et al. (2015) proposed a further revision of the
definition as “a tidally-influenced subterranean estuary located within crevicular
and cavernous karst and volcanic terrains that extends inland to the limit of seawater
penetration.” Future diving exploration and scientific investigations of anchialine
caves are likely to further broaden our understanding of this concept such that new
and expanded definitions will result.
Anchialine caves occur primarily on oceanic islands and peninsulas in tropical to
subtropical regions around the globe (Fig. 18.1). However, the greatest numbers of
anchialine caves are in the Caribbean and Western Atlantic, especially the Bahamas
Archipelago and Mexico’s Yucatan Peninsula. Although most anchialine caves form
in limestone by solution, they can also occur in volcanic rock as submerged lava
T. M. Iliffe (*)
Department of Marine Biology, Texas A&M University at Galveston, Galveston, TX, USA
F. Alvarez
Colección Nacional de Crustáceos, Instituto de Biología, UNAM, México, México

https://doi.org/10.1007/978-3-319-98852-8_18
384 T. M. Iliffe and F. Alvarez
Fig. 18.1 World map showing the general locations (light gray circles) of regions where anchialine
ecosystems occur
tubes (e.g., the Canary Islands and Hawaii) or as tectonic cracks (e.g., Galapagos
Islands and Iceland). The world’s longest underwater caves are found along the
Caribbean coast of the Yucatan Peninsula where entrances to such caves are locally
referred to as “cenotes.” In this area, more than 1300 km of submerged cave passages
has been explored and surveyed. Two of the longest are Sistema Sac Actun at
347 km and Sistema Ox Bel Ha at 270 km (QRSS 2018). Similar underwater
caves in the Bahamas and Belize, known as “blue holes,” occur both on land and
beneath the seafloor. While many blue holes are formed by solution, some occur in
straight line and are thought to originate through slump faulting (linear fractures
originating from mass wasting by glacio-eustatic changes and gravitational stresses,
then enlarged by karstic dissolution along the margin of shallow-water limestone
platforms with the deep ocean (Palmer 1986).
One of the defining characteristics of anchialine caves is their subterranean tidal
exchange with the ocean, typically with lower salinity water outflowing on a falling
tide, while higher salinity water floods inland on the rising tide. Characteristically,
tides in anchialine cave pools have diminished amplitude and occur later than the
peaks for the corresponding tide in the adjacent ocean.
Another distinguishing feature is the presence of moderate to well-developed
haloclines and resulting vertical stratification with meteoric fresh to brackish surface
waters overlying denser seawater. One or more salinity interfaces or haloclines
separate the cave water column into multiple layers, each with their own distinctive
water chemistry and biology. In coastal aquifers, the thickness of the freshwater lens
and resulting depth of the halocline increase with distance inland.
In most Yucatan cenotes, water temperature increases below the halocline in the
saltwater layer, while in many Bahamian blue holes, the opposite occurs, with
temperatures dropping beneath the halocline. In some caves, a sharp spike in
temperature is found at the halocline, possibly as a result of increased microbial
metabolism of particulate organic matter settling out on the density interface. Open
sinkhole-type caves often receive large inputs of organic matter (e.g., leaves, etc.)
such that the water becomes anoxic for several meters or more below the halocline,
18 Research in Anchialine Caves 385
clouds of hydrogen sulfide form, and a strong “rotten egg” odor is apparent to divers.
In those caves with horizontal circulation of seawater from the ocean, dissolved
oxygen levels recover below the hydrogen sulfide, but in most cases, the underlying
seawater clears but remains anoxic with only obligate anaerobic microbes able to
exist. For those caves in which the influx of organic matter is lower, the saltwater
layer tends to be hypoxic, but fauna with specialized metabolic adaptations are able
to survive and sometimes flourish (Bishop et al. 2004).
Low pH is commonly associated with haloclines in anchialine caves, likely as a
result of CO2 produced there by microbial oxidation of particulate organic matter.
Passages in anchialine caves commonly occur at haloclines due to dissolution of
limestone by low-pH waters, in conjunction with mixing corrosion, whereby the
combining of two water masses (e.g., fresh- and saltwater), both saturated with
dissolved CaCO3, results in an undersaturated mixture, capable of dissolving more
limestone.
Marine caves containing conduits connecting the open ocean with neighboring,
more restricted water bodies such as nearly enclosed bays (e.g., Harrington Sound in
Bermuda), shallow-water interior of carbonate platforms (e.g., the Bahamas), or
saltwater lakes in the interior of islands (e.g., Palau) can exhibit strong tidal currents.
In such cave systems, it may only be possible for divers to enter during a brief period
at slack high tide when currents in the cave slow, stop, and then change direction.
The exact time when slack cave tides occur depends on a complex combination of
factors including nature and size of the cave passages, hydrography and distance
between the ocean and adjacent water bodies, wind direction and speed, phase of the
moon, etc.
18.2 Exploration and Investigation of Anchialine Caves
Submerged caves are one of the few remaining global environments that can only be
explored and studied by direct human participation, while cave diving, especially for
the untrained or ill equipped, is considered one of the most dangerous activities
(Potts et al. 2016). Cave diving involves all of the hazards of open-water diving but
adds the risks of becoming lost, running out of breathing gas, getting stuck, or not
being able to make a direct ascent to the surface. Conducting scientific research
during cave dives further increases the complexity and potential danger (Iliffe 2018).
Cave divers use a guideline, marked with directional arrows, to navigate through
complex underwater labyrinths. They reserve at least two-thirds of their initial gas
supply to exit and thus need additional air tanks. Due to depth and long duration of
cave dives, many of them involve decompression stops to allow accumulated inert
gases, e.g., nitrogen or helium, to slowly and safely escape from divers’ tissues.
Many cave divers move scuba tanks from the back to positions under each arm so
that they can negotiate their way through low areas in the cave passage. An
increasing number of cave divers are using closed-circuit rebreathers which recycle
exhaled gas through a carbon dioxide scrubber and then add additional oxygen
and/or diluent gas to maintain the PO2 in an appropriate and safe range. Rebreathers
release no exhaust bubbles so are less disruptive to the cave environment and cause
fewer disturbances to cave fauna.
18.3 Diversity of Anchialine Fauna
A wide variety of invertebrates and vertebrates have invaded and colonized

anchialine habitats (Fig. 18.2), becoming specialized to live either in the freshwater
or in the marine layer. Only a few species occur both above and below the halocline,
although this varies according to the salinity gradient between the layers. The
majority of species found in anchialine environments are adapted to cave life,
showing the common responses of pigment loss, reduction of eyes, elongation of
appendages, increased number of sensory structures such as setae or aesthetascs,
reduced fecundity, and absence of dispersing larvae. Other adaptations, to hypoxic
conditions or to withstand food deprivation, may include varying oxygen consump-
tion, enzyme activity, and lipid concentration (Bishop and Iliffe 2012).
Stygobitic groups that have been recorded from anchialine habitats include pro-
tists, sponges, chaetognaths, annelids, molluscs, crustaceans, mites, echinoderms,
and fishes. Cnidarians, ctenophores, nematodes, priapulids, echiurids, and insects
have also been collected in anchialine environments but are considered as accidental
occurrences. The most diversified group in anchialine habitats is the crustaceans with
more than 500 species representing 4 classes, 16 orders, and 57 families. Crustaceans
are remarkable in that radiation into this habitat has resulted in the differentiation of
major lineages, originating from both freshwater and marine ancestors (Iliffe and
Bishop 2007). Characteristic styogobitic groups (see also Fig. 18.2) inhabiting
anchialine caves include:
Remipedia The Remipedia, a class of crustaceans exclusively found in anchialine
caves, are eyeless, unpigmented, predatory crustaceans with a head and elongate
trunk composed of up to 42 similar body segments. Swimming appendages are
located laterally on each segment, with animals slowly swimming on their backs.
Paired, hollow-tip fangs, connected to secretory glands, can inject venom composed
of hydrolytic enzymes and a neurotoxin into their prey (von Reumont et al. 2014).
The preys, typically small shrimp or other crustaceans, are held by the mouthparts
while being digested externally. Although they have a generally primitive body plan,
their brain is highly organized and well differentiated with a large olfactory region.
Remipedes are a prime example of an anchialine stygobite with a Tethyan
distribution, inhabiting caves on opposite side of the Atlantic Ocean and from the
eastern side of the Indian Ocean (Koenemann and Iliffe 2013). Of the 29 described
species, 20 are from the Bahamas Archipelago, 3 from the Yucatan Peninsula,
2 from the Canary Islands, and 1 each from Belize, Cuba, the Dominican Republic,
and Western Australia. A recent molecular phylogenetic revision has subdivided
extant remipedes into 8 families containing 12 genera (Hoenemann et al. 2013;
Fig. 18.2 Characteristic anchialine species: (a) Stygiomysis cokei (Stygiomysida, Stygiomysidae),
(b) Pelagomacellicephala iliffei (Polychaeta, Polynoidae), (c) Parhippolyte sterreri (Decapoda,
Barbouriidae), (d) Exumalana reptans (Isopoda, Cirolanidae), (e) Spelaeoecia capax (Ostracoda,
Halocyprididae), (f) Bomburiella gigas (Calanoida, Epactriscidae), (g) Mictocaris halope
(Mictacea, Mictocarididae), (h) Speonebalia sp. (Leptostraca, Nebaliidae), (i) Tulumella grandis
(Thermosbaenacea, Tulumellidae), (j) Remipedia larva—Pleomothra apletocheles, (k)
Tuluweckelia cernua (Amphipoda, Hadziidae), (l) Typhliasina pearsei (Ophidiiformes, Bythitidae),
(m) Godzillius fuchsi (Remipedia, Godzilliidae), (n) Cryptocorynetes elmorei (Remipedia,
Cryptocorynetidae), (o) Pleomothra apletocheles (Remipedia, Pleomothridae), (m) and (o)—ante-
rior ventral view
Olesen et al. 2017). Furthermore, multiple lines of morphological and molecular

evidence indicate that remipedes are the closest living crustacean sister group of the
hexapods or insects (Moura and Christoffersen 1996; Ertas et al. 2009; von Reumont
et al. 2011; Misof et al. 2014; Oakley et al. 2012; Stemme et al. 2013, 2015; Christie
2014).
While many remipede species occur only in a single cave, for yet unknown
reasons, seven or more species inhabit Dan’s Cave on Abaco, Bahamas. Typically,
remipedes occur in low density, although there a few exceptions such as Cenote
Crustacea in Yucatan, Mexico, where many hundreds of remipedes inhabit a
restricted section of this anchialine cave. Larvae of only one species of remipede,
Pleomothra apletocheles, are known (Olesen et al. 2014a), having been collected
exclusively from Dan’s Cave. At least nine and likely more developmental stages
have been observed, all free-living, nonfeeding, and carrying out only small changes
between stages.
Mictacea The peracarid crustacean order Mictacea is represented by a single species
from anchialine caves in Bermuda. Although originally grouped with several similar
crustaceans from the deep sea, it has now been separated (Poore 2015). Ovigerous
females of Mictocaris halope from Bermuda have a typical ventral marsupium, but
little else is known about the larvae or their development (Olesen et al. 2014b).
Copepoda Calanoida with Anchialine Representatives The basal calanoid family
Epacteriscidae contains 18 anchialine genera inhabiting caves in widely distributed
locations (Fosshagen et al. 2001; Bradford-Grieve et al. 2014). They include
Azygonectes from the Caicos Islands and the Bahamas; Balinella from the Bahamas
and Yucatan; Bofuriella, Bomburiella, Caiconectes, and Erebonectoides from the
Caicos Islands; Bunderia from Western Australia; Cryptonectes, Enantronoides,
Minnonectes, and Oinella from the Bahamas; Edaxiella from Jamaica; Enantronia
from Lanzarote, Canary Islands; Enantiosis from the Bahamas, Bermuda, Belize,
Galapagos, Fiji, and Palau; Epacteriscus from Belize and Bermuda; Erebonectes
from Bermuda; and Gloinella and Iboyella from Cuba. The calanoid family
Pseudocyclopidae contains 11 anchialine genera including stygobitic species of
Badijella from Croatia; Boholina from Korea, the Philippines, and Sulawesi;
Brattstromia from Belize; Exumella from the Bahamas, Yucatan, and Balearic
Islands; Exumellina, Normancavia, Robpalmeria, and Stargatia from the Bahamas;
Hondurella from the Bay Islands of Honduras; Ridgewayia from Bermuda, the
Galapagos Islands, and Palau; and Stygoridgewayia from Western Australia
(Bradford-Grieve et al. 2014).
Copepoda Cyclopoida with Anchialine Representatives The cyclopoid family
Speleoithonidae contains three species of Speleoithona from anchialine caves in
the Bahamas and Bermuda (da Rocha and Iliffe 1991, 1993); Smirnovipinidae with
the genus Costanzoia inhabits anchialine caves from Italy and the Balearic Islands
(Zagami and Brugnano 2012); Cyclopinidae with the monotypic genus Oromiina
occurs in an anchialine lava tube cave in the Canary Islands; and Cyclopidae with
two monotypic genera, Troglocyclops, occurs in a single cave in the Bahamas and,
Prehendocyclops, in multiple caves in the Yucatan Peninsula.
Copepoda Harpacticoida with Anchialine Representatives The harpacticoid family
Superornatiremidae includes three genera and nine species from anchialine caves in
Bermuda, the Canary Islands, and the Balearic Islands (Huys 1996; Jaume 1997),
while Novocriniidae contains the monotypic genus Novocrinia inhabiting anchialine
and marine caves on the Belize Barrier Reef.
Copepoda Misophrioida Nine genera of misophrioid copepods are found exclu-
sively in anchialine habitats: Boxshallia, Dimisophria, and Palpophria from
Lanzarote; Expansophria from the Galapagos, Lanzarote, Palau, and Sardinia;
Huysia and Protospeleophria from the Bahamas; Mexicophria from Yucatan;
Speleophria from the Balearic Islands, Bermuda, Croatia, northern Western
Australia, the Nullarbor in southern Western Australia, Palau and Yucatan; and
Speleophriopsis from the Balearic Islands, Bermuda, the Canary Islands, Croatia,
and Palau (Boxshall et al. 2014).
Copepoda Platycopioda Two anchialine genera of platycopid copepods,
Antrisocopia and Nanocopia, are known only from a single Bermuda cave
(Fosshagen and Iliffe 1985, 1988).
Ostracoda Halocyprida with Anchialine Representatives Most cave-adapted spe-
cies from the ostracod order Halocyprida belong to four exclusively anchialine
genera. The genus Danielopolina from the family Thaumatocyprididae has recently
been revised by Iglikowska and Boxshall (2013) such that anchialine cave species
were separated into Humphreysella with 11 species from the Bahamas, Canary
Islands, Christmas Island, Cuba, Galapagos, Jamaica, and Yucatan and the mono-
typic Welesina from Western Australia. The family Deeveyinae contains Deeveya
with seven anchialine species all from the Bahamas Archipelago and Spelaeoecia
with 11 species from the Bahamas, Bermuda, Cuba, Jamaica, and Yucatan
(Kornicker et al. 2007).
Mysida Noteworthy anchialine mysids include the genera Aberomysis from Palau;
Antromysis from Cuba, Jamaica, and Yucatan; Bermudamysis and Platyops from
Bermuda; Burrimysis from the Balearic Islands; Heteromysoides from the Bahamas,
Cayman Islands, Canary Islands, and Okinawa; Palaumysis from the Bahamas,
Okinawa, Palau, and the Philippines; Spelaeomysis from Cuba, Italy, and Zanzibar;
and Stygiomysis from Italy, Jamaica, and Mexico (Pesce and Iliffe 2002).
Isopoda with Anchialine Representatives Atlantasellidae contains a single genus,
Atlantasellus, with two anchialine species from Bermuda and the Dominican Repub-
lic. Anchialine species from the family Cirolanidae are clustered in Mexico and the
Caribbean region (Iliffe and Botosaneanu 2006) and include Cirolana (Anopsilana)
from Cuba, Grand Cayman Island, Haiti, Indonesia, Jamaica, Madagascar, Palau, the
Philippines, and Yucatan; Arubolana from Aruba, Bermuda, and Jamaica; Bahalana
from the Bahamas and Cuba; Creaseriella from Yucatan; Exumalana from the
Bahamas; Haptolana from Belize, Cuba, Somalia, Yucatan, and Western
Australia; Lucayalana from the Bahamas; and Yucatalana from Yucatan. The
Leptanthuridae includes the monotypic Bunderanthura from Western Australia
(Poore and Humphreys 2013). The Paranthuridae contains Curassanthura from
Bermuda, Canary Islands, and Curaçao. The Stenetriidae includes Stenobermuda
from the Bahamas and Bermuda, while the Gnathostenetroididae has
Neostenetroides from the Bahamas and Cuba.
Amphipoda with Anchialine Representatives Anchialine members of the amphipod
suborder Gammaridea include the families Hadziidae with Bahadzia from the Bahamas,
Cuba, Dominican Republic, Haiti, and Yucatan and Liagoceradocus from Fiji,
Lanzarote, Solomon Islands, Western Australia, and Western Samoa; Pardaliscidae
with Spelaeonicippe from the Bahamas and Lanzarote; and Pseudoniphargidae with
Pseudoniphargus from groundwater around the Mediterranean Basin, in Atlantic
drainage systems of Portugal and Spain, on Madeira, Azores, Canary and Balearic
Islands, as well as Morocco, France, and Bermuda. The suborder Ingolfiellidea includes
Ingolfiellidae with anchialine species of Ingolfiella from Bermuda, Lanzarote, the
Maldives, and Socotra.
Stygiomysida This order has recently been removed from mysids and placed in its
own order, the Stygiomysida (Meland et al. 2015). The genus Stygiomysis contains
seven species—one from the Mediterranean and the others from anchialine caves
and coastal groundwater in the Caribbean region.
Thermosbaenacea Two genera of anchialine thermosbaenaceans are noteworthy.
The genus Halosbaena is represented by nine species (only four of them
described)—five inhabiting anchialine and inland caves in Western Australia, plus
one species each from Ryukyu, Christmas and Canary Islands, as well as interstitial
beach environments around the Caribbean Basin (Page et al. 2016). Tulumella on the
other hand is more geographically restricted with three anchialine species, two from
the Bahamas and one from Yucatan (Wagner 1994).
A unique feature of thermosbaenaceans is their dorsal brood pouch, significant
since all of their presumed close relatives have a ventral brood pouch (Olesen et al.
2014b). The collection from a Yucatan cave of a Tulumella female with a brood
pouch containing 12 embryos in an advanced developmental stage provided an
opportunity to observe previously unknown structures (Olesen et al. 2015).
Phyllocarida A blind, unpigmented leptostracan, Speonebalia cannoni, occurs in
anchialine caves in the Caicos Islands.
Decapoda Anchialine shrimps include at least 18 genera from 10 families
(De Grave and Sakihara 2011):
Agostocarididae with Agostocaris from the Bahamas and Yucatan;
Alpheidae with Bermudacaris from Bermuda and the Balearic Islands;
Metabetaeus from Christmas Island, Funafuti, Hawaii, Jaluit and Arno Atolls,
Kiribati, Loyalty Islands, Rapa Nui, Ryukyu Islands, Sulawesi, Tokelau, and Tuvalu
(Anker 2010); and the monotypic Yagerocaris from Yucatan;
Anchialocarididae with the monotypic Anchialocaris from Cozumel Island

(Mexico);
Atyidae with Antecaridina from Daito and Ryukyu Islands (Japan), Entedebir
Island (Red Sea), Europa Island, Fiji, Guam, Hawaii, Mozambique, the Philippines,
Saudi Arabia, and the Solomon Islands; Halocaridina including two species from
Hawaii; Stygiocaris from Western Australia; Typhlatya from Ascension Island, the
Bahamas, Barbuda, Bermuda, Cuba, Curaçao, the Dominican Republic, Galapagos,
Herzegovina, Honduras, Puerto Rico, Spain, and Yucatan;
Barbouriidae with Barbouria from the Bahamas, Bermuda, Cayman Islands,
Cuba, Jamaica, and Yucatan; Calliasmata from Funafuti Atoll, Hawaii, Ryukyu
Islands, Sinai Peninsula, and Yucatan; Janicea from Antigua, the Bahamas, Ber-
muda, Brazil, Canary Islands, Cape Verde Islands, São Tomé, and Yucatan; and
Parhippolyte from Aldabra, the Bahamas, Bermuda, Fiji, Funafuti Atoll, Hawaii,
Indonesia, Loyalty Islands, Mexico (Gulf of California and Yucatan), Molucca
Islands, the Philippines, and Sulawesi;
Laomediidae with Naushonia from the Bahamas, Cuba, and Palau;
Palaemonidae with the monotypic Creaseria from Yucatan (Anker 2014);
Macromaxillocarididae with the monotypic Macromaxillocaris from the
Bahamas;
Pontoniinae with Periclimenes from Fiji, Hawaii, Loyalty Islands, the Seychelles,
and Sulawesi,
Procarididae with Procaris from Ascension Island, Bermuda, Cozumel, and
Hawaii and the monotypic Vetericaris from Hawaii.
Within the Anomura, the normally deep-sea family Munidopsidae contains only a
single shallow-water species from Munidopsis that inhabits an anchialine lava tube
in the Canary Islands (Wilkens et al. 2009).
Other Invertebrates After the very diverse crustaceans, the annelids and molluscs
follow as groups with moderate levels of diversity in anchialine systems. Annelids are
represented in anchialine habitats by polychaetes of the families Fauveliopsidae with
Fauveliopsis from the Canary Islands; Nerillidae with Leptonerilla from Bermuda
and the Canary Islands, Speleonerilla from the Bahamas, Bermuda, the Canary
Islands, Cuba and Yucatan, and Meganerilla and Mesonerilla from the Canary
Islands; Polynoidae with Gesiella from the Canary Islands and Pelagomacellicephala
from the Bahamas; Protodrilidae with Megadrilis from the Canary Islands;
Scalibregmatidae with Speleobregma and Axiokebuita from the Canary Islands; and
Spionidae with Prionospio from Western Australia and the oligochaete family
Tubificidae with Phallodriloides from Bermuda. The molluscan diversity in
anchialine habitats is composed of both truly anchialine species and marine species
that exploit anchialine environments close to the sea. Stygobitic anchialine molluscs
belong to the gastropod families Tornidae with Teinostoma from Yucatan, Caecidae
with Caecum from Bermuda, and Neritiliidae with Laddia from Christmas Island,
Hawaii, Nauru, Saipan, and Tinian; Micronerita from Palau, the Philippines, Saipan,
and Tinian; Pisulina from Christmas and Cocos Islands; Siaesella from Palau; and
Teinostomops from Indonesia, Palau, and Tahiti.
Cave Fish The only fish families that have become established in anchialine
habitats are the Bythitidae with the genera Ogilbia from the Galapagos, Lucifuga
from the Bahamas and Cuba, and Typhliasina from Yucatan; the Synbranchidae
with Ophisternon from Western Australia and Yucatan; and the Eleotridae with
Milyeringa from Western Australia.
Most anchialine species have a very narrow range that can be as small as a single
cave or even a specific section of one cave. In many areas, flooded passages or caves
are not always interconnected, and they may often be hydrologically isolated,
creating patterns of high endemicity. This isolation and limited distribution of
anchialine fauna, coupled with their proximity to the coastline where active touristic
development is commonly occurring, means that anchialine caves and their fauna are
frequently under serious threat from construction activities, water pollution, and
limestone quarrying. As such, a number of anchialine species are currently on the
IUCN Red List of endangered species.
Although anchialine systems are found throughout the world, diversity is highest
in areas such as the Bahamas Archipelago, Caribbean Basin, Bermuda, and the
Canary and Balearic Islands, where in general, the levels of biodiversity correspond
to the size of the cave systems. At a regional scale, these locations represent
biodiversity hot spots for anchialine fauna. The extremely fragmented distributions
of anchialine fauna pose intriguing questions about their origin and distribution
patterns while presenting challenges for adequate protection and conservation. In
geographical regions where anchialine systems are better developed (e.g., Bermuda,
the Bahamas, Yucatan, Australia), beta diversity can be high since species replace-
ment over short distances may be high. Even though the same groups are usually
present in distant and geographically isolated localities, the species representing each
group are usually different.
18.4 Anchialine Cave Food Webs and Functioning
One of the most important questions regarding the functioning of anchialine systems
is where the energy comes from since there are no obvious primary producers.
Organic matter enters cave systems through natural entrances either from the sea
or from the land above the cave, as well as through smaller crevices and cracks in the
bedrock. Some anchialine caves with air-filled chambers above the water level may
host large colonies of bats, with their guano providing a rich food source. Cave-
adapted crustaceans living deep within anchialine caves have conspicuous stable
carbon isotopic values suggesting that carbon and energy from chemoautotrophic
pathways linked to a microbial loop shuttling methane and dissolved organic carbon
are fueling the cave food web within the extensive coastal cave systems in the
Yucatan Peninsula (Pohlman et al. 1997; Brankovits et al. 2017) and in an anchialine
sinkhole in Australia (Humphreys 1999). The concept of channeling carbon and
energy from dissolved organic compounds into heterotrophic bacterial biomass to
higher-order metazoans is a departure from the classical view that subterranean
ecosystems are a linear chain from plant detritus to consumers (Dickson 1975) or are
based on chemolithoautotrophic processes derived from thermomineral sources
(Sarbu et al. 1996; see also Chaps. 11 and 15).
18.5 Future Studies
A series of unique questions have arisen from the initial studies of anchialine
ecosystems. Here we outline some that are of considerable scientific significance.
Based on the discovery of chemosynthesis-based trophic webs in anchialine habitats
(Pohlman 2011), future research in the biogeochemistry of anchialine systems
should aim at describing the steps involved in the cycling of organic compounds.
A great diversity of species and of higher taxonomic categories has been
described from anchialine systems. Although some species were known from the
first half of the twentieth century, most discoveries have occurred in the last 20–30
years. It is interesting to note that while exploration of these flooded caves has
advanced greatly, increasing the number and extent of systems known throughout
the world, biological reconnaissance and, more so, detailed specific studies of single
species have only just began. For example, it has been empirically estimated that less
than 5% of the topographically recognized anchialine systems of the Yucatan
Peninsula have been explored. Improved diving techniques and equipment and a
growing number of scientifically trained cave divers will advance biological explo-
ration in the near future.
From the biogeographic and phylogenetic point of view, anchialine fauna repre-
sent an element of enormous importance for several reasons, some of which have
been outlined already. Anchialine systems may have operated as refuges through
geologic time conserving relict groups that are absent from other aquatic ecosystems.
The Remipedia is one example, with species in the Greater Caribbean, the Canary
Islands, and Australia that serve as a model to define types of anchialine fauna in
different parts of the world and to link distant geographic areas that were connected
in the past geologic eras (Hoenemann et al. 2013). Atyid shrimps of the genus
Typhlatya also show a highly disjunct global distribution suggesting a very old
origin with the separation of the main lineages occurring by both vicariance (the
Atlantic opening) and dispersal (Jurado-Rivera et al. 2017). The affinities of some
anchialine species, like the misophrioid copepods of the genus Expansophria, have
linked species present in shallow coastal anchialine caves with deep-sea species.
These unique relationships have prompted novel hypothesis about the vertical
separation of ancestral stocks to produce the observed pattern (Boxshall and Iliffe
1986).
Since anchialine fauna is generally restricted to caves in a band no more than
10 km inland from the coastline, such species would have been forced to migrate in
pace with tectonic uplift and glacio-eustatic sea-level change, thus implying the
persistence of a vertical continuum of suitable habitats in both time and space.
Sea-level position has been a controlling factor in the formation and subsequent
development of coastal anchialine caves. The average water depth of anchialine cave
systems along the Yucatan’s Caribbean coast parallels increasing halocline depth
with distance inland. This indicates such caves formed when sea level was at or near
its current position, i.e., during the last interglacial (Marine Isotope Stage 5e).
Subsequently, the caves drained as sea level receded and were completely dry during
the last glacial minimum, as evidenced by the prevalence and size of now submerged
speleothems. Alternate habitats, at least 100+ m below current sea level, must have
provided a refuge for anchialine fauna at that time. At sites in Yucatan and the
Bahamas, massive collapse has broken though to remnant cave passages nearing that
depth, but further explorations are required to determine if these caves served as an
ice age retreat for anchialine fauna.
Acknowledgments We extend our sincere appreciation to numerous taxonomists, ecologists, and

other biologists from around the world who over the years have generously contributed their time
and effort to these studies. Since exploration of deep, extensive, and potentially dangerous under-
water caves is an essential component to these studies, we gratefully recognize the many cave divers
who have supported our research endeavors, especially cave divers and close friends Sheck Exley
(1949–1994), Rob Palmer (1952–1997), Rob Parker (1962–1997), Wes Skiles (1958–2010), and
Agnes Milowka (1981–2011), all of whom tragically perished in diving accidents. This research has
been supported by grants from the US National Science Foundation (#03-15903, 87-00079,
84-17494), National Oceanic and Atmospheric Administration (U.S. Department of Commerce),
National Geographic Society (4725-92, 3412-86), and others to TMI and awards from CONACYT
[#155644(546)] to FA as well as Texas A&M University—CONACYT collaborative science grants
(#10655) to both authors.
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Chapter 19
Volcanic Anchialine Habitats of Lanzarote
Alejandro Martínez and Brett C. Gonzalez
19.1 Introduction
Anchialine systems predominantly occur in karstic limestone and volcanic rock

throughout tropical locales (Iliffe et al. 2000; Iliffe and Kornicker 2009; see also
Chap. 18). However, these systems can be found as far north as Iceland and as far
south as Australia. Most anchialine habitats occur in caves, but they may also be
present in a variety of other geological formations such as small depressions,
submerged faults, and even in areas of uplifted geology (Iliffe and Bishop 2007).
The most extensive anchialine systems are known from the Caribbean. The
largest of these systems are present in the Yucatán Peninsula of México, but large
systems are also known from elsewhere, including the Bahamas, Bermuda, and the
Mediterranean. Caves in these areas are all formed in limestone by solutional
processes, being further shaped by mixing corrosion, and typically containing both
fresh meteoric and intruding marine waters. Karstic regions are known for their
scarcity of surface water features, making nearly all drainage subterranean (Iliffe
et al. 2000). Since most of these caves lie perpendicular to the coast, they are thought
to be major drainage conduits into the surrounding marine waters (Iliffe et al. 2000).
Sistema Sac Actun (Yucatán Peninsula) is the largest submerged anchialine system,
which has more than 347 km of surveyed passages interconnecting with more than
100 entrance pools. However, the majority of anchialine caves are much smaller,
ranging in lengths from several meters to few kilometers. Small caves are typically
associated with locations outside the Caribbean and Mediterranean, mostly found
A. Martínez (*)
Institute of Ecosystems Studies, National Research Council of Italy, Verbania, Italy
B. C. Gonzalez
Marine Biological Section, Department of Biology, University of Copenhagen, Copenhagen,
Denmark

https://doi.org/10.1007/978-3-319-98852-8_19
400 A. Martínez and B. C. Gonzalez
throughout the Indo-Pacific (Iliffe and Bishop 2007). In general, karstic caves are
hundreds of thousands if not many millions of years old, persisting far longer than
those caves formed in volcanic rock.
Anchialine volcanic caves are known from several island locations including the
Canary, Galapagos, and the Hawaiian Islands. Most of these caves are in the form of
lava tubes formed during eruptions of fluid basaltic pahoehoe lava, in which the
slow-moving surface of the lava is cooled, forming a conduit below that becomes the
tube after the lava flow ceases (Bravo 1964; Carracedo et al. 2003; Martínez et al.
2016a). Typically, lava tubes are formed on land and flow toward lower elevations.
In island settings, when lava reaches the coastline it is suddenly cooled by the
surrounding marine waters, preventing the tube from forming further (Iliffe and
Bishop 2007). Under special conditions lava tubes may also be formed along the sea
floor or in submarine environments (Fornari et al. 1985). However, these two
conditions are not present in Lanzarote. Lava tubes are often relatively short-lived
(tens of thousands of years) due to their proximity to the surface where they are
exposed to damaging elements (i.e., erosion) (Iliffe and Bishop 2007).
19.2 Anchialine System in Lanzarote
Lanzarote is one of seven major volcanic islands situated off the west coast of
Western Sahara (~160 km), being the second oldest among the Canary Islands
(about 15 million years) and harboring the most diverse anchialine ecosystems in
the Eastern Atlantic. There are three types of surface features providing access to the
anchialine systems throughout Lanzarote, namely, exposed lava tubes, pools, or
hand dug wells scattered throughout the island.
The main anchialine environment in Lanzarote lies within La Corona lava tube
(Fig. 19.1). This cave was formed from a lateral vent off La Corona Volcano at the
Fig. 19.1 Schematic cross section of the anchialine sections of La Corona lava tube. (A) Cueva de
Los Lagos; (B) Los Jameos del Agua. Vertical dashed lines represent the approximate area occupied
by the tourist complex; (C) Lago Escondido, upper section of the Túnel de la Atlántida; (D) Túnel
de la Atlántida; (E) Montaña de Arena. Areas shaded in light gray represent the current sea level;
horizontal dashed line indicates possible position of the sea level during the formation of the lava
tube. Horizontal black scale bar 500 m; vertical scale on left axis exaggerated. Modified from
Wilkens et al. (2009)
19 Volcanic Anchialine Habitats of Lanzarote 401
northern tip of the island. La Corona lava tube traverses over 6.2 km in a southeast-
erly direction, whereby it continues an additional 1.6 km into the Atlantic Ocean
beyond the present coastline of the island. The occurrence of upper and lower
sections throughout the lava tube suggests that several volcanic episodes were
involved during speleogenesis (Jantschke et al. 1994). Secondary collapses or
“jameos” currently divide the cave into several sections, however, only three of
them are flooded by anchialine waters (Martínez et al. 2016a). The most inland of
them, known as Cueva de Los Lagos, opens 600 m from the coastline into a partially
flooded passageway ending in a short sump. Downstream, this sump leads through
non-navigable galleries to the second known anchialine section, represented by the
tidal lake of Los Jameos del Agua, which has been developed into a major tourist
attraction (Fig. 19.2a). The third anchialine section, known as Túnel de la Atlántida,
opens nearby Los Jameos del Agua and is completely submerged, extending over
1.6 km under the sea floor without any conspicuous connection to the overlying
ocean. Túnel de la Atlántida ends abruptly at its maximum depth of 64 m (Martínez
et al. 2016a). Maximum extension and depth of Túnel de la Atlántida fits with
palaeocoastlines of Lanzarote, and age estimates based on Ar/K radiometric methods
date formation of La Corona lava tube to the last glacial maximum (~21,000 6500
years) (Carracedo et al. 2003). This estimate suggests that the formation of the lava
tube was subaerial (formed on the surface) and stopped when the lava flow was
abruptly interrupted due to the sudden cooling by contact with the Atlantic Ocean.
Flooding of La Corona lava tube was more recent and after the last glacial maxima
when sea level rose to its present level.
Anchialine environments of Lanzarote are not limited to those of the flooded
portions of La Corona lava tube but can also be found at the surface around the island
in the form of landlocked bodies of water known as anchialine pools or ponds
(Fig. 19.2b). These occur mostly in two localities, Montaña Bermeja on the south-
west coast, and Órzola on the northeastern tip of the island (Martínez et al. 2016a).
Typically, the anchialine pools of Lanzarote consist of small depressions that
penetrate the subterranean marine waters of the island. Similarly, in the anchialine
habitats in La Corona, these anchialine ponds fluctuate with respect to the surround-
ing tides. Given the proximity to abandoned cinder mines, it is not currently known
if all anchialine pools in Lanzarote are natural, or if some are man-made (Wilkens
et al. 1993).
In addition, several other man-made access points to anchialine waters exist in
Lanzarote (Fig. 19.2c). Hand dug wells were built during the early eighteenth and
nineteenth centuries along the coastline, pumping the subterranean anchialine
marine waters into saltpans for salt production (Martínez et al. 2016a). Most of
these wells are relatively small, ranging between 2 and 3 m in diameter and
averaging 10 m in depth. Since construction, the majority of these wells have been
destroyed and those that remain are mostly abandoned. Active wells can only be
found in the town of Costa Teguise and in the saltworks of Guatiza and Los
Agujeros.
Fig. 19.2 Types of anchialine environments seen throughout Lanzarote including several well-
known anchialine endemic species. (a) Los Jameos del Agua anchialine lake during early morning;
(b) Charcos de Luis anchialine ponds, near Órzola, at the northern tip of Lanzarote; (c) an anchialine
well in Los Agujeros near Mala on the northeast coast of Lanzarote; (d) Galatheid squat lobster
Munidopsis polymorpha at the bottom of Los Jameos del Agua lake; (e) Remipede Morlockia
sp. swimming in the water column of Túnel de la Atlántida; (f) Polynoid annelid Gesiella jameensis
on the bottom of Los Jameos del Agua lake; (g) Summary of the interactions between the different
types of anchialine habitats and species in Lanzarote (adapted from Martínez et al. 2016b). Numbers
19.3 Biodiversity and Ecological Studies in La Corona Lava

Tube: A Hotspot of Diversity for Anchialine
Subterranean Fauna
La Corona lava tube has been, and continues to be, the focus for most of the faunistic
studies in anchialine ecosystems throughout Lanzarote. Studies in La Corona started
with the description of the squat lobster Munidopsis polymorpha from Los Jameos
del Agua by Koelbel in 1892 (Fig. 19.2d) and continued with several additional
species descriptions by various European scientists during the first half of the
twentieth century (Calman 1904; Harms 1921; Fage and Monod 1936). Parzefall
and Wilkens (1975) conducted the first systematic investigation focusing on the
biology and behavior of Munidopsis polymorpha in Los Jameos del Agua (Wilkens
et al. 1990). However, it was the scientific cave diving explorations of Túnel de la
Atlántida that yielded the most impressive discoveries of several endemic stygobites
including annelids, copepods, thermosbaenaceans, and remipedes (Iliffe et al. 1984).
Most of these endemic stygobites occur in the water column of the aphotic
anchialine portions of La Corona, restricted to Túnel de la Atlántida and Cueva de
Los Lagos. The water column represents the most specialized habitat in anchialine
cave systems. In La Corona, the water column is characterized by low and stable
temperatures (~18 C) and dissolved oxygen (~3.7–5.7 mg/L), with minimal cur-
rents due to tidal exchange (Wilkens et al. 2009). In contrast to other anchialine
systems, the water column exhibits no stratification in salinity, temperature, or
oxygen, yet deployed water profilers (automated water quality samplers that collect
data through the water column from quality sensors) do show fluctuation in all
parameters that coincide with tidal exchange (Martínez et al. 2016a). La Corona lava
tube, like numerous other anchialine cave systems, is regarded as a detritus-based
system (see also Chap. 14), where particulate organic matter is introduced through
tidal exchange or infiltration across the overlying lava rock. The presence of
suspended organic matter within the water column favors a rich assemblage of
endemic suspension feeders dominated by stygobitic crustaceans such as
thermosbaenaceans, mysids, and amphipods, as well as a few highly specialized
annelid species (Martínez et al. 2016b; Worsaae, Martínez, and Gonzalez, pers.
obs.). The water column of La Corona is also home to several species of endemic
predators (see also Table 19.1), such as the remipedes Morlockia ondinae
(Fig. 19.2e) and M. atlantida, the annelid Gesiella jameensis (Fig. 19.2f), and the
⁄
Fig. 19.2 (continued) correspond to the following species (see also Table 19.1): 1 Boxshallia
bulbantennulata, 2 Parhyale multispinosa, 3 Calanoida, 4 Halosphiloscia, 5 Syllidae, 6 Tubiluchus
lemburgi, 7 Meganerilla cesari, 8 Bogidiella uniramosa, 9 Heteromysoides cotti, 10 Munidopsis
polymorpha, 11 Humphreysella sp., 12 Halosbaena fortunata, 13 Hadzia acutus, 14 Harpacticoida,
15 Gesiella jameensis, 16 Spelaeonicippe buchi, 17 Mesonerilla n. sp. 1, 18 Leptonerilla
diatomeophaga, 19 Cumacea, 20 Caryophyllia smithii, 21 Bonellia viridis; see Table 19.1 for
more information on the species systematics. Photos by © Brett C. Gonzalez (A, C) and © Juan
Valenciano (D, E, F)
Table 19.1 Summary of the stygobiont and endemic species recorded from the anchialine ecosystems from Lanzarote
404
Taxonomy Area Ecology Origin

Family Species C J T MA W P St In En Tet De Sha
Platyhelminthes
Cheliplanidae Cheliplana cavavulcanica – – T MA – – – In En – – Sha
Schizorhynchidae Schizochilus lanzarotensis – – T MA – – – In En – – Sha
Annelida
Acrocirridae Macrochaeta n. sp.a – J T – – – St In En – ? ?
Fauveliopsidae Fauveliopsis jameoaquensis – J T – – – St In En ? ? ?
Nerillidae Mesonerilla n. sp. 1b – – T – – – In En – – Sha
Mesonerilla n. sp. 2b C J T – – – St In En – – Sha
Speleonerilla isa C – T – – – St – En – – Sha
Leptonerilla diatomeophaga – J T – – – St In – Tet ? ?
Polynoidae Gesiella jameensis C J T – – – St – En – De –
Protodrilidae Megadrilus pelagicus C – T – – – St – En – – Sha
Scalibregmatidae Speleobregma lanzaroteum – – T – – – St – En – De –
Spionidae Prionospio n. sp.c C – T – – – St – En Tet – –
Syllidae Sphaerosyllis iliffei – – T MA – – St In En – – Sha
Crustacea, Calanoida
Arietellidae Paramisophria reducta – – T – – – St – En – ? –
Epacteriscidae Enantronia canariensis – – T – – – St – En – ? –
Pseudocyclopiidae Stygocyclopia balearica C – T – – – St – – – ? –
Stephidae Stephos canariensis C – T – – P St – En – – Sha
Crustacea, Cyclopoida
Cyclopinidae Oromiina fortunata – J – – – – St – En – ? –
Cyclopinidae Muceddina multispinosa C – – – – – St – – – De –
Crustacea, Harpacticoida
Superornatiremidae Neoechinophora karaytugi – – T – – – St – En Tet – –
A. Martínez and B. C. Gonzalez
19
Crustacea, Misophrioida
Misophriidae Dimisophria cavernicola – – T – – – St – En – De –
Expansophria dimorpha C – T – – – St – En Tet De –
Palpophridae Palpophria aestheta – – T – – – St – En – De –
Spelophriidae Speleophriopsis canariensis C – – – – – st – En Tet De –
Boxshallia bulbantennulata – – – – – P St – En – ? ?
Crustacea, Tantulocarida
Basipodellidae Stygotantulus stocki – – – – – P St – En – – ?
Crustacea, Ostracoda
Polycopidae Eupolycope pnyx – – T – – – St – En – – –
Thaumatocyprididae Humphreysella phalanx – – T – W – St – En Tet De –
Humphreysella wilkensi C – T – W – st – En Tet De –
Sarsiellidae Eusarsiella bedoyai – – T MA – – – – En – – –
Volcanic Anchialine Habitats of Lanzarote
Crustacea, Amphipoda
Bogidiellidae Bogidiella uniramosa – J – – W – St In En Tet – –
Hyalidae Parhyale multispinosa C J T – – P – – En – – –
Pardaliscidae Spelaeonicippe buchi C J T – – – St – En – De –
Talitridae Hadzia acutus – J T MA W – St – En Tet – –
Crustacea, Isopoda
Paranthuridae Curassanthura canariensis – J – – – – St – En Tet – –
Crustacea, Mysida
Mysidae Heteromysoides cotti – J T – W P St – En Tet – –
Crustacea, Thermosbaenacea
Halosbaenidae Halosbaena fortunata – – T – W – St – En Tet – –
Crustacea, Decapoda
Galatheidae Munidopsis polymorpha C J T – W P St – En – De –
(continued)
405
406
Taxonomy Area Ecology Origin

Family Species C J T MA W P St In En Tet De Sha
Crustacea, Remipedia
Speleonectidae Morlockia atlantida C – T – – – St – En Tet – –
Morlockia ondinae C – T – – – St – En Tet – –
C Cueva de los Lagos, J Jameos del Agua, T Túnel de la Atlántida, MA Montaña de Arena, W Wells, P ponds, St stygobite, In interstitial, En endemic, Tet
Tethyan origin suggested, De suggested marine deep-sea affinity, Sha attributed marine shallow water affinity, ? currently unknown
a
In Núñez et al. (1997)
b
In Worsaae et al. (2009)
c
In Martínez et al. (2016)
A. Martínez and B. C. Gonzalez
copepod Enantronia canariensis (Martínez et al. 2016a). In contrast, anchialine

systems throughout the Caribbean are known to support chemoautotrophic produc-
tion from low concentrations of dissolved inorganic compounds (Pohlman et al.
1997; Brankovits et al. 2017). However, it remains unknown if such processes are, or
capable of, occurring within the dark remote sections of La Corona lava tube.
The benthic environments in La Corona lava tube are spatially more complex
than those of the water column and include rock surfaces as well as patches of
different types of sediments that host crevicular or interstitial habitats depending on
their origin and grain size. Patches of lava debris are distributed throughout the lava
tube, although they are more common in areas of breakdown such as the entrance of
Túnel de la Atlántida or in several parts within Cueva de Los Lagos. While these
patches might superficially resemble interstitial environments, they mainly comprise
lava pebbles, resulting in similar permeability to that of the surrounding subterranean
crevicular environment. As a consequence, they do not host typical interstitial
species but rather a few stygobites in low abundances, including several species of
copepods, the isopod Curassanthura canariensis, and several nerillid annelids
(Martínez et al. 2009; Worsaae et al. 2009).
True interstitial environments are characterized by the presence of smaller sized
sediment particles. In La Corona, they are restricted to Montaña de Arena; a 30 m
high sand dune formed approximately 750 m from the entrance of Túnel de la
Atlántida. This accumulation of marine coarse sand has been entering the cave
ceiling through a non-visible crack for an unknown length of time and contains
both marine and cave endemic species (Martínez et al. 2009). In contrast to the
patches of lava pebbles, this environment is colonized by a rich fauna and includes
typical interstitial meiofaunal groups such as annelids, platyhelminthes, gastrotrichs,
gnathostomulids, priapulids, and crustaceans (García-Valdecasas 1985; Núñez et al.
2009; Worsaae et al. 2009; García-Herrero et al. 2017; Gobert et al. 2017). As a
result of tidal exchange through this connection to the overlying marine environ-
ment, several marine species referred to as “accidentals” or stygoxenes are also
present. This connection also contributes nutrients into the system as areas surround-
ing Montaña de Arena are characterized by an increase in particulate organic matter
in both the water column and the dune itself, favoring the rich interstitial diversity.
Within the La Corona lava tube, indirect solar insolation is restricted to the
anchialine lake of Los Jameos del Agua. The intensity of light in Los Jameos del
Agua varies across the lake and favors the presence of primary production in the
form of microscopic algae as well as dense beds of benthic diatoms. This primary
production sustains large populations of mysids, copepods, ostracods, and other
suspension feeders within the water column and in the benthos where several
interstitial cave endemic annelids are present (Núñez et al. 1997; Worsaae et al.
2009). However, the opportunistic squat lobster Munidopsis polymorpha is by far
the most abundant stygobiont in both Los Jameos and the entrance pool of Túnel de
la Atlántida (Wilkens et al. 1990). Stygophilic and accidental stygoxenic species are
also common in these localities, taking advantage of the increased concentration of
organic matter. In particular, the stygophilic annelid Bonellia viridis (Bonellidae,
Echiura) has increased its population size in recent decades, enriching the deposits of
cinders and lava debris in the lake with organic matter derived from fecal deposits
(Brito et al. 2009).
19.4 Biodiversity Studies in Anchialine Pools and Wells

in Lanzarote
Anchialine pools host a very particular combination of marine and stygobitic species
(see also Table 19.1), some of them exclusive to these pools such as the copepod
Boxshallia bulbantennulata and the parasitic crustacean Stygotantulus stocki. His-
torically, research on these pools has mostly consisted of species descriptions.
Faunal surveys of these pools have often recorded species otherwise known only
from La Corona lava tube, providing evidence of connectivity among the island’s
anchialine habitats, thereby strengthening the foundation for theories regarding
dispersal between distant anchialine caves by means of crevicular habitats or
“spelean corridors” (Hart et al. 1985; Wilkens et al. 1986; Gonzalez et al. 2017).
Some species present in these anchialine pools, such as the amphipod Parhyale
multispinosa, can be considered “pond specialists”, capable of coping with extreme
changes in both temperature and salinity while forming permanent populations. In
contrast, true stygobites, such as Munidopsis polymorpha and Heteromysoides cotti,
migrate in and out of the ponds, taking advantage of trophic resources during the
most favorable nighttime conditions when temperatures and risk of predation are
typically lower. The bottom of these anchialine pools are quite different from the
bottom of La Corona, having several layers of fine sediment covered by varying
degrees of decomposing organic matter (including terrestrial leaf litter), green algae,
and cyanobacteria. Often, several benthic species, many of which also occur in
marine intertidal environments, occur in these bottom pool sediments. One of the
most extraordinary records from these anchialine pools is that of the endangered
European eel Anguilla anguilla (Actinopterygii, Anguillidae), which has been fre-
quently observed in these pools in northern Lanzarote (Wilkens et al. 1986).
Hand dug wells are also connected to the subterranean waters of Lanzarote
through crevicular spaces and are often directly affected by the surrounding tides.
However, the limited water present in these wells is less exposed than that of
anchialine pools, being protected by the steep walls from not only the direct sunlight,
but also the extreme temperatures occurring daily. The bottoms of these wells mostly
consist of gravel and mud, but natural or anthropogenic debris is often present. Few
stygobites are known from hand dug wells, but the stygobitic amphipod Hadzia
acutus can always be found and baited traps have been shown to attract additional
stygobiont species from the surrounding crevicular spaces. Again, this illustrates the
connectivity (see Fig. 19.2g) across the various subterranean areas scattered among
the island of Lanzarote (Wilkens et al. 1986).
19.5 Evolutionary Studies in Lanzarote: Origin

of the Anchialine Fauna
The origin of many anchialine lineages remains an open and fascinating evolutionary
and biogeographical question. While most anchialine lineages have an unequivo-
cally marine origin, they have been placed often in new genera, families, or even
higher taxonomic ranks that are restricted to caves distributed across broad geo-
graphic ranges (Wilkens et al. 2009; see also Chap. 18) (Fig. 19.2). Such disjunct
global distributions were first described in stygobitic crustaceans, interpreted as the
result of cave colonization by their marine ancestors thought to be present along the
coasts of the Tethys Sea during the Mesozoic. These ancestral cave populations
would have subsequently been divided by plate tectonic vicariance (Stock 1993).
This so-called Tethyan origin has been credited to several stygobitic groups, includ-
ing remipedes, thermosbaenaceans, atyid shrimp, and thaumatocyprid ostracods.
However, while this hypothesis may explain the presence and distribution of
stygobionts in caves throughout the Caribbean, Australia, and other locations
along margins of continental plates, it cannot explain the presence of stygobionts
in geologically young oceanic islands of volcanic origin. This evolutionary origin
conundrum includes fauna present in Lanzarote, as well as several other oceanic
islands such as the Galapagos, Bermuda, and Christmas Island. The presence of
several species previously attributed to a Tethyan origin in anchialine habitats in
these geologically young islands cannot be explained without addressing alternative
forms of dispersal (Jurado-Rivera et al. 2017). Molecular phylogenetic studies
attempting to address colonization alternatives are still missing or incomplete for
many of these groups in La Corona, but those already published have resulted in
topologies incongruent with the known vicariant scenarios (see also Chap. 12), such
as Halosbaena fortunata (Page et al. 2016), or have yielded very old divergence
times compared with the geological ages estimated for the island that the species
inhabits (e.g., Gesiella jameensis; Gonzalez et al. 2017).
Alternatively, a deep-sea origin hypothesis has also been proposed for numerous
anchialine stygobites. This hypothesis offers an alternative to the Tethyan origin as it
includes the possibility of dispersal among caves through cave-like habitats in the
deep sea, including crevices (i.e., spaces among rocks). Several stygobites endemic
to La Corona lava tube have a clear deep-sea affinity, as they belong to groups never
preset in shallow water (Martínez et al. 2009; Gonzalez et al. 2017). This affinity,
however, does not necessarily indicate that cave colonization occurred from the
deep, as independent phylogenetic analyses have actually favored a shallow water
origin for some of these species (i.e., several misophrioids and Paramisophria
copepods; Boxshall and Jaume 2000; Jaume et al. 2000). In other instances (i.e.,
Speleobregma lanzaroteum), a deep-sea origin cannot be unequivocally attributed
until more sampling is performed (Martínez et al. 2013). The only case in which
phylogenetic analyses cannot reject colonization from the deep are those involving
the polynoid Gesiella jameensis, and the squat lobster Munidopsis polymorpha
(Ahyong et al. 2011; Gonzalez et al. 2017).
19.6 Evolutionary Studies in Lanzarote: Adaptations

to Lava Cave Anchialine Environments
Adaptations to cave-specific environments, including subterranean anchialine hab-

itats, are known as troglomorphism (see also Chap. 4). However, traits associated
with troglomorphism may also be present in non-cave environments that have
similar ecological conditions to those of caves. For example, loss of eyes or
pigmentation is two of the most iconic regressive troglomorphic adaptations, yet
they are conditions commonly seen among deep-sea or interstitial lineages
(Danielopol et al. 1996; Giere 2009). Therefore, unraveling true troglomorphic traits
often demands specific comparative analyses.
Comparative analyses from two endemic cave annelids from La Corona lava tube
have shown that some traits were indeed present prior to cave colonization, thus
reducing the number of features that can be regarded as troglomorphic. For instance,
Speleobregma lanzaroteum is an endemic scalibregmatid annelid that bears palps
and is found suspension feeding within the water column of La Corona lava tube.
This species belongs to a family dominated by burrowers lacking head appendages
(Martínez et al. 2013), and it was thought that the presence of palps was a
troglomorphic adaptation to cave suspension feeding. However, comparative ana-
lyses showed that these palps were not correlated with cave colonization but were
already present in the ancestor shared by Speleobregma and the species of the genus
Axiokebuita, dwelling both in deep-sea and cave crevicular habitats (Martínez et al.
2014). Similarly, recent comparative studies on the endemic polynoid Gesiella
jameensis, also including the Bahamian anchialine polynoid species complex of
Pelagomacellicephala iliffei, showed that the loss of eyes was already present in
their deep-sea ancestors but the elongation of dorsal cirri, a sensory projection
located on the parapodium, in both of these cave polynoids evolved in association
with cave colonization (Gonzalez et al. 2018). Additionally, two lineages of pri-
marily interstitial annelids in La Corona, belonging to the families Nerillidae and
Protodrilidae, are often interpreted as providing other examples of troglomorphic
adaptation (Worsaae 2014; Martínez et al. 2015). Endemic species from both these
groups inhabit the water column of La Corona lava tube and exhibit elongated
ciliated palps and ciliary bands along their body in conjunction with their adaptation
to suspension feed in the water column (Martínez et al. 2016b).
19.7 Conservation of Anchialine Habitats
Anchialine habitats, like many other natural splendors, are highly susceptible to
anthropogenic encroachment. One of the largest impacts on anchialine habitats is
land use, whether it is for commercialization and construction, mineral exploitation,
or water resources (Iliffe and Kornicker 2009). Due to the vastness and interconnec-
tivity of the subterranean realm, even activities several kilometers away from
anchialine caves or pools may have detrimental effects, especially with regard to
those contaminants capable of being flushed through the system by tidal pumping.
Any anthropogenic access to subterranean or anchialine waters via bore holes, wells,
quarries, or deep well injection sites have the potential to introduce contamination,
ultimately impacting the environmental health of the system accessed and those
neighboring it. This is likely to have conservation implications because the majority
of species living within these habitats have limited distributions and highly specific
habitat requirements. As a result, the majority of anchialine species automatically
qualify for inclusion on endangered or protected lists because of these characteristics
(Iliffe and Bishop 2007).
The push for development of tourism throughout regions containing anchialine
habitats (i.e., tropical oceanic islands and coastal regions) puts subterranean ecosys-
tems at a heightened risk of contamination. Recent booms in tourism, combined with
the lack of effective environmental laws or implementation of laws in many of these
locations, have complicated conservation efforts, even in areas with developed
educational outreach and conservation programs. Fortunately, the entire island of
Lanzarote has been protected as a Biosphere Reserve since 1993, and more recently
(2015), part of Lanzarote, including the Chinijo Islands, became a UNESCO
Geopark. For the most part, urbanization and land use in Lanzarote have been
strongly controlled, largely because of the efforts of local artist César Manrique.
Los Jameos del Agua and Túnel de la Atlántida have been a major touristic
attraction in Lanzarote since 1966, receiving thousands of visitors each year. While
the tourist center attempts to protect these anchialine ecosystems from uncontrolled
visitation, dumping of trash, acts of vandalism, and the existence of the center itself
expose these fragile ecosystems to other types of anthropogenic threats. These
threats mostly revolve around the intentional tossing of coins into the lake by visitors
who regard this water body as a natural “wishing well”, despite the posted placards
forbidding such activities. Additionally, the occasional dropping of random objects,
evening musical events, and periodical festivals may also potentially have detrimen-
tal effects on these fragile ecosystems. While to date the populations of many of the
endemic species in the lake show no signs of stress, ongoing research supported by
the Tourist Center as well as the Governments of Lanzarote and the Canary Islands is
attempting to understand the cumulative effects of all these disturbances, with aims
of finding ways to further minimize anthropogenic impacts.
Acknowledgements This chapter is dedicated to the pioneering works of Thomas M. Iliffe, Pedro
Oromí, Jorge Núñez, and Horst Wilkens for their support and friendship throughout our careers.
Their countless discoveries and contributions continue to drive young scientists underground,
pushing the boundaries of science and “evolution in the dark” as we know it. We are forever
grateful to Katrine Worsaae for her willingness to open up her lab in order to make this research and
numerous others a reality. Exploration and discovery is not without risks, and for this we are
indebted to all the cave divers and support teams, for without their support, these investigations
would never have been possible. Several scientific grants, including those from Denmark,
Germany, Spain, and the USA, have supported our exploration and research over the years. We
would also like to personally thank Elena Mateo, UNESCO Geopark of Lanzarote, and the Chinijo
Archipelago, for their continued support in providing scientific access to these remarkable sites.
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Chapter 20
Research in Calcretes and Other Deep
Subterranean Habitats Outside Caves
Stuart Halse
20.1 Introduction
Box 20.1
The outstanding difference between traditional subterranean fauna studies and
those carried out recently in Australia is the emphasis in Australia on the fauna
outside caves within the network of small cavities that occur deep underground
across large parts of the landscape. Much of the arid zone, especially in the
western half of Australia, is rich in subterranean fauna. Despite the ancient age
of the land mass in which many species occur, it appears that most of the species
(or at least the lineages from which they have evolved) moved underground
during the past 15 million years seeking moisture as the Australian continent
moved north and became increasingly arid (Byrne et al. 2008).
This chapter deals with the subterranean fauna occurring outside caves in the
Australian arid zone (Fig. 20.1). It focuses on the characteristics of the habitats in
which stygofauna and troglofauna occur, as well as the taxonomic structure of these
subterranean fauna communities and some of the more general characteristics of
subterranean fauna outside caves. Two other important texts on Australian subter-
ranean fauna are Humphreys (2016), which focuses on biogeography and the origin
of the fauna, and Hose et al. (2015), which provides more information about eastern
Australia.
Despite the large areas of karstic habitats in Australia, the continent has very few
large caves. This is especially so in the arid zone, where there are arguably only two
S. Halse (*)

https://doi.org/10.1007/978-3-319-98852-8_20
416 S. Halse
Fig. 20.1 Locations of the Pilbara and Yilgarn regions (or cratons) and other places mentioned
in text
large cave systems. One is on the Nullarbor Plain (Webb and James 2006) and the
other is around Camooweal, north-west of Mount Isa (Grimes 1988; Eberhard 2003)
(Fig. 20.1). Despite their large size, both systems have relatively depauperate
subterranean faunas (Richards 1971; Eberhard 2003). The same is true of the
20 Research in Calcretes and Other Deep Subterranean Habitats Outside Caves 417
Judbarra/Gregory karst area on the edge of the arid zone in the Northern Territory
(Moulds and Bannink 2012) (Fig. 20.1).
In very general terms, stygofauna in the Australian arid zone occurs mostly in
unconfined, surficial regional aquifers that extend across the landscape. These
aquifers mostly lie within alluvium (sometimes containing calcrete bodies), other
detrital geologies, or in fractured or weathered rock. Troglofauna species mostly
occur in the vadose (or unsaturated) zone, but there is no commonly used terminol-
ogy to define their habitat, which extends downward from a meter or more below-
ground surface to the water table. Troglofauna are found mainly in mineralized or
weathered rock, calcrete, and detritals (consisting of scree, alluvium, or colluvium).
The formations in the vadose zone containing troglofauna in Australia share some
features with the milieu souterrain superficial (MSS) or shallow superficial habitats
(SSH) of the northern hemisphere (Mammola et al. 2016), although the overall scale
and geological setting of the Australian formations is quite different.
20.2 Subterranean Habitat Other than Caves
Box 20.2
While the fauna of caves can be collected relatively easily by skilled cavers
and these cavers can see where the animals are and what they are doing at the
time of collection, sampling areas outside caves is a blind process. Either hauls
nets or traps are used to collect stygofauna and troglofauna. The operator is
usually remote from the sampling device, and it is rarely possible to collect
information about the exact habitat occupied by the animals. Information on
their behavior is never available. As a result, the ecological preferences of
stygofauna and troglofauna occupying the broader landscape are very poorly
understood even at the level of the types of spaces used by the animals and the
degree of interconnectedness of these spaces.
The main feature of aquifer and vadose zone habitats used by both stygofauna and
troglofauna is that the available subterranean spaces are relatively small and mostly
comprise what Howarth (1983) termed microcaverns (<5 mm in width) and
mesocaverns (5–500 mm) (Fig. 20.2; see also Chap. 3). Mesocaverns occur mostly
in calcrete and in the upper layers of extensively weathered rock formations. Most of
the calcrete bodies that have been investigated are of groundwater origin, with the
production of calcrete being principally the result of a shallow depth to the water
table combined with a climatic regime that comprises low annual rainfall with
occasional very heavy rain events and high rate of evaporation (Mann and Horwitz
1979). As groundwater levels rise following recharge from the heavy rain events,
calcium and carbonate are transported in groundwater into areas of high evaporation
where calcium carbonates are precipitated. As a consequence of changing baseline
418 S. Halse
Fig. 20.2 Habitats in which subterranean fauna species occur. (a) Palaeochannel containing
extensive subterranean calcrete in the Yilgarn; (b) drill core through saturated calcrete; (c) sche-
matic illustration of subterranean habitat where calcrete is present; (d) iron ore range in the Pilbara,
with deep gullies and an exposed face of hardcap containing mesocaverns; (e) drill core through
mineralized iron ore formation; (f) schematic illustration of subterranean habitat in iron ore range
with a deep water table (not illustrated)
groundwater levels over time because of long-term climatic variation, there is

re-working and re-forming of calcrete during wet and dry periods, which creates
mesocaverns and smaller spaces both above and below the water table in the
calcrete. Spaces tend to be largest around the water table; deeper sections of calcrete
are often quite compact and may lack any spaces. It is also common for areas of
calcrete to contain substantial pods of clay and silt that lack spaces, so that overall the
habitat structure within a calcrete body can be quite heterogeneous. Figure 20.2
shows a 30 cm length of calcrete core from just below the water table; it is composed
of porcelain-like clasts of calcrete bound together by cementing carbonate and
partially infilled with cream-colored clay and fine sand. The resulting unit has a
discontinuous, vuggy texture.
The amount of weathering in rock formations in the Pilbara and Yilgarn is
relatively high because of the very old age of these two cratons (Johnson 2009;
see also Fig. 20.1). Weathering breaks down rocks and may lead to formation of
spaces within the rock (vugginess). The surface of many rock formations in the
Pilbara consists of a ferricrete duricrust (commonly called hardcap; Fig. 20.2d) that
has resulted from weathering of the exposed host rock and may extend as deep as
60 m. Especially in its upper layers, the hardcap is frequently vuggy and may also
contain mesocaverns and even small caves. Occasionally, the hardcap has been
folded as a result of tectonic activity and so may occur at depth below unmodified
rock formations. The other process that leads to vugginess in rock formations is
mineralization, whereby various substances are leached from fresh rock with the
consequent enrichment of iron or other mineral elements (Morris 1983). Minerali-
zation can be substantially deeper than weathering because many of the processes
leading to mineralization occur at depth during rock formation (Evans et al. 2013).
As already mentioned, the vadose zone habitats in Australia (or at least the upper
strata) have some similarities to the MSS zone described by Juberthie (1983) and
Ortuño et al. (2013). This is particularly the case in and around low ranges
containing iron ore formations, especially where detritals form an important com-
ponent of the landscape profile (Morris and Ramanaidou 2007), and in palaeovalleys
filled with alluvium/colluvium and calcretes (Morgan 1993). There are also analo-
gies between the hardcap in Australia and the canga of Brazilian iron formations (see
also Chap. 21). However, a large proportion of the subterranean habitat in most iron
ore formations occurs in what is most appropriately regarded as vuggy bedrock.
The relationship between high numbers of stygofauna and the occurrence of
calcrete in palaeochannels in the Yilgarn region of Western Australia is well
documented (Humphreys 2001, 2008; Guzik et al. 2010), with up to 75 species
recorded from an individual calcrete body or cluster of calcrete deposits (EPA 2016).
Another relatively well-studied relationship is the occurrence of high numbers of
troglofauna species in mineralized banded iron formations and channel iron deposits
farther north in the Pilbara region (EPA 2007, 2011, 2012), with more than 100 spe-
cies having been recorded from sections of the banded iron formation of the
Hamersley Range and about 25 species per mesa (flat-topped hill) from individual
small mesas in the Robe Valley (unpublished data).
It should also be noted that it is not uncommon for a site to yield high numbers of
stygofauna and low numbers of troglofauna, or vice versa. This is sometimes the
result of different geologies occurring in the vadose zone and in the underlying
groundwater aquifer; in other situations, the flow of water may have kept spaces
open in the aquifer, whereas they have been filled by fine sediment in the
vadose zone.
420 S. Halse
Box 20.3
The sampling methods used to collect stygofauna and troglofauna from
subterranean habitats across the landscape have been described by Eberhard
et al. (2009) and Halse and Pearson (2014). Stygofauna are sampled using
groundwater monitoring bores to access the water table and underlying aquifer
(s). The bores usually have a slotted PVC casing. This casing prevents the
bores collapsing below the water table, while the vertical slots (usually
1–3 mm wide and extending the full depth of the bore below water table)
allow stygofauna to migrate into the bore void from the surrounding aquifer.
Stygofauna are collected by dropping a weighted haul net made of very fine
mesh to the bottom of the bore, agitating the sediments at the bottom, and then
slowly retrieving the net back through the water column. Troglofauna are
usually sampled in holes drilled for mineral exploration. These holes are
uncased and may be open at the ground surface, although sometimes they
have a short PVC collar to reduce the likelihood of the hole collapsing.
Troglofauna are collected either by trapping or scraping. Traps consist of
short PVC cylinders that have slightly smaller diameter than the hole and
moderate-sized perforations along their length to allow entry of troglofauna.
The traps are baited with leaf material, lowered to the desired depth on a piece
of cord and left in place for 6–8 weeks before being retrieved. Scraping
consists of lowering a weighted, reinforced haul net to the bottom of the
hole (or just into the water table) and then pulling it back to the surface
along the wall of the hole, thus scraping troglofauna from the wall.
20.3 Subterranean Fauna Outside Caves
Arid and semiarid regions of Western Australia contain very high richness of
stygofauna and troglofauna. Eberhard et al. (2009) estimated, based on a regional
sampling program, that 500–550 stygofauna species occur in the Pilbara region
alone, while Guzik et al. (2010) used expert opinion to estimate that 4140 subterra-
nean fauna species, comprising 2680 stygofauna and 1460 troglofauna species,
occur in the western half of Australia, mostly in the arid zone. More recently,
Halse (2016) proposed, based on a combination of sampling results and extrapola-
tion of the pattern of increasing richness from further sampling (mostly for environ-
mental impact assessment), that nearly 3000 species of subterranean fauna occur in
the Pilbara (Table 20.1).
Other environmental impact assessment sampling suggests that the Yilgarn has
stygofauna richness similar to the Pilbara but fewer troglofauna species. On this
basis, it is considered likely that more than 4500 stygofauna and troglofauna species
occur in the Pilbara and Yilgarn. This figure more-or-less matches that proposed
independently by Guzik et al. (2010) for the larger western half of Australia, but
undoubtedly most of the richness is on the Western Shield where the Pilbara and
Table 20.1 Number of No. of species

subterranean fauna species in
Faunal group Collected by BEC Estimated
the Pilbara, as collected by
Bennelongia Environmental Stygofauna
Consultants (BEC) or Crustacea
estimated to be present based Amphipoda 106 200
on extrapolation of the Isopoda 31 75
collecting results to date by
Syncarida 70 300
BEC and other environmental
consultants Copepoda 130 250
Ostracoda 194 300
Other 4 4
Hydracarina 23 40
Annelida 74 150
Mollusca 5 10
Total 637 1329
Troglofauna
Isopoda 81 200
Pseudoscorpiones 66 150
Schizomida 59 120
Araneae 53 130
Palpigradi 18 40
Diplopoda 24 50
Chilopoda 51 120
Symphyla 38 80
Pauropoda 27 60
Diplura 90 200
Thysanura 47 100
Blattodea 27 40
Hemiptera 23 50
Coleoptera 69 150
Other 7+diptera 21
Total 680 1511
Yilgarn occur (Fig. 20.1). As is a regular feature of subterranean fauna species with
their localized distributions, nearly all stygofauna and troglofauna species in the
Pilbara and Yilgarn are endemic to the region in which they occur (Humphreys et al.
2008; Halse and Pearson 2014; Halse et al. 2014).
422 S. Halse
20.3.1 Stygofauna
Box 20.4
Most stygofauna species are crustaceans. The aquifers they use in the
Australian arid zone occupy a variety of geologies that provide suitable spaces
for animals. Alluvial and colluvial aquifers are important habitat because of
their widespread occurrence and frequent high species richness, while areas of
saturated calcrete (usually within alluvial or colluvial aquifers) are important
habitat because of high species richness and very fine-scale endemism. Some
aquifers in iron formations, especially channel iron deposit, may also support
moderate numbers of stygofauna species. However, depth to the water table is
a constraint on stygofauna occurrence, with assemblages usually being sparse
where depth to the water table is much more than 30 m.
A detailed account of the stygofauna of the Pilbara is given by Halse et al. (2014).
Although information on the Yilgarn is extensive, it is less consolidated and the first
moderately comprehensive overview is provided here. General information about
the communities in Yilgarn calcretes is provided by Humphreys (2001) and
Humphreys et al. (2008).
A significant feature of both the Pilbara and Yilgarn is the relatively small overlap
in species composition of the hyporheic fauna of streams and the stygofauna
communities of deeper groundwater aquifers (Halse et al. 2002). While some species
typical of the hyporheos are found in regional aquifers, such as darwinulid ostracods,
the candonid ostracod Candonocypris tenuis, many cyclopoid copepods, and possi-
bly phreatoicid isopods (Knott and Halse 1999; Pinder et al. 2010; Schön et al.
2010), the reverse rarely occurs. The low overlap is probably partly a result of the
water associated with the alluvium of the ephemeral rivers and creeks being poorly
connected to regional groundwater (Dogramaci et al. 2012), but the absence of
deeper groundwater species in the hyporheos also suggests that the ecological and
life history characteristics of these stygobitic groundwater species make them
unsuited to hyporheic conditions.
Overall, the higher level taxonomic composition of stygofauna assemblages in
the Pilbara and Yilgarn is similar, despite some differences in the proportions of
major taxonomic groups (Fig. 20.3). Six groups are considered here in more detail.
Copepods dominate the fauna of both areas, comprising approximately 60% of the
animals in the Yilgarn and 40% in the Pilbara. However, individual species are often
represented by large numbers of animals and copepods comprise only 44% and 20%
of species in the Yilgarn and Pilbara, respectively (Halse et al. 2014; unpublished
data), which is similar to the representation of 20–40% of species in European
communities (Galassi et al. 2009). Perhaps of most interest, there appears to have
been explosive speciation of harpacticoid copepods in some Yilgarn calcretes where
Fig. 20.3 Proportions of stygofauna in the Pilbara and Yilgarn belonging to different taxonomic
groups. (a) Pilbara species, (b) Pilbara abundance, (c) Yilgarn species, and (d) Yilgarn abundance.
Based on collecting results of Bennelongia Environmental Consultants
copepod species may represent almost half the fauna (Karanovic and Cooper 2011,
2012).
Ostracods represent 24% and 13%, respectively, of the animals in the Pilbara and
Yilgarn (Fig. 20.3) and 30% and 10% of the species. The greater contribution of
ostracods to the fauna of the Pilbara reflects the enormous radiation of candonid
ostracods in this region, consisting of 11 described endemic genera and more than
108 collected species (Karanovic 2007; Reeves et al. 2007, unpublished data). By
global standards, where ostracods typically constitute about 3% of all species
(Eberhard et al. 2005), both the Pilbara and Yilgarn are rich in ostracods, but the
Pilbara has exceptional diversity.
Another group showing high species richness is dytiscid beetles in Yilgarn
calcretes and some other parts of the arid zone (Watts and Humphreys 2009;
Eberhard et al. 2016). Strangely, only one dytiscid species has been recorded from
424 S. Halse
the Pilbara (Watts and McRae 2013). The large number of stygofauna dytiscid
beetles collected to date from the western half of Australia (approx. 100), despite
single calcretes almost never containing more than three species, is a consequence of
the high species turnover between calcretes. Beetles are estimated to represent 2.2%
of the animals in the Yilgarn and 4.5% of the species.
Amphipod species have much the same pattern of occurrence in the Yilgarn as
dytiscids, but they are more speciose and occur in higher abundance. They are also
abundant in the Pilbara and represent 16% and 7%, respectively, of the animals in the
Pilbara and Yilgarn (Fig. 20.3) and 17% and 20% of the species (Halse et al. 2014;
unpublished data). This is similar to the overall representation of amphipod species
in stygofaunal assemblages globally (19%, Eberhard et al. 2005). Much of the
stygofaunal research in the Yilgarn and Pilbara has been on amphipods, with species
in the Yilgarn considered to be confined to single calcretes (although these may
sometimes be more accurately described as a cluster of adjacent calcrete bodies),
while species in the Pilbara mostly have ranges confined to the catchments of
individual tributaries of major rivers (Finston et al. 2004, 2007; Cooper et al.
2007; Bradford et al. 2010, 2013; King et al. 2012).
Based on limited taxonomic and genetic work to define species units (e.g., Guzik
et al. 2008), syncarids comprise 3.1% and 4.1%, respectively, of the animals in the
Pilbara and Yilgarn and 11% of the species in both regions. This is a substantially
higher proportion of species than recorded globally (Eberhard et al. 2005). Limited
surveys have shown that syncarids are also ubiquitous in alluvial aquifers of better
watered coastal areas of Australia (Cho et al. 2005; Camacho and Hancock 2012;
Cook et al. 2012), and it is likely that more survey will show the Australian fauna is
at least as rich as that of Europe (see Camacho and Valdecasas 2008) and with
perhaps less of an arid zone focus than most groups of Australian stygofauna.
Oligochaetes represent 9% and 8%, respectively, of the animals in the Pilbara and
Yilgarn (Fig. 20.3) and 11% and 8% of the species compared with a global average
of 2% of species (Eberhard et al. 2005). As in other parts of the world (Creuzé des
Châtelliers et al. 2009), many oligochaetes in the Pilbara and Yilgarn are quite
widespread and also have surface occurrences, so that they should be treated as
stygophiles (and sometimes possibly stygoxenes). The greater number of stygal
species in the Pilbara and Yilgarn is principally attributable to the collection of
relatively large numbers of enchytraeid species during sampling (31% of Pilbara and
50% of Yilgarn species compared with 11% of European species). It is also of
interest that phreodrilids are quite common as stygofauna in the arid Pilbara and
Yilgarn (Pinder 2008; Brown et al. 2015).
20.3.2 Troglofauna
Information on the occurrence of troglofauna outside caves comes almost entirely

from environmental impact assessment surveys associated with mining proposals
and so data are strongly biased toward the sampling of hard rock geologies. Areas of
calcrete have usually been sampled at low intensity, if at all, because of the difficulty
maintaining open holes for sampling in soft substrata. Halse and Pearson (2014)
have provided a description of the taxonomic composition of troglofauna in the
Pilbara, but the first account of the overall composition of Yilgarn troglofaunal
assemblages is presented here.
Box 20.5
Troglofauna in the Australian arid zone are taxonomically much more diverse
than stygofauna. Sampling has been strongly biased toward iron formations
where exploration drill holes (for mining) provide access to subterranean
habitat. Nevertheless, it is likely that iron formations provide some of the
most important troglofaunal habitat. Areas of unsaturated calcrete also seem to
provide important habitat, provided the water table is not too shallow and soil
salinity is not too high. While species ranges are still being documented, one of
the outstanding characteristics of Australian arid zone troglofauna species is
that they appear to have very small ranges, which may sometimes be <1 km2.
One of the peculiarities of the information on troglofauna in Pilbara and Yilgarn

is that there has been no attempt to assess the occurrence of troglofaunal mites and
collembolans. Both groups comprise significant components of the fauna in other
parts of the world (Ducarme et al. 2004; Kováč et al. 2016), and troglofauna species
belonging to these groups have been observed frequently in Pilbara and Yilgarn
samples (Greenslade 2002).
Several of the groups that are prominent in troglofaunal assemblages of the
Pilbara (cockroaches, schizomids, dipterans) are absent, or nearly so, from the
Yilgarn (Fig. 20.4). Isopods are the dominant group in assemblages of the Yilgarn.
Based on the number of animals collected, they represent 6% and 43% of the fauna
in the Pilbara and Yilgarn, respectively, and 12% and 30% of species. Isopod
occurrence is globally variable with 12% of species in the Balkan Peninsula (Sket
et al. 2004) and 26% of species in Portugal (Reboleira et al. 2013), and the variation
between regions in Western Australia reflects this.
Hemipterans (mostly Meenoplidae) appear to have variable ranges (Fig. 20.5).
The group is relatively abundant, representing 23% and 10% of animals in the
Pilbara and Yilgarn, respectively, but little more than 3% of species in each region
(Fig. 20.4). Some troglophilic species appear to have ranges extending over hun-
dreds of kilometers, while other potentially troglobitic species appear to have small
ranges (JM McRae, unpublished data). Culver and Pipan (2008) considered
troglobitic hemipterans to be more common in shallow subterranean habitats than
caves. Records from the Pilbara, in particular, suggest that hemipterans may occur at
considerable depths (Halse and Pearson 2014; unpublished data).
Cockroaches, mostly belonging to the family Nocticolidae, are also abundant in
the Pilbara, where they represent 19% of animals but only 4% of species, although
cockroaches are one of the many groups in which the use of genetic species concepts
426 S. Halse
Fig. 20.4 Proportions of troglofauna in the Pilbara and Yilgarn belonging to different taxonomic
groups. (a) Pilbara species, (b) Pilbara abundance, (c) Yilgarn species, and (d) Yilbara abundance.
Based on collecting results of Bennelongia Environmental Consultants
is likely to substantially increase the number of species recognized (Trotter et al.

2017; Fig. 20.5). A single cockroach has been collected from the Yilgarn. While
comparative data are difficult to obtain, the diversity of troglofaunal cockroaches in
the Pilbara appears to be unusually high (Roth 1991; Moulds and Bannink 2012).
In contrast to the abundance of hemipterans and cockroaches, the proportions of
beetles in the troglofauna assemblages of the Pilbara and Yilgarn are surprisingly
low, especially when the Yilgarn is comparatively rich in stygofaunal beetles. Only
6% and 3% of troglofaunal animals and 10% and 9% of troglofaunal species are
beetles in the Pilbara and Yilgarn, respectively. Typically, beetles comprise more
than a third of the species in troglofaunal communities elsewhere in the world
(Culver and Sket 2000; Sket et al. 2004; Niemiller and Zigler 2013). While more
taxonomic investigation is likely to substantially increase the number of beetles
known from the Pilbara and Yilgarn (e.g., Baehr and Main 2016; Table 20.1), their
proportion of the known fauna is not expected to change greatly.
Fig. 20.5 Degrees of troglomorphy in meenoplid hemipterans and nocticolid cockroaches. (a)
Troglobitic meenoplid, (b) troglophilic meenoplid, (c) troglobitic nocticolid, and (d) troglophilic
nocticolid with eyespot
Millipedes represent 9% and 6% of animals in the Pilbara and Yilgarn, respec-

tively (Fig. 20.4), as a result of the widespread occurrence of the circumtropical
troglophilic Lophoturus madecassus (see Car et al. 2013). The group comprises only
3.5%and 1.7% of species known from these regions compared with 10% of the fauna
in the Balkan Peninsula (Sket et al. 2004).
Perhaps the most iconic troglofaunal group in the Australian arid zone is the
minor arachnid order Schizomida. Its occurrence is indicative of a taxonomically
rich troglofauna community, and collection of schizomid species in mesas of the
Robe Valley in the Pilbara led to the first troglofauna-based recommendations
against mine approval by the Environmental Protection Authority in Western
Australia (EPA 2007). Schizomids occur moderately often in the vadose zone and
in caves across northern Australia (e.g., Harvey 2001), as well as in humid surface
habitats of the tropics more generally (Monjaraz-Ruedas 2013). They have been
collected in high abundance in the iron formation ranges of the central Pilbara and
comprise 7% of animals and 9% of species in troglofauna assemblages of the Pilbara
as a whole (Fig. 20.4). Some of the diversity of schizomids in the Robe Valley of the
428 S. Halse
Pilbara has been documented in detail by Harvey et al. (2008) and Harms
et al. (2018).
Diplurans usually comprise a small to moderate proportion of troglofauna assem-
blages (1.1% in the Balkan Peninsula, Sket et al. 2004; 1.4–6% in superficial
subterranean habitats, Culver and Pipan 2008; 7% in Portugal, Reboleira et al.
2013). In contrast, they comprise 13% and 7% of species in the Pilbara and Yilgarn,
respectively, despite accounting for only 3% of the animals in each region. Some of
these species are certainly troglophiles, and determining the proportion of troglobites
is likely to require detailed taxonomic investigations and, ideally, life history studies
to understand species ranges. However, the estimated median range of 16 km2 for
Pilbara species (Halse and Pearson 2014) suggests the proportion of troglobites may
be quite high.
For pauropods, symphylans, and, to a lesser extent, palpigrads, it is difficult to
distinguish troglofaunal from epigean species, because all animals of these three
groups lack eyes and pigment. Furthermore, collection from subterranean habitats
does not necessarily mean a species is troglofauna because most animals collected in
drill holes are clearly identifiable as epigean species that have “fallen” into the drill
hole. Many holes lack collars and are open at the surface with nothing to prevent
surface species falling in. Even when holes are collared with PVC pipe, there is often
subsidence around the collar and space for surface species to enter the hole. Bearing
in mind the uncertainties associated with interpreting captures of the three groups in
the Western Australian context, palpigrads and symphylans that are clearly
troglobitic are regularly recorded in other parts of the world (e.g., Sket et al. 2004)
and the described palpigradid Eukoenenia guzikae from the Yilgarn is considered to
be troglobitic (Barranco and Harvey 2008). Halse and Pearson (2014) suggested that
at least some of the pauropod species collected from the Pilbara are also likely to be
troglobites because of their small ranges and, more particularly, the hostile surface
soil conditions in the arid Pilbara. Currently, pauropods, symphylans, and palpigrads
are considered to comprise 4.9%, 5.6%, and 2.6% of animals and 6.3%, 13.6%, and
2.3% of species in the Pilbara and Yilgarn, respectively (Fig. 20.4).
20.4 Species Distributions
As a group, subterranean fauna species are characterized by small ranges. This is

especially so for troglofauna species (Halse and Pearson 2014), which in the Pilbara
appear to have ranges that are mostly at least an order of magnitude smaller than
those of stygofauna species (Eberhard et al. 2009; Halse et al. 2014). Linear ranges
of <1 km sometimes occur and ranges of 1–2 km are probably quite common among
arid zone troglofauna in the Pilbara (Table 20.2). In contrast only about 5% of
Pilbara stygofauna species are likely to have linear ranges of <30 km (Halse et al.
2014). There is probably less difference between ranges of stygofauna and
troglofauna in calcretes in the Yilgarn where a habitat feature (i.e., the calcrete
Table 20.2 Median linear ranges (recalculated from Halse and Pearson 2014) of different groups
of troglofauna species in the Pilbara and the main geologies from which the groups are known in the
Pilbara and Yilgarn
Troglofauna group Median linear range (km) Major habitats
Pseudoscorpiones 5.3 Mineralized rock, detritals (incl. calcrete)
Palpigradida 21 Mineralized rock, detritals (incl. calcrete)
Schizomida 2.6 Mineralized rock
Araneae 2.2 Mineralized rock (incl. calcrete)
Chilopoda 6.2 Mineralized rock, detritals (incl. calcrete)
Diplopoda 4.5 Mineralized rock, detritals (incl. calcrete)
Pauropoda 6.6 Detritals, mineralized rock (incl. calcrete)
Symphyla 3.2 Detritals, mineralized rock (incl. calcrete)
Isopoda 1.8 Mineralized rock, detritals (incl. calcrete)
Diplura 4.5 Mineralized rock, detritals (incl. calcrete)
Thysanura 3.7 Mineralized rock, detritals (incl. calcrete)
Blattodea 6.1 Mineralized rock
Hemiptera 68 Mineralized rock, detritals (incl. calcrete)
Coleoptera 8.7 Mineralized rock, detritals
Diptera 159 Mineralized rock
body) is often the factor limiting ranges rather than characteristics of the species
themselves or the distribution of subtle habitat differences within the calcrete.
The pattern of subterranean fauna species being restricted to single calcretes or
calcrete clusters in the Yilgarn led to Steven Cooper and others proposing the
calcrete island hypothesis in relation to stygofauna (Cooper et al. 2002, 2007). It
also seems to apply to troglofauna species (Javidkar et al. 2016). Under this
hypothesis, most species in calcretes of the Yilgarn region are expected to be
restricted to individual calcrete bodies that may have linear ranges of only tens of
kilometers at most. The areas between calcrete bodies, which include intervening
sections of the palaeochannel valleys hosting the calcretes, are unsuitable for
stygofauna and troglofauna because of high salinity (Humphreys et al. 2008), lack
of suitable voids and spaces, or otherwise inhospitable habitat. A series of papers by
Tomas Karanovic on the copepods of the Yeelirrie calcrete illustrate the extreme
levels of geographic replacement and local endemism that may occur within
calcretes, with some stygofauna species appearing to have linear ranges of <5 km
(Karanovic and Cooper 2011, 2012; Karanovic et al. 2015).
Another generalization is that weathered and mineralized iron ore deposits
provide rich troglofauna habitat. The occurrence of rich troglofauna communities
in iron ore ranges in Australia is analogous to the occurrence of troglofauna in iron
ore mining areas of Brazil (Silva et al. 2011; see Chap. 21), although in Brazil the
animals have mostly been collected from caves rather than from microcaverns within
areas of vuggy iron ore (Fig. 20.2). That said, the factors affecting the importance of
different types of iron ore deposits for troglofauna in Western Australia are still
being studied. For reasons still to be explained, banded iron formations and channel
430 S. Halse
iron deposits in the Pilbara support greater numbers of troglofauna species than
banded iron formations in the Yilgarn, with Pilbara communities being more com-
plex and, as already mentioned, supporting groups such as schizomids and cock-
roaches that are absent (or very nearly so) from the Yilgarn.
The vuggy habitats found in banded iron and other rock formations, especially if
the available spaces are mostly microcaverns, probably provide few pathways for
significant lateral underground dispersal (Fig. 20.2). Therefore, the troglobitic spe-
cies in rock habitats would be expected to have smaller ranges than species
inhabiting various types of detritals (scree and alluvium/colluvium) where the
potential for dispersal through the matrix is likely to be greater. Despite this, and
based on the current very limited understanding of the habitats that species occupy, it
appears that the ranges of most troglofauna species in the Pilbara (Table 20.2) are
small and determined by factors other than the broad type of geology in which the
species occurs. For example, species occurring in mineralized rock do not consis-
tently have smaller ranges than species in detritals. Probably the most important
factor affecting range is whether species are troglophilic and have a surface dispersal
phase, rather than relying on below-ground dispersal as troblobites do, but other
intrinsic biological differences between groups may also affect species’ ranges. It
should be emphasized that groups for which median ranges are relatively large
because they include some widespread troglophiles, such as palpigrads and hemip-
terans, also contain some presumed troglobitic species with very small ranges.
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Chapter 21
Subterranean Biodiversity in Ferruginous
Landscapes
Rodrigo Lopes Ferreira, Marcus Paulo Alves de Oliveira,

and Marconi Souza Silva
21.1 Introduction
When both their epigean and subterranean components are considered, ferriferous
formations are certainly among the most unknown ecosystems of the world. Such
formations are widely distributed, even in small portions, covering approximately
5% of the world’s land surface (Ollier and Galloway 1990). Important deposits of
these iron ore formations, which are of pre-Cambrian origin, are located in Brazil,
Australia, India, and South Africa and various other countries (Costa 1993).
Although many of these areas have been targeted by mineral exploration for decades,
recent studies conducted in Brazil and Australia have revealed a high richness and
singularity of the fauna and flora of these ferruginous formations (Jacobi et al. 2007;
Souza-Silva et al. 2011; Gibson et al. 2015).
R. L. Ferreira (*)
Centro de Estudos em Biologia Subterrânea, Departamento de Biologia,
Universidade Federal de Lavras, Lavras, Brazil
Programa de Pós-graduação em Ecologia Aplicada, Departamento de Biologia,
e-mail: [email protected]fla.br
M. P. A. de Oliveira
Programa de Pós-graduação em Ecologia Aplicada, Departamento de Biologia,
M. S. Silva
Centro de Estudos em Biologia Subterrânea, Departamento de Biologia,
e-mail: [email protected]fla.br

https://doi.org/10.1007/978-3-319-98852-8_21
436 R. L. Ferreira et al.
Fig. 21.1 Schematic profile of the ferruginous habitats: (a) superficial ferruginous breccia (canga
formation), (b) metallophilic savannah, (c) tropical rainforest, (d) banded iron formation (BIF), (e)
ferricretes, (f) cave at the contact between the BIF and the canga, (g) cave entirely in the canga, (h)
cave in banded iron formation (BIF), (i) cave in ferricretes, (j) voids, (k) roots from the external
vegetation, (l) water stored in voids, (m) actinomycetes on the cave walls
21.2 Subterranean Environments in Iron Ore Landscapes
In Brazil, the two largest ferriferous formations differ in their structure. In Carajás
(Pará state), the formations are represented by jaspilite interspersed by ore bodies
rich in iron. In this area, extensive iron ore plateaus comprising superficial ferru-
ginous breccia (denominated as canga formations; Fig. 21.1a) cover the banded
ferriferous formations (Piló et al. 2015). The vegetation covering those formations
(800 m asl) is metallophilic savannah, and the slopes are covered by rainforest with
medium and large-size trees typical of the Amazon region (Fig. 21.1b, c). In the Iron
Quadrangle (Minas Gerais state), the banded formations are represented by itabirites
and the landforms are more pronounced, with cangas occurring until 2000 m asl
(Salgado and Carmo 2015). The vegetation of the range tops and slopes comprises
grass with scrub and small-size trees, sparse or grouped in small forests, while the
valleys are dominated by rainforest typical of Atlantic forest (Salgado and Carmo
2015).
The original banded iron formation (BIF), due to its long history of diastrophism
and weathering, is rarely observed unaltered near the surface (Fig. 21.1d). The canga
formations are usually the main surface layer on top of plateaus and mountains
(Fig. 21.1a), unlike the ferricretes (Fig. 21.1e) which are located on the middle and
basal portions of the cliffs (Piló et al. 2015). Chemical alterations occur at different
depths, thus creating subterranean spaces with a volume and distribution that is
controlled by the low rates of dissolution and chemical change found in iron ore,
21 Subterranean Biodiversity in Ferruginous Landscapes 437
even in tropical regions of high rainfall. The high porosity of the iron ore formations
results from the removal of silica and carbonates, which are more soluble than iron
(Schuster et al. 2012).
Caves formed in iron ore found in Brazil (more than 2500) are usually small
(average size of 30 m), although there are records of caves with 1500 m of extension
(Piló et al. 2015). A great number of caves are inserted at the contact between BIF
and canga (Fig. 21.1f), but they may occur entirely in the canga (Fig. 21.1g), in the
BIF (Fig. 21.1h), and in ferricretes (Fig. 21.1i) (Auler et al. 2014). According to
Auler et al. (2014), the higher frequency of caves in the contact zone of canga/BIF
may reflect that the documentation of caves is biased toward shallower caves in
erosional areas. There are a significant number of voids (or caves) in deep portions of
the rock that lack natural entrances for humans. Such facts are indicative of
hypogenic speleogenesis linked to slow biospeleogenetic processes (e.g., microbial
reducibility of Fe) that require a long time to occur, thus attesting the old age of these
subterranean spaces (Parker et al. 2013).
The most relevant characteristics of ferruginous subterranean ecosystems are their
old age, occurring relatively close to surface, and also the presence of several
interconnected small spaces in the canga formations—the voids in Fig. 21.1j
(Ferreira 2005), which differ from other shallow subterranean habitats described in
most parts of the world (epikarst, MSS, calcrete, and hypothelminorheic) (Culver
and Pipan 2014). They are also found in BIF in Australia (see also Chap. 20). The
voids in canga formations represent a distinct type of shallow subterranean habitat
because of their similarity to the structural and microclimate conditions found in
macro-caves (permanent, old aphotic spaces within the matrix rock), other than their
small dimensions (Ferreira 2005). Such voids also differ from other shallow subter-
ranean habitats in the availability of trophic resources. Once the roots of overlying
vegetation penetrate the voids (Fig. 21.1k), they are not necessarily oligotrophic
environments. Furthermore, the voids networks in canga formations allow the
storage and circulation of water (Fig. 21.1l), thus favoring the coexistence of
terrestrial and aquatic fauna which move from the surface to the innermost subter-
ranean habitats, frequently accessing the macro-caves. In epikarst, water circulation
is limited to small and semi-isolated wet areas, while MSS is usually formed by a
mosaic of isolated and unconsolidated rock fragments (Culver and Pipan 2014).
Ferruginous voids are connected to the edaphic epigean environment and its com-
ponents, such as soil, lapidicolous compartments, and leaf litter.
The ecological and evolutionary importance of ferruginous voids is evident in the
diversity of the species they harbor, including relict and endemic species, many of
them troglobite. The voids play an important role as refugees and corridor for the
fauna, thus acting as a fundamental habitat for the conservation of the biodiversity in
ferruginous ecosystems.
Fig. 21.2 Some troglobitic species found in Brazilian iron ore caves: (a) Glomeridesmus spelaeus
(Glomeridesmidae), (b) Coarazuphium tapiaguassu (Carabidae), (c) Pseudonannolene spelaea
(Pseudonannolenidae), (d) Ferricixius davidi (Cixiidae), (e) Pseudochthonius sp. (Chthoniidae),
(f) Brasilomma enigmatica (Prodidomidae), (g) Pirassunungoleptes sp. (Zalmoxidae), (h) Carajas
paraua (Caponiidae), (i) Charinus ferreus (Charinidae), (j) Circoniscus carajasensis
(Scleropactidae), (k) Trichorhina sp. (Plathyarthridae), (l) Hyalella sp. (Hyalellidae)
21.3 Troglobitic Species
Ferruginous caves certainly stand out in terms of the high richness of troglobitic
species they support, especially when compared with caves in other lithologies
(Souza-Silva et al. 2011; Ferreira et al. 2015). The troglobitic species found in iron
ore caves belong to a variety of invertebrate groups, as shown in Fig. 21.2. Among
the factors that apparently contributed to the evolution of troglobites in these
environments can be mentioned the superficiality of the systems, the external
environmental severity, and the time (Ferreira et al. 2015). The superficiality of
many ferruginous subterranean spaces allows the access of roots from the external
vegetation, what provides trophic resource for several species. Furthermore, the
relative severity of epigean environments above these caves (scarce vegetation
covering the canga and high exposure of the soil and rock to solar radiation)
contributes to the active colonization of subterranean environments. Finally, other

important component for the evolution of troglobites in ferruginous caves is the age
of the formations and their internal changes. Schuster et al. (2012) conducted a study
on the stability of cangas in the Carajás region of Brazil and showed that the canga of
this region had a very slow rate of erosion throughout the last million years. The
canga surface is extremely resistant to erosional processes, in spite of a humid
tropical environment. However, details of the deep profile of the canga suggest
that it is internally dynamic with translocation of materials as well as processes of
dissolution and precipitation (Schuster et al. 2012).
Similarly, Auler et al. (2014) suggested that the speleogenetic processes through
which the Brazilian ferruginous caves developed have many indications of
hypogenesis associated with long-term biospeleogenetic processes performed by
bacteria (Fig. 21.1m) and other organisms over hundreds of millions of years,
demonstrating that dynamic sub-superficial spaces are extremely old in these envi-
ronments. Many of the spaces may have sheltered ancestral species of several current
troglobites. The ancestral species may have colonized these spaces earlier than
speculated for the evolution of troglobites in environments such as limestone caves.
The level of endemism of troglobitic species associated with ferruginous land-
scapes is quite variable. There are species of wide distributions, which may extend
beyond the limits of the ferruginous lithology (e.g., the Prodidomidae spider
Brasilomma enigmatica—Fig. 21.2a—found in ferruginous, limestone, and quartzite
caves with a linear range of approximately 300 km) as well as species with restricted
distributions, such as the planthopper Ferricixius davidi (Fig. 21.2b—Cixiidae) that
is endemic to one single cave.
Such differences in the level of endemism result from several factors. The first
(and maybe one of the most important) is the local geomorphology, which may favor
(or not) subterranean contact among macro-caves, thus increasing (or reducing) the
range of a given species. For example, there is a clear difference in patterns of
similarity of the troglobitic fauna associated with caves in two important Brazilian
ferruginous regions. Figure 21.3 presents two graphs of multidimensional scaling
(MDS) ordination based on the composition of the troglobitic fauna of some caves of
Carajás (Pará state) and Iron Quadrangle (Minas Gerais state). Such graphs do not
consider unique species since they would lead to some stress in the analyses because
they only occur in one cave. It is noticeable that caves of Iron Quadrangle do not
form distinct groups despite the apparent geomorphological compartmentalizing of
this formation. Thus, it is assumed that there are subterranean connections among
these macro-caves, allowing faunal migrations along almost the entire Iron Quad-
rangle, as demonstrated by the distribution of the troglobitic spider Tisentnops
mineiro (Brescovit and Sánchez-Ruiz 2016). On the other hand, caves located in
Carajás present a distinct pattern. Cavities located in a given unit (plateau) are highly
similar regarding the troglobitic fauna, indicating the existence of species movement
among the macro-caves of the same plateau, but the dissimilarity observed in caves
of different units is remarkable and demonstrates that there is no movement of
troglobites among plateaus. Therefore, troglobitic species that exist in one plateau
are completely different from those that exist in other plateaus of Carajás region.
Fig. 21.3 MDS analysis performed for some ferruginous caves of Carajás (a) and the Iron
Quadrangle (b), considering only troglobitic species. The symbols on the graph represent caves,
and the colors indicate from which unit they belong to. The units (areas) present in each formation
are highlighted in colors (corresponding to the graph) in hypsometric maps of each area: (a) Carajás
region (Pará state, Brazil): green triangles: Serra Leste unit; blue triangles: Serra Norte Unit; light
blue squares: Serra Sul unit; (b) Iron quadrangle (Minas Gerais state, Brazil): blue triangles: Serra
da Piedade unit; light blue squares: Escarpa Oriental Unit; red diamond: Quadrilátero Oeste unit;
pink circle: Morrarias de Dom Bosco unit; gray cross: Serra do Gandarela unit. See further
explanations in the text
The similarity of troglobitic species composition observed within each plateau in

the Carajás region is probably the result of species migration among caves through
voids in the canga. Such spaces may even represent, for many species, their main
habitat. Figure 21.4b shows the distribution of some troglobitic species present in a
plateau in Carajás. The caves in which a given species occurs (represented by small
circles) were grouped by lines indicating a minimum distribution for each species,
and this distribution is much wider than the macro-caves per se. Thus, each plateau
in that area may, in a sense, represent a great and unique cave formed by macro-
galleries interconnected by huge systems of voids.
Another important factor that may determine general patterns of distribution of
troglobitic species is biological or environmental preferences. In the Carajás region,
analyses performed using troglobitic species present in the same ferruginous plateau
revealed patterns of cave use that may reflect preferences for distinct micro-habitats.
Figure 21.4a represents a multidimensional scaling analysis conducted considering
troglobitic species present in a plateau of Carajás. Each cave is represented by a dot
in the graph. Three groups were formed, each one with highly similar caves
regarding troglobitic species. Sub-superficial connections that allow the migration
Fig. 21.4 (a) MDS analysis performed for the ferruginous caves present in a plateau in the Carajás
region, considering only troglobitic species. The caves are represented in the graph by colored dots,
and an aerial image of the area was incorporated in the graph, so that each cluster generated by the
analysis is linked to their correspondent caves in the aerial image. See explanations in the text; (b)
Distribution of some troglobitic species in a ferruginous plateau in the Carajás region. Circles in the
aerial phonograph indicate the caves in the area. Each color refers to a species (orange—Charinus
sp.; yellow—Carajas paraua; blue—Circoniscus buckupi) and the lines connecting the caves
indicate the minimum distribution of each species shown in the figure. See further explanations
in the text
among macro-caves exist connecting all the system, although not all species are
widely or uniformly distributed. An aerial photograph of the area (including the
location of caves) was overlapped to the graph, and each group defined by the MDS
was associated with the corresponding cave in the aerial image. It is noted that one of
these groups comprises more internally located caves in the plateau (yellow group),
while other group comprises caves preferentially associated with the breaks in the
canga border (green group). Finally, a small group is formed by two isolated caves
(blue group). Such pattern suggests that specific traits of each micro-region (maybe
related to micro-climate or availability and type of trophic resources) are important
in determining which species will occur on each of these “units.” Thus, it is noted
that even considering each plateau as a large cave, different regions exhibit distinct
traits that determine the differentiation in the occurrence and distribution of the cave
fauna. In short, these “mega-caves” are not homogeneous in biological terms.
21.4 Iron Ore Formations in Brazil and Australia
Australia and Brazil have the largest ferruginous geosystems in the world, but the
characteristics of the systems in each country lead to different habitats being present
and, consequently, differences in their subterranean faunas. In Brazil, the occurrence
of iron ore macro-caves is extremely common, and they are usually dry, with rare
temporary drainages resulting from drip or percolation of pluvial waters through
voids or geological discontinuities (Piló et al. 2015). Caves are the main target of
subterranean studies in Brazil, and most of the known fauna for Brazilian ferriferous
areas came from these habitats. However, in Australia, the lower intensity of erosive
and dissolution processes hampered the formation of macro-caves, and the subter-
ranean fauna has been sampled using geological exploration drill holes and bores to
access much smaller subterranean habitats (Eberhard et al. 2009; Guzik et al. 2011;
Halse and Pearson 2014; see also Chap. 20).
Consequently, the subterranean fauna of Brazilian ferruginous geosystems is
almost totally represented by terrestrial invertebrates, while in Australia there are a
significant number of stygobiotic species as well in ferruginous formations, although
most stygofauna occur in alluvium and a range of other geologies (Halse et al. 2014).
Currently, there are approximately 150 troglomorphic species associated with
Brazilian ferruginous caves, of which 21 are formally described and only one is
aquatic (Ferreira et al. 2015; Brescovit and Sánchez-Ruiz 2016; Zeppelini and
Oliveira 2016; Giupponi and de Miranda 2016; Asenjo et al. 2018; Souza and
Ferreira 2018). While there are more than 403 formally described species in the
western part of Australia (Guzik et al. 2011), most of these species are stygobitic
copepods, ostracods, and beetles found in alluvium or calcrete. A few of these
species occur in aquifers in ferruginous formations (Halse et al. 2014). There are
about 50 described troglobitic species from ferriferous formations, although almost
700 ferriferous troglobites and troglophiles have been collected from these forma-
tions and about 1500 species are estimated to occur (see also Chap. 20).
The distance from coastal regions may be an important factor determining the
differences in the number of stygobiotic species between Brazil and Australia. The
Hamersley Range and sections of the Robe Valley, which together probably embrace
the highest concentrations of stygobites in Australian ferruginous areas (Halse et al.
2014), are located within 50 km of the coast at their western extent. Thus, it is
plausible to consider a previous connection between the aquifers present in these
areas and the ocean that may have favored the migration and evolution of ancestral
species, which currently are represented by many endemic stygobites found therein.
By contrast, in Brazil, the Iron Quadrangle and Carajás regions are at least 270 km
and 1000 km, respectively, from the coast, which may preclude the occurrence of
species derived from oceanic ancestors. However, it is important to highlight that the
lack of studies regarding the fauna of Brazilian ferruginous aquifers unfortunately
prevents any consistent comparison regarding evolutionary processes that have led
to differences between these two different ferruginous systems.
21.5 Ecology
The relation between the size of a cave and its invertebrate richness is well known
(Souza-Silva et al. 2011; Ferreira et al. 2015; Simões et al. 2015; Jaffé et al. 2016).
Large caves tend to be more heterogeneous, which probably results in an increase of
microhabitats, and consequently more species may establish themselves in these
conditions. Souza-Silva et al. (2011) demonstrated that the relationship between
species richness and cave size tends to be more pronounced in ferruginous caves
than in other lithologies. Probably the larger ferruginous caves accumulate more
organic resources, besides the fact that they potentially can connect to more voids
(due to the increase of the subterranean volume). Such conditions make the macro-
caves “attractors” of epigean and interstitial fauna, and this attraction may occur
exponentially, with a small increase in linear development leading to a large increase
in connections and substantially increasing species richness above what is found in
other lithologies where such voids do not occur (Souza-Silva et al. 2011; Ferreira
et al. 2015).
This cave size/species richness relationship was also demonstrated by Ferreira
et al. (2015) in a set of 240 caves sampled in the region of Carajás. Thus, the size of
ferruginous caves is important not only for the maintenance of high species richness,
but also because it allows the evolution and coexistence of more troglobitic species.
Ferreira et al. (2015) also demonstrated that total species richness and richness of
troglobitic species are related. This suggests that some of the conditions favoring the
occurrence and evolution of troglobites in the subterranean environment also favor
the settlement and current occurrence of other non-troglobitic species. When a cave
rich in troglobitic species is protected, a significant number of non-troglobitic
species associated with these systems are also preserved.
Ferreira et al. (2015) determined the turnover of species through time for the same
previously mentioned set of caves in relation to the dry and rainy seasons in the
region. The temporal turnover was calculated from data of presence/absence of
species through the index of Harrison et al. (1992), modified from Whittaker
(1960). In general, caves sampled in the same plateau in Carajás had high turnover
values, which suggest there tends to a considerable replacement of species between
the dry and rainy seasons. For the analyzed set, 89.8% of caves had turnover values
higher than 60%, 56.1% had values higher to 70%, and 19.4% had values higher
than 80% (Ferreira et al. 2015). Ferreira et al. (2015) also conducted a multiple
regression between the species turnover and the size and number of entrances of the
caves. The turnover was negatively related to size of the cave and positively related
to the number of entrances. Thus, this species replacement is higher in caves with
many entrances, but lower for larger caves. This model corroborates what was
expected, since a large amount of entrances increases the contact with the external
environment, thus increasing the chances of colonization of the cave by accidental or
transient species.
Thus, caves present in the Carajás region tend to have highly variable species
composition through time, especially small caves which are continuously accessed
by several species from the epigean environment. Finally, it is important to highlight
the negative relationship observed between the number of troglobitic species and the
turnover (Ferreira et al. 2015). Caves with more troglobitic species are those with
lower turnover values, which tend to be considered more ecologically stable.
21.6 Threats
Ferruginous regions present in Brazil and Australia comprise some of the main areas
of mineral exploration in the world. Mining extraction removes the ferruginous crust
(canga formation) that covers the mineral reserves, thus modifying the landscape and
having a potentially huge impact on the local and regional biodiversity (Jacobi and
Carmo 2008). According to Gibson et al. (2015), the Australian ferruginous land-
scapes are characterized by the complexity of fauna and flora, with endemic taxa
including cryptic and subterranean species with restricted geographical distributions.
The loss of habitat in these areas as a result of mining may lead to irreversible
changes in biological communities (see also Chap. 22). In Brazilian ferruginous
geosystems, the level of endemism and complexity are similar; however, the sce-
nario is still more alarming, since most of the ferruginous macro-caves occur in the
shallowest part of the deposits (Auler and Piló 2005). Due to the depth of mine
excavations, sometimes below groundwater, the contamination of groundwater and
watercourses by mineral wastes, heavy metals, and toxic elements also must be
considered a potentially important impact (Veado et al. 2006).
In November 2015, a huge waste dam associated with an iron ore mine collapsed
in central Brazil, causing what has being considered one of the biggest environmen-
tal disasters ever registered in the country (Yoemans and Bowater 2016; Porto 2016).
The impacts include social problems (an entire village was buried by the waste) as
well as environmental problems (a basin of an important Brazilian river—Doce
River—suffered silting, heavy metals contamination and fish mortality, as well as
other impacts). Although the media and authorities highlighted different impacts, all
of them were visible impacts occurring in epigean habitats. To date there have been
no studies examining the potentially serious impacts of the accident on ferruginous
subterranean environments, which were certainly affected.
In addition to mining, the urban expansion and other anthropogenic activities also
pose important threats to the subterranean biodiversity in ferruginous landscapes. In
Brazil, the main ferruginous areas are located close to large metropolitan centers or
areas of that receive a large number of migrant workers and their families (Ferreira
et al. 2015). In the Carajás region, many of the impacts documented for the Amazon
region are present, like livestock, agriculture, cutting, and fire associated with the
timber harvesting (Veríssimo et al. 1996; Arima et al. 2005; Ferreira et al. 2005). For
Australia there are alterations caused by the introduction of feral animals and exotic
plants (McKenzie et al. 2006). Besides direct anthropic impacts, the fire may be
considered a natural disturbance in these areas, enhanced by the human occupation
that increases the frequency of focus of forest fires (Ferreira et al. 2015; Gibson et al.
2015).
The geoferruginous system in Brazil is characterized by the expressive hydric
potential, which is essential for the preservation of the fauna with many endemic
stygobite species, and also for the public supply, since these areas may be located in
arid regions and/or next to large urban centers. The uncontrolled use of water from
these systems may alter the balance between the recharge of the aquifer and the
residence time of subterranean water, thus precluding the hydric recovery of the
system (Gama and Matias 2015). Such condition leads to alterations in the hydric
balance with the reduction of water levels and decrease of free flow, thus impacting
the whole associated ecosystem. According to Mourão (2007), the time needed for
the renewal of an aquifer of Iron Quadrangle (Brazil) may be superior to 500 years,
demonstrating that the time of water permanence in these ferruginous systems is
high. Such situation demonstrates the stability of these environments, what may be
essential for the development of the stygobites, which have been increasingly
sampled in ferriferous regions.
Finally, despite the growing number of publications on the biology of caves in
iron ore, the knowledge about them is still in its very early stages. It is still important
to study the fauna present in ferruginous caves in order to conserve them. Future
research will provide more information about the environment factors controlling
species occurrence, the processes structuring the communities, and the distribution
pattern of troglobitic species found in these caves.
Acknowledgements We are grateful to Ana Clara Moreira Viana for producing the figure
regarding the schematic profile of the ferruginous habitats. We are also thankful to the whole
team of the Center of Studies on Subterranean Biology from the Federal University of Lavras for
their assistance in several field works in ferrugineous caves. We are finally grateful to all institutions
that supported distinct research projects regarding ferrugineous cave fauna, providing funds,
scholarships, and infrastructure (CNPq—Conselho Nacional de Desenvolvimento Cientifico e
Tecnológico; FAPEMIG—Fundação de Amparo à Pesquisa do Estado de Minas Gerais;
CAPES—Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; CECAV—Centro
Nacional de Pesquisa e Conservação de Cavernas; UFLA—Universidade Federal de Lavras and
VALE company).
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Part V
Conservation and Protection of Cave
Habitats and Cave Fauna
Chapter 22
Conservation of Cave Fauna,
with an Emphasis on Europe
and the Americas
Matthew L. Niemiller, Steven J. Taylor, and Maria Elina Bichuette
22.1 Introduction
There has been an increasing awareness and concern for subterranean biodiversity
over the past two decades (Culver et al. 2000; Danielopol et al. 2000; Elliott 2000;
Gibert and Deharveng 2002; Culver and Pipan 2009, 2014; Gibert and Culver 2009).
Although the subterranean environment traditionally has been considered species
poor, an exceptionally species-rich and phylogenetically diverse community of
organisms exists in caves, groundwater, and other subterranean habitats of Europe,
North America, Central America, and South America (Culver et al. 2000; Ferreira
et al. 2007; Trajano and Bichuette 2009; Cordeiro et al. 2014; Gallão and Bichuette
2015). Subterranean biodiversity is particularly diverse in Europe where some 5000
troglobionts have been described (Gibert and Culver 2009; Deharveng et al. 2012).
Most local hotspots of subterranean biodiversity globally, defined by Culver and
Sket (2000) as caves or wells with 20 or more troglobionts, are known from southern
Europe (Culver and Sket 2000; Culver and Pipan 2009). Five of the six caves with
40 or more species occur in Europe, including Postojna-Planina Cave System in
Slovenia and Vjetrenica in Bosnia-Herzegovina, which are the two most biodiverse
caves in the world with almost 100 species (Culver and Pipan 2009). The sixth cave
M. L. Niemiller (*)
Department of Biological Sciences, The University of Alabama in Huntsville, Huntsville, AL,
USA
S. J. Taylor
Office of General Studies, Colorado College, Colorado Springs, CO, USA
M. E. Bichuette
Departamento de Ecologia e Biologia Evolutiva, Universidade Federal de São Carlos,
São Carlos, Sao Paulo, Brazil

https://doi.org/10.1007/978-3-319-98852-8_22
452 M. L. Niemiller et al.
is the Mammoth Cave System in Kentucky, USA. For South America, specifically
Brazil, there are at least three cave systems with high diversity of troglobionts
(Deharveng and Bedos 2012). More recently, four cave systems have been identified
as areas of high diversity of troglobionts in Brazil: Areias Cave System (26+
troglobionts), Alambari Cave System (16 species), Parede Vermelha Cave (12+
species), and Olhos d’Água Cave (11+ species) (Trajano et al. 2016).
Several life history traits common to many troglobionts, and to some extent, cave-
roosting bats, are associated with increased risk of extinction, including low repro-
ductive rates and limited dispersal ability (Culver and Pipan 2009, 2014). Thus,
population rescue is often much slower and risk of extinction much greater relative
to populations of related surface species. Moreover, many troglobionts may be
particularly sensitive to small fluctuations in abiotic variables such as temperature,
humidity, dissolved oxygen, and concentrations of heavy metals, among others.
Obligate cave/subterranean fauna, as well as many facultative cave/subterranean
species (such as bats), rely heavily on subterranean habitats and thus are highly
vulnerable to threats that result in environmental change and habitat disturbance and
degradation. These threats vary with respect to scope, source, severity, and timing
among species, karst regions, and continents. Some threats, such as climate change
and groundwater pollution, are global in scope (Culver and Pipan 2009). However,
effects of climate change and sources of groundwater pollution vary at regional and
local scales. Other threats affect cave/subterranean populations and species at a local
or regional scale, such as mining and quarrying, impoundments, groundwater
extraction, commercialization of caves, and amateur and scientific collection. Impor-
tant threats to subterranean communities in caves, groundwater, and other subterra-
nean habitats in Europe, North America, Central America, and South America have
been reviewed and discussed in Juberthie (1995), Elliott (2000), Tercafs (2001),
Culver and Pipan (2009, 2014), Reboleira et al. (2011), Trajano (2010), Simoes et al.
(2014), and Gallão and Bichuette (2015, 2018), among others, and we direct those
interested in specific threats to subterranean biodiversity in general to these publi-
cations (Fig. 22.1).
Box 22.1
Several biological factors common to many troglobionts are associated with
increased vulnerability to threats and risk of extinction (reviewed in Culver
and Pipan 2009, 2014). First, most subterranean species are geographically
rare, often having small, highly restricted geographic ranges (Culver et al.
2006; Zagmajster et al. 2008; Deharveng et al. 2009; Gallão and Bichuette
2018). These animals often are endemic to a single or few cave systems
(Christman et al. 2005; Deharveng et al. 2009; Niemiller and Zigler 2013).
Many species also may be numerically rare. Several troglobionts are known
from only one or a few specimens (e.g., Niemiller et al. 2017). However, it is
often unclear whether such species are actually comprised of very small
(continued)
22 Conservation of Cave Fauna, with an Emphasis on Europe and the Americas 453
Fig. 22.1 Locations of caves, systems, features, and areas mentioned specifically in this chapter.
1 Caves of the Demänová valley; 2 Postojna-Planina Cave System; 3 Skocjanske Jame; 4 Dinaric
Karst; 5 Wind Cave; 6 Vjetrenica; 7 Cave Creek; 8 Mammoth Cave; 9 Buffalo National River; 10
Hubbard’s Cave; 11 Carlsbad Caverns; 12 Anillo de Cenotes; 13 Chiquibul Cave; 14 Parede
Vermelha Cave; 15 Olhos d’Água Cave; 16 Alambari Cave System; 17 Areias Cave System

populations (i.e., observed rarity reflects actual rarity) or whether rarity reflects
undersampling or sampling of suboptimal habitat. For example, some species
may be common in epikarst but are very infrequently observed in caves.
Herein, we identify and review several important international and national

legislation, policies, and conservation initiatives that have been implemented or
proposed related to the protection and conservation of cave/subterranean
biodiversity.
Box 22.2
Despite these biological factors and many documented and suspected threats,
only a small fraction of subterranean biodiversity receives any direct protec-
tion under international, national, or regional legislation. Compared to verte-
brates, subterranean invertebrates and other organisms have largely been
neglected in conservation studies, assessments, and policy decisions, despite
their significantly greater diversity, roles in groundwater and subterranean
ecosystem services, sometimes high levels of endemicity, and benefits to
mankind.
22.2 International Legislation
Four major international conventions that focus on biodiversity issues are relevant to
the protection and conservation of subterranean biodiversity and ecosystems: Con-
vention on Biological Diversity (CBD), Convention on International Trade in
Endangered Species of Wild Fauna and Flora (CITES), Ramsar Convention on
Wetlands of International Importance, and World Heritage Convention (WHC).
Each of these biodiversity-related conventions aims to implement conservation
actions at the international, national, and regional levels. However, few subterranean
species are currently protected under the auspices of these conventions.
The Convention on Biological Diversity (CBD) is an international treaty among
196 countries today that entered into force in 1993 to develop strategies for the
conservation and sustainable use of biodiversity. All countries in Europe, North
America, Central America, and South America are member parties, except for the
United States. CBD has three main goals: conservation of biodiversity, sustainable
use of its components, and fair and equitable sharing of benefits arising from genetic
resources. These objectives are of particular importance for developing countries.
The convention requires countries to prepare a national biodiversity strategy, called
National Biodiversity Strategies and Action Plans (NBSAPs), and to ensure that
NBSAPs are implemented into all relevant planning and activities that may have a
positive or negative impact on biodiversity. Of the 196 member parties, 94% have
developed NBSAPs. Cave and groundwater biodiversity and ecosystems are specif-
ically addressed in several countries’ NBSAPs. For example, the Slovenian NBSAPs
has a specific objective on cave habitat types “to maintain subterranean habitat types
in ecologically important areas, and the entire subterranean fauna, at favorable
conservation status.” However, the focus on subterranean fauna and ecosystems in
NBSAPs appears to be highly variable among countries.
The Convention on International Trade in Endangered Species of Wild Fauna
and Flora (CITES) is an international agreement signed in 1973 and entered into
force in 1975 between governments to ensure that international trade of wild animals
and plants of conservation concern does not further threaten their continued survival.
Today, 181 countries, including most countries in Europe, North America, Central
America, and South America (Table 22.1), are member parties. Some 5600 species
of animals are protected by CITES. However, only about 40% of the species listed
are invertebrates. These species are listed in one of the three CITES Appendices
according to how threatened they are by international trade. No troglobionts from
Europe, North America, Central America, or South America are listed; however,
some non-troglobiotic taxa that rely on caves are included (e.g., cave-roosting birds,
Steatornis caripensis).
The Convention on Wetlands of International Importance (Ramsar Convention)
is the oldest global international environmental agreement adopted in 1971 and
entered into force in 1975. Its mission is the conservation and wise use of all
wetlands through local and national actions and international cooperation. Some
169 countries are contracting parties to the convention, including most countries in
Table 22.1 Important international and national legislation relevant to the conservation and protection of subterranean biodiversity and ecosystems in countries
22
of Europe, North America, Central America, and South America

Country Bern Bonn CBD CITES RC WHC Legislation
Europe
Albania 1999 2001* 1994 2003 1996 1989 Law on Biodiversity Protection (2006)
Law on Protected Areas (2002, 2008)
Law on Wild Fauna Protection (2008)
Andorra 2001 2015 2012 1997 Law of Protected Species (2001)
Austria 1983 2005 1994 1982 1983 1992 Flurverfassungsgrundsatzgesetz (1951)
Belarus 2003 1993 1995 1991 1988 Law on Protection of the Environment (2002)
Law on the Animal World (2007)
Belgium 1990 2003* 1997 1983 1986 1996 Regional policy
Bosnia and Herzegovina 2009 2002 2009 1992 1993 Law on Environmental Protection (2013)
Bulgaria 1991 1999* 1996 1991 1976 1974 Nature Protection Act (1967, 1991)
Biological Diversity Act (2002, 2011)
Croatia 2000 2000* 1997 2000 1991 1992 Cave Protection Act (1900)
Nature Protection Act (2005)
Regulation on Protection of Wild Species (2006)
Cyprus 1988 2001* 1996 1974 2001 1975 Law on the Protection and Management of Nature and Wildlife (2003)
Czech Republic 1998 1994* 1994 1993 1993 1993 Act on the Protection of Nature and Landscape (1992, 2004)
Denmark 1983 1983* 1994 1977 1978 1979 Nature Conservation Act (2009, 2013)
Estonia 1992 2008* 1994 1992 1994 1995 Animal Protection Act (2000)
Nature Conservation Act (2004)
European Union 1982 1983 1994 2015
Conservation of Cave Fauna, with an Emphasis on Europe and the Americas
Finland 1986 1989* 1994 1976 1975 1987 Nature Conservation Act (1923, 1996)
Nature Conservation (Amendment) Act (1991)
France 1990 1990* 1994 1978 1986 1975 Environment Code (2010)
Germany 1985 1984* 1994 1976 1976 1976 Federal Nature Conservation Act (2010)
Greece 1983 1999 1994 1992 1975 1981 Biodiversity Law (2011)
455
(continued)
456

Hungary 1990 1983* 1994 1985 1979 1985 Act on Nature Conservation (1996)
Iceland 1993 1994 2000 1978 1995 Nature Conservation Act (1999)
Ireland 1982 1983* 1996 2002 1985 1991 Wildlife Act (1976)
Wildlife (Amendment) Act (2000)
Italy 1982 1983* 1994 1979 1977 1978 Law on Protected Areas (1991)
Latvia 1997 1999* 1996 1997 1995 1995 Law on the Conservation of Species and Biotypes (2000, 2005)
Law on Specially Protected Nature Territories (1993, 2007)
Law on Subterranean Depths (1996)
Liechtenstein 1982 1997 1998 1979 1991 Law on the Protection of Nature and Landscape (2004)
Lithuania 1997 2002* 1996 2001 1993 1992 Law on the Protected Fauna, Flora and Fungi Species and Commu-
nities (1997)
Law on Protected Areas (2001)
Underground Law (1995)
Luxembourg 1982 1983* 1994 1983 1998 1983 Law on Nature Protection and Natural Resources (2004)
Malta 1994 2001* 2001 1989 1989 1978 Flora, Fauna, and Natural Habitats Protection Regulations (2006,
[amended] 2013)
Moldova 1994 2001* 1996 2001 2000 2002 Law on the Animal Kingdom (1995)
Montenegro 2010 2009* 2006 2007 2006 2006 Law on Nature Protection (1977, 1989)
Netherlands 1982 1983* 1994 1984 1980 1992 Nature Conservation Act (1998)
Flora and Fauna Act (2002)
Norway 1986 1985 1993 1976 1975 1977 Nature Diversity Act (2009)
Poland 1996 1996* 1996 1989 1978 1976 Nature Conservation Act (2004)
Portugal 1982 1983* 1994 1980 1981 1980 Nature Conservation Act (2008)
Romania 1993 1998* 1994 1994 1991 1990 Law no. 49 (2011) for the approval of Government Emergency Ordi-
nance no. 57 on the regime of natural protected areas, conservation of
natural habitats, wild flora and fauna (2007)
Serbia 2008 2008 2002 2006 1992 2001 Law on Nature Conservation (2010)
M. L. Niemiller et al.
22
Slovakia 1997 1995* 1994 1993 1993 1993 Act on Nature and Landscape Protection (2002)
Slovenia 2000 1999* 1996 2000 1991 1992 Nature Conservation Act (1999)
Cave Protection Act (2004)
Spain 1986 1985 1994 1986 1982 1982 Law on Natural Heritage and Biodiversity (2007)
Sweden 1983 1983 1994 1974 1975 1985 Environmental Code (1998)
Switzerland 1982 1995* 1995 1974 1976 1975 Federal Act on the Protection of Nature and Cultural Heritage (1966)
The Former Yugoslav Republic 1999 1999* 1998 2000 1991 1997 Law on Nature Protection (2004)
of Macedonia
Ukraine 1999 1999* 1995 1999 1991 1988 Law on Animals (2001)
United Kingdom 1982 1985* 1994 1976 1976 1984 Wildlife and Countryside Act (1981)
Countryside Rights of Access Act (2000)
Nature Conservation (Scotland) Act (2004)
Protected Species Act (2003)—Bermuda
North and Central America
Antigua and Barbuda 2007 1993 1997 2005 1983 Environmental Protection and Management Act (2014)
Bahamas 1993 1979 1997 2014 Forestry Act (2010)
Barbados 1994 1992 2006 2002
Belize 1994 1986 1998 1990 Wildlife Protection Act (1981)
Canada 1993 1975 1981 1976 Species at Risk Act (2002)
Costa Rica 2007 1994 1975 1992 1977 Biodiversity Law (1997)
Law on Wildlife Conservation (2012)
Cuba 2008 1994 1990 2001 1981 Wild Animals Protection Act (1968)
Dominica 1994 1995 1995 Forestry and Wildlife Act (1976)
Dominican Republic 1997 1986 2002 1985 General Law on Environmental and Natural Resources (2000)
El Salvador 1994 1987 1999 1991 Wildlife Conservation Law (1994)

Grenada 1994 1999 2012 1998 Birds and Other Wildlife Protection Act (1957)
Guatemala 1995 1979 1990 1979 Forestry Law (1996)
Haiti 1996 1980
457
(continued)
458

Honduras 2007 1995 1985 1993 1979 Forest, Protected Areas, and Wildlife Law (2007)
Jamaica 1995 1997 1998 1983 Endangered Species Act (2000)
Wildlife Protection Act (1945)
Mexico 1993 1991 1986 1984 General Wildlife Act (2000)
Nicaragua 1996 1977 1997 1979 Law on Use and Conservation of Biodiversity (2012)
Panama 1989 1995 1978 1990 1978 General Environmental Law (1998)
Wildlife Law (1995)
Saint Kitts and Nevis 1993 1994 1986 National Conservation and Environment Protection Act (1987)
Saint Lucia 1993 1982 2002 1991 Wildlife Protection Act (1980)
Saint Vincent and the Grenadines 1996 1988 2003 Wildlife Protection Act (1987)
United States 1974 1986 1973 Endangered Species Act (1973)
South America
Argentina 1992 1995 1981 1992 1978 Law on Wildlife Conservation (1997)
Bolivia 2003 1995 1979 1990 1976 Law on the Rights of Mother Earth (2010)
Supreme Decree 22641 (1990)
Brazil 2015 1994 1975 1993 1977 Environmental Crimes Law (1999)
Chile 1983 1994 1975 1981 1980 Environmental Law (1994)
Hunting Law (1996)
Columbia 1995 1981 1998 1983 General Environmental Law (1993)
Ecuador 2004 1993 1975 1991 1975 Forestry and Conservation of Natural Areas and Wildlife Law (1981)
Biodiversity Law (2004)
Guyana 1994 1977 1977 Environmental Protection Act (1996)
Paraguay 1999 1994 1976 1995 1988 Law of Wild Life (1992)
Peru 1997 1993 1975 1992 1982 Forest and Wildlife Act (1975)
Biological Diversity Conservation and Sustainable Use Act (1997)
Suriname 1996 1980 1985 1997 Nature Conservation Law (1954)
M. L. Niemiller et al.
22
Trinidad and Tobago 1996 1984 1993 2005 Conservation of Wildlife Act (1958, 1980)
Uruguay 1990 1994 1975 1984 1989
Venezuela 1994 1977 1988 1990 Protection of Wildlife Act (1970)
Biological Diversity Act (2000)
International legislation includes several important conventions: the Bern Convention (Bern), Bonn Convention (Bonn), Convention on Biological Diversity
(CBD), Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), Ramsar Convention (RC), and World Heritage Convention
(WHC). EUROBATS member parties are denoted by an asterisk (*) under the Bonn Convention column. Entry dates for each country are presented
459
Europe, North America, Central America, and South America. At the time of joining
the convention, each contracting party must designate at least one wetland site for
inclusion in the List of Wetlands of International Importance. Ramsar Sites are
designated based on nine criteria, eight of which are related to biodiversity. When
a wetland is officially included in the list, it is recognized as being of significant
value for humanity as a whole. Each contracting party must take the necessary
actions to ensure that the ecological character of the Ramsar Site is preserved.
Many Ramsar Sites are also protected under other international and national protec-
tion conventions and schemes, such as the World Heritage List under the UNESCO
World Heritage Convention. Karst and other subterranean hydrological systems are
broadly defined as wetlands, including marine, inland freshwater, and man-made
types. Thirty-seven Ramsar Sites are inland karst wetlands that cover over
850,000 ha and occur in Europe, including notable cave systems such as Skocjanske
Jame in Slovenia and Caves of the Demänová valley in Slovakia. In North and
Central America, 44 karst and subterranean wetland Ramsar Sites occur that cover
over 3.4 million ha, predominantly in Mexico, including the Anillo de Cenotes in the
Yucatan. Only two Ramsar Sites in Chile are karst or subterranean wetlands in South
America.
The Convention Concerning the Protection of the World Cultural and Natural
Heritage or the World Heritage Convention (WHC) was adopted by the United
Nations Educational, Scientific and Cultural Organization (UNESCO) General Con-
ference in 1972 and came into force in 1975. WHC aims to promote cooperation
among nations to protect cultural and natural heritage globally that is of outstanding
universal value to humanity. Today, 191 parties have agreed to identify, protect, and
conserve World Heritage Sites. A site has outstanding universal value, as defined by
the Operational Guidelines for the Implementation of the World Heritage Conven-
tion if it has cultural and/or natural significance that is so exceptional that it
transcends national boundaries and is of importance to current and future generations
of all humanity. Nomination and inclusion in the World Heritage List represents a
formal pledge by the host country to take steps necessary to protect a site, which
includes the appropriate legal, scientific, administrative, and financial measures for
the identification, protection, conservation, preservation, and rehabilitation of sites
with outstanding universal value. Listing as a World Heritage Site is often accom-
panied by increased tourism at some sites and prioritization for funding and technical
assistance, but also controversial debates regarding how to protect some threatened
sites.
Several sites included in the UNESCO World Heritage List contain significant
cave and karst systems (Table 22.2). Of these sites, seven are specifically recognized
for their outstanding biodiversity value under the biodiversity criteria (ix) and/or (x):
criterion (ix) “to be outstanding examples representing significant on-going eco-
logical and biological processes in the evolution and development of terrestrial,
freshwater, coastal, and marine ecosystems and communities of plants and animals”,
and criterion (x) “to contain the most important and significant natural habitats for
in-situ conservation of biological diversity, including those containing threatened
species of outstanding universal value from the point of view of science and
Table 22.2 List of UNESCO World Heritage Sites and Tentative List Sites (T) within cave and
karst terrains in Europe, North America, Central America, and South America
Year Natural
Continent Country World heritage site inscribed criteria
Europe Bulgaria Pirin National Park 1983 vii, viii
Bulgaria Vratsa Karst Nature Reserve T vii, viii,
ix, x
Croatia Kornati National Park and Telascica Nature T vii, viii,
Park x
Croatia Plitvice Lakes National Park* 1979, vii, viii,
2000 ix
Croatia Velebit Mountain T vii, viii,
ix, x
France Ensemble de grottes à concretions du Sud de T vii, viii,
la France ix
France/ Pyrenees-Mont Perdu* 1997, vii, viii
Spain 1999
Greece Meteora 1988 vii
Hungary Hydrothermal Caves and Thermal Karst Sys- T viii
tems of the Rozsadomb Area
Hungary/ Caves of Aggtelek and Slovak Karst* 1995, viii
Slovakia 2000
Italy The Murge of Altamura T vii, viii
Montenegro Durmitor National Park 1980, vii, viii,
2005 x
Norway Svalbard Archipelago T vii, viii,
ix, x
Serbia The Tara National Park with the Drina River T x
Canyon
Slovenia Skocjan Caves* 1986 vii, viii
North Canada Canadian Rocky Mountain Parks* 1984 vii, viii
America Canada Nahanni National Park* 1978 vii, viii
Cuba Alejandro de Humboldt National Park 2001 ix, x
Cuba Desembarco del Granma National Park* 1999 vii, viii
Mexico Reserve de la Biosphere Selva El Ocote T n
Mexico Sian Ka’an 1987 vii, x
United Carlsbad Caverns National Park* 1995 vii, viii
States
United Grand Canyon National Park 1979 vii, viii,
States ix, x
United Mammoth Cave National Park* 1981 vii, viii,
States x
South Brazil Canyon du Rio Peruaçu, Minas Gerais T vii, viii,
America ix, x
Venezuela Canaima National Park* 1994 vii, viii,
ix, x
Natural criteria for each site are listed and sites with outstanding universal value are denoted with an
asterisk (*)
conservation.” There are several additional areas not listed in Table 22.2 that have
cave and karst of national rather international significance or have cultural rather
than natural value (Williams 2008). Although caves and karst are well represented in
World Heritage sites in humid tropical and temperate regions, particularly in the
Northern Hemisphere, representation of caves and karst in World Heritage sites is
deficient in several regions (Williams 2008), particularly the arid to semiarid zone of
the tropics to subtropics, the periglacial zone, and in the Southern Hemisphere,
including South America.
22.3 Conservation Organizations
Several organizations are important partners and leaders for conservation world-
wide, working to protect and conserve biodiversity, including subterranean fauna.
These organizations, highlighted by the International Union for the Conservation of
Nature and Natural Resources and NatureServe, work at global, national, and
regional scales to assist government agencies and other conservation organizations
on many biodiversity conservation issues, setting priorities for funding, research,
and conservation efforts and developing sound conservation policies, legislation,
and practices.
The International Union for the Conservation of Nature and Natural Resources
(IUCN) was founded in 1948 and is the world’s oldest international environmental
and conservation organization, which today includes 200+ government and 900+
nongovernment organizations as members. IUCN assists governments and other
conservation organizations with national and international biodiversity policies and
initiatives. The IUCN Global Species Programme, in conjunction with the IUCN
Species Survival Commission (SSC), produces, maintains, and manages the IUCN
Red List of Threatened Species. The IUCN SSC is a group of more than 10,000
volunteer experts that provide valuable information and advice on biodiversity to
IUCN. Most experts belong to the more than 140 Specialist Groups, Red List
Authorities, Task Forces, and Subcommittees. Although most Specialist Groups
focus on particular taxa, some groups are more ecosystem focused, including the
Cave Invertebrate Specialist Group. Information on the life history, ecology, status,
trends, and threats of species provided by these experts is used in the IUCN Red List,
which is a list that highlights species that are at greatest risk of extinction and greatest
need of conservation. The IUCN Red List is widely recognized as the most com-
prehensive, objective global approach for evaluating the conservation status of flora
and fauna (Lamoreaux et al. 2003). The IUCN Red List is used to help raise
awareness about threatened species, set priorities for and guide conservation efforts
and funding, and influence environmental policies and legislation (Rodrigues et al.
2006; Baillie et al. 2008; Cardoso et al. 2011a, b). A species may be classified on the
IUCN Red List as critically endangered (CR), endangered (EN), or vulnerable
(VU) on a global scale if it meets specific conditions under any one of these five
criteria (IUCN 2001): (A) past, present, or projected reduction in population size
Fig. 22.2 Percentage of species assessed under IUCN Red List criteria (a), troglobiont diversity for
various groups of invertebrate taxa (b), and percentages of troglobionts listed under the United
States Endangered Species Act as endangered or threatened (c)
over three generations; (B) small geographic range in combination with fragmenta-
tion, population decline, or fluctuations; (C) small population size in combination
with decline or fluctuations; (D) very small population or very restricted distribution;
and (E) a quantitative analysis of extinction risk. Species should be assessed against
all criteria, when possible, to confirm that the most accurate threat classification is
obtained (IUCN 2001).
Although the IUCN Red List has been widely accepted and has many strengths
(Rodrigues et al. 2006), several biases and limitations have been noted (Cardoso
et al. 2011a). Taxa that have been evaluated are biased toward those occurring in
terrestrial ecosystems and those which are vertebrates (IUCN 2010). Most mammals,
birds, and amphibians have been evaluated, yet only 0.5% of described arthropods
have been assessed (Cardoso et al. 2011a, b). Subterranean fauna of Europe (outside
of Croatia and Slovenia), North America, Central America, and South America are
poorly represented in the IUCN Red List, with only 10% of subterranean species
having been evaluated. Of the subterranean species evaluated, taxonomic compo-
sition is biased toward fishes, salamanders, and decapods, where >75% of taxa
within these groups have been evaluated (Fig. 22.2a). In contrast, just 14% of
arachnids and hexapods, which collectively account for 48% of subterranean bio-
diversity overall in the United States, have been evaluated.
Cardoso et al. (2011a) cited two primary reasons for the taxonomic bias against
invertebrates on the IUCN Red List. First, even if most necessary biological data
could be gathered for Red List criteria, thresholds suggest that they were defined
based on the requirements of large vertebrate species and, thus, may not be appro-
priate for most invertebrates, Second, current Red List criteria are difficult to apply to
invertebrates because of four main shortfalls (Cardoso et al. 2011b): (1) most species
are undescribed (the Linnean shortfall); (2) the full distributions of most species are
unknown (the Wallacean shortfall); (3) the abundances of most species and how they
change spatially and temporally are unknown (the Prestonian shortfall); and (4) the
ecology of species and sensitivity to habitat changes are largely unknown (the
Hutchinsonian shortfall). These shortfalls are the consequence of limited research
on—and funding for—invertebrates, especially related to taxonomy and life history
(Cardoso et al. 2011b). Consequently, it is difficult to gather the necessary informa-
tion to classify most subterranean invertebrate species under the IUCN Red List
criteria. These same shortfalls apply not only globally, but also to most subterranean
species in most karst regions of Europe, North America, Central America, and South
America. To address these issues, recommendations have been suggested to modify
current IUCN Red List criteria to increase the feasibility and adequacy of the
assessment process, in hopes of achieving greater representation of invertebrates
on priority species lists (Martin et al. 2010; Cardoso et al. 2011a).
IUCN Red List Categories and Criteria also are applied at smaller spatial
scales—including regions and countries—which are more practical for management
and conservation planning. To date, Regional and National Red Lists have been
developed in 26 regions, 113 countries, and 45 subnational entities. A database of
these Regional and National Red Lists is maintained by the IUCN National Red List
project (http://www.nationalredlist.org). As is the case for the global IUCN Red
List, few subterranean species are included in Regional and National Red Lists. An
exception is the subterranean fauna of Croatia, which includes over 450 cave
obligate species from caves in the richest cave biodiversity region in the world,
the Dinaric Karst (Culver and Sket 2000; Gottstein-Matocec 2002; Ozimec 2011).
The Croatian Red Book of Cave Fauna is the first Red List assessment of
troglobionts and stygobionts of its kind in the world, covering 186 taxa spanning
16 classes, 29 orders, and 54 families. Thirty-five percent (60 taxa) of the taxa
included were assessed as Critically Endangered. Several specific threats to Croatian
cave fauna have been identified, such as threats associated with urbanization and
heavy tourism, groundwater pollution and exploitation, and illegal collection
(Ozimec 2011).
Brazil has 158 described troglobionts distributed in 13 states, with the greatest
diversity in Bahia (Serra do Ramalho karst area and São Desidério region—part of
Bambuí group, Una-Irecê and Rio Pardo groups, Canudos supergroup, and sand-
stone Chapada Diamantina) and São Paulo (part of Açungui group) (Gallão and
Bichuette 2018). Only 33 troglobionts were included in the Brazilian Red List of
2004 and another 30 species known as of 2003 were not evaluated. From 2004 to
2014, the number of troglobionts included in the Brazilian Red List increased to
83 species. Many of these species are listed as Endangered or Critically Endangered.
Mining and hydroelectric projects are the main threats to subterranean biodiversity
in Brazil, but other impacts, such as pollution of aquifers by pesticides and defor-
estation for agriculture and pastureland, also represent significant threats (Gallão and
Bichuette 2018).
There is a pressing need to address higher levels of biodiversity, such as habitats,
communities, and ecosystems (Nicholson et al. 2009; Rodriguez et al. 2011, 2012;
Keith et al. 2015). IUCN is currently developing categories and criteria for a Red List
of Ecosystems (RLE) as a global standard for ecosystem risk assessment at multiple
scales (Rodriguez et al. 2011, 2012; Keith et al. 2015). Much like the IUCN Red List
of Threatened Species, RLE has eight categories of risk for each ecosystem based on
five quantitative criteria designed to evaluate symptoms of risk in terrestrial, subter-
ranean, freshwater, and marine ecosystems (Keith et al. 2013). The RLE will
establish an objective, robust, and repeatable international standard for ecosystem
risk assessment and losses of ecosystem functions and services that will allow for
comparisons among regions and time periods. Subterranean ecosystems are being
considered in the development of RLE criteria, which has a target date of 2025 to
achieve global coverage of ecosystems.
NatureServe is a nonprofit organization that provides scientific expertise,
resources, and data for conservation action. The NatureServe Network includes
over 80 Natural Heritage programs and conservation data centers throughout the
Western Hemisphere, including the United States, Canada, Mexico, Brazil, and
several other countries and territories in the Caribbean, Central America, and
South America. NatureServe offers several tools and services for conservation
science and biodiversity assessment, which includes standards and methods for
collecting, managing, mapping, and sharing biodiversity data and conducting assess-
ments of species conservation status, ecosystem ecological integrity, and climate
change vulnerability, among others. NatureServe maintains biodiversity data for
over 77,000 species and 870 ecosystems in the United States and Canada, which
can be accessed through a web-based product, NatureServe Explorer (http://
explorer.natureserve.org). A similar product, NatureServe Infonatura (http://
infonatura.natureserve.org), provides information for some 8700 species of birds,
mammals, and amphibians in the Caribbean, Central America, and South America.
NatureServe also partners with IUCN by providing data and coordination of their
own assessments to IUCN Red List assessments, as well as working together on
conservation assessments.
Of particular usefulness are standards and methods for conservation status rank
assessment to determine relative extinction risk of a species or ecosystem (Faber-
Langendoen et al. 2009; Master et al. 2009). NatureServe conservation status ranks
are based on a one to five scale, from most to least at risk of extinction: G1 (Critically
Imperiled), G2 (Imperiled), G3 (Vulnerable), G4 (Apparently Secure), and G5
(Secure). Two additional ranks associated with extinction exist: GH (Possibly
Extinct) and GX (Presumed Extinct). Status ranks can be assessed at three geo-
graphic scales: global (G), national (N), and state (S). Conservation ranks are based
on ten primary factors grouped into three main categories: rarity, trends, and threats
(Master et al. 2009). Rarity factors include range extent of occurrence (EOO), area of
occupancy (AOO), number of occurrences, number of occurrences with good
viability or ecological integrity, population size, and environmental specificity.
Trend factors include both short-term and long-term trends in population size,
EOO, AOO, number of occurrences, and viability or ecological integrity of occur-
rences. Threat factors include threat impact and intrinsic vulnerability to threats.
The comprehensive and rigorous biodiversity information data maintained and

the tools and services developed by NatureServe have made NatureServe Conser-
vation Status Assessment, like the IUCN Red List, a key tool in conservation from
regional to global scales in the Western Hemisphere. Approximately 12% of
troglobiotic taxa in the United States and Canada remain to be evaluated, which is
a large improvement over taxa that remain to be evaluated under IUCN Red List
criteria. Seventy-four percent of taxa have been assessed at an elevated risk of
extinction (G1 to G3). Two species, Bactrurus cellulanus and Pseudanophthalmus
krekeleri, may have already been lost and are presumed extinct (GX) (Elliott 2000;
Taylor and Niemiller 2016), while ten additional species may possibly be extinct
(GH). This conservation tool, however, is also not without its shortcomings with
respect to subterranean biodiversity. The conservation status of most subterranean
fauna has not been reviewed in 10+ years, and much subterranean biodiversity
remains to be assessed in many regions, including Mexico, the Caribbean, Central
America, and South America. Only mammals, birds, and amphibians have been
assessed to date in these regions. Moreover, biological surveys are likely needed for
many taxa assessed as possibly (GH) or presumed extinct (GX) in the wild, as there
is hope that some of these taxa have been lost already. For example, a recent study by
Niemiller et al. (2017) rediscovered two species of cave carabid beetles,
Pseudanophthalmus insularis and P. paulus, which were classified as Possibly
Extinct (GH), as they had not been observed in 60 and 50 years, respectively.
22.4 National Legislation
Most countries in Europe, North America, Central America, and South America are
member parties to the conventions mentioned at Sect. 22.2, although there are
notable exceptions (Table 22.1). Relatively few countries have enacted national
endangered species legislation, despite worldwide threats and decline in biodiver-
sity. Even when enacted, few subterranean taxa are directly protected under endan-
gered species legislation. Important legislation related to the conservation and
protection of subterranean biodiversity and ecosystems in Europe, North America,
Central America, and South America are listed in Table 22.1. Several of these new
pieces of national legislation are related to specific provisions of international
conventions listed above. Unfortunately, there is considerable variation among
countries in the level of protection enacted for subterranean biodiversity and eco-
systems. Numerous local and regional ordinances and regulations exist in several
countries in Europe, North America, Central America, and South America that may
also protect cave systems and cave and groundwater fauna (Juberthie 1995;
Lamoreaux et al. 1997; Tercafs 2001; Huppert 2006). This includes the establish-
ment of city parks or ordinances to protect groundwater from karst groundwater
pollution.
22.4.1 European Legislation
European bats were protected under the Bonn Convention by the Agreement on the
Conservation of Populations of European Bats (EUROBATS). However, with
notable exceptions, troglobiotic invertebrates receive limited if any specific protec-
tion in most European countries. For example, no subterranean invertebrates are
specifically protected under the Biodiversity Diversity Act in Bulgaria, while in
Slovenia, which possesses one of the most diversity subterranean faunas in the world
(Culver and Sket 2000; Sket and Zagmajster 2004), the subterranean environment in
total, including biodiversity, is protected by the national Cave Protection Act and
other legislation. Many stygobionts and troglobionts are also strictly protected under
the Regulation on Protection of Wild Species in Croatia.
Although some cave systems and associated fauna are locally protected in their
respective countries, in general no national conservation programs directed specifi-
cally for subterranean habitats and diversity have been established in most European
countries. In contrast, subterranean waters—and by proxy their biodiversity—are
universally protected, mainly for reasons associated with public health (e.g., Bel-
gium). Many European cave systems are directly protected by national legislation
and the establishment of protected areas such as national parks, nature reserves, and
Natura 2000 sites (reviewed in Juberthie 1995). In Belgium, several nature reserves
have been established by the Royal Commission of Monuments and Sites to protect
karst environments, including caves, and in Greece, caves are considered natural and
cultural heritage monuments and therefore protected. Similar cave protection legis-
lation exists in other European countries such as Hungary and Italy.
The Council of Europe’s Convention on the Conservation of European Wildlife
and Natural Habitats of 1979, also known as the Bern Convention, was the first
international treaty to protect species and habitats in Europe. To date, 50 European
countries, as well as some African countries, and the European Union have acceded
to the convention, which came into force in 1982. The Convention aims to ensure
conservation and protection of species and habitats by imposing restrictions on the
take and trade of protected flora and fauna and constitutes a commitment to protect
habitats of imperiled species. The Convention imposes legal obligations of
contracting parties for the protection of over 500 plants and over 1000 animal species
and their habitats, listed in Appendices I, II, and III. The only troglobiont listed on
the Bern Convention is Proteus anguinus (Appendix II); however, some
non-troglobiotic vertebrates that rely on caves are listed, such as Atylodes genei,
Speleomantes spp., and most species of European cave-dwelling bats.
The Convention on the Conservation of Migratory Species of Wild Animals, also
called the Bonn Convention or CMS, aims to conserve species that regularly move
across national borders. The Bonn Convention was adopted in 1972 and came into
force in 1985. Some 122 parties, including the European Union, work cooperatively
to conserve endangered migratory species and their habitats. The Agreement on the
Conservation of Populations of European Bats (EUROBATS) under the Bonn
Convention came into force in 1994. This agreement aims to conserve all 53 European
bat species through legislation, education, and other conservation actions with
international cooperation of the 36 parties of EUROBATS.
The European Union Habitats Directive (EUHD) is a primary piece of legislation
adopted in 1992 to protect habitats and species in freshwater, terrestrial, and marine
habitats in Europe as a European Union response to the Bern Convention. Many
provisions of the Ramsar Convention and Bonn Convention also have been incor-
porated in the EUHD. The EUHD contains a series of annexes that identify species
and habitats of conservation concern in the 27 Member States of the European
Union. In addition, the EUHD provides for the creation of a network of Special
Areas of Conservation (SACs), called the Natura 2000 Network. The Natura 2000
Network also includes Species Protected Areas designated under the Birds Directive.
Over 26,000 Natura 2000 sites have been designated across Europe to date, includ-
ing SACs designated for subterranean biodiversity. For example, 55 localities for the
olm (Proteus anguinus) have been protected within 26 SACs in the Slovenian
Natura 2000 Network (Hudoklin 2011).
The EUHD aims to protect some 230 habitats and over 1000 species listed in the
Directive’s five annexes. Annex I contains a list of priority habitat types. In relation
to subterranean biodiversity and ecosystems, the following habitat types are listed:
caves not open to the public (Natura 2000 code 8310), fields of lava and natural
excavations (Natura 2000 code 8320), and submerged or partially submerged sea
caves (Natura 2000 code 8330). Annex II is a list of species whose core areas of
habitats are designated as Sites of Community Importance (SCIs) that ultimately
must be protected under the Natura 2000 Network as SACs. A strict protection
protocol must be implemented for species listed in Annex IV across their entire
distribution both within and outside of Natura 2000 sites. Annex V includes a list of
species whose exploitation and taking in the wild may be subject to management
actions. Some species appear on multiple annex lists.
Very few troglobionts and stygobionts are included in Annexes II, IV, or V but
include the salamander Proteus anguinus (II and IV), the beetle Leptodirus
hochenwartii (II and IV), and the bivalve Congeria kusceri (II and IV). All
European bats species as well as six species of the salamander genus Speleomantes
are listed on Annex II and/or IV. Several biases and criticisms have been noted in the
list of species protected under the EUHD (Cardoso 2012). As in many aspects of
conservation (Clark and May 2002), vertebrates dominate over invertebrate taxa on
the list. Even among the list of arthropod taxa that are included in Annexes II, IV,
and V, 94% of taxa belong to the more “charismatic” insect orders Lepidoptera,
Coleoptera, Odonata, and Orthoptera (Cardoso 2012). Cardoso (2012) also noted
other bias in the listing of arthropod species related to taxonomy, geography, range
size, body size, and aesthetic value. These biases in the species lists have significant
legal and financial implications. Species that are not listed in the Annexes cannot be
used as justification for support for LIFE Nature sub-program conservation projects
(Cardoso 2012). Consequently, non-listed species are neglected in conservation
policy and funding.
The EU Groundwater Directive of 2006 (GWD) establishes a regime that sets
groundwater quality standards and introduces measures to prevent or limit inputs of
pollutants into groundwater, which complement the EU Water Framework Direc-

tive. The GWD emphasizes the importance of protective measures for groundwater
ecosystems by proposing increased monitoring and research to provide better stan-
dards for groundwater quality (Griebler et al. 2010; Stein et al. 2012). This directive
has led to new research on ascertaining and evaluating biological criteria as indica-
tors for groundwater quality assessment (Griebler et al. 2010; Korbel and Hose 2011;
Stein et al. 2010, 2012).
22.4.2 North, Central, and South American Legislation
In North and Central America, the primary legislation for species protection is the
Endangered Species Act of 1973 (ESA) in the United States and similar legislation,
the Species at Risk Act of 2002 (SARA), in Canada. In contrast to the European
Union Habitats Directive (see above), which focuses more on habitats, species are
the primary focus of the ESA and SARA. Similar endangered species legislation has
been passed in Mexico (General Wildlife Act of 2000), Costa Rica (Biodiversity
Law of 1998), and other countries (Table 22.1). In Bermuda, 24 species of cave
invertebrates are protected under the Protected Species Act (2003). As in Europe,
several additional pieces of legislation afford at least indirect protection of cave and
karst landscapes and, in turn, subterranean biodiversity in North America, the
Caribbean, and Central America (reviewed in Kueny and Day 2002). However,
little legislation directly targets the conservation and protection of caves. In Central
America, 18% of karst is afforded some protection, with the highest level of karst
protection in Belize (Kueny and Day 2002).
Brazil is the only country with a government agency devoted specifically to the
study, protection, and management of caves, the National Cave Research and
Conservation Center (CECAV), created in 1997. However, no current legislation
specifically protects caves and karst areas in Brazil. In 1996, a Decree (99556) was
published that effectively offered some protection to Brazilian caves by stating that
the “the use of caves is restricted to speleological activities, tourism, education,
scientific, and preserving its physical integrity.” In the past decade, however,
changes to existing laws, due to pressure from the mineral and other sectors, have
significantly weakened protections. Decree 99556 was replaced by Decree 6640 in
2008, which requires that caves be classified according to criteria proposed in the
Instruction Normative no. 2 of 2009, and in its revised form of 2017, as low,
medium, high, and maximum relevance. Caves of maximum relevance are
completely protected, but caves of lesser relevance are subject to lesser protection
and can possibly be destroyed. However, other existing legislation can sometimes be
applied to protect caves and cave biodiversity, such as laws related to groundwater
aquifer and fauna protection.
The Endangered Species Act (ESA) of 1973 is the foremost tool for the protection
of biodiversity in the United States. The primary purpose of the ESA is to protect and
recover imperiled species and their associated habitats and ecosystems. Under the
ESA, species may be listed as either endangered or threatened. “Endangered” status
is warranted when a species is at high risk of extinction throughout all or a significant

portion of its range, whereas “Threatened” status is warranted when a species is
likely to become endangered within the foreseeable future. The U.S. Fish and
Wildlife Service (USFWS) oversees the listing and protection of all terrestrial
animals and plants and freshwater animals, while the National Marine Fisheries
Service oversees marine animals. The ESA prohibits “take” of listed species as well
as interstate and international trade. Take includes such actions as harassing,
harming, hunting, trapping, capturing, trapping, killing, or collecting. Protections
also include prohibition of acts that result in significant habitat modification or
degradation that may result in the death or injury of wildlife. The ESA also requires
federal agencies to use their legal authorities to conserve listed species and consult
with USFWS to ensure that any actions authorized, funded, or implemented by a
federal agency do not jeopardize the continued existence of a listed species. USFWS
is required to develop recovery plans for each listed species unless it is determined
that such a plan will not promote the conservation of a species. Recovery plans serve
as essential guides in the management and recovery of listed species. The ESA also
requires the designation of critical habitat of some listed species. Critical habitat is
defined as specific geographic areas that contain features critical to the conservation
of a listed species that may require special management and protection. The desig-
nation of critical habitat provides protections of listed species by prohibiting federal
agencies (but not private landowners) from activities that adversely modify desig-
nated areas.
Of the 1200+ troglobionts in the United States, only 35 species are listed as
federally endangered or threatened under the ESA, including seven species of
beetles, seven spiders, three harvestmen, one pseudoscorpion, two crayfish, three
shrimps, four amphipods, two isopods, one snail, three fish, and two salamanders.
Six additional taxa are Candidate species, which are taxa under consideration for
listing under the ESA. Candidate species are not afforded protection under the ESA.
Seven bats that regularly use caves, either as summer roosts or winter hibernacula,
are listed under the ESA. In addition to species native to the United States, the ESA
offers protections for foreign species, which includes bans on the importation and
sale in the United States of foreign species listed. The only foreign troglobiont listed
on the U.S. ESA is the Mexican Blindcat (Prietella phreatophila), a catfish from
Mexico.
The small percentage of troglobionts listed or considered for listing under the
ESA is somewhat surprising, given that most troglobionts have restricted distribu-
tions or are known from just a few occurrence records (Culver et al. 2000; Elliott
2007; Niemiller and Zigler 2013). Rarity, as well as presumed low reproductive
rates, poor dispersal ability, and susceptibility to environmental change, renders
these species extremely vulnerable to anthropogenic threats (Culver and Pipan
2009). There are marked biases in diversity and geography of listed species
(Fig. 22.2). Five of the 19 vertebrate troglobionts (26.3%) are listed, despite com-
prising just 1.4% of all troglobionts in the United States. In contrast, just 2.2% of
invertebrate troglobionts, which comprise 98.6% of troglobionts in the United
States, are listed. In fact, the only invertebrate group with >5% of species listed
are decapods (crayfish and shrimp) at 11.6% (Fig. 22.2b). Some critics have argued
that the ESA and USFWS are biased against insects and other invertebrates in the
listing of species (Greenwald et al. 2005) and in the amount of money spent on
conservation when compared to listings and expenditures for vertebrate species
(Bossart and Carlton 2002; Male and Bean 2005). It has also been suggested that
the paucity of invertebrates listed under the ESA may be attributed more to a lack of
scientific data and lack of qualified biologists to file and review listing petitions
(Lugo 2007). This may also hold true for some groups of invertebrate troglobionts
for which few experts exist.
Geographically, 57% (20 of the 35) of the troglobionts listed under the ESA occur
in central Texas karst, including the Edwards Aquifer. The high number of listed
species from this karst region does not reflect greater diversity and endemism
relative to other karst regions in the United States. Rather, it reflects greater vulner-
ability and risk of extinction from higher levels of development and demands for
groundwater associated with continued growth of the cities of San Antonio, Austin,
and areas in between relative to other karst regions (Culver and Pipan 2009).
Other U.S. federal laws have been passed that provide varying degrees of
protection to caves and karst on federal and private lands, and—directly or indi-
rectly—subterranean biodiversity. This legislation has been reviewed in depth by
Huppert (1995, 2006), Lera (2002), Seiser (2013), and others. Caves designated as
“significant caves” that occur on federally owned lands in the United States are
protected by the Federal Cave Resources Protection Act of 1988. This act requires
federal land managers to account for all cave resources under their jurisdiction
(Huppert 2006). However, only significant caves under the Department of Agri-
culture and Department of the Interior are covered by the act. Federal lands under the
jurisdiction of the Department of Defense and other extensive tracts of land are not
included under provisions of this act (Huppert 1995, 2006). In addition, there is
considerable debate regarding the definition of a “significant cave” and levels of
protection for caves not deemed significant. The U.S. National Park Service Act of
1916 protects caves that occur on National Park Service lands, including prominent
national parks where the protection of specific caves is the primary focus, such as
Mammoth Cave National Park in Kentucky, Carlsbad Caverns National Park in New
Mexico, and Wind Cave National Park in South Dakota. This act also protects caves
that occur in national parks and other lands managed by the U.S. National Park
Service (national monuments, national scenic rivers, etc.).
The Wilderness Act of 1964 established a means to designate wilderness areas
where activities such as logging, mining, and even road development are prohibited
and created the National Wilderness Preservation System. This system now protects
some 110 million acres of wilderness areas in the United States. Caves within the
boundaries of these designated wilderness areas are protected. However, this legis-
lation has largely focused on the surface environment and does not specifically
define which ecosystems are and are not to be considered (Seiser 2013). Conse-
quently, there have been efforts to designate select cave systems as Cave Wilderness.
Unfortunately, despite four attempts since 1967 to create a Cave Wilderness desig-
nation, including two by the Cave Research Foundation, one by the National
Speleological Society, and by a unit within the National Park Service, no Wilderness
designation has been made to date to protect and preserve a specific cave or area
within a karst region in the United States (Seiser 2013).
Some caves that occur along rivers designated as Scenic Rivers are protected by
the Wild and Scenic Rivers Act of 1968. Notable Scenic Rivers important for cave
conservation include the Buffalo National River in Arkansas and the Ozarks
National Science River in Missouri (Huppert 2006). A bill, the Oregon Caves
Revitalization Act, was introduced in the United States Senate in 2013, which
included a Scenic River designation for the subterranean section of Cave Creek,
also known as River Styx. Unfortunately, this bill passed the Senate but was never
passed by the House of Representatives. If passed, this legislation would have
represented the first time that the Wild and Scenic Rivers Act was specifically
applied to a subterranean stream or river (Seiser 2013).
Several other federal acts exist that may have a bearing on the protection of cave
resources in the United States, including biodiversity (reviewed in Huppert 2006).
These include the Archaeological Resources Protection Act of 1979, Antiquities Act
of 1906, Eastern Wilderness Act of 1975, Endangered American Wilderness Act of
1978, Historical Sites Act of 1935, National Environmental Policy Act of 1969,
National Forest Management Act of 1976, National Historic Preservation Acts of
1966 and 1976, National Parks and Recreation Act of 1978, National Wildlife
Refuge System Administration Act of 1966, and several others. Moreover, several
“clean water acts” may be used to help protect karst aquifers, and, in turn, ground-
water fauna (Jones et al. 2003), such as the Resource Conservation Recovery Act,
the Comprehensive Environmental Resource Compensation Liability Act, and the
Safe Drinking Water Act.
In the United States, cave-dwelling species may be afforded protection under
state endangered species acts. For example, some species are protected under the
Virginia Endangered Species Act of 1972 and the Virginia Endangered Plant and
Insect Species Act of 1979. Species that are listed under the federal U.S. Endangered
Species Act of 1973 are automatically listed under state law in Virginia. Both state
and federal agencies have regulatory authority, while the Virginia Natural Heritage
Program in the Virginia Department of Conservation and Recreation provides
technical expertise to aid in the recovery and long-term protection of a species
culminating in its removal from the list of protected species (Orndorff 2005).
Although scope, details, enforcement, and punishments vary by state, in general it
is unlawful to kill, harm, collect, possess, or traffic imperiled species listed as
protected in their respective states. Most of the existing state endangered acts simply
provide a measure for listing and prohibition of taking and trafficking of listed
species, but lack mechanisms for the recovery, consultation, or critical habitat
designation (George and Snape 2010). Just six US states have a provision requiring
critical habitat designation, and only five states require recovery plans for listed
species. Penalties for violation of state endangered species acts range from a
misdemeanor with fines up to $1000 and/or 90 days imprisonment to up to
$10,000, 180 days imprisonment, and possible seizure and forfeiture of property in
some states.
Twenty-eight US states have laws aimed at the protection of caves and their
resources, with most passed since 1976 (Huppert 1995, 2006; LaMoreaux et al.
1997; Lera 2002). Puerto Rico and the Cherokee Nation also have similar legislation.
Other US states, in addition to the 28 states that have cave protection legislation,
have passed resource protection legislation that mentions cave resources. These laws
vary in their definition of a cave, application, and effectiveness in cave protection.
Many state cave protection laws only apply to caves on state lands, while those that
also cover caves occurring on private lands usually require landowner consent to
seek prosecution (Huppert 1995, 2006). Caves fall under the definition of wilderness
in some states and may be afforded varying levels of protection under state wilder-
ness acts. Caves may be protected in some states if they occur on specific state-
owned or managed lands, such as state parks, wildlife management, or natural areas.
In addition, several states have passed legislation prohibiting the contamination and
pollution of surface waters and groundwater. Similar legislation has been passed in
some Canadian provinces to protect caves and cave resources, such as the Cave
Protection Act in British Columbia.
The Nature Conservancy (TNC) is a US-based nonprofit organization dedicated to
the conservation of lands and waters of ecological importance. TNC is the largest
environmental nonprofit organization in terms of assets and revenue in the Western
Hemisphere. TNC has helped protect almost 48 ha of land and 8000 km of rivers in all
50 US states and more than 35 countries worldwide. TNC owns or manages several
properties with significant cave and karst resources, including some where the
primary features are cave resources. An example is Hubbard’s Cave in Tennessee,
which contains one of the largest hibernacula of the federally endangered Gray Bat
(Myotis grisescens). TNC also works with state, federal, and other organizations and
researchers to monitor bat populations, to document subterranean biodiversity, and to
develop strategies to protect endangered species and subterranean ecosystems. For
example, TNC staff and Chiquibul National Park officials in Belize developed a
5-year management plan for the Chiquibul Cave system, the largest cave in Belize
and longest in all of Central America. TNC in Tennessee constructed an artificial cave
to assist in bat and White-Nose Syndrome research.
22.4.3 Regional and Local Conservation Organizations
There are organizations that work at national and regional scales to assist govern-
ment agencies and other conservation organizations on many biodiversity conser-
vation issues, setting priorities for funding, research, and conservation efforts and
developing sound conservation policies, legislation, and practices. The National
Speleological Society (NSS) is the world’s largest organization dedicated to the
exploration, study, and conservation of caves and their environments, with over
10,000 members and 250 grottos. NSS and affiliated cave conservancies own or
manage over 100 caves in the United States, which includes several biologically
significant caves. NSS has also been actively involved in response to White-Nose
Syndrome since its discovery in New York in 2007, including raising grant money to
support WNS research (e.g., NSS White-Nose Syndrome Rapid Response Fund),
participating and organizing WNS conferences and symposia, and participating and
planning in WNS-related research and state and federal planning. The Brazilian
Speleological Society (SBE—http://www.cavernas.org.br), founded in 1969, has
ca. 1800 members, including speleological organizations. SBE and other inde-
pendent speleological organizations in Brazil (e.g., Grupo Bambuí de Pesquisas
Espeleológicas/GBPE and Grupo Pierre Martin de Pesquisas Espeleológicas/
GPME) conduct projects related to cave discovery and documentation, as well as
cave conservation. SBE maintains a catalog of more than 10,000 caves in Brazil.
Recent conservation-based projects involving SBE include the creation of Conser-
vation Units, such as Natural Heritages and Parks to protect caves and cave fauna,
such as the karst of Mambaí, central Brazil). Another Brazilian organization is the
Boticario Foundation of Nature Protection (FBPN), which provides grants for
conservation projects to project many environments and biodiversity, including
projects related to caves and karst.
22.5 Conclusions
A variety of legislation has been proposed and enacted worldwide that may directly
(e.g., endangered species acts) or indirectly (e.g., water quality legislation) provide
some protection for subterranean fauna. Conservation efforts have largely focused
on protecting sites of exceptional species richness or phylogenetically unique bio-
diversity (Culver and Sket 2000; Danielopol et al. 2009; Gibert et al. 2009). Levels
of protection for subterranean life offered by international and national and regional
legislation vary greatly throughout Europe and the Americas. At the international
level, subterranean fauna appearing on lists and in appendices as protected species,
including CITES, IUCN Red List, Bern Convention, and EU Habitats Directive, are
largely biases toward vertebrates, particularly bats. This is despite the fact that the
overwhelming biodiversity of subterranean ecosystems comprises invertebrates. The
importance of and threats to subterranean invertebrate fauna in Europe have been
known for some time (Juberthie 1995), specifically recognized in Recommendation
no. 36 of the Bern Convention (1992) in which it is recommended that national
inventories of subterranean invertebrates and subterranean habitats to protect be
compiled and that species of conservation concern be identified. Unfortunately, this
recommendation remains to be completed (Haslett 2007). This marked taxonomic
bias is also observed in national and regional legislation, such as the list of species
protected under the United States Endangered Species Act. The taxonomic biases
favoring conservation and protection of vertebrates over invertebrates are not limited
just to subterranean ecosystems (Cardoso et al. 2011a, b). The four main shortfalls in
invertebrate conservation identified by Cardoso et al. (2011b), as well as other
impediments, apply to the conservation of subterranean biodiversity and ecosystems.
In particular, the Linnean shortfall (i.e., much subterranean biodiversity remains to
be described) is the primary driver behind the other three shortfalls: the Wallacean,
Prestonian, and Hutchinsonian shortfalls in the conservation of subterranean inver-
tebrate faunas. In some regions, these shortfalls also apply to vertebrate subterranean
biodiversity. Gallão and Bichuette (2012) emphasized the importance of the IUCN
Red List for protection of cavefishes in Brazil and highlighted limitations of some
criteria in the inclusion of taxa on the IUCN Red List. These shortfalls also can have
significant political implications, as the presence of IUCN Red Listed species is one
of the most important tools to prioritize caves based on biological criteria for
protection in Brazil, for example.
A major factor behind taxonomic bias in subterranean biodiversity conservation
is the lack of biologists qualified to study subterranean taxa and ecosystems. This is
particularly evident in the continuing decrease in taxonomic experts that can
describe and properly identify often difficult groups, such as springtails, flatworms,
and annelids. Correct species identification, generally requiring a high degree of
familiarity with taxon-specific morphological characters, is paramount to addressing
the other major shortfalls in subterranean biodiversity conservation, including elu-
cidating accurate species distributions and habitats, determining abundance and
population size, and predicting species responses to habitat change. Some progress
has been made in recent years to address the Wallacean shortfall in subterranean
conservation of Europe and the Americas, highlighted by the PASCALIS project
(Gibert 2005). The PASCALIS (Protocols for the Assessment and Conservation of
Aquatic Life in the Subsurface) project was a 3-year (2002–2004) large-scale
groundwater bioinventory initiative that developed standard sampling protocols for
comparing stygobiont biodiversity in six European regions in five countries:
Belgium, France, Italy, Slovenia, and Spain. Unfortunately, limited prospects for
securing employment for newly trained taxonomic experts, reflecting a lack of value
society places on invertebrate taxonomy, continues to discourage young scientists
from placing emphasis on taxonomy during their graduate training, especially in the
United States.
Acknowledgements We thank JE Gallão for sharing unpublished data for Brazilian cave fauna,
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), and Fundação o Boticário de Proteção à
Natureza (FBPN) for grants to MEB.
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Chapter 23
Conservation and Impact Assessment
of Subterranean Fauna in Australia
Stuart Halse
23.1 Introduction
This chapter examines some of the difficulties encountered when trying to protect
subterranean species occurring in the broad landscape, rather than in caves, in
Western Australia. We refer to these species as landscape subterranean fauna. A
particular focus of the chapter is the protection of landscape subterranean fauna in
areas outside nature reserves in situations where land development is likely
(e.g. Henle et al. 2008), particularly as part of the environmental impact assessment
process. Assessments usually deal with mining and water supply developments
(e.g. Sheppard et al. 2009; Mudd 2010).
Box 23.1
Subterranean fauna can be protected by legislation in two ways. Often the
focus of the legislation is maintaining their subterranean habitat, as happens
for many subterranean species occurring in caves that have high aesthetic and
tourism values and a relatively easily defined spatial extent. In other cases, the
legislation is directly concerned with protecting the subterranean species
themselves. This usually happens for species with strong public appeal, such
as cave nesting birds and bats (although, ironically, almost all high profile
subterranean species occur in caves). Most Australian subterranean fauna
species occur outside caves in the groundwater aquifers or in the vadose
zone deep below the ground surface. While standard biodiversity legislation
has a theoretical capacity to protect subterranean species in these habitats, their
(continued)
S. Halse (*)

https://doi.org/10.1007/978-3-319-98852-8_23
480 S. Halse

unique ecology and the complexities associated with protecting their habitat
present significant logistical challenges for both species and habitat-based
protection policies.
The classical method of protecting surface fauna, at least in Australia, is to create

nature reserves where populations of all (or most) species can be conserved
(e.g. Margules and Pressey 2000; Scott et al. 2001). This can work well for cave
fauna, although in most cases the motivation for creating reserves has been a strong
public desire to protect the cave itself, rather than its subterranean fauna (Bryan
2002). It is frequently only when protected caves are used for tourism (as commonly
happens; see Chap. 22) that there is focus on the protection of the subterranean fauna
within the cave system as visitor use threatens to alter cave conditions (Russell and
MacLean 2008; Pellegrini and Ferreira 2012). Most of the few exceptions to cave
reservation being based on the aesthetic values of caves are those caves supporting
large colonies of bats or birds (Hall 1990).
Achieving protection of landscape subterranean fauna, whether by reservation or
by constraining threatening or inappropriate land use, is more challenging than
protecting cave fauna. There has been no reservation of land in Western Australia
for the explicit protection of landscape subterranean fauna, despite there probably
being more focus on landscape subterranean fauna in Western Australia than
anywhere else in the world (see Chap. 20 and references therein). However, land-
scape subterranean fauna has received a unique level of attention in the environ-
mental impact assessment process in Western Australia since the mid-1990s.
The original reasons for subterranean fauna being included in environmental
impact assessments in Western Australia are threefold. First, considerable work
had been done showing the occurrence of rich stygofauna communities in ground-
water aquifers of north-western Australia (e.g. Humphreys 1993, 1999; Pesce and de
Laurentiis 1996). Second, several submissions lodged during the public comment
period of several development proposals, both for mining and water supply in north-
western Australia, suggesting there might be significant impacts on stygofauna,
caused re-scoping of the assessments (e.g. EPA 1996, 1998). Third, and related to
the second point, the object of Environment Protection Act 1986 is “to protect the
environment of [Western Australia], having regard to [five] principles”, one of
which is that “Conservation of biological diversity and ecological integrity should
be a fundamental consideration”, and accordingly the Environmental Protection
Authority has recognized that subterranean fauna constitutes a part of Western
Australia’s biodiversity to be included in their assessment of projects. Ultimately,
however, all environmental assessment decisions are based on a consideration of
social and economic issues as well as environmental ones. As such, there have been
cases where the recommendations of the Environmental Protection Authority against
approval of a project because of threats to subterranean fauna have been overturned
23 Conservation and Impact Assessment of Subterranean Fauna in Australia 481
by the Minister for the Environment for social or economic reasons, but with
conditions set to minimize environmental harm (EPA 2006, 2007, 2016a).
While the focus of actions to protect landscape subterranean fauna species has
been based on the Environment Protection Act, more specific biodiversity legislation
provides broad support for the actions of the Environmental Protection Authority.
The Wildlife Conservation Act 1950, section 14, states “Except to the extent which
the Minister declares by notice . . . all fauna is wholly protected throughout the whole
of the State at all times”. This section is usually interpreted to mean that actions
(including removal of habitat by proposed development projects) that would rea-
sonably be considered likely to result in the extinction of a species cannot be legally
approved. The critical issue is that the action being considered must threaten the
persistence of the whole species. Protection is stronger for the 258 species that are
listed as endangered or vulnerable under the Wildlife Conservation Act. For these
species, both individual animals and recognized habitats of the species receive
protection. Forty-one of these listed species are subterranean, with three of them
being fish and 38 being invertebrates (Government of Western Australia 2017).
Currently, there is a transfer occurring from the Wildlife Conservation Act to the
Biodiversity Conservation Act 2016. The objects of the new Act are “(a) to conserve
and protect biodiversity and biodiversity components in [Western Australia]; and
(b) to promote the ecologically sustainable use of biodiversity components in the
State. In the pursuit of the objects of this Act, regard must be had to the principles of
ecologically sustainable development . . .”.
23.2 Complexities of Subterranean Fauna Assessment
Box 23.2
The key issue in relation to environmental impact assessments is that both
stygofauna and, more particularly, troglofauna species may have ranges that
are smaller than the footprints of proposed developments. Furthermore, there
is usually a high level of uncertainty associated with the estimated ranges of
species and a bias towards underestimation, so that many apparently restricted
species probably occur beyond the project footprint. Given that the economic
and social costs of refusing development approval may be very high, decision-
making is complex. The difficulties of accurately estimating species ranges are
unsurprising, given that landscape subterranean fauna began to be studied in
Western Australia only about 20 years ago and little is understood about the
ecology of these species.
While agricultural operations in Australia (horticulture, broad-acre cropping, animal

feedlots and, probably to a lesser extent, grazing) are likely to have cumulative
impacts on the habitat of many subterranean fauna species (Korbel and Hose 2011),
482 S. Halse
in most of these situations the species would be expected to persist at reduced

densities rather than being sent extinct (Di Lorenzo and Galassi 2013; Di Lorenzo
et al. 2014). The more threatening impacts come from excavation of open-cut mine
pits and large-scale water abstraction, especially the dewatering of mines to allow
safe mining below the water table, abstraction to provide large volumes of water for
ore processing and abstraction for irrigation and urban water supply. Some mine pits
extend more or less continuously for many tens of kilometres (EPA 2014, 2016b),
some have depths below the ground surface (and water table) of several hundreds of
metres (e.g. EPA 2002), and many borefields that dewater mine pits or supply mine
process water result in groundwater drawdown over distances of many tens of
kilometres (EPA 2014, 2016b, c). In contrast, many troglofauna species have linear
ranges of only a few kilometres at most (Halse and Pearson 2014; see also Chap. 20).
While stygofauna species usually have larger ranges than troglofauna in the Pilbara,
sampling suggests that approximately 10% of species have linear ranges smaller than
25 km (Halse et al. 2014), and some species in catchment headwaters appear to have
linear ranges of less than 5 km. Many stygofauna species in calcretes of the Yilgarn
region, in inland central and southern Western Australia, appear to have even smaller
ranges (Karanovic and Cooper 2011, 2012; Karanovic et al. 2015), as probably do
troglofauna species. Thus, the entire distributions of some troglofauna and
stygofauna species are likely to lie within large mine pits or areas where the water
table has been drawn down significantly by groundwater abstraction. However,
defining the ranges of individual species and determining which species are actually
confined to these impact areas is difficult.
The main challenges to defining species’ ranges are the inefficiency of subterra-
nean fauna sampling, lack of information about the microhabitats used by species
and the very limited information about the pattern of occurrence of these microhab-
itats in the landscape, especially outside proposed areas of impact (where there has
usually been little geological work as well as little subterranean fauna sampling). A
more simplistic way of looking at the problem is that most of the difficulty in
defining ranges results from the species living underground and sampling being a
blind, below-ground process. A useful analogy is to think of subterranean fauna as
occurring in a forest below ground, where soil faunas inhabit the understorey (near
the surface) and stygofauna and troglofauna occur in the canopy at depth (see Hose
et al. 2017). These canopy species are difficult to observe and are underrepresented
in the data collected. Furthermore, there is spatial variation in the structure of this
forest that often results in the occurrence of different stygofauna and troglofauna
species in adjacent areas. Currently, sampling is usually undertaken without any
understanding of variation in the forest structure or the species preferences for
particular forest types. Sampling is done blindly through bores or drill holes that
run from the forest floor (close to the surface) to outer sections of canopy (at deep
depths). A further complication is that in many geological formations the subterra-
nean forest is a patchy one and many drill holes intersect clearings in the forest rather
than the subterranean voids or spaces constituting the subterranean canopy in which
stygofauna and troglofauna occur (Fig. 23.1).
Fig. 23.1 Drill hole being sampled for troglofauna by scraping. Hole has short PVC collar to
prevent collapse at the surface and a metal “sleeve” has been inserted in the collar to reduce friction
when retrieving the sample. Deeper sections of the hole are uncased
However, while the technical challenges involved in undertaking impact assess-

ment of subterranean fauna are considerable, perhaps the biggest challenge to the
continued inclusion of subterranean fauna in environmental impact assessment is the
low level of empathy for this group of animals amongst policymakers, the public at
large and many zoologists working on surface fauna. This is principally related to a
low level of knowledge, but, nevertheless, it should be recognized that there is no
impetus to protect microscopic species that have poorly documented, and sometimes
not obvious, ecological roles.
23.2.1 Sampling Framework
Effective impact assessment requires a well-defined framework. The Environmental

Protection Authority in Western Australia has issued a series of guidelines for
subterranean fauna assessment since 2003, with the most recent describing the
principles of assessment (EPA 2016d). Elsewhere in Australia, the Queensland
Government released a subterranean fauna assessment guideline in 2014 (DSITI
2015), while South Australia released a discussion document in 2015 (Goonan et al.
484 S. Halse
Fig. 23.2 Framework for environmental impact assessment in Western Australia, based on EPA
(2015). Subterranean fauna is a factor that may lead to recommendation that a project should not
receive environmental approval. EPA, Environmental Protection Authority
2015). Subterranean fauna has been identified as an issue requiring assessment in

many development projects in New South Wales, but the framework for assessment
is less formal than in states such as Western Australia and Queensland.
The main elements of the Western Australian framework for assessment are
shown in Fig. 23.2. It consists of an initial desktop investigation to determine
whether subterranean fauna is likely to occur in the vicinity of the project and
whether the project may potentially have a deleterious impact on any subterranean
fauna present. This may be followed by limited fieldwork to confirm the conclusions
of the desktop investigation or more intensive field study aimed at identifying the
species in the impact area(s) of the project and whether or not they are likely to be
restricted to the impact area(s). This stage may also include geological habitat
mapping to help define the likely ranges of species and the finer scale distributions
of species within the project impact area(s). Based on the lateral and vertical
distribution of species in relation to project impacts, the likely effect of the project
on subterranean fauna conservation values can be determined. If persistence of some
species may be threatened, the potential effectiveness of management measures to
mitigate impacts is investigated before a final assessment is made of threat to
subterranean fauna. Some of the details of the assessment approach have been
criticized (e.g. Karanovic et al. 2013) or supported (e.g. Harvey et al. 2011)
elsewhere, and further discussion of technical aspects of the framework is provided
below.
23.2.2 Sampling Efficiency
As described in Chap. 20, sampling subterranean fauna in the broad landscape

occurs via various types of drill holes. While this sampling is often considered to
be inefficient because relatively few animals are collected and repeatability is quite
low, there is in fact relatively little information about the relationship between
capture rates and how completely the species present in the holes and surrounding
matrix are documented.
Repeated sampling of stygofauna at selected bores in the Pilbara region in the
north-west of Western Australia has shown that the first sample from a bore captures
46% of species occurring in high abundance and 23% of species found in low
abundance (Eberhard et al. 2009). Based on species richness estimates generated
by Chao 2 or ICE, six samples collected over three to four years captured more than
80% of all species present at the bore holes sampled and more than 90% of the
abundant species. These results suggest that, contrary to general perceptions, it is
possible to document most of the stygofauna species at a site through conventional
sampling techniques. It is also realistic to expect to show through sampling that all
(or nearly all) species known from the impact area of a project occur outside this
area, provided of course that they actually do so. However, the level of sampling
effort required (at least six sampling events) represents a cost and period of time that
is frequently seen as unaffordable in impact assessments, especially if the project is
in a remote area.
There is no reliable information on the number of samples required to collect
most of the troglofauna species occurring in a drill hole or its immediate vicinity.
While assessment results suggest it is relatively straightforward to show by sampling
that the abundant species known from the impact area of a project also occur outside
486 S. Halse
Table 23.1 Numbers of troglofauna species collected from 150 drill holes sampled three times in
relation to maximum capture abundance of the species in any sampling event and how often the
species was collected at each bore (SA Halse, unpublished data)
Maximum No. of times the species collected from a bore Probability of being collected
abundance 1 2 3 twice (or more) (%)
3 animals 12 15 4 61
2 animals 15 3 0 17
1 animal 76 5 0 6
it, the effort required to show wider occurrence of low abundance species will be
considerable. For example, preliminary results from 150 drill holes in the Pilbara
sampled three times each show that while abundant species (defined as those for
which at least three animals were collected in one of the sampling events) had 61%
probability of being collected at least twice, the species always collected as single
animals had only 6% probability of being collected twice (Table 23.1). A further
challenge is that in most survey programmes many of the holes sampled for
troglofauna yield no animals, even if repeatedly sampled in areas that generally
yield troglofauna. The most likely reason for this is that the holes do not intersect the
vugs and other subterranean spaces that house animals. This is a consequence of the
relatively small volume of troglofauna habitat within even troglofauna-rich land-
scapes (Fig. 23.3).
Karanovic et al.’s (2013) contention that the levels of sampling recommended by
the Environmental Protection Authority (40 stygofauna and 60 troglofauna samples
from impact areas) are too low, especially for troglofauna (see Halse and Pearson
2014), is correct in most situations, as can be seen in the collectors’ curves shown in
environmental impact reports. However, notwithstanding the need for greater sam-
pling effort, the assessment of impacts of development in Western Australia is
probably more thorough than practised anywhere else.
23.2.3 Inferring Distributions
While assessments of the potential impacts of development on subterranean fauna

usually focus on threats to species, another important aspect of the conservation
impact is the richness and biological interest of the subterranean fauna assemblage in
the development area. Regional surveys provide a framework of information that
enables the characteristics of the assemblage to be assessed. They also enable the
likely values of an area to be predicted with moderate confidence in advance of
survey, based on geological information and broad patterns of subterranean fauna
occurrence. Another benefit of regional surveys is that they provide information
about the characteristic ranges of species of various taxonomic groups (especially
Fig. 23.3 Schematic illustration of the location of a drill hole in relation to the subterranean meso-
and microcaverns providing habitat for troglofauna. Hardcap is a ferricrete duricrust found above
many rock formations in the Pilbara. It is usually vuggy and often contains mesocaverns and even
caves
families and genera) in different geologies, and the extent to which a species’ range
can be predicted (or not) from that of related species. This information will be better
refined over time as general ecological and life history studies of species are
undertaken.
The main factors affecting the occurrence of stygofauna species in the Pilbara and
Yilgarn regions of Western Australia are hydrological connectivity (including to the
surface), salinity, dissolved oxygen levels and geology/lithology (Halse et al. 2014),
but factors other than hydrological connectivity and salinity do not predict occur-
rence well. A major issue when trying to predict species ranges from environmental
characteristics is that these characteristics often vary substantially within an aquifer
over vertical and spatial scales of a few metres as a result of differences in the size
and spacing of voids that affect transmissivity (see Larned 2012; Bradford et al.
2013) or stratification of the aquifer if groundwater is moderately saline (e.g. Halse
et al. 2014). Fine-scale changes can rarely be mapped well from the surface.
However, it may be said that in general both calcrete and alluvial aquifers are likely
to contain areas of high transmissivity and calcrete aquifers usually contain halo-
clines. Alluvial habitats often occur within the dewatered zone around mine pits, and
both calcrete and alluvial habitats are potential sources of mine processing water.
The factors affecting troglofauna occurrence in the Pilbara and Yilgarn are poorly
documented, but the most important factor is probably the frequency of subterranean
488 S. Halse
spaces over a threshold size (this is analogous to hydrological connectivity being

important for stygofauna). Troglofauna are common in vuggy habitats, such as those
occurring in weathered or mineralized iron ore formations and calcretes. Mineralized
iron ore formations are areas of potential mine development. The limited information
available suggests that while broad geological categories are important determinants
of the occurrence of troglofauna, subtle variations in geological classification (in the
sense of mineral composition and origin) do not affect troglofauna occurrence
greatly if lithology (general physical characteristics including texture) remains
similar.
23.2.4 Identifying Species
Another challenge in determining species ranges and, more particularly, whether a

species has been collected both inside and outside a project impact area is the species
identification process. The key issue is usually whether animals found inside and
outside the impact area represent a single species or two sister species. Identification
of subterranean fauna is especially challenging because of the very short history of
study (approximately 20 years for stygofauna and 10 years for troglofauna in north-
western Western Australia). It is often suggested that morphological convergence
towards a subterranean life form (loss of pigment and eyes, elongated appendages
and sensory structures) contributes to difficulty in distinguishing species morpho-
logically, but the high degree of plasticity in many subterranean species is also an
issue, whereby the numbers of setae and segments in structures such as antennae can
vary between left and right side.
Use of DNA sequencing to identify species in the assessment process is becoming
routine, but, just as with morphology, there are many situations when it is unclear
from sequence results whether one or two species are present. The main reason for
this is that the potential for dispersal and intermingling of animals in the subterra-
nean environment is limited. This applies even to alluvial aquifers where dispersal
would be expected to be comparatively easy, but genetic differentiation may be
observed over distances as small as 50 m (Asmyhr et al. 2014). Genetic differenti-
ation also occurs within calcrete aquifers in the absence of obvious barriers, perhaps
as a result of areas within the calcrete becoming isolated during (geologically) dry
periods and reuniting with the remainder of the calcrete during wetter periods (Guzik
et al. 2011; see also Chap. 20). In some cases, sequence divergence in mitochondrial
DNA can be high while nuclear DNA shows little difference (Javidkar et al. 2016),
and it has been suggested the important units for conservation may be the genetic
lineages that have at least some morphological support to suggest they are separate
species (De Queiroz 1998; Carstens et al. 2013). However, the likely high future
emphasis on the use of environmental DNA (e.g. Jerde et al. 2011) may lead to
greater use of a genetic species concept, with a consequent increase in the numbers
of restricted species recognized. It is interesting to note that, given the patterns of
diversity revealed by morphologically and genetically defined species are similar
(Eme et al. 2017), many of the objectives of conserving subterranean fauna, espe-
cially at the assemblage level, can probably be achieved by morphological species
identifications alone. However, if environmental DNA can improve the detection
rate of species (and there are challenges to be faced—see Korbel et al. 2017), the
level of information about species ranges will be improved and there will be a
consequent increase in confidence that environmental impact assessments are
accurate.
23.2.5 Determining Impacts
In situations such as a mine pit being excavated to the water table (or below)
throughout the range of a restricted troglofauna species, it is clear that all of the
species’ habitat will be lost and that extinction is highly likely. Similarly, if ground-
water drawdown occurs to bedrock (or substantially greater than 30 m below the
ground surface) across the range of a restricted stygofauna species, extinction would
be expected to occur. In many cases, however, possibly suitable habitat will remain
in parts of the aquifer. This is particularly the case at the edges of the groundwater
drawdown cone, where only a small part of the aquifer will be drawn down.
Assessing the degree of impact in such situations is complex because of limited
information about species’ vertical distributions and the vertical distribution of the
different habitats the species may occupy.
The issue of impact being unlikely at the margin of the zone of dewatering can be
partly addressed by setting a threshold of drawdown (often 2 m) that must be
exceeded before groundwater abstraction is considered potentially threatening. In
uniform alluvial aquifers, the safe drawdown may be much greater than 2 m because
there will be a considerable depth of similar habitat remaining unaffected by
drawdown. Many borefields designed to produce sustainable yields of processing
water will probably contain significant remaining stygofauna habitat across the
borefields. However, in calcrete aquifers the preferred habitat of at least some species
may be the more karstic habitat associated with water table fluctuations and draw-
downs of 2 m from current level will result is significant loss of habitat for species.
It is often suggested another reason to think stygofauna will usually survive
groundwater drawdown is that pockets of water remain in the subterranean matrix
after drawdown. This is easy to conceptualize in rock aquifers but may be less likely
in calcrete and alluvial aquifers where the matrix is more porous and may drain more
completely over time. Hose et al. (2017) have examined the likelihood of stygofauna
surviving in water films in more detail.
This very brief review of determining impacts in environmental assessment has
focused on mining. Some other potential impacts are considered in the next section,
but the main message is that understanding likely impacts and, more importantly,
how they might be mitigated through management actions requires an understanding
of the biology of the species involved or, at the very least, improved information
about the ecology of troglofauna or stygofauna in general.
490 S. Halse
23.3 Broad Threats
The broad threats to subterranean fauna across the general landscape have not been
considered in Western Australia outside environmnetal impact assessments. However,
there is more information on the threats to stygofauna than troglofauna. Reviews have
examined the possible threats from mining and the water chemistry changes associated
with mining, especially increased salinity, and reduced carbon and nutrient inputs
(Hancock et al. 2005; Humphreys 2009; Nevill et al. 2010). Elsewhere, it has been
shown that nutrient overload, from sewerage treatment works, can substantially alter
the abundances of some stygofauna species (Scarsbrook and Fenwick 2003). How-
ever, studies of the impacts of agriculture on stygofauna have shown mixed results,
with not all impacts being deleterious (Korbel et al. 2013); just as the effects of metals
and pesticides on stygofauna appear to be similar or less than on surface aquatic
invertebrates (Hose 2005; Hose et al. 2016), although Humphreys (2007) has cau-
tioned against this conclusion. In line with Hose’s findings, Marmonier et al. (2013)
also showed similar sensitivities of surface and groundwater species to pollution.
Thus, it is suggested here that the main differences between stygofauna and surface
aquatic invertebrates in their responses to pollution perhaps relate mostly to the time
and method of exposure rather than innate physiological differences.
In the absence of other information, it is tentatively suggested that the situation
for troglofauna is analogous to stygofauna. Some papers have suggested that
troglofauna will be sensitive to many impacts, such as pesticides (Souza Silva and
Ferreira 2015), but there is currently little empirical support for these conclusions in
troglofauna across the broad landscape.
23.4 Awareness of Subterranean Fauna
Box 23.3
Other than a few fish species, the subterranean fauna of the broad landscape in
Australia consists of microscopic invertebrate species about which relatively
little is known. The subterranean environment itself is out of sight and
subterranean species will always be low on the list of environmental concerns
unless it can be clearly shown that they are important to maintaining ecosys-
tem function in a way that benefits the human population. This work is well
advanced for stygofauna but needs to be a focus of troglofauna research. It
would also be useful if the role of subterranean fauna species in the landscape,
and the ecological services they provide, were better understood.
It is difficult to envisage strong community support in Western Australia for

subterranean fauna conservation without a greater appreciation of the role of sub-
terranean fauna in the ecosystem. This includes a scientific consensus that support is
warranted. At present, while authors such as Tomlinson et al. (2007) argue strongly
for greater inclusion of subterranean fauna in environmental monitoring and man-
agement systems, others such as Majer (2009) question the existing level of funding
for subterranean research and argue it should be spent on surface systems. This view
was rebutted by Harvey et al. (2011), but it illustrates the need to justify the
protection of subterranean fauna beyond the oft-cited requirement of the Wildlife
Conservation Act to ensure the persistence of all species. While there are strong
administrative, as well as conservation, reasons for retaining the position that all
species should be protected, rather than finding a rationale for protecting some
species and not others, it would be much easier if ecological justifications for the
protection of subterranean fauna could be provided.
As with the understanding of threats, identifying ecological reasons for protection
is more advanced for stygofauna than troglofauna, especially in relation to the
ecosystem services they provide (e.g. Danielopol et al. 2003; Steube et al. 2009;
Griebler and Avramov 2015). It is anticipated, however, that further study over the
next decade is likely to provide compelling justification for the preservation of both
troglofauna and stygofauna across the broad landscape and around development
projects.
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Scott JM, Davis FW, McGhie RG et al (2001) Nature reserves: do they capture the full ranges of
America’s biodiversity? Ecol Appl 11:999–1007
Sheppard G, Gallardo A, Hall J et al (2009) Managing the cumulative groundwater impact s of
multiple iron ore projects at Cape Preston (or . . . one plus one plus one plus one does not equal
four). In: Water in Mining Conference, 15–17 Sept 2009, Perth, Australasian Institute of
Mining and Metallurgy, pp 169–177
Souza Silva M, Ferreira RL (2015) Cave invertebrates in Espírito Santo state, Brazil: a primary
analysis of endemism, threats and conservation priorities. Subterr Biol 16:79–102
Steube C, Richter S, Griebler C (2009) First attempts towards an integrative concept for the
ecological assessment of groundwater ecosystems. Hydrogeol J 17:23–35
Tomlinson M, Boulton AJ, Hancock PJ et al (2007) Deliberate omission or unfortunate oversight:
should stygofaunal surveys be included in routine groundwater monitoring programs?
Hydrgeol J 15:1317–1320
Part VI
Cave Ecology for the Twenty-First Century
Chapter 24
Where Angels Fear to Tread: Developments
in Cave Ecology
William F. Humphreys
24.1 Introduction
The preceding chapters describe the state of knowledge of cave ecology across a
wide range of habitats and geographies. They address the substantial advances made
over the last several decades in our understanding of subterranean biology generally
and of subterranean ecology in particular, and many identify outstanding areas of
research specific to their subject that need to be addressed. However, we are still far
from understanding such fundamental issues as adaptive mechanisms, species dis-
tribution patterns, trophic interactions and ecosystem functioning.
The advances have been made, variously, by fundamental reappraisal of accepted
theory (Pipan and Culver 2012) and paradigms (Culver and Pipan 2014) or as a result of
novel applications of established methods in both laboratory and field experiments; new
experimental approaches; large-scale, interdisciplinary collaborative studies; and the
application of molecular genetics (Griebler et al. 2014). The latter, especially, has
benefitted hugely from a culture of open-access data—often driven by funding agencies
and journal publication guidelines—such as GenBank and others within the Inter-
national Nucleotide Sequence Database Collaboration—and by ProteomeXchange for
proteomics data (Vizcaíno et al. 2014). This trend of increasing openness in scientific
culture is seen also in the numerous biodiversity data bases such as Global Biodiversity
Information Facility (GBIF; Robertson et al. 2014) and numerous specialised data bases
(https://en.wikipedia.org/wiki/List_of_biodiversity_databases) including the IUCN Red
List for conservation status. Likewise, the availability of standardised procedures and
shared data storage of ecological data will also accelerate ecological understanding
“Where angels fear to tread” was first written by Alexander Pope in his 1711 poem “An Essay on
Criticism”
W. F. Humphreys (*)
School of Biological Sciences, University of Western Australia, Crawley, WA, Australia

https://doi.org/10.1007/978-3-319-98852-8_24
498 W. F. Humphreys
although its broader scope presents much greater challenges (Reichman et al. 2011;
Thomas et al. 2012). This progress will build upon itself with further application of
current methodological, technological and analytical advances to test hypotheses in
subterranean ecology, but we will also see the application of emerging techniques to
the same end.
In this chapter, I have selected some emerging or recently emerged techniques
and equipment and suggest how they may be applied to advance subterranean
ecology. The choice is, of course, idiosyncratic, but I hope by so doing to encourage
multidisciplinary collaborative studies (Wicks and Humphreys 2011) using a wider
variety of techniques to advance understanding of subterranean ecology.
24.2 Methodological and Technological Innovations
A few methods and technologies have been game changers for subterranean ecology,
as they have in other areas, but some have addressed issues specifically pertaining to
subterranean ecology, to single out a few amongst the former: stable isotope ratio
analysis permitting both the source and pathways of energy supporting subterranean
ecosystems (Martínez del Rio et al. 2009), the analysis of continental- and regional-
scale distributions (Christman and Culver 2001; Culver et al. 2003, 2006) and
molecular methods assisting the recognition of cryptic species and informing
about the ecological and biogeographic history of the species present (Juan et al.
2010; Jurado-Rivera et al. 2017). Those techniques specifically addressing subter-
ranean studies include sampling the MSS (Juberthie et al. 1980; see also Chap. 3)
which promoted extensive development of research in shallow subterranean systems
(Culver and Pipan 2014), the invention of the Bou-Rouch pump for sampling
hyporheic and stygofauna habitats in sediments (Bou and Rouch 1967; Hunt and
Stanley 2000), sampling fauna from drips in caves to study the biogeography of the
fauna of epikarst (Culver et al. 2012a; Pipan 2005) and cave diving, especially in
anchialine ecosystems, later enhanced by the use of rebreathing apparatus (Iliffe and
Bowen 2001).
24.3 Natural History: Basic Biology
The natural history of many European and some North American and Japanese
troglobionts has been studied by generations of scientists and amateur naturalists—citizen
scientists. Together, they have established a broad background to the natural history of
troglobionts, knowledge well beyond that of the diversity and distributional information
lately being recorded in other parts of the world. It is hard to envisage such natural history
information emerging elsewhere in the context of contemporary social and scientific
funding mores. For example, the enormous increase in knowledge of the biodiversity and
phylogeography of the Australian subterranean fauna has not been accompanied by any
great increase in natural history knowledge. Indeed, there is probably not a single
24 Where Angels Fear to Tread: Developments in Cave Ecology 499
subterranean species in Australia where the basic life history parameters have been well
established—unlike in Brazil where single-species studies are still pursued (Gnaspini
1996; Moracchioli 2002; Bichuette and Trajano 2003; Trajano and Bichuette 2008). In
consequence, the general paradigms drawn from the Northern Hemisphere are widely
applied to the Australian subterranean fauna despite so doing with other fauna, such as
birds, having shown to be totally misleading (Christidis and Boles 2008). Indeed, the late
blossoming of subterranean biology in Australia may be attributed to the application of
the contemporary northern paradigms to the Southern Continent leading to attention
being focussed on karst and cooler, wetter areas and those most recently influenced by
glaciation, rather than arid and tropical regions now known for their globally exceptional
subterranean biodiversity. The lack of basic natural history is a serious impediment to the
development of ecological research in those areas where subterranean biological research
has developed more recently. The compilation of information on subterranean natural
history and its formal publication remains a critical issue in developing subterranean
ecology across much of the globe. While there may be few outlets in mainstream
publications, it is facilitated by dedicated journals such as Subterranean Biology and
online publications such as Speleobiology Notes.
24.4 Enumerating Numbers and Movements of the Fauna
Emerging and improved technologies that permit individual animals to be identified

and tracked are important aids to ecological studies and biodiversity conservation
(Pimm et al. 2015), but they are often inappropriate, largely due to size, for
application to research on the invertebrates typical of subterranean ecosystems.
Mark-release-recapture is a routine method of gathering data on population size
and movement of animals, and marking individuals, rather than batches, provides
data of much greater resolution. The method is limited by the ability to unambigu-
ously mark individuals, and this becomes increasingly difficult with smaller indi-
viduals. Bee tags have been used to mark queen bees by apiarists, and the scope of
this method has been greatly extended by the availability of individual microdots
(0.5 mm diameter) containing up to 26 characters of information (Whitehead and
Peakall 2012) and which have been successfully applied to 9 mm parasitoid wasps.
This inexpensive method is widely applicable if there is a suitable non-toxic
adhesive—due to the slight sclerotisation and greater permeability of troglobionts
than epigean species (Hadley et al. 1981; Robinson 2005; Vittori et al. 2012), toxin
penetration is potentially a greater issue for cave studies.
Progressive improvement in the size, cost and diversity of microsensor techno-
logy is making available a range of sensors that will allow for the identification and
movement of individuals; indeed, it may also extend to the detailed recording of
microenvironments. Currently the size (2.5 mm2) and passive response means the
use of microsensors is restricted to species the size of honeybees and depends on the
carrier returning in close proximity to an RFID (radio frequency identification)
reader. Research is scoped to greatly reduce the size of the chips to permit the
tagging of mosquitoes, to monitor environmental conditions such as gases or
500 W. F. Humphreys
temperature and to derive power from the locomotion of the arthropod carrier to
transmit information allowing monitoring from a greater distance (https://blog.csiro.
au/tiny-technology-creates-a-buzz/). Amongst the questions such technology could
resolve is the degree to which cave animals utilise the meso- and microvoids and the
attributes of these environments. The application of any of these techniques to
troglobionts is likely to be more favourable than when used on epigean species
because the marks have the potential to persist for longer, and recaptured rates
should be much greater and represent a greater proportion of the population. This
is based on the expectation that cave animals are long-lived and slow growing (but
see Vernarski et al. 2012), have modest population sizes and live in clearly bounded
habitats (but see Trontelj et al. 2009).
Population size and structure are fundamental attributes affecting the ecology of
species and also their evolution because both genetic drift and the phenotypic effect
for efficient selection are inversely related to population size (Koonin 2015). Sub-
terranean habitats, however, are often difficult, even impossible, to access and
sample to obtain estimates of population size, and consequently such estimates in
aquatic and terrestrial subterranean habitats are few (Jugovic et al. 2015). This is
rather surprising given the oft repeated claim of rarity in subterranean animals and
the relationship between the size and variation in populations and risk of extinction
(Legendre et al. 2008). Conversely, the utility of population size as a measure of
endangerment against Red List criteria (Cardoso et al. 2011) has been challenged as
inappropriate and impractical for invertebrates.
Jugovic et al. (2015) estimated Troglocaris anophthalmus populations of above
1000 individuals in a 180 m2 lake in Vipavska jama using mark-release-recapture
(MRC, marking by amputation) but with wide confidence intervals and, unsurpris-
ingly, detected no seasonal variation. MRC (marked using fluorescent pigments) was
used to determine if cave populations of millipedes (Stygiochiropus communis) and
schizomids (Draculoides vinei) were sufficient to sustain experiments; analysis using
Bayesian statistics indicated that the apparently modest populations of both species in
two small caves in Australia proved to have unexpectedly large populations
(Humphreys and Shear 1993; Humphreys et al. 1989) indicating exchange between
the cave and the mesovoids in the limestone. Simon and Buikema (1997) reported
densities of the isopod Caecidotea recurvata (marked by staining) of up to 74.6 m2
in moderately polluted cave pools. Knapp and Fong (1999) estimated a population of
amphipods (marked with ink) in a headwater cave stream of about 3000–4200
individuals. However, many, perhaps most populations in subterranean systems are
more rarely encountered leaving too few individuals to achieve adequate recaptures
following marking, or else the species has attributes making it inappropriate or
impossible to mark. This can potentially be resolved using genetic criteria to establish
the effective population size (Ne) of the subterranean populations using a sample of
the individuals and achievable using a number of estimators (Do et al. 2014).
However, owing to the effects on gene flow of the patchy nature of populations and
subterranean habitat (Bradford et al. 2013), such analyses could be enhanced, as in
another context (Hillary et al. 2018), through the application of a close-kin mark-
recapture framework and demographic models to estimate actual populations.
Population estimates could also potentially be complimented by the use of (e)DNA,

such as being trialled in lake fisheries (http://dna-barcoding.blogspot.com.au/2016/
01/edna-to-measure-fish-abundance-in-lake.html), and which proved useful for the
olm (Proteus) in confined but inaccessible subterranean systems (Gorički et al. 2017).
24.5 Sampling of Aquatic and Terrestrial Animals
Laser scanning (Azmy et al. 2012), thermal imaging (Betke et al. 2008) and remote
infrared cameras can be useful for sampling undisturbed populations in caves, and
the latter have been successfully used for a prolonged period both for public
education and for ecological studies of the southern bent-wing bat Miniopterus
schreibersii bassanii, in Australia (Codd et al. 2003). Mini-video cameras have
been used to describe groundwater habitats (Niederreiter and Danielopol 1991),
but high resolution of digital cameras and low-cost mass storage of digital data
now make possible the observation of even quite small species over prolonged
periods. Provided with a suitable power source, such cameras could be a useful
adjunct to other methods in behavioural ecology, for example, category and period-
icity of behaviours and the frequency of interactions within and between species.
Considerable research into subterranean faunas now occurs in habitats only accessi-
ble through bores (tube wells) (Danielopol 1989; Humphreys 2001, 2008; Eberhard
et al. 2009). Although dedicated shallow bores may be established for a research project
(Danielopol 1984), deep bores used to sample subterranean fauna—with notable excep-
tions (Christner et al. 2014) owing to their high cost—have typically been established for
other purposes (water supply, seismic survey, mineral exploration or orebody delinea-
tion), and they may or may not be wholly or partly cased (lined). The natural vertical
location in the substrate of individuals sampled from bores is typically uncertain because
sampling is often conducted by hauling a phreatobiological net through the water
column or by traps. In both cases individuals may have moved naturally or accidentally
down the tubes, else swum up the bore from a deeper aquifer. While a number of
methods have been developed to accurately locate samples in relatively superficial
bores, both for aquatic (Hahn 2005) and terrestrial samples (Lopez and Oromí 2010;
Rendoš et al. 2016), accurate location in deep bores has rarely been achieved. More
recently, techniques such as nested bores and Snap Samplers® (ProHydro, Inc., Fairport,
NY) allow accurate discrete interval sampling (Parker and Clark 2002), while pneumatic
syringe sampling can provide simultaneous sampling at 5 cm vertical intervals (Seymour
et al. 2007) (Fig. 24.1). Sorensen et al. (2013) deployed packers in dedicated boreholes
to isolate segments of the bore at defined geological features to investigate both the type,
location and density of stygobiont populations in Great Britain. This allowed them to
determine that invertebrates and microbes were more abundant in boreholes than in the
aquifer that bores were sites of enhanced biogeochemical cycling and that in the chalk
aquifer, the ecosystem extended from the surface to a water depth of 70 m. Such
collaborative research mobilising the resources of a major agency (in this case the
British Geological Survey) comprising at least hydrogeologists and ecologists and the
502 W. F. Humphreys
Fig. 24.1 Snap Samplers® can be chained to take simultaneous samples of undisturbed water from
different depths. Image courtesy of Sandy Britt, ProHydro, Inc. (left); pneumatic sampler taking
simultaneous samples at 5 cm intervals over a depth of 3 m (Seymour et al. 2007). Photo by
© William F Humphreys (right)
use of packers, or bores to dedicated depths, will greatly enhance understanding of the
ecology of deep aquifers.
24.6 Sampling of Fauna in Anchialine Systems
Stygobionts have long been collected from emergent streams and springs in conti-
nental waters (Rouch and Carlier 1985; Spangler and Barr 1995; Danielopol et al.
1997; Halse et al. 2002; Galassi et al. 2014), including from submerged springs
(Hutchins et al. 2013). The recent collection of stygobionts from surface anchialine
springs, which are possibly rare, revealed several species of decapod larvae, identi-
fied using the CO1 gene, as well as unexpected genetic diversity and biogeography
(Weese et al. 2016). The revelation of this obligate hypogean component of
anchialine pool-dwelling shrimps will prompt further efforts to sample comparable
habitats that should provide new insights on the ecology of anchialine systems
globally. Anchialine systems have been characterised as subterranean estuaries
(Bishop et al. 2015) which loosely link them with the chemical estuaries discharging
as submarine groundwater discharge (SGD) (Moore 1999). Discharge from the latter
can be substantial, being estimated to average 4.5 m3 m1 day1 along the 86 km
shoreline of Christmas Island, Indian Ocean (Humphreys 2014), a carbonate-cover
island (sensu Mylroie et al. 2001). Where discharge occurs through submarine
springs, rather than diffused through sediments, there is potential to sample adults
and larvae of any metazoan that may be associated with subterranean estuaries. The
suitability is unknown for sampling of submarine groundwater discharge at springs,

known as wonky holes, arising in drowned palaeochannels in the Great Barrier Reef,
Australia, up to 60 km offshore (Stieglitz 2005, 2016).
24.7 Remotely Operated Vehicles (ROVs) in Air, on Land

and in Water
Although ROVs—drones, dare I mention them—may seem inapplicable to cave

ecology, they do have potential utility in caves both to gain access and reduce
disturbance to cave ecosystems, especially compaction due to the passage of people,
and to be guided remotely, by tether, or autonomously. Starting as a novelty and
becoming progressively smaller, more sophisticated and customised, drones will no
doubt be exploited in caves in a number of applications, such as accessing otherwise
inaccessible areas—variously too small, dangerous, difficult or time consuming—to
observe, enumerate or sample fauna, substrates or the environment. Indeed, 3D point
cloud maps within a cave generated by 2D laser scanner mounted in a micro-
quadcopter have already demonstrated their utility and precision (Kaul et al.
2016). Drones moving across surfaces by means of appendages, inching, tracks or
wheels have similar potential and could be especially useful for confined space
photography and manipulations such as setting up experiments in inaccessible sites
to protect them from interference. Propeller-driven drones are applicable to both
aerial sites, for which a plethora of models is available, and aquatic sites for which
drones are still few and under development. Control of the latter is typically via
tethers (e.g. trident www.openrov.com; http://fathomdrone.com/) which is challeng-
ing in confined systems, but they do have great potential for recording and sampling,
and they still possess limited mission capability (video and depth) (Fig. 24.2).
However, the autonomy (https://ibubble.camera/) and environmental recording
capability (http://www.hydromea.com/) of underwater drones are being developed
rapidly, and they will especially facilitate sampling in highly stratified water col-
umns, such as anchialine systems, that may be disturbed by divers and at depths too
great to dive. A video and depth recording drone was deployed in Hranicka Abyss,
Czech Republic, to a depth of 404 m, well beyond the depth limit of the diver
(K. Starnawski at 265 m) (http://phys.org/news/2016-09-explorer-deepest-underwa
ter-cave.html).
24.8 Diversity of Subterranean Fauna
There is a profuse literature on the diversity of troglobionts, and, as recently as the

turn of the millennium, subterranean sites were considered as biodiversity ‘hotspots’
having as few as 20 troglobiont plus stygobiont species (range 20–84, n ¼ 18 caves
504 W. F. Humphreys
Fig. 24.2 Clockwise from top left. 1 TILT Ranger (http://inkonova.se/tilt-ranger/) is rugged, flies,
climbs and swims and can carry a payload (https://www.youtube.com/watch?v¼X5TfsJWz7RY);
2 Trident underwater remotely operated vehicle (ROV) is compact, 410 mm long, and dives to a depth
of 100 m with a 3–4 h duration (https://www.youtube.com/watch?v¼rnVpXb_1RhA; https://www.
openrov.com/products/trident/#specs); 3 Small underwater ROVs are being fitted with equipment
racks that will increase their task diversity (https://www.fathomdrone.com/); 4 Aerial drones, such as
Micro Drone 3.0, can be tiny, avoid collisions and fly into fine spaces to take stabilised HD video
(https://microdrone.co.uk/products/micro-drone-3plus, https://www.google.com.au/search?q¼Micro
+Drone+3.0.+Stabilised+HD+video.&rlz¼1C1CAFB_enAU610AU610&oq¼Micro+Drone+3.0.
+Stabilised+HD+video.&aqs¼chrome..69i57.647j0j8&sourceid¼chrome&ie¼UTF-8)
and wells—sites, Culver and Sket 2000). Of these only seven caves contained more
than 25 troglobionts (26–36 species) and nine with >25 stygobionts (27–48 species)
(Culver and Pipan 2013) at sites that encompassed carbonate karst, lava caves and
chemoautotrophic systems. Many other types of subterranean environments, not
always shallow subterranean systems (Culver and Pipan 2014), also support high,
even exceptional, levels of troglobiont diversity. Ten lava caves on the Canary
archipelago have more than ten cave-adapted species (range 11–37 species) (see
Chap. 17). For perspective, Mammoth Cave (Kentucky, USA) has 26 troglobiont
species in more than 540 km passages.
The diversity of subterranean fauna has been considered to be the greatest in the
temperate regions, particularly the Northern Hemisphere on account of the presence of
extensive limestone formations, especially the Dinaric karst region. It has been proposed
that a mid-(northern) latitudinal ridge of biodiversity exists (at about 34 N in North
America and 42–46 in Europe) and that this pattern of distribution reflects something
fundamental about cave ecology—reflecting long-term surface productivity at these
latitudes (Culver et al. 2006)—rather than merely the distribution of favourable habitat.
Indeed, Culver and Sket (2000) were puzzled by the scarcity of high diversity
caves in the tropics, and this issue was partly addressed by Deharveng and Bedos
(2012) who showed that the tropical subterranean fauna can be modestly diverse,
reporting between 3 and 28 obligate subterranean species from 17 caves in Southeast
Asia. Similarly, hotspots of cave species from the Neotropical region have been
reported from Brazilian limestone caves containing 28 and 22 species (Silva and
Ferreira 2016), a much greater diversity than found in Brazilian iron ore caves
(Souza-Silva et al. 2011), although the extent of the latter means they contain a
high number of troglobionts.
Recently sampling of highly diverse subterranean faunas in atypical,
non-carbonate substrates lacking karst and surface access, located in the arid tropics
and subtropics, has raised fundamental questions about the biogeographic patterns
described above and the ecological requirements for subterranean ecosystems. On
the Western Shield of Australia, subterranean habitats are only accessible through
boreholes, but rich subterranean faunas occur in a wide range of unconventional
habitats, such as groundwater calcretes, and Precambrian-banded iron formations
(BIF) and its derivatives (Halse and Pearson 2014) such as fractured rock, scree,
valley fill, river gravels and goethite pisolites. Investigations for the development of
a proposed uranium mine at Yeelirrie, Western Australia (27 180 S), recorded at
least 70 species of stygofauna and 45 species of troglofauna (115 subterranean
species in total) mostly from groundwater calcretes, with one bore contained
27 stygobiont species with another 13 bores contained 10 or more species
(Bennelongia 2015b; Subterranean Ecology Pty Ltd. 2011). Seventy-eight species
of stygobionts are reported from the Ethel Gorge calcrete (23 180 S, within the
tropics) (Bennelongia 2015a), now a threatened ecological community that was
originally assessed during the development of an iron ore mine in the Pilbara region
of Western Australia (Eberhard and Humphreys 1999). Elsewhere in the Pilbara,
54 stygobiont species have been collected from a single borehole (known as PSS016,
~21 180 S; Halse et al. 2014). Halse and Pearson (2014) claimed to have collected
549 troglofauna species or morphospecies in the Pilbara working in 65 sample areas
mostly smaller than 10 km2 and 90% located within the tropical Pilbara. The efforts
of numerous other environmental consultancy firms have certainly produced many
additional species to those collected by Halse and Pearson (2014) [e.g. 21 species of
troglobionts were described by Harvey et al. (2008), Baehr et al. (2012) and Smith
et al. (2012)]. In the same area, Halse et al. (2014) recorded about 350 species of
stygofauna. Hence, in the space of several years, sampling a small fraction of the arid
tropical Pilbara has yielded approaching 1000 species of subterranean fauna.
The generality of the Australian findings to other continents, especially the
Gondwanan terranes, remains to be established. Iron ore bodies in Brazil contain
fauna in caves (above), but whether the fauna is more deeply infused within the
orebodies and detrital fields remains open. Similarly, although groundwater calcretes
are widespread in arid areas, especially in South Africa, Namibia and Botswana
(Shaw and de Vries 1988; Weaver et al. 1993; Pickford et al. 1999), there have been
no reports there of diverse subterranean faunas either below or above the water table.
Globally, comparison will only be made by the widespread sampling of subterranean
506 W. F. Humphreys
habitats accessed through boreholes while having an open mind as to which sub-
strates are suitable subterranean habitats. The consensus that the main subterranean
habitats are in carbonate karst, lava fields and submerged granular sediments (Sket
2008) is much too restrictive. On the Western Shield of Australia, connected voids in
any substrate are significant habitats of subterranean biodiversity.
24.9 Metagenomics and Environmental DNA (eDNA)
Metagenomics has emerged as a powerful tool that can be used to analyse genetic
material recovered directly from environmental samples. The field is most fully
developed as microbial metagenomics on account of its utility to identify microbial
lineages regardless of the ability of member organisms to be cultured in the labora-
tory (Allen and Banfield 2005; Thomas et al. 2012), but it is increasingly being
developed as a tool for general biological survey and monitoring. An excellent
primer on the use of eDNA for aquatic biodiversity and biomonitoring surveys is
provided by Shaw et al. (2017). However, the full impact of eDNA on subterranean
ecology is yet to be developed; when eDNA-derived data are combined with
compound-specific stable isotopes and stable isotope probing, it has the potential
to progress from community genomic data sets to the knowledge of gene expression
and proteomics to reveal how species contribute to the net activity of the community
(Allen and Banfield 2005; Dumont and Murrell 2005).
Environmental DNA―eDNA―is a DNA shed into the environment by its living
or recently dead inhabitants. It is normally present at extremely low concentrations
but can be detected using PCR amplification and then characterised. Conversely,
both detection and identification can be performed directly from the water using
microarrays containing the appropriate molecular probes. While eDNA of aquatic
systems is most commonly analysed using samples of water, it is more concentrated,
sometimes by several orders of magnitude, and more persistent in sediments (fish:
Turner et al. 2015). Consequently, there may be potential to detect cave fauna from
DNA in soil samples. While eDNA from sediments is more detectable, it is probably
inappropriate to determine contemporary occupancy from this source.
eDNA is potentially useful in subterranean ecology in characterising the taxo-
nomic composition of the ecosystem, particularly owing to the rarity of the fauna
(or low abundance), or where the system is only remotely and sparsely accessible,
such as aquifer ecosystems. In parallel with species richness estimates, this could
provide a measure of how completely the taxonomic assemblage in an ecosystem/
habitat has been sampled and how many of the indicated taxa are known species and
the taxonomic load remaining to be addressed in that ecosystem. At the community
level, even if a ‘taxon’ has been placed only at an unknown terminal in a molecular
phylogeny, generated say from GenBank, it may provide information pertinent to the
trophic structure of the community. However, the utility of eDNA in initial broad-
scale biological survey is limited owing to the difficulties in characterising the
species involved. In addition to providing information on species diversity and
species composition in subterranean systems, when combined with metabarcoding,

eDNA has the potential to provide information, at low cost and from the same
samples, on nutrient cycling, ecosystem health and connectivity (Thomsen and
Willerslev 2015).
Perhaps the greatest potential for eDNA is for ecological monitoring, especially
in groundwater systems, both in terms of efficacy and efficiency, permitting consid-
erable improvements in confidence of the results. Let me expand on one such
circumstance that associated with stygofauna in the Pilbara region of Western
Australia where, if there are likely to be ‘significant stygofauna or troglofauna
values’, subterranean fauna surveys are required to be undertaken associated with
proposed major resource developments (EPA 2013). Forty stygofauna samples from
at least 10 bores (and 60 troglofauna samples from at least 30 bores) are required to
be sampled across the impact zone of the development—it includes an interim
recommendation for troglofauna sampling from at least 60 samples from 30 bores
(EPA 2007). The aim of the process is minimal, namely, to ascertain if any species
are restricted to the developmental footprint in order to guide the environmental
review process. If subterranean fauna is present and species are found to be restricted
to the footprint of the development, then the project may not gain approval, or else
some method of mitigation may be required to limit the threat to those subterranean
species, and ‘a monitoring process may be enshrined’ under the ministerial condi-
tions allowing the project to proceed.
Without going into the problems associated with arriving at the requirement to
undertake monitoring (e.g. Karanovic et al. 2013; Halse et al. 2014), which needs a
lot more ecological understanding, subsequently there are at least three parties with
an interest in the veracity of the monitoring, namely, the regulators (Environmental
Protection Authority and public; EPA 2013), the mining tenement holder (owner)
and any environmental consultants engaged to do the monitoring. Each would
benefit in their own way by having accurate information from the monitoring as it
would reduce risks as they affect each party. As stygofauna are mostly rare and
sparsely accessible, direct monitoring, as outlined below, poses possibly insur-
mountable difficulties.
The Pilbara has an extraordinarily diverse subterranean fauna (Eberhard et al.
2009; Halse et al. 2014), and, as elsewhere, additional species continue to be collected
with increased sampling effort. For example, one sampling event (six net hauls in one
bore) collected 33% and six samples 82% of the species known to be present.
However, as only about 60% of the species in the region were known—based on
rarefaction statistics—these initial captures represent about 20% and 50%, respec-
tively, of the species probably present in the system. ‘Currently, it is unusual for bores
in Australia to be sampled more than twice in . . . environmental assessment
programmes . . .’ (Eberhard et al. 2009). Sampling bores at a rate to detect even half
the species would deplete the fauna while providing no party with the confidence they
need in the veracity of the data. Indeed, it is worthless as a method of detecting
temporal change in the stygofauna―detecting risk of extinction―the purpose of
monitoring.
508 W. F. Humphreys
Conversely, eDNA taken from pumped water samples potentially could detect the
entire suite of species in the community at a single pass without depleting the fauna.
Should the method prove suitable, it would be most effective if used to monitor only
those species known to be present using custom microarrays—additional molecular
probes could be added as additional species are detected. The successful develop-
ment of eDNA methodology in this context could provide a high degree of certainty
to all parties involved in the rather limited ambition of the monitoring but one that
currently imposes considerable costs but yields no effective conservation outcome.
Owing to the potential for greatly increased certainty, efficacy and efficiency, the
development of eDNA methods for monitoring groundwater systems is likely to
attract considerable research effort that could lead to substantial improvements in the
technique and would be applicable across a broad range of ecological research and of
global import.
24.10 Determining Predator-Prey Relationships
Bradford et al. (2013) used molecular methods to detect prey items in the guts of
three different sized sympatric species of subterranean diving beetles in a simple
aquifer ecosystem in the Australian arid zone and showed differential prey choice
between the species. In more complex systems, in which the target prey may be
unknown, PCR amplification may be undesirable owing to amplification biases and
cross reactivity with predators and with related species genomes. These potential
problems may be overcome using alternative methods, for example, those applied to
epigean beetles by Paula et al. (2014) who used PCR-free direct shotgun sequencing
of total DNA isolated as a time series from harlequin beetle guts and then identified
prey items by matching sequences to reference data bases. By this means, they were
able to reveal the dynamics of the complex interactions amongst predators, prey and
their symbionts. Methods such as these, especially when combined with various
stable isotope methods, such as the emerging compound-specific stable isotope
techniques, can do much to inform on food webs, food selectivity and niche
segregation in subterranean systems in which direct observation is typically not
possible.
Despite the elegance of these approaches, it may be still being problematic to
distinguish predation from scavenging on carcasses, owing to the long duration that
DNA derived from cadavers may be detected in gut contents, as was found in soil
insect predator-prey systems (Juen and Traugott 2005). This is an issue of special
relevance to subterranean ecology because many troglobiont predators opportunis-
tically scavenge, as pointed out by Racovitza (1907) and widely reiterated (Culver
2012). However, the transition to other feeding types in epigean and troglobiontic
lineages may occur in other ways (Poulson 2012); various trogloxenic opilionid
species typically scavenge, while the troglobiotic forms are more commonly pred-
ators (Hobbs 2012).
The truncated nature of subterranean biodiversity in both functional and evolu-

tionary aspects results from the typical absence of primary production (in the
absence of chemoautotrophy) and the scarcity of top predators (Gibert and
Deharveng 2002). However, many authors note the high prevalence of predators
amongst troglobiont communities, a disparity that may have resulted from (Gibert
and Deharveng 2002) emphasis on aquatic systems. Oromí (Chap. 17) points to the
‘remarkably high diversity’ of predators amongst the troglobiont community in
Tenerife, a total of 37 predatory species, nearly three quarters of the 50 troglobionts
recorded. In addition, he notes the diversification there of lineages, especially the
nine species of Dysdera, with up to five species per cave. This is analogous to the
stygobiont diving beetles in Australian calcrete aquifers which have up to four
species in a single borehole (Watts and Humphreys 2009), even three sister species
(Guzik et al. 2009).
Not only is the main taxonomic composition of subterranean systems funda-
mentally different from the epigean systems, but the trophic and biodiversity struc-
ture may differ between aquatic and terrestrial subterranean systems. Such
differences may have profound or subtle influence on the sensitivity of subterranean
ecosystems to, for example, the dynamics of trophic cascades (sensu Ripple et al.
2016) which may be dampened (Finke and Denno 2004).
24.11 Are Parasites and Commensals of Subterranean

Fauna So Rare?
These two categories have rarely been reported for subterranean species (Culver
2012), and this may be an accurate reflection of the rarity of these interactions for
ecological reasons pertaining to the subterranean habitat. It may to some extent,
however, be the result of under-sampling owing to the relative scarcity of individuals
of subterranean species. A sulphur-oxidising filamentous bacteria—a Thiothrix
phylotype—lives only as an epibiont on Niphargus amphipods in the sulphide-rich
Frasassi Cave complex in Italy (Dattagupta et al. 2009). Surprisingly, there is a
possible evidence of chemosynthetic ectosymbiont microbes on Xibalbanus
tulumensis, a member of the Remipedia which are considered to be top predators
(Pakes and Mejia-Ortiz 2014). Firmer evidence has been presented for the atyid
shrimp, Typhlatya pearsei, which harbour endosymbiont mutualistic bacteria and
show host-mediated adaptations both to the symbionts and to sulphide toxicity in
their extreme anchialine environment (Pakes et al. 2014). The latter example is
especially interesting for both current and palaeoecology because some clades within
Typhlatya diverged more than 100 Mya (Jurado-Rivera et al. 2017). It is unknown
whether such endosymbiosis is characteristic of the genus or else an ecotype induced
in response to sulphidic water present in some anchialine systems. If such endosym-
biosis is a characteristic of Typhlatya, then the phylogeography of the microbial
510 W. F. Humphreys
consortium may be concordant with that of the host or else driven by host ecology in
response to sulphidic or other ecosystem inhabited by a given species of Typhlatya.
24.12 Ecotone and the Transition to Subterranean Life
The adaptation of an epigean lineage to subterranean life, which I will refer to as

trogloneogenesis, must involve the ecotone between the epigean and subterranean
realm. The photic environment across this ecotone was used as the basis of the
earliest classification of cave animals (Schiödte 1849; Racovitza 1907).
Caves and other subterranean habitats may come to be occupied by species
through active or passive means (Danielopol and Rouch 2012), but there have
been minimal studies of ecological processes in the entrance (twilight) zone of
caves (Hobbs 2012), and there are remarkably few studies examining the ecological
relationships between epigean and troglobiont species, such as the study of amphi-
pods by Luštrik et al. (2011). This type of study could be extended to examine
interactions between species of trogloxenes, troglophiles and troglobites.
The hypothesis that light is the prime environmental factor that separates the
surface from the subsurface realm (Pipan and Culver 2012), and the comprehensive
review of the wide variety of shallow subterranean habitats and their faunas (Culver
and Pipan 2014), has refocused attention on that ecotone. This has the added
advantage of facilitating experimental research owing to the diversity and accessi-
bility of shallow subterranean habitats and greater population densities. These
factors will lead to a proliferation of research into the ecological and evolutionary
processes that lead to the separation of surface and subsurface populations, of which
the ecology of light may make a significant contribution (Tierney et al. 2016). For
example, Fišer et al. (2014) reviewed limits to the vertical extent of groundwater
metazoans. Subsequently, they investigated how the response to light of ten
co-occurring pairs of surface and stygobiont amphipods in springs influenced habitat
choice and ultimately the geographic extent of subterranean amphipods (Fišer et al.
2016); light triggered habitat choice of the eyeless subterranean species but not of the
eyed surface species. No comparable multispecies study has been conducted on
troglobionts; however, millipedes of the highly troglomorphic genus Stygiochiropus
(Paradoxosomatidae) may be found in full tropical sunlight if the outflowing air
results in a condensing atmosphere, as in Papillon Cave in Cape Range, Australia
(Humphreys and Shear 1993). This indicates, as was suggested for surface amphi-
pods by Fišer et al. (2016), that habitat choice by troglobionts may also be affected
by factors besides light, in this case by humidity.
The extreme ecotone marking the transition from epigean to subterranean life has
always suggested a zone of marked selective pressure gradients. Yet there is
mounting evidence that the development of many troglomorphies arises by neutral
processes not selection (Wilkens and Strecker 2017). The common occurrence of
shallow subterranean environments will facilitate research in the evolutionary
process involved, a long contentious area of subterranean biology (Culver and

Wilkens 2000).
24.13 Invasive Species in Subterranean Systems
As climate change is considered the major driver of trogloneogenesis, the current

anthropogenic climate change is expected to increase the rate of species crossing the
ecotone between surface and subterranean realms. As we have seen above (Fišer
et al. 2014), the maintenance of ecotonal separation between surface and subterra-
nean amphipods was achieved by their response to light, the major signal at the
ecotone. But this tells us little about those fundamental questions in the discipline:
what are the drivers of trogloneogenesis, and how do species colonise already
inhabited subterranean spaces? The structural and numerical simplicity of subterra-
nean ecosystems would seem to provide a good model from which to explore the
broader ecological questions of ecotonal and niche transition, hampered perhaps by
the sensitivity and slow life of subterranean species. Although cave animals are
considered to have been derived from surface lineages which must have undergone
trogloneogenesis, examples are rare of even epigean species impacting on subterra-
nean lineages. Romero et al. (2002) recorded the replacement of troglomorphic
populations of the silver catfish fish, Rhamdia quelen, by epigean populations of
the same species in Trinidad. Some species have invaded extremophile environ-
ments such as Poecilia spp. that are prolific in the sulphide spring complexes at the
Baños del Azufre in Mexico (Plath et al. 2007). This has raised concerns about the
ability of P. reticulata, established in a karst window in Cape Range, Western
Australia, to adapt to subterranean life, including the sulphidic parts, and threaten
the rich anchialine fauna of the region. These examples show that invasive species
may possibly have direct ecological implications to subterranean fauna and ecolog-
ical studies are needed in order to inform potential mitigation measures. One such
study showed that invasive fish in anchialine pools in Hawaii preyed on ‘ōpae‘ula’,
the atyid shrimp Halocaridina rubra, and displaced the survivors to deeper more
cryptic depths (Havird et al. 2013).
24.14 Is Carbon Dioxide Concentration Significant to Cave

Ecology?
The thesis that areas with elevated levels of carbon dioxide—bad-air zones—in high
humidity cave passages would ‘be found to harbour unique communities of obligate
cave species’ (Howarth and Stone 1990) and that this zone may be the principal zone
present in mesocavernous cracks and voids (Howarth 1993) has been sustained in
the literature (Deharveng 1988; Deharveng and Bedos 2000; Stone et al. 2012,
512 W. F. Humphreys
Chap. 3), questioned (Humphreys 2012) but never formally been tested or refuted
despite its standing for more than a quarter of a century. This is surprising given the
implications that such a widespread zone would have on the ecology of subterranean
animals, affecting the evolution, habitat selection, physiology and energetics. It is
notable that the original paper does not provide strong support for the thesis as the
data from Bayliss Cave shows a marked increase in troglomorphic species coinci-
dent with a rise to near-saturated relative humidity before the region of elevated CO2
occurs. Thereafter, the number of troglobiont species decreases with increasing
concentration of CO2 (see Fig. 23 in Stone (2010) and Howarth (1988)). The high-
stress environment thesis proposes, amongst others, that elevated CO2 levels may be
a common feature of habitats in mesocavernous cracks and voids (Howarth 1993)
although no additional evidence is provided of the role of elevated CO2 in this formal
presentation. Few biologists routinely measure CO2 in caves, other than in show
caves for the purpose of conservation and public safety (Šebela 2011), and when
measured (Deharveng 1988), it is rarely associated with other parameters, such as
oxygen, to determine the source. Further, the carbon dioxide level of soil atmo-
spheres is typically 0.3–3% (Lavelle and Spain 2001); soils on karst may not be very
elevated (Czech Republic, annual variation of CO2 ~0.1–0.43%; Faimon et al. 2012)
but are similar to those in some species-rich tropical caves such as in Cape Range,
which had mean carbon dioxide concentrations of 0.18% (range 0.06–0.5%; s.
d. ¼ 0.16, n ¼ 7) (Humphreys 1989). In a single entrance cave, Trinh et al. (2018)
found seasonal variation in CO2 between 1000 and 8000 ppm by volume. The CO2
concentration resulted from mixing background atmospheric CO2 with CO2 pro-
duced from soil, water and cave biota including humans in show-cave sections. As in
most such studies, the focus of the research is on the influence of CO2 concentration
on karst processes. If sufficient data are available, a meta-analysis may help to
resolve whether carbon dioxide concentration is a significant issue in subterranean
ecology.
24.15 Understanding the Dynamics of Cave Climate
Although cave environments are often considered the epitome of stability, there are
regions within caves that are profoundly variable, and these relate to the size of the
openings, the distance from entrances and the weather outside. Caves breathe largely
owing to changes in barometric pressure and temperature, and the resulting mass
movement of air transfers heat and moisture into and out of a cave depending on the
vectors of the pressure differential. Even in the absence of air movement, caves may
lose or gain moisture owing to water vapour pressure gradients that cause excessive
drying of caves in the temperate winter and at night in the tropics when external
temperature falls below cave temperature (the tropical winter effect; Howarth 1980).
Recording cave climate is especially difficult in remote areas where changes are
frequent (diurnal) and remote (deep into cave in distance or time) or the magnitude of
change is great (closer to entrances). Cave climate is an important ecological factor,
but the recording of an adequate number of parameters at sufficient spatial and

temporal scales is challenged by the harsh environment and difficulty of access.
Recent advances in fibre-optical communication and sensors, mainly designed for
extreme subterranean environments, offer new opportunities (Ahuja and Parande
2012) which, integrated with wireless communications, could facilitate real-time
distributed monitoring throughout caves. This would enable recording of cave
climate and enable researchers to take advantage of rare events signalled from any
of a wide range of parameters (e.g. temperature, pressure, density, flow rate, etc.),
trace gases (De Freitas et al. 1982) or potential metabolic signals using biomedical
sensors (simultaneous measurement of CO2, O2, pH and temperature: Ferguson et al.
1997). Such information could provide the climatological context, at a range of
spatial scales, to fundamental cave ecology and applied ecology, especially micro-
bial ecology, for cave management of tourist caves and the preservation of archae-
ological sites, especially sensitive Palaeolithic art sites such as Niaux Cave, Ariège
(Andrieux 1990).
24.16 Cave Faunas as Indicators of Climate Change
Climate change has been predicted to have profound effects on karst systems
generally (Day 2011), and limestone caves themselves have been a major focus of
climate change studies, particularly using sediments and speleothems as retrospec-
tive proxies for climate change (Denniston et al. 2015, 2016) and correlative species
distribution models to the same end (Mammola and Leroy 2017). Climate change is
often invoked to explain the colonisation by epigean species of subterranean habitats
and their distribution, variously, due to the associated aridity, glaciation and changes
in sea level (e.g. Leys et al. 2003; Faille et al. 2013; Stock 1980; Moritsch et al. 2014,
respectively). While prospective studies of climate change on subterranean systems
are lacking, they are likely to be established in relation to subterranean biology as
they have been for other disciplines. However, it is important to recognise that the
entire subterranean communities may be at risk from climate change because
subterranean species have minimal chance of relocating and characteristically
forming communities comprising small-range species, precisely those species that
are at greatest risk from climate change (Ohlemüller et al. 2008).
Conversely, it has been posited that subterranean systems are exceptionally
resilient to climate change, and the evidence for this comes from the age of
subterranean lineages and their restricted dispersal (Faille et al. 2015) in addition
to the presumed property of being confined to their geological context by dint of their
adaptation to subterranean life (Humphreys 2009). Proposed for the anchialine
assemblage in Cape Range, Australia (Humphreys 2000a, b, c), the persistence of
subterranean lineages has been established more firmly by molecular clock dating
for numerous old subterranean lineages with small-range endemicity (Leys et al.
2003; Abrams et al. 2012; Humphreys 2017). In addition, some stygobiont lineages
have apparently persisted from the fragmentation of Gondwana (Poore and
514 W. F. Humphreys
Humphreys 1998; Wilson 2008) and would have endured extreme changes in
climate from rainforest to desert.
Temperature is relatively stable in subterranean habitats, and subterranean species
are presumed to be stenothermal (adapted to a narrow range of temperature).
However, some subterranean lineages adapt to a wide range of thermal conditions,
and there is no reason to posit that the ecophysiology of subterranean lineages is
evolutionarily fixed. Subterranean invertebrates occur at temperatures ranging from
ice caves (Coleoptera Leptodirini; Racovita 2000) to hot springs (48 C,
Thermosbaenacea; Barker 1959), and a given species may complete its life at
constant temperature in caves covering a temperature range of 11 C (1829 C;
Humphreys and Shear 1993). Cave temperatures are commonly close to the mean
annual surface temperature (Wigley and Brown 1976), and so, although buffered
from short-term temperature variation, the cave temperature will ultimately track the
climatic mean temperature. Consequently, old subterranean lineages will have
already been subjected to repeated and sometimes severe change in climate and
tracked profound millennial-scale changes in mean annual temperature (Humphreys
2000b). In the context of mitigation, the presumption may be unfounded that
protection from variation in temperature may be beneficial because local adaptation
may result from a complex interplay of ecological trade-offs depending on both the
absolute temperature and the temperature range as has been shown for rock-pool
copepods by Hong and Shurin (2015). Indeed, owing to their ubiquity and density in
subterranean waters and epikarst (Pipan 2005; Galassi et al. 2009), copepods are
likely to be good models for climate change adaptation in stygobionts.
24.17 Biofilms, the Drivers of Cave Ecology?
Knowledge of microbial communities in caves and other subterranean ecosystems is

rudimentary (Northup 2011) despite being an important, perhaps the major, source of
food available in some subterranean ecosystems. This applies both to aquatic and to
subaerial subterranean systems (Simon et al. 2003, 2007) where there is a similar
phylogenetic diversity on cave walls around the world (Engel 2012b). Biofilms serve
to concentrate organic matter by capturing dissolved organic carbon (Simon and
Benfield 2001) to form the foundation of groundwater food webs (Hartland et al.
2011), or else they are formed by sulphur-oxidising bacteria or microbes supported by
one or more of a plethora of other chemolithotrophic metabolic pathways (Engel
2012a). Although biofilms are dominated by microbes in both aquatic and subaerial
habitats, they can be components of complex and structured ecological communities,
comprising archaea, bacteria, protists, viruses and fungi, displaying a full array of
ecological processes (see Humphreys (2006) and Engel (2012b)). While much of the
energy captured may be consumed within the biofilm ecosystem itself, the biofilm
serves as the food base for the stygofauna community, and some taxa are specialised
to harvest it directly, such as harpacticoid copepods (Dahms et al. 2007) and some
atyid shrimps with their brushlike setae (Page et al. 2007). Hartland et al. (2011) used
emission-excitation matrix (EEM) plots of dissolved organic carbon (DOC) fluores-

cence (Baker and Spencer 2004) to follow changes along the water flow path to
explore the ecological significance of biofilms in New Zealand. They demonstrated
that changes in biofilm resulting from sewage-enriched groundwater had profound
effects on stygofauna communities—there were fewer species, larger populations and
greater dominance, greatly changing community structure with perhaps irreversible
consequences.
As discussed by Hershey and Barton (Chap. 5), recent advances in molecular
biology have profoundly changed the detail in which microbial ecology can be
studied in nature. The application of metagenomics—possibly extended into stable
isotope probing and the coupling of molecular biological methods with stable
isotope abundance in biomarkers (Radajewski et al. 2000)—provides cultivation-
independent means of identifying microbial function. Combined with the quantifi-
cation of gene expression using real-time PCR (qPCR) (Smith and Osborn 2009),
these approaches have greatly improved understanding of the diversity, distribution,
community structure and function of individual taxa. For example, Suzuki et al.
(2000) were able to determine the spatial and temporal quantitative difference in the
distributions of bacteria (cocci) and archaea in marine water, while Takai and
Horikoshi (2000) quantified archaea in deep-sea hydrothermal vents, hot springs
and freshwater sediments. Using 16S rRNA to distinguish the functional part of the
microbial community in hyporheic zone biofilms, Wagner et al. (2014) were able to
partition the effects of terrestrial dissolved organic matter (allochthonous DOM) and
that produced in situ (autochthonous DOM). They found that only allochthonous
DOM drove changes in community composition but that neither source of DOM had
any clear effect on the function of the hyporheic biofilms, suggesting this conferred
stability to the stream ecosystem. Such methods have great potential to contribute to
research on the ecology of biofilms in groundwater over a range of spatial scales to
elucidate the functional and dynamic role of biofilm in aquifer systems to provide the
secure foundation necessary to elucidate the functional role of stygofauna in aquifers
(Boulton et al. 2008). Studies of the human microbiome have revealed the structural
complexity of biofilms organised in microbial consortia at the micron scale (Welch
et al. 2016), an approach quite applicable to aquatic biofilm. The complex interplay
amongst the microbiota reveals their effect on human nutrition and susceptibility to
disease (Robinson et al. 2010), and, by analogy, such profound interactions are
likely to inform on the ecology of subterranean fauna, particularly stygobionts.
Prosser et al. (2007) argue that understanding of microbial ecology will be advanced
by a much greater application of theory to harness the predictive power from the
unprecedented accumulation of biological information from metagenomics and to
test the generality of ecological theory.
As a subset of microbial ecology, it is important to note that mycology of
subterranean ecosystems, as elsewhere, is sparsely studied, particularly that in
groundwater despite the diversity occurring there. Caves support an array of fungi,
yeast and slime moulds, with 1029 species recorded from caves and mines, but only
6% of these are from water, and these are mostly sampled from temperate regions
(Vanderwolf et al. 2013). Lategan et al. (2012) reported from 14 to 24 taxa per
516 W. F. Humphreys
shallow aquifer in eastern Australia, and Kuehn and Koehn (1988) recorded 25 gen-
era from the artesian Edwards Aquifer in Texas and foreshadowed their potential
significance in heterotrophic energy conversion. The application of the techniques
appropriate to prokaryotes to the fungi present in caves and groundwater will
accelerate and start to elucidate their functional role within subterranean ecosystems.
24.18 Biogeochemical Processes as Drivers of Cave Ecology
The finding of rich chemosynthetic microbial populations associated with deep

terrestrial systems (Stevens and McKinley 1995) and deep-sea hydrothermal vents
(‘smokers’; Deming and Baross 1993) has led to a flurry of related work on
subterranean systems, for example, in caves, including anchialine systems, in
Europe, Mexico and Australia, and deep aquifers in the USA (Engel 2012a).
Chemoautotrophy may be aerobic (oxidation of hydrogen, sulphur, iron, manganese,
ammonia or methane) or anaerobic (by methanogenesis, acetogenesis, denitrification
or the reduction of sulphate, iron or ammonia) with multiple co-occurring
chemolithotrophic metabolic processes reported from most systems—between
three and seven processes in about half of 23 systems (Engel 2012a, Table 2).
Investigations into chemoautotrophy in subterranean ecosystems are expected to
develop rapidly and lead to recognition of the diversity of energy sources in
subterranean systems and an understanding that energy sources may switch
according to the development stage of the individual and the ecosystem or show
periodic change according to the state of ecological development or on diurnal,
seasonal or climatic factors.
The presence of 13C-depleted biomass in cave-adapted fauna of Ox Bel Ha, an
anchialine system in Yucatán (Pohlman et al. 1997), indicated the presence of
chemoautotrophic pathways through methane-linked carbon cycling, as found in
the Edwards Aquifer, Texas (Bishop et al. 2014). Brankovits et al. (2017) presented
strong evidence that methane and DOC are indeed ecologically important parts of
the carbon cycle within the Ox Bel Ha anchialine ecosystem. Further, they showed
that rain provides external forcing of both the delivery and distribution of methane in
the system. Brankovits et al. (2017) achieved this outcome by means of an integrated
study combining water chemistry and fauna sampling, compound-specific stable
isotope ratio analysis (CSIA) and long-term continuous sampling of water using
electrically independent osmotic pumps. This study demonstrated the presence of
high concentrations of methane and methane oxidation in the freshwater section of
the anchialine system. Further, methanotrophic bacteria were a large part of the diet
of atyid shrimps as indicated by 13C-depleted acid biomarkers (13C values as low as
54.1%) and deuterium-depleted δD values of shrimp tissue (Brankovits et al.
2017).
This study highlights the benefit of using CSIA to determine the 13C signature
solely of the C16:C17 fatty acid component; phospholipid-derived fatty acids
(PLFA) are widely used as chemotaxonomic markers of bacteria. In contrast, the
regular SIA data for whole tissue of Palaemonetes antrorum from the Edwards
Aquifer, which had 13C as low 44 (Bishop et al. 2014), did not allow further
partitioning of the stable isotope signals for 15N, 13C and 34S. As such facilities
become more readily available, the increasing use of CSIA in subterranean ecology
may be expected to refine greatly the understanding of the range of trophic resources
and trophic networks within subterranean systems.
Secondly, the application of osmotic pumps (OsmoSamplers: Jannasch et al.
2004) to subterranean ecology signals the way forwards as they can provide contin-
uous sampling for as long as 2 years in hostile environments, even in deep-sea
hydrothermal vents (Wheat et al. 2000), independent of mechanical or electrical
systems, and achieving a temporal resolution in the order of 1 day. These attributes
make the samplers ideal to recover samples associated with rare and episodic
climatic events, such as found widely in arid regions (Humphreys 2006) that can
have such profound impacts on ecological systems (Davis et al. 2013). The long-
term nature and multidisciplinary approach of this exemplar study have allowed
profound insight into the functioning of this anchialine system and present a
benchmark for further studies on the ecology of subterranean systems.
24.19 Ecosystem Functions (Services)
The ecological functions provided by subterranean ecosystems are poorly

characterised and even less quantified in contexts other than microbiota and bio-
remediation work (Humphreys 2000c; Colwell and D’Hondt 2013). It is largely
unknown whether they have a role in hydrogeology and water quality (Herman et al.
2001) or whether groundwater invertebrates alone or in community (Griebler et al.
2014) are ‘ecosystem engineers’ helping to maintain aquifer hydrodynamics
(Boulton et al. 2008; Griebler and Avramov 2015). However, it has been demon-
strated that invertebrate bioturbation of sediments serves to maintain hydrologic
conductivity in clogged interfaces enhancing biogeochemical and microbial pro-
cesses (Nogoro et al. 2006). The role of soil perturbation in terrestrial systems in
driving ecological processes (Herrick and Jones 2012) is even less studied amongst
troglobionts than amongst stygobionts despite the conspicuous tunnelling and soil
pellet movement shown by many species, including earthworms, millipedes and
schizomids. There is still a dearth of information on these oft discussed but rarely
studied issues, and new experimental approaches are required to better understand
the contribution of groundwater fauna to ecosystem dynamics.
While bioremediation by microbes can be ascribed as monetary value under
ecosystem services models, the same cannot be done for groundwater invertebrates
but that does not mean their function has no value (Silvertown 2015). Analysis of the
dynamics of macroinvertebrates in moving carbon and chemotrophic energy through
subterranean ecosystems will provide the basis for understanding their functional
significance and an appraisal of the ecological services they provide for the ecosys-
tem and beyond. To this end, the recent paper by Smith et al. (2016) is enlightening
518 W. F. Humphreys
because they examined how stygobionts interact with the microbial community, an
especially cryptic area. They calculated that by hitch-hiking on stygobionts—an
undescribed neoniphargid amphipod—the numbers of prokaryotes transported were
up to five orders of magnitude greater and, most notably, moved 34 times faster than
in the surrounding groundwater allowing them potentially to move much further in
the bulk groundwater than those travelling by advection alone. How and which
microbes are transported within groundwater is crucial to understanding their role in
maintaining ecosystem health because prokaryote communities play a crucial role in
the turnover of biomass and energy and in purifying groundwater (Danielopol et al.
2003).
24.20 Age of Subterranean Lineages as Indicators of Past

Conditions
The age of subterranean lineages is principally of interest in the context of

phylogeography and evolution, but it is also pertinent to ecology because it may
help to characterise the ancestral ecological conditions. Cave animals have selective
similarities that lead to the ecomorph termed troglomorphs that most easily makes
them identifiable as cave animals (Culver et al. 1995). That the degree of
troglomorphism is related to the age of cave species has been contentious, but
Derkarabetian et al. (2010) established that it is possible to predict the taxon age
from morphology in troglobiontic Laniatores harvestmen (Opiliones) that diverged
from surface populations between 8.2 and 2.9 Mya. This finding that morphology
can be used to predict the age of subterranean lineages remains to be more widely
tested, but it is likely that the information in such character divergence would
become ‘saturated’ in older lineages.
Climatic vicissitudes during the Pleistocene (2.6 million to 11,700 years ago) had
long been considered the driving force isolating subterranean lineages underground
(Pleistocene effects model; Barr 1968; Barr and Holsinger 1985). In the last decade,
largely owing to molecular clock estimates, it is increasingly appreciated that
trogloneogenesis of most subterranean lineages occurred several million years ago
(see Trontelj (2007)), or even the late Miocene, driven by aridity (Leys et al. 2003),
or Oligocene (possibly the Eocene) but continuing into the Plio-Pleistocene
(Ribera et al. 2010; Faille et al. 2013). There is even increasing evidence that
some stygobiont species have even persisted beneath continental ice sheets
(Taylor and Niemiller 2016). Hence, the climatic conditions favourable to
trogloneogenesis may have been quite varied.
Despite the advances mentioned above, the age of hypogean fauna remains, as
always, ‘contentious’ (Humphreys 2000a), especially in respect of the hypothesis
that intercontinental vicariance results from the migration of tectonic plates. This is
despite evidence that microplate dispersal of subterranean lineages occurred in the
Miocene between Sardinia and Iberia at ~33 Mya (Juberthie 1988; Sbordoni 1982;
Ribera et al. 2010; Faille et al. 2011, 2013). The contentious area is the proposed
Mesozoic vicariance in the Tethys Sea (Wagner 1994), a debate now largely
associated with the core anchialine higher taxa of crustaceans such as remipedes,
thermosbaenaceans, some atyid shrimps (Typhlatya and close relatives) and some
thaumatocyprid ostracods (Iglikowska and Boxshall 2013).
Molecular phylogenies of varying coverage, taxon sampling density and gene
spread have been proffered as evidence pertinent to the hypothesis of interconti-
nental vicariance in anchialine stygobionts. Some proponents of intercontinental
vicariance (fish: Chakrabarty et al. 2012) have been challenged on the basis of
inappropriate calibration (De Bruyn et al. 2013) and molecular evidence (Larson
et al. 2013). Others (amphipods: Bauzà-Ribot et al. 2012) on the basis of calibration,
modelling rates across branches and substitution saturation (Phillips et al. 2013).
These critiques all suggest much shorter estimates of divergence times that are
inconsistent with the intercontinental vicariance proposed criticisms that have been
counterargued by Bauzà-Ribot et al. (2013). This debate cannot be resolved with
current data and has led to a joint project to reappraisal of the entire evidence using
greater taxon sampling and genomic methods (Jurado-Rivera et al. 2017). Resolu-
tion of the historical biogeography of anchialine faunas requires a multi-taxon
approach to seek congruent temporal and spatial patterns such as was started using
allozymes in the 1990s by Adams and Humphreys (1993) (Page et al. 2016) to
understand local biogeography. Already, the presence of some core anchialine taxa
on isolated seamounts in the Indo-Pacific—Halosbaena and Humphreysella on
Christmas Island (Humphreys and Danielopol 2005; Page et al. 2016) and
Halosbaena on Minamidaitōjima, Ryukyu Islands, Japan (Shimomura and Fujita
2009)—the topology of phylogenies (Iglikowska and Boxshall 2013; Hoenemann
et al. 2013) and the age of some taxa (Botello et al. 2012; Page et al. 2008, 2016)
indicate that the resolution will be multifactorial. The common ecology of at least
part of the ‘core’ anchialine taxa may prove to be a result of post-drift community
assembly over a long period.
24.21 Conservation Ecology
Conservation ecology is dependent on the information about the number and

distribution of species to inform, amongst other things, assessment of endangerment
(IUCN Red List of Threatened Species listings; Vié et al. 2008; see also Chap. 22),
as well as the establishment and management of protected areas to sustain commu-
nities and ecosystems (Michel et al. 2009). The Global Biodiversity Information
(GBIF) is capturing records and species across the globe for open access (www.gbif.
org), while the World Register of Marine Species (WoRMS) (www.marinespecies.
org) is developing a subset comprising the fauna of marine caves (WoRCS) (www.
marinespecies.org/about.php). Several other areas are progressing that pertain spe-
cifically to subterranean ecology.
520 W. F. Humphreys
Regional knowledge has advanced through, for example, the compilation of

existing information (Culver et al. 2003) and by its rigorous analysis (Zagmajster
et al. 2014). In addition, de novo regional surveys of stygobiont species have been
conducted at both regional (Pilbara, Australia; Halse et al. 2014) and international
scales (six Western European countries, PASCALIS project; Gibert and Culver
2009), although with very different taxonomic rigour owing to differential maturity
of subterranean biology research between the regions. These exemplars can inform
researchers planning regional studies elsewhere because in most parts of the world,
regional knowledge of subterranean biodiversity is largely lacking, and they would
benefit from standardisation to allow comparison (Culver et al. 2012b).
The corollary to regional surveys is what species are present and how compre-
hensive is taxonomic knowledge for the subterranean fauna in a region. Such
knowledge varies massively between regions, especially between hemispheres. In
Europe and the USA, there is a high level of knowledge owing to a long history of
subterranean biology research although many undescribed species are still being
recovered (Zakšek et al. 2009). By comparison, in Australia, where such studies are
recent, very large numbers of unknown taxa are being found continually (Guzik et al.
2010; Eberhard and Giachino 2011) often recognised initially from DNA studies
(Javidkar et al. 2015, 2018).
Owing to convergent morphologies associated with adaptation to subterranean
life, both troglobionts and stygobionts seem to be especially rich in cryptic species,
one of the reasons that molecular methods have routinely been used to provide
distribution limits of putative troglobiont species for environmental impact assess-
ment in Western Australia. Although the proportion of cryptic species does not seem
to vary between the major metazoan taxa (Pfenninger and Schwenk 2007), it is not
known whether the proportion of cryptic species is related to habitat, in this context,
between epigean and subterranean members of taxonomic lineages or between
interstitial and free-swimming members of a lineage. This is significant because it
could affect key information in conservation ecology and influence how conserva-
tion effort is allocated. However, any such analysis would need to be treated with
caution because cryptic species are commonly determined as a result of molecular
studies (Zakšek et al. 2009; Zhang and Li 2014), but whether species are considered
cryptic (e.g. King et al. 2012; Karanovic et al. 2016), indeed, whether a taxonomic
type II error (mistakenly not recognising that more than one species is present)
places species in synonymy, is dependent on the adequacy of taxonomic knowledge;
where taxonomic knowledge is weak, taxon-focused conservation efforts must
prioritise modern species delimitation research (Hedin 2015).
24.22 Be Alert for Opportunistic Experiments
The information available from background monitoring of subterranean ecosystems

allows unexpected or episodic events (Boero 1996) to be used to explore additional
attributes of a system, as a ‘natural experiment’ not otherwise possible, such as the
Episodic Events: Great Lakes Experiment (e.g. Chen et al. 2004). I provide a
subterranean example from each of an aquatic and terrestrial system. Major earth-
quakes may markedly affect groundwater, including large and sustained changes to
water level and water quality, even a 1000s km distant from the epicentre (Sneed
et al. 2003), both of which may impact on groundwater communities. The only
detailed study of an earthquake on an aquifer community is from the karstic Gran
Sasso Aquifer following the 2009 L’Aquila earthquake, Italy; the aquifer subse-
quently became almost devoid of stygobiont life, markedly reducing the distribution
and abundance of obligate groundwater copepod species, but not of epigean species
(Galassi et al. 2014). In a terrestrial context, the monitored decline of populations of
several species of tropical troglobionts in an episodically recharged cave in arid
Australia provided an opportunity to establish an experiment to determine the effect
of water and organic matter on the re-establishment of populations of several species
in the cave (Humphreys 1991). These studies were possible only because there had
been detailed monitoring of the fauna and its environment prior to the event that
provided the opportunity. Such background information would be a natural outcome
of the establishment of subterranean biodiversity monitoring systems on permanent
plots, as has been widely undertaken on epigean ecosystems worldwide, a compo-
nent of the implementation of the Convention on Biological Diversity (Dogsé 1998).
Acknowledgements Brian Vine who first showed me what became Draculoides vinei (Harvey),
took me caving in Cape Range and changed forever my research focus. Darren Brook of Exmouth,
who has been a constant caving companion. Mark Adams and Steve Cooper of the South Australian
Museum and Adelaide University and the succession of researchers through their labs. Julianne
Waldock of the Western Australian Museum who has been there throughout. To each and everyone,
my appreciation.
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Glossary
Aa lava High-viscosity, lumpy lava flow

Adaptive shift hypothesis Which supports that parapatric speciation occurs in the
face of gene flow, with divergent selection between cave and surface habitats
driving differentiation
Admixed In population genetics, describing the state of formerly isolated populations
that started interbreeding
Aesthetascs A small cuticular process with a chemosensory function placed on the
first antennae of most crustaceans
Allele Any kind of variant of the genetic information at a given genetic locus, from
single nucleotide to entire chromosomic regions on two homologous chromo-
somes. Most commonly to denote different genes found at the same position
(locus) on homologous chromosomes in a single individual or in a population of
individuals
Allochthonous Having an origin distant from the site it was found (here, external to
the cave environment)
Allopatric Used of taxa occupying different, mutually exclusive geographical areas
Allozyme Allelic variant of an enzyme, typically distinguished from other such
variants by difference in electrophoretic mobility; analogues of alleles at the level
of enzymes
Alluvial Comprised of or related to alluvium
Alluvium Unconsolidated sediments deposited by streams, usually consisting of
silt, sand, gravel and detrital material
Autotrophy The synthesis of organic carbon from CO2, using energy from inorganic
chemical reactions
Biofilm A microbial community that adheres to itself and surfaces through the
formation of a sticky, extracellular substance
Biomass Total quantity or weight of organisms in a given area or volume, at a
specific point in time (e.g. mg dry mass m2)
Biospeleology (speleobiology, cave biology) The science that studies living organ-
isms in caves, their origin, phylogeny, adaptations, ecology, distribution, etc.

https://doi.org/10.1007/978-3-319-98852-8
534 Glossary
Boulder choke A place where the cave passage is blocked by coarse, often
breakdown, blocks; similarly, a silt choke consists of very fine-grained material
Breccia Rock formed by predominantly angular clasts of several sizes that pre-
dominate on the finer matrix
Breccia pipe A collapsed cave roof that has been extended upwards by stoping, until
all of the original cave volume is filled with breccia material; breccia pipes may
penetrate through all kinds of overlying rocks and eventually reach the surface
Canga formations Wide coverings of iron ore breccias located on the iron forma-
tions and that function as a support element on tops of dissected plateaus
Carbon dioxide equilibria Chemical equilibria between carbon dioxide, water and
dissolved carbonate
Cavernicole An organism that lives in caves
Chemolithoautotrophy A metabolic mode of inorganic substrates which gives
energy to an organism from light-independent chemical reactions and enables
biochemical fixation of carbon dioxide for most or all organism’s carbon
requirements
Chemolithotrophy Metabolic energy production from electrons obtained from the
oxidation of inorganic compounds derived from rocks
Chemosynthesis The synthesis of organic compounds in the absence of sunlight in
which the energy is derived from inorganic compounds
Chlorophyll a The main photosynthetic pigment in oxygenic phototrophs which
has two absorption maxima: in the violet part of the spectrum at 440 nm (γ peak)
and in the red part of the spectrum at 680 nm (α peak)
Chloroplast A semi-autonomous and double-membrane organelle known as plas-
tid containing chlorophyll and also various accessory pigments in the cells of
eukaryotic photosynthetic organisms
Chlorosis The loss or disintegration of chlorophyll in the tissue of a plant which
commonly appears yellowish due to various causes, e.g. microbial infection,
activity of phytotoxins, deficiency of light, magnesium or iron
Climatic relict hypothesis Which invokes allopatric speciation after preadapted
species become stranded in caves during climatic change
Cloned The process of placing a piece of genetic information (usually a gene) into a
vector to allow multiple copies to be made for sequence analysis
Colluvial Comprised of or related to colluvium
Colluvium Unconsolidated sediments deposited at the base of hillslopes or in
depressions through sheetwash or downslope creep; they may consist of silt,
sand, gravel and rocks
Congelation ice It forms as water freezes on the bottom of the ice cover, and the
latent heat of crystallization is conducted upwards through the ice and snow to the
atmosphere. It forms as a stable ice sheet with a smooth bottom surface and may
develop to massive sheets of different thicknesses that mantle bottoms of ice
caves. Ice of this type occurs in caves with dynamic microclimates
Contig A contiguous piece of genetic information assembled from overlapping,
smaller pieces of genetic information acquired through DNA sequencing
Glossary 535
Convergent evolution As habitually understood, when in two species a character

evolves independently from different ancestral states to a similar derived state
under the same environmental or ecological conditions. For example, the inde-
pendent development of an aphaenopsian habitus in two independent lineages of
beetles from respective ancestors with different body shapes or the development
of fins in cetaceans and fish
Core microbiome The microbial community (along with its genetic information)
that remains invariant between ecosystems found in the same conditions
Diastrophism It designates tectonism (orogenic, epirogenetic and tafrogenetic
tectonics), affecting the terrestrial crust and resulting in the formation of geolog-
ical basins, mountain chains, folds, faults, fractures, joints, etc.
Doline From Slavic languages “dolina” ¼ small valley. Technical term for all
closed depressions in karst formed through dissolution, suffusion of unconsoli-
dated sediment or collapsing a cave roof. Dolines are important interfaces
between surface and underground habitats. The American term sinkhole is used
on any kind of hole formed by sinking ground
Ectotherm An animal whose body temperature depends on external sources of
heat, in contrast to endotherms
Endoparasite An internal parasite
Epibenthic An organism that lives on the surface of sediments at the bottom of
water
Epibiontic An organism that lives attached to another organism without benefit or
detriment for the host
Epigean An organism living above the ground, on the surface—in contrast to
subterranean or below the ground organisms
Epigenic caves Caves formed by meteoric water, primarily in the vadose zone
Epikarst A weathered zone of enhanced porosity near the surface, at the soil/
bedrock contact of many karst landscapes
Ferricrete duricrust Referred to as hardcap in the Pilbara region of Western
Australia; it is similar to laterite and consists of a hardened layer where soil
particles are cemented by iron oxides released by leaching of underlying iron
formations
Genomic sequencing Sequencing of the entire genetic information within a cell,
including the chromosomes and extrachromosomal genetic information (such as
plasmids)
Georeferencing Defining the spatial position of the locality with coordinates
Glacier caves The caves formed in glaciers by melting water along crevices in the
ice and along its contact with bedrock; some glacier caves form by volcanic heat
released at fumaroles
Grana (singular: granum) A stack of thylakoids in chloroplasts containing a
number of flattened membranous sacks interconnected by membranous bridges
to adjacent thylakoids
Hygrophilous An organism living in moist and wet habitats
Hypogean Subterranean, underground
536 Glossary
Hypogenic caves Caves formed by rising groundwater or production of deep-seated

solutional aggressiveness
Hypogenic speleogenesis The formation of solution-enlarged permeability struc-
tures by waters ascending to a cave-forming zone from below, where deeper
groundwaters in regional or intermediate flow systems interact with shallower
and local groundwater flow systems
Hyporheic Refers to the zone of saturated sediments below the river bed that is
usually viewed as an ecotone between the stream and underlying groundwater
Hyporheos The assemblage of organisms living in the hyporheic zone
Internode A section of stem at plants responsible for transport of water, hormones
and other necessary compounds to nodes
Interstitial habitat Small spaces filled with water between grains of sand found in
sediments below lakes and wetlands, gravel bars in rivers and sand below streams
Itabirites A finely stratified metamorphic rock with iron contents of 50–55%; it has
a banded structure composed of silica and iron
Karren and grikes (lapiés or lapiaz) Weathered and exposed limestone surfaces
found in karst regions and consisting of different forms of rock pinnacles
separated by deep grooves
Karst A terrain formed by the dissolution of soluble rocks such as limestone,
dolomite and gypsum and characterized by rocky ground, caves, sinkholes,
underground rivers, etc.
Lavicole An organism living exclusively on barren lavas and feeding on fallout of
aerial plankton
Macroecology Discipline of ecology that studies patterns and processes on large
spatial scales
Macroevolution Major evolutionary change regarding the evolution of whole
taxonomic groups over long periods of time
Meiofauna Small benthic invertebrates that live in both marine and fresh water
environments; organisms that can pass through a 1 mm mesh but will be retained
by a 45 μm mesh
Microarthropods Small invertebrates (<2 mm) in the phylum Arthropoda, the
most frequent microarthropods are mites (Acari) and springtails (Collembola)
Microfauna Animals with the body size up to 0.2 mm such as nematodes, rotifers
and small arthropods, also covering protozoans (protist kingdom)
Microbivorous An animal eating microorganism, such as bacteria, algae and
microfungi
Mesa Flat-topped hill
Metadata Data about data; information on the scope of the data, geographic area,
geographic projections, etc.
Metallophilic savannah Type of vegetation occurring in the plateaus of the
Amazonian ferruginous formations, represented by a gradient of open brush
vegetation with intense deciduousness, resembling semiarid vegetation
Metaproteomics The study of all protein samples recovered directly from environ-
mental samples
Glossary 537
Metatranscriptomics The study of the function and activity of the complete set of
transcripts (RNA-seq) from environmental samples
Meteoric water Water from the earth surface water cycle, in contrast to endogenic
or hypogenic water that originates from the mantle
Microfabrics Structure or arrangement of mineral and organic particles on a
microscopic scale
Microphthalmic With reduced eye development, which can be functional or not; in
comparison with related species having well-developed, large eyes
Microsatellite A stretch of DNA consisting of a repetition of short nucleotide
motifs of up to six nucleotides; the number of repeated motifs is highly variable.
Because microsatellites at a given genetic locus behave as alleles that can be
distinguished by length, they are used as genetic markers at the level of individual
organisms, population or very closely related species
Morphocytological adaptations Changes in a plant’s morphology and cytology to
enable survival in a certain habitat or a response to a certain stimulus, which
includes macroscopic changes such as elongation of stems, leaves and internodes,
fewer leaves and microscopic changes such as weakening of cell walls and
alterations of chloroplasts, grana and thylakoids
MSS “Milieu souterrain superficiel”, superficial underground compartment (also
“mesovoid shallow substratum”, “upper hypogean zone”, or—in part—“subter-
ranean shallow environments”). Originally, the network of empty voids and
cracks found at the junction of the soil and rock layer, generally covered by
topsoil and with environmental conditions similar to those of deeper fissures or
caves
Neoteny Retention of some larval or immature characters by adults in a species
Nivicolous An organism that occurs on snow or in its proximity
Notch A geomorphological term for a V-shaped cut or hole in a surface, which
formation probably includes a combination of chemical, physical and biological
processes
Oligotrophic Derived from the Greek “little to eat” to describe nutrient-limited
environments with less than 0.2 mg/L of organic carbon
Ombrophilous A plant capable of thriving or withstanding in areas with wet and
rain conditions
Omnivorous An animal combining different food sources, i.e. decaying organic
material, carcasses, microorganisms, living plant tissues and liquids, animals as
prey, etc.; feeding on a variety of food of both plant and animal origin
Orthogenesis The development of a fixed evolutionary program independently of
the particular environmental or ecological conditions experienced by the species.
This fix program can be similar in different lineages, resulting in parallel or
convergent evolution
Panmictic Describing the state of unrestricted random mating within a population
Pahoehoe lava Low-viscosity lava that flows like a river (“rope lava”)
538 Glossary
Parallel evolution As habitually understood, when in two species a character

evolves independently from a similar ancestral state to a similar derived state
under the same environmental or ecological conditions; for example, the inde-
pendent development of elongated appendages in Leptodirini beetles from related
ancestral species with the same plesiomorphic condition or the development of
wings in bats and pterosaurs from the anterior extremities
PCR amplification Amplification of DNA sequences using the polymerase chain
reaction
Percolation Slow gravitational movement of water through intergranular pores or
rock joints
Phanerophyte A plant with perennating buds well above the surface of the ground,
typically referred to trees, shrubs or climbing plants
Photoneutral An organism unaffected by light
Photophilic An organism receptive to, that seeks or thrives in light
Photophobic An organism intolerant to or avoiding light
Phreatic zone The zone below the water table where all spaces are saturated with
water
Phylogenetic diversity (PD) Diversity of the species that includes information on
phylogenetic relations among them; it can be calculated either per site (alpha PD)
or as a measure of comparing species assemblies (beta PD)
Phylogeny A genetic family tree (or phylogram) that describes the evolutionary
history between groups of organisms
Phylotype An organism identified (or grouped) by its genetic 16S rRNA gene
sequence; phylotypes differ from species in that, while a species has been
successfully cultured and classified within the laboratory, a phylotype is only
identified through its DNA sequence
Phylum (pl. phyla) A level of classification or taxonomic rank below kingdom and
above class
Physiography The study of landforms, synonymous with geomorphology; physi-
ography of a cave refers to its formation, morphology and internal environment
Primer A small fragment of single-stranded DNA that is used to determine the start
sites for PCR amplification
Productivity The rate of production of new biomass by an individual, population or
community in a given area or volume over a specified time period (e.g. mg dry
mass m2 year1)
Protonema (pl. protonemata) A filamentous and branched multicellular structure
formed after spore germination which represents the juvenile gametophyte in a
life cycle of mosses and liverworts
Pteridophyte In the valid nomenclature, an informal denomination adopted from
older literature for flowerless and seedless vascular plant which reproduces with
spores referred as Pteridophyta (ferns)
Raphe A longitudinal fissure through the valve in some pennate diatoms
(Bacillariophyceae) involved in gliding locomotion
Glossary 539
Redox active compounds Compounds with valency states that makes them likely
to participate in energetic oxidation/reduction reactions
Regelation The phenomenon of ice melting under pressure and freezing again
when the pressure is reduced
Rhizophagous Feeding on roots
Sapsucking Feeding on sap
Sensu lato In taxonomy used to denote the named taxon along with additional
groups usually related or confused with the name, in contrast with sensu stricto
which refers to only the precise taxon
Sequencing The process of decoding the individual base nucleotide sequence of
DNA
Single nucleotide polymorphism (SNP) Allelic variation of a single nucleotide
position homologous between chromosomes in an individual or a population of
individuals
Speleogenesis The process of cave formation
Streamsink A place where a surface stream enters a cave
Stygal Having the characteristics of stygofauna
Stygobiont/stygobite An animal that lives only in groundwater
Stygofauna A general term covering all animals living in groundwater associated
with caves, streams and the broad landscape
Stygophile An animal that lives both on the surface and in groundwater habitats
Stygoxene An animal that is only rarely living in groundwater habitats, accidentally
or in search for shelter on short periods of time
Subglacial refuge A habitat under an ice sheet that preserves aquatic subterranean
fauna during climate changing periods
Sulphurous ecosystems An ecosystem that exists and functions in sulphurous
waters or depends on sulphurous waters
Syphon Section of a cave passage that is completely filled with water
Taxon (pl. taxa) The group of one or more populations that is considered as a
taxonomical unit; it can be a species, an order, a class, etc.
Thylakoid A flat, disc-like vesicle resembling a coin, bounded on each side by
double-plasma membranes, which run close to each other and contain photosyn-
thetic pigments
Transposon An element of DNA that, through the use of enzymes, can jump
around (transpose) within a DNA sequence
Troglobiont/troglobite An animal that lives only in terrestrial subterranean habitats
Troglofauna A general term covering all air-breathing (terrestrial) animals living
underground in caves and in the subterranean matrix of the broad landscape
Troglomorphism (troglobiomorphism) The morphological adaptation of an ani-
mal characterized by loss of pigment, reduced eye structures or blindness,
elongated body and appendages, etc.
Troglophile An animal that can live both on the surface and in subterranean habitats
Trogloxene An animal that is only rarely entering caves, accidentally or in search
for shelter on short periods of time
540 Glossary
Trophic webs The connection between different food chains (one-way flow of
trophic relationships) in a biological community
Vadose zone The unsaturated zone of the groundwater environment between the
surface and the phreatic—saturated zone
Venturi effect Pumping effect around a fluid jet
Vicariants Closely related taxa isolated geographically from one another by a
biogeographical barrier
Vuggy Refers to rocks containing cavities, voids or large pores
Weathering Set of combined chemical, physical and/or biological processes of
disintegration, degradation and decomposition of rocks caused by several agents
near the surface of the earth’s crust
Index
A B
Aa lava, 19 Bacteria, 60, 80, 84, 174, 258, 259, 301, 303, 310,
Acari, mites, 123–124 313, 314, 316, 317, 335, 342, 351, 353,
Actinobacteria, 71, 74, 76, 78, 79, 298 358–361, 364, 365, 439, 509, 514–516
Adaptive shift hypothesis, 245, 379 Bat guano, 129, 131, 198, 259, 297, 312–314,
Admixed, 279, 281 317, 365
Aesthetascs, 56, 185, 186, 386 Bathynellacea, bathynellaceans, 56, 176, 187,
Algae, 91–94, 96, 98, 99, 301, 310, 335, 345, 197, 277
407, 408 Bayliss Cave, Australia, 26, 302, 512
Allele, 273–275, 287–289, 533, 537 Biofilm, 71, 91, 98, 99, 101, 301, 311, 313, 317,
Allochthonous, 25, 69, 70, 515, 533 318, 353, 357, 359–361, 364, 365,
Allopatric, 271, 281, 284, 291, 379, 533, 534 514–516, 533
Allozyme, 273–275, 277, 283, 285, 519, 533 Biomass, 73, 77, 84, 95, 98, 99, 157, 297, 301,
Alluvial, 277, 278, 422, 424, 487–489, 533 302, 310, 313, 315, 335, 351, 392, 516,
Alluvium, 417, 419, 422, 430, 442, 533 518, 533, 538
Amblyopsid fishes, Amblyopsis, 54, 55, 304 Biospeleology (speleobiology, cave biology), 2,
Amblypygi, amblypygids, 117, 125–126, 283, 41, 186, 269, 274, 499, 533
302, 364 Bivalvia, bivalves, 175, 178, 182, 468
Amphibians, 52, 304, 463, 465, 466 Blattaria, cockroaches, 143, 377
Amphipoda, amphipods, 133, 176, 186, 196, Boulder choke, 12, 534
201, 387, 390, 405, 421 Breccia, 436, 534
Anchialine, 61, 181, 183, 185–187, 272, 369, Breccia pipe, 12, 534
374, 375, 383–394, 399–411, 498, Bryophyta, bryophytes, mosses, 91–94, 96,
502–503, 509, 511, 513, 516, 517, 519 99, 122
Annelida, annelids, 116, 175, 180, 358,
404, 421
Araneae, spiders, 111, 117, 124–125, 376, C
421, 429 Canga formations, 436, 437, 444, 534
Archaea, 74, 80–81, 84, 301, 335, 359–361, Cape Range, Australia, 111, 298, 510–513
514, 515 Carajás region, Brazil, 439–444
Asellus, 44, 53, 54, 56, 57, 60, 186, 279, 280, Carbon dioxide equilibria, 17, 534
287, 289, 290, 358 Carlsbad Caverns, USA, 453, 461, 471
Astyanax, 53–56, 58–61, 234, 280, 287–289 Castleguard Cave, Canada, 336
Autotrophy, 84, 533 Cavernicole, 41–43, 45, 47–52, 95, 339, 534
Ayyalon Cave, Israel, 359 Cenote, 102, 384, 388, 453, 460

https://doi.org/10.1007/978-3-319-98852-8
542 Index
Chaetognatha, chaetognaths, 386 D

Chemolithoautotrophy, 95, 297, 301, 310, Decapoda, decapods, 132–133, 175, 185, 186,
319–321, 345, 534 387, 390, 405
Chemolithotrophy, 69, 73, 259, 351, 353, 393, Demänovská Cave System (Demänovská Ice
514, 516, 534 Cave, Caves of the Demänová Valley),
Chemosynthesis, 301, 353, 359, 534 Slovakia, 335
Chiroptera, bats, 27, 29, 50, 82, 85, 116, 123, Diastrophism, 436, 535
143, 145, 261, 301, 304, 362, 364, 365, Diplopoda, millipedes, 47, 109, 113, 114, 127,
392, 452, 467, 468, 470, 474, 479, 480 128, 131, 132, 140, 303, 376, 421, 429
Chlorophyll a, 98, 534 Diplura, diplurans, 108, 114, 128, 129,
Chloroplast, 94, 99, 534, 535, 537 137–138, 421, 428, 429
Chlorosis, 94, 534 Diptera, dipterans, 108, 140, 145–146, 244,
Cixiidae, cixiid planthoppers, 144, 377, 257, 303, 336, 338, 339, 364, 425, 429
438, 439 Dobšinská Ice Cave, Slovakia, 332, 341, 343, 344
Climatic relict hypothesis, 379, 534 Doline, 13, 16, 243, 334, 337, 344, 535
Cloned, 73–75, 534 Dysdera, 125, 376, 378, 509
Coleoptera, beetles, 26, 44, 52, 53, 108, 111,
112, 139–157, 176, 188, 229, 230, 235,
237, 245, 336, 339, 377, 421, 429, E
468, 514 Echinodermata, echinoderms, 179, 386
Collembola, springtails, 44, 45, 47, 51, 108, Echiuridae, echiurids, 386
109, 112–114, 122, 128, 129, 134–137, Ectotherm, 304, 535
245, 258, 302–304, 338, 345, 377, 425 Edwards Aquifer, USA, 321, 471, 516, 517
Colluvial, 422, 534 Eisriesenwelt Ice Cave, Austria, 332, 341
Colluvium, 417, 419, 430, 534 Endemism, endemicity, 58, 78, 130, 206, 222,
Congelation ice, 332, 534 259, 422, 429, 439, 444, 471
Contig, 77, 534 Endoparasite, 535
Convergent evolution, 113, 114, 235, 269, 280, Epibenthic, 282, 535
520, 535, 537 Epibiontic, 174, 535
Copepoda, copepods, 175, 179, 182, 183, 388, Epigean, 33, 34, 42, 43, 45, 48, 50–52, 55,
389, 421 57–60, 112, 114, 115, 124, 129–131, 134,
Core microbiome, 70, 535 135, 139, 142, 143, 149–153, 157, 173,
Crabs, 132–133, 186, 187, 364 177, 179, 182, 185–188, 230, 232–234,
Crayfishes, 57, 59, 186, 187, 281, 315, 470, 471 236–239, 241, 244–246, 257–261, 265,
Crustacea, crustaceans, 33, 44, 53, 54, 56–58, 274, 298, 302–304, 373, 374, 377–379,
129, 132–134, 175, 179, 182–187, 196, 428, 435, 437, 438, 443, 444, 499, 500,
200–208, 211, 214, 216, 217, 219, 221, 508–511, 513, 520, 521, 535
236, 277–280, 303, 313, 336, 338, 345, Epigenic caves, 69, 352, 353, 535
358, 386, 388, 391, 392, 403–409, 421, Epikarst, 12–15, 31, 32, 298, 311, 437, 453,
422, 519 498, 514, 535
Cueva de Felipe Reventón, Canary Islands,
369, 371, 373, 375
Cueva de los Roques, Canary Islands, 369, 371, F
373–375, 378 Ferricrete duricrust, 419, 487, 535
Cueva de Villa Luz (Cueva del Azufre, Baños Fishes, 33, 44, 53–60, 173, 188–189, 201, 217,
del Azufre, Las Sardinas Cave), Mexico, 233, 234, 270, 280–282, 285, 302–304,
60, 353, 362–365 362, 365, 386, 392, 444, 463, 470, 481,
Cueva del Viento-Sobrado, Canary Islands, 490, 506, 511, 519, 535
369, 371–375 Frasassi cave complex, Italy, 509
Cyanobacteria, 76, 79, 91–99, 101, 102, 301, Fungi, microfungi, 80, 82–84, 174, 245, 258,
360, 408 300, 301, 313, 314, 316, 335, 344, 345,
Cyclopoida, 175, 182, 183, 185, 388, 404 353, 359, 361, 456, 514–516
Index 543
G Lepidoptera, lepidopterans, 108, 145, 260, 261,

Gammarus, 57, 186, 279, 285, 289, 290 339, 468
Gastropoda, gastropods, snails, 116–119, 175, Leptodirini, 45, 47, 52, 53, 111, 154, 155, 230,
178, 182 231, 238–241, 243, 245–247, 261, 336,
Gastrotricha, gastrotrichs, 175, 180 514, 538
Genomic sequencing, 77, 535 Leptodirus, 43, 148, 155, 247, 468
Georeferencing, 195, 535 Lukina Jama–Trojama cave system, Croatia,
Glacier caves, 331, 343, 535 299, 303
Gnathostomulids, 407
Grana granum, 94, 535, 537
Grylloblattodea, 108, 142, 338 M
Macroecology, 195, 218–222, 536
Macroevolution, 242, 247, 536
H Mammoth Cave, USA, 71, 174, 375, 452, 453,
Hadesia, 26, 47, 155, 284, 303 461, 471, 504
Harpacticoida, 56, 175, 182–184, 389, 403, 404 Meiofauna, 298, 536
Hygropetric, 25, 26, 47, 130, 155, 284, 303 Mesa, 419, 427, 536
Hygrophilous, 94, 535 Mesocavernous shallow substratum (MSS), 25,
Hypogean, 33, 42, 107, 110, 123, 177, 185, 27, 107, 115, 129, 137, 141, 150, 152,
238, 257, 260, 261, 297, 298, 302, 502, 154, 231, 241, 243, 244, 246, 283, 344,
518, 535, 537 375, 379, 417, 419, 437, 498, 537
Hypogenic caves, 8, 13, 14, 300, 352–354, 536 Metadata, 197, 536
Hypogenic speleogenesis, 437, 536 Metalophilic savannah, 436, 536
Hyporheic, 32, 174, 180, 187, 270, 278, 422, Metaproteomics, 84, 361, 536
498, 515, 536 Metatranscriptomics, 84, 361, 537
Hyporheos, 422, 536 Metazoa, metazoans, 359, 392, 502, 510, 520
Meteoric water, 14, 336, 340, 535, 537
Microarthropods, 122, 298, 365, 536
I Microbivorous, 345, 536
Internode, 94, 536, 537 Microfabrics, 83, 537
Interstitial habitat, 107, 115, 186, 187, 270, 277, Microfauna, 536
338, 407, 536 Microphthalmic, 54, 129, 133, 136, 147, 151,
Iron Quadrangle region, Brazil, 436, 439, 440, 152, 156, 232, 537
442, 445 Microsatellite, 275, 278–280, 282, 288–290, 537
Isopoda, isopods, 128, 133–134, 175, 184, 186, Mollusca, mollusks, 175, 182, 358, 391, 421
376, 387, 389, 405, 421, 429 Morphocytological adaptations, 537
Itabirites, 436, 536 Movile Cave, Romania, 174, 177, 179, 181,
186, 352, 354–362, 375
MSS, see Mesocavernous shallow substratum
K Mt Erebus ice caves, 345
Karren and grikes (lapiés or lapiaz), 12, 536
Karst, 7, 25, 72, 95, 151, 173, 199, 240, 270, 300,
311, 333, 375, 383, 400, 417, 452, 499 N
Kartchner Caverns, USA, 76, 77 Nematoda, nematodes, 118, 175, 179, 201, 358
Krubera–Voronja cave system, Georgia, 257, 299 Neoteny, 52, 61, 189, 304, 537
Kungurskaya Ice Cave, Russia, 332, 333, 338 Niphargus, 53, 57–61, 186, 190, 196, 264, 280,
281, 339, 509
Nivicolous, 338, 537
L Notch, 100, 537
La Corona lava tube, Canary Islands, 400, 401,
403–410
Lava tube caves, 370, 372, 388 O
Lavicole, 373, 536 Oliarus, 284, 304
Lechuguilla Cave, USA, 71, 78, 80, 83 Oligochaeta, oligochetes, 175, 178, 180, 181
544 Index
Oligotrophic, 69, 70, 76–78, 118, 120, 122–124, Pseudoscorpiones, pseudoscorpions, 33, 117,
131–138, 143, 145, 147, 151, 153, 154, 122, 244, 376, 421, 429, 471
297–304, 331, 344, 345, 437, 537 Pteridophyte, 91, 94, 96, 538
Ombrophilous, 94, 537 Ptomaphagus, 43, 46, 148, 154
Omnivorous, 132, 139, 537
Opiliones, harvestmen, 117–121, 518
Orthogenesis, 238, 537 Q
Orthoptera, crickets, 108, 113, 139–142, Quartzite caves, rocks, 300, 439
300, 468
Ostracoda, ostracods, 175, 184, 185, 387, 389,
405, 421 R
Raphe, 98, 538
Redox active compounds, 69, 70, 539
P Regelation, 332, 539
Pahoehoe lava, 19, 369–373, 400, 537 Remipedia, remipedes, 175, 185, 386–388, 393,
Palpigradi, palpigrades, 114, 122, 421, 428, 429 403, 406, 409, 509, 519
Panmictic, 279, 537 Rhaphidophoridae, rhaphidophorid, 139–141,
Parallel evolution, 60, 230, 269, 538 283, 301
PCR amplification, 73, 77, 506, 508, 538 Rhizophagous, 144, 373, 377, 539
PD, see Phylogenetic diversity Rotifera, rotifers, 175, 179, 358, 359
Percolation, 13, 32, 33, 298, 335, 441, 538
Phanerophyte, 91, 93, 94, 538
Photoneutral, 58, 538 S
Photophilic, 58, 538 Salamanders, 33, 53, 61, 189, 303, 304, 314,
Photophobic, 58, 538 315, 463, 468, 470
Phreatic zone, 13–15, 32, 174, 311, 538 Sandstone caves, rocks, 185
Phylogenetic diversity (PD), 221, 514, 538 Sapsucking, 373, 539
Phylogeny, 119, 221–222, 235, 239, 243, 247, Scărişoara Ice Cave, Romania, 340–343
271, 506, 533, 538 Schizomida, schizomids, 118, 126, 421,
Phylotype, 71, 73–75, 509, 538 427, 429
Phylum/phyla, 73, 74, 76, 78, 82, 118, 175, Scoliopteryx, 260, 334, 336
176, 358, 536, 538 Sensu lato, 281, 539
Physiography, 7–20, 538 Sequencing, 73, 75–79, 81, 84, 360–362, 488,
Pilbara region, Australia, 185, 419, 420, 485, 508, 534, 535, 539
505, 507 Shrimps, 56, 61, 185–187, 281, 383, 386, 390,
Platyhelminthes, flatworms, 177–179, 358, 393, 409, 470, 471, 502, 509, 511, 514,
404, 407 516, 519
Polychaeta, polychaetes, 135, 175, 178, Sierra de El Abra caves, Mexico, 280
181, 387 Silická ľadnica Ice Cave, Slovakia, 332–334, 338
Porifera, sponges, 175, 177, 178 Single nucleotide polymorphism (SNP), 275,
Postojna–Planina Cave System, Slovenia, 257, 280, 282, 539
279, 289, 302, 375, 451, 453 Sistema Ox Bel Ha, Mexico, 384
Priapula, priapulids, 386, 407 Sistema Sac Actun, Mexico, 384, 399
Primer, 77, 506, 538 Škocjanske jame, Slovenia, 453, 460
Proasellus, 53, 61, 186 Speleogenesis, 11–12, 214, 352, 364, 401, 437,
Productivity, 78, 98, 217, 222, 309–311, 536, 539
314–317, 319, 321, 322, 504, 538 Speonomus, 43, 154, 155, 238, 239, 261, 283
Proteus, 53, 54, 56–59, 61, 173, 189, 220, 232, Streamsink, 13, 14, 16, 539
233, 237, 467, 468, 501 Stygal, 424, 539
Protista (Protozoa), protists (protozoans), Stygobiont/stygobite, 2, 31–33, 43, 44, 53–60,
174–176 132, 173, 174, 177–185, 188, 189, 203,
Protonema/plural protonemata, 96, 99, 538 206, 209, 229, 231, 236, 257, 269, 282,
Pseudanophthalmus, 111, 150, 283, 466 301–303, 336, 345, 374, 375, 386, 403,
Pseudokarst caves, 7 404, 406–409, 442, 444, 445, 464, 467,
Index 545
468, 475, 501–505, 509, 510, 513–515, 442, 443, 451, 452, 454, 463, 464, 467,
517–521, 539 468, 470, 471, 498–500, 503–505,
Stygobromus, 186, 221, 336, 338 508–510, 512, 517, 518, 520, 521, 539
Stygofauna, 415, 417, 419–426, 428, 429, 442, Troglocaris, 53, 56, 57, 187, 281, 500
480–482, 485–491, 498, 505, 507, 514, Troglofauna, 415, 417, 419–421, 424–430,
515, 539 481–483, 485–492, 505, 507, 539
Stygophile, 2, 53, 173, 177, 179, 424, 539 Troglomorphism/troglobiomorphism, 42, 186,
Stygoxene, 173, 177, 407, 424, 539 237, 239–241, 302, 378, 410, 518, 539
Subglacial refuge, 336, 539 Troglophile, 2, 29, 30, 41, 42, 107, 108, 112,
Sulphurous ecosystems, 539 113, 118–120, 123–127, 130, 131, 135,
Syncarida, 176, 187, 421 136, 141–146, 152–155, 157, 198, 218,
Syphon, 13, 539 257, 261, 262, 264, 304, 315, 317, 339,
375, 428, 430, 442, 510, 539
Trogloxene, 29, 30, 42, 107, 113, 299, 315,
T 339, 373, 374, 510, 539
Taxon/taxa, 23, 44, 45, 56, 77, 95, 96, 107, Trophic webs, 353–354, 364, 393, 540
109–112, 114, 120, 127, 147, 153, 173,
180–182, 186, 188, 196, 201, 202, 214,
216, 218, 222, 235, 269, 273, 289, 311, V
316, 320, 338, 377–379, 444, 454, Vadose zone, 3, 12, 13, 15, 31–33, 69, 298,
462–464, 466, 468, 470, 475, 506, 514, 311–313, 417, 419, 427, 479, 535
515, 518–520, 533, 539, 540 Venturi-effect, 13
Thermosbaenacea, 184–186, 387, 390, 403, Vjetrenica Cave, Bosnia and Herzegovina, 375,
405, 409, 514, 519 451, 453
Thylakoid, 94, 535, 537 Vicariants, 271–273, 279, 281, 284, 378, 409
Transposon, 77 Vuggy, 419, 429, 430, 487, 488
Trechini, 45, 111, 147, 149, 150, 230, 235,
237–240, 243, 245
Tricladida, triclads, 175, 177, 178 W
Troglobiont/troglobite, 2, 23, 29, 31, 41–44, Weathering, 3, 20, 29, 32, 70, 83, 100, 102,
46–51, 107–113, 116, 118–122, 333, 346, 417, 419, 429, 436, 488, 512,
124–127, 129–139, 141–147, 149–156, 535, 536
198, 201–203, 206, 209, 212, 218,
255–257, 259, 260, 262, 264, 269, 271,
273, 275, 277, 281–285, 287, 288, 291, Y
298, 299, 301–304, 310, 315, 336, 338, Yilgarn region, Australia, 416, 419, 429,
339, 345, 369, 373–379, 428, 437–439, 482, 487

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Ecological Studies 235

Oana Teodora Moldovan

Analysis and Synthesis

More information about this series at http://www.springer.com/series/86

ISSN 0070-8356 ISSN 2196-971X (electronic)

Library of Congress Control Number: 2018962260

© Springer Nature Switzerland AG 2018

Part I Into Karst, Caves and Cave Habitats

Part II There is Rich and Diverse Life in Caves

10 Historical and Ecological Factors Determining Cave Diversity . . . . 229

Part III About Communities, Populations and Food

Part IV Particular Cave Ecosystems

Part V Conservation and Protection of Cave Habitats and Cave Fauna

Part VI Cave Ecology for the Twenty-First Century

Oana Teodora Moldovan, Ľubomír Kováč, and Stuart Halse

Wherefore each of you, when his turn comes, must go down to

1.1 On Caves, Cave Ecology, and Cave Inhabitants

© Springer Nature Switzerland AG 2018 1

1.2 Cave Ecology as the Center of Biospeleological

1.3 What Can Bring a Multi-author Book on Cave

Juberthie C, Decu V (eds) (1994) Encyclopaedia biospeologica, Tome I. Société de Biospéologie,

S.-E. Lauritzen (*)

© Springer Nature Switzerland AG 2018 7

Fig. 2.1 Conceptual cross section of a karst system

2.2 Cave Patterns

Depending on bedrock structure, and on hydrologic regimes, solutional caves

2.3 Size Distribution of Cave Voids

Cave Void Distribution

where n is the number of elements of diameter δ, a is a scaling constant and D is the

Fig. 2.4 The carbonate

2.4 Karst Caves

too large and collapses

inception birth growth stagnation ‹‹death››

2.4.2 Hydromorphic Zones in Karst

Fig. 2.6 Flow chart

vadose flow vadose flow

(thermal) hypogenic inlet boundary

outlet boundary (springs)

2.5 The Physics and Chemistry of Caves

2.5.1 Cave Ventilation

2.5.2 Attenuation of Surface Temperature

L0 ¼ 100 d 1:2 v0:2 ,

2.5.3 The Chemistry of the Cave Environment

(~400 ppm) to >104 ppm or more in closed chambers. Respiration-derived CO2

petroleum reservoirs or from sulphate reduction may produce quite low pH in

2.6 Non-karstic Caves

Francis G. Howarth and Oana Teodora Moldovan

Subterranean habitats support discrete ecosystems composed of communities that

© Springer Nature Switzerland AG 2018 23

There is a continuum between caves and the other subterranean habitats

Table 3.1 Examples of Microhabitats Caves MSS

dispersed. Cave microhabitats were deﬁned in studies on cave invertebrates and

3.2 Terrestrial Cave Habitats

3.2.1 Abiotic Factors Characteristic of Cave Communities

3.2.2 Environmental Zones

Entrance-zone Twilight-zone Transition-zone Deep-zone Stagnant-zone

3.3 Aquatic Cave Habitats

Cave Surface Hypo- Perched Water Anchialine Littoral

3.3.1 The Aquatic Living Space

Groundwater habitats in unconsolidated sediments and in various rocks (Table 3.3)