Protist, Vol.

164, 842–860, November 2013

http://www.elsevier.de/protis
Published online date 6 November 2013

PROTIST TOOLS

A Short Guide to Common Heterotrophic

Flagellates of Freshwater Habitats Based
on the Morphology of Living Organisms
Alexandra Jeuck, and Hartmut Arndt 1

Department of General Ecology, Zoological Institute, Cologne Biocenter,

University of Cologne, Zülpicher Straße 47b, D-50674 Cologne, Germany

Introduction Kim and Archibald 2013). In contrast, the domi-

nant taxonomic groups within different marine and
Heterotrophic flagellates (HF) are very likely the freshwater pelagic communities (e.g. stramenopile
most abundant eukaryotes on Earth, hundreds of taxa, dinoflagellates, choanoflagellates, kathable-
specimens occur in each droplet of water even pharids) and benthic communities (e.g. euglenids,
in groundwater and the deep sea. As the main free-living kinetoplastids, cercozoans) seem to be
feeders on bacteria they play an essential role in surprisingly similar (Arndt et al. 2000). Their modes
aquatic and terrestrial food webs (Arndt et al. 2000; of movement as important taxonomic character-
Azam et al. 1983; Bonkowski 2004). In addition, istics comprise gliding or free-swimming forms or
they can act as important herbivores (Arndt and they may be temporarily or permanently attached to
Mathes 1991; Nauwerck 1963; Sherr and Sherr a substrate (Fenchel 1987). Feeding modes include
1994), detritivores (Scherwass et al. 2005) and true filter-feeding (e.g. choanoflagellates), direct
osmotrophs (Christoffersen et al. 1997; Sanders interception feeding (e.g. chrysomonads) or rapto-
et al. 1989; Sherr 1988) as well as mixotrophs (Bird rial feeding (e.g. most benthic forms) (Boenigk and
and Kalff 1986; Sanders 1991). The relative contrib- Arndt 2002; Fenchel 1991).
utions to these different modes of feeding can vary One important prerequisite to estimate the role of
within taxonomic groups and even within one and HF for the flux of matter in ecosystems is the deter-
the same organism, e.g. Ochromonas sp. (Jones mination of their abundance. In the early days of HF
2000; Wilken et al. 2013). Furthermore, bacterial quantification, the so called “HNF” were counted
communities are not only grazed by protozoans exclusively in fluorescently stained fixated sam-
but are also structured by protistan grazers (e.g. ples (e.g. Porter and Feig 1980). However, fixation
Boehme et al. 2009; Güde 1979; Jürgens and Matz bears the problem that many species are disrupted
2002; Pernthaler 2005). upon the fixation process (Choi and Stoecker 1989;
HF are a very heterogenous group with an Sonntag et al. 2000) or are misidentified as small
enormous size range between 1-450 ␮m (some naked amoebae, zoospores, yeasts, or disrupted
authors refer to the species smaller than 15 ␮m as cells from a range of eukaryotes, since flagella are
heterotrophic nanoflagellates “HNF”, Arndt et al. not always adequately preserved (Patterson and
2000). High tolerances to changes in salinity Larsen 1991). Especially benthic HF are affected
allow several species to live both in marine as by the above explained problems because they are
well as in freshwater habitats, though several additionally masked by sediment particles. Further,
phylogenetic studies have also indicated clearly large HF can easily be overlooked on membrane
separated marine and freshwater clades (e.g. filters due to their low contribution to total HF abun-
dance and their special sensitivity to fixatives. This
1
Corresponding author; fax +49 221 470 5932 is crucial since they may contribute to about half of
e-mail [email protected] (H. Arndt). the HF biovolume (Arndt et al. 2000). Therefore, the

© 2013 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.protis.2013.08.003
 Guide to Free-living Heterotrophic Freshwater Flagellates 843

J bid
Jacobids
Jacob ds Free living
Free-livin
kinetoplasds

Euglenids

Bicosoecids
Bi id p
Diplomonads
Chrysomonads P
Preaxostyla
l
Discoba
Malawimonas
Ciliophryids
p y
Metamonads
Dictyostelians
Algae – group
yceae Chloroplasda,
(Rhodophyceae, da Mulcilia
Gl koph
Glau k phyta)
y ) ?
b l
Tubulineans
Dinoflagellates
Stramenopiles
? Phalansteriids
Alveolates F i
Fungi
C
Cercozoans Nucleariids
Ichthyosporeans
Foraminiferans
Polycysneans Metazoans Choanoflagell
g ates
C
Cercomonads
d

Spongomonads ?
PROTISTS OF UNCERTAIN
RTAIN POSITION
Thaumatomonads
Cryptophyceans Apusomonads Spironemids
Glissomonads Ancyromonads
A d
d
Cryomonads Kathablepharids Phyll
y omonas
Collodictyonids

Figure 1. Selective overview of the systematic position (according to Adl et al. (2012) of flagellate groups
considered in the guide (see Charts 1–15).

taxonomic resolution of fixation processes is often freshwater flagellates due to its polytomous organi-
very low, most quantitative studies even ignored the zation, the help of drawings and video sequences
variety of HF. (Supplementary Material). According to our experi-
In contrast, live counting is an essential and reli- ence in quantitative studies of morphotypes, about
able method for determining HF abundances. It 80-90 per cent of the dominant heterotrophic flag-
allows a combination of taxonomic and quantita- ellates found in freshwater habitats should be
tive studies for understanding the role of HF in identifiable at least to rough taxonomic categories
microbial food webs (Leadbeater and Green 2000; (and functional guilds). The term morphotype is
Patterson and Larsen 1991). Unfortunately, the tax- used here for taxa that can be differentiated by
onomic resolution of HF studies is hampered by the means of their morphology when only analysed
fact that the literature is often focused on only a few by light microscopy. It means that e.g. genotypes
taxonomic groups. Therefore, we designed a (hope- may be hidden among certain morphotypes. A typ-
fully) user-friendly guide for ecologists to unravel ical example is the species complex of Neobodo
the “HNF” black box. In contrast to the few already designis (Scheckenbach et al. 2006). The guide
existing taxonomic keys/reviews (e.g. Bass et al. comprises all the main groups, so that a large num-
2009; Cavalier-Smith and Chao 2010; Lee et al. ber of species morphotypes can be determined at
2000; Patterson and Hedley 1992; Patterson and least to the level of genus. Special attention was
Larsen 1991; Zhukov 1993), the present guide is given to the most interesting and ecologically rel-
compact and focuses on the dominant and common evant forms. The taxonomic categories were used
forms of HF occurring in pelagic and benthic fresh- according to the recently revised classifications by
water habitats. Following the idea of Foissner and Adl et al. (2012).
Berger (1996) with regard to ciliates, we designed Within the next decade, next generation
a guide which should be useful also for biolo- sequencing of field samples will give more detailed
gists not specialized in the study of heterotrophic insights into the structure of HF field communities
844 A. Jeuck and H. Arndt

Protists

With 1 or 2 flagella With Without flagella, but with Without flagella/cilia, but Without flagella/cilia, Without
(=relatively
( l ti l llong cilia more numerous relatively short with rigid star-shaped but capable of flagella/cilia, but
compared to the cell size).
size) than 2 cilia for locomotion and/or arranged axopods producing with plastids
Cell size ranges from less flagella ingestion (=pseudopodia, stiffened pseudopodia for (photosynthetic)
than 15μm to more than by microtubules) or ingestion and
50μm. Colonies and tentacles locomotion
aggregations may occur.

Flagellates C
Chart 6 C
Ciliates Heliozoans, Amoebae Algae
sucto a s
suctorians

Not considered here

Not to confuse with

other very small and
often free-moving
Autotrophic; Heterotrophic; organisms which are
compact and clearly chloroplasts not clearly visible, difficult to differentiate between not protists: e.g.
visible chloroplast ingested and functional chloroplast; groups containing also large bacteria, and
mixotrophic or autotrophic forms are asterisked ( ) also metazoans
(e.g. turbellarians,
rotifers)
See literature
referring to
autotroph
t t hiic
flagellates Fastt-track to the main groups of heterotrophic flagellates

Chart 8 & 9 Chart 4 Chart 2 Chart 5 Chart 12 Chart 12 Chart 10

Important
character
h t

Choanoflagellates Chrysomonads Ciliophryids Bicosoecids Euglenids Euglenids Free-living

Monosiga Spumella Pteridomonas Bicosoeca Entosiphon Petalomonas kinetoplastids
Neobodo

Cryptophyceans Thaumatomonads Dinoflagellates Apusomonads Cercomonads Spironemids Diplomonads

Goniomonas Thaumatomonas Gyrodinium Apusomonas Cercomonas Spironema Hexamita

Chart 14 Chart 3 Chart 15 Chart 13 Chart 11 Chart 6 Chart 6

Chart 1. User-friendly guide to common heterotrophic freshwater flagellates. All scale bars in the guide indicate
10 ␮m.
 Guide to Free-living Heterotrophic Freshwater Flagellates 845

Heterotrophic flagellates

Single cells Colonial, cells often

Colonial often
living
g in a consortium Chart 7

At least 1 flagellum visible More than

than 2 flagella visible

2 flagell
fl lla (sometimes
( ti l 1 visible)
only i ibl ) 1 flagellum visible Chart 6

Presence
P off collar
ll
Chart 3

With collar, funnel-shaped (use phase contrast); the flagellum Without collar ((use phase contrast))
creates
t a filter
filt current;
t protoplast
t l t (witho
( ith utt the
th collar)
ll )
< 20 μm Flagellum gliding or non
gliding;
lidi presence off
C ll b
Cell d
body tentacles
Choanoflagellates Ciliophryids
often
Collar shape varies; collar Tentacles appear
spherical;
i t off a corona off
consists ll unde
as a collar d r Phalansterium
g
flagellum 3-5
microvilli; protoplast normally the microscope Cienkowski 1870
times longer
egg-shaped or spherical;
than cell; cell
commonly y sessile ((attached This Chart Amoebozoans
upp to 17
17μmm
to the substrate), some free-
swimming

Gliding flagellum; no tentacles

tentacles Non gliding flagellum; with/without
with/without te
tentacles
ntacles
Chart 8

Cell shape/length Cell shape/length/with or without tentacles

P t i end
Posterior d Flagellum (relatively Second gliding flagellum Leaf-
L f With ttentacles,
t l sometimes
ti Without tentacles,
of the cell body thick) rigid while sometimes under cell body shaped
shaped cell with optical re
refracting
fracting some with a
is turned gliding except from the and hard to see; originating body; granules,
granules sometimes plasmatic stalk at the
upwards
p at a p; dorsoventrally
tip; from a proboscis edges
g of g around the
in one ring p
posterior end;; manyy
sharp angle; up flattened or metabolic (=trunk-like protrusion of the cell flagellum; some with mixotrophic forms;
to 10μm (=changeable cell the cell) at the anterior end curled up; a stalk at the posterior second flagellum
h
shape);) 6 - 100μm (=anterior
( ll ) up tto
t i flflagellum); up to
t 10
10μm endd (swarmers
( possible);
ibl ) often hard to see;
10μm mixotrophic; egg- to apple- leaf- to apple-
shaped ca. 10 -15μm15μm shaped; ca. 5 -15μm
Some specific E l id
Euglenids Shape of the Phyllomonas contorta
Cercozoans anterior Klebs 1892 Ciliophryids Chrysomonads
g
flagellum;;
Common species Chart 12 cell length Protista incertae sedis
examples: Chart 4

Common genera examples:

Allantion tachyploon
Sandon 1924
Glissomonads Proboscis ((“nose”) directed Beating proboscis ((“nose”)) at the anterior
Ciliophrys Pteridomonas
anteriorlyy or more laterally;
y end (small
( anterior flagellum attached to
Cienkowski 1876 Penard 1889
sometimes capable of this, not visible using light microscopy),
Metopion fluens producing pseudopodia; sometimes bulbous at the end of the
Larsen & ca 7-10μm proboscis; 1 well visible gliding flagellum
Patterson 1990 directed posteriorly; 3 – ca.7μm
7μm
Metromonads
Free-living kinetoplastids Actinomonas
Apusomonads
Kent 1880
Kiitoksia
Vørs 1992 Chart 13 Chart 10
Protista incertae sedis

Chart 2.
846 A. Jeuck and H. Arndt

2 flagella
(second flagellum not always
visible)

Without lorica With lorica (shell-like

covering)
Chart 5

Non gliding Gliding (some species (e.g. Bodo saltans)) also

temporarily attached to substrate); 1 anterior
flagellum and 1 gliding flagellum; heterodynamic:
Chart 4 anterior flagellum beats, gliding flagellum trails

Flagelllla/cell
Fl / ll shape/m
h / ovementt/l/length;
th position of flagella; changeability of cell shape;
capable or incapable of producing pseudopodia

Flagella originate ventrally to Fl

Flagella
ll circa
i equall Flagella inserting subapically Posterior edge of Relatively thick Second gliding flagellum
slightly subapically in
i a in length; capable of in groove; usually with the cell body is flagella, originate sometimes under cell
longitudinal groove (hard to producing acronem ((!)) ((=diminution at moving up and apically in body and hard to see;
see) beating to the left and pseudopodia; the end of the flagella); cell down; slightly emargination anterior flagellum lies in
right constantly; egg-shaped strong metabolic body is oval, ellipsoidal, egg- metabolic (=depression); a proboscis (=trunk-like
to leaf-shaped and partially (=changeable cell shaped
h d or b
bean-shaped;
h d (=changeable cell cell body egg- protrusion of the cell);
flattened, partially with shape); amoeboid; relatively constant cell shape shape); < 8μm shaped
h d to
t metabolic
t b li ((=changeabl
h ble
verrucous surface; posterior 5- 60μm
5 slightly metabolic ellipsoidal and cell shape) and some
end lifted; capable of (=changeabl
g e cell shape);
p flattened; rigid produce pseudopodia;
producing pseudopodia (not usually < 10μm cell shape; up to 10μm
alwaysy visible);
); 10 - 25μm 6-100μm
Cercomonas Free-living Capable or incapable
Dujardin 1841 k
kinetopl
i t lastids
tid of producing
Flagella length pseudopodia; with or
Cercomonads without amoeba-like Euglenids Apusomonads
Chart 10 movements;; with or
without emargination C a t 12
Chart Chartt 13
C 3
Flagella equal Flagella different Chart 11 (=depression)
in length in length

Common
C genera example
l s:

Partly leaf- Egg-shaped cell Leaf-shaped cell body Egg-shaped to Pseudopodia capab e of
Incapabl o produ
p oduccing
g
shaped cell body; with scales oval or pointed at the spherical and posteriorly along the pseudopodia; with emargination
body;y; without on the cell p
posterior end;; without flattened cell gliding flagellum;; (=depression; at the flagellar
scales on the surface (visible scales on the cell surface, body; without amoeboid movements; i
insertion
ti along
l the
th edge (“
d (“nos e”));
”))
cell surface; using high complex life-cycle with scales on the without emargination anteriorly depressed laterally
threadlike magnification)
ifi ti ) thick cyst walls; anterior cellll surface
f ((=depression)
d i ) compressed; anterior flagellum thin
pseudopodia flagellum typically waving often difficult to see
may be formed

Gyromitus Thaumatomastix Thaumatomonas Protaspis Bodomorpha Ancyromonas

Skuja 1939 Lauterborn 1899 De Saedeleer 1931 Skuja 1939 Hollande 1942 Kent 1880

Th t d
Thaumatomonads Th
Thaumatomonads
t d Thaumatomonads
Th t d C
Cryomonads
d Gli d
Glissomonads A
Ancyromonads
d
Common species examples: Common species examples:

G. disomatus T. setifera T. lauterborni P. verrucosa B. reniformis B. minima Ancyromonas

y
Skuja 1939 Lauterborn 1899 De Saedeleer 1931 Larsen & Zhukov 1978 (Hollande 1942) Kent 1880
Patterson 1990 Hollande 1952

Chart 3.
 Guide to Free-living Heterotrophic Freshwater Flagellates 847

Non gliding

Flagella length/position; presence of refracting particles/stalk/grooves; cell length

Flagella different in length (longer Flagella equal or different in 2 flagella equal or different in 1 equatorial (usually hard to
flagellum sometimes up to five length; cell attached by one length; cells always in see) and 1 longitudinal
times longer than the cell body; flagellum to the substrate; suspension; with refracting flagellum; clearly
shorter flagellum often hard to see); actively swimming (rotating); particles (ejectisomes; only distinguishable from all other
if attached, then by a plasmatic very tiny cell, 1.5-4μm visible using high flagellates by 1 longitudinal
stalk (never attached by the magnification with phase and 1 equatorial groove;
flagellum=different to bicosoecids;; contrast); anterior end laterally ca. 8-ca.400μm
2-30μm flattened; 5-40μm

Chrysomonads Naked bicosoecids Cryptophyceans Dinoflagellates

Chart 14 Chart 15

Common group, several not yet described

species, often only distinguishable by
molecular techniques (Arndt et al., in prep.)

Occurring benthically; with plastids: 2 green, gold-

coloured, sometimes olive-green, red or blue plastids
(only visible using high magnification with phase contrast);
cortex slightly metabolic (=changeable cell shape)

Presence of scales
(usually only visible using phase contrast)

Without scales With scales

Mode of nutrition

Heterotrophic Mixotrophic (slightly greenish or yellowish

chloroplasts are often visible)

Spumella Ochromonas Paraphysomonas

Cienkowski 1870 Wyssotzki 1887 De Saedeleer 1929

Common species examples:

Common species examples:

Single cells of Scales

Anthophysa and
Siderodendron and Schematic drawing of a
other genera scale as visible under the
(usually colonial) electron microscope
are difficult to
Variable
distinguish from
i
iin size
Spumella

Spumella vulgaris Ochromonas oligochrysis Paraphysomonas vestita

Cienkowski 1870 Ettl 1978 (Stokes) De Saedeleer 1930
Occurring pelagically and benthically; some living in colonies;
some species with lorica;; sometimes attached to the substrate by
a plasmatic stalk ((or exopolymeric matrix)) at the posterior end

Chart 4.
848 A. Jeuck and H. Arndt

With lorica
(shell-like housing, often only
i ibl using
visible i phah se contrt astt))

Not attached to the lorica by the flagellum, Attached to the lorica by the posterior flagellum
(sometimes attached by plasmatic thread)

Lorica shape; flagella; lorica length Lorica shape; flagella; lorica length

Common genera examples:

Lorica very thin; 1 Cone-shaped lorica very Lorica attached by Cone-shaped and pointed Lorica laterally compressed
long
g and 1 short thin; cell bodyy with stalk to substrate; cell lorica; second flagellum rounded at the bottom,, pointed,,
emergent flagellum; chromatophore (mixotroph); body attached to lorica hard to see (free- ending in a thin stalk; second
10-15μm 1 long and 1 short emergent by plasmatic thread; swimming
i i specimens:
i flagellum often lying in a ventral
flagellum; sometimes 1 long and 1 short clearly distinct flagellum); groove; cell not forming a large
pseudopodia; 7--20μm emergent flagellum; cell forming a large lip to lip to one side; 8-12μm
5-28μm one side;; 10-15μm

Calycomonas Poteriochromonas Stokesiella Histiona Reclinomonas

Lohmann 1908 Scherffel 1901 Lemmermann 1910 Voigt 1902 Flavin & Nerad 1993

Chrysomonads Chrysomonads Chrysomonads Jakobids Jakobids

Bicosoecids

Common species examples:

Aqueous lorica, Bell-shaped lorica; cytostomal Bell-shaped lorica; cytostomal Ellipsoidal, egg-shaped to Cylindrical lorica;
transparent; 7-10μm area usually slightly narrowed; area strongly enlarged; h
vase-shaped d lorica
l i ; flagellum up to ca. 40μm
posterior end button-like posterior end rounded and is been unreeled in a jerking
thickened; 12-14μm pointed slightly; 20-40μm manner (cf. tongue of a
chameleon)); ca. 30μm μ

Bicosoeca paropsis
p p Bicosoeca p
planctonica Bicosoeca campanulat
p a Bicosoeca lacustris Bicosoeca cylindri
y ca
Skuja 1956 Kisselew 1931 (=B. crystallina Skuja James-Clark 1867 (Lackey) Bourrelly
1956) 1951
((Lackey)
L k )B Bourrelly
ll 1951

Other common species examples:

B. lacustris
i ht also
might l fform
colonies see
colonies,
Chart 7

B. exilis Penard 1921 B. conica Lemmermann 1912 B. kepneri Reynolds 1927 B. ovata Lemmermann 1914

Chart 5.
 Guide to Free-living Heterotrophic Freshwater Flagellates 849

More than 2
flagella

Number of flagella

Number of flagella; cell shape/length

Other number
of flagella,
4 flagella 2-16 flagella
2 rotation-
symmetric
Egg-shaped to ellipsoid; Spherical, cells often covered 2-16 flagella (asynchronous), Clubbed, drop-shaped or comma-
20-30μm by a layer of mucus; 8-20μm star-shaped inserting; 5-20μm shaped; obligate anaerobic; 8-30μm

Collodictyon Quadricilia Multicilia lacustris Trimastix

Carter 1865 (Skuja) Vørs 1992 Cienkowsky 1881 Kent 1880

Collodictyonids Protista incertae sedis Amoebozoans Preaxostyla

Number of flagella; cell shape/length

12-24 flagella; Many short flagella (asynchronous), all on tip 2 rows of 12 flagella; egg-shaped with a slightly
ca. 20μm at the cytostomal area; conical cell; 10-20μm conical posterior and a truncated anterior end;
obligate anaerobic; 14-20μm

Paramastix conifera Spironema multiciliatum Hemimastix

Skuja 1948 Klebs 1893 Foissner, Blatterer & Foissner 1988

Protista incertae sedis Spironemids Spironemids

2 distinct, posterior flagella Presence of posterior flagella Without posterior flagella

Cell shape; flagella/cell length

length Cell shape; number of flagella; cell length

Irregular posterior cell projection; 6 Oval, spindle-shaped, cigar-shaped

h d Almost triangular, spindle- Egg-shaped or pear-
anterior flagella slightly shorter than cell or flattened; 6 anterior flagella circa shaped or spiral; 6 flagella; shaped, flattened; 8 flagella;
body; obligate anaerobic; 16-22μm as long as cell body; 10-35μm obligate anaerobic; 12-35μm obligate anaerobic; 7-30μm

Urophagus Hexamita Trigonomonas Trepomonas

Klebs 1892 Dujardin 1838 Klebs 1892 Dujardin 1841

Di l d
Diplomonads Di l d
Diplomonads Diplomonads Di l d
Diplomonads

Chart 6.
850 A. Jeuck and H. Arndt

Colonial

P
Presence off collar;
ll number of flagella;
presence of lorica (housing); presence of
stalk

Without collar; 1 visible flagellum Without collar; 2 flagella; With collar; 1 flagellum; Without collar; 2 flagella (second
(second sed to attach to the lorica); without lorica; without stalk with or without stalk flagellum hard to see); without lorica;;
with or without stalk with stalk

Form off the colony;

F l Form of the colony Form of the colony Form of the colony
with or without stalk

Colonies star-shaped: Colonies shaped Colonies first Colonies branched or Colonies branched or Colonies
6-30 cells attached byy like a tree, branched shaped like a unbranched, cells unbranched, cells not branched;
the bottom of the and stuck together cushion or attached or not attached attached to a stalk, young
lorica to each other; (up to five- sponge-like, to a stalk, some attached colonies disc-shaped to colonies are
without stalk ( free- storeyed);
storeyed with long older colonies by intercellular bridges to spherical or tubular colourless,
swimming) stalk ( attached) often llob
bate
t each other,
other othe
otherr cell
cellss finger-shape
- d to linear; older ones
radially, spongee-like or flagellum 2-3 times often
tubelike arranged longer than cell bodies brownish

see Chart
Chart 9: Colonial
choanoflagellates

Bi
Bicosoeca lacustri
l t is Bi
Bicosoec a petiolata
i l S
Spongomonas Phalansterium
Ph l i
James-Clark 1867 (Stein) Bourrelly 1951
1951 Stein 1878 Cienkowski 1870

Bicosoecids Bicosoecids Spongomonads

p g Amoebozoans

With
Withoutt C
Common species
i example
l s:
stalk
see also Phalansterium
Chart 5 digitatum
Stein 1878

Number of cells per colony

Spongomonas uvella
Stein 1878

Bicosoeca ‘socialis‘=colony
form of Bicosoeca lacustris
Common species examples:
Common species example:
Cluster of up to 8 Number of cells often
cells or single < 60, spherical or
ll iirregularly
cells; l l hemispherical
branched system colonies; umbel-like
of rigid stalks with a branched stalk
Cells also free-
swimming; if
Siderodendron Anthophysa with
ith red
d stigma
ti
Pringsheim 1946 Bory 1822 (eyespot
(eyespot)
A. steini Senn
y
Chrysomonads y
Chrysomonads 1900
g
Siderodendron manganiferum p y vegetans
Anthophysa g
Pringsheim 1946 (Müller) Stein 1878
Single cells are difficult to distinguish from Spumella – like flagellates

Chart 7.
 Guide to Free-living Heterotrophic Freshwater Flagellates 851

Choanoflagellates are polymorphic species which build different life-cycle-forms (see Chart 9)

Choanoflagellates
g ((Crasp
pedida))

Presence of theca
((=membranous layyer,, enclosing
g the cell body)
y)
Without theca With theca
Number of collars, presence of stalk

One collar Two collars One collar;; stalked

M
Monosiga
i K Kentt 1880
1880 Diplosiga
Di l i F Frenzel 18 92
1892 Codosiga
C d i
James Clark 1868
James-Clark
Co o spe
Common spec
cies
es examples
e a p es:

Monosiga angustata
Kent 1880

Monosiga ovata
Kent 1880
Diplosiga socialis
F
Frenzell 18
1892
92 Colony form
form of Codosiga:
Monosiga fusiformis see next Chart
K
Kent 1880

Latest investigations showed: Depending on life-cycle, several species might form swarmers easily mistaken for Monosiga – like forms.

Form of th
thee theca

Cup-shaped
p p theca Flask-shaped
p theca Tube-shaped
p theca

Non structured theca Structured theca (w

( ith spike
p s))

g
Salpingoeca Salpingoeca g
Lagenoeca g
Salpingoeca Aulomonas
James-Clark 1868 James-Clark 1868 Kent 1880 James-Clark 1868 Lackey 1942

Common species examples:

This might be the
Common single cell of the
Type species
species: colony
l forming
f i
p
example: Stelexomonas
dichotoma (see
Chart 9))

S. schilleri S. amphoridium Lagenoeca poculiformis S. cylindrica Aulomonas purdyi

(Schiller
(S hill ) St
Starmach 1968 James-Clark
J 186
Cl k 18 688 Schiller
S 1953
hill 1953 Fott 1953
F tt 1953 Lackey
L k 194 2
1942

S. urceolata S. oblonga S. vaginicola S. gracilis S. frequentissima S. globulosa S. variabilis (=Lagenoeca

Kent 1880 Stein 1868 Stein 1878 James-Clark 1868 (Zacharias) Lemmermann 1913 Zhukov 1978 variabilis) Skuja 1956

Chart 8.
852 A. Jeuck and H. Arndt

Life-cycl
y e of choanoflagellate
g s,,
example for Salpingoeca (after Dayel et al. 2011)
Desmarella – like colony Sphaeroeca – like colony

Colonial cells
C
Colonial choanofflagellates (C
(Craspedida))

Form of colony

Unbranched colonies Branched colonies

Solitary cells
Slow swimmer Fast swimmer

Salpingoeca – like
thecate cell

Sitting separately
separately or in 4 15 cells sitting together
Usually 4-15 M
Many stalked
t lk d cells
ll radially
di ll 6
6-60
60 cells may
may be
groups
group s at the tip
tip of the stalk, like a pear
pearll necklace;
necklace; single cells arranged in a gelatinou
arranged gelatinouss held together by a
stalk is 2-10 times longeg r attached byy intercellular bridges,
g matrix gelatinous cluster
g
than cell body free-swimming

Codosiga Desmarella-like forms Sphaeroeca-like forms Proterospongia-like forms of

James-Clark
J Cl k 18
186
688 off choanoflagellates
h fl ll t off choanoflagellate
h fl ll t s choanoflagellate
h fl ll t s

Desmarella/Sphaeroeca/Proterospongia – like forms might be life-cycle forms of other species

C
Common i
species
example:

Si
Single
l cellll

If occurring as single cells (detached from the colony),

Codosiga botrytis those are capable of producing pseudopodia at the
(Ehrenberg)
(Eh b ) Kent
K t 1880 t i end
posterior d.

Common species example:

2 to several
several cells, attache
cells at tached by Cells not attached
attached to a
long and thin individual stalks to stalk, surrounded by a
a common stalk;; branched like dichotomous tube of
an umbel or bifurcated organici matter

Codosiga
C d i Stelexomonas dichotoma
James-Clark 1868
James-Clark 1868 Lackey 1942

According to latest molecular investigations,

Codosiga umbellata
this morphotype is present in several cryptic
Stein 1878
species
i which
hi h currently
tl might
i ht nott yett be
b
distinguished

Chart 9.
 Guide to Free-living Heterotrophic Freshwater Flagellates 853

Free-living kinetoplastids
2 flagella; agitated 1 visible flagellum and 1
Number of flagella; movement; position of the ingestion apparatus
swimming; ingestion moveable anterior proboscis
apparatus at the tip of the („nose“ =trunk-like
anterior end, partially (e.g. protrusion of the cell) (with
Rhynchomonas nasuta (Stokes 1888)
Bodo saltans) attached to smaller flagellum); proboscis
– species complex
substrate beating frequently; ingestion
Neobodonids
apparatus located in
proboscis
Movement
Common species examples:

Cell body swings abruptly around the base Anterior flagellum is swinging
Anterior flagellum Fast and equal swimmer;
of the flagellum; cell is ca. 5-10μm distant around the anterior part of the
‚paddles‘ and when crawling on the
from the substrate, attachment by the tip of cell like a lasso; attachment by
appears hook - like substrate, then cell is
the recurrent flagellum; anterior flagellum the recurrent flagellum is trembling and swinging;
creates a whirl to transport the bacteria into frequently changing browsing the surface with
the ingestion apparatus
the anterior flagellum

Bodo saltans Neobodo designis Neobodo curvifilus Parabodo

Ehrenberg 1832 - species complex (Skuja) Vickerman 2004 - species complex Griessmann 1913 Skuja 1939

Eubodonids Neobodonids Neobodonids Parabodonids

Common species
example:

Other common genera examples of neobodonids:

Dimastigella Rhynchobodo
Lackey 1940 Parabodo nitrophilus
Sandon 1928 Skuja 1939

Chart 10.

(e.g. Pawlowski et al. 2011; Stoeck et al. 2010; knowledge of HF (recommended literature: e.g.
and Domonell et al., unpubl. observ.). However, Hausmann et al. 2003; Leadbeater and Green
their application still bears a lot of problems 2000; Patterson and Larsen 1991). The systematic
concerning the following points: 1) many specific position of flagellate groups considered in the guide
primers are necessary to cover the whole HF is given in Figure 1 following the recent revision by
diversity due to the wide molecular diversity of Adl et al. (2012). Following the idea of Foissner and
HF (some groups will be overlooked), 2) active Berger (1996) with regard to ciliates, we designed a
and inactive forms are difficult to differentiate, 3) polytomous guide. The first Chart contains a gen-
databases are incomplete (Will and Rubinoff 2004) eral overview of the most important characters of
and contain high numbers of errors (Prosdocimi the main groups of protists. The user is led to a
et al. 2013). Quantitative morphotype studies will “fast-track” regarding the main groups of HF (Charts
provide helpful information to increase reliability 1–15) by schematic drawings with arrows point-
of databases and offer important knowledge on ing to the important characters. If identification is
the ecology and evolution of HF groups significant already sufficient the user is led to the special
in ecosystems (Pawlowski et al. 2012). We would Chart of the organism. If not, he should proceed
like to increase the resolution of these quantitative to Chart 2. The user is led from obvious characters
morphotype studies by this taxonomic guide. (e.g. colonial or single cells, number of flagella) to
more detailed descriptions. Features of organisms
which are used to discriminate between different
How to Use the Guide taxa are set within boxes while the discriminator for
the subsequent path of identification is shown with-
The present guide is intended to help also unex- out boxes. Figures are added to the descriptions of
perienced researchers to identify HF morphotypes, characters to accelerate and increase the chance
although it is recommended to have a general of identification. All scale bars in the guide indicate
854 A. Jeuck and H. Arndt

Cercomonads Ultrastructural and

molecular
l l analysis
l i isi
often needed for a
Cell/pseudopodia
p p shape
p /leng
gth;; flagella
g movement/leng
gth;; changeability
g y of cell shape
p distinct differentation

Comprising small, but also largest known Small and compact cell body, Often smaller cell body; various Spherical or oval cell body;
cercomonads; pseudopodia mostly when moving, then spindle- forms of pseudopodia; anterior various forms of pseudopodia;
rounded or finger-like;
- often with long shaped; short flagella, flagellum is slowly rowing, very long anterior flagellum,
flagella, posterior trailing; slightly posterior trailing; 5-15μm posterior trailing; 3-18μm posterior often not attached to
metabolic; 5-60μm cell body; ca. 10-13μm

Cercomonas Eocercomonas Paracercomonas Nucleocercomonas

Dujardin 1841 emend. Karpov et al. 2006 Karpov et al. 2006 Karpov et al. 2006 Arndt et al. 2012
Common species examples: Other
forms

Cercomonas plasmodialis Eocercomonas ramosa Paracercomonas metabolica Nucleocercomonas praelonga

Mylnikov 1985 Karpov et al. 2006 (Zhukov and Mylnikov 1987) Brabender et al. 2012
Karpov et al. 2006

Ovoid cell body, dorsoventrally Highly and fast varying cell shape; various forms of Oval or drop-like cell body; various forms of
compressed; rigid cell shape; pseudopodia
pseudopo d a an
a ywher
y eeo on tthe
e cell;
ce ; aanteri
te oor flagell
age u um pseudopodia; very short anterior flagellum,
3-9μm beats very often; fast gliding cell; extremely metabolic; not tapering, directed to the left, posterior is
9-13μm longer; ca. 5-8μm

Cavernomonas Metabolomonas Brevimastigomonas

Vickerman 2009 Kiss et al. 2012 Nitsche et al. 2012

For detailed descriptions of these genera see: Bass et al. 2009; Brabender et al. 2012; Karpov et al. 2006

Chart 11.

10 ␮m. The guide leads only to the species or genus 5-20 ␮l on a prepared microscope slide or an Utermöhl cham-
morphotype. It is widely accepted that many pro- ber (Utermöhl 1958; HydroBios GmbH, Kiel, Germany; Fig. 2).
The HF composition has to be analysed within one hour after
tistan morphotypes contain a few up to hundreds sampling by means of a phase contrast microscope equipped
of genotypes (typical examples are the common with high resolution video-recording which is helpful for further
species Neobodo designis (Scheckenbach et al. and later identification. 20x, 40x objectives are helpful for rough
2006) or Codosiga botrytis (Stoupin et al. 2012)), morphological observations and quantitative counts. Individual
see above. If video sequences (Supplementary samples can be analysed within a few minutes (particularly
critical for pelagic samples). The use of a 63x long distance
Material) are available for the organisms, they are objective or water immersion objectives (63x and 100x; with a
marked by a video icon ( ). Some additional lit- long working distance) are recommended for morphotype iden-
erature is given on the Charts (Cavalier-Smith and tifications. Phase contrast equipment is mostly indispensible to
Chao 2010; Bass et al. 2009; Brabender et al. 2012; analyse position and movement of flagella, the presence of col-
Karpov et al. 2006). Due to the fact that major forms lar, lorica, or stalk of some flagellates. Differential interference
contrast (DIC) equipment might additionally help to identify cell
of heterotrophic flagellates are ubiquitous in marine structures such as paramylon, ingestion apparatus, vacuoles or
and freshwater environments, this guide may also extrusomes.
be useful in parts for an orientation regarding flag- A realistic compromise should be a combination of counts
ellate groups in brackish water systems. by the living-droplet method with counts of chemically fixed
samples and analysis by epifluorescence microscopy. The fol-
lowing fixatives are commonly in use: 2% glutaraldehyde (e.g.
Caron 1983; Choi and Stoecker 1989), 2% formaldehyde (e.g.
Methods Porter and Feig 1980; Sherr et al. 1989), buffered formalde-
hyde (e.g. Børsheim and Bratbak 1987; Sherr and Sherr 1983)
The guide is based on an analysis using the classical living- and 0.5% acidic Lugol’ solution (10 g I2 , 20 g KI, 10 g sodium
droplet method by phase contrast/DIC light microscopy (e.g. acetate in 140 ml aq. dest.) plus 3% formaldehyde (e.g. Sherr
Arndt and Mathes 1991; Gasol 1993; Massana and Güde et al. 1989).
1991). This method is reliable to detect morphological and The fixed samples need to be kept at 4 ◦ C in the dark until
behavioural characteristics of even very small HF regarding staining. Fluorescent dyes (e.g. DAPI (= 4 6-diamidino-2-
their main taxonomic groups – in contrast to hidden charac- phenylindole, Sigma Lot 47H4107, Porter and Feig 1980) are
teristics within samples treated by fixatives. Pelagic flagellates suitable for staining. After staining the sample should be filtered
(undiluted samples) and benthic flagellates (diluted by a fac- onto a 0.2-␮m membrane filter using vacuum not exceeding
tor of 5- >20 with filtered water) can be studied in droplets of 150 mm Hg. For an even distribution of the particles, another
 Guide to Free-living Heterotrophic Freshwater Flagellates 855

Euglenids
g

1 visible flagellum; second flagellum reduced or fused to 2 flagella,, different in leng

gth,, heterodynami
y c ((the flagella
the side of the cell;; storage
g granule
g (p
(paramylon
y )p present have different beating patterns)

Changeability of the cell shape

Metabolic ((=changeable cell shape) Relatively rigid

Form/movement of flagellum/cell
g Form/movement of flagellum/cell
g body;
y;
body; cell shape/length cell shape/length
Common genera examples: Common genera examples:

Well developed Relatively thick flagellum Relatively thick flagellum, Flagellum stiffed in front Flagellum most active
flagellum, most active beating in whiplash fashion;
fashion; beats in a lashingg manner; of the cell, most active p front end
near the tip;
near the tip; cell body cell body actively swimming sickle-shaped or bean- and vibrating near the of the cell enlarged to
squirms actively; (rotating) or metabolically shaped, laterally or tip; egg-shaped, ‘flickering’ funnel-
cigar-, cone-shaped gliding; spindle-shaped to d
dorsoventr ll flattened
t ally tt d; spindle-shaped oval to shaped frontal region,
to spherical (often cylindrical; 16 - 80μm 14-100μm triangular; rigid cells and often striated;
deforming) often dorsoventrally flattened; 25 - 60μm
flattened;; 20 -100μm
μ 6-50μm

Peranema Astasia Menoidium Petalomonas Urceolus

Dujardin 1841
1841 Dujardin 1841 Perty 1852 Stein 1859 Mereschkowsky 1879

Confusion with
Notosolenus possible:
p
observe carefully if
P
there is a second
Paramylon (P)
P fl ll
flagellum ( h the
(when th
cell turns) P

P
Confusion with
Heteronema possible:
observe carefully if there Ingestion
g ap
ppparatus; form/movement of flagella;
g changeability
g y of
iis a second fl ll
d flagellum cell shape; cell length; presence of storage granule (paramylon)
(when the cell turns)
Some common genera examples:

With ingestion apparatus (often hard to With ingestion apparatus With ingestion ap apparatus
paratus With ingestion ap apparatus
paratus
see); swimming anterior flagellum weaker (small); swimming anterior
(small); (often hard to see); swimming
swimming (well develope
developed d and
and often shorter than posterior,
p , beating
g g
flagellum ( p in tail motion))
(tip anterior flagellum
g (most
( active visible);
); p
posterior
normally; posterior trailing, contracts to jerk often longer than trailing near the tip) longer than flagellum trailing behind
the cell backwards; cells dorsoventrally posterior; often metabolic trailing posterior; cells the cell when moving;
flattened
fl tt d; slightly
li htl metabolic
t b li (=changeable
( h bl ((=changeabl
h ble cellll sha
h pe);
) dorsoventr
d ll flattened;
t ally tt d rigid;
i id cells
ll d
dorsoventrally
t ll
cell shape) or rigid; 14-100
100μm 25 100μm
25-100μ 10-30μ
10 30μm flattened; rigid;
rigid; 10 -30μm
30μm

Anisonema Dujardin 1841 Heteronema Dujardin 1841 Notosolenus Stokes 1884, Entosiphon Stein 1878
em. Sk j 1939
Skuja 1939

Ingestion
Confusion with Confusion with
Confusion apparatus
t
Peranema possible Petalomonas P
if the second possible if the
flagellum is second flagellum is
overlooked overlooked
P
Second flagell
g um

Chart 12.
856 A. Jeuck and H. Arndt

Apusomonads

Position of the anterior and recurrent flagellum; changeability of the cell shape

Anterior flagellum lies in a proboscis (=trunk-like protrusion of the Anterior trailing flagellum protrudes from the proboscis (=trunk-like
cell); recurrent flagellum often hard to see; cells are metabolic protrusion of the cell); recurrent flagellum lies directed posteriorly
p y under
(=changeable cell shape) and some produce pseudopodia the cell; flattened cell body is slightly metabolic (=changeable cell shape)

Apusomonas
Alexeieff 1924

Common species example:

Easily
confused with Apusomonas proboscidea
Rhynchomonas Alexeieff 1924
nasuta!

Amastigomonas Manchomonas Podomonas Thecamonas Multimonas

De Saedeleer 1931 Cavalier-Smith 2010 Cavalier-Smith 2010 Larsen & Patterson 1990 Cavalier-Smith 2010

For detailed descriptions of these genera see: Cavalier-Smith and Chao 2010

Chart 13.

Cryptophyceans

Flagella length; presence of dark refracting particles ((=ejectisomes) (partly hard to see); presence of bright
refracting storage granulum; presence of striations; cell shape; mode of movement; cell length

Flagella approximately equal in length; Flagella approximately equal in length; ejectisomes Flagella different in length; ejectisomes
ejectisomes orientated in a row around the are clustered around a emargination (=depression) scattered within the cell body (only visible
anterior end of the cell (only visible using high at the anterior end (only visible using high using high magnification with phase
magnification with phase contrast); without magnification with phase contrast); with storage contrast); with storage granulum; without
storage granulum; with 3 - 6 striations; clearly granulum; without striations; circular section; striations; circular section; swimming and
dorsoventrally flattened; swimming close to the swimming fast in the water column; 15 - 30μm jumping in the water column; 10 -18μm
substrate; 5 -12μm

Goniomonas Cryptomonas Kathablepharis

(Fresenius) Stein 1878 Ehrenberg 1831 emend. Hoef-Emden & Melkonian 2003 Skuja 1939
Synonym: Cyathomonas Fromentel 1874 Synonym: Chilomonas Ehrenberg 1831

Cryptomonads Cryptomonads Kathablepharids

Common species examples: Common species example: Common species example:

Movement:
Similar species fast, rotating in
occur in brackish Refracting a waving
Many food
water: ejectisomes manner, shorter
vacuoles
e.g. flagellum
fl ll beats
b t
G. amphinema around the
Larsen & Cryptomonas paramaecium anterior end;
Patterson 1990:: Ehrenberg 1832 emend. yp
only pelagically
g y
Hoef-Emden & Melkonian occuring.
2003
Kathablepharis
Goniomonas truncata ovalis
(Fresenius) Stein 1878 Skuja 1948

Chart 14.
 Guide to Free-living Heterotrophic Freshwater Flagellates 857

Dinoflagellates

Cell shape

Egg-shaped,, ellipsoidal to spherical Other cell shape

Position of the equatorial groove (cingulum)

Oth position
Other iti off the
th Anterior position of
cingulum the cingulum
C ll sha
Cell h pe; form
f off grooves; cellll llength
th

Common g
genera examples:
p

Often with longitudinal Almost bilateral symmetrical; With plates, sometimes also Distinct theca (=membranous
striations on the surface; cingulum at the cell’s ornamented with spines, teeth-like layer, enclosing the cell body);
longitudinal groove (sulcus)
( l ) posterior
t i end, d the
th two
t ends
d projections or wings; cingulum
i l almost
l t edges
d of the grooves are
partially spirals to cingulum,
cingulum of the cingulum meet; horizontally divides the body into two clearly visible due to ridges;
the two ends of the cingulum
cingulum morphology chang
morphology change ess due to halfs of different length; 20-100μm
do not meet;1 ; 0-50μμm ingested
g food items;; 10- 16-55μμm
50μm

Gyrodinium Gymnodinium Peridiniopsis Peridinium

Kofoid & Swezy
y 1921 Stein 1878 Lemmermann 1904 Ehrenbergg 1832

With With
Withoutt
Withou Without
chlo
hl roplast
l ts chlo
hl roplast
l ts
p
chloroplast s,, chloroplasts,
but may but may
contain t i
contain
colou
l red d coloured
food food
vacuoles vacuoles

Common genera examples:

l

Cell shap
pe; p
position/form of g
grooves; cell lengt
gh

Cell body often egg-shaped, usually the

Sometimes egg- or mushroom-shaped cell llongitudinal
it di l groove (sulcus
( l ) exten
t dsd from
f
body, o
oftte
en do
dorsoventrall
so e t a y flattene
atte edd;; 8
8-50μ
50μm without
the cingulum witho ut torsion; 6 50μm
torsion; 6-50μ
Without With
p
chloroplasts chloroplasts Amphidinium
Katodinium Claparède & Lachmann
Fott 1957
1957 1858/61

Cell shape; position/form

n/form of grooves; cell length

Common genera examples:

With longer or shorter horn-likelike Lens-shaped to round,

Lens round almost Elongate elliptical or lunate,
p
projection
j s (anterior
( end with Common y
symmetrical; ; equatorial
q groove
g sometimes rounded or
one horn, posterior end with p
species (cingulum) almost spherical; apiculate, with large spines;
several), asymmetrically example: longitudinal groove (sulcus) only at 40-450μm
shaped
h d; equatorial
t i l groove the posterior
th t i sideid off the
th cellll body
b d ;
(cingulum
(cingulum)) lies horizo
horizontally
ntally, 16 40μm
16-40μ With
divides the cell body into two chloroplasts
halfs of nearlyy eq
qual length;
g Without
40-450μm chloroplasts,
hl l t
but may
With contain
chloroplasts coloured
C ti
Ceratium Diplopsalis food Cystodinium
Ceratium hirundinella
Schrank 1793 Bergh 1881 Klebs 1912
(O.F. Müller) Dujardin 1841 vacuoles

Chart 15.
858 A. Jeuck and H. Arndt

Figure 2. Schematic drawing of some helpful counting chambers. a) Simple microchamber: Two cover slips
are placed on the slide (each on every end). A 10 ␮l droplet is placed between them and a third cover slip above
the droplet and the two slips. b) Utermöhl chamber for counting living flagellates or sedimented, chemically
fixed samples.

membrane (0.6 ␮m pore size) may be placed underneath the Appendix A. Supplementary data
filter. The filters are generally stored deep-frozen at –20 ◦ C
until examination by an epifluorescence microscope.
It is additionally recommended to establish cultures of HF
Supplementary data associated with this arti-
isolated from the samples (e.g. simply by adding autoclaved cle can be found, in the online version, at
wheat grains etc. as a carbon source for food bacteria to http://dx.doi.org/10.1016/j.protis.2013.08.003.
parts of raw samples). This should aid in morphotype iden-
tification of bacterivorous forms and to check whether the
determined morphotypes are life-cycle forms (swarmer, colony
etc.) of other types, as it is often the case with e.g. choanoflag-
ellates where extrinsic factors can induce the formation of References
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