Molecular Phylogenetics and Evolution

Molecular Phylogenetics and Evolution 76 (2014) 110–126
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev
Phylogeny of Eutardigrada: New molecular data and their morphological

support lead to the identification of new evolutionary lineages
Roberto Bertolani a, Roberto Guidetti b,⇑, Trevor Marchioro b, Tiziana Altiero a, Lorena Rebecchi b,
Michele Cesari b
a
Department of Educational and Human Sciences, University of Modena and Reggio Emilia, Reggio Emilia, via Allegri 9, 42121 Reggio Emilia, Italy
b
Department of Life Sciences, University of Modena and Reggio Emilia, Modena, via Campi 213/D, 41125 Modena, Italy
a r t i c l e i n f o a b s t r a c t
Article history: An extensive study of the phylogeny of Eutardigrada, the largest class of Tardigrada, has been performed
Received 11 September 2013 analyzing one hundred and forty sequences (eighty of which newly obtained) representative of one hun-
Revised 18 December 2013 dred and twenty-nine specimens belonging to all families (except Necopinatidae) of this class. The molec-
Accepted 7 March 2014
ular (18S and 28S rRNA) results were compared with new and previous morphological data, allowing us
Available online 19 March 2014
to find new phylogenetic relationships, to identify new phylogenetic lineages, to erect new taxa for some
lineages, and to find several morphological synapomorphies supporting the identified clusters. The class
Keywords:
Eutardigrada has been confirmed and, within it, the orders Apochela and Parachela, the superfamilies
Tardigrada
Molecular phylogeny
Macrobiotoidea, Hypsibioidea, Isohypsibioidea, and Eohypsibioidea, and all the families and subfamilies
Molecular systematics considered, although with emended diagnoses in several cases. In addition, new taxa have been erected:
Morphology the new subfamily Pilatobiinae (Hypsibiidae) with the new genus Pilatobius, as well as an upgrading of
Integrative taxonomy Diphascon and Adropion to genus level, previously considered subgenera of Diphascon. Our results dem-
Pilatobius gen. nov onstrate that while molecular analysis is an important tool for understanding phylogeny, an integrative
Pilatobiinae subfam. nov and comparative approach using both molecular and morphological data is necessary to better elucidate
evolutionary relationships.
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Guil et al., 2013b) revealed some incongruence with the traditional
morphological systematics of the phylum. In particular, the papers
With the application of DNA sequencing to tardigrades, new by Sands et al. (2008), and by Marley et al. (2011) revealed differ-
information has been obtained on the phylogenetic position of ent evolutionary lines within Parachela that were only partially in
both the phylum and its main evolutionary lines. Tardigrades have agreement with those revealed by the previous morphological
been included in the clade Ecdysozoa by Aguinaldo et al. (1997), classical approaches. In particular, four main clusters were identi-
within the Panarthropoda, together with Onychophora and fied for which four superfamilies were proposed, which were par-
Arthropoda (Rota-Stabelli et al., 2010; Campbell et al., 2011), tially confirmed in further studies (Jørgensen et al., 2010; Guil and
although the position of Tardigrada within this last group is still Giribet, 2012). The superfamilies were initially without any mor-
undetermined (Nielsen, 2012). phological support, which was presented only later by Marley
Molecular analyses within the phylum confirmed the subdivi- et al. (2011). The most surprising conclusion of Sands et al.
sion of the higher taxa of Tardigrada, namely Heterotardigrada
(2008) was the attribution of Hypsibius and Isohypsibius to two dif-
(Jørgensen and Kristensen, 2004; Jørgensen et al., 2010) and Eutar-
ferent superfamilies, since these two genera were previously con-
digrada (Guidetti et al., 2005; Nichols et al., 2006). In contrast, re-
sidered belonging to the same sub-family (Hypsibiinae).
cent molecular studies at the genus and species levels (Guidetti
Considering that several eutardigrade families and genera were
et al., 2005, 2009; Kiehl et al., 2007; Møbjerg et al., 2007; Sands
not included in previous papers based on molecular analysis, we
et al. 2008; Jørgensen et al., 2010, 2011; Guil and Giribet, 2012;
carried out a further phylogenetic study analyzing two molecular
markers expanding the study to all eutardigrade families (except
⇑ Corresponding author. Fax: +39 0592055548. Necopinatidae) and increasing the number of analyzed genera
E-mail addresses: [email protected] (R. Bertolani), roberto.guidetti@ and species. In addition, considering that Eutardigrada, the largest
unimore.it (R. Guidetti), [email protected] (T. Marchioro), tiziana.altiero@
unimore.it (T. Altiero), [email protected] (L. Rebecchi), [email protected]
class of the phylum, is generally characterized by a low level of
(M. Cesari). morphological diversity (especially compared with Heterotardigra-
http://dx.doi.org/10.1016/j.ympev.2014.03.006
1055-7903/Ó 2014 Elsevier Inc. All rights reserved.
R. Bertolani et al. / Molecular Phylogenetics and Evolution 76 (2014) 110–126 111
da; Fig. 1), and the identification of synapomorphies for each of the a Leitz DM RB microscope, using differential interference contrast –
phylogenetic lineages has been a particularly difficult task, we ana- DIC – and phase contrast – PhC, with 40 and 100 immersion oil
lyzed morphological characters of the studied taxa more in depth, objectives) and identified according to its morphological characters
utilizing light and scanning electron microscopy, in order to (Guidetti and Bertolani, 2005; Pilato and Binda, 2010). Before DNA
acquire better knowledge of eutardigrade phylogeny and system- extraction, pictures of the sclerified structures of in vivo specimens
atics. Therefore, for classifying organisms within a phylogenetic were also taken by using a Nikon DS-Fj1 photocamera, following
systematic framework, we used an integrative approach (as in the protocols described in Cesari et al. (2011) and Bertolani et al.
Bertolani et al., 2011a), analyzing molecular phylogenies and com- (2011b).
pare them with morphological characters. With our integrative
approach our goals were both to verify the current evolutionary
2.2.2. DNA extraction, PCR amplification, and sequencing
lineages (or to identify new ones) and to provide further morpho-
Genomic DNA extraction was carried out from 80 single speci-
logical support for those taxa, which to date have a taxonomic des-
mens (Table S1) through a rapid salt and ethanol precipitation
ignation but are insufficiently described and thus not easily
(Cesari et al., 2009). Regions of the nuclear ribosomal subunit
identifiable.
genes 18S and 28S rRNA were amplified using the following primer
combinations: SSU F04 (50 -GCT TGT CTC AAA GAT TAA GCC-30 ) and
2. Materials and methods SSU R26 (30 -CAT TCT TGG CAA ATG CTT TCG-50 ; Kiehl et al., 2007)
for 18S, and 28S 1274 (50 -GAC CCG TCT TGA AAC ACG GA-30 ) and
2.1. Species sampling 28S 689 (50 -ACA CAC TCC TTA GCG GA-30 ) for 28S. For both genes,
the polymerase chain reaction was carried out in 20 ll of reaction
Eighty eutardigrade specimens belonging to 26 genera were used. volume, which consisted of 2 ll reaction buffer (including 20 mM
The full list of specimens, including collecting information, is pro- of MgCl2), 2.5 mM of each dNTP, 10 pmol (final concentration) of
vided in Table S1. Tardigrades were extracted from different sub- each primer, 1 U of DreamTaq polymerase (Fermentas) and 2 ll
strates (Table S1) collected in Europe and in USA. Moss, grass and of template DNA. A negative control lacking template DNA was car-
leaf litter were placed in water for about half an hour. Then, animals ried out to test the possibility of contamination with foreign DNA.
were isolated from all substrates, including freshwater sediments, PCR was performed in a PCR Sprint Thermal Cycler (Hybaid). The
using sieves. Finally, tardigrades were individually picked up using protocol for 18S consisted of 35 cycles with 1 min at 94 °C, 35 s at
a glass pipette under a stereomicroscope and processed. 52 °C and 2 min at 72 °C, with a final elongation step at 72 °C for
10 min. The protocol for 28S consisted of 40 cycles with 45 s at
2.2. Molecular analyses 96 °C, 1 min at 48 °C and 1 min at 72 °C, with a final elongation step
at 72 °C for 10 min. The amplified products were gel purified using
2.2.1. Species identification for molecular analysis the Wizard Gel and PCR cleaning kit (Promega). Sequencing reactions
Before DNA extraction, each tardigrade specimen used in were performed using the ABIPRISMÒ BigDye™ Terminator Version
molecular analysis was observed in vivo by light microscopy (with 1.1 Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on
Fig. 1. Scanning electron micrographs of tardigrades. A: Batillipes (Arthrotardigrada) (picture kindly donated by C. Schulze and A. Schmidt-Rhaesa). B: Echiniscus
(Echiniscoidea). C: Milnesium (Apochela). D: Thulinius (Parachela, Isohypsibioidea). E: Ramazzottius (Parachela, Hypsibioidea). F: Bertolanius (Parachela, Eohypsibioidea). G:
Paramacrobiotus (Parachela, Macrobiotoidea). Scale bars: A = 20 lm; B–G = 100 lm.
112 R. Bertolani et al. / Molecular Phylogenetics and Evolution 76 (2014) 110–126
purified amplicons. Each sequencing reaction contained 0.2 lM of a using the program RAxML v7.2.4 (Stamatakis, 2006) using the
single PCR primer to initiate the sequencing reaction, 2 ll of Big- GTR + Gamma model. Bootstrap resampling with 1000 replicates
Dye™, 70 ng of purified products, 4 ll of 5 BigDye™ Terminator was undertaken via the rapid bootstrap procedure of Stamatakis
Version 1.1 Sequencing Buffer and distilled H2O for a final volume et al. (2008) to assign support to branches in the ML tree.
of 20 ll. Cycling conditions for sequencing reactions consisted of RNA secondary structure, protein secondary/tertiary structure,
25 cycles of 96 °C for 10 s, 50 °C for 5 s and 60 °C for 4 min. Both and other functional considerations all influence the rate and pat-
strands were sequenced using an ABI Prism 3100 (Applied Biosys- tern of evolution: variability characterizing both slowly and rap-
tems, Foster City, CA, USA). The nucleotide sequences of the newly idly evolving stems is a direct consequence on the identity of the
analyzed specimens have been submitted to GenBank (Table S1). bases; tertiary structure and protein interactions (Gutell et al.,
2002; Hickson et al., 1996; Pagel and Meade, 2004). To account also
2.2.3. Phylogenetic analysis for this variability, a Bayesian mixture model (Pagel and Meade,
To achieve the best evolutionary tree and the most reliable phy- 2004) was tested and used on the aligned dataset. Mixture model
logenetic inference, phylogenetic analyses were focused on a com- do not require a priori specification of partitions (as for the doublet
bined dataset (18S and 28S rRNA) to prevent stochastic errors and model). The parameters of each evolution model (substitution
to yield an evolutionary inference based on the most complete rates, base frequencies) are estimated from the data and, in the
dataset for both taxon and gene sampling. end, to each character can be assigned a probability of membership
Sixty-nine 18S rRNA sequences and 19 28S rRNA sequences of in each model. This method produces different categories among
tardigrades belonging to different genera and species of heterotar- the alignment to which appropriate models (inferred from data)
digrades and eutardigrades were retrieved from GenBank are applied. The CAT model (mixture model) implementation was
(Table S2), when available the complete 18S and 28S sequences achieved by using the program Phylobayes 3.2e (Lartillot and
were the preferred source of data mining. Sequences of 10 out- Philippe, 2004) and the alignment was analyzed using the site-
groups belonging to Arthropoda, Priapulida and Kinorhyncha heterogeneous CAT + Gamma model. For all Phylobayes analyses
where carefully selected (to avoid contaminant and sequences two chains were run. Chains where considered to have converged
from misidentified species) and downloaded from the NCBI data- when the ‘‘Maxdiff’’ between the two independent chains was <0.2
base to complete the dataset (Table S2). The combined dataset (see Phylobayes manual). The ‘‘bpcomp’’ program in the Phyloba-
was assembled to achieve the most complete taxon sampling yes suite was used to test convergence. For each analysis, burn in
rather than the most homogeneous gene sampling. However, to and subsampling frequencies were estimated in order to minimize
ensure to the phylogenetic software the possibility to explore the ‘‘maxdiff’’.
entire ‘‘tree space’’ (potential trees topology given an alignment),
at least one common gene was always granted; specifically the 2.3. Morphological analysis
18S gene sequence, which was therefore present for every consid-
ered taxa. Preliminary analyses on the single gene datasets allowed 2.3.1. Light microscopy
to identify redundant sequences or poorly curated data. Tardigrade specimens were mounted on slide in polyvinyl lac-
The multigene dataset was aligned with the Muscle algorithm tophenol fluid or in Faure-Berlese fluid. All mounted animals were
implemented in MEGA 5 (Tamura et al., 2011). Default parameters observed up to 100 immersion oil objective magnification, using
were used because they are designed for the average best accuracy DIC and PhC as cited above for an accurate analysis of the taxo-
(Edgar, 2004). The resulting alignment was accurately inspected by nomic characters (e.g. claws, mouth, buccal-pharyngeal apparatus,
searching for software homology misinterpretations. The GBlocks cuticle). Pictures of the sclerified structures of mounted animals
program (Catresana, 2000) was used for applying relaxed settings have been taken using the same photocamera cited above.
and parameters (minimum number of sequences for a conserved po- Specimens originating from the same samples used for molecu-
sition: 27; minimum number of sequences for a flank position: 30; lar analysis (Table S1) were mounted in Faure-Berlese fluid as
maximum number of contiguous non-conserved positions: 20; min- paragenophore vouchers (Pleijel et al., 2008; Cesari et al., 2013).
imum length of a block: 2; allowed gap positions: all) and aiming to Further detailed observations were carried out on specimens of
discard uninformative regions of the alignment. Alignment concate- Acutuncus antarcticus (Richters, 1904), Calohypsibius ornatus (Rich-
nation was accomplished through Seaview alignment editor (Gouy ers, 1900), Hebesuncus conjungens (Thulin, 1911), Microhypsibius
et al., 2010). The complete combined alignment included 129 tardi- bertolanii Kristensen, 1982, Microhypsibius sp., Mixibius saracenus
grade taxa and a maximum of 3701 characters in length. (Pilato, 1973), Hexapodibius micronyx Pilato, 1969, Hexapodibius
Bayesian inference (BI) was conducted using the program sp., Parhexapodibius ramazzottii Manicardi and Bertolani, 1987, Par-
MRBAYES 3.1.2 (Huelsenbeck and Ronquist, 2001) using the hybrid hexapodibius pilatoi Bernard, 1977, Ramajendas sp. and Ramazzot-
MPI/OpenMP version (Pratas et al., 2009) on the CIPRES Science tius sp. from Bertolani’s collection (Department of Life Sciences,
Gateway Portal (http://www.phylo.org/sub_sections/portal/). Best University of Modena and Reggio Emilia, Italy) and specimens of
fitting model evaluations for each analysis were performed taking Parhexapodibius lagrecai Binda and Pilato, 1969 from Binda and Pil-
into account AIC, BIC and hLRT (jModeltest 0.0.1, Posada, 2008) ato’s collection (Department of Biological, Geological and Environ-
resulting in the GTR + Gamma + I model to be most suitable for mental Sciences, University of Catania, Italy), mounted in polyvinyl
all the datasets. Tracer 1.5 (Rambaut and Drummond, 2007) was lactophenol fluid or in Faure-Berlese fluid.
used to compute and compare Bayes factors for the two models
that scored better in the AIC and BIC tests. After this analysis the 2.3.2. Scanning electron microscopy
best fitting model resulted in the GTR + Gamma, which has been Paragenophores (adult animals) of several species used for molec-
used in the final analysis. In MrBayes, two independent runs, each ular analysis were prepared for scanning electron microscopy (SEM)
of four Metropolis-coupled Markov chains Montecarlo (MCMC), observations of the mouth opening, cuticle surface and claws. To pre-
were launched for 40 106 generations, trees were sampled every vent animal contraction during fixation, animals were first relaxed in
1000 generations. The obtained parameter value files of each run warm water (50 °C) until they were completely relaxed. Then tardi-
were analyzed with Tracer 1.5 to verify the convergence of the grades were fixed in boiling absolute ethanol for a few minutes and
MCMC runs and their effective sample sizes. rinsed three times in absolute ethanol. Finally animals were dehy-
A phylogenetic analysis on the dataset was also computed in a drated by evaporation of boiling absolute ethanol, mounted on stubs
maximum likelihood (ML) framework. ML analysis was performed and sputter-covered with a thin layer of gold.
Table 1
Taxonomic summary.
Order Apochela Schuster, Nelson, Grigarick and Christenberry, 1980

Family Milnesiidae Ramazzotti, 1962
Composition: Milnesium (type genus), Bergtrollus, Limmenius, Milnesioides.
Order Parachela Schuster, Nelson, Grigarick and Christenberry, 1980
Superfamily Eohypsibioidea Bertolani and Kristensen, 1987
Family Eohypsibiidae Bertolani and Kristensen, 1987
Composition: Eohypsibius (type genus), Austeruseus, Bertolanius.
Superfamily Macrobiotoidea Thulin, 1928 (emended)
Family Macrobiotidae Thulin, 1928 (emended)
Composition: Macrobiotus (type genus), Adorybiotus, Biserovus, Calcarobiotus, Famelobiotus, Insuetifurca, Minibiotus, Minilentus, Paramacrobiotus, Pseudohexapodibius,
Richtersius, Schusterius, Tenuibiotus, Xerobiotus.
Family Murrayidae Guidetti, Rebecchi and Bertolani, 2000
Composition: Murrayon (type genus), Dactylobiotus, Macroversum.
Superfamily Isohypsibioidea Sands, McInnes, Marley, Goodall-Copestake, Convey and Linse, 2008 (emended in this paper)
Family Isohypsibiidae Sands, McInnes, Marley, Goodall-Copestake, Convey and Linse, 2008
Composition: Isohypsibius (type genus), Apodibius, Dastychius, Doryphoribius, Eremobiotus, Halobiotus, Haplohexapodibius, Haplomacrobiotus, Hexapodibius,
Paradiphascon, Parhexapodibius, Pseudobiotus, Thulinius.
Superfamily Hypsibioidea Pilato, 1969 (emended in this paper)
Family Ramazzottiidae Sands, McInnes, Marley, Goodall-Copestake, Convey and Linse, 2008
Composition: Ramazzottius, Hebesuncus, probably Ramajendas and Thalerius.
Family Hypsibiidae Pilato, 1969 (emended in this paper)
Subfamily Hypsibiinae Pilato, 1969 (emended in this paper)
Composition: Hypsibius (type genus), Borealibius.
Subfamily Itaquasconinae Pilato, 1969 (emended in this paper)
Composition: Itaquascon (type genus), Adropion, Astatumen, Bindius, Mesocrista, Parascon, Platicrista.
Subfamily Diphasconinae Dastych, 1992 (emended in this paper)
Composition: Diphascon.
Subfamily Pilatobiinae subfam. nov.
Composition: Pilatobius gen. nov.
Incerta subfamilia
Genera: Acutuncus, Mixibius.
Family Calohypsibiidae Pilato, 1969 (emended in this paper)
Composition: Calohypsibius.
Family Microhypsibiidae Pilato, 1998
Composition: Microhypsibius (type genus), Fractonotus.
Incerta superfamilia
Family Necopinatidae Ramazzotti and Maucci, 1983
Composition: Necopinatum.
The analysis of the morphology of the male gametes of the four superfamilies: Isohypsibioidea, Hypsibioidea, Eohypsibioidea,
eutardigrades Minibiotus furcatus (Eherenberg, 1859) and Parama- and Macrobiotoidea (Figs. 2 and 3). Only the tree obtained with
crobiotus areolatus (Murray, 1911) was carried out. The spermato- the BI, under GTR model, shows the Isohypsibioidea basal to the
zoa were extracted from the testis and prepared for SEM analysis other superfamilies and the Hypsibioidea as sister-group of the
according to the technique perfected by Rebecchi and Guidi (1991). clade with the other two superfamilies (Eohypsibioidea, Macrobio-
All SEM observations were carried out using a FEI XL 40 SEM toidea) being closely related (Fig. 2). The BI (under CAT model) and
(Fei Company – Oxford Instruments) in the ‘‘Centro Interdiparti- ML analyses are not able to solve the relationships among super-
mentale Grandi Strumenti’’ of the University of Modena and Reggio families (Fig. 3).
Emilia, Italy. Within the phyletic lineage of Isohypsibioidea (Fig. 4), species
attributed to seven genera are present (i.e. Isohypsibius, Pseudobio-
3. Results tus, Doryphoribius, Eremobiotus, Halobiotus, Thulinius, Hexapodibius),
whose relationships are not always well-resolved. Some of these
3.1. Molecular analysis genera are not monophyletic (i.e. Isohypsibius, Eremobiotus, Dory-
phoribius). Within this cluster, there are Hexapodibius species (Cal-
Phylogenetic molecular analyses of combined sequences (18S ohypsibiidae) currently attributed to Hypsibioidea.
rRNA + 28S rRNA) carried out with the Bayesian inference (BI), un- The second evolutionary line, corresponding to the Hypsibioi-
der GTR + Gamma model (Fig. 2) and CAT + Gamma model (Fig. 3), dea (Fig. 5), presents two well supported main clusters, one formed
or Maximum Likelihood (ML, under GTR + Gamma model; Fig. 2) by the monophyletic genera Ramazzottius and Hebesuncus (both
display a very similar topology for the relationships of the genera belonging to Ramazzottiidae, incorrectly named Ramazzottidae
within the families, although the BI under GTR + Gamma model by Marley et al., 2011), the other formed by species of several gen-
tree shows higher branch supports. era of Hypsibiidae (i.e. Hypsibius, Borealibius, Mixibius, Acutuncus,
Overall results confirm the Eutardigrada and Heterotardigrada Diphascon, Astatumen, Itaquascon, Platicrista), Microhypsibiidae
classes as well as their orders Parachela and Apochela (Eutardigra- (Microhypsibius), and Calohypsibiidae (Calohypsibius). Within this
da) and Echiniscoidea (Heterotardigrada) (Figs. 2 and 3). The order last line, a specimen attributed to Calohypsibius by Sands et al.
Arthrotardigrada (Heterotardigrada) is not supported in any of the (2008) is basal to a cluster of four phyletic lineages (Fig. 5) com-
performed analyses resulting in a paraphyletic group (Figs. 2 and 3). posed of: (i) Calohypsibius ornatus (Calohypsibiidae), Acutuncus ant-
Within Eutardigrada, Milnesium species (Apochela) are in a sis- arcticus (Hypsibiidae), Microhypsibius species (Microhypsibiidae)
ter-group relationship with the cluster grouping all the Parachela and Mixibius species (a genus currently tentatively attributed to
species (Figs. 2 and 3). The main parachelan clade is formed by four the superfamily Isohypsibioidea; Marley et al., 2011); (ii) Diphas-
well supported evolutionary lines that can be identified with the con (Diphascon) patanei, Diphascon (Diphascon) nodulosum and
Fig. 2. Phylogenetic reconstruction (Bayesian and Maximum Likelihood analyses) based on combined dataset (18S + 28S rRNA sequences) under the GTR + Gamma model.
Values above branches indicate posterior probability values (Bayesian inference); while values under branches show bootstrap values (Maximum Likelihood analysis). Clades
A–D are represented in Figs. 4–6, respectively.
Fig. 3. Phylogenetic reconstruction (Bayesian analysis) based on combined dataset (18S + 28S rRNA sequences) under the CAT + Gamma model. Values above branches
indicate posterior probability values. Clades A–D are represented in Figs. 4–6, respectively.
Diphascon (Diphascon) ramazzottii; (iii) Diphascon species of the The phylogenetic lineage corresponding to the Eohypsibioidea
subgenus Adropion (characterized by the absence of a drop-like (represented by the only family Eohypsibiidae) is composed by
thickening between the buccal and flexible pharyngeal tube) and species of Eohypsibius and Bertolanius (Fig. 6).
species of three genera (i.e. Platicrista, Astatumen, Itaquascon) Within the last phyletic lineage, corresponding to the Macrobio-
belonging to the subfamily Itaquasconinae; iv) two well-supported toidea, two main phylogenetic lineages are present (Fig. 6). One
clusters, one formed by several Diphascon species of the subgenus line contains a well supported cluster composed of species of
Diphascon, the other by Hypsibius and Borealibius species (Fig. 5). Murrayon and Dactylobiotus (both belonging to Murrayidae) in
Fig. 4. Clade A. Phylogenetic reconstruction within the superfamily Isohypsibioidea based on combined dataset (18S + 28S rRNA sequences) and inferred by Bayesian and
Maximum Likelihood analyses, under the GTR + Gamma model (left topology), and Bayesian analysis under CAT + Gamma model (right topology). Values above branches
indicate posterior probability values (Bayesian inference), while values under branches show bootstrap values (Maximum Likelihood analysis). Taxa in bold indicate newly
analyzed specimens.
Fig. 5. Clade B. Phylogenetic reconstruction within the superfamily Hypsibioidea based on combined dataset (18S + 28S rRNA sequences) and inferred by Bayesian and
Maximum Likelihood analyses, under the GTR + Gamma model (left topology), and Bayesian analysis, under CAT + Gamma model (right topology). Values above branches
indicate posterior probability values (Bayesian inference), while values under branches show bootstrap values (Maximum Likelihood analysis). Taxa in bold indicate newly
analyzed specimens.
relationship with Adorybiotus granulatus and Richtersius coronifer joannae, Macrobiotus sapiens, Macrobiotus polonicus) are in close
(both currently belonging to Macrobiotidae). The second lineage relationship with Macrobiotus cf. nelsonae, and a second cluster
is formed by all the other species of Macrobiotidae, which results that encompasses Macrobiotus furciger and Macrobiotus harmsworthi
in a polyphyletic lineage. This last clade is composed of two main (Fig. 6).
clusters, one constituted by Paramacrobiotus and Minibiotus spe-
cies, in which Minibiotus appears to be polyphyletic for the sis- 3.2. Morphological analysis
ter-group relationship of Minibiotus furcatus with Paramacrobiotus
species (Fig. 6). The other cluster groups Xerobiotus pseudohufelandi The claws of Hexapodibius, Parhexapodibius and Calohypsibius
and the species of the Macrobiotus hufelandi group (i.e. Macrobiotus genera, currently attributed to Calohypsibiidae, are very small
hufelandi, Macrobiotus vladimiri, Macrobious macrocalix, Macrobiotus and look very similar (Fig. 7), but are not equal. In all examined
Fig. 6. Clades C and D. Phylogenetic reconstruction within the superfamily Eohypsibioidea (C) and Macrobiotoidea (D) based on combined dataset (18S + 28S rRNA
sequences) and inferred by Bayesian and Maximum Likelihood analyses, under the GTR + Gamma model (left topology), and Bayesian analysis, under CAT + Gamma model
(right topology). Values above branches indicate posterior probability values (Bayesian inference), while values under branches show bootstrap values (Maximum Likelihood
analysis). Taxa in bold indicate newly analyzed specimens.
species of Hexapodibius and Parhexapodibius, a suture between the secondary branch always forms a continuous arc in both claws
primary and secondary branch arising from the base of the claw is (Fig. 10N; be careful to look at several claws) and not a sort of an-
clearly visible (Fig. 7A and B respectively). In Calohypsibius the gle, as in Isohypsibius type claws (Fig. 10M).
proximal part of the claw, when observed in frontal view, looks The presence of a paired elliptical organ on the head reported in
large, as the sum of the width of the two branches (Figs. 7C, and some species of Calohypsibiidae and Microhypsibiidae has not
10K), but the suture between primary and secondary branch of been identified in any examined species of Calohypsibius, Hexapodi-
the claw is not detectable. This pattern can be interpreted as a claw bius, Parhexapodibius and Microhypsibius.
without a basal (common) tract and without a suture between pri- With regard to Macrobiotidae, the analysis of the mouth of
mary and secondary branch, or as a claw with an unusually large Minibiotus furcatus with SEM shows the presence of ten very short
common tract (basal tract). In Microhypsibius (currently attributed lamellae (Fig. 8A). These structures were not recognizable in our
to Microhypsibiidae) the claws are also small: the basal tract of the specimens by observations with light microscopy.
claw is always well recognizable, but it is as thin as one of the The testicular spermatozoon of M. furcatus (Fig. 9A–D), observed
branches (Figs. 7D, and 10J). The claws of Hebesuncus and Ramajen- by SEM, is thin and about 65.0 lm in length. It is made up of three
das (Figs. 7E and 10I) have similarities with the Hypsibius type regions: the head, including acrosome and nuclear region, the
claws (Fig. 10G), but in the shape of the main branch on the exter- middle piece and the terminal tufted tail (Fig. 9A-D). The acrosome
nal (or posterior) claw they look similar to those of the Ramazzot- is about 22.0 lm in length, has a smooth surface and rounded tip
tius (Figs. 7F and 10H). With phase contrast, in Hebesuncus, (Fig. 9B). The nuclear region (about 18.0 lm in length) is heli-
Ramajendas and Ramazzottius the main branch of the external (or cal and tightly coiled (Fig. 9B–D). Its coils increase in width cau-
posterior) claw looks dark for the most part but its proximal part dally, towards the middle piece. The midpiece (about 3.0 lm in
looks always clear (Fig. 7E), whereas in Hypsibius the entire claw length) is kidney-shaped and exhibits hemispherical protuber-
looks dark with phase contrast. The claws of Acutuncus and Mixibi- ances on its surface (Fig. 9D). The tail (about 23.0 lm in length)
us look similar in the two genera (Fig. 7G and H) and, in our opin- has a constant diameter and splits terminally into a tuft of fila-
ion, they are not attributable to the Isohypsibius type, but rather to ments (each about 5.5 lm in length; Fig. 9A). The male gamete
a modified Hypsibius type. In fact, the two claws on the same leg of M. furcatus looks very similar to that of Paramacrobiotus areola-
are quite different in size, and the convex curvature of the tus (Fig. 9E).
Fig. 7. Micrographs of eutardigrade claws (phase contrast). A: Hexapodibius mycronyx (third pair of legs); arrows indicate the suture between the two branches (insert shows
a different focus). B: Parhexapodibius ramazzottii (third pair of legs); arrows indicate the suture between the two branches. C: Calohypsibius ornatus (first pair of legs). D:
Microhypsibius sp. (fourth pair of legs) E: Hebesuncus ryani (fourth pair of leg). F: Ramazzottius sp. (fourth pair of leg). G: Acutuncus antarcticus (third pair of legs). H: Mixibius
saracenus (third pair of legs). Scale bars: A–C = 5 lm, D–H = 10 lm.
4. Discussion compared for the first time with a molecular approach. Molecular
data were integrated and supported with morphological data
Using a combined dataset of 18S and 28S rRNA sequences, we obtained from new observations and from references. The integra-
were able to obtain data on the phylogenetic relationships among tion between molecular and morphological information is impor-
eight of the nine current (according to Guidetti and Bertolani, tant in understanding phylogeny and as a general principle to
2011; Degma et al., 2013) eutardigrade families (only Necopinati- define the taxa. The erection of new taxa (at any level) only on
dae is lacking), comprising 31 current genera, 10 of which were the basis of molecular data should be avoided, in agreement with
Fig. 8. Scanning electron micrographs of mouth openings. A: Minibiotus furcatus. B:

Paramacrobiotus gr. richtersi. Scale bars = 2.5 lm. Asterisks = peribuccal lamellae.
a recommendation of the International Commission on Zoological Fig. 9. Scanning electron micrographs of spermatozoa. Male gametes of Minibiotus
Nomenclature (1999). Without morphological support, the risk of furcatus (A–D). A: In toto cell. B: Acrosome region. C: Transition area between
acrosome and nuclear regions. D: Kidney-shaped middle piece and helical nuclear
a mistake is high and the information for identifying the taxa is
region. E: Two male gametes of Paramacrobiotus areolatus. Scale bars: A, B, E = 5 lm;
substantially lacking, especially when micrometazoans, such as C, D = 1 lm. a = acrosome region, mp = middle piece, n = nuclear region, t = tail,
tardigrades, are considered. tt = terminal tuft.
Our molecular results confirm the monophyly of Eutardigrada
and its position as the sister group of Heterotardigrada. Within
the Eutardigrada, Parachela is supported as the sister group of Considering morphology, with respect to the median plane of
Apochela. From a morphological point of view, Parachela are char- the leg, the two double claws are symmetrical in the superfamily
acterized by the synapomorphy of two double claws (i.e. claws Macrobiotoidea (Fig. 10C–F) and asymmetrical in the superfamilies
composed of a primary and a secondary branch very often joined Eohypsibioidea (Fig. 10O), Isohypsibioidea (Fig. 10L and M) and
through a common tract), normally two per leg (Fig. 10C–O). Hypsibioidea (Fig. 10G–K), even though each taxon has a unique
Parachela normally also have cuticular placoids in the pharynx claw structure based on other characters (see below and taxo-
(secondarily lost in very few cases; Fig. 11B–L) that are always nomic account).
missing in Apochela (Fig. 11A). The phylogenetic relationships among superfamilies identified
At the higher phylogenetic level of the tree, Parachela are di- by Bayesian analysis under the GTR model (Fig. 2) is completely
vided into four main clusters (Fig. 2 and 3). Three of these cluster in agreement with those found by Marchioro et al. (2013) obtained
correspond to the evolutionary lines identified by Kiehl et al. by a total evidence analysis of the muscular architectures and
(2007) and by Sands et al. (2008) and one corresponding to the 18S + 28 S rRNA of species representative of the four superfamilies.
family Eohypsibiidae (recently proposed as superfamily by Marley
et al., 2011). Based only on molecular evidence, Sands et al. (2008) 4.1. Phylogeny of eutardigrade superfamilies
proposed superfamily ranks for the first three evolutionary lines,
namely Macrobiotoidea, Hypsibioidea (names derived from previ- The superfamily Eohypsibioidea has been recently proposed
ous families) and Isohypsibioidea, a new proposed name. Follow- (Marley et al., 2011), raising in taxonomic rank the family Eohyp-
ing the taxonomic scheme by Pilato and Binda (2010), a second sibiidae, but unfortunately on the basis of only one molecular da-
paper by Marley et al. (2011) was aimed to complete the molecular tum from GenBank. Our molecular data (Fig. 2 and 6) confirm
based scenarios providing some morphological support. the validity of Eohypsibiidae (the only family of Eohypsibioidea),
Fig. 10. Graphic representation of claw morphologies in tardigrades. A: Carphania (Heterotardigrada). B: Milnesium. C: Macrobiotus. D: Dactylobiotus. E: Murrayon. F:
Xerobiotus. G: Hypsibius. H: Ramazzottius. I: Ramajendas. J: Microhypsibius. K. Calohypsibius. L: Hexapodibius. M: Isohypsibius. N: Thulinius. O: Bertolanius. A, B–E, G, N–P:
redrawn from Bertolani, 1982; F: redrawn from Bertolani and Biserov, 1996; H: redrawn from Bertolani et al., 1993; I: redrawn from Pilato and Binda, 1990; J: redrawn from
Pilato, 1998.
with the relationship between its genera Eohypsibius and Bertola- Sands et al., 2008; Marley et al., 2011; Guil and Giribet, 2012). Mar-
nius (=Amphibolus) very well supported. Our data confirm Eohypsi- ley et al. (2011) report as diagnostic characters of the Isohypsibioi-
bioidea as possible sister-group of Macrobiotoidea. This dea the asymmetrical Isohypsibius type claws (Fig. 10M), and
relationship was suggested on molecular basis by Jørgensen et al. ridged AISM (Fig. 11H), but our molecular data suggest that also
(2010), Marley et al. (2011) and Guil et al. (2013b) and confirmed Hexapodibius (two clearly different species have been examined
in the total evidence analyses by Guil et al. (2013a) and Marchioro by us from a molecular point of view) should be attributed to this
et al. (2013). The relationship between Eohypsibius and Bertolanius superfamily, even though its claws are very reduced (as are those
has strong morphological support by peculiar characters (synapo- of other genera, such as Parhexapodibus, examined here from a
morphies); other than the asymmetrical double claw structure of morphological point of view). We consider as an autoapomorphy
Eohypsibiidae type (Fig. 10O), and the peculiar ability of the claws of the Isohypsibioidea the Isohypsibius type claws (asymmetrical
to rotate on their own bases, verified by Bertolani and Kristensen double claws with similar shape and size on each leg, see taxo-
(1987) and reported by Marley et al. (2011); the mouth shape nomic account) (Fig. 10L-M), secondarily modified in some genera
(Fig. 11D) and the number and shape of peribuccal lamellae (see above and further discussion). An extreme reduction should
(Bertolani and Kristensen, 1987; see taxonomic account). We be happened in Apodibius, which is attributable to this superfamily
therefore conclude that our new molecular data and the identified from a molecular point of view (Dabert et al., 2014). Not all Isohyp-
synapomorphies support the evolutionary line of Eohypsibioidea. sibioidea share ridge-shaped AISM, e.g. Halobiotus, Doryphoribius
The Macrobiotoidea are supported by our molecular results (Fig. 11G), Hexapodibius (Fig. 11E), Parhexapodibius, Haplomacrobi-
(Fig. 2, 3 and 6), confirming the data by Sands et al. (2008) and otus and Apodibius, the last five genera have a ventral lamina.
those of other recent papers (Marley et al., 2011; Guil and Giribet, The cluster Hypsibioidea (Fig. 2, 3 and 5) is well-supported from
2012). According to Marley et al. (2011), Macrobiotoidea are char- molecular point of view (present study, Sands et al., 2008; Marley
acterized by the double claw pairs being symmetrical with respect et al., 2011; Guil and Giribet, 2012), and includes taxa with claws
to the median plane of the leg (Fig. 10C–F), and the presence of a of the Hypsibius type or Ramazzottius type (Fig. 10G-H) (see taxo-
ventral lamina on the buccal tube (Fig. 11B and C) that generally nomic account). According to Marley et al. (2011), Hypsibioidea
leads to asymmetrical apophyses for the insertion of the stylet are characterized by asymmetrical claws of the Hypsibius type
muscles (AISM). Even though Macrobiotoidea are the only para- (Ramazzottius-type is considered a derived Hypsibius type), and
chelan superfamily characterized by symmetrical claws, we should hooked AISM (or, if the buccal tube is elongated, the AISM can be
remember that symmetrical claw organization is present also in all broad ridges). We consider as a synapomorphy of the superfamily
Heterotardigrada (Fig. 10A) and in the apochelan Milnesiidae the presence of asymmetrical double claws that share an evident
(Fig. 10B) and could represent a plesiomorphic character (Guidetti difference in size and shape on the same leg (Fig. 10G–K), a situa-
et al., 2005). In addition, the ventral lamina or other characters tion not found in other Parachela. This synapomorphy is in line
shared by all Macrobiotoidea (e.g. ornamented eggs) are also pres- with molecular support and together with morphological data al-
ent in other superfamilies (see taxonomic account), other than in lows us to support the superfamily Hypsibioidea. The small claws
the heterotardigrade Oreella (Bertolani et al., 1996). We consider of the Microhypsibius type and the Calohypsibius type (Figs. 7C–D,
the presence of ten peribuccal structures (lamellae or papulae; and 10J and K) present within the superfamily Hypsibioidea appear
Fig. 8) and double claws characterized by a peculiar stalk with different from the Hypsibius type claws, and they should be consid-
cylindrical or laminar shape (Fig. 10C–E) as clear synapomorphic ered secondarily reduced (according to Pilato, 1989; Dastych and
characters of this superfamily (Guidetti and Bertolani, 2001). Alberti, 1990, and in our opinion too), similar to what indepen-
According to our molecular data, the clade Isohypsibioidea also dently happens in Hexapodibius. Most Hypsibioidea have a narrow
includes Hexapodibius (Fig. 2–4). This superfamily is very well sup- buccal tube (Fig. 11I–M), flexible main branch of the external
ported from a molecular point of view, as first shown by Kiehl et al. claws, but these characters are shared with other evolutionary
(2007) and subsequently by other authors (Møbjerg et al., 2007; lines.
Fig. 11. Graphic representation of buccal-pharyngeal apparatus morphologies in eutardigrades. A: Milnesium (dorsal view). B: Paramacrobiotus (ventro-lateral view). C:
Minibiotus (lateral view). D: Eohypsibius (dorsal view). E: Hexapodibius (lateral view). Thulinius (dorsal view). G: Doryphorybius (lateral view). H: Isohypsibius (lateral view). I:
Hypsibius (dorsal view). J: Diphascon (ventral view). K: Adropion (dorsal view). L: Pilatobius gen. nov. (dorsal view). M. Platicrista (dorsal view). A: redrawn from Pilato et al.,
2002; B, F–M: redrawn from Bertolani, 1982; C: redrawn from Pilato et al., 2003; D: redrawn from Bertolani and Kristensen, 1987; E: redrawn from Binda and Pilato, 1992.
4.2. Phylogeny within each eutardigrade superfamily view, Adorybiotus and Richtersius appear related to this family,
but morphological support for this relationship is not evident. Both
Considering the evolutionary lineages within the superfamilies, genera seem to belong to independent lineages, and both are well-
many clades can be identified within each of them (Fig. 4–6). supported with both morphological data (Pilato and Binda, 1987)
and molecular data. All the remaining genera of Macrobiotoidea
4.2.1. Macrobiotoidea analyzed in this study cluster together, but they could represent
Macrobiotoidea (Fig. 6) consists of clusters of several lineages. different families. Lacking morphological evidence sufficient to
Murrayidae family, which includes Dactylobiotus and Murrayon as characterize each of them, provisionally we prefer to maintain
genera analyzed from a molecular point of view, is supported both the family Macrobiotidae for all of them, even though this family
by molecular (see results; Guidetti et al., 2005; Marley et al., 2011; clearly appears polyphyletic and must be reconsidered in the fu-
Guil and Giribet, 2012) and morphological data (Guidetti and Ber- ture. Within this cluster of Macrobiotidae, two main evolutionary
tolani, 2001; Guidetti et al., 2005) (mainly peculiar claws, Fig. 10D lineages are recognized: one with Minibiotus and Paramacrobiotus,
and E; see also taxonomic account). Within this family, the genus the other with Xerobiotus and Macrobiotus. Macrobiotus and Mini-
Murrayon appears to be polyphyletic. From a molecular point of biotus are polyphyletic, as also shown by Guil and Giribet (2012).
In Minibiotus, one species (M. furcatus) is closer to Paramacrobiotus (Fig. 11E), whereas Calohypsibius does not, and the placoids of Cal-
than to the other Minibiotus species. In reality, M. furcatus shows ohypsibius are quite different in shape and number from those of
very short lamellae around the mouth opening (Fig. 8A) instead Hexapodibius and the other genera here cited above, which are very
of papulae as should be in Minibiotus. Moreover, most Minibiotus similar. A possible polyphyletic status of the actual Calohypsibiidae
species show buccal-pharyngeal apparatus characters (Guil and and a possible transfer of some genera to another superfamily were
Guidetti, 2005; Fig. 11C) different from those of M. furcatus. Since also questioned by Guil et al. (2013a) based on their morphological
the spermatozoan morphology has a phylogenetic signal (Guidi analyses. For these reasons we propose to move Hexapodibius and
and Rebecchi, 1996; Rebecchi and Bertolani, 1999; Rebecchi related genera (i.e. Parhexapodibius, Haplomacrobiotus, Haplohexa-
et al., 2000, 2003, 2011), it has been considered in M. furcatus podibius) to the superfamily Isohypsibioidea (see taxonomic ac-
and P. areolatus. The male gamete of M. furcatus shows strong sim- count). We want to emphasize that the taxa belonging to
ilarity with the particular spermatozoon of Paramacrobiotus spe- Isohypsibioidea share a male gamete morphology without the
cies (our results; Guidi and Rebecchi, 1996; Rebecchi et al., midpiece, which differs from that of the other tardigrade super-
2011), above all the head shape, thin, slightly coiled, much longer families (Wolburg-Buchholz and Greven, 1979; Rebecchi and
than the tail and with a long and thin acrosome. These morpholog- Bertolani, 1999; Rebecchi, 2001; Nelson et al., 2010). To date, the
ical data confirm the relationships between Paramacrobiotus and superfamily Isohypsibioidea includes only one family, Isohypsibii-
M. furcatus supported by sequence analyses. Nonetheless, we do dae, but the evolutionary lineages within it are often unclear. The
not have sufficient morphological support and molecular data for genus Isohypsibius is certainly polyphyletic, confirming previous
moving M. furcatus to another genus. molecular results (Kiehl et al., 2007; Møbjerg et al., 2007). The
From our molecular data related to the second evolutionary genus Doryphoribius, too, appears polyphyletic, and similarly
line, we can note that: a. Macrobiotus harmsworthi and Macrobiotus Pseudobiotus, but morphological information are insufficient for
furciger (belonging to the harmsworthi group according to distinguishing the different evolutionary lineages among them
Ramazzotti and Maucci, 1983) are related but clearly separated and to support the erection of new taxa. The situation of Eremobio-
from Paramacrobiotus, even though some evident similarities can tus requires further analysis and comparison with the specimens
be noted in the type of buccal armature and in M. harmsworthi in used for obtaining the sequences available in GenBank.
the morphology of the egg shell processes; b. the Macrobiotus
hufelandi group (Bertolani and Rebecchi, 1993; Guidetti et al., 4.2.3. Hypsibioidea
2013a) and Xerobiotus form a cluster of species having in common The cluster of Hypsibioidea is subdivided in two main branches
the shape of the buccal-pharyngeal apparatus, type of egg shell (Fig. 5). The first branch includes the genera Ramazzottius and
processes (in most cases) and spermatozoa (Guidi and Rebecchi, Hebesuncus (the two genera of Ramazzottiidae) in agreement with
1996; Rebecchi et al., 2000, 2011; Guidetti et al., 2005). Nevertheless, previous results (Kiehl et al., 2007; Sands et al., 2008; Marley et al.
Xerobiotus clearly differs from the species of the M. hufelandi group 2011; Guil and Giribet 2012). There is no agreement regarding the
by morphological characters: the shape of the claws (Fig. 10F), type of claws of these two genera. Pilato and Binda (2010) attribute
which are reduced, and also the absence of ‘‘pearls’’ (pores) in the homonymous type of claws only to Ramazzottius (Fig. 10H),
the cuticle. The presence of Macrobiotus nelsonae (characterized whereas they attribute the claws of Hebesuncus (Fig. 7E) to the
by placoids similar to those of M. hufelandi group and ‘‘pearls’’ on Hypsibius type (Fig. 10G). In contrast, Marley et al. (2011) attribute
the cuticle, but by different large and conical egg processes) within to both genera the same type of claws: the Ramazzottius type
the M. hufelandi group (generally characterized by inverted goblet- (Fig. 10H). In reality, the claws look different in the two genera
like egg processes) indicates that this group has more common and the statement of Pilato and Binda (2010) seem more correct.
characters in the animal morphology than in the egg shell Nevertheless, as reported in our results and suggested by Guil
morphology. According to our molecular data (Fig. 6), we should et al. (2013a), we also find a significant similarity among the claws
consider the specimen associated with the GenBank sequence of the two genera and Ramajendas (Fig. 10I), which are different
U32393 to belong not to Macrobiotus but to Paramacrobiotus. The from the Hypsibius type claw. We consider the peculiarity of the
two Macrobiotus clades identified with molecular methods within Ramazzottius and Hebesuncus claws (the claw with the primary
the second evolutionary line of Macrobiotidae should represent branch connected to basal tract with a thin, flexible tract) as a syn-
separate genera, but there is not yet sufficient morphological infor- apomorphy of the Ramazzotiidae family, together with asymmetric
mation to support a formal description. Further studies on this AISM (Pilato and Binda, 2010). Moreover, both genera lay free eggs
genus are necessary. with ornamented shells, but the polarity of this character should be
verified. According to Dastych (2009), the genus Thalerius could be
4.2.2. Isohypsibioidea related to Ramajendas.
Isohypsibioidea superfamily includes several evolutionary lin- The other branch of Hypsibioidea (Fig. 5) is similarly monophy-
eages (Fig. 5). First of all, according to molecular data, the presence letic and includes genera characterized by Hypsibius type claws (or,
of Hexapodibius and the exclusion of Calohypsibius in Isohypsibioi- in our opinion, derived from it, such as in Acutuncus, Fig. 7G, and
dea mean that the two genera cannot belong to the same family, in Mixibius, Fig. 7H; see below) and Microhypsibius type (Fig. 7D,
contrast with previous morphological studies (Pilato, 1969a,b). The 10J) and Calohypsibius type (Fig. 7C, 10K) claws, which are exclu-
claws of both genera appear similar, due to a strong reduction in sively in this clade (for the description of Microhypsibius and Hyp-
the length of the claw basal tract and branches (Fig. 7A and C). Dif- sibius type claws see Bertolani et al., 2009; Pilato and Binda, 2010;
ferences have also been detected between the two claw types: for Calohypsibius type claw see our results and taxonomic account).
Hexapodibius claws show a suture between the primary and sec- Pilato and Binda (2010) attribute the external claw of both Acut-
ondary branch arising from the base of the claw (Hexapodibius type uncus and Mixibius to the Isohypsibius type, and the internal one
claws; Figs. 7 and 10L; see taxonomic account), while this suture is to the Hypsibius type and to a modified Isohypsibius type, respec-
absent in Calohypsibius (Calohypsibius type claws; Figs. 7 and 10K; tively. These claws appear as such but in our opinion, as stated
see also taxonomic account). Moreover, the buccal-pharyngeal in the results, none of them can really be attributed to the Isohyp-
apparatus is quite different in the two genera: Hexapodibius and re- sibius type. Rather they can be attributed to a modified Hypsibius
lated genera (i.e. genera with the same kind of claws: Parhexapodi- type. For this reason, one of us (Bertolani, 1982), before the molec-
bius, or derived from it by the loss of one branch: Haplomacrobiotus, ular approach to phylogeny existed, moved Isohypsibius saracenus
Haplohexapodibius) have a ventral lamina on the buccal tube to the genus Hypsibius; this species was subsequently considered
as the type species of the genus Mixibius. Most species of this clade amended and raised to genus level (see taxonomic account).
lay eggs with smooth shells within the exuvium, with the excep- Diphascon (A.) sp. 1 (HQ604931) certainly does not have the
tion of Acutuncus and a few Hypsibius, which have very short pro- drop-like thickening but it does not cluster with the other two
cesses on the egg shell. Mixibius eggs are unknown. An Adropion species considered here. Therefore, our provisional attri-
autoapomorphy of this second clade of Hypsibioidea is difficult bution of Diphascon (A.) sp. 1 to the genus Adropion should be
to identify. A particular structure present only in this clade and studied more in depth. The second clade within the large cluster
in several (but not all) of its lineages is the septulum, a posterior shows two different branches: one includes many species of the
cuticular thickening within the pharynx that alternates with the subgenus Diphascon (Diphascon), i.e. with pharyngeal tube and
lines of placoids. Within the clade, Calohypsibius (with its typical drop-like thickening (Fig. 11J), and with parallel macroplacoids
claws) represents one independent lineage or maybe two lineages, within the pharyngeal bulb, while the second branch includes
but a better morphological study is necessary, given that the mor- species without a pharyngeal tube (Hypsibius and Borealibius) that
phology of Calohypsibius sp. is not described by Sands et al. (2008). in this case should be considered a synapomorphy of the two gen-
The position of Microhypsibius in another evolutionary lineage with era (Fig. 11I). The first branch (monophyletic line) corresponds to
respect to Calohypsibius confirms the differences between these a redefined subfamily Diphasconinae (see taxonomic account).
genera (Thulin, 1928; Kristensen, 1982; Bertolani and Kristensen, Diphascon higginsi is correctly included in this subfamily (and
1987). Other clear morphological differences between Microhypsi- not in Itaquasconinae), having the drop-like structure even
bius and Calohypsibius can be noted in the buccal-pharyngeal appa- though this thickening is flat and not always very visible
ratus (see Pilato and Binda, 2010). In conclusion, the families (Bertolani, 1982; Dastych, 1988; Pilato and Binda, 1988). The
Calohypsibiidae (now including only the genus Calohypsibius) and second branch can be considered as the redefined subfamily
Microhypsibiidae should be maintained. The presence of Mixibius Hypsibiinae (see taxonomic account). Within Hypsibiinae, Hypsibius
and Acutuncus in the same cluster with Microhypsibius is very dif- and Borealibius appear close from a molecular point of view, but
ficult to explain on a morphological basis, as the claws and the buc- they clearly differ in the shape of the AISM (Pilato et al., 2006).
cal-pharyngeal apparatuses are quite different. However, the From a molecular point of view, a very well supported small
presence of Mixibius saracenus within the Hypsibioidea and cluster within Hypsibiidae (Fig. 5) is represented by D. (D.) patanei,
Hypsibiidae is in line with the first transfer of this species from D. (D.) ramazzottii and D. (D.) nodulosum. These species have a pha-
Isohypsibius to Hypsibius on the basis of its morphology (Bertolani, ryngeal tube and drop-like thickening, but differ from the rede-
1982), followed by a further transfer to the genus Mixibius fined Diphasconinae by the shape of the pharyngeal bulb (which
subsequently erected by Pilato (1992). Acutuncus antarcticus was is oval, but more rounded in these three species) and macroplac-
also previously considered a Hypsibius and then transferred to a oids that are positioned like brackets, each line forming a
new genus (Pilato and Binda 1997). The presence of M. saracenus parenthesis (Fig. 11L). On the basis of our molecular and
within the Hypsibioidea is also in agreement with the results of morphological analysis of Hypsibiidae, we conclude that the genus
morphological and total evidence analyses of Guil et al. (2013a). Diphascon and the subgenus Diphascon are not monophyletic and
Considering the Hypsibiidae without pharyngeal (annulate) that a new genus, Pilatobius gen. nov., and a new subfamily,
tube, Mixibius and Acutuncus have a particular position in the Pilatobiinae subfam. nov. must be erected for D. patanei and related
molecular tree. The type of claw, similar but not identical to that species (see taxonomic account).
of all other Hypsibiidae genera, could possibly support the inclu- The genus Apodibius has been studied by a molecular point of
sion of these two genera in the Hypsibiidae family, but the data view very recently (Dabert et al., 2014) and placed within Iso-
are insufficient to attribute them to a specific subfamily (see taxo- hypsibiidae family. Instead, we do not have molecular informa-
nomic account). The other Hypsibiidae (most of the considered tion on Necopinatum (Necopinatidae). The buccal-pharyngeal
taxa of this family) clearly display Hypsibius type claws and should apparatus of Necopinatum suggests that it could belong to Isoh-
represent a monophyletic family, here redefined (see taxonomic ypsibioidea, but to a different evolutionary lineage with respect
account). to Apodibius, lacking Necopinatum the strengthening bar, which
The remaining Hypsibiidae lineage of the molecular tree is instead is present in the other genus. Molecular data should clar-
characterized by the presence of a pharyngeal (annulate) tube ify the situation.
(Fig. 11J-M), except for the Hypsibius-Borealibius lineage (Fig. 5). Finally, our results identify new phylogenetic relationships
It is not yet possible to understand if this character is an apomor- among eutardigrades and further demonstrate that molecular
phy of this clade (adaptive convergence with genera of other analysis is a fundamental tool to understand phylogeny and to con-
superfamilies), or a plesiomorphy, maybe of the Eutardigrada, firm (often it is so) or to modify (as sometimes happens in this pa-
being present in most parachelan superfamilies (or all; see com- per) the evolutionary considerations previously inferred by
ments on Paradiphascon in taxonomic account) and in Milnesiidae morphological analysis. The integrative approach applied in this
(see Guidetti et al., 2012, 2013b). This lineage is clearly subdi- study allowed us to find morphological support (synapomorphies)
vided into a small and a large cluster, the latter subdivided in for the new clusters here identified by molecular data and to find
two clades. One clade of the larger cluster includes only species further characters to support previous taxa erected on molecular
having a pharyngeal (annulate) tube, but without a drop-like evidence. This kind of approach should also open new perspectives
thickening between the buccal and pharyngeal tube (Fig. 11K in the understanding of the evolution of biological features in tar-
and M), in addition to a pharyngeal bulb that is frequently partic- digrades, such as cryptobiotic phenomena, different reproductive
ularly long and with thin, long and parallel macroplacoids. Among modes and the biogeographical distribution that characterizes
them are D. scoticum and D. belgicae, belonging to the subgenus the species.
Adropion, and species belonging to other genera of Itaquasconinae
(i.e. Platicrista, Astatumen, Itaquascon). The spermatozoa of P.
angustata and D. scoticum, the only species of this family in which 5. Taxonomic account
the male gamete has been studied to date, show a similar mor-
phology (Guidi and Rebecchi, 1996; Rebecchi et al., 2000). This The taxonomic account of the Eutardigrada taxa, including the
monophyletic clade should be identified with a redefined subfam- taxa emended or newly erected, is presented here (see Table 1).
ily Itaquasconinae (see taxonomic account). On the basis of The emendations and definitions follow the conclusions of our
molecular and morphological data, the Adropion subgenus is here discussion.
molecular studies to verify their eventual position in the

Class EUTARDIGRADA Richters, 1926
Murrayidae. In this case, the characters identifying the
Claws with a primary and a secondary branch fused or
family Murrayidae should be emended.
positioned one behind the other. Without cephalic filaments
(such as cephalic appendages, only papillae can be present). Family Murrayidae Guidetti, Rebecchi and Bertolani, 2000
Double claws V-shaped or L-shaped, with the two branches
Order Apochela Schuster, Nelson, Grigarick and
diverging immediately after a short common basal section;
Christenberry, 1980
ventral deep indentation on the ventral lamina; buccal tube
Eutardigrada having claws with completely separated
completely rigid; epicuticular layer with pillar-like
primary and secondary branches; papillae around the
structures, sometime visible only at ultrastructural level.
mouth (peribuccal papillae) and 2 lateral papillae on the
Composition: Murrayon (type genus), Dactylobiotus,
head (cephalic papillae); elongated pharyngeal bulb
Macroversum.
completely without placoids.
Superfamily Isohypsibioidea Sands, McInnes, Marley,
Family Milnesiidae Ramazzotti, 1962
Goodall-Copestake, Convey and Linse, 2008 (emended
The same characters as Apochela.
diagnosis)
Composition: Milnesium (type genus), Bergtrollus, Limmenius,
Double claws asymmetrical with respect to the median plane
Milnesioides.
of the leg (2121), normally with similar shape and size on
Order Parachela Schuster, Nelson, Grigarick and each leg; double claws of the Isohypsibius type (secondary
Christenberry, 1980 branch of the external claw inserted perpendicularly on the
Eutardigrada with double claws on the legs (or derived from claw basal tract), or reduced from it: Hexapodibius type
them, rarely completely lost) with secondary branch (very short, without common basal tract, with a base as
normally joined to the primary branch; pharyngeal bulb large as the sum of the primary and secondary branch
with placoids (very rarely lost). widths, and with an evident suture between primary and
secondary branch); Haplomacrobiotus type (one branch
Superfamily Eohypsibioidea Bertolani and Kristensen, 1987 only); completely absent (Apodibius). Buccal tube
Double claws of Eohypsibius type (i.e. subdivided into three completely rigid (apart Paradiphascon; see below) and often
distinct sections, one on top of the other), and asymmetrical relatively large, without (Dastychius, Eremobiotus,
with respect to the median plane of the leg (sequence Halobiotus, Isohypsibius, Paradiphascon, Pseudobiotus,
2121), with the ability to rotate on their base; double claws Thulinius) or with (Apodibius, Doryphoribius,
similar in size and shape on the same leg; 14 peribuccal Haplomacrobiotus, Haplohexapodibius, Hexapodibius,
lamellae; buccal tube completely rigid, or caudally Parhexapodibius) ventral lamina. Eggs with smooth shell
annulated, in any case with dorsal and ventral crest-shaped laid within the exuvium.
apophyses for the insertion of the stylet muscles. Eggs laid
freely and surrounded by an ornamented shell. Family Isohypsibiidae Sands, McInnes, Marley, Goodall-
Copestake, Convey and Linse, 2008
Family Eohypsibiidae Bertolani and Kristensen, 1987 The same characters of the superfamily.
The same characters as the superfamily. Composition: Isohypsibius (type genus), Apodibius, Dastychius,
Composition: Eohypsibius (type genus), Austeruseus, Doryphoribius, Eremobiotus, Halobiotus, Haplohexapodibius,
Bertolanius. Haplomacrobiotus, Hexapodibius, Paradiphascon,
Superfamily Macrobiotoidea Thulin, 1928 Parhexapodibius, Pseudobiotus, Thulinius.
Double claws symmetrical with respect to the median plane The type of claws of Ramajendas and Paradiphascon have been
of the leg (sequence 2112); double claw of each leg similar variously interpreted (Pilato and Binda, 1990; Dastych,
in shape and size; each double claw characterized by the 1992; Marley et al., 2011). In our opinion and in agreement
presence of a peculiar stalk (peduncle) with cylindrical or with Guil et al. (2013a), due to the type of claws,
laminar shape; 10 peribuccal lamellae or papulae; buccal Paradiphascon should belong to the Isohypsibiidae,
tube strengthened by a ventral lamina. Eggs laid freely and although further molecular and morphological studies are
always surrounded by an ornamented shell. required to confirm its position. In agreement with the
finding of Guil et al. (2013a), we consider Ramajendas to
Family Macrobiotidae Thulin, 1928 belong to the Hypsibioidea. For the claw type characterized
Double claws Y-shaped, with the two branches forming an by a disconnected main branch on the external claws, as in
evident common basal tract of variable length. Buccal tube the Hebesuncus claw type, we suggest provisionally to place
completely rigid, or caudally annulated. the genus in the family Ramazzottidae.
Composition: Macrobiotus (type genus), Adorybiotus,
Biserovus, Calcarobiotus, Famelobiotus, Insuetifurca, Superfamily Hypsibioidea Pilato, 1969 (emended diagnosis)
Minibiotus, Minilentus, Paramacrobiotus, Pseudohexapodibius, Double claws asymmetrical with respect to the median plane
Richtersius, Schusterius, Tenuibiotus, Xerobiotus of the leg (2121), the external (or posterior) claw often with
(Pseudodiphascon is considered a genus dubius; Guidetti and flexible main branch; double claws different in size and
Pilato 2003). shape on the same leg (Hypsibius and Ramazzottius type, or
modified), or very reduced in size (Calohypsibius and
Remarks: According to our molecular and morphological data, Microhypsibius type); buccal tube often very narrow.
M. furcatus does not belong to Minibiotus and it is
provisionally retransferred to Macrobiotus, pending new Family Ramazzottiidae Sands, McInnes, Marley, Goodall-
data. The genera Richtersius and Adorybiotus are Copestake, Convey and Linse, 2008
provisionally placed in this family following their Double claws different in shape and size on the same leg, the
traditional classification, waiting further morphological and external (or posterior) claw with the primary branch
(continued on next page)

connected to basal tract with an evident, thin, flexible tract Genus Pilatobius gen. nov.
(Ramazzottius or oberhaeuseri type, or modified), or The same characters as the subfamily.
completely disconnected. Eggs laid freely and with Composition: Pilatobius bullatus (Murray, 1905) (type species),
ornamented shell. Pilatobius aculeatus (Maucci, 1951–1952), Pilatobius
Composition: Ramazzottius (type genus), Hebesuncus and very bisbullatus (Iharos, 1964), Pilatobius borealis (Biserov, 1996),
probably Ramajendas and Thalerius. Pilatobius brevipes (Marcus, 1936), Pilatobius burti (Nelson,
1991), Pilatobius elongatus (Mihelčič, 1959), Pilatobius
Family Hypsibiidae Pilato, 1969 (emended diagnosis)
gerdae (Mihelčič, 1951), Pilatobius granifer (Greven, 1972),
Double claws different in shape and size on the same leg, the
Pilatobius halapiense (Iharos, 1964), Pilatobius iltisi (Schuster
external (or posterior) of Hypsibius type claw with the
and Grigarick 1965), Pilatobius latipes (Mihelčič, 1959),
secondary branch forming a continuous hook with its basal
Pilatobius nodulosus (Ramazzotti, 1957), Pilatobius
tract and the primary branch connected to the basal tract
nonbullatus (Mihelčič, 1951), Pilatobius oculatus (Murray,
with a flexible part. Septulum present in the pharynx of some
1906), Pilatobius patanei (Binda and Pilato, 1971), Pilatobius
genera. Eggs smooth (but rarely weakly ornamented) laid
ramazzottii (Robotti, 1970), Pilatobius rugosus (Bartoš,
within the exuvium, in some cases eggs ornamented laid free.
1935), Pilatobius secchii (Bertolani and Rebecchi, 1996),
Subfamily Hypsibiinae Rudescu, 1964 Pilatobius sexbullatus (Ito, 1995), Pilatobius trachidorsatus
Completely rigid buccal tube. Apophyses for the insertion of (Bartoš, 1935).
the stylet muscles hook-shaped. Other species not sufficiently described are placed in this new
Composition: Hypsibius (type genus), Borealibius. genus pending further morphological and/or molecular
analyses: Pilatobius recamieri (Richters, 1911) and Pilatobius
Subfamily Itaquasconinae Pilato, 1969 (emended diagnosis) rugocaudatus (Rodriguez Roda, 1952).
Buccal tube followed by a flexible portion, without cuticular
Etymology: dedicated to professor Giovanni Pilato, University
thickening between them; the flexible portion is an of Catania, Italy.
annulated pharyngeal tube in all genera (Parascon
Remarks: most of the known species with cuticular bars on
excluded); pharyngeal bulb very elongated; placoids very legs and (or) with a sculptured cuticle.
long and in line, sometime reduced to a bulbar lining.
Composition: Itaquascon (type genus), Adropion, Astatumen, Incerta subfamilia Claws of modified Hypsibius type: the two
Bindius, Mesocrista, Parascon, Platicrista. claws of the same leg are quite different in size from each
other and the convex curvature of the secondary branch
Genus Adropion Pilato, 1987 (taxonomic rank raised to genus
always forms a continuous arc in both claws.
level) Genera: Acutuncus, Mixibius.
Evident placoids and stylet supports.
Composition: Adropion scoticum (Murray, 1905) (type species) Family Calohypsibiidae Pilato, 1969 (emended diagnosis)
and the species already included in the subgenus Adropion Double claws of the Calohypsibius type (small, rigid, in frontal
(Guidetti and Bertolani, 2005). view with a base as large as the sum of the primary and
Remarks: From a molecular point of view the genus appears secondary branch widths, but without a suture between the
polyphyletic, but further studies are necessary to clarify the two branches); double claws similar in size and shape on
situation. the same leg; buccal tube completely rigid; apophyses for
the insertion of the stylet muscles on the buccal tube
Subfamily Diphasconinae Dastych, 1992 (emended
asymmetrical with respect to the frontal plane.
diagnosis) Composition: Calohypsibius.
Buccal tube followed by an annulated pharyngeal tube, with a
cuticular thickening between them (often drop-shaped, Family Microhypsibiidae Pilato, 1998
sometimes barely evident); pharyngeal bulb is an elongated Double claws of the Microhypsibius type (small, rigid, with an
oval, containing always 3 macroplacoids in a line (and evident thin basal tract continuous with the primary
sometimes with a microplacoid and/or septulum). branch); buccal tube completely rigid; double claws similar
Composition: Diphascon. in size and shape on the same leg; apophyses for the
insertion of the stylet muscles on the buccal tube
Genus Diphascon Plate, 1888 (emended)
asymmetrical with respect to the frontal plane. Pharynx
The same characters as the subfamily. with a septulum in some species.
Composition: Diphascon chilenense Plate, 1889 (type species)
Composition: Microhypsibius (type genus), Fractonotus.
and the species already included in the subgenus Diphascon
(Guidetti and Bertolani, 2005), excluding those attributed Incerta superfamilia
to Pilatobius gen. nov. Family Necopinatidae Ramazzotti and Maucci, 1983
Excluding the species here attributed to Pilatobius gen. nov., all Claws absent in each leg of the first pair substituted by a small
the species belonging to the previous genus Diphascon but not cuticular forcep; buccal tube rigid, without ventral lamina.
yet attributed to a subgenus (see Guidetti and Bertolani, 2005) Egg smooth laid within the exuvium.
are placed in this taxon, pending further investigations. Composition: Necopinatum.
Subfamily Pilatobiinae subfam. nov.

Buccal tube followed by an annulated pharyngeal tube, with a
drop-like thickening between them; pharyngeal bulb
roundish or slightly oval, always containing 2 Acknowledgments
macroplacoids similar in length and in rows that look as
parentheses, and a septulum. The authors thank Diane R. Nelson, East Tennessee State Uni-
Composition: Pilatobius gen. nov. versity, Johnson City, Tennessee (U.S.A.) for the English revision
of the manuscript and for helping in collecting specimens, and the Degma, P., Bertolani, R., Guidetti, R,. 2013. Actual Checklist of Tardigrada Species.
pp. 38. <http://www.tardigrada.modena.unimo.it/miscellanea/Actual%20
anonymous referees for their suggestions. Special thanks to the
checklist%20of%20Tardig rada.pdf>. (Ver. 23: 15-07-2013).
Laboratory of Sequencing (Labgen), Department of Biomedical Sci- Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and
ence, University of Modena and Reggio Emilia (Italy) for the high throughput. Nucleic Acids Res. 32, 1792–1797.
sequencing service. The research is part of the project MoDNA Gouy, M., Guindon, S., Gascuel, O., 2010. SeaView version 4: a multiplatform
graphical user interface for sequence alignment and phylogenetic tree building.
(Morphology and DNA: DNA barcoding and phylogeny of tardi- Mol. Biol. Evol. 27 (2), 221–224.
grades, basic research and applications) supported by Fondazione Guidetti, R., Bertolani, R., 2001. Phylogenetic relationships in the Macrobiotidae
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the online version, at http://dx.doi.org/10.1016/j.ympev.2014.03. Macrobiotidae (Eutardigrada, Parachela): a combined morphological and
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Geonemy, ecology, reproductive biology and morphology of the tardigrade
Hypsibius zetlandicus (Eutardigrada: Hypsibiidae) with erection of Borealibius
Further reading
gen. n. Polar Biol. 29, 595–603.
Pleijel, F., Jondelius, U., Norlinder, E., Nygren, A., Oxelman, B., Schander, C., Bertolani, R., Marley, N.J., Nelson, D.R., 1999. Re-description of the genus Thulinia
Sundberg, P., Thollesson, M., 2008. Phylogenies without roots? A plea for the (Eutardigrada: Hypsibiidae) and of Thulinia augusti (Murray, 1907) comb. nov.
use of vouchers in molecular phylogenetic studies. Mol. Phyl. Evol. 48, 369–371. Zool. Anz. 238, 139–145.
Posada, D., 2008. JModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25,
1253–1256.
Pratas, F., Trancoso, P., Stamatakis, A., Sousa, L., 2009. Fine-grain parallelism using
Multi-core, Cell/BE, and GPU systems: accelerating the Phylogenetic Likelihood
Function. In: Proceedings of ICPP, Vienna, Austria, September 2009.

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Molecular Phylogenetics and Evolution 76 (2014) 110–126

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Molecular Phylogenetics and Evolution

Phylogeny of Eutardigrada: New molecular data and their morphological

Order Apochela Schuster, Nelson, Grigarick and Christenberry, 1980

Fig. 8. Scanning electron micrographs of mouth openings. A: Minibiotus furcatus. B:

molecular studies to verify their eventual position in the

(continued on next page)

Subfamily Pilatobiinae subfam. nov.

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