Phylogeny of Eutardigrada: New molecular data and their morphological support lead to the identification of new evolutionary lineages

Molecular Phylogenetics and Evolution 76 (2014) 110–126

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/locate /ympev

Phylogeny of Eutardigrada: New molecular data and their morphologicalsupport lead to the identification of new evolutionary lineages

http://dx.doi.org/10.1016/j.ympev.2014.03.0061055-7903/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Fax: +39 0592055548.E-mail addresses: [email protected] (R. Bertolani), roberto.guidetti@

unimore.it (R. Guidetti), [email protected] (T. Marchioro), [email protected] (T. Altiero), [email protected] (L. Rebecchi), [email protected](M. Cesari).

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, Italyb 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

Article history:Received 11 September 2013Revised 18 December 2013Accepted 7 March 2014Available online 19 March 2014

Keywords:TardigradaMolecular phylogenyMolecular systematicsMorphologyIntegrative taxonomyPilatobius gen. novPilatobiinae subfam. nov

a b s t r a c t

An extensive study of the phylogeny of Eutardigrada, the largest class of Tardigrada, has been performedanalyzing one hundred and forty sequences (eighty of which newly obtained) representative of one hun-dred and twenty-nine specimens belonging to all families (except Necopinatidae) of this class. The molec-ular (18S and 28S rRNA) results were compared with new and previous morphological data, allowing usto find new phylogenetic relationships, to identify new phylogenetic lineages, to erect new taxa for somelineages, and to find several morphological synapomorphies supporting the identified clusters. The classEutardigrada has been confirmed and, within it, the orders Apochela and Parachela, the superfamiliesMacrobiotoidea, Hypsibioidea, Isohypsibioidea, and Eohypsibioidea, and all the families and subfamiliesconsidered, although with emended diagnoses in several cases. In addition, new taxa have been erected:the new subfamily Pilatobiinae (Hypsibiidae) with the new genus Pilatobius, as well as an upgrading ofDiphascon and Adropion to genus level, previously considered subgenera of Diphascon. Our results dem-onstrate that while molecular analysis is an important tool for understanding phylogeny, an integrativeand comparative approach using both molecular and morphological data is necessary to better elucidateevolutionary relationships.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction Guil et al., 2013b) revealed some incongruence with the traditional

With the application of DNA sequencing to tardigrades, newinformation has been obtained on the phylogenetic position ofboth the phylum and its main evolutionary lines. Tardigrades havebeen included in the clade Ecdysozoa by Aguinaldo et al. (1997),within the Panarthropoda, together with Onychophora andArthropoda (Rota-Stabelli et al., 2010; Campbell et al., 2011),although the position of Tardigrada within this last group is stillundetermined (Nielsen, 2012).

Molecular analyses within the phylum confirmed the subdivi-sion of the higher taxa of Tardigrada, namely Heterotardigrada(Jørgensen and Kristensen, 2004; Jørgensen et al., 2010) and Eutar-digrada (Guidetti et al., 2005; Nichols et al., 2006). In contrast, re-cent molecular studies at the genus and species levels (Guidettiet al., 2005, 2009; Kiehl et al., 2007; Møbjerg et al., 2007; Sandset al. 2008; Jørgensen et al., 2010, 2011; Guil and Giribet, 2012;

morphological systematics of the phylum. In particular, the papersby Sands et al. (2008), and by Marley et al. (2011) revealed differ-ent evolutionary lines within Parachela that were only partially inagreement with those revealed by the previous morphologicalclassical approaches. In particular, four main clusters were identi-fied for which four superfamilies were proposed, which were par-tially confirmed in further studies (Jørgensen et al., 2010; Guil andGiribet, 2012). The superfamilies were initially without any mor-phological support, which was presented only later by Marleyet al. (2011). The most surprising conclusion of Sands et al.(2008) was the attribution of Hypsibius and Isohypsibius to two dif-ferent superfamilies, since these two genera were previously con-sidered belonging to the same sub-family (Hypsibiinae).

Considering that several eutardigrade families and genera werenot included in previous papers based on molecular analysis, wecarried out a further phylogenetic study analyzing two molecularmarkers expanding the study to all eutardigrade families (exceptNecopinatidae) and increasing the number of analyzed generaand species. In addition, considering that Eutardigrada, the largestclass of the phylum, is generally characterized by a low level ofmorphological diversity (especially compared with Heterotardigra-

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da; Fig. 1), and the identification of synapomorphies for each of thephylogenetic lineages has been a particularly difficult task, we ana-lyzed morphological characters of the studied taxa more in depth,utilizing light and scanning electron microscopy, in order toacquire better knowledge of eutardigrade phylogeny and system-atics. Therefore, for classifying organisms within a phylogeneticsystematic framework, we used an integrative approach (as inBertolani et al., 2011a), analyzing molecular phylogenies and com-pare them with morphological characters. With our integrativeapproach our goals were both to verify the current evolutionarylineages (or to identify new ones) and to provide further morpho-logical support for those taxa, which to date have a taxonomic des-ignation but are insufficiently described and thus not easilyidentifiable.

2. Materials and methods

2.1. Species sampling

Eighty eutardigrade specimens belonging to 26 genera were used.The full list of specimens, including collecting information, is pro-vided in Table S1. Tardigrades were extracted from different sub-strates (Table S1) collected in Europe and in USA. Moss, grass andleaf litter were placed in water for about half an hour. Then, animalswere isolated from all substrates, including freshwater sediments,using sieves. Finally, tardigrades were individually picked up usinga glass pipette under a stereomicroscope and processed.

2.2. Molecular analyses

2.2.1. Species identification for molecular analysisBefore DNA extraction, each tardigrade specimen used in

molecular analysis was observed in vivo by light microscopy (with

Fig. 1. Scanning electron micrographs of tardigrades. A: Batillipes (Arthrotardigrada(Echiniscoidea). C: Milnesium (Apochela). D: Thulinius (Parachela, Isohypsibioidea). E: RaParamacrobiotus (Parachela, Macrobiotoidea). Scale bars: A = 20 lm; B–G = 100 lm.

a Leitz DM RB microscope, using differential interference contrast –DIC – and phase contrast – PhC, with 40� and 100� immersion oilobjectives) and identified according to its morphological characters(Guidetti and Bertolani, 2005; Pilato and Binda, 2010). Before DNAextraction, pictures of the sclerified structures of in vivo specimenswere also taken by using a Nikon DS-Fj1 photocamera, followingthe protocols described in Cesari et al. (2011) and Bertolani et al.(2011b).

2.2.2. DNA extraction, PCR amplification, and sequencingGenomic DNA extraction was carried out from 80 single speci-

mens (Table S1) through a rapid salt and ethanol precipitation(Cesari et al., 2009). Regions of the nuclear ribosomal subunitgenes 18S and 28S rRNA were amplified using the following primercombinations: SSU F04 (50-GCT TGT CTC AAA GAT TAA GCC-30) andSSU 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) and28S 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 reactionvolume, which consisted of 2 ll reaction buffer (including 20 mMof MgCl2), 2.5 mM of each dNTP, 10 pmol (final concentration) ofeach primer, 1 U of DreamTaq polymerase (Fermentas) and 2 llof template DNA. A negative control lacking template DNA was car-ried out to test the possibility of contamination with foreign DNA.PCR was performed in a PCR Sprint Thermal Cycler (Hybaid). Theprotocol for 18S consisted of 35 cycles with 1 min at 94 �C, 35 s at52 �C and 2 min at 72 �C, with a final elongation step at 72 �C for10 min. The protocol for 28S consisted of 40 cycles with 45 s at96 �C, 1 min at 48 �C and 1 min at 72 �C, with a final elongation stepat 72 �C for 10 min. The amplified products were gel purified usingthe Wizard Gel and PCR cleaning kit (Promega). Sequencing reactionswere performed using the ABIPRISM� BigDye™ Terminator Version1.1 Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on

) (picture kindly donated by C. Schulze and A. Schmidt-Rhaesa). B: Echiniscusmazzottius (Parachela, Hypsibioidea). F: Bertolanius (Parachela, Eohypsibioidea). G:

112 R. Bertolani et al. / Molecular Phylogenetics and Evolution 76 (2014) 110–126

purified amplicons. Each sequencing reaction contained 0.2 lM of asingle PCR primer to initiate the sequencing reaction, 2 ll of Big-Dye™, 70 ng of purified products, 4 ll of 5� BigDye™ TerminatorVersion 1.1 Sequencing Buffer and distilled H2O for a final volumeof 20 ll. Cycling conditions for sequencing reactions consisted of25 cycles of 96 �C for 10 s, 50 �C for 5 s and 60 �C for 4 min. Bothstrands were sequenced using an ABI Prism 3100 (Applied Biosys-tems, Foster City, CA, USA). The nucleotide sequences of the newlyanalyzed specimens have been submitted to GenBank (Table S1).

2.2.3. Phylogenetic analysisTo achieve the best evolutionary tree and the most reliable phy-

logenetic inference, phylogenetic analyses were focused on a com-bined dataset (18S and 28S rRNA) to prevent stochastic errors andto yield an evolutionary inference based on the most completedataset for both taxon and gene sampling.

Sixty-nine 18S rRNA sequences and 19 28S rRNA sequences oftardigrades belonging to different genera and species of heterotar-digrades and eutardigrades were retrieved from GenBank(Table S2), when available the complete 18S and 28S sequenceswere the preferred source of data mining. Sequences of 10 out-groups belonging to Arthropoda, Priapulida and Kinorhynchawhere carefully selected (to avoid contaminant and sequencesfrom misidentified species) and downloaded from the NCBI data-base to complete the dataset (Table S2). The combined datasetwas assembled to achieve the most complete taxon samplingrather than the most homogeneous gene sampling. However, toensure to the phylogenetic software the possibility to explore theentire ‘‘tree space’’ (potential trees topology given an alignment),at least one common gene was always granted; specifically the18S gene sequence, which was therefore present for every consid-ered taxa. Preliminary analyses on the single gene datasets allowedto identify redundant sequences or poorly curated data.

The multigene dataset was aligned with the Muscle algorithmimplemented in MEGA 5 (Tamura et al., 2011). Default parameterswere used because they are designed for the average best accuracy(Edgar, 2004). The resulting alignment was accurately inspected bysearching for software homology misinterpretations. The GBlocksprogram (Catresana, 2000) was used for applying relaxed settingsand parameters (minimum number of sequences for a conserved po-sition: 27; minimum number of sequences for a flank position: 30;maximum number of contiguous non-conserved positions: 20; min-imum length of a block: 2; allowed gap positions: all) and aiming todiscard uninformative regions of the alignment. Alignment concate-nation was accomplished through Seaview alignment editor (Gouyet al., 2010). The complete combined alignment included 129 tardi-grade taxa and a maximum of 3701 characters in length.

Bayesian inference (BI) was conducted using the programMRBAYES 3.1.2 (Huelsenbeck and Ronquist, 2001) using the hybridMPI/OpenMP version (Pratas et al., 2009) on the CIPRES ScienceGateway Portal (http://www.phylo.org/sub_sections/portal/). Bestfitting model evaluations for each analysis were performed takinginto account AIC, BIC and hLRT (jModeltest 0.0.1, Posada, 2008)resulting in the GTR + Gamma + I model to be most suitable forall the datasets. Tracer 1.5 (Rambaut and Drummond, 2007) wasused to compute and compare Bayes factors for the two modelsthat scored better in the AIC and BIC tests. After this analysis thebest fitting model resulted in the GTR + Gamma, which has beenused in the final analysis. In MrBayes, two independent runs, eachof four Metropolis-coupled Markov chains Montecarlo (MCMC),were launched for 40 � 106 generations, trees were sampled every1000 generations. The obtained parameter value files of each runwere analyzed with Tracer 1.5 to verify the convergence of theMCMC runs and their effective sample sizes.

A phylogenetic analysis on the dataset was also computed in amaximum likelihood (ML) framework. ML analysis was performed

using the program RAxML v7.2.4 (Stamatakis, 2006) using theGTR + Gamma model. Bootstrap resampling with 1000 replicateswas undertaken via the rapid bootstrap procedure of Stamatakiset al. (2008) to assign support to branches in the ML tree.

RNA secondary structure, protein secondary/tertiary structure,and other functional considerations all influence the rate and pat-tern of evolution: variability characterizing both slowly and rap-idly evolving stems is a direct consequence on the identity of thebases; tertiary structure and protein interactions (Gutell et al.,2002; Hickson et al., 1996; Pagel and Meade, 2004). To account alsofor this variability, a Bayesian mixture model (Pagel and Meade,2004) was tested and used on the aligned dataset. Mixture modeldo not require a priori specification of partitions (as for the doubletmodel). The parameters of each evolution model (substitutionrates, base frequencies) are estimated from the data and, in theend, to each character can be assigned a probability of membershipin each model. This method produces different categories amongthe alignment to which appropriate models (inferred from data)are applied. The CAT model (mixture model) implementation wasachieved by using the program Phylobayes 3.2e (Lartillot andPhilippe, 2004) and the alignment was analyzed using the site-heterogeneous CAT + Gamma model. For all Phylobayes analysestwo chains were run. Chains where considered to have convergedwhen the ‘‘Maxdiff’’ between the two independent chains was <0.2(see Phylobayes manual). The ‘‘bpcomp’’ program in the Phyloba-yes suite was used to test convergence. For each analysis, burn inand subsampling frequencies were estimated in order to minimize‘‘maxdiff’’.

2.3. Morphological analysis

2.3.1. Light microscopyTardigrade specimens were mounted on slide in polyvinyl lac-

tophenol fluid or in Faure-Berlese fluid. All mounted animals wereobserved up to 100� immersion oil objective magnification, usingDIC and PhC as cited above for an accurate analysis of the taxo-nomic characters (e.g. claws, mouth, buccal-pharyngeal apparatus,cuticle). Pictures of the sclerified structures of mounted animalshave been taken using the same photocamera cited above.

Specimens originating from the same samples used for molecu-lar analysis (Table S1) were mounted in Faure-Berlese fluid asparagenophore vouchers (Pleijel et al., 2008; Cesari et al., 2013).

Further detailed observations were carried out on specimens ofAcutuncus antarcticus (Richters, 1904), Calohypsibius ornatus (Rich-ers, 1900), Hebesuncus conjungens (Thulin, 1911), Microhypsibiusbertolanii Kristensen, 1982, Microhypsibius sp., Mixibius saracenus(Pilato, 1973), Hexapodibius micronyx Pilato, 1969, Hexapodibiussp., Parhexapodibius ramazzottii Manicardi and Bertolani, 1987, Par-hexapodibius pilatoi Bernard, 1977, Ramajendas sp. and Ramazzot-tius sp. from Bertolani’s collection (Department of Life Sciences,University of Modena and Reggio Emilia, Italy) and specimens ofParhexapodibius lagrecai Binda and Pilato, 1969 from Binda and Pil-ato’s collection (Department of Biological, Geological and Environ-mental Sciences, University of Catania, Italy), mounted in polyvinyllactophenol fluid or in Faure-Berlese fluid.

2.3.2. Scanning electron microscopyParagenophores (adult animals) of several species used for molec-

ular analysis were prepared for scanning electron microscopy (SEM)observations of the mouth opening, cuticle surface and claws. To pre-vent animal contraction during fixation, animals were first relaxed inwarm water (50 �C) until they were completely relaxed. Then tardi-grades were fixed in boiling absolute ethanol for a few minutes andrinsed three times in absolute ethanol. Finally animals were dehy-drated by evaporation of boiling absolute ethanol, mounted on stubsand sputter-covered with a thin layer of gold.

http://www.phylo.org/sub_sections/portal/

Table 1Taxonomic summary.

Order Apochela Schuster, Nelson, Grigarick and Christenberry, 1980Family Milnesiidae Ramazzotti, 1962Composition: Milnesium (type genus), Bergtrollus, Limmenius, Milnesioides.

Order Parachela Schuster, Nelson, Grigarick and Christenberry, 1980Superfamily Eohypsibioidea Bertolani and Kristensen, 1987

Family Eohypsibiidae Bertolani and Kristensen, 1987Composition: 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, 2000Composition: 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, 2008Composition: 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, 2008Composition: 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 subfamiliaGenera: Acutuncus, Mixibius.

Family Calohypsibiidae Pilato, 1969 (emended in this paper)Composition: Calohypsibius.Family Microhypsibiidae Pilato, 1998Composition: Microhypsibius (type genus), Fractonotus.

Incerta superfamiliaFamily Necopinatidae Ramazzotti and Maucci, 1983Composition: Necopinatum.


The analysis of the morphology of the male gametes of theeutardigrades Minibiotus furcatus (Eherenberg, 1859) and Parama-crobiotus areolatus (Murray, 1911) was carried out. The spermato-zoa were extracted from the testis and prepared for SEM analysisaccording to the technique perfected by Rebecchi and Guidi (1991).

All SEM observations were carried out using a FEI XL 40 SEM(Fei Company – Oxford Instruments) in the ‘‘Centro Interdiparti-mentale Grandi Strumenti’’ of the University of Modena and ReggioEmilia, Italy.

3. Results

3.1. Molecular analysis

Phylogenetic molecular analyses of combined sequences (18SrRNA + 28S rRNA) carried out with the Bayesian inference (BI), un-der GTR + Gamma model (Fig. 2) and CAT + Gamma model (Fig. 3),or Maximum Likelihood (ML, under GTR + Gamma model; Fig. 2)display a very similar topology for the relationships of the generawithin the families, although the BI under GTR + Gamma modeltree shows higher branch supports.

Overall results confirm the Eutardigrada and Heterotardigradaclasses as well as their orders Parachela and Apochela (Eutardigra-da) and Echiniscoidea (Heterotardigrada) (Figs. 2 and 3). The orderArthrotardigrada (Heterotardigrada) is not supported in any of theperformed analyses resulting in a paraphyletic group (Figs. 2 and 3).

Within Eutardigrada, Milnesium species (Apochela) are in a sis-ter-group relationship with the cluster grouping all the Parachelaspecies (Figs. 2 and 3). The main parachelan clade is formed by fourwell supported evolutionary lines that can be identified with the

four superfamilies: Isohypsibioidea, Hypsibioidea, Eohypsibioidea,and Macrobiotoidea (Figs. 2 and 3). Only the tree obtained withthe BI, under GTR model, shows the Isohypsibioidea basal to theother superfamilies and the Hypsibioidea as sister-group of theclade with the other two superfamilies (Eohypsibioidea, Macrobio-toidea) being closely related (Fig. 2). The BI (under CAT model) andML analyses are not able to solve the relationships among super-families (Fig. 3).

Within the phyletic lineage of Isohypsibioidea (Fig. 4), speciesattributed to seven genera are present (i.e. Isohypsibius, Pseudobio-tus, Doryphoribius, Eremobiotus, Halobiotus, Thulinius, Hexapodibius),whose relationships are not always well-resolved. Some of thesegenera are not monophyletic (i.e. Isohypsibius, Eremobiotus, Dory-phoribius). Within this cluster, there are Hexapodibius species (Cal-ohypsibiidae) currently attributed to Hypsibioidea.

The second evolutionary line, corresponding to the Hypsibioi-dea (Fig. 5), presents two well supported main clusters, one formedby the monophyletic genera Ramazzottius and Hebesuncus (bothbelonging to Ramazzottiidae, incorrectly named Ramazzottidaeby Marley et al., 2011), the other formed by species of several gen-era of Hypsibiidae (i.e. Hypsibius, Borealibius, Mixibius, Acutuncus,Diphascon, Astatumen, Itaquascon, Platicrista), Microhypsibiidae(Microhypsibius), and Calohypsibiidae (Calohypsibius). Within thislast line, a specimen attributed to Calohypsibius by Sands et al.(2008) is basal to a cluster of four phyletic lineages (Fig. 5) com-posed of: (i) Calohypsibius ornatus (Calohypsibiidae), Acutuncus ant-arcticus (Hypsibiidae), Microhypsibius species (Microhypsibiidae)and Mixibius species (a genus currently tentatively attributed tothe superfamily Isohypsibioidea; Marley et al., 2011); (ii) Diphas-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). CladesA–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 branchesindicate posterior probability values. Clades A–D are represented in Figs. 4–6, respectively.


Diphascon (Diphascon) ramazzottii; (iii) Diphascon species of thesubgenus Adropion (characterized by the absence of a drop-likethickening between the buccal and flexible pharyngeal tube) andspecies of three genera (i.e. Platicrista, Astatumen, Itaquascon)belonging to the subfamily Itaquasconinae; iv) two well-supportedclusters, one formed by several Diphascon species of the subgenusDiphascon, the other by Hypsibius and Borealibius species (Fig. 5).

The phylogenetic lineage corresponding to the Eohypsibioidea(represented by the only family Eohypsibiidae) is composed byspecies of Eohypsibius and Bertolanius (Fig. 6).

Within the last phyletic lineage, corresponding to the Macrobio-toidea, two main phylogenetic lineages are present (Fig. 6). Oneline contains a well supported cluster composed of species ofMurrayon 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 andMaximum Likelihood analyses, under the GTR + Gamma model (left topology), and Bayesian analysis under CAT + Gamma model (right topology). Values above branchesindicate posterior probability values (Bayesian inference), while values under branches show bootstrap values (Maximum Likelihood analysis). Taxa in bold indicate newlyanalyzed specimens.

Fig. 5. Clade B. Phylogenetic reconstruction within the superfamily Hypsibioidea based on combined dataset (18S + 28S rRNA sequences) and inferred by Bayesian andMaximum Likelihood analyses, under the GTR + Gamma model (left topology), and Bayesian analysis, under CAT + Gamma model (right topology). Values above branchesindicate posterior probability values (Bayesian inference), while values under branches show bootstrap values (Maximum Likelihood analysis). Taxa in bold indicate newlyanalyzed specimens.


relationship with Adorybiotus granulatus and Richtersius coronifer(both currently belonging to Macrobiotidae). The second lineageis formed by all the other species of Macrobiotidae, which resultsin a polyphyletic lineage. This last clade is composed of two mainclusters, one constituted by Paramacrobiotus and Minibiotus spe-cies, in which Minibiotus appears to be polyphyletic for the sis-ter-group relationship of Minibiotus furcatus with Paramacrobiotusspecies (Fig. 6). The other cluster groups Xerobiotus pseudohufelandiand the species of the Macrobiotus hufelandi group (i.e. Macrobiotushufelandi, Macrobiotus vladimiri, Macrobious macrocalix, Macrobiotus

joannae, Macrobiotus sapiens, Macrobiotus polonicus) are in closerelationship with Macrobiotus cf. nelsonae, and a second clusterthat encompasses Macrobiotus furciger and Macrobiotus harmsworthi(Fig. 6).

3.2. Morphological analysis

The claws of Hexapodibius, Parhexapodibius and Calohypsibiusgenera, currently attributed to Calohypsibiidae, are very smalland 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 rRNAsequences) 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 Likelihoodanalysis). Taxa in bold indicate newly analyzed specimens.


species of Hexapodibius and Parhexapodibius, a suture between theprimary and secondary branch arising from the base of the claw isclearly visible (Fig. 7A and B respectively). In Calohypsibius theproximal part of the claw, when observed in frontal view, lookslarge, as the sum of the width of the two branches (Figs. 7C, and10K), but the suture between primary and secondary branch ofthe claw is not detectable. This pattern can be interpreted as a clawwithout a basal (common) tract and without a suture between pri-mary and secondary branch, or as a claw with an unusually largecommon tract (basal tract). In Microhypsibius (currently attributedto Microhypsibiidae) the claws are also small: the basal tract of theclaw is always well recognizable, but it is as thin as one of thebranches (Figs. 7D, and 10J). The claws of Hebesuncus and Ramajen-das (Figs. 7E and 10I) have similarities with the Hypsibius typeclaws (Fig. 10G), but in the shape of the main branch on the exter-nal (or posterior) claw they look similar to those of the Ramazzot-tius (Figs. 7F and 10H). With phase contrast, in Hebesuncus,Ramajendas and Ramazzottius the main branch of the external (orposterior) claw looks dark for the most part but its proximal partlooks always clear (Fig. 7E), whereas in Hypsibius the entire clawlooks dark with phase contrast. The claws of Acutuncus and Mixibi-us look similar in the two genera (Fig. 7G and H) and, in our opin-ion, they are not attributable to the Isohypsibius type, but rather toa modified Hypsibius type. In fact, the two claws on the same legare quite different in size, and the convex curvature of the

secondary branch always forms a continuous arc in both claws(Fig. 10N; be careful to look at several claws) and not a sort of an-gle, as in Isohypsibius type claws (Fig. 10M).

The presence of a paired elliptical organ on the head reported insome species of Calohypsibiidae and Microhypsibiidae has notbeen identified in any examined species of Calohypsibius, Hexapodi-bius, Parhexapodibius and Microhypsibius.

With regard to Macrobiotidae, the analysis of the mouth ofMinibiotus furcatus with SEM shows the presence of ten very shortlamellae (Fig. 8A). These structures were not recognizable in ourspecimens by observations with light microscopy.

The testicular spermatozoon of M. furcatus (Fig. 9A–D), observedby SEM, is thin and about 65.0 lm in length. It is made up of threeregions: the head, including acrosome and nuclear region, themiddle piece and the terminal tufted tail (Fig. 9A-D). The acrosomeis about 22.0 lm in length, has a smooth surface and rounded tip(Fig. 9B). The nuclear region (about 18.0 lm in length) is heli-cal and tightly coiled (Fig. 9B–D). Its coils increase in width cau-dally, towards the middle piece. The midpiece (about 3.0 lm inlength) is kidney-shaped and exhibits hemispherical protuber-ances on its surface (Fig. 9D). The tail (about 23.0 lm in length)has a constant diameter and splits terminally into a tuft of fila-ments (each about 5.5 lm in length; Fig. 9A). The male gameteof M. furcatus looks very similar to that of Paramacrobiotus areola-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 showsa 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: Mixibiussaracenus (third pair of legs). Scale bars: A–C = 5 lm, D–H = 10 lm.


4. Discussion

Using a combined dataset of 18S and 28S rRNA sequences, wewere able to obtain data on the phylogenetic relationships amongeight of the nine current (according to Guidetti and Bertolani,2011; Degma et al., 2013) eutardigrade families (only Necopinati-dae is lacking), comprising 31 current genera, 10 of which were

compared for the first time with a molecular approach. Moleculardata were integrated and supported with morphological dataobtained from new observations and from references. The integra-tion between molecular and morphological information is impor-tant in understanding phylogeny and as a general principle todefine the taxa. The erection of new taxa (at any level) only onthe 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.

Fig. 9. Scanning electron micrographs of spermatozoa. Male gametes of Minibiotusfurcatus (A–D). A: In toto cell. B: Acrosome region. C: Transition area betweenacrosome and nuclear regions. D: Kidney-shaped middle piece and helical nuclearregion. E: Two male gametes of Paramacrobiotus areolatus. Scale bars: A, B, E = 5 lm;C, D = 1 lm. a = acrosome region, mp = middle piece, n = nuclear region, t = tail,tt = terminal tuft.


a recommendation of the International Commission on ZoologicalNomenclature (1999). Without morphological support, the risk ofa mistake is high and the information for identifying the taxa issubstantially lacking, especially when micrometazoans, such astardigrades, are considered.

Our molecular results confirm the monophyly of Eutardigradaand its position as the sister group of Heterotardigrada. Withinthe Eutardigrada, Parachela is supported as the sister group ofApochela. From a morphological point of view, Parachela are char-acterized by the synapomorphy of two double claws (i.e. clawscomposed of a primary and a secondary branch very often joinedthrough a common tract), normally two per leg (Fig. 10C–O).Parachela normally also have cuticular placoids in the pharynx(secondarily lost in very few cases; Fig. 11B–L) that are alwaysmissing in Apochela (Fig. 11A).

At the higher phylogenetic level of the tree, Parachela are di-vided into four main clusters (Fig. 2 and 3). Three of these clustercorrespond to the evolutionary lines identified by Kiehl et al.(2007) and by Sands et al. (2008) and one corresponding to thefamily Eohypsibiidae (recently proposed as superfamily by Marleyet al., 2011). Based only on molecular evidence, Sands et al. (2008)proposed superfamily ranks for the first three evolutionary lines,namely Macrobiotoidea, Hypsibioidea (names derived from previ-ous families) and Isohypsibioidea, a new proposed name. Follow-ing the taxonomic scheme by Pilato and Binda (2010), a secondpaper by Marley et al. (2011) was aimed to complete the molecularbased scenarios providing some morphological support.

Considering morphology, with respect to the median plane ofthe leg, the two double claws are symmetrical in the superfamilyMacrobiotoidea (Fig. 10C–F) and asymmetrical in the superfamiliesEohypsibioidea (Fig. 10O), Isohypsibioidea (Fig. 10L and M) andHypsibioidea (Fig. 10G–K), even though each taxon has a uniqueclaw structure based on other characters (see below and taxo-nomic account).

The phylogenetic relationships among superfamilies identifiedby Bayesian analysis under the GTR model (Fig. 2) is completelyin agreement with those found by Marchioro et al. (2013) obtainedby a total evidence analysis of the muscular architectures and18S + 28 S rRNA of species representative of the four superfamilies.

4.1. Phylogeny of eutardigrade superfamilies

The superfamily Eohypsibioidea has been recently proposed(Marley et al., 2011), raising in taxonomic rank the family Eohyp-sibiidae, but unfortunately on the basis of only one molecular da-tum from GenBank. Our molecular data (Fig. 2 and 6) confirmthe 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 fromPilato, 1998.


with the relationship between its genera Eohypsibius and Bertola-nius (=Amphibolus) very well supported. Our data confirm Eohypsi-bioidea as possible sister-group of Macrobiotoidea. Thisrelationship was suggested on molecular basis by Jørgensen et al.(2010), Marley et al. (2011) and Guil et al. (2013b) and confirmedin the total evidence analyses by Guil et al. (2013a) and Marchioroet al. (2013). The relationship between Eohypsibius and Bertolaniushas strong morphological support by peculiar characters (synapo-morphies); other than the asymmetrical double claw structure ofEohypsibiidae type (Fig. 10O), and the peculiar ability of the clawsto rotate on their own bases, verified by Bertolani and Kristensen(1987) and reported by Marley et al. (2011); the mouth shape(Fig. 11D) and the number and shape of peribuccal lamellae(Bertolani and Kristensen, 1987; see taxonomic account). Wetherefore conclude that our new molecular data and the identifiedsynapomorphies support the evolutionary line of Eohypsibioidea.

The Macrobiotoidea are supported by our molecular results(Fig. 2, 3 and 6), confirming the data by Sands et al. (2008) andthose of other recent papers (Marley et al., 2011; Guil and Giribet,2012). According to Marley et al. (2011), Macrobiotoidea are char-acterized by the double claw pairs being symmetrical with respectto the median plane of the leg (Fig. 10C–F), and the presence of aventral lamina on the buccal tube (Fig. 11B and C) that generallyleads to asymmetrical apophyses for the insertion of the styletmuscles (AISM). Even though Macrobiotoidea are the only para-chelan superfamily characterized by symmetrical claws, we shouldremember that symmetrical claw organization is present also in allHeterotardigrada (Fig. 10A) and in the apochelan Milnesiidae(Fig. 10B) and could represent a plesiomorphic character (Guidettiet al., 2005). In addition, the ventral lamina or other charactersshared by all Macrobiotoidea (e.g. ornamented eggs) are also pres-ent in other superfamilies (see taxonomic account), other than inthe heterotardigrade Oreella (Bertolani et al., 1996). We considerthe presence of ten peribuccal structures (lamellae or papulae;Fig. 8) and double claws characterized by a peculiar stalk withcylindrical or laminar shape (Fig. 10C–E) as clear synapomorphiccharacters of this superfamily (Guidetti and Bertolani, 2001).

According to our molecular data, the clade Isohypsibioidea alsoincludes Hexapodibius (Fig. 2–4). This superfamily is very well sup-ported from a molecular point of view, as first shown by Kiehl et al.(2007) and subsequently by other authors (Møbjerg et al., 2007;

Sands et al., 2008; Marley et al., 2011; Guil and Giribet, 2012). Mar-ley et al. (2011) report as diagnostic characters of the Isohypsibioi-dea the asymmetrical Isohypsibius type claws (Fig. 10M), andridged AISM (Fig. 11H), but our molecular data suggest that alsoHexapodibius (two clearly different species have been examinedby us from a molecular point of view) should be attributed to thissuperfamily, even though its claws are very reduced (as are thoseof other genera, such as Parhexapodibus, examined here from amorphological point of view). We consider as an autoapomorphyof the Isohypsibioidea the Isohypsibius type claws (asymmetricaldouble claws with similar shape and size on each leg, see taxo-nomic account) (Fig. 10L-M), secondarily modified in some genera(see above and further discussion). An extreme reduction shouldbe happened in Apodibius, which is attributable to this superfamilyfrom a molecular point of view (Dabert et al., 2014). Not all Isohyp-sibioidea share ridge-shaped AISM, e.g. Halobiotus, Doryphoribius(Fig. 11G), Hexapodibius (Fig. 11E), Parhexapodibius, Haplomacrobi-otus and Apodibius, the last five genera have a ventral lamina.

The cluster Hypsibioidea (Fig. 2, 3 and 5) is well-supported frommolecular point of view (present study, Sands et al., 2008; Marleyet al., 2011; Guil and Giribet, 2012), and includes taxa with clawsof the Hypsibius type or Ramazzottius type (Fig. 10G-H) (see taxo-nomic account). According to Marley et al. (2011), Hypsibioideaare characterized by asymmetrical claws of the Hypsibius type(Ramazzottius-type is considered a derived Hypsibius type), andhooked AISM (or, if the buccal tube is elongated, the AISM can bebroad ridges). We consider as a synapomorphy of the superfamilythe presence of asymmetrical double claws that share an evidentdifference in size and shape on the same leg (Fig. 10G–K), a situa-tion not found in other Parachela. This synapomorphy is in linewith molecular support and together with morphological data al-lows us to support the superfamily Hypsibioidea. The small clawsof the Microhypsibius type and the Calohypsibius type (Figs. 7C–D,and 10J and K) present within the superfamily Hypsibioidea appeardifferent from the Hypsibius type claws, and they should be consid-ered secondarily reduced (according to Pilato, 1989; Dastych andAlberti, 1990, and in our opinion too), similar to what indepen-dently happens in Hexapodibius. Most Hypsibioidea have a narrowbuccal tube (Fig. 11I–M), flexible main branch of the externalclaws, but these characters are shared with other evolutionarylines.

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

Considering the evolutionary lineages within the superfamilies,many clades can be identified within each of them (Fig. 4–6).

4.2.1. MacrobiotoideaMacrobiotoidea (Fig. 6) consists of clusters of several lineages.

Murrayidae family, which includes Dactylobiotus and Murrayon asgenera analyzed from a molecular point of view, is supported bothby molecular (see results; Guidetti et al., 2005; Marley et al., 2011;Guil and Giribet, 2012) and morphological data (Guidetti and Ber-tolani, 2001; Guidetti et al., 2005) (mainly peculiar claws, Fig. 10Dand E; see also taxonomic account). Within this family, the genusMurrayon appears to be polyphyletic. From a molecular point of

view, Adorybiotus and Richtersius appear related to this family,but morphological support for this relationship is not evident. Bothgenera seem to belong to independent lineages, and both are well-supported with both morphological data (Pilato and Binda, 1987)and molecular data. All the remaining genera of Macrobiotoideaanalyzed in this study cluster together, but they could representdifferent families. Lacking morphological evidence sufficient tocharacterize each of them, provisionally we prefer to maintainthe family Macrobiotidae for all of them, even though this familyclearly appears polyphyletic and must be reconsidered in the fu-ture. Within this cluster of Macrobiotidae, two main evolutionarylineages are recognized: one with Minibiotus and Paramacrobiotus,the other with Xerobiotus and Macrobiotus. Macrobiotus and Mini-biotus are polyphyletic, as also shown by Guil and Giribet (2012).


In Minibiotus, one species (M. furcatus) is closer to Paramacrobiotusthan to the other Minibiotus species. In reality, M. furcatus showsvery short lamellae around the mouth opening (Fig. 8A) insteadof papulae as should be in Minibiotus. Moreover, most Minibiotusspecies show buccal-pharyngeal apparatus characters (Guil andGuidetti, 2005; Fig. 11C) different from those of M. furcatus. Sincethe spermatozoan morphology has a phylogenetic signal (Guidiand Rebecchi, 1996; Rebecchi and Bertolani, 1999; Rebecchiet al., 2000, 2003, 2011), it has been considered in M. furcatusand P. areolatus. The male gamete of M. furcatus shows strong sim-ilarity with the particular spermatozoon of Paramacrobiotus spe-cies (our results; Guidi and Rebecchi, 1996; Rebecchi et al.,2011), above all the head shape, thin, slightly coiled, much longerthan the tail and with a long and thin acrosome. These morpholog-ical data confirm the relationships between Paramacrobiotus andM. furcatus supported by sequence analyses. Nonetheless, we donot have sufficient morphological support and molecular data formoving M. furcatus to another genus.

From our molecular data related to the second evolutionaryline, we can note that: a. Macrobiotus harmsworthi and Macrobiotusfurciger (belonging to the harmsworthi group according toRamazzotti and Maucci, 1983) are related but clearly separatedfrom Paramacrobiotus, even though some evident similarities canbe noted in the type of buccal armature and in M. harmsworthi inthe morphology of the egg shell processes; b. the Macrobiotushufelandi group (Bertolani and Rebecchi, 1993; Guidetti et al.,2013a) and Xerobiotus form a cluster of species having in commonthe shape of the buccal-pharyngeal apparatus, type of egg shellprocesses (in most cases) and spermatozoa (Guidi and Rebecchi,1996; Rebecchi et al., 2000, 2011; Guidetti et al., 2005). Nevertheless,Xerobiotus clearly differs from the species of the M. hufelandi groupby morphological characters: the shape of the claws (Fig. 10F),which are reduced, and also the absence of ‘‘pearls’’ (pores) inthe cuticle. The presence of Macrobiotus nelsonae (characterizedby placoids similar to those of M. hufelandi group and ‘‘pearls’’ onthe cuticle, but by different large and conical egg processes) withinthe M. hufelandi group (generally characterized by inverted goblet-like egg processes) indicates that this group has more commoncharacters in the animal morphology than in the egg shellmorphology. According to our molecular data (Fig. 6), we shouldconsider the specimen associated with the GenBank sequenceU32393 to belong not to Macrobiotus but to Paramacrobiotus. Thetwo Macrobiotus clades identified with molecular methods withinthe second evolutionary line of Macrobiotidae should representseparate genera, but there is not yet sufficient morphological infor-mation to support a formal description. Further studies on thisgenus are necessary.

4.2.2. IsohypsibioideaIsohypsibioidea superfamily includes several evolutionary lin-

eages (Fig. 5). First of all, according to molecular data, the presenceof Hexapodibius and the exclusion of Calohypsibius in Isohypsibioi-dea mean that the two genera cannot belong to the same family, incontrast with previous morphological studies (Pilato, 1969a,b). Theclaws of both genera appear similar, due to a strong reduction inthe length of the claw basal tract and branches (Fig. 7A and C). Dif-ferences have also been detected between the two claw types:Hexapodibius claws show a suture between the primary and sec-ondary branch arising from the base of the claw (Hexapodibius typeclaws; Figs. 7 and 10L; see taxonomic account), while this suture isabsent in Calohypsibius (Calohypsibius type claws; Figs. 7 and 10K;see also taxonomic account). Moreover, the buccal-pharyngealapparatus is quite different in the two genera: Hexapodibius and re-lated genera (i.e. genera with the same kind of claws: Parhexapodi-bius, or derived from it by the loss of one branch: Haplomacrobiotus,Haplohexapodibius) have a ventral lamina on the buccal tube

(Fig. 11E), whereas Calohypsibius does not, and the placoids of Cal-ohypsibius are quite different in shape and number from those ofHexapodibius and the other genera here cited above, which are verysimilar. A possible polyphyletic status of the actual Calohypsibiidaeand a possible transfer of some genera to another superfamily werealso questioned by Guil et al. (2013a) based on their morphologicalanalyses. For these reasons we propose to move Hexapodibius andrelated genera (i.e. Parhexapodibius, Haplomacrobiotus, Haplohexa-podibius) to the superfamily Isohypsibioidea (see taxonomic ac-count). We want to emphasize that the taxa belonging toIsohypsibioidea share a male gamete morphology without themidpiece, which differs from that of the other tardigrade super-families (Wolburg-Buchholz and Greven, 1979; Rebecchi andBertolani, 1999; Rebecchi, 2001; Nelson et al., 2010). To date, thesuperfamily Isohypsibioidea includes only one family, Isohypsibii-dae, but the evolutionary lineages within it are often unclear. Thegenus Isohypsibius is certainly polyphyletic, confirming previousmolecular results (Kiehl et al., 2007; Møbjerg et al., 2007). Thegenus Doryphoribius, too, appears polyphyletic, and similarlyPseudobiotus, but morphological information are insufficient fordistinguishing the different evolutionary lineages among themand to support the erection of new taxa. The situation of Eremobio-tus requires further analysis and comparison with the specimensused for obtaining the sequences available in GenBank.

4.2.3. HypsibioideaThe cluster of Hypsibioidea is subdivided in two main branches

(Fig. 5). The first branch includes the genera Ramazzottius andHebesuncus (the two genera of Ramazzottiidae) in agreement withprevious results (Kiehl et al., 2007; Sands et al., 2008; Marley et al.2011; Guil and Giribet 2012). There is no agreement regarding thetype of claws of these two genera. Pilato and Binda (2010) attributethe homonymous type of claws only to Ramazzottius (Fig. 10H),whereas they attribute the claws of Hebesuncus (Fig. 7E) to theHypsibius type (Fig. 10G). In contrast, Marley et al. (2011) attributeto both genera the same type of claws: the Ramazzottius type(Fig. 10H). In reality, the claws look different in the two generaand the statement of Pilato and Binda (2010) seem more correct.Nevertheless, as reported in our results and suggested by Guilet al. (2013a), we also find a significant similarity among the clawsof the two genera and Ramajendas (Fig. 10I), which are differentfrom the Hypsibius type claw. We consider the peculiarity of theRamazzottius and Hebesuncus claws (the claw with the primarybranch connected to basal tract with a thin, flexible tract) as a syn-apomorphy of the Ramazzotiidae family, together with asymmetricAISM (Pilato and Binda, 2010). Moreover, both genera lay free eggswith ornamented shells, but the polarity of this character should beverified. According to Dastych (2009), the genus Thalerius could berelated to Ramajendas.

The other branch of Hypsibioidea (Fig. 5) is similarly monophy-letic and includes genera characterized by Hypsibius type claws (or,in our opinion, derived from it, such as in Acutuncus, Fig. 7G, andMixibius, Fig. 7H; see below) and Microhypsibius type (Fig. 7D,10J) and Calohypsibius type (Fig. 7C, 10K) claws, which are exclu-sively in this clade (for the description of Microhypsibius and Hyp-sibius type claws see Bertolani et al., 2009; Pilato and Binda, 2010;for Calohypsibius type claw see our results and taxonomic account).Pilato and Binda (2010) attribute the external claw of both Acut-uncus and Mixibius to the Isohypsibius type, and the internal oneto the Hypsibius type and to a modified Isohypsibius type, respec-tively. These claws appear as such but in our opinion, as statedin the results, none of them can really be attributed to the Isohyp-sibius type. Rather they can be attributed to a modified Hypsibiustype. For this reason, one of us (Bertolani, 1982), before the molec-ular approach to phylogeny existed, moved Isohypsibius saracenusto the genus Hypsibius; this species was subsequently considered


as the type species of the genus Mixibius. Most species of this cladelay eggs with smooth shells within the exuvium, with the excep-tion of Acutuncus and a few Hypsibius, which have very short pro-cesses on the egg shell. Mixibius eggs are unknown. Anautoapomorphy of this second clade of Hypsibioidea is difficultto identify. A particular structure present only in this clade andin several (but not all) of its lineages is the septulum, a posteriorcuticular thickening within the pharynx that alternates with thelines of placoids. Within the clade, Calohypsibius (with its typicalclaws) represents one independent lineage or maybe two lineages,but a better morphological study is necessary, given that the mor-phology of Calohypsibius sp. is not described by Sands et al. (2008).The position of Microhypsibius in another evolutionary lineage withrespect to Calohypsibius confirms the differences between thesegenera (Thulin, 1928; Kristensen, 1982; Bertolani and Kristensen,1987). Other clear morphological differences between Microhypsi-bius and Calohypsibius can be noted in the buccal-pharyngeal appa-ratus (see Pilato and Binda, 2010). In conclusion, the familiesCalohypsibiidae (now including only the genus Calohypsibius) andMicrohypsibiidae should be maintained. The presence of Mixibiusand Acutuncus in the same cluster with Microhypsibius is very dif-ficult to explain on a morphological basis, as the claws and the buc-cal-pharyngeal apparatuses are quite different. However, thepresence of Mixibius saracenus within the Hypsibioidea andHypsibiidae is in line with the first transfer of this species fromIsohypsibius to Hypsibius on the basis of its morphology (Bertolani,1982), followed by a further transfer to the genus Mixibiussubsequently erected by Pilato (1992). Acutuncus antarcticus wasalso previously considered a Hypsibius and then transferred to anew genus (Pilato and Binda 1997). The presence of M. saracenuswithin the Hypsibioidea is also in agreement with the results ofmorphological and total evidence analyses of Guil et al. (2013a).Considering the Hypsibiidae without pharyngeal (annulate)tube, Mixibius and Acutuncus have a particular position in themolecular tree. The type of claw, similar but not identical to thatof all other Hypsibiidae genera, could possibly support the inclu-sion of these two genera in the Hypsibiidae family, but the dataare insufficient to attribute them to a specific subfamily (see taxo-nomic account). The other Hypsibiidae (most of the consideredtaxa of this family) clearly display Hypsibius type claws and shouldrepresent a monophyletic family, here redefined (see taxonomicaccount).

The remaining Hypsibiidae lineage of the molecular tree ischaracterized by the presence of a pharyngeal (annulate) tube(Fig. 11J-M), except for the Hypsibius-Borealibius lineage (Fig. 5).It is not yet possible to understand if this character is an apomor-phy of this clade (adaptive convergence with genera of othersuperfamilies), or a plesiomorphy, maybe of the Eutardigrada,being present in most parachelan superfamilies (or all; see com-ments on Paradiphascon in taxonomic account) and in Milnesiidae(see Guidetti et al., 2012, 2013b). This lineage is clearly subdi-vided into a small and a large cluster, the latter subdivided intwo clades. One clade of the larger cluster includes only specieshaving a pharyngeal (annulate) tube, but without a drop-likethickening between the buccal and pharyngeal tube (Fig. 11Kand M), in addition to a pharyngeal bulb that is frequently partic-ularly long and with thin, long and parallel macroplacoids. Amongthem are D. scoticum and D. belgicae, belonging to the subgenusAdropion, 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 whichthe male gamete has been studied to date, show a similar mor-phology (Guidi and Rebecchi, 1996; Rebecchi et al., 2000). Thismonophyletic clade should be identified with a redefined subfam-ily Itaquasconinae (see taxonomic account). On the basis ofmolecular and morphological data, the Adropion subgenus is here

amended and raised to genus level (see taxonomic account).Diphascon (A.) sp. 1 (HQ604931) certainly does not have thedrop-like thickening but it does not cluster with the other twoAdropion species considered here. Therefore, our provisional attri-bution of Diphascon (A.) sp. 1 to the genus Adropion should bestudied more in depth. The second clade within the large clustershows two different branches: one includes many species of thesubgenus Diphascon (Diphascon), i.e. with pharyngeal tube anddrop-like thickening (Fig. 11J), and with parallel macroplacoidswithin the pharyngeal bulb, while the second branch includesspecies without a pharyngeal tube (Hypsibius and Borealibius) thatin this case should be considered a synapomorphy of the two gen-era (Fig. 11I). The first branch (monophyletic line) corresponds toa redefined subfamily Diphasconinae (see taxonomic account).Diphascon higginsi is correctly included in this subfamily (andnot in Itaquasconinae), having the drop-like structure eventhough this thickening is flat and not always very visible(Bertolani, 1982; Dastych, 1988; Pilato and Binda, 1988). Thesecond branch can be considered as the redefined subfamilyHypsibiinae (see taxonomic account). Within Hypsibiinae, Hypsibiusand Borealibius appear close from a molecular point of view, butthey clearly differ in the shape of the AISM (Pilato et al., 2006).

From a molecular point of view, a very well supported smallcluster within Hypsibiidae (Fig. 5) is represented by D. (D.) patanei,D. (D.) ramazzottii and D. (D.) nodulosum. These species have a pha-ryngeal tube and drop-like thickening, but differ from the rede-fined Diphasconinae by the shape of the pharyngeal bulb (whichis oval, but more rounded in these three species) and macroplac-oids that are positioned like brackets, each line forming aparenthesis (Fig. 11L). On the basis of our molecular andmorphological analysis of Hypsibiidae, we conclude that the genusDiphascon and the subgenus Diphascon are not monophyletic andthat a new genus, Pilatobius gen. nov., and a new subfamily,Pilatobiinae subfam. nov. must be erected for D. patanei and relatedspecies (see taxonomic account).

The genus Apodibius has been studied by a molecular point ofview very recently (Dabert et al., 2014) and placed within Iso-hypsibiidae family. Instead, we do not have molecular informa-tion on Necopinatum (Necopinatidae). The buccal-pharyngealapparatus of Necopinatum suggests that it could belong to Isoh-ypsibioidea, but to a different evolutionary lineage with respectto Apodibius, lacking Necopinatum the strengthening bar, whichinstead is present in the other genus. Molecular data should clar-ify the situation.

Finally, our results identify new phylogenetic relationshipsamong eutardigrades and further demonstrate that molecularanalysis is a fundamental tool to understand phylogeny and to con-firm (often it is so) or to modify (as sometimes happens in this pa-per) the evolutionary considerations previously inferred bymorphological analysis. The integrative approach applied in thisstudy allowed us to find morphological support (synapomorphies)for the new clusters here identified by molecular data and to findfurther characters to support previous taxa erected on molecularevidence. This kind of approach should also open new perspectivesin the understanding of the evolution of biological features in tar-digrades, such as cryptobiotic phenomena, different reproductivemodes and the biogeographical distribution that characterizesthe species.

5. Taxonomic account

The taxonomic account of the Eutardigrada taxa, including thetaxa emended or newly erected, is presented here (see Table 1).The emendations and definitions follow the conclusions of ourdiscussion.


Class EUTARDIGRADA Richters, 1926Claws with a primary and a secondary branch fused or

positioned one behind the other. Without cephalic filaments(such as cephalic appendages, only papillae can be present).

Order Apochela Schuster, Nelson, Grigarick andChristenberry, 1980

Eutardigrada having claws with completely separatedprimary and secondary branches; papillae around themouth (peribuccal papillae) and 2 lateral papillae on thehead (cephalic papillae); elongated pharyngeal bulbcompletely without placoids.

Family Milnesiidae Ramazzotti, 1962The same characters as Apochela.Composition: Milnesium (type genus), Bergtrollus, Limmenius,

Milnesioides.

Order Parachela Schuster, Nelson, Grigarick andChristenberry, 1980

Eutardigrada with double claws on the legs (or derived fromthem, rarely completely lost) with secondary branchnormally joined to the primary branch; pharyngeal bulbwith placoids (very rarely lost).

Superfamily Eohypsibioidea Bertolani and Kristensen, 1987Double claws of Eohypsibius type (i.e. subdivided into three

distinct sections, one on top of the other), and asymmetricalwith respect to the median plane of the leg (sequence2121), with the ability to rotate on their base; double clawssimilar in size and shape on the same leg; 14 peribuccallamellae; buccal tube completely rigid, or caudallyannulated, in any case with dorsal and ventral crest-shapedapophyses for the insertion of the stylet muscles. Eggs laidfreely and surrounded by an ornamented shell.

Family Eohypsibiidae Bertolani and Kristensen, 1987The same characters as the superfamily.Composition: Eohypsibius (type genus), Austeruseus,

Bertolanius.

Superfamily Macrobiotoidea Thulin, 1928Double claws symmetrical with respect to the median plane

of the leg (sequence 2112); double claw of each leg similarin shape and size; each double claw characterized by thepresence of a peculiar stalk (peduncle) with cylindrical orlaminar shape; 10 peribuccal lamellae or papulae; buccaltube strengthened by a ventral lamina. Eggs laid freely andalways surrounded by an ornamented shell.

Family Macrobiotidae Thulin, 1928Double claws Y-shaped, with the two branches forming an

evident common basal tract of variable length. Buccal tubecompletely rigid, or caudally annulated.

Composition: Macrobiotus (type genus), Adorybiotus,Biserovus, Calcarobiotus, Famelobiotus, Insuetifurca,Minibiotus, Minilentus, Paramacrobiotus, Pseudohexapodibius,Richtersius, Schusterius, Tenuibiotus, Xerobiotus(Pseudodiphascon is considered a genus dubius; Guidetti andPilato 2003).

Remarks: According to our molecular and morphological data,M. furcatus does not belong to Minibiotus and it isprovisionally retransferred to Macrobiotus, pending newdata. The genera Richtersius and Adorybiotus areprovisionally placed in this family following theirtraditional classification, waiting further morphological and

molecular studies to verify their eventual position in theMurrayidae. In this case, the characters identifying thefamily Murrayidae should be emended.

Family Murrayidae Guidetti, Rebecchi and Bertolani, 2000Double claws V-shaped or L-shaped, with the two branches

diverging immediately after a short common basal section;ventral deep indentation on the ventral lamina; buccal tubecompletely rigid; epicuticular layer with pillar-likestructures, sometime visible only at ultrastructural level.

Composition: Murrayon (type genus), Dactylobiotus,Macroversum.

Superfamily Isohypsibioidea Sands, McInnes, Marley,Goodall-Copestake, Convey and Linse, 2008 (emendeddiagnosis)

Double claws asymmetrical with respect to the median planeof the leg (2121), normally with similar shape and size oneach leg; double claws of the Isohypsibius type (secondarybranch of the external claw inserted perpendicularly on theclaw basal tract), or reduced from it: Hexapodibius type(very short, without common basal tract, with a base aslarge as the sum of the primary and secondary branchwidths, and with an evident suture between primary andsecondary branch); Haplomacrobiotus type (one branchonly); completely absent (Apodibius). Buccal tubecompletely rigid (apart Paradiphascon; see below) and oftenrelatively large, without (Dastychius, Eremobiotus,Halobiotus, Isohypsibius, Paradiphascon, Pseudobiotus,Thulinius) or with (Apodibius, Doryphoribius,Haplomacrobiotus, Haplohexapodibius, Hexapodibius,Parhexapodibius) ventral lamina. Eggs with smooth shelllaid within the exuvium.

Family Isohypsibiidae Sands, McInnes, Marley, Goodall-Copestake, Convey and Linse, 2008

The same characters of the superfamily.Composition: Isohypsibius (type genus), Apodibius, Dastychius,

Doryphoribius, Eremobiotus, Halobiotus, Haplohexapodibius,Haplomacrobiotus, Hexapodibius, Paradiphascon,Parhexapodibius, Pseudobiotus, Thulinius.

The type of claws of Ramajendas and Paradiphascon have beenvariously interpreted (Pilato and Binda, 1990; Dastych,1992; Marley et al., 2011). In our opinion and in agreementwith Guil et al. (2013a), due to the type of claws,Paradiphascon should belong to the Isohypsibiidae,although further molecular and morphological studies arerequired to confirm its position. In agreement with thefinding of Guil et al. (2013a), we consider Ramajendas tobelong to the Hypsibioidea. For the claw type characterizedby a disconnected main branch on the external claws, as inthe Hebesuncus claw type, we suggest provisionally to placethe genus in the family Ramazzottidae.

Superfamily Hypsibioidea Pilato, 1969 (emended diagnosis)Double claws asymmetrical with respect to the median plane

of the leg (2121), the external (or posterior) claw often withflexible main branch; double claws different in size andshape on the same leg (Hypsibius and Ramazzottius type, ormodified), or very reduced in size (Calohypsibius andMicrohypsibius type); buccal tube often very narrow.

Family Ramazzottiidae Sands, McInnes, Marley, Goodall-Copestake, Convey and Linse, 2008

Double claws different in shape and size on the same leg, theexternal (or posterior) claw with the primary branch

(continued on next page)


connected to basal tract with an evident, thin, flexible tract(Ramazzottius or oberhaeuseri type, or modified), orcompletely disconnected. Eggs laid freely and withornamented shell.

Composition: Ramazzottius (type genus), Hebesuncus and veryprobably Ramajendas and Thalerius.

Family Hypsibiidae Pilato, 1969 (emended diagnosis)Double claws different in shape and size on the same leg, the

external (or posterior) of Hypsibius type claw with thesecondary branch forming a continuous hook with its basaltract and the primary branch connected to the basal tractwith a flexible part. Septulum present in the pharynx of somegenera. Eggs smooth (but rarely weakly ornamented) laidwithin the exuvium, in some cases eggs ornamented laid free.

Subfamily Hypsibiinae Rudescu, 1964Completely rigid buccal tube. Apophyses for the insertion of

the stylet muscles hook-shaped.Composition: Hypsibius (type genus), Borealibius.

Subfamily Itaquasconinae Pilato, 1969 (emended diagnosis)Buccal tube followed by a flexible portion, without cuticular

thickening between them; the flexible portion is anannulated pharyngeal tube in all genera (Parasconexcluded); pharyngeal bulb very elongated; placoids verylong and in line, sometime reduced to a bulbar lining.

Composition: Itaquascon (type genus), Adropion, Astatumen,Bindius, Mesocrista, Parascon, Platicrista.

Genus Adropion Pilato, 1987 (taxonomic rank raised to genuslevel)

Evident placoids and stylet supports.Composition: Adropion scoticum (Murray, 1905) (type species)

and the species already included in the subgenus Adropion(Guidetti and Bertolani, 2005).

Remarks: From a molecular point of view the genus appearspolyphyletic, but further studies are necessary to clarify thesituation.

Subfamily Diphasconinae Dastych, 1992 (emendeddiagnosis)

Buccal tube followed by an annulated pharyngeal tube, with acuticular thickening between them (often drop-shaped,sometimes barely evident); pharyngeal bulb is an elongatedoval, containing always 3 macroplacoids in a line (andsometimes with a microplacoid and/or septulum).

Composition: Diphascon.

Genus Diphascon Plate, 1888 (emended)The same characters as the subfamily.Composition: Diphascon chilenense Plate, 1889 (type species)

and the species already included in the subgenus Diphascon(Guidetti and Bertolani, 2005), excluding those attributedto Pilatobius gen. nov.

Excluding the species here attributed to Pilatobius gen. nov., allthe species belonging to the previous genus Diphascon but notyet attributed to a subgenus (see Guidetti and Bertolani, 2005)are placed in this taxon, pending further investigations.

Subfamily Pilatobiinae subfam. nov.Buccal tube followed by an annulated pharyngeal tube, with a

drop-like thickening between them; pharyngeal bulbroundish or slightly oval, always containing 2macroplacoids similar in length and in rows that look asparentheses, and a septulum.

Composition: Pilatobius gen. nov.

Genus Pilatobius gen. nov.The same characters as the subfamily.Composition: Pilatobius bullatus (Murray, 1905) (type species),

Pilatobius aculeatus (Maucci, 1951–1952), Pilatobiusbisbullatus (Iharos, 1964), Pilatobius borealis (Biserov, 1996),Pilatobius brevipes (Marcus, 1936), Pilatobius burti (Nelson,1991), Pilatobius elongatus (Mihelcic, 1959), Pilatobiusgerdae (Mihelcic, 1951), Pilatobius granifer (Greven, 1972),Pilatobius halapiense (Iharos, 1964), Pilatobius iltisi (Schusterand Grigarick 1965), Pilatobius latipes (Mihelcic, 1959),Pilatobius nodulosus (Ramazzotti, 1957), Pilatobiusnonbullatus (Mihelcic, 1951), Pilatobius oculatus (Murray,1906), Pilatobius patanei (Binda and Pilato, 1971), Pilatobiusramazzottii (Robotti, 1970), Pilatobius rugosus (Bartoš,1935), Pilatobius secchii (Bertolani and Rebecchi, 1996),Pilatobius sexbullatus (Ito, 1995), Pilatobius trachidorsatus(Bartoš, 1935).

Other species not sufficiently described are placed in this newgenus pending further morphological and/or molecularanalyses: Pilatobius recamieri (Richters, 1911) and Pilatobiusrugocaudatus (Rodriguez Roda, 1952).

Etymology: dedicated to professor Giovanni Pilato, Universityof Catania, Italy.

Remarks: most of the known species with cuticular bars onlegs and (or) with a sculptured cuticle.

Incerta subfamilia Claws of modified Hypsibius type: the twoclaws of the same leg are quite different in size from eachother and the convex curvature of the secondary branchalways forms a continuous arc in both claws.

Genera: Acutuncus, Mixibius.

Family Calohypsibiidae Pilato, 1969 (emended diagnosis)Double claws of the Calohypsibius type (small, rigid, in frontal

view with a base as large as the sum of the primary andsecondary branch widths, but without a suture between thetwo branches); double claws similar in size and shape onthe same leg; buccal tube completely rigid; apophyses forthe insertion of the stylet muscles on the buccal tubeasymmetrical with respect to the frontal plane.

Composition: Calohypsibius.

Family Microhypsibiidae Pilato, 1998Double claws of the Microhypsibius type (small, rigid, with an

evident thin basal tract continuous with the primarybranch); buccal tube completely rigid; double claws similarin size and shape on the same leg; apophyses for theinsertion of the stylet muscles on the buccal tubeasymmetrical with respect to the frontal plane. Pharynxwith a septulum in some species.

Composition: Microhypsibius (type genus), Fractonotus.

Incerta superfamiliaFamily Necopinatidae Ramazzotti and Maucci, 1983Claws absent in each leg of the first pair substituted by a small

cuticular forcep; buccal tube rigid, without ventral lamina.Egg smooth laid within the exuvium.

Composition: Necopinatum.

Acknowledgments

The authors thank Diane R. Nelson, East Tennessee State Uni-versity, Johnson City, Tennessee (U.S.A.) for the English revision


of the manuscript and for helping in collecting specimens, and theanonymous referees for their suggestions. Special thanks to theLaboratory of Sequencing (Labgen), Department of Biomedical Sci-ence, University of Modena and Reggio Emilia (Italy) for thesequencing service. The research is part of the project MoDNA(Morphology and DNA: DNA barcoding and phylogeny of tardi-grades, basic research and applications) supported by FondazioneCassa di Risparmio di Modena (Italy) and the University of Modenaand Reggio Emilia (Italy).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2014.03.006.

References

Aguinaldo, A.M.A., Turbeville, J.M., Linford, L.S., Rivera, M.C., Garey, J.R., Raff, R.A.,Lake, S.A., 1997. Evidence for a clade of nematodes, arthropods and othermoulting animals. Nature 387, 489–493.

Bertolani, R., 1982. Tardigradi (Tardigrada). Guide per il riconoscimento delle specieanimali delle acque interne italiane. Quaderni CNR, AQ/1/168 15, pp. 1–104.

Bertolani, R., Altiero, T., Nelson, D.R., 2009. Tardigrada (Water Bears). In: Likens, G.E.(Ed.), Encyclopedia of Inland Waters 2. Elsevier, Oxford, pp. 443–455.

Bertolani, R., Kristensen, R.M., 1987. New records of Eohypsibius nadjae Kristensen,1982, and revision of the taxonomic position of two genera of Eutardigrada(Tardigrada). In: Bertolani, R. (Ed.), Biology of Tardigrades, Sel. Symp. Monogr.U.Z.I. 1. Mucchi, Modena, Italy, pp. 359–372.

Bertolani, R., Rebecchi, L., 1993. A revision of the Macrobiotus hufelandi group(Tardigrada, Macrobiotidae), with some observations on the taxonomiccharacters of eutardigrades. Zool. Scr. 22, 127–152.

Bertolani, R., Biserov, V.I., 1996. Leg and claw adaptations in soil tardigrades, witherection of two new genera of Eutardigrada, Macrobiotidae: Pseudohexapodibiusand Xerobiotus. Invertebr. Biol. 115, 299–304.

Bertolani, R., Guidetti, R., Rebecchi, L., 1993. Tardigradi dell’Appennino umbro-marchigiano. Biogeographia 17, 223–245.

Bertolani, R., Rebecchi, L., Claxton, S.K., 1996. Phylogenetic significance of egg shellvariation in tardigrades. Zool. J. Linn. Soc. 116, 139–148.

Bertolani, R., Biserov, V., Rebecchi, L., Cerari, M., 2011a. Taxonomy andbiogeography of tardigrades using an integrated approach: new results on thespecies of the Macrobiotus hufelandi group. Inv. Zool. 8, 23–26.

Bertolani, R., Rebecchi, L., Giovannini, I., Cesari, M., 2011b. DNA barcoding andintegrative taxonomy of Macrobiotus hufelandi C.A.S. Schultze 1834, the firsttardigrade species to be described, and some related species. Zootaxa 2997, 19–36.

Binda, M.G., Pilato, G., 1992. Minibiotus furcatus, nuova posizione sistematica perMacrobiotus furcatus, Ehrenberg, 1859 e descrizione di due nuove specie(Eutardigrada). Animalia 19, 111–120.

Campbell, L.I., Rota-Stabelli, O., Edgecombe, G.D., Marchioro, T., Longhorn, S.J.,Telford, M.J., Philippe, H., Rebecchi, L., Peterson, K.J., Pisani, D., 2011. MicroRNAsand phylogenomics resolve the relationships of Tardigrada and suggest thatvelvet worms are the sister group of Arthropoda. PNAS 108, 15920–15924.

Cesari, M., Bertolani, R., Rebecchi, L., Guidetti, R., 2009. DNA barcoding inTardigrada: the first case study on Macrobiotus macrocalix Bertolani &Rebecchi 1993 (Eutardigrada, Macrobiotidae). Mol. Ecol. Res. 9, 699–706.

Cesari, M., Giovannini, I., Bertolani, R., Rebecchi, L., 2011. An example of problemsassociated with DNA barcoding in tardigrades: a novel method for obtainingvoucher specimens. Zootaxa 3104, 42–51.

Cesari, M., Guidetti, R., Rebecchi, L., Giovannini, I., Bertolani, R., 2013. A DNAbarcoding approach in the study of tardigrades. J. Limnol. 72 (s1), 182–198.

Catresana, J., 2000. Selection of conserved blocks from multiple alignments for theiruse in phylogenetic analyses. Mol. Biol. Evol. 17, 540–552.

Dabert, M., Dastych, H., Hohberg, K., Dabert, J., 2014. Phylogenetic position of theenigmatic clawless eutardigrade genus Apodibius Dastych, 1983 (Tardigrada),based on 18S and 28S rRNA sequence data from its type species A. confusus. Mol.Phyl. Evol. 70, 70–75.

Dastych, H., 1988. The Tardigrada of Poland. Pol. Akad. Nauk, Monogr. Fauny Polski16, 1–255.

Dastych, H., 1992. Paradiphascon manningi gen. n. sp. n., a new water-bear fromSouth Africa, with the erecting of a new subfamily Diphasconinae (Tardigrada).Mitt. Hamburg Zool. Mus. Inst. 89, 125–139.

Dastych, H., 2009. Thalerius konradi gen. nov., sp. nov., a new tardigrade from theperiglacial area of the Oetztal Alps, Austria (Tardigrada). Contr. Nat. Hist. 12,391–402.

Dastych, H., Alberti, G., 1990. Redescription of Macrobiotus xerophilus (Dastych,1978) comb. nov., with some phylogenetic notes (Tardigrada, Macrobiotidae).Mitt. Hamburg Zool. Mus. Inst. 87, 157–169.

Degma, P., Bertolani, R., Guidetti, R,. 2013. Actual Checklist of Tardigrada Species.pp. 38. <http://www.tardigrada.modena.unimo.it/miscellanea/Actual%20checklist%20of%20Tardig rada.pdf>. (Ver. 23: 15-07-2013).

Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy andhigh throughput. Nucleic Acids Res. 32, 1792–1797.

Gouy, M., Guindon, S., Gascuel, O., 2010. SeaView version 4: a multiplatformgraphical user interface for sequence alignment and phylogenetic tree building.Mol. Biol. Evol. 27 (2), 221–224.

Guidetti, R., Bertolani, R., 2001. Phylogenetic relationships in the Macrobiotidae(Tardigrada: Eutardigrada: Parachela). Zool. Anz. 240, 371–376.

Guidetti, R., Pilato, G., 2003. Revision of the genus Pseudodiphascon (Tardigrada,Macrobiotidae), with the erection of three new genera. J. Nat. Hist. 37, 1679–1690.

Guidetti, R., Bertolani, R., 2005. Tardigrade taxonomy: an updated check list of thetaxa and a list of characters used in their identification. Zootaxa 845, 1–46.

Guidetti R., R. Bertolani. 2011. Phylum Tardigrada Doyère, 1840. In: Zhang, Z.-Q.(Ed.) Animal Biodiversity: An Outline of Higher-level Classification and Surveyof Taxonomic Richness. Zootaxa, vol. 3148, pp. 96–97.

Guidetti, R., Gandolfi, A., Rossi, V., Bertolani, R., 2005. Phylogenetic analysis inMacrobiotidae (Eutardigrada, Parachela): a combined morphological andmolecular approach. Zool. Scr. 34, 235–244.

Guidetti, R., Schill, R.O., Bertolani, R., Dandekar, T., Wolf, M., 2009. New moleculardata for tardigrade phylogeny, with the erection of Paramacrobiotus gen. n. J.Zool. Syst. Evol. Res. 47, 315–321.

Guidetti, R., Altiero, T., Marchioro, T., Sarzi Amadè, L., Avdonina, A.M., Rebecchi, L.,2012. Form and function of the feeding apparatus in Eutardigrada (Tardigrada).Zoomorphology 131, 127–148.

Guidetti, R., Peluffo, J.R., Rocha, A.M., Cesari, M., Moly de Peluffo, M.C., 2013a. Themorphological and molecular analyses of a new South American urbantardigrade offer new insights on the biological meaning of the Macrobiotushufelandi group of species (Tardigrada: Macrobiotidae). J. Nat. Hist. 47, 2409–2426.

Guidetti, R., Bertolani, R., Rebecchi, L., 2013b. Comparative analysis of the tardigradefeeding apparatus: adaptive convergence and evolutionary pattern of thepiercing stylet system. J. Limnol. 72 (s1), 24–35.

Guidi, A., Rebecchi, L., 1996. Spermatozoan morphology as a character fortardigrade systematics: comparison with sclerified parts of animals and eggsin eutardigrades. Zool. J. Linn. Soc. 116, 101–113.

Guil, N., Guidetti, R., 2005. A new species of Tardigrada (Eutardigrada:Macrobiotidae) from Iberian Peninsula and Canary Islands (Spain). Zootaxa889, 1–11.

Guil, N., Giribet, G., 2012. A comprehensive molecular phylogeny of tardigrades—adding genes and taxa to a poorly resolved phylum-level phylogeny. Cladistics28, 21–49.

Guil, N., Machordom, A., Guidetti, R., 2013a. High level of phenotypic homoplasyamong eutardigrades (Tardigrada) based on morphological and total evidencephylogenetic analyses. Zool. J. Linn. Soc. 169, 1–26.

Guil, N., Jørgensen, A., Giribet, G., Kristensen, R.M., 2013b. Congruence betweenmolecular phylogeny and cuticular design in Echiniscoidea (Tardigrada,Heterotardigrada). Zool. J. Linn. Soc. 169, 713–736.

Gutell, R.R., Lee, J.C., Cannone, J.J., 2002. The accuracy of ribosomal RNA comparativestructure models. Curr. Opin. Struct. Biol. 12 (3), 301–310.

Hickson, R.E., Simon, C., Cooper, A.C., Spicer, G.S., Sullivan, J., Penny, D., 1996.Conserved sequence motifs, alignment, and secondary structure for the thirddomain of animal 12S rRNA. Mol. Biol. Evol. 13, 150–169.

Huelsenbeck, J.P., Ronquist, F., 2001. MrBAYES: Bayesian inference of phylogenetictrees. Bioinformatics 17, 754–755.

International Commission on Zoological Nomenclature 1999. International Code ofZoological Nomenclature, fourth ed. The International Trust for ZoologicalNomenclature, London.

Jørgensen, A., Faurby, S., Hansen, J.G., Møbjerg, N., Kristensen, R.M., 2010. Molecularphylogeny of Arthrotardigrada (Tardigrada). Mol. Phyl. Evol. 54, 1006–1015.

Jørgensen, A., Kristensen, R.M., 2004. Molecular phylogeny of Tardigrada –investigation of the monophyly of Heterotardigrada. Mol. Phyl. Evol. 32,666–670.

Jørgensen, A., Møbjerg, N., Kristensen, R.M., 2011. Phylogeny and evolution of theEchiniscidae (Echiniscoidea, Tardigrada) – an investigation of the congruencebetween molecules and morphology. J. Zool. Syst. Evol. Res. 49, 6–16.

Kiehl, E.T., Dastych, H., D’Haese, J., Greven, H., 2007. The 18S rRNA sequencessupport polyphyly of the Hypsibiidae (Eutardigrada). J. Limnol. 66 (Suppl. 1),21–25.

Kristensen, R.M., 1982. New aberrant eutardigrades from homothermic springs onDisko Island, West Greenland. In: Nelson, D.R. (Ed.), Proceedings of the ThirdInternational Symposium on Tardigrada. East Tennessee State University Press,Johnson City, Tennessee, USA, pp. 203–220.

Lartillot, N., Philippe, H., 2004. A Bayesian mixture model for across-siteheterogeneities in the amino-acid replacement process. Mol. Biol. Evol. 21 (6),1095–1109.

Marchioro, T., Rebecchi, L., Cesari, M., Hansen, J.G., Viotti, G., Guidetti, R., 2013.Somatic musculature of Tardigrada: phylogenetic signal and metamericpatterns. Zool. J. Linn. Soc. 169, 580–603.

Marley, N.J., McInnes, S.J., Sands, C.J., 2011. Phylum Tardigrada: a re-evaluation ofthe Parachela. Zootaxa 2819, 51–64.

Møbjerg, N., Jørgensen, A., Eibye-Jacobsen, J., Halberg, K.A., Persson, D., Kristensen,R.M., 2007. New records on cyclomorphosis in the marine eutardigradeHalobiotus crispae (Eutardigrada: Hypsibiidae). J. Limnol. 66 (Suppl. 1), 132–140.



http://refhub.elsevier.com/S1055-7903(14)00098-0/h0005






















































http://www.tardigrada.modena.unimo.it/miscellanea/Actual%20checklist%20of%20Tardig%20rada.pdf

http://www.tardigrada.modena.unimo.it/miscellanea/Actual%20checklist%20of%20Tardig%20rada.pdf















































































Nelson, D., Guidetti, R., Rebecchi, L., 2010. Tardigrada. In: Thorp, J.H., Covich, A.P.(Eds.), Ecology and Classification of North American Freshwater Invertebrates,third ed. Academic Press (Elsevier), San Diego.

Nichols, P.B., Nelson, D.R., Garey, J.R., 2006. A family level analysis of tardigradephylogeny. Hydrobiology 558, 53–60.

Nielsen, C., 2012. Animal Evolution: Interrelationships of the Living Phyla, third ed.Oxford University Press.

Pagel, M., Meade, A., 2004. A phylogenetic mixture model for detecting pattern-heterogeneity in gene sequence or character-state data. Syst. Biol. 53, 571–581.

Pilato, G., 1969. Evoluzione e nuova sistemazione degli Eutardigrada. Boll. Zool. 36,327–345.

Pilato, G., 1969b. Schema per una nuova sistemazione delle famiglie e dei generidegli Eutardigrada. Boll. Accad. Gioenia Sci. Nat., Catania, Ser. 10, 181–193.

Pilato, G., 1989. Phylogenesis and systematic arrangement of the familyCalohypsibiidae Pilato, 1969 (Eutardigrada). Z. Zool. Syst. Evolutionsforsch. 27,8–13.

Pilato, G., 1992. Mixibius, nuovo genere di Hypsibiidae (Eutardigrada). Animalia 19,121–125.

Pilato, G., 1998. Microhypsibiidae, new family of eutardigrades, and description ofthe new genus Fractonotus. Spixiana 21, 129–134.

Pilato, G., Binda, M.G., 1987. Richtersia, nuovo genere di Macrobiotidae e nuovadefinizione di Adorybiotus Maucci e Ramazzotti, 1981 (Eutardigrada). Animalia14, 147–152.

Pilato, G., Binda, M.G., 1988. Precisazioni e rettifiche alla descrizione di alcunespecie di Tardigradi. Terza nota. Animalia 15, 45–55.

Pilato, G., Binda, M.G., 1990. Tardigradi dell’Antartide. I. Ramajendas, nuovo generedi Eutardigrado. Nuova posizione sistematica di Hypsibius remaudi Ramazzotti,1972 e descrizione di Ramajendas frigidus n. sp. Animalia 17, 61–71.

Pilato, G., Binda, M.G., 1997. Acutuncus, a new genus of Hypsibiidae (Eutardigrada).Entomol. Mitt. Zool. Mus., HamG. 12, 159–162.

Pilato, G., Binda, M.G., 2010. Definition of families, subfamilies, genera andsubgenera of the Eutardigrada, and keys to their identification. Zootaxa 2404,1–54.

Pilato, G., Binda, M.G., Lisi, O., 2002. Notes on tardigrades of the Seychelles with thedescription of two new species. Boll. Accad. Gioenia Sci. Nat., Catania 35, 503–517.

Pilato, G., Binda, M.G., Lisi, O., 2003. Remarks on some species of tardigrades fromSouth America with the description of Minibiotus sidereus n. sp. Zootaxa 195, 1–8.

Pilato, G., Guidetti, R., Rebecchi, L., Lisi, O., Hansen, J.G., Bertolani, R., 2006.Geonemy, ecology, reproductive biology and morphology of the tardigradeHypsibius zetlandicus (Eutardigrada: Hypsibiidae) with erection of Borealibiusgen. n. Polar Biol. 29, 595–603.

Pleijel, F., Jondelius, U., Norlinder, E., Nygren, A., Oxelman, B., Schander, C.,Sundberg, P., Thollesson, M., 2008. Phylogenies without roots? A plea for theuse of vouchers in molecular phylogenetic studies. Mol. Phyl. Evol. 48, 369–371.

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 usingMulti-core, Cell/BE, and GPU systems: accelerating the Phylogenetic LikelihoodFunction. In: Proceedings of ICPP, Vienna, Austria, September 2009.

Rambaut, A., Drummond, A.J., 2007. Tracer v1.5. <http://beast.bio.ed.ac.uk/Tracer>.Ramazzotti, G., Maucci, W., 1983. II Phylum Tardigrada. III Edizione riveduta e

aggiornata. Mem. Ist. Iatl. Idrobiol. 41, 1–1012.Rebecchi, L., 2001. The spermatozoon in tardigrades: evolution and relationships

with the environment. Zool. Anz. 240, 525–533.Rebecchi, L., Altiero, T., Guidi, A., 2011. The ultrastructure of the tardigrade

spermatozoon: a comparison between Paramacrobiotus and Macrobiotus species(Eutardigrada, Macrobiotidae). Invert. Zool. 8 (1), 63–67.

Rebecchi, L., Bertolani, R., 1999. Spermatozoon morphology of three species ofHypsibiidae (Tardigrada, Eutardigrada) and phylogenetic evaluation. Zool. Anz.238, 319–328.

Rebecchi, L., Guidi, A., 1991. First SEM studies on tardigrade spermatozoa. Invertebr.Reprod. Dev. 19, 151–156.

Rebecchi, L., Guidi, A., Bertolani, R., 2000. Tardigrada. In: Jamieson, B.G.M. (Ed.),Progress in Male Gamete Biology. Series edited by Adiyodi, K.G., Adiyodi, R.G.,IX, Part B. Oxford and IBH, New Delhi, pp. 267–291.

Rebecchi, L., Guidi, A., Bertolani, R., 2003. The spermatozoon of the Echiniscidae(Tardigrada, Heterotardigrada) and its phylogenetic significance.Zoomorphology 122, 3–9.

Rota-Stabelli, O., Kayal, E., Gleeson, D., Daub, J., Boore, J., Telford, M., Pisani, D.,Blaxter, M., Lavrov, D., 2010. Ecdysozoan mitogenomics: evidence for a commonorigin of the legged invertebrates, the Panarthropoda. Genome Biol. Evol. 2,425–440.

Sands, C.J., McInnes, S.J., Marley, N.J., Goodall-Copestake, W.P., Convey, P., Linse, K.,2008. Phylum Tardigrada: an ‘‘individual’’ approach. Cladistics 24, 1–11.

Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogeneticanalyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–2690.

Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for theRaxML web servers. Syst. Biol. 57, 758–771.

Tamura, K., Peterson, D., Peterson, N., Stechler, G., Nei, M., Kumar, S., 2011. MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28(10), 2731–2739.

Thulin, G., 1928. Über die Phylogenie und das system der Tardigraden. Hereditas 11,207–266.

Wolburg-Buchholz, K., Greven, H., 1979. On the fine structure of the spermatozoonof Isohypsibius granulifer Thulin 1928 (Eutardigrada) with reference to itsdifferentiation. Zesz. Nauk. Uniw. Jagiellon. Prace Zool. 25, 191–197.

Further reading

Bertolani, R., Marley, N.J., Nelson, D.R., 1999. Re-description of the genus Thulinia(Eutardigrada: Hypsibiidae) and of Thulinia augusti (Murray, 1907) comb. nov.Zool. Anz. 238, 139–145.

















































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Phylogeny of Eutardigrada: New molecular data and their morphological support lead to the identification of new evolutionary lineages

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