Zoologica Scripta Phylogeny of Chaetonotidae and other Paucitubulatina (Gastrotricha: Chaetonotida) and the colonization of aquatic ecosystems TOBIAS KÅNNEBY, M. ANTONIO TODARO & ULF JONDELIUS Submitted: 22 March 2012 Accepted: 25 June 2012 doi:10.1111/j.1463-6409.2012.00558.x Kånneby, T., Todaro, M. A., Jondelius, U. (2012). Phylogeny of Chaetonotidae and other Paucitubulatina (Gastrotricha: Chaetonotida) and the colonization of aquatic ecosystems. —Zoologica Scripta, 00, 000–000. Chaetonotidae is the largest family within Gastrotricha with almost 400 nominal species represented in both freshwater and marine habitats. The group is probably non-monophyletic and suffers from a troubled taxonomy. Current classification is to a great extent based on shape and distribution of cuticular structures, characters that are highly variable. We present the most densely sampled molecular study so far where 17 of the 31 genera belonging to Chaetonotida are represented. Bayesian and maximum likelihood approaches based on 18S rDNA, 28S rDNA and COI mtDNA are used to reconstruct relationships within Chaetonotidae. The use of cuticular structures for supra-specific classification within the group is evaluated and the question of dispersal between marine and freshwater habitats is addressed. Moreover, the subgeneric classification of Chaetonotus is tested in a phylogenetic context. Our results show high support for a clade containing Dasydytidae nested within Chaetonotidae. Within this clade, only three genera are monophyletic following current classification. Genera containing both marine and freshwater species never form monophyletic clades and group with other species according to habitat. Marine members of Aspidiophorus appear to be the sister group of all other Chaetonotidae and Dasydytidae, indicating a marine origin of the clade. Halichaetonotus and marine Heterolepidoderma form a monophyletic group in a sister group relationship to freshwater species, pointing towards a secondary invasion of marine environments of these taxa. Our study highlights the problems of current classification based on cuticular structures, characters that show homoplasy for deeper relationships. Corresponding author: Tobias Kånneby, Department of Invertebrate Zoology, Swedish Museum of Natural History, PO Box: 50007, SE-104 05 Stockholm, Sweden. E-mail: tobias.kanneby@nrm.se M. Antonio Todaro, Department of Life Sciences, University of Modena and Reggio Emilia, via Campi, 213 ⁄ d, I-41100 Modena, Italy. E-mail: antonio.todaro@unimore.it Ulf Jondelius, Department of Invertebrate Zoology, Swedish Museum of Natural History, PO Box: 50007, SE-104 05 Stockholm, Sweden. E-mail: ulf.jondelius@nrm.se Introduction Gastrotricha is a phylum of aquatic invertebrates with approximately 775 species. Gastrotrichs are very small, ranging from 70 lm up to 3.5 mm in total body length. Like many other meiofaunal groups (e.g. rotifers and tardigrades) they have a cosmopolitan distribution (Artois et al. 2011). The group is common, can be found in high densities and plays an important role in the meiofauna community as links between the microbial loop and larger invertebrate predators (Todaro & Hummon 2008). Gastrotrichs are considered a monophyletic group (but see Manylov et al. 2004) and are morphologically character- ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters ized by: (i), a multilayered cuticle; (ii) locomotory and sensory cilia covered by epicuticle; (iii), outlet of adhesive duo-gland system covered by cuticle (except Neodasys, but see also Hochberg & Litvaitis (2000)); and (iv), helicoidal muscles surrounding the alimentary canal (Hochberg & Litvaitis 2001; Todaro et al. 2006). The cuticle can be elaborately sculptured into various arrangements of scales and ⁄ or spines, but can also lack these structures altogether. Gastrotrichs are currently classified as Platyzoa within Lophotrochozoa (Todaro et al. 2006; Dunn et al. 2008; Hejnol et al. 2009) and are in turn divided into the two 1 Phylogeny of Chaetonotidae (Gastrotricha) d T. Kånneby et al. orders Chaetonotida and Macrodasyida. Chaetonotida is divided into the two suborders Multitubulatina and Paucitubulatina (d’Hondt 1971). The former contains a single genus Neodasys, whereas the latter contains the rest of Chaetonotida. Neodasys is strictly marine and shares characters with Macrodasyida (e.g. vermiform shape and lateral adhesive tubes), but because of the organization of the pharynx it has been classified as a member of Chaetonotida (Remane 1961). Recent studies based on 18S rDNA have found Neodasys to be nested within Macrodasyida (Todaro et al. 2006; Petrov et al. 2007) while others have proposed it as the sister group of the Paucitubulatina based on morphology (Zrzavý 2003). Paucitubulatina has been found to be monophyletic based on morphology and molecular data (Hochberg & Litvaitis 2000; Todaro et al. 2003, 2006; Zrzavý 2003; Manylov et al. 2004; Petrov et al. 2007) and is divided into seven families of which Xenotrichulidae and Muselliferidae are strictly marine and hypothesized to have retained plesiomorphic character states within Paucitubulatina (Hochberg & Litvaitis 2000; Kieneke et al. 2008; Leasi & Todaro 2008). Dasydytidae, Dichaeturidae, Neogosseidae and Proichthydidae are strictly freshwater. The seventh and largest family within Chaetonotida, Chaetonotidae, contains almost 400 nominal species and can be found in both marine and freshwater environments (Todaro & Hummon 2008; Balsamo et al. 2009; Hummon & Todaro 2010). The group suffers from a troubled taxonomy and the interrelationships are poorly understood. It is most likely that the group is a non-monophyletic assemblage (Hochberg & Litvaitis 2000; Zrzavý 2003; Todaro et al. 2006; Kieneke et al. 2008; Kånneby et al. 2011). So far phylogenetic studies based on morphological character matrices or molecular data dealing fully or in part with Chaetonotidae are scarce. Molecular studies are usually poorly sampled and are based on a single gene. Generic relationships in Chaetonotidae are even more obscure and many of the long established genera are probably nonmonophyletic (Kånneby et al. 2011). None of the current genera have been established based on phylogenetic analyses and generic classification relies heavily on shape and distribution of cuticular structures, which have been shown to vary even among isogenic clones (Amato & Weiss 1982). In addition, Chaetonotidae contains both marine and freshwater species. Some genera (e.g. Aspidiophorus, Chaetonotus and Heterolepidoderma) even have representatives in both environments. Gastrotricha are generally regarded as primarily marine but the diversification from marine to freshwater environments is not fully understood (Todaro et al. 2012). Members of Xenotrichulidae, the hypothesized sister taxon of Chaetonotidae, are marine but also known to thrive in brackish environments 2 (Hummon 2008). Chaetonotidan gastrotrichs have successfully colonized many different freshwater habitats but whether invasion to freshwater happened only once or repeatedly remains an open question. Hochberg & Litvaitis (2000) used an 81 character matrix to analyse relationships of genera within Chaetonotidae and found it non-monophyletic. Kieneke et al. (2008) proposed a new classification of the deeper relationships of Gastrotricha based on a similar approach with an extended character matrix of 135 characters dealing with species of all the known genera. Their study also indicated a non-monophyletic Chaetonotidae, although with low statistical support at most nodes. After comprehensive studies based on morphology, Kisielewski (1991) speculated on the relationships within Chaetonotidae. He regarded the homogeneity of dorsal and ventral scales together with spines, characters present in Lepidochaetus and Polymerurus, as plesiomorphic character states. Recently an early divergence of Polymerurus based on plesiomorphic character states of the muscular system has been proposed (Leasi et al. 2006; Leasi & Todaro 2008). Previous molecular studies were mostly based on the 18S rDNA gene and not aimed at resolving phylogenetic relationships within Paucitubulatina; consequently, major taxa are not well sampled including the numerically dominant Chaetonotidae. For example, Todaro et al. (2006) performed one of the most densely sampled studies on Gastrotricha, but none of the groups previously considered to be nesting within Chaetonotidae (e.g. Dasydytidae and Neogosseidae), based on phylogenies from morphological character matrices were included in the analysis. Kånneby et al. (2011) performed the first multi-gene approach and found members of Dasydytidae nested within Chaetonotidae. Very few molecular studies are supported by sufficient sampling to evaluate monophyly of individual genera within Paucitubulatina. Todaro et al. (2006) indicated a non-monophyletic Chaetonotus, a group with more than 200 species defined by the presence of spined scales. Chaetonotus has been scrutinized before and Kisielewski (1997) proposed a subgeneric classification of the group based on distribution of scales and spines as well as their morphology. Dealing with species delimitation in freshwater Gastrotricha Kånneby et al. (2011) presented a preliminary phylogeny of Chaetonotidae and Dasydytidae, in which only three genera, Lepidochaetus, Polymerurus and Stylochaeta, of six sampled genera were monophyletic, thus indicating the need for taxonomic revisions within the group. A phylogenetic study based on extensive sampling of species and several molecular markers will help understand the phylogenetic relationships within and between the taxa currently referred to as Chaetonotidae and other Paucitubulatina, and will form a solid foundation for future ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters T. Kånneby et al. studies dealing with gastrotrich evolution. In this study, we aim to: (i), give a reliable hypothesis on relationships within the highly diversified Paucitubulatina based on Bayesian as well as maximum likelihood approaches of 18S rDNA, 28S rDNA and COI mtDNA and test of prior morphological hypotheses with the approximately unbiased test (AU-test); (ii), investigate the debated relationship between Chaetonotidae and Dasydytidae; (iii), explore the invasion of different aquatic ecosystems (e.g. marine vs. freshwater) chaetonotid gastrotrichs; and (iv), test the current subgeneric classification of Chaetonotus (see Schwank 1990; Kisielewski 1997; Weiss 2001). B A F Phylogeny of Chaetonotidae (Gastrotricha) Material and methods Collection and documentation We present the most densely sampled phylogeny for Paucitubulatina where 17 of the 31 recognized genera within Chaetonotida are sampled. Chaetonotidae (Fig. 1) is the most densely sampled family with 54 species, while Dasydytidae (Fig. 2) and Xenotrichulidae (Fig. 3) are represented by 7 and 6 species, respectively. Two species of Neodasys, Neodasys chaetonotoideus and Neodasys uchidai (Multitubulatina) were also sampled and used as outgroups. In total, 123 terminals are included in the analysis. The rarer Dichaeturidae, Neogosseidae, Muselliferidae, D C G d H E I Fig. 1. Gastrotrichs of the family Chaetonotidae. —A. Arenotus strixinoi, lateral view. —B. Aspidiophorus ophiodermus, habitus. —C. Aspidiophorus paramediterraneus, habitus. —D. Chaetonotus (Primochaetus) heteracanthus, dorsal view. —E. Chaetonotus (Wolterecka) uncinus, dorsal view. —F. Halichaetonotus paradoxus, dorsal view. —G. Ichthydium squamigerum, habitus. —H. Lepidochaetus zelinkai, dorsal view. —I. Polymerurus nodicaudus, habitus. Scale bars: A–E, 50 lm; F and G, 20 lm; H, 40 lm; I, 100 lm. ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters 3 Phylogeny of Chaetonotidae (Gastrotricha) A d T. Kånneby et al. B C D E F Fig. 2. Gastrotrichs of the family Dasydytidae. —A. Dasydytes carvalhoae, anterior ventral view. —B. Dasydytes papaveroi, habitus. —C. Haltidytes crassus, dorsal view. —D. Haltidytes squamosus, dorsal view. —E. Ornamentula paraënsis, dorsal view of anterior portion. —F. Stylochaeta scirtetica, habitus. Scale bars: A, C, D, E, 20 lm; B, 25 lm; F, 40 lm. Proichthydidae and Undulinae are not included in our phylogeny because of lack of material. Freshwater specimens were collected by means of a plankton net with a mesh size of 25 lm or by scooping up benthic material or plants by hand. Collection in marine environments was done in the littoral or sub-littoral with a 0.5 l plastic jar. Freshwater samples were kept in aerated aquaria while marine samples were kept in a refrigerator. Subsamples were treated with a 1–7% MgCl2-solution depending on original salinity (higher concentration for higher original salinity) and subsequently studied under a dissecting microscope. Individual gastrotrichs were picked out with a micropipette, mounted on a slide and documented alive with a Nikon Eclipse 80i DIC microscope equipped with a Nikon Digital Sight DS-Fi1 digital camera. For molecular studies, specimens were recovered from the slide and put in 95% EtOH and stored at )18º to 20C until further treatment. A full list of specimens can be found in Table 1. Many of the specimens used in this study have been photographed and the photos have been deposited as collection 799280 in Morphbank (http:// www.morphbank.net/799280) (Table 1). 4 DNA extraction, amplification of 18S rDNA, 28S rDNA and COI mtDNA and sequencing follow the protocols presented in Todaro et al. (2011) and Kånneby et al. (2011). Alignment To detect conflicts in the phylograms and arrive at a strongly corroborated phylogenetic hypothesis two sets of the nuclear genes (18S rDNA and 28S rDNA) were aligned separately. The first set was aligned with Muscle v3.6 (Edgar 2004) and the second with Mafft (online version) (Katoh et al. 2002, 2005). For Mafft, the Q-INS-i strategy, taking the secondary structure of rDNA into account, was used. COI mtDNA was aligned according to amino acids using the general invertebrate mitochondrial code and then back-translated to nucleotides using Translator X with Muscle (Abascal et al. 2010). This ensures that gaps in the alignment correspond to the translated amino acid sequence. Ambiguously aligned positions for the 18S rDNA and 28S rDNA alignments were filtered using Aliscore v.2 with default settings and treating gaps as missing data (Misof & Misof 2009). ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters T. Kånneby et al. A B d Phylogeny of Chaetonotidae (Gastrotricha) C Fig. 3. Gastrotrichs of the family Xenotrichulidae. —A. Draculiciteria tesselata, dorsal view; —B. Heteroxenotrichula squamosa, ventral view; —C. Xenotrichula velox, ventral view. Scale bars: A, B, 25 lm; C, 50 lm. For individual genes, the combined nuclear genes (18S rDNA and 28S rDNA) and the combined data set (18S rDNA, 28S rDNA and COI mtDNA) Neodasys was used as outgroup. For the 28S rDNA alignment, no sequences for Neodasys had been obtained, and hence, Xenotrichulidae was used as outgroup. Models of nucleotide substitution were tested for each data set with jModelTest0.1 (Guindon & Gascuel 2003; Posada 2008) under the Akaike Information Criterion (AIC). Phylogenetic analyses All data sets were analysed with MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003; Huelsenbeck & Ronquist 2005) using the freely available Oslo University Bioportal Cluster (http://www.bioportal.uio.no) (see Kumar et al. 2009) under the six parameter general time reversible (GTR) model with estimated proportion of invariable sites and gamma distributed rate variation across sites. All analyses were run for 40 million generations with default settings of priors. Convergence was ascertained by checking the log likelihood graphs, the average standard deviation of split frequencies and the potential scale reduction factor (PSRF). After a burnin of 10 million generations, chains were sampled every 1000th generation. As chain mixing was very low under the default settings (four MCMC chains for each run and a heating parameter of 0.2) the combined data set was analysed using eight MCMC chains in each run with a heating parameter of 0.1 to ensure sufficient mixing and a reliable sample from the posterior ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters distribution. Consensus trees were built from two independent runs of 30 000 samples each for all Bayesian analyses. Maximum likelihood analyses using RaxML v7.0.4 (Stamatakis 2006) with 1000 bootstrap replicates were run on the combined data set to check for congruence with the Bayesian analysis. These analyses were run for both Muscle and Mafft alignments under the GTR+I+G model of nucleotide substitution with Neodasys as outgroup. Moreover, to test for congruence between phylogenetic hypotheses based on molecular vs. morphological characters the tree-topology obtained from the combined data set based on the Mafft alignment and Bayesian analysis was constrained to the topology presented by Hochberg & Litvaitis (2000), which is based on a morphological data matrix. This constrained topology was analysed by maximum likelihood using RaxML v.7.0.4 with 100 bootstrap replicates and subsequently tested against the topology presented in Fig. 4, using the approximately unbiased (AU) test (Shimodaira 2002) implemented in Tree-Puzzle v5.2 (Schmidt et al. 2002) and Consel v0.1i (Shimodaira & Hasegava 2001). Results Alignment and phylogenetic analyses The original combined data set aligned with Mafft consisted of 6043 and 5972 nucleotide positions for the outgroups Neodasys and Xenotrichulidae, respectively. The corresponding numbers for the data sets aligned with 5 Phylogeny of Chaetonotidae (Gastrotricha) d T. Kånneby et al. Table 1 Taxa used for phylogenetic analyses in this study as well as their sampling locations and GenBank accession numbers GenBank Accession number Taxon (Morphbank Id.) Suborder Paucitubulatina d’Hondt, 1971 Family Chaetonotidae Gosse, 1864 Genus Arenotus Kisielewski, 1987 Arenotus strixinoi Kisielewski, 1987 (791776) Genus Aspidiophorus (Voigt, 1902) Aspidiophorus ophiodermus Balsamo, 1983 (791777–791778) Aspidiophorus paramediterraneus Hummon, 1974 (791779) Aspidiophorus polystictos Balsamo & Todaro, 1987 Aspidiophorus polystictos Balsamo & Todaro, 1987 Aspidiophorus tentaculatus Wilke, 1954 (791780) Aspidiophorus tentaculatus Wilke, 1954 (791780) Aspidiophorus tetrachaetus Kisielewski, 1986 (791781) Aspidiophorus sp. 3 Genus Chaetonotus (Chaetonotus) Ehrenberg, 1830 Chaetonotus daphnes Balsamo & Todaro, 1995 (791782–791784) Chaetonotus daphnes Balsamo & Todaro, 1995 (791785–791787) Chaetonotus cf. laroides Marcolongo, 1910 (791812–791813) Chaetonotus cf. laroides Marcolongo, 1910 (791788–791789) Chaetonotus laroides Marcolongo, 1910 (791790–791792) Chaetonotus cf. maximus Ehrenberg, 1838 (791793) Chaetonotus cf. maximus Ehrenberg, 1838 (791794–791795) Chaetonotus microchaetus Preobrajenskaja, 1926 (791796) Chaetonotus microchaetus Preobrajenskaja, 1926 (791797) Chaetonotus microchaetus Preobrajenskaja, 1926 (791798–791799) Chaetonotus microchaetus Preobrajenskaja, 1926 (791800) Chaetonotus microchaetus Preobrajenskaja, 1926 (791801) Chaetonotus cf. oculifer Kisielewski, 1981 (791802) Chaetonotus cf. oculifer Kisielewski, 1981 (791803) Chaetonotus polyspinosus Greuter, 1917 (791804–791805) Chaetonotus polyspinosus Greuter, 1917 (791806–791807) 6 Sampling location Coordinates 18S 28S COI Represa do Lobo-Broa, Brazil S 22º 11¢10"; W 47º 54¢ 02" JQ798537 JQ798608 JQ798677 Fiskebäckskil, Sweden N 58º 14¢ 39"; E 11º 27¢ 16" JN185463 JN185510 Na Ilha Bela, Brazil S 23º 50¢ 30"; W 45º 24¢ 14" JQ798538 JQ798609 JQ798678 Mediterranean Sea Na JQ798597 JQ798664 JQ798726 Mediterranean Sea Na JQ798598 JQ798665 JQ798727 Hawksnest Bay, US Virgin Islands Hawksnest Bay, US Virgin Islands Fiskebäckskil, Sweden N 18º 20¢ 46"; W 64º 46¢ 49" JQ798553 JQ798625 JQ798690 N 18º 20¢ 46"; W 64º 46¢ 49" JQ798591 JQ798659 JQ798721 N 58º 14¢ 39"; E 11º 27¢ 16" JN185505 JN185540 JN185576 Little Lameshur Bay, US Virgin Islands N 18º 19¢ 11"; W 64º 43¢ 34" JQ798559 JQ798629 JQ798694 Skarvesäter, Sweden N 58º 14¢ 23"; E 11º 22¢ 01" JQ798545 JQ798617 JQ798683 Skarvesäter, Sweden N 58º 14¢ 23"; E 11º 22¢ 01" JQ798549 JQ798621 JQ798687 Abisko, Sweden N 68º 21¢ 14"; E 18º 48¢ 90" JQ798579 Na JQ798711 Saltö, Sweden N 58º 52¢ 21"; E 11º 07¢ 34" JQ798602 JQ798669 JQ798731 Abisko, Sweden N 68º 21¢ 14"; E 18º 48¢ 90" JQ798580 Na JQ798712 Abisko, Sweden N 68º 25¢ 58"; E 18º 23¢ 00" JQ798574 JQ798646 JQ798706 Abisko, Sweden N 68º 21¢ 23"; E 18º 47¢ 59" JQ798577 Na JQ798709 Askö, Sweden N 58º 49¢ 23"; E 17º 38¢ 32" JQ798605 JQ798672 JQ798734 Skarvesäter, Sweden N 58º 14¢ 23"; E 11º 22¢ 01" JQ798546 JQ798618 JQ798684 Abisko, Sweden N 68º 21¢ 18"; E 18º 48¢ 40" JQ798575 Na JQ798707 Abisko, Sweden N 68º 26¢ 04"; E 18º 14¢ 53" JQ798582 JQ798650 JQ798713 Runmarö, Sweden N 59º 17¢ 22"; E 18º 47¢ 56" JQ798583 JQ798651 JQ798714 Fiskebäckskil, Sweden N 58º 14¢ 39"; E 11º 27¢ 16" JQ798576 JQ798647 JQ798708 Abisko, Sweden N 68º 25¢ 58"; E 18º 23¢ 00" JQ798581 JQ798649 Na Cotejuba, Brazil S 01º 16¢ 01"; W 48º 33¢ 45" JQ798563 JQ798633 JQ798698 Lago Bolonha, Belem, Brazil S 01º 25¢ 31"; W 48º 25¢ 55" JQ798586 JQ798654 JQ798717 ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters T. Kånneby et al. d Phylogeny of Chaetonotidae (Gastrotricha) Table 1 Continued GenBank Accession number Taxon (Morphbank Id.) Sampling location Coordinates 18S 28S COI Chaetonotus similis Zelinka, 1889 (791808–791809) Chaetonotus cf. similis Zelinka, 1889 (791810–791811) Chaetonotus cf. similis Zelinka, 1889 (791814–791816) Chaetonotus cf. sphagnophilus Kisielewski, 1981 Chaetonotus sp. 1 (791817–791818) Genus Chaetonotus (Hystricochaetonotus) Schwank, 1990 Chaetonotus aemilianus Balsamo, 1978 (791819) Chaetonotus hystrix Metschnikoff, 1865 (791820) Chaetonotus cf. hystrix Metschnikoff, 1865 (791821) Chaetonotus cf. novenarius Greuter, 1917 (791822–791823) Genus Chaetonotus (Marinochaetus) Kisielewski, 1997 Chaetonotus mariae Todaro, 1992 (791824) Genus Chaetonotus (Primochaetus) Kisielewski, 1997 Chaetonotus acanthocephalus Valkanov, 1937 (791825–791826) Chaetonotus acanthodes Stokes, 1887 (791827) Chaetonotus acanthodes Stokes, 1887 (791828–791830) Chaetonotus acanthodes Stokes, 1887 (791831) Chaetonotus armatus Kisielewski, 1981 (791832–791835) Chaetonotus heideri Brehm, 1917 (791836–791837) Chaetonotus heideri Brehm, 1917 (791838–791839) Chaetonotus heteracanthus Remane, 1927 (791840–791842) Chaetonotus sp. 2 (791843) Genus Chaetonotus (Schizochaetonotus) Schwank, 1990 Chaetonotus cf. dispar Wilke, 1954 (791844–791846) Chaetonotus neptuni Wilke, 1954 Chaetonotus neptuni Wilke, 1954 Chaetonotus schultzei Metschnikoff, 1865 Genus Chaetonotus (Wolterecka) Mola, 1932 Chaetonotus uncinus Voigt, 1902 (791847) Abisko, Sweden N 68º 20¢ 92"; E 19º 02¢ 21" JQ798578 JQ798648 JQ798710 Skarvesäter, Sweden N 58º 14¢ 23"; E 11º 22¢ 01" JQ798548 JQ798620 JQ798686 Concordia Pond, US Virgin Islands Saltö, Sweden N 18º 18¢ 46"; W 64º 42¢ 24" JQ798592 JQ798660 JQ798722 N 58º 52¢ 21"; E 11º 07¢ 34" JQ798604 JQ798671 JQ798733 Saltö, Sweden N 58º 52¢ 21"; E 11º 07¢ 34" JQ798601 JQ798668 JQ798730 Torneträsk, Sweden N 68º 21¢ 19"; E 18º 49¢ 21" JQ798556 JQ798626 JQ798693 Torneträsk, Sweden N 68º 21¢ 19"; E 18º 49¢ 21" JQ798557 JQ798627 Na Saltö, Sweden N 58º 52¢ 21"; E 11º 07¢ 34" JQ798603 JQ798670 JQ798732 Lago Preta, Belem, Brazil S 01º 25¢ 46"; W 48º 24¢ 00" JQ798566 JQ798636 JQ798699 Little Lameshur Bay, US Virgin Islands N 18º 19¢ 11"; W 64º 43¢ 34" JQ798558 JQ798628 Na Nova Ipixuna, Brazil S 04º 50¢ 38"; W 49º 06¢ 48" JQ798569 Na JQ798701 Fiskebäckskil, Sweden N 58º 14¢ 39"; E 11º 27¢ 16" JQ798544 JQ798616 JQ798682 Askö, Sweden N 58º 49¢ 23"; E 17º 38¢ 32" JQ798552 JQ798624 Na Hållö, Sweden N 58º 20¢ 00"; E 11º 12¢ 50" JQ798585 JQ798653 JQ798716 Lake Ånnsjön, Sweden N 63º 15¢ 35"; E 12º 26¢ 51" JQ798594 Na JQ798723 Fiskebäckskil, Sweden N 58º 14¢ 39"; E 11º 27¢ 16" JQ798547 JQ798619 JQ798685 Belém, Brazil S 01º 27¢ 14"; W 48º 28¢ 36" JQ798590 JQ798657 JQ798720 Fiskebäckskil, Sweden N 58º 14¢ 39"; E 11º 27¢ 16" JQ798543 JQ798615 JQ798681 Torneträsk, Sweden N 68º 21¢ 19"; E 18º 49¢ 21" JQ798555 Na JQ798692 Östersidan, Sweden N 58º 15¢ 07"; E 11º 27¢ 57" JQ798561 JQ798631 JQ798696 Ilha Bela, Brazil S 23º 50¢ 30"; W 45º 24¢ 14" JQ798539 JQ798610 JQ798679 Mediterranean Sea Na JQ798595 JQ798662 JQ798724 Lago Pratignano, Italy N 44º 10¢ 51"; E 10º 49¢ 12" JQ798596 JQ798663 JQ798725 Represa do Lobo-Broa, Brazil S 22º 11¢10"; W 47º 54¢ 02" JQ798540 JQ798611 Na ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters 7 Phylogeny of Chaetonotidae (Gastrotricha) d T. Kånneby et al. Table 1 Continued GenBank Accession number Taxon (Morphbank Id.) Genus Chaetonotus (Zonochaeta) Remane, 1927 Chaetonotus bisacer Greuter, 1917 (791848–791849) Chaetonotus bisacer Greuter, 1917 (791850–791851) Chaetonotus (Zonochaeta) sp. 1 (791852–791853) Chaetonotus (Zonochaeta) sp. 2 (791854–791856) Genus Halichaetonotus Remane, 1936 Halichaetonotus aculifer (Gerlach, 1953) Halichaetonotus paradoxus (Remane, 1927) Halichaetonotus euromarinus Hummon & Todaro, 2010 (791857–791859) Halichaetonotus sp. 4 Halichaetonotus sp. 2 Genus Heterolepidoderma Remane, 1927 Heterolepidoderma loricatum Schrom, 1972 Heterolepidoderma macrops Kisielewski, 1981 (791860–791863) Heterolepidoderma ocellatum (Metschnikoff, 1865) (791864–791866) Heterolepidoderma ocellatum (Metschnikoff, 1865) (791867–791868) Heterolepidoderma acidophilum Kånneby, Todaro & Jondelius, 2012 (791869–791870) Heterolepidoderma acidophilum Kånneby, Todaro & Jondelius, 2012 (791871–791872) Heterolepidoderma acidophilum Kånneby, Todaro & Jondelius, 2012 Heterolepidoderma acidophilum Kånneby, Todaro & Jondelius, 2012 Heterolepidoderma sp. 1 (791873–791874) Heterolepidoderma sp. 2 (791875–791876) Heterolepidoderma sp. 3 (791877–791879) Genus Ichthydium Ehrenberg, 1830 Ichthydium skandicum Kånneby, Todaro & Jondelius, 2009 (791880–791881) Ichthydium skandicum Kånneby, Todaro & Jondelius, 2009 (791880–791881) Ichthydium squamigerum Balsamo & Fregni, 1995 (791882–791883) Genus Lepidochaetus Kisielewski, 1991 Lepidochaetus brasilense Kisielewski, 1991 Lepidochaetus brasilense Kisielewski, 1991 (791884–791887) 8 Sampling location Coordinates 18S 28S COI Lago Bolonha, Belém, Brazil Belém, Brazil S 01º 25¢ 31"; W 48º 25¢ 55" JQ798565 JQ798635 Na S 01º 27¢ 14"; W 48º 28¢ 36" JQ798589 Na Na Nybro, Sweden N 56º 44¢ 56"; E 15º 54¢ 14" JQ798593 JQ798661 Na Lago Bolonha, Belem, Brazil S 01º 25¢ 31"; W 48º 25¢ 55" JQ798587 JQ798655 JQ798718 Saltö, Sweden Åhus, Sweden Östersidan, Sweden N 58º 52¢ 41"; E 11º 07¢ 29" N 55º 54¢ 22"; E 14º 17¢ 41" N 58º 15¢ 07"; E 11º 27¢ 57" JQ798550 JQ798599 JQ798551 JQ798622 JQ798666 JQ798623 JQ798688 JQ798728 Na Waterlemon Bay, US Virgin Islands Åhus, Sweden N 18º 21¢ 47"; W 64º 43¢ 26" JQ798560 JQ798630 JQ798695 N 55º 54¢ 22"; E 14º 17¢ 41" JQ798600 JQ798667 JQ798729 Cagliari, Italy N 39º 12¢ 01"; E 09º 09¢ 43" JQ798541 JQ798612 Na Torneträsk, Sweden N 68º 21¢ 19"; E 18º 49¢ 21" JN185469 JN185515 JN185548 Hållö, Sweden N 58º 20¢ 00"; E 11º 12¢ 50" JN185475 JN185519 JN185554 Islandsberg, Sweden N 58º 12¢ 38"; E 11º 25¢ 09" JN185476 JN185520 JN185555 Skeppsmyra, Sweden N 59º 49¢ 25"; E 19º 03¢ 50" JN185500 JN185535 JN185572 Islandsberg, Sweden N 58º 12¢ 38"; E 11º 25¢ 09" JN185462 JN185509 JN185543 Lake Rudan, Sweden N 59º 09¢ 27"; E 18º 07¢ 31" JN185477 JN185521 JN185556 Abisko, Sweden N 68º 20¢ 53"; E 18º 58¢ 09" JN185480 JN185524 JN185559 Trunk Bay, US Virgin Islands Abisko, Sweden Mata da Pirelli, Belem, Brazil N 18º 21¢ 08"; W 64º 46¢ 08" JQ798554 Na JQ798691 N 68º 21¢ 29"; E 18º 46¢ 38" S 01º 21¢ 18"; W 48º 20¢ 31" JN185485 JQ798572 JQ798644 JQ798641 JN185563 JQ798704 Fiskebäckskil, Sweden N 58º 14¢ 39"; E 11º 27¢ 16" JQ798573 JQ798645 JQ798705 Fiskebäckskil, Sweden N 58º 14¢ 39"; E 11º 27¢ 16" JQ798606 JQ798673 JQ798735 Fiskebäckskil, Sweden N 58º 14¢ 39"; E 11º 27¢ 16" JQ798607 JQ798674 JQ798736 Represa do Lobo-Broa, Brazil Belem, Brazil S 22º 11¢ 20"; W 47º 52¢ 54" JN185458 JN185507 JQ798680 S 01º 27¢ 34"; W 48º 26¢ 05" JN185495 JQ798658 JN185568 ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters T. Kånneby et al. d Phylogeny of Chaetonotidae (Gastrotricha) Table 1 Continued GenBank Accession number Taxon (Morphbank Id.) Sampling location Coordinates 18S 28S COI Lepidochaetus zelinkai (Grünspan, 1908) Lepidochaetus zelinkai (Grünspan, 1908) (791888–791891) Lepidochaetus zelinkai (Grünspan, 1908) (791892–791893) Lepidochaetus zelinkai (Grünspan, 1908) (791894–791896) Lepidochaetus zelinkai (Grünspan, 1908) (791897–791899) Lepidochaetus zelinkai (Grünspan, 1908) (791900–791901) Lepidochaetus zelinkai (Grünspan, 1908) Lepidochaetus zelinkai (Grünspan, 1908) (791902–791903) Genus Lepidodermella Blake, 1933 Lepidodermella intermedia Kånneby, Todaro & Jondelius, 2012 (791904–791906) Lepidodermella minor minor (Remane, 1936) (791907–791908) Lepidodermella squamata (Dujardin, 1841) (791909) Lepidodermella squamata (Dujardin, 1841) (791910–791911) Lepidodermella squamata (Dujardin, 1841) (791912–791914) Lepidodermella squamata (Dujardin, 1841) (791915–791917) Lepidodermella squamata (Dujardin, 1841) Lepidodermella squamata (Dujardin, 1841) (791918–791919) Lepidodermella squamata (Dujardin, 1841) Lepidodermella squamata (Dujardin, 1841) (791920–791922) Genus Polymerurus Remane, 1927 Polymerurus nodicaudus (Voigt, 1901) Polymerurus nodicaudus (Voigt, 1901) Polymerurus nodicaudus (Voigt, 1901) Polymerurus nodicaudus (Voigt, 1901) (791923–791924) Polymerurus nodicaudus (Voigt, 1901) (791925–791926) Polymerurus nodicaudus (Voigt, 1901) Polymerurus rhomboides (Stokes, 1887) (791927–791928) Polymerurus rhomboides (Stokes, 1887) Polymerurus rhomboides (Stokes, 1887) (791931–791933) Family Dasydytidae Daday, 1905 Genus Dasydytes Gosse, 1851 Dasydytes carvalhoae Kisielewski, 1991 (791934–791937) Dasydytes elongatus Kisielewski, 1991 (791938–791941) Dasydytes elongatus Kisielewski, 1991 (791942–791944) Lago Pratignano, Italy Snasahögarna, Sweden N 44º 10¢ 51"; E 10º 49¢ 12" N 63º 12¢ 39"; E 12º 18¢ 19" JN185459 JN185498 JN185508 Na Na JN185571 Askö, Sweden N 58º 49¢ 23"; E 17º 38¢ 32" JN185503 JN185538 JN185574 Torneträsk, Sweden N 68º 21¢ 19"; E 18º 49¢ 21" JN185483 JQ798643 Na Fiskebäckskil, Sweden N 58º 14¢ 39"; E 11º 27¢ 16" JN185486 JN185527 JN185564 Abisko, Sweden N 68º 26¢ 04"; E 18º 14¢ 53" JN185487 JN185528 JN185565 Abisko, Sweden Runmarö, Sweden N 68º 20¢ 53"; E 19º 02¢ 16" N 59º 17¢ 22"; E 18º 47¢ 56" JN185496 JN185497 Na JN185534 JN185569 JN185570 Mount Njulla, Sweden N 68º 21¢ 36"; E 18º 43¢ 02" JN185468 JN185514 JN185547 Fiskebäckskil, Sweden N 58º 14¢ 51"; E 11º 27¢ 04" JN185474 Na JN185553 Fiskebäckskil, Sweden N 58º 14¢ 51"; E 11º 27¢ 04" JN185504 JN185539 JN185575 Belem, Brazil S 01º 27¢ 14"; W 48º 28¢ 36" JN185472 JN185518 JN185551 Hållö, Sweden N 58º 20¢ 00"; E 11º 12¢ 50" JN185478 JN185522 JN185557 Hållö, Sweden N 58º 20¢ 00"; E 11º 12¢ 50" JN185479 JN185523 JN185558 Abisko, Sweden Torneträsk, Sweden N 68º 26¢ 04"; E 18º 14¢ 53" N 68º 21¢ 19"; E 18º 49¢ 21" JN185481 JN185482 JN185525 JN185526 JN185560 JN185561 Abisko, Sweden Lake Trösvattnet, Sweden N 68º 21¢ 11"; E 18º 48¢ 54" N 59º 33¢ 21"; E 14º 29¢ 23" JN185484 JN185489 Na JN185530 JN185562 Na Represa do Lobo-Broa, Brazil Lago Pratignano, Italy Lago Pratignano, Italy Petroglyphs, US Virgin Islands S 22º 11¢ 20"; E 47º 52¢ 54" N 44º 10¢ 51"; E 10º 49¢ 12" N 44º 10¢ 51"; E 10º 49¢ 12" N 18º 19¢ 58"; W 64º 44¢ 44" JN185460 JN185501 JN185502 JN185465 JQ798614 JN185536 JN185537 JN185512 JN185542 Na JN185573 JQ798689 Cotejuba, Brazil S 01º 16¢ 01"; W 48º 33¢ 45" JN185473 JQ798642 JN185552 Nybro, Sweden Nybro, Sweden N 56º 44¢ 56"; E 15º 54¢ 14" N 56º 44¢ 56"; E 15º 54¢ 14" JN185490 JQ798584 JN185531 Na Na JQ798715 Petroglyphs, US Virgin Islands Lago Bolonha, Belem, Brazil N 18º 19¢ 58"; W 64º 44¢ 44" S 01º 25¢ 31"; W 48º 25¢ 55" JN185467 JN185493 JN185513 JN185533 JN185546 JN185567 Mata da Pirelli, Belem, Brazil S 01º 21¢ 18"; W 48º 20¢ 31" JQ798570 JQ798639 JQ798702 Mata da Pirelli, Belem, Brazil S 01º 21¢ 18"; W 48º 20¢ 31" JQ798568 JQ798638 JQ798700 Lago Preta, Belem, Brazil S 01º 25¢ 46"; W 48º 24¢ 00" JQ798588 JQ798656 JQ798719 ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters 9 Phylogeny of Chaetonotidae (Gastrotricha) d T. Kånneby et al. Table 1 Continued GenBank Accession number Taxon (Morphbank Id.) Sampling location Coordinates 18S 28S COI Dasydytes papaveroi Kisielewski, 1991 (791945–791947) Dasydytes papaveroi Kisielewski, 1991 (791948–791950) Genus Haltidytes Remane, 1936 Haltidytes squamosus Kisielewski, 1991 (791951–791954) Genus Ornamentula Kisielewski, 1991 Ornamentula paraënsis Kisielewski, 1991 (791955–791956) Genus Stylochaeta Hlava, 1904 Stylochaeta fusiformis (Spencer, 1890) (791957–791959) Stylochaeta scirtetica Brunson, 1950 (791960–791962) Stylochaeta scirtetica Brunson, 1950 (791960–791962) Stylochaeta scirtetica Brunson, 1950 (791960–791962) Family Xenotrichulidae Remane, 1927 Genus Draculiciteria Hummon, 1974 Draculiciteria tesselata (Renaud-Mornant, 1968) (791963) Draculiciteria tesselata (Renaud-Mornant, 1968) (791964–791967) Genus Heteroxenotrichula Wilke, 1954 Heteroxenotrichula squamosa Wilke, 1954 (791968) Genus Xenotrichula Remane, 1927 Xenotrichula intermedia Remane, 1934 Xenotrichula cf. intermedia Remane, 1934 Xenotrichula punctata Wilke, 1954 Xenotrichula velox Remane, 1927 (791969–791971) Xenotrichula velox Remane, 1927 (791969–791971) Xenotrichula sp. 1 (791972–791975) Cotejuba, Brazil S 01º 16¢ 01"; W 48º 33¢ 45" JQ798564 JQ798634 Na Lago Preta, Belem, Brazil S 01º 25¢ 46"; W 48º 24¢ 00" JQ798571 JQ798640 JQ798703 Lago Preta, Belem, Brazil S 01º 25¢ 46"; W 48º 24¢ 00" JQ798567 JQ798637 Na Lago Bolonha, Belem, Brazil S 01º 25¢ 31"; W 48º 25¢ 55" JQ798562 JQ798632 JQ798697 Lago Preta, Belem, Brazil S 01º 25¢ 46"; W 48º 24¢ 00" JN185471 JN185517 JN185550 Nybro, Sweden N 56º 44¢ 56"; E 15º 54¢ 14" JN185494 Na Na Nybro, Sweden N 56º 44¢ 56"; E 15º 54¢ 14" JN185491 Na JN185566 Nybro, Sweden N 56º 44¢ 56"; E 15º 54¢ 14" JN185492 JN185532 Na Punta Ala, Italy N 42º 48¢ 42"; E 10º 44¢ 46" JN185457 JN185506 JN185541 Trunk Bay, US Virgin Islands N 18º 21¢ 12"; W 64º 46¢ 05" JN185470 JN185516 JN185549 Punta Ala, Italy N 42º 48¢ 42"; E 10º 44¢ 46" JQ798542 JQ798613 Na Mahdia, Tunisia N 35º 30¢ 57"; E 11º 03¢ 00" JF357664 JF357712 JF432047 Failaka, Kuwait N 29º 23¢ 38"; E 48º 24¢ 07" JN185461 Na Na Östersidan, Sweden N 58º 15¢ 07"; E 11º 27¢ 57" JN185464 JN185511 Na Åhus, Sweden N 55º 54¢ 22"; E 14º 17¢ 41" JN185499 Na Na Åhus, Sweden N 55º 54¢ 22"; E 14º 17¢ 41" JN185488 JQ798652 Na Maho Bay, US Virgin Islands N 18º 21¢ 27"; W 64º 44¢ 42" JN185466 Na JN185545 Tramore, Ireland N 52º 09¢ 29"; W 07º 08¢ 38" JQ798535 Na JQ798675 Malaga, Spain N 36º 43¢ 04"; W 04º 21¢ 30" JQ798536 Na JQ798676 Suborder Multitubulatina d’Hondt, 1971 Family Neodasyidae Remane, 1929 Genus Neodasys Remane, 1927 Neodasys chaetonotoideus Remane, 1927 Neodasys uchidai Remane, 1961 The Morphbank Id number is given in parentheses for most specimens and images can be accessed at http://www.morphbank.net/791xxx, where xxx is the last part of the Id number for a certain specimen. All images can be viewed as a collection at http://www.morphbank.net/799280. Na, Not available. Muscle were 6385 and 6329 nucleotide positions. After ambiguously aligned positions had been filtered with Aliscore, the combined data set aligned with Mafft consisted of 5264 and 5227 for Neodasys and Xenotrichulidae, 10 respectively. The corresponding numbers for the data sets aligned with muscle were 5260 and 5234. The 95% consensus trees based on the two different alignments, with Neodasys as outgroup, were congruent for ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters T. Kånneby et al. d Phylogeny of Chaetonotidae (Gastrotricha) Fig. 4. Phylogenetic relationships of Chaetonotidae inferred from Bayesian analysis of 18S rDNA, 28S rDNA and COI mtDNA. All nodes except those labelled with * or ** are above 0.95 or 0.70 referring to posterior probability and bootstrap replicates inferred from maximum likelihood analysis, respectively. Branches labelled with red colour indicate marine lineages. ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters 11 Phylogeny of Chaetonotidae (Gastrotricha) d T. Kånneby et al. the combined data set. The phylogeny based on Mafft alignments showed the highest resolution (Fig. 4). For individual genes, there were no major conflicts between alignments. The gene tree of 18S rDNA based on the Mafft alignment was better resolved than the gene tree based on Muscle. Both trees showed low resolution of deeper nodes. The 28S rDNA gene tree showed higher resolution and posterior probability at deeper nodes than the 18S rDNA gene tree. However, Dasydytidae is nonmonophyletic in the 28S gene tree based on the muscle alignment while these nodes are not resolved in gene trees based on the Mafft alignment. The COI mtDNA gene tree is only well resolved close to the terminals and the obtained clades are congruent with those of the nuclear gene trees. A minor conflict is that the sister group relation between Chaetonotus (Chaetonotus) microchaetus and Lepidodermella squamata was not obtained (Fig. 5). The maximum likelihood analysis was based on the combined data set and congruent with Bayesian analyses (Fig. 4). The AU-test gives high statistical support (P < 0.001) for the topology obtained in our analyses compared with the topology based on morphological generic characters obtained by Hochberg & Litvaitis (2000). Phylogeny The most important results of our study show that all of the combined data set and individual gene trees provide strong support for a clade containing Dasydytidae nested within Chaetonotidae (Fig. 4). Hence, Chaetonotidae is non-monophyletic with members of Dasydytidae in close relationship to Polymerurus or Chaetonotus (Zonochaeta). Xenotrichulidae is retained as monophyletic in a sister group relationship to the Chaetonotidae + Dasydytidae clade in all analyses where Neodasys serves as outgroup. Dasydytidae is retained as monophyletic in both alignments based on the combined nuclear and mitochondrial data set. Our study also indicates that marine representatives of Aspidiophorus appear to be the sister group of all other Chaetonotidae + Dasydytidae. The deeper relationships of the Chaetonotidae + Dasydytidae clade were poorly resolved in the phylogenies based on the combined nuclear and mitochondrial data set. With regard to the monophyly of individual genera only three appear monophyletic, Lepidochaetus, Polymerurus and Stylochaeta. Chaetonotus is polyphyletic and clusters with various groups and Chaetonotus (Zonochaeta) is the only subgenus obtained as monophyletic (Fig. 4). Chaetonotidae Marine members of Aspidiophorus are indicated as the sister group of all other Chaetonotidae + Dasydytidae (Fig. 4). Within the family, only two of nine sampled genera 12 appear monophyletic, Lepidochaetus and Polymeurus, both represented by two species each. Genera containing both marine and freshwater representatives are not monophyletic and group with other species according to habitat. One such clade contains the strictly marine Halichaetonotus and marine representatives of Heterolepidoderma. The freshwater representatives of Heterolepidoderma cluster with other freshwater species which is also true for freshwater Aspidiophorus. Ichthydium, characterized by a more or less naked smooth cuticle, is polyphyletic. Arenotus also has a smooth cuticle and does not group with any of the sampled Ichthydium but instead with Chaetonotus (Chaetonotus) polyspinosus. Moreover, the polyphyly of the morphologically diverse Chaetonotus, with representatives scattered all over in the phylogeny, comes as no surprise (Fig. 4). Nodes closer to the terminals usually have high support and some interesting clades emerge in our analyses. The more densely sampled Chaetonotus (Chaetonotus) is polyphyletic and members group with various genera in Chaetonotidae. One clade contains large C. (Chaetonotus) species from the maximus-similis group, for example, Chaetonotus (Chaetonotus) similis, Chaetonotus (Chaetonotus) laroides, Chaetonotus (Chaetonotus) maximus and C. (C.) microchaetus that cluster with L. squamata (Figs 4 and 5). Another clade contains small representatives of three genera, Chaetonotus, Heterolepidoderma and Ichthydium, while another contains Aspidiophorus ophiodermus, an unidentified Heterolepidoderma and Chaetonotus (Chaetonotus) daphnes in a sister group relation to C. (C.) polyspinosus and Arenotus strixinoi (Fig. 4). Chaetonotus (Hystricochaetonotus) is only represented by three species but it appears to be a polyphyletic taxon. Chaetonotus (Hystricochaetonotus) hystrix and Chaetonotus (Hystricochaetonotus) aemilianus form a highly supported clade while the position of Chaetonotus (Hystricochaetonotus) cf. novenarius is unresolved. Likewise the position of the single species of Chaetonotus (Marinochaetus), Chaetonotus (Marinochaetus) mariae is unresolved in all phylogenies (Figs 4 and 5). Chaetonotus (Primochaetus) is represented by six species in the phylogeny. All representatives of the group, except Chaetonotus (Primochaetus) heteracanthus (Fig. 1D), emerge in a single clade together with species of Ichthydium, Lepiodermella and Aspidiophorus based on the Mafft alignment. The deeper nodes within this clade are poorly resolved. C. (P.) heteracanthus is in a sister group relation to the monophyletic C. (Zonochaeta). Marine species of Chaetonotus (Schizochaetonotus) appear to be a sister group to Chaetonotidae + Dasydytidae except marine Aspidiophorus, although the group itself is not well resolved. It is also important to notice that the single freshwater species included in this subgenus and our analysis, Chaetonotus (Schizochaetonotus) schultzei, is nested in a well ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters T. Kånneby et al. d Phylogeny of Chaetonotidae (Gastrotricha) Fig. 5. Phylogenetic relationships of Chaetonotidae inferred from Bayesian analysis of COI mtDNA. Only nodes with a posterior probability above 0.95 are shown. ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters 13 Phylogeny of Chaetonotidae (Gastrotricha) d T. Kånneby et al. supported clade containing Chaetonotus (Wolterecka) uncinus, marine Heterolepidoderma and Halichaetonotus (Fig. 4). Dasydytidae Dasydytidae appears monophyletic and nested within Chaetonotidae in close alliance with members of Chaetonotus (Zonochaeta) and Polymerurus. The resolution of the phylogeny based on the Muscle alignment does not allow one or the other to be singled out as the sister group of the family. However, the combined analysis based on Mafft suggests members of Chaetonotus (Zonochaeta) and C. (P.) heteracanthus to be the sister group of Dasydytidae. Stylochaeta is the only genus retained as monophyletic while Dasydytes is non-monophyletic with Dasydytes carvalhoae in a sister group relation to a clade containing Stylochaeta, Dasydytes elongatus and Dasydytes papaveroi (Fig. 4). Xenotrichulidae The two current subfamilies Draculiciterinae and Xenotrichulinae are strongly supported in our phylogeny, where Draculiciteria appears as the sister group of all other Xenotrichulidae. Within Xenotrichulinae, monophyly of Xenotrichula is not retained because the single species of Heteroxenotrichula, Heteroxenotrichula squamosa, included in the analysis is nested together with Xenotrichula intermedia and Xenotrichula velox (Fig. 4). Discussion This study is the first attempt to resolve the phylogeny of Chaetonotidae using both nuclear and mitochondrial genetic data. The results are in general congruence with earlier molecular studies but give new insights regarding the evolution and interrelationships of Chaetonotidae. The major finding of our study is that Chaetonotidae (sensu Leasi & Todaro 2008) is non-monophyletic as members of Dasydytidae form a highly supported clade within the family. This has already been suggested in morphological (Hochberg & Litvaitis 2000; Kieneke et al. 2008) and molecular analyses (Kånneby et al. 2011). There is need of taxonomic revisions within the Chaetonotidae because only Polymerurus and Lepidochaetus appear monophyletic following current classification for the group (see also Kånneby et al. 2011). We also confirm the results of Todaro et al. (2006) where Chaetonotus is a polyphyletic taxon. Only one of the proposed subgenera, C. (Zonochaeta), appears monophyletic. There is also a hint on the evolution of Gastrotricha regarding dispersal between marine and freshwater habitats. Marine members of Aspidiophorus still retain the ancestral character of marine habitat and appear as the sister group of all other Chaetonotidae and Dasydytidae. The phylogeny also shows that representatives of nominal genera with both marine and freshwater 14 representatives do not form monophyletic groups, but instead group with other species from similar habitats. This seems likely due to the broad taxonomic boundaries used to define genera (and subgenera) and to the practice of squeezing species into a given genera, when the same species also possesses traits that belong to another genus. However, current results are not conclusive as to a separation into a marine vs. freshwater phyletic line of all species belonging to a given genus (e.g. Aspidiophorus and Chaetonotus). The most plausible hypothesis points towards a marine origin of Chaetonotidae as the sister group Xenotrichulidae is a strictly marine group. One character that may indicate this is the pedunculated scales present both in marine Aspidiophorus and members of Xenotrichulinae. Further morphological studies of marine Aspidiophorus can confirm the homology of these structures and may provide important information of the ground pattern of Chaetonotidae. The position of marine Aspidiophorus is in conflict with previous morphological studies, suggesting a basal position of the strictly freshwater taxon Polymerurus (Leasi & Todaro 2008) but congruent with previous analyses of 18S rDNA (Todaro et al. 2006). Although not well resolved marine members of C. (Schizochaetonotus) also appear primarily marine (Fig. 4). A secondary invasion of certain Chaetonotidae into marine environments seems likely as Halichaetonotus and marine Heterolepidoderma are nested in a sister group relation to freshwater species of Chaetonotus. This may be further studied by investigating putative adaptations of the protonephridial system in marine and freshwater chaetonotidans. Our results imply that the use of cuticular structures for defining groups should be treated with caution as many of these characters are in conflict with our phylogeny and need critical evaluation. However, at the species level the cuticular structures are crucial characters when identifying and diagnosing new species. But when applied at higher levels of classification these characters are not consistent. Since Chaetonotidae appear as a fairly homogenous group, at least at gross anatomy level, it might prove difficult to assign phylogenetically informative morphological characters that can be used for higher level classification. To give an idea, the two species of Polymerurus analysed in our study form a highly supported monophyletic group. Looking only at the dorsal covering, however, the two species appear very different: Polymerurus nodicaudus has spined scales while Polymerurus rhomboides has stalked scales. However, the most important diagnostic character for Polymerurus is the segmented nature of the furca carrying reduced adhesive tubes (Fig. 1I). In this light, it becomes apparent that there are major problems in genera diagnosed on the nature of cuticular structures alone, for example, Aspidiophorus, Chaetonotus and Heterolepidoderma ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters T. Kånneby et al. to name a few, all non-monophyletic in our study. Another example is Ichthydium, a group based on a negative character, a cuticle covering without scales and spines (Fig. 1G). However, ultrastructural studies have revealed that many species indeed possess minute cuticular structures. Our result indicating polyphyly of Ichthydium is also supported by Kieneke et al. (2008). Arenotus strixinoi also has an apparently smooth naked cuticle but is separated from Ichthydium on the basis of the soft homogenous cuticle separated from the epidermis, a larger body size and the complicated structure of the mouth ring (Fig. 1A). This monotypic genus is very rare and has so far only been found at the type locality in Brazil (Kisielewski 1987; current study). From this evidence, it seems likely that scale reduction has evolved multiple times, and consequently this trait alone can not be used as a diagnostic character at generic level. With very few exceptions, Todaro et al. (2011) found classification of the diverse family Thaumstodermatidae to be congruent with molecular data. Classification within Macrodasyida thus seems more reliable as it is based on characters such as shape of the caudal end, distribution of adhesive tubes and reproductive system and to a lesser extent on cuticular structures. Future studies can benefit from the phylogenetic hypothesis generated here and characters can be optimized and evaluated in a phylogenetic context and subsequently used in classification. Chaetonotidae With almost 400 species, Chaetonotidae is the most specious group of the phylum. The overall morphological similarity among species belonging to different genera, and the widespread occurrence of traits supposed to be diagnostic at generic level (e.g. pedunculated scales diagnostic for Aspidiophorus (Fig. 1B, C) but also present in Polymerurus, the hydrofoil scales diagnostic for Halichaetonotus but also present in Heterolepidoderma and Chaetonotus) makes it hard to discern ingroup phylogenetic relationships without molecular data. Chaetonotus with its more than 200 species, many of which are morphologically very similar, is especially hard to work with. We have sampled all recognized subgenera within Chaetonotus except for Chaetonotus (Captochaetus), a small group of rather large predatory gastrotrichs (Kisielewski 1997). Our finding of a non-monophyletic Chaetonotus agrees with previous studies (Todaro et al. 2006; Kieneke et al. 2008). Chaetonotus (Zonochaeta) is the only subgenus within Chaetonotus retained as monophyletic; the group is morphologically diverse and characterized by a transverse row of longer spines dorsally and the presence of a pair of long terminal spines (Kisielewski 1997). Our phylogeny supports the exclusion of Chaetonotus (Primochaetus) acanthodes ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters d Phylogeny of Chaetonotidae (Gastrotricha) and its affiliation to a different group (Kisielewski 1997). However, its current subgenus, Chaetonotus (Primochaetus) appears polyphyletic as one species, C. (P.) heteracanthus (Fig. 1D) is resolved in a sister group relationship to C. (Zonochaeta), and the remaining four species, Chaetonotus (P.) heideri, C. (P.) acanthodes, Chaetonotus (P.) acanthocephalus and Chaetonotus (P.) armatus, appear distributed in two different clades. Chaetonotus (Hystricochaetonotus), characterized by threelobed scales with spines carrying denticles, is represented by three species, C. (H.) hystrix, C. (H.) aemilianus and C. (H.) cf. novenarius. The position of C. (H.) cf. novenarius is not resolved. The sister group relationship to Heterolepidoderma macrops is puzzling but was obtained in analyses of both alignments of the combined data set as well as combined analyses of the nuclear genes. In the individual nuclear gene trees, the position of H. macrops was unresolved. In the COI mtDNA gene tree, H. macrops clustered in a clade together with C. (C.) maximus and L. squamata. Chaetonotus (Chaetonotus) is the largest subgenus within the group and is obtained as polyphyletic with one or several species distributed in three separate clades. One of these clades contains large C. (Chaetonotus) and L. squamata. Chaetonotus (Chaetonotus) laroides, C. (C.) similis and C. (C.) maximus are morphologically very similar and proper identification of these species requires a careful evaluation of the shape of the scales and spines. Chaetonotus (Chaetonotus) microchaetus is easily separated from the rest in that it has significantly smaller scales and shorter spines, as well as a peculiar arrangement of scales in the interciliary area. The relationship between C. (C.) microchaetus and L. squamata stands out as the morphological differences are many, especially the dorsal scales that are small and spined in the former and large and unspined in the latter. But there are also similarities, most notably in the covering of the interciliary area. Both species share the transverse scale bars in the anterior end of the interciliary area, moreover the distribution of scales in the posterior area shows resemblance. An interesting clade also contains large chaetonotids, including A. strixinoi, A. ophiodermus, C. (C.) polyspinosus, C. (C.) daphnes and Heterolepidoderma sp. 3. Except for A. strixinoi all species within the clade are characterized by their minute dorsal scales with keels. It may be that the absence of cuticular structures in Arenotus derives from a reduction process already started in some of the species of the same clade (e.g. C. (C.) polyspinosus and C. (C.) daphnes). Within the clade, two highly resolved subclades are present, one containing C. polyspinosus and A. strixinoi and the other A. ophiodermus, Heterolepidoderma sp. 3 and C. (C.) daphnes. Chaetonotus (Chaetonotus) daphnes have dorsal scales that are very similar to those scales found in Heterolepidoderma 15 Phylogeny of Chaetonotidae (Gastrotricha) d T. Kånneby et al. species. A difference is that in C. (C.) daphnes the keels of the scales end in a short spine, and it has thus been classified as a Chaetonotus. Aspidiophorus ophiodermus on the other hand have keeled scales, much like the ones found in Heterolepidoderma, but most of them are borne on short peduncles. The posteriormost dorsal side of A. ophiodermus is covered with larger keeled but unstalked scales much resembling those found in Heterolepidoderma. Another character in common for all the sampled species in this clade is that they have a granulated foregut (Fig. 1B), which has also been reported in Heterolepidoderma majus and Aspidiophorus schlitzensis (Schwank 1990). There is also a clade containing small members of Chaetonotus (Chaetonotus), Heterolepidoderma and Ichthydium. Within this clade, Chaetonotus (Chaetonotus) oculifer is in a close relationship with Heterolepidoderma ocellatum and Heterolepidoderma acidophilum. Kånneby et al. (2011) reported the tendency of spine development in Swedish specimens of H. ocellatum and H. acidophilum which may point to a closer relationship between species of Heterolepidoderma and Chaetonotus with ocellar granules than previously thought. Another unidentified species of Heterolepidoderma that possess the typical keeled dorsal scales of the genus, but have the ventrolateral areas covered by trilobed scales with rather short simple spines is the sister group of this clade. Dasydytidae Dasydytidae forms a well supported clade in a sister group relationship to members of C. (Zonochaeta). Dasydytidae are separated from Chaetonotidae in that they have a different arrangement of locomotory cilia adapted to a semiplanktonic lifestyle and they also possess long and strong movable spines used for floatation and defence (Fig. 2). Moreover, Kieneke et al. (2008) proposed an apomorphy for Dasydytidae consisting of an antagonistic system of segmented muscles and oblique somatic muscle pairs that aid movements of the long spines. The phylogeny based on the Mafft alignment gave high support for a sister group relationship between C. (Zonochaeta) and Dasydytidae, also suggested by Kisielewski (1991), while the phylogeny based on Muscle alignments do not resolve this node. In our study, four of the seven recognized genera have been sampled, among them three species of the subgenus Prodasydytes, hypothesized to have retained plesiomorphic character states within Dasydytidae (Kisielewski 1991). Our phylogenetic hypothesis agrees well with the cladogram based on morphological characters proposed by Kisielewski (1991): fig. 131. Haltidytes squamosus (Fig. 2D) and Ornamentula paraënsis (Fig. 2E) form a clade in a sister group to Dasydytes and Stylochaeta. Both Ornamentula and H. squamosus have long spines as well as dorsal covering 16 consisting of very large scales as opposed to the smaller scales or naked cuticle in Dasydytes and Stylochaeta (Fig. 2A–C, F). Xenotrichulidae The position of Xenotrichulidae as a sister group of Chaetonotidae and Dasydytidae is well supported. Although Musellifer was not included in our analysis results from previous studies including both Musellifer and Xenotrichulidae point to a sister group relationship between Xenotrichulidae and Chaetonotidae + Dasydytidae (Todaro et al. 2006). The newly obtained sequences of D. tesselata show that this species is indeed a group within Xenotrichulidae as opposed to earlier results based on 18S rDNA (Zrzavý 2003; Manylov et al. 2004; Todaro et al. 2006) where Draculiciteria grouped with members of Chaetonotidae. The previous studies all used the same sequence for D. tesselata, available from GenBank. Todaro et al. (2006) discusses the potential contamination of this sequence and it seems like their suspicion was well grounded. In our study, two specimens of D. tesselata (Fig. 3A) obtained from Italy and US Virgin Islands, respectively, form a sister group to Xenotrichulinae. Within Xenotrichulinae, Xenotrichula is non-monophyletic since H. squamosa (Fig. 3B) is nested within the genus, as previously suggested by Marotta et al. (2005) when analysing characters based on sperm ultrastructure. Data set The data sets presented in this study are based on three genes, 18S rDNA, 28S rDNA and COI mtDNA. Using a multigene approach to reconstruct phylogenetic relationships is important as conflicts between, for example, mitochondrial and nuclear data can easily be detected. The hypothesis suffers from low support of deeper nodes within Chaetonotidae, but the three genes proved useful at resolving very deep relationships and terminal ones. To better understand the relationships within Chaetonotidae additional genetic markers should be sequenced and utilized. Protein coding nuclear genes may prove to be useful as they can be translated into amino acids and more easily aligned. Acknowledgements The authors wish to thank Keyvan Mirbakhsh for the technical assistance. We are also grateful to Drs. Diego Fontaneto and Sven Boström for their advice and discussions. Collection in Brazil was made possible by a permit from CNPq (Portaria MCT No. 972) to Dr Carlos E. F. Rocha. The trip to St. John, US Virgin Islands, was made possible thanks to a grant from US-NSF (Grant No DEB 0918499 to Dr. Rick Hochberg). The authors wish to ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters T. Kånneby et al. thank two anonymous reviewers for their insightful comments. This study was supported by grants from the Swedish Taxonomy Initiative (to UJ), the Royal Swedish Academy of Sciences, Riksmusei Vänner, Helge Ax:son Johnson foundation, Lennanders foundation and Abisko Scientific Research Station (to TK). References Abascal, F., Zardoya, R. & Telford, M. J. (2010). TranslatorX: multiple alignment of nucleotide sequences guided by amino acid translations. Nucleic Acids Research, 38, W7–W13. Amato, A. J. & Weiss, M. J. (1982). Developmental flexibility in the cuticular pattern of a cell-constant organism, Lepidodermella squammata (Gastrotricha). Transactions of the American Microscopical Society, 101, 229–240. Artois, T., Fontaneto, D., Hummon, W. D., McInnes, S. J., Todaro, M. A., Sørensen, M. V. & Zullini, A. (2011). Ubiquity of microscopic animals? Evidence from the morphological approach in species identification. In D. Fontaneto (Ed.) Biogeography of Microscopic Organisms: Is Everything Small Everywhere? (pp. 245–249). New York: Cambridge University Press. Balsamo, M., d’Hondt, J. L., Pierboni, L. & Grilli, P. (2009). Taxonomic and nomenclatural notes on freshwater Gastrotricha. Zootaxa, 2158, 1–19. Dunn, C. W., Hejnol, A., Matus, D. Q., Pang, K., Browne, W. E., Smith, S. A., Seaver, E., Rouse, G. W., Obst, M., Edgecombe, G. D., Sørensen, M. V., Haddock, S. H. D., Schmidt-Rhaesa, A., Okusu, A., Kristensen, R. M., Wheeler, W. C., Martindale, M. Q. & Giribet, G. (2008). Broad phylogenomic sampling improves resolution of the animal tree of life. Nature, 452, 745–749. Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792–1797. Guindon, S. & Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology, 52, 696–704. Hejnol, A., Obst, M., Stamatakis, A., Ott, M., Rouse, G. W., Edgecombe, G. D., Martinez, P., Bagũna, J., Bailly, X., Jondelius, U., Wiens, M., Müller, W. E. G., Seaver, E., Wheeler, W. C., Martindale, M. Q., Giribet, G. & Dunn, C. W. (2009). Assessing the roots of bilaterian animals with scalable phylogenomic methods. Proceedings of the Royal Society B, 276, 4261–4270. Hochberg, R. & Litvaitis, M. K. (2000). Phylogeny of Gastrotricha: a morphology-based framework of gastrotrich relationships. Biological Bulletin, 198, 299–305. Hochberg, R. & Litvaitis, M. K. (2001). A muscular double helix in Gastrotricha. Zoologischer Anzeiger, 240, 61–68. d’Hondt, J. L. (1971). Gastrotricha. In H. Barnes (Ed.) Oceanography and Marine Biology: An Annual Review, vol 9 (pp. 141–192). London: George Allen and Unwin Ltd. Huelsenbeck, J. P. & Ronquist, F. (2005). Bayesian analysis of molecular evolution using MrBayes. In R. Nielsen (Ed.) Statistical Methods in Molecular Evolution (pp. 183–232). New York: Springer. Hummon, W. D. (2008). Brackish-Water Gastrotricha of the Polish Baltic Coast. Meiofauna Marina, 16, 109–116. ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters d Phylogeny of Chaetonotidae (Gastrotricha) Hummon, W. D. & Todaro, M. A. (2010). Analytic taxonomy and notes on marine, brackish-water and estuarine Gastrotricha. Zootaxa, 2392, 1–32. Kånneby, T., Todaro, M. A. & Jondelius, U. (2011). A phylogenetic approach to species delimitation in freshwater Gastrotricha from Sweden. Hydrobiologia, 683, 185–202. (2012). Katoh, K., Misawa, K., Kuma, K. & Miyata, T. (2002). MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research, 30, 3059–3066. Katoh, K., Kuma, K., Toh, H. & Miyata, T. (2005). MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research, 33, 511–518. Kieneke, A., Riemann, O. & Ahlrichs, W. H. (2008). Novel implications for the basal internal relationships of Gastrotricha revealed by an analysis of morphological characters. Zoologica Scripta, 37, 429–460. Kisielewski, J. (1987). New records of marine Gastrotricha from the French coasts of Manche and Atlantic I. Macrodasyida with descriptions of seven new species. Bulletin de la Musee d’Histoire Naturelle de Paris, 4, 837–877. Kisielewski, J. (1991). Inland-water Gastrotricha from Brazil. Annales Zoologici Warszawa, 43(Suppl. 2), 1–168. Kisielewski, J. (1997). On the subgeneric division of the genus Chaetonotus Ehrenberg (Gastrotricha). Annales Zoologici, 46, 145–151. Kumar, S., Skjæveland, Å. & Orr, R. J. S. (2009). AIR: a batchoriented web program package for construction of supermatrices ready for phylogenomic analyses. BMC Bioinformatics, 10, 357. Leasi, F. & Todaro, M. A. (2008). The muscular system of Musellifer delamarei (Renaud-Mornant, 1968) and other chaetonotidans with implications for the phylogeny and systematization of the Paucitubulatina (Gastrotricha). Biological Journal of the Linnean Society, 94, 379–398. Leasi, F., Rothe, B. H., Schmidt-Rhaesa, A. & Todaro, M. A. (2006). he musculature of three species of gastrotrichs surveyed with confocal laser scanning microscopy (CLSM). Acta Zoologica (Stockholm), 87, 171–180. Manylov, O. G., Vladychenskaya, N. S., Milyutina, I. A., Kedrova, O. S., Korokhov, N. P., Dvoryanchikov, G. A., Aleshin, V. V. & Petrov, N. B. (2004). Analysis of 18S rRNA gene sequences suggests significant molecular differences between Macrodasyida and Chaetonotida (Gastrotricha). Molecular Phylogenetics and Evolution, 30, 850–854. Marotta, R., Guidi, L., Pierboni, L., Ferraguti, M., Todaro, M. A. & Balsamo, M. (2005). Sperm ultrastructure of Macrodasys caudatus (Gastrotricha: Macrodasyida) and a sperm-based phylogenetic analysis of Gastrotricha. Meiofauna Marina, 14, 9–21. Misof, B. & Misof, K. (2009). A Monte Carlo approach successfully identifies randomness in multiple sequence alignments: a more objective means of data exclusion. Systematic Biology, 58, 21–34. Petrov, N. B., Pegova, A. N., Manylov, O. G., Vladychenskaya, N. S., Mugue, N. S. & Aleshin, V. V. (2007). Molecular phylogeny of Gastrotricha on the basis of a comparison of the 18S rRNA genes: rejection of the hypothesis of a relationship between Gastrotricha and Nematoda. Molecular Biology, 41, 445–452. Posada, D. (2008). jModelTest: phylogenetic model averaging. Molecular Biology and Evolution, 25, 1253–1256. 17 Phylogeny of Chaetonotidae (Gastrotricha) d T. Kånneby et al. Remane, A. (1927). Beiträge zur Systematik der Süsswassergastrotrichen. Zoologische Jahrbücher Abteilung für Systematik Oekologie und Geographie der Tiere, 53, 269–320. Remane, A. (1961). Neodasys uchidai nov. spec., eine zweite Neodasys-Art (Gastrotricha Chaetonotoidea). Kieler Meeresforschungen, 17, 85–88. Ronquist, F. & Huelsenbeck, J. P. (2003). MRBAYES 3: bayesian phylogenetic inference under mixed models. Bioinformatics, 19, 1572–1574. Schmidt, H. A., Strimmer, K., Vingron, M. & von Haesler, A. (2002). TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics, 18, 502–504. Schwank, P. (1990). Gastrotricha. In J. Schwoerbel & P. Zwick. (Eds) Süsswasserfauna von Mitteleuropa, Band 3. Gastrotricha und Nemertini (pp. 1–252). Gustav Stuttgart, Jena, New York: Fischer Verlag. Shimodaira, H. (2002). An approximately unbiased test of phylogenetic tree selection. Systematic Biology, 51, 492–508. Shimodaira, H. & Hasegava, M. (2001). CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics, 17, 1246–1247. Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22, 2688–2690. Todaro, M. A. & Hummon, W. D. (2008). An overview and a dichotomous key to genera of the phylum Gastrotricha. Meiofauna Marina, 16, 3–20. Todaro, M. A., Kånneby, T., Dal Zotto, M. & Jondelius, U. (2011). Phylogeny of Thaumastodermatidae (Gastrotricha: Macrodasyida) inferred from nuclear and mitochondrial sequence data. PLoS ONE, 6, e17892. Todaro, M. A., Littlewood, D. T. J., Balsamo, M., Herniou, E. A., Cassanelli, S., Manicardi, G., Wirz, A. & Tongiorgi, P. (2003). The interrelationships of the Gastrotricha using nuclear small rRNA subunit sequence data, with an interpretation based on morphology. Zoologischer Anzeiger, 242, 145–156. Todaro, M. A., Telford, M. J., Lockyer, A. E. & Littlewood, D. T. J. (2006). Interrelationships of the Gastrotricha and their place among the Metazoa inferred from 18S rRNA genes. Zoologica Scripta, 35, 251–259. 18 Todaro, M. A., Dal Zotto, M., Jondelius, U., Hochberg, R., Hummon, W. D., Kånneby, T. & Rocha, C. E. F. (2012). Gastrotricha: a marine sister for a freshwater puzzle. PLoS One, 7, e31740. Weiss, M. J. (2001). Widespread hermaphroditism in freshwater gastrotrichs. Invertebrate Biology, 120, 308–341. Zrzavý, J. (2003). Gastrotricha and metazoan phylogeny. Zoologica Scripta, 32, 61–81. Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. 18S28SCOIMuscleNeofilt.pdf. Phylogenetic relationships of Chaetonotidae inferred from Bayesian analysis of 18S rDNA, 28S rDNA and COI mtDNA based on the Muscle alignment. Numbers at nodes correspond to posterior probabilities. Fig. S2. 18S28SMafftNeofilt.pdf. Phylogenetic relationships of Chaetonotidae inferred from Bayesian analysis of 18S rDNA and 28S rDNA based on the Mafft alignment. Numbers at nodes correspond to posterior probabilities. Fig. S3. 18SMafftNeofilt.pdf. Phylogenetic relationships of Chaetonotidae inferred from Bayesian analysis of 18S rDNA based on the Mafft alignment. Numbers at nodes correspond to posterior probabilities. Fig. S4. 28SMafftXenofilt.pdf. Phylogenetic relationships of Chaetonotidae inferred from Bayesian analysis of 28S rDNA based on the Mafft alignment. Numbers at nodes correspond to posterior probabilities. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters