CSIRO PUBLISHING www.publish.csiro.au/journals/is Invertebrate Systematics, 2010, 24, 560–572 Evolution in the deep sea: a combined analysis of the earliest diverging living chitons (Mollusca : Polyplacophora : Lepidopleurida) Julia D. Sigwart A,F, Enrico Schwabe B, Hiroshi Saito C, Sarah Samadi D and Gonzalo Giribet E A Queen’s University Belfast, School of Biological Science, Marine Laboratory, Portaferry, Northern Ireland, BT22 1PF, UK. B Zoologische Staatssammlung, Mollusca Section, 81247 Munich, Germany. C National Museum of Nature and Science, Department of Zoology, Tokyo 169-0073, Japan. D Muséum National d’Histoire Naturelle, Départment Systématique et Evolution, UMR7138 UPMC-IRD-MNHNCNRS Paris 6, France. E Museum of Comparative Zoology, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA. F Corresponding author. Email: j.sigwart@qub.ac.uk Abstract. Lepidopleurida is the earliest diverged group of living polyplacophoran molluscs. They are found predominantly in the deep sea, including sunken wood, cold seeps, other abyssal habitats, and a few species are found in shallow water. The group is morphologically identified by anatomical features of their gills, sensory aesthetes, and gametes. Their shell features closely resemble the oldest fossils that can be identified as modern polyplacophorans. We present the first molecular phylogenetic study of this group, and also the first combined phylogenetic analysis for any chiton, including three gene regions and 69 morphological characters. The results show that Lepidopleurida is unambiguously monophyletic, and the nine genera fall into five distinct clades, which partly support the current view of polyplacophoran taxonomy. The genus Hanleyella Sirenko, 1973 is included in the family Protochitonidae, and Ferreiraellidae constitutes another distinct clade. The large cosmopolitan genus Leptochiton Gray, 1847 is not monophyletic; Leptochiton and Leptochitonidae sensu stricto are restricted to North Atlantic and Mediterranean taxa. Leptochitonidae s. str. is sister to Protochitonidae. The results also suggest two separate clades independently inhabiting sunken wood substrates in the south-west Pacific. Antarctic and other chemosynthetic-dwelling species may be derived from wood-living species. Substantial taxonomic revision remains to be done to resolve lepidopleuran classification, but the phylogeny presented here is a dramatic step forward in clarifying the relationships within this interesting group. Introduction Polyplacophora (chitons) represent a distinctive molluscan clade living in marine environments worldwide, with a fossil record extending 500 million years (Runnegar et al. 1979; Sigwart and Sutton 2007). The earliest derived living order (sister group to all other taxa), Lepidopleurida comprises a large assemblage of chitons that share features with fossil shells, and are morphologically supported by their special (usually posterior) adanal gill arrangement, simple gamete structures, and aesthete innervation (Sirenko 1993, 2006; Buckland-Nicks 2006; Sigwart 2008). These features separate Lepidopleurida from all other living chitons, which are in the order Chitonida (Sirenko 2006). Approximately 130 living species are known within Lepidopleurida, all within the extant suborder Lepidopleurina (Sirenko 2001, 2006); however, genera or other subgroups often lack consistent morphological synapomorphies (Fig. 1). Molecular studies on chitons are scarce. To date, a single study has focussed on higher-level relationships within Polyplacophora
CSIRO 29 April 2011 using DNA sequence data (Okusu et al. 2003). Other studies have centred on species identification particularly within the genus Mopalia Gray, 1847, which excludes lepidopleuran taxa (Kelly et al. 2007; Kelly and Eernisse 2008), or incidentally included multiple chitons in investigating the higher-level relationships within Mollusca (e.g. Passamaneck et al. 2004; Giribet et al. 2006; Wilson et al. 2010). Lepidopleuran taxa in these studies are usually limited to Lepidopleurus cajetanus (Poli, 1791) and Leptochiton asellus (Gmelin, 1791), which are shallow water, European species and common compared with most species in the group. The aim of this study was to focus on one manageable aspect of chiton phylogeny, the order Lepidopleurida, by testing the internal relationships within this clade with a far larger taxon sampling than has been included in any previous study. We included nine of the ten putative lepidopleuran genera, which are primarily deep sea species (Schwabe 2008a). The sequencing efforts focussed on three phylogenetically informative regions: 10.1071/IS10028 1445-5226/10/060560 Combined analysis of primitive living chitons (Lepidopleurida) Invertebrate Systematics 561 Fig. 1. Examples of chitons in the order Lepidopleurida, representing the major groups resolved in the present analysis. In all images, the anterior end is to the left, or top. (A) Leptochitonidae s. str.: Leptochiton asellus, Strangford Lough, Northern Ireland, intertidal. (B) Clade I: Leptochiton rugatus, Sooke, Vancouver Island, Canada, intertidal. (C) Clade I: Leptochiton boucheti, Vanuatu, 667–750 m. (D) Protochitonidae: Hanleyella oldroydi, Cortes Bank, CA, USA, 367–389 m. (E) Ferreiraellidae: Ferreiraella plana, Vanutau, 630–705 m. (F) Clade II: Nierstraszella lineata, Solomon Islands, 490–520 m. Photos by J. D. Sigwart, except D, photo by G. Giribet. complete 18S rRNA (~1800 bp), a large fragment of 28S rRNA (~2200 bp, compared with the ~300 bp used by Okusu et al. 2003), and the mitochondrial protein-coding gene cytochrome c oxidase subunit I (COI; 650 bp). We also utilised a morphological data matrix for the sampled taxa and combined the morphological and molecular data in the first combined analysis for the class Polyplacophora. Materials and methods Taxon selection In total, 57 specimens from 38 ingroup species were treated for this study, including museum specimens fixed in ethanol, and original field collections of live animals (Table 1). Species level identifications for all specimens were verified by their 562 Invertebrate Systematics J. D. Sigwart et al. Table 1. Taxonomic arrangement of the polyplacophoran suborder Lepidopleurina (order Lepidopleurida) This table includes only living genera and families; genera in bold are included in the present study. Modified from Sirenko (2006) Suborder Family Lepidopleurina Thiele, 1909 Ferreiraella Sirenko, Ferreiraellidae 1988 Dell’Angelo & Palazzi, 1991 Hanleyidae Bergenhayn, Hanleya Gray, 1857 1955 Lepidopleurus Risso, Leptochitonidae Dall, 1889 1826 Leptochiton Gray, 1847 Parachiton Thiele, 1909 Pilsbryella Nierstrasz, 1905 Nierstraszellidae Sirenko, Nierstraszella Sirenko, 1993 1993 Protochitonidae Ashby, Deshayesiella Carpenter 1925 in Dall, 1879 Oldroydia Dall, 1894 A Hanleyella Sirenko, 1973 A Genus Based on the results of the present study, Hanleyella is tentatively included in Protochitonidae rather than Leptochitonidae. Table 2. Universal primer sequences used for DNA amplification Each of the three fragments for the two ribosomal genes was maintained as an independent input file (see also Table 3). The relative position of primers for 18S rRNA are based on the sequence of Limulus polyphemus (GenBank accession L81949) and for 28S rRNA are based on the complete sequence of L. polyphemus (AF212167) (see map of 28S rRNA primers in Giribet and Shear 2010) Gene fragment and primer name 18Sa: 1F 18Sb: 3F 18Sa: 4R 18Sb: 7R 18Sb: 18Sbi 18Sc: 18Sa2.0 18Sc: 9R 28Sa: 28S rd1a 28Sa: 28S rd4b 28Sa: 28Sb 28Sb: 28Sa 28Sb: 28S rd5b 28Sc: 28S rd4.8a 28Sc: 28S rd7b1 COI: LCO1490 COI: HCOout morphology. All specimens were fixed in 70–99% EtOH and preserved in 80–99% EtOH at
80C. Additional outgroup taxa representing Chitonida (Chitonina and Acanthochitonina) were selected to represent uncontroversial major groups, as well as the genus Callochiton Gray, 1847, which has previously been resolved as the immediate sister group to Lepidopleurida (Okusu et al. 2003), or sister to the remaining Chitonida (Buckland-Nicks 2006, 2008; Giribet et al. 2006; Wilson et al. 2010). Two specimens of Leptochiton medinae (Plate, 1899) were combined into a single terminal for the molecular study, as they did not provide overlapping in the amplified fragments. DNA extraction, amplification, and sequencing A small tissue sample was removed for each specimen from the muscle tissue of the foot or girdle. For small-bodied taxa (<6 mm long) a large portion of the animal body was used for DNA extraction. Total DNA was extracted using the DNeasy Tissue Kit (QIAGEN, Valencia, CA) using the standard protocol for extraction and purification recommended by the supplier. The purified total DNA was amplified in the target gene fragments using polymerase chain reaction (PCR; see primers in Table 2). Two nuclear ribosomal genes (nearly complete 18S rRNA and a 2 Kb fragment of 28S rRNA) were amplified in three overlapping fragments each using the primers described in Edgecombe and Giribet (2006). In addition, the mitochondrial protein-coding gene cytochrome c oxidase subunit I (COI) was amplified as a single fragment using the primer pair LCO1490/ HCO2198 (Folmer et al. 1994). Polymerase chain reactions were performed in 50 mL volume, including: 2 mL of the purified template DNA, 1 mM of each primer (0.5 mL of 20 mm stock), 200 mM of each dNTP (Invitrogen), 1 PCR buffer containing 1.5 mM MgCl2 (Perkin Sequence position 1 bp 376 bp 569 bp 1421 bp 1319 bp 1120 bp 1781 bp 26 bp 888 bp 1220 bp 888 bp 1419 bp 1328 bp 2222 bp Primer sequence (50 –30 ) TAC CTG GTT GAT CCT GCC AGT AG GTT CGA TTC CGG AGA GGG A GAA TTA CCG CGG CTG CTG G GCA TCA CAG ACC TGT TAT TGC GAG TCT CGT TCG TTA TCG GA ATG GTT GCA AAG CTG AAA C GAT CCT TCC GCA GGT TCA CCT AC CCC SCG TAA YTT AAG CAT AT CCT TGG TCC GTG TTT CAA GAC TCG GAA GGA ACC AGC TAC GAC CCG TCT TGA AGC ACG CCA CAG CGC CAG TTC TGC TTA C ACC TAT TCT CAA ACT TTA AAT GG GAC TTC CCT TAC CTA CAT GGT CAA CAA ATC ATA AAG ATA TTG G CCA GGT AAA ATT AAA ATA TAA ACT TC Elmer), 1.25 units of AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, CT), and ddH2O. The PCR were performed on a GeneAmp PCR System 9700 thermal cycler, using a thermal cycling regime based on the protocol developed by Okusu et al. (2003). The cycle included an initial denaturation step (5 min at 95C) followed by 35 cycles of denaturation (95C for 30 s), annealing (30 s at 44–46C, experimentally determined for each sample), and extension (72C for 1 min). After the 35 cycles were completed there was a final extension step at 72C for 1 min. Polymerase chain reaction products were visualised by electrophoresis in a 1% agarose gel. Successfully amplified products were then purified using the QIAquick PCR purification kit (QIAGEN). Purification was followed by a sequence reaction to generate single-stranded purified products for direct sequencing. Each sequence reaction, of a total volume of 10 mL, was made up of: 2 mL of the PCR product, 1 mL of one of the PCR primer pairs, 2 mL of halfTERM Dye Terminator Reagent (Genpak, Stony Brook, NY), and 2 mL of ABI BigDye Terminator v3.0 (Applied Biosystems, Foster City, CA), and ddH2O. The sequence reactions, performed using the thermal cycler described above, involved an initial denaturation step for 3 min at 95C, and 25 cycles (95C for 10 s, 50C for 5 s, 60C for 4 min). The BigDye labelled, single-stranded PCR products were finally cleaned with AGTC® Gel Filtration Cartridges (Edge BioSystems, Gaithersburg, MD). The sequence reaction products were then analysed using an ABI Prism 3100 Genetic Analyser (Applied Biosystems). The chromatograms were visualised using the software Sequencher 4.0 (Gene Codes Corporation, Ann Arbor, MI). Combined analysis of primitive living chitons (Lepidopleurida) Forward and reverse fragments were assembled to form double-stranded products and chromatograms were compared for consistency. For 28S and 18S rRNA, the three amplicons obtained for each gene were merged into a single sequence. Exemplars from consistent homologous regions were tested using NCBI BLAST (National Center for Biotechnology Information basic local alignment search tool) to confirm that they corresponded with known polyplacophoran sequences deposited in GenBank. Any oddities or strikingly inconsistent regions were also checked this way to ensure there was no contamination. Individual amplicon analyses were also conducted to check for possible contaminant sequences. Final sequences were edited and aligned using the software MacGDE (Smith et al. 1994; Linton 2005). The datasets included additional sequences obtained from GenBank as outgroups (see Table 3). All sequences were then split into fragments using internal primers and secondary structure features (Giribet and Wheeler 2001; Giribet 2002) for subsequent analyses. From each final sequence, known external primers were excluded. Due to the lack of amplicons for some ribosomal fragments due to poor tissue preservation (mostly of the deep sea species), each of the three fragments for the two ribosomal genes was maintained as an independent input file (see also Appendix 1). The protein-coding gene COI showed no length variation among the taxa studied. Morphology Morphological features were coded according to the published matrix of Sigwart (2009), including 69 characters for shell, girdle, radula, and gill arrangement. All characters were non-additive. Additional outgroup taxa were coded from specimens in the Royal BC Museum (Victoria, Canada). Five ingroup taxa used by Sigwart (2009) were not included here because suitable material was unavailable: Leptochiton alveolus (Sars MS, Lovén, 1846), L. binghami (Boone, 1928), L. inquinatus (Reeve, 1847), L. scabridus (Jeffreys, 1880), and L. thandari Sirenko, 2001. Material coded as L. americanus Kaas & Van Belle, 1985 by Sigwart (2009) has subsequently been reidentified by one of the authors (ES) as L. laurae Schwabe & Sellanes, 2010. The present study also added four new ingroup taxa to the analysis: Leptochiton cf. giganteus (Nierstrasz, 1905), an undescribed Leptochiton sp. from the Gulf of Mexico, Parachiton hodgsoni Sirenko, 2000, and Hanleyella oldroydi (Bartsch MS, Dall, 1919). For details and discussion on the morphological characters see Sigwart et al. (2007) and Sigwart (2009a). Analyses Phylogenetic analysis was conducted in the program POY ver. 4 (Varón et al. 2010) for the molecular and combined analyses of morphology and molecules using parsimony under direct optimisation (Wheeler 1996). Analysis of the morphological dataset alone did not differ from the results obtained by Sigwart (2009a). All genes were analysed independently and in combination under a set of 10 analytical parameters varying the indel : change ratio and the transversion : transition ratio in a sensitivity analysis fashion (Wheeler 1995). One parameter set also explored different costs for opening and extending indels (De Laet Invertebrate Systematics 563 2005). The morphological characters received a weight of 1 each when combined with the molecular data. All phylogenetic analyses were run in a cluster of Dell Blades (8 processors per blade, 32 Gb of RAM) using 20–40 processors. A typical analysis consisted of a timed search (driven search) of two hours each with up to 100 Wagner trees. The timed search of POY implements a default search strategy that effectively combines tree building with TBR branch swapping, parsimony ratchet, and tree fusing (see Goloboff 1999). Nodal support was calculated via bootstrapping. The optimal parameter set was obtained according to a modified Mickevich–Farris character incongruence metric (ILD; Mickevich and Farris 1981). Results Extraction of usable DNA from Lepidopleurida was problematic. During the course of this work, DNA was extracted from more than 80 specimens representing 45 ingroup taxa; however, amplification was truly successful in only 38 ingroup species. In some cases samples did appear to amplify for some regions, but the relatively low annealing temperatures required often resulted in poor quality sequences. This poor DNA quality was most likely due to the deep sea habitat of many of the specimens and the time spent between collection and preservation of tissues, as well as the current lack of specific primers that could improve amplification quality. In all analyses, the order Lepidopleurida is monophyletic relative to the species sampled from Chitonida, and most closely related to species in Callochiton. The large ingroup genus Leptochiton Gray, 1847 is clearly not monophyletic. Comparing the results from analyses under 10 different parameter sets, equal weights (i.e. 1 : 1 for both transversion : transition and indel : transversion ratios) minimised incongruence in the combined molecular analysis (Table 4). This combined analysis of three gene regions resulted in a single most parsimonious tree of length 6077. However, when the data were analysed including morphological characters, the optimal parameter set was 3221 (indel opening = 3; transversions = transitions = 2; indel extension = 1). This combined analysis resulted in a single most parsimonious tree of length 13 282. These two trees are shown in Fig. 2. Additional investigation of the trees resulting from single gene phylogenies had limited phylogenetic signal, but the 18S rRNA tree was most similar to that resulting from combined analyses. These two resulting trees, from the combination of three genes (Fig. 2A), and three genes plus morphology (Fig. 2B), consistently resolve several internal clades. Ferreiraellidae, represented by two species in the genus Ferreiraella Sirenko, 1988, is monophyletic. The family Protochitonidae includes Deshayesiella Carpenter MS, Dall, 1879 and Oldroydia Dall, 1894 – the clade resolved here, which we label Protochitonidae also includes Hanleyella Sirenko, 1973. The clade that we label Leptochitonidae sensu stricto includes the type species of the family (Leptochiton asellus (Gmelin, 1791)) and other species sampled from the North Atlantic and Mediterranean. Clade I includes the genus Parachiton Thiele, 1909 as well as several primarily Pacific Leptochiton species; however, also in this clade, L. intermedius (Salvini-Plawen, 1968) is from the Aegean Sea, and Leptochiton ‘sp.’ is an undescribed species collected from 564 Lepidopleurida : Ferreiraellidae Ferreiraella plana Ferreiraella xylophaga karenae A Ferreiraella xylophaga karenae B Ferreiraella xylophaga karenae C Lepidopleurida : Hanleyidae Hanleya nagelfar Lepidopleurida : Leptochitonidae Lepidopleurus cajetanus Leptochiton aequispinus Leptochiton algesirensis Leptochiton asellus A 18S 28S COI General region Specimen locality MNHN – Boa1 CP2465 MNHN – Boa1 CP2432 MNHN – Boa1 CP2433 MNHN – Solomon2 CP2212 HQ907740 HQ907739 HQ907738 HQ907741 HQ907795 HQ907796 HQ907798 HQ907797 HQ907844 HQ907845 HQ907846 SW Pacific SW Pacific SW Pacific SW Pacific Vanuatu; 770–799 m; 2005 Vanuatu: Big Bay; 630–705 m; 2005 Vanuatu: Big Bay; 593–630 m; 2005 Solomon Islands: Sta Isabel; 400–475 m; 2004 Sneli HQ907742 HQ907799 N Atlantic/Mediterranean Iceland: Bioice stn. 3589; 2002 MCZ DNA100108 Saito Dell’Angelo Sneli AF120502 HQ907743 HQ907744 HQ907747 HQ907802 HQ907803 HQ907804 HQ907807 HQ907847 HQ907848 HQ907849 HQ907851 N Atlantic/Mediterranean Japan N Atlantic/Mediterranean N Atlantic/Mediterranean ZSM 20050590 ZSM 20008014 HQ907808 HQ907806 AY145414 AY377662 HQ907809 HQ907810 HQ907853 HQ907854 HQ907852 HQ907855 HQ907856 HQ907857 N Atlantic/Mediterranean N Atlantic/Mediterranean N Atlantic/Mediterranean N Atlantic/Mediterranean SW Pacific SW Pacific SW Pacific N Atlantic/Mediterranean SW Pacific SW Pacific Spain: Tossa de mar, Girona; ~10 m; 1997 Japan: Sagami Bay; 240–418 m; 2002 Italy: Sardinia, S’Archittu; 2003 Norway: Aksnestangen, Trondheim; 50–200 m; 2004 Sweden: Gullmarsundfjord; 30 m; 2003 Sweden: Tjärnö; 2000 Sweden: Kristineberg MRS Sweden: Tjärnö; 2000 Vanuatu: Big Bay; 773–900 m; 2005 Vanuatu: Malo; 373–800 m; 2005 Vanuatu: Big Bay; 773–900 m; 2005 France: Bretagne, off Roscoff; 8 m; 2003 Vanuatu: Big Bay; 773–900 m; 2005 Philippines: Bohol/Sulu seas sill; 679–740 m; 2005 Angola: 179’S 1121’E; 2004 Philippines: Bohol/Sulu seas sill, Dipolog Bay; 150–163 m; 2005 Philippines: Bohol Sea, off Pamilacan Island; 273–356 m; 2005 USA: California, Cortes Bank; 367–389 m; 2007 Japan: Shibasaki, Miura Peninsula, Japan, intertidal; 2006 Croatia: Istira, Rovinje, Punta Corente; 0–4 m; 2004 Japan: Sagami Bay; 94–95 m; 2002 Vanuatu; 618–641 m; 2005 South Georgia and South Sandwich Islands, 42.55’S 27 57.02’W; 332.3–356.0m; 2002 Chile: off Concepcion, 3621.650 S 7344.42’W; 900–904 m; 2004 Leptochiton asellus B Leptochiton asellus C Leptochiton asellus D Leptochiton asellus E Leptochiton boucheti A Leptochiton boucheti B Leptochiton boucheti C Leptochiton cancellatus Leptochiton deforgesi A Leptochiton deforgesi B MCZ DNA100830; ZSM 20008014 MNHN – Boa1 CP2435 MNHN – Boa1 CP2412 MNHN – Boa1 CP2435 ZSM 20034176 MNHN – Boa1 CP2435 MNHN – Panglao CP2362 HQ907748 HQ907746 AY145382 AY377631 HQ907750 HQ907751 HQ907749 HQ907752 HQ907753 HQ907754 Leptochiton denhartogi Leptochiton foresti A ZSM 20034402 MNHN – Panglao CP2380 HQ907755 HQ907756 HQ907813 HQ907814 HQ907858 HQ907859 E Atlantic SW Pacific Leptochiton foresti B MNHN – Panglao CP2343 HQ907757 HQ907815 HQ907860 SW Pacific Leptochiton cf. giganteusM MCZ DNA102583 HQ907779 HQ907801 HQ907873 E Pacific/N Pacific Leptochiton hirasei Saito HQ907758 HQ907816 HQ907861 Japan Leptochiton intermedius ZSM 20040266 HQ907759 HQ907817 Leptochiton japonicus Leptochiton juvenis Leptochiton kerguelensis Saito MNHN – Boa1 CP2462 ZSM 20021483 HQ907760 HQ907761 HQ907762 HQ907818 HQ907819 HQ907820 HQ907862 HQ907863 HQ907864 Japan SW Pacific Antarctica Leptochiton laurae ZSM 20041460 HQ907745 HQ907805 HQ907850 Antarctica Taxa that were not included in the morphological cladistic analysis of Sigwart (2009a). HQ907811 HQ907812 N Atlantic/Mediterranean (continued next page ) J. D. Sigwart et al. M Specimen number/origin Invertebrate Systematics Table 3. GenBank accession numbers and collection and locality data for specimens used in this study All specimens are deposited in museum collections: MNHN, Muséum national d’Histoire naturelle; MCZ, Museum of Comparative Zoology, Harvard University; ZSM, Zoologische Staatssammlung München; all others in National Museum of Ireland, Natural History Division, Dublin. The ‘general region’ refers to the coloured biogeographic regions illustrated in Fig. 2 18S 28S COI General region Specimen locality Leptochiton medinae ZSM 20021117 (=MCZ DNA100876); ZSM 20050450 HQ907763; HQ907764 HQ907821 HQ907865 Antarctica Leptochiton cf. pergranatus FMNH – GC 234–4435 HQ907773 HQ907829 Leptochiton rugatus A Sirenko HQ907769 HQ907826 Leptochiton rugatus B Leptochiton saitoi A Leptochiton saitoi B Leptochiton vanbellei Leptochiton vaubani Sirenko MNHN – Panglao CP2356 MNHN – Boa1 CP2466 MNHN – Boa1 CP2435 MNHN – Solomon2 CP2246 HQ907770 HQ907771 HQ907772 HQ907775 HQ907768 HQ907827 HQ907828 HQ907831 HQ907825 Leptochiton vietnamensis A MNHN – Panglao CP2385 HQ907776 HQ907832 Leptochiton vietnamensis B MNHN – Panglao CP2385 HQ907777 HQ907833 Leptochiton vietnamensis C Leptochiton n. sp. 4 A Leptochiton n. sp. 4 B MNHN – Panglao CP2356 MNHN – Boa1 CP2479 MNHN – Panglao CP2380 HQ907778 HQ907765 HQ907766 HQ907834 HQ907822 HQ907823 Leptochiton n. sp. 5 Leptochiton sp.M MNHN – Boa1 CP2433 FMNH 306049 HQ907767 HQ907774 HQ907824 HQ907830 Parachiton acuminatus ZSM 20033088 HQ907787 Saito ZSM 20050798 Saito HQ907788 HQ907789 HQ907790 South Georgia and Sandwich Islands, 5844.35’S 2510.48’W, 725–815 m (ZSM 20021117); Chile: Fuerto Bulnes, S of Punta Arenas; 2005 (ZSM 20050450) USA: Gulf of Mexico, Bush Hill vent area; 2005 Russia: Ussuriyskiy Bay, Sea of Japan; 2–4 m; 2004 Russia: Vostok Bay; 2.0–2.5 m; 2003 Philippines: Bohol Sea; 1764 m; 2005 Vanuatu; 786–800 m; 2005 Vanuatu: Big Bay; 773–900 m; 2005 Solomon Islands: Sta Isabel; 664–682 m; 2004 Philippines: Bohol/Sulu seas sill; 982–989 m; 2005 Philippines: Bohol/Sulu seas sill; 982–989 m; 2005 Philippines: Bohol Sea; 1764 m; 2005 Vanuatu; 350–358 m; 2005 Philippines: Bohol/Sulu seas sill, Dipolog Bay; 150–163 m; 2005 Vanuatu: Big Bay; 593–630 m; 2005 USA: Gulf of Mexico, Bush Hill vent area; 2005 Indonesia: Sulawesi, Mantehage Island; 7.5 m Japan: Gahi-jima, Kerama Islands; 9 m; 2006 South Africa: Cape Aguthas; 2005 Japan: Gahi-jima, Kerama Islands; 9 m; 2006 MNHN – Panglao CP2385 Nierstraszella lineata A Nierstraszella lineata B E Pacific/N Pacific (Japan) HQ907869 HQ907870 HQ907871 HQ907867 E Pacific/N Pacific (Japan) SW Pacific SW Pacific SW Pacific SW Pacific HQ907872 SW Pacific SW Pacific HQ907866 SW Pacific SW Pacific SW Pacific SW Pacific W Atlantic/Gulf of Mexico SW Pacific HQ907841 HQ907880 HQ907881 SW Pacific (Japan) E Atlantic SW Pacific (Japan) HQ907781 HQ907835 HQ907875 SW Pacific MNHN – Panglao CP2380 HQ907782 HQ907836 HQ907876 SW Pacific MNHN – Solomon2 CP2211 HQ907783 HQ907837 Saito ZSM 20034397 HQ907784 HQ907785 HQ907838 HQ907839 HQ907877 SW Pacific (Japan) SW Pacific (Japan) Sirenko HQ907737 HQ907794 HQ907843 E Pacific/N Pacific (Japan) Hanleyella oldroydi*M MCZ DNA102582 HQ907780 HQ907800 HQ907874 E Pacific/N Pacific Oldroydia percrassa ZSM 20040613 HQ907786 HQ907878 E Pacific/N Pacific Nierstraszella lineata C Nierstraszella lineata D Lepidopleurida : Protochitonidae Deshayesiella curvata HQ907840 SW Pacific Philippines: Bohol/Sulu seas sill; 982–989 m; 2005 Philippines: Bohol/Sulu seas sill, Dipolog Bay; 150–163 m; 2005 Solomon Islands: Sta Isabel; 313–387 m; 2004 Japan: Suruga Bay; ~500 m; 2003 Japan: Suruga Bay; 1999 Russia: Ussuriyskiy Bay, Sea of Japan; 2–4 m; 2004 USA: California, Cortes Bank; 367–389 m; 2007 USA: California, off La Jolla; 1972 (continued next page ) 565 Taxa that were not included in the morphological cladistic analysis of Sigwart (2009a). Revision based on present analysis (previously in Leptochitonidae). * HQ907868 HQ907879 Parachiton communis Parachiton hodgsoniM Parachiton politus Lepidopleurida : Nierstraszellidae Nierstraszella andamanica M W Atlantic/Gulf of Mexico Invertebrate Systematics Specimen number/origin Combined analysis of primitive living chitons (Lepidopleurida) Table 3. (continued ) 566 Table 3. (continued ) Lorica volvox Mopalia muscosa Tonicella lineata 18S 28S COI General region MCZ DNA100109 MCZ DNA101902 MCZ DNA100833 (partim) MCZ DNA100592 MCZ DNA100837; DNA101109 AF120503 HQ907736 AY377636 AY377655 AY377656 DQ279957 HQ907792 AY145398 AY377686 AY145402 AF120627 AY377704 AY377720 HQ907842 N Atlantic/Mediterranean W Atlantic/Gulf of Mexico W Atlantic/Gulf of Mexico E Pacific/N Pacific Japan MCZ DNA100873 MCZ DNA100831 HQ907735 AY377632 HQ907791 DQ279952 AY377700 Antarctica N Atlantic/Mediterranean MCZ DNA100579 MCZ DNA100157 AY377645 AY377651 DQ279953 AY377682 AY377712 AY377716 Australia N Atlantic/Mediterranean MCZ DNA100834 AY145380 AY145412 AY377709 Japan MCZ DNA100599 AY377650 MCZ DNA100571 MCZ DNA100522 MCZ DNA100580 AY377647 AY377648 AY377635 AY377681; HQ907793 DQ279954 DQ279956 AY377665 Specimen locality Invertebrate Systematics Chitonida : Acanthochitonidae Acanthochitona crinita Acanthochitona rhodea Chaetopleura apiculata Cryptochiton stelleri Cryptoplax japonica Chitonida : Callochitonidae Callochiton bouveti Callochiton septemvalvis Chitonida : Chitonidae Callistochiton antiquus Chiton olivaceus Chitonida : Ischnochitonidae Ischnochiton comptus Chitonida : Mopaliidae Katharina tunicata Specimen number/origin E Pacific/N Pacific AY377713 AY377702 Australia E Pacific/N Pacific E Pacific/N Pacific J. D. Sigwart et al. Combined analysis of primitive living chitons (Lepidopleurida) Invertebrate Systematics 567 Table 4. Tree lengths and ILD results The first numeral used in the parameter set (leftmost) column corresponds to the ratio between indel : transversion and the following two numbers correspond with the ratio between transversion : transition; e.g. 111 is equal weights, 121 corresponds to an indel : transversion ratio of 1 and a transversion : transition ratio of 2 : 1 111 121 141 211 221 241 411 421 441 3221 18S 28S COI MOL MOR TOT ILD MOL ILD TOT 721 1059 1714 789 1185 1965 903 1404 2390 1472 2470 3646 5911 2730 4104 6809 3152 4903 8358 5156 2713 3959 6331 2719 3962 6342 2719 3962 6346 5460 6077 8926 14 436 6444 9593 15739 7053 10 773 18 057 12 465 594 594 594 594 594 594 594 594 594 594 6875 9741 15 252 7256 10 411 16 557 7862 11 589 18 896 13 282 0.02847 0.02935 0.03325 0.03197 0.03565 0.03958 0.03956 0.04678 0.05333 0.03024 0.05484 0.04958 0.04603 0.05843 0.05437 0.05116 0.06283 0.06265 0.06393 0.04517 cold seep habitats in the Gulf of Mexico, reported by Cordes et al. (2005) as L. alveolus. Clade II is primarily made up of species found living in sunken wood deposits and from the tropical West Pacific. The habitats of two species also included in Clade II are not well documented: L. medinae (Chile), and L. kerguelensis Haddon, 1886 (Antarctica). The two species of Nierstraszella Sirenko, 1992, included in Clade II, do not form a single clade and Nierstraszella may include the specimen identified as L. vietnamensis A Sirenko, 1998. The two other genera represented by multiple species in this analysis, Ferreiraella and Parachiton, are monophyletic, but Parachiton includes L. intermedius. There are a small number of taxa that also fall outside these groupings. Hanleya Gray, 1857 is clearly within Lepidopleurina but does not resolve with any of the larger clades. The same is true for the species pair Leptochiton japonicus (Thiele, 1909) and L. aequispinus (Bergenhayn, 1933). The relationships between these clades are different between the two resulting trees (Fig. 2). Sister relationships between Protochitonidae and Leptochitonidae s. str., and between Ferreiraellidae and Clade I, are supported by both trees and effectively every permutation of the analysis. Discussion This study, although taxonomically focussed on one clade within Polyplacophora, is substantially larger both in taxon sampling and in genetic sampling than any previous work on chitons. All nine accepted genera within Lepidopleurida were represented. Four additional genera or subgenera that are of interest to the definition of this group were not included here because specimens were unavailable or did not yield good quality DNA. The monotypic Pilsbryella was excluded from Sirenko’s (2006) classification, but has several distinctive morphological characteristics that separate it from the ‘typical’ Leptochiton (Kaas and Van Belle 1985). Hemiarthrum Carpenter in Dall, 1876, Weedingia Kaas, 1988, and Choriplax Pilsbry, 1894 have been historically placed in Lepidopleurida, but more recent classifications have included them in the order Chitonida (e.g. Sirenko 2006 contra Kaas and Van Belle 1985). The 57 ingroup specimens were selected to represent 38 nominal species, which differ slightly from those sampled by Sigwart (2009). The results demonstrate several instances of probable cryptic species: Leptochiton vietnamensis, L. deforgesi Sirenko, 2001, and L. boucheti Sirenko, 2001. Other species that were represented by a single specimen may also hide species complexes and this may apply to any of the species included. We have presented two preferred trees, one from molecular data and the second including morphological characters: both resolve the same clades, but propose different relationships between them. Distribution, habitats, and biogeography The Japanese specimens included in this analysis demonstrate that the lepidopleuran fauna of Japan does not represent a single biogeographic province. Taxa from the southern islands of Japan (Parachiton communis, P. politus, Nierstraszella lineata C and D) group with other species from the tropical south-west Pacific. Those from the northern part of the Sea of Japan, on the Russian coast (Leptochiton rugatus, Deshayesiella curvata) have sister relationships with taxa from the Eastern Pacific. The fauna of central Japan consists of three different elements, northern, tropical, and temperate, in a mixing zone between the Kuroshio and Oyashio currents (Ekman 1953; Okutani 1969). The three ingroup species that we examined from central Japan do not form a clade, and the pair L. japonicus and L. aequispinus do not resolve a clear relationship with the other major clades. Substantial work remains to be done to understand the biogeography of the central Japanese fauna. The analysis is dominated by taxa from the tropical south-west Pacific, comprising half of the ingroup terminals. These taxa occur in three areas of the tree, with the majority of taxa in Clade II, but separate from a few in Clade I, and the Ferreiraellidae. Those in Clade I are found only north of Papua New Guinea, in the Philippines (Leptochiton foresti) and southern Japan (Parachiton communis, P. politus). Another species, Parachiton acuminatus is known primarily from the Bismarck Sea but specimens have also been recovered from New Caledonia (Enrico Schwabe, unpubl. data). Eight other terminals in Clade II are also from the Philippines, but all in species that have ranges extending south to the Solomon Islands or as far as New Caledonia (Table 3). Clade II has a biogeographic origin in the south-west Pacific, with subsequent radiation to Antarctica and Japan. Nierstraszella 568 Invertebrate Systematics J. D. Sigwart et al. Fig. 2. Two alternative phylogenetic trees illustrating relationships within Lepidopleurida. We identified five ingroup clades: Leptochitonidae (Lepto), Protochitonidae (Proto), Ferreiraellidae (Ferreira), and two others numbered I and II. Dotted lines in the ingroup indicate species that are specialist on sunken wood substrates. Coloured dots show general geographic regions of the range of each species, as indicated in inset map. Where multiple exemplars of a species were included they are noted A, B, C (for specimen information, see Table 3). Numbers on branches indicate jackknife support values. (A) Combined analysis of molecular data from three loci (MOL) analysed under the optimal parameter set 111, single most parsimonious tree (MPT) length 6077 steps. (B) Combined analysis of all molecular data and morphological data (TOT) under the optimal parameter set 3221, single MPT length 13 282. Combined analysis of primitive living chitons (Lepidopleurida) lineata and Leptochiton vietnamensis occur in Japan and in the South China Sea, so it is not surprising that this clade could also encompass species such as L. hirasei, which is known only from Japan. The Antarctic species L. kerguelensis has a circumpolar distribution in the Southern Ocean (Schwabe 2008b), whereas L. medinae is known from Tierra del Fuego and both coasts of Patagonia (Schwabe and Sellanes 2010). Clade I contains the other Antarctic species of Leptochiton s.l. included in our analysis, indicating there have been at least two separate invasions of lepidopleuran chitons to the Southern Ocean, in contrast with the Antarctic as a source of radiation in other molluscs (Strugnell et al. 2008). Sirenko (2004) postulated that Ferreiraella plays a pivotal role in the ancient origins of lepidopleuran taxa, in its morphological affinity with some of the earliest neoloricate fossils, and further that this was evidence for sunken wood as the ancestral habitat of lepidopleurans as a group. Our data suggest two separate colonisations of sunken wood habitats, with Ferreiraellidae separate from Leptochiton s.l. in Clade II (Fig. 2). But the wood dwelling taxa consistently occur as the earliest derived members of the local part of the tree. Sunken wood may be a critical factor in the origin and radiation of species in the south-west Pacific (in Clade II), although other members of this clade in Antarctica and possibly the Atlantic have adapted to other habitat substrates. Sunken wood has been postulated in the origins of chemosynthetic deep sea habitats (Distel et al. 2000). We include three species from cold seep habitats: Leptochiton sp. and L. laurae in Clade I, and L. cf. pergranatus in Clade II. These terminals consistently resolve in close proximity to sunken wood species, but without strong support. Resolving molecules and morphology Lepidopleuran shells typically lack insertion plates, lateral extensions of the ventral shell that anchor the shell to the girdle muscle block. But this shell feature is partially expressed in several taxa. Three genera in Lepidopleurina (sensu Sirenko 2006), Ferreiraella, Deshayesiella, and Hanleya, have shells with unslit insertion plates. Sirenko (1997, 2006) has discussed the potential for multiple evolutionary origins of shell insertion plates within Polyplacophora. Our trees (Fig. 2A, B) indicate that there have been (at least) three separate origins of insertion plates within Lepidopleurida, as these three genera occur in disparate parts of the tree. Ferreiraella species have well developed, unslit insertion plates on both terminal valves. The genus is restricted to sunken wood habitats and is also characterised by having a ‘naked’ ventral girdle, not covered in spicules, and distinctive spatulate lateral teeth on the radula (Sirenko 1988; Saito 2006). Two of the eight described species in this genus were included in the present analysis. The family Ferreiraellidae includes only one living genus, Ferreiraella, and several Carboniferous fossil chitons that share the affinity for sunken wood (Sirenko 2004, 2006). The living species encompass a worldwide distribution (Caribbean, Eastern and Western Pacific) and a more detailed molecular phylogeny of this genus could test Sirenko’s (2004) hypothesis about the ancient origin of this family. Invertebrate Systematics 569 Hanleya is the only genus in the family Hanleyidae, although historical classifications have included other morphologically disparate genera that also have unslit insertion plates. This analysis has not clearly resolved the position of Hanleya relative to other taxa included. Hanleya nagelfar is interesting because it is very large for the group (up to 60 mm long, whereas the majority of lepidopleurans are less than 20 mm) and spongivorous (Todt et al. 2009). Its relationship to proposed congeners is worth further study (Warén and Klitgaard 1991). This genus is distinctly different from other lepidopleurans based on morphological and now also molecular data, but still resolves within Lepidopleurida. Hanleya and Deshayesiella are known to differ from Leptochiton in several features of gamete morphology. The former two have egg hulls with a jelly coat punctured by macropores that serve as specific sites for sperm entry, whereas Leptochiton eggs have a completely smooth jelly coat without specific sites for sperm penetration (Buckland-Nicks 2008). The present analysis did not support a grouping that would include both Hanleya and Deshayesiella. But gamete data are not yet available for many species, and it would not be surprising to determine that Oldroydia and Hanleyella also share the same egg morphology and that this is a consistent character of Hanleyidae and Protochitonidae. Recent work by Sirenko and Clark (2008) highlights the similarity between a resurrected species of Deshayesiella, and the monotypic Oldroydia percrassa, which have very similar shell morphology. These two genera were included as the only living genera in the family Protochitonidae in the revised taxonomy of Sirenko (2006) – we suggest that Hanleyella is also a member of this family. Hanleyella oldroydi is one of the most abundant deep water chitons in the Southern California Bight (Stebbins and Eernisse 2009); most other species in this clade are quite rarely encountered. Nierstraszella is comprehensively defined by morphological features, particularly the characteristic fleshy proteinaceous layer that covers the dorsal shell surface (Sirenko 1992). Nierstraszella is also endemic to sunken wood substrates. Sigwart (2009b) recently revised the description of the species in Nierstraszella, identifying two distinct but broadly distributed species, which are both included here. Our consensus trees do not recover a monophyletic Nierstraszella, although some other parameter sets of the combined analysis do recover a monophyletic Nierstraszella including the exemplar of Leptochiton vietnamensis A (not figured). Although we believe this is not contamination it may represent cryptic or problematic identifications in L. vietnamensis. Parachiton is identified by a dramatically enlarged tail valve and distinctive radular morphology; however, our results show a species of Leptochiton within the genus. Morphological cladistic analysis also failed to resolve a Parachiton clade with the three species examined (Sigwart 2009), and the radular morphology is not consistent in all species (Sirenko 1999). The species pair Leptochiton japonicus and L. aequispinus are clearly closely related on the basis of morphological data. Our results further suggest that they are sister taxa and both significantly diverged from other Leptochiton taxa. Both species were considered to be junior synonyms of L. belknapi (Ferreira 1979; Kaas and Van Belle 1987), but have been reinstated 570 Invertebrate Systematics (Saito 1997). There are a number of wide ranging species of Leptochiton that are anecdotally accepted to contain multiple cryptic species, including particularly L. belknapi Dall, 1878 and allies (Ferreira 1979; Wu and Okutani 1984) and the species lumped wth L. rugatus (Carpenter in Pilsbry, 1892) (Ferreira 1979; Saito 2000; Stebbins and Eernisse 2009). These species complexes would particularly benefit from closer examination with molecular methods, and results could also illuminate the degree of morphological variability found within true species. Leptochiton was anticipated to be non-monophyletic, on the basis of rather vague anatomical descriptions in the genus definition, but this analysis has also highlighted other areas in need of taxonomic revision. The species currently included in Leptochiton are resolved across three major clades. The type species, L. asellus, is included in the clade that we consider to represent Leptochitonidae sensu stricto. Similarly the species of Leptochiton in this clade are considered to be Leptochiton s. str., but the clade also includes the monotypic Lepidopleurus Risso, 1826. The taxonomic relationship between Leptochiton and Lepidopleurus has created problems since 1892 and may continue to do so. Lepidopleurus was the first genus name proposed for lepidopleuran chitons. The genus was presented as a list including the monotypic L. cajetanus and two other unrelated species. Nearly twenty years later the genus name Leptochiton was established by Gray (1847). Both of these species were included in the family Leptochitonidae Dall, 1889 with Leptochiton asellus as the type species. Lepidopleurus cajetanus and Leptochiton asellus are both contained in our clade Leptochitonidae s. str. Only three years later, Pilsbry (1892) listed Leptochiton as a junior subjective synonym of Lepidopleurus, and changed the family name to Lepidopleuridae. The two generic names and family names have been used more or less interchangeably for the past 100 years. Sirenko (1979) argued for the reinstatement of Leptochitonidae by priority. This convention has been followed by most workers since that time, but some contemporary authors have advocated use of Lepidopleuridae (Dell’Angelo and Palazzi 1991). The higher ranks Lepidopleurida (order) and Lepidopleurina (suborder) are used universally. The nomenclature is further confused by colloquial use of the term ‘lepidopleurids’ to refer to members of the order, even by workers who use Leptochitonidae as the preferred family name. To circumvent a small part of this confusion we support the use of the common name ‘lepidopleuran’ as an alternative. The results of this analysis indicate that there is potentially not sufficient evidence to separate Lepidopleurus and Leptochiton s. str. as separate genera. The same topology is recovered by morphological characters alone (Sigwart 2009). Lepidopleurus has very distinctive shell morphology with pronounced concentric ridges on the lateral areas and terminal valves. The shell shape is in contrast with the typical flat and plain shells of most species of Leptochiton that might be marked with patterns of granules but generally lack strong raised sculpture. The morphological definitions of genera and families within Lepidopleurida are described from animals that differ from the norm set by Leptochiton asellus. The question remains, how to interpret relationships between these very different generic J. D. Sigwart et al. groups as well as within the majority of relatively plain and character-poor species. Morphological features clearly can resolve phylogenetic signal; however, the interpretation of morphology has not provided a suite of taxonomic characters that reliably split Lepidopleurida into subgroups. Any group that is so widespread, both in terms of geographic range and depth, and purportedly mostly belongs in a single genus, raises immediate doubts about monophyly and accuracy of classification. The phylogenetic hypotheses generated by this study will enable future testing of the taxonomy and classification within Lepidopleurida. The major genus, Leptochiton, contains most of the species named, but it is not supported by morphological synapomorphies and results as paraphyletic in all molecular analyses. The phylogeny proposed here will also provide a baseline to develop further studies and interpret evolutionary patterns within the order and within Polyplacophora. Acknowledgements For providing specimens, we thank: Boris Sirenko (St Petersburg, Russia), Hermann Strack (Britanny, France), Bruno Dell’Angelo (Bologna, Italy), and Jon Arne Sneli (Tromso, Norway). University of California Ship Funds to Nerida Wilson (California, United States) allowed for an invitation to Gonzalo Giribet on a cruise to the Cortes Bank that also provided specimens. Muséum national d’Histoire naturelle (MNHN) specimens were collected onboard the RV M/V DA-BFAR in the Philippines (expedition led by Philippe Bouchet and the National Fisheries Research and Development Institute Ludivina Labe), and the RV ALIS in Vanuatu and Solomon Islands (funding by the Institut de la Recherche pour le Dévelopement to Bertrand Richer de Forges, Sarah Samadi, and Philippe Bouchet). Two anonymous reviewers provided constructive comments that improved this paper. References Buckland-Nicks, J. (2006). Fertilization in chitons: morphological clues to phylogeny. Venus 65, 51–70. Buckland-Nicks, J. (2008). Fertilization biology and the evolution of chitons. American Malacological Bulletin 25, 97–111. doi:10.4003/0740-278325.1.97 Cordes, E. E., Hourdez, S., Predmore, B. L., Redding, M. L., and Fischer, C. R. (2005). Succession of hydrocarbon seep communities associated with the long-lived foundation species Lamellibranchia luymesi. Marine Ecology Progress Series 305, 17–29. doi:10.3354/meps305017 Dall, W. H. (1889). Report on the results of dredging, under the supervision of Alexander Agassiz, in the Gulf of Mexico (1877–78) and in the Caribbean Sea (1879–80), by the United States Coast Survey Steamer “Blake,” Lieutenant-Commander C. D. Sigsbee, U.S.N., and Commander J. R. Bartlett, U.S.N., commanding. Report on the Mollusca, Pt. 2: Gastropoda and Scaphopoda. Bulletin of the Museum of Comparative Zoology 18, 1–492. De Laet, J. E. (2005). Parsimony and the problem of inapplicables in sequence data. In ‘Parsimony, Phylogeny, and Genomics’. (Ed. V. A. Albert.) pp. 81–116. (Oxford University Press: Oxford.) Dell’Angelo, B., and Palazzi, S. (1991). Considerazioni sulla famiglia “Leptochitonidae” Dall 1889 (Mollusca, Polyplacophora). IV. Aggiunte e correzioni. Bollettino Malacologico 27, 35–38. Distel, D. L., Baco, A. R., Chuang, E., Morrill, W., Cavanaugh, C., and Smith, C. R. (2000). Do mussels take wooden steps to deep-sea vents? Nature 403, 725–726. doi:10.1038/35001667 Edgecombe, G. D., and Giribet, G. (2006). A century later – a total evidence re-evaluation of the phylogeny of scutigeromorph centipedes (Myriapoda: Chilopoda). Invertebrate Systematics 20, 503–525. doi:10.1071/IS05044 Combined analysis of primitive living chitons (Lepidopleurida) Ekman, S. (1953). ‘Zoogeography of the Sea.’ pp. 1–417. (Sidwick and Jackson: London, UK.) Ferreira, A. J. (1979). The family Lepidopleuridae (Mollusca: Polyplacophora) in the eastern Pacific. Veliger 22, 145–165. Folmer, O., Black, M., Hoeh, W., Lutz, R., and Vrijenhoek, R. C. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3, 294–299. Giribet, G. (2002). Relationships among metazoan phyla as inferred from 18S rRNA sequence data: a methodological approach. In ‘Molecular Systematics and Evolution: Theory and Practice’. (Eds R. DeSalle, G. Giribet, and W. C. Wheeler.) pp. 85–101. (Birkhäuser Verlag: Basel, Switzerland.) Giribet, G., and Shear, W. A. (2010). The genus Siro Latreille, 1796 (Opiliones, Cyphophthalmi, Sironidae), in North America with a phylogenetic analysis based on molecular data and the description of four new species. Bulletin of the Museum of Comparative Zoology 160, 1–33. doi:10.3099/0027-4100-160.1.1 Giribet, G., and Wheeler, W. C. (2001). Some unusual small-subunit ribosomal RNA sequences of metazoans. American Museum Novitates 3337, 1–14. doi:10.1206/0003-0082(2001)337<0001:SUSSRR>2.0.CO;2 Giribet, G., Okusu, A., Lindgren, A. R., Huff, S. W., Schroedl, M., and Nishiguchi, M. K. (2006). Evidence for a clade composed of molluscs with serially repeated structures: monoplacophorans are related to chitons. Proceedings of the National Academy of Sciences of the United States of America 103, 7723–7728. doi:10.1073/pnas.0602578103 Gmelin, J. F. (1791). Vermes, Testacea, genus 300: Chiton. In ‘C. von Linnaeus’ Systema Naturae, Tome 1, Pars 6. Edn. 13’. pp. 1106–1107. (Homiae: Stockholm, Sweden.) Goloboff, P. A. (1999). Analyzing large data sets in reasonable times: solutions for composite optima. Cladistics 15, 415–428. doi:10.1111/ j.1096-0031.1999.tb00278.x Gray, J. E. (1847). Additional observations on Chitones. Proceedings of the Zoological Society of London 15, 126–127. Kaas, P., and Van Belle, R. A. (1985). ‘Monograph of Living Chitons (Mollusca: Polyplacophora), Vol. 1.’ (E.J.Brill/Dr W. Bakhuys: Leiden.) Kaas, P., and Van Belle, R. A. (1987). ‘Monograph of Living Chitons (Mollusca: Polyplacophora), Vol. 3.’ (E.J.Brill/Dr W. Bakhuys: Leiden.) Kelly, R. P., and Eernisse, D. J. (2008). Reconstructing a radiation: the chiton genus Mopalia in the north Pacific. Invertebrate Systematics 22, 17–28. doi:10.1071/IS06021 Kelly, R. P., Sarkar, I. N., Eernisse, D. J., and DeSalle, R. (2007). DNA barcoding using chitons (genus Mopalia). Molecular Ecology Notes 7, 177–183. doi:10.1111/j.1471-8286.2006.01641.x Linton, E. W. (2005). MacGDE: Genetic Data Environment for MacOS X ver. 2.0. Software available at http://macgde.bio.cmich.edu/ [Accessed 1 September 2010]. Mickevich, M. F., and Farris, J. S. (1981). The implications of congruence in Menidia. Systematic Zoology 30, 351–370. doi:10.2307/2413255 Okusu, A., Schwabe, E., Eernisse, D. J., and Giribet, G. (2003). Towards a phylogeny of chitons (Mollusca, Polyplacophora) based on combined analysis of five molecular loci. Organisms, Diversity & Evolution 3, 281–302. doi:10.1078/1439-6092-00085 Okutani, T. (1969). Systematics, ecological distribution and paleoecological implications of archbenthal and abyssal mollusca from Sagami Bay and adjacent areas. Journal of the Faculty of Science, the University of Tokyo, section II 17, 1–98. Passamaneck, Y. J., Schander, C., and Halanych, K. M. (2004). Investigation of molluscan phylogeny using large-subunit and small-subunit nuclear rRNA sequences. Molecular Phylogenetics and Evolution 32, 25–38. doi:10.1016/j.ympev.2003.12.016 Pilsbry, H. A. (1892). Monograph of the Polyplacophora. In ‘Manual of Conchology’. (Ed. G. W. Tryon.) pp. 1–128. (Academy of Natural Sciences: Philadelphia, PA.) Invertebrate Systematics 571 Poli, J. X. (1791). Genus 1, Chiton. Testacea utriusque Siciliae corumque historia et anatome. Tomus 1, 1–11. [Ex Regio Typographeio: Parma, Italy.] Risso, A. (1826). ‘Histoire Naturelle des Principales Productions de I’Europe Méridionale et Particulièrement de Celles des Environs de Nice et des Alpes Maritimes 4.’ pp. 1–439. (Schoell: Paris.) Runnegar, B., Pojeta, J., Taylor, M. E., and Collins, D. (1979). New species of the Cambrian and Ordovician chitons Mattevia and Chelodes from Wisconsin and Queensland: evidence for the early history of polyplacopohran mollusks. Journal of Paleontology 53, 1374–1394. Saito, H. (1997). Deep-sea chiton fauna of Suruga Bay (Mollusca: Polyplacophora) with descriptions of six new species. National Science Museum Monographs 12, 31–58. Saito, H. (2000). Polyplacophora. In ‘Marine Mollusca of Japan’. (Ed. T. Okutani.) pp. 9–17. (Tokai University Press: Tokyo.) Saito, H. (2006). A new species of Ferreiraella Sirenko, 1988 (Mollusca: Polyplacophora) from the Philippine Basin. Venus 65, 91–96. Schwabe, E. (2008a). A summary of reports of abyssal and hadal Monoplacophora and Polyplacophora (Mollusca). Zootaxa 1866, 205–222. Schwabe, E. (2008b). Discovery of the South African polyplacophoran Stenosemus simplicissimus (Thiele, 1906) (Mollusca, Polyplacophora, Ischnochitonidae) in the Southern Ocean. American Malacological Bulletin 24, 71–77. doi:10.4003/0740-2783-24.1.71 Schwabe, E., and Sellanes, J. (2010). Revision of Chilean bathyal chitons (Mollusca: Polyplacophora) associated with cold-seeps, including description of a new species of Leptochiton (Leptochitonidae). Organisms, Diversity & Evolution 10, 31–55. doi:10.1007/s13127009-0002-6 Sigwart, J. D. (2008). Gross anatomy and positional homology of gills, gonopores, and nephridiopores in “basal” living chitons (Polyplacophora: Lepidopleurina). American Malacological Bulletin 25, 43–49. Sigwart, J. D. (2009a). Morphological cladistic analysis as a model for character evaluation in primitive living chitons (Polyplacophora, Lepidopleurina). American Malacological Bulletin 27, 95–104. doi:10.4003/006.027.0208 Sigwart, J. D. (2009b). The deep-sea chiton Nierstraszella (Mollusca: Polyplacophora: Lepidopleurida) in the Indo-West Pacific: taxonomy, morphology and a bizarre ectosymbiont. Journal of Natural History 43, 447–468. doi:10.1080/00222930802604157 Sigwart, J. D., and Sutton, M. D. (2007). Deep molluscan phylogeny: synthesis of palaeontological and neontological data. Proceedings of the Royal Society B 274, 2413–2419. doi:10.1098/rspb.2007.0701 Sigwart, J. D., Andersen, S. B., and Schnetler, K. I. (2007). First record of a chiton from the Palaeocene of Denmark (Polyplacophora: Leptochitonidae) and its phylogenetic affinities. Journal of Systematic Palaeontology 5, 123–132. doi:10.1017/S1477201906001982 Sirenko, B. I. (1979). On the composition of the family Leptochitonidae Dall, 1889 (= Lepidopleuridae Pilsbry, 1892) (Polyplacophora) with description of a new bathyal species. In ‘Proceedings of the Zoological Institute, USSR Academy of Sciences’. Vol. 80, pp. 116–121. Sirenko, B. I. (1988). A new genus of deep sea chitons Ferreiraella gen. n. (Lepidopleurida, Leptochitonidae) with a description of a new ultraabyssal species. Zoological Journal 67, 1776–1786. Sirenko, B. I. (1992). Nierstraszellidae fam. nov. – a new family of chitons (Polyplacophora, Lepidopleurida) from the bathyal Western Pacific. Ruthenica 2, 81–90. Sirenko, B. I. (1993). Revision of the system of the order Chitonida (Mollusca: Polyplacophora) on the basis of the correlation between the type of gills arrangement and the shape of the chorion processes. Ruthenica 3, 93–117. Sirenko, B. I. (1997). The importance of the development of articulamentum for taxonomy of chitons (Mollusca, Polyplacophora). Ruthenica 7, 1–24. 572 Invertebrate Systematics J. D. Sigwart et al. Sirenko, B. I. (1999). A new and unusual species of Parachiton (Mollusca: Polyplacophora) from South Africa. African Zoology 35, 93–98. Sirenko, B. I. (2001). Deep-sea chitons (Mollusca, Polyplacophora) from sunken wood off New Caledonia and Vanuatu. Mémoires du Muséum national d’Histoire naturelle 185, 39–71. Sirenko, B. I. (2004). The ancient origin and persistence of chitons (Mollusca, Polyplacophora) that live and feed on deep submerged land plant matter (xylophages). Bollettino Malacologico 111–116. Sirenko, B. I. (2006). New outlook on the system of chitons (Mollusca: Polyplacophora). Venus 65, 27–49. Sirenko, B., and Clark, R. (2008). Deshayesiella spicata (Berry, 1919) (Mollusca: Polyplacophora), a valid species. Ruthenica 18, 1–7. Smith, S. W., Overbeck, R., Woese, C. R., Gilbert, W., and Gillevet, P. M. (1994). The Genetic Data Environment: an expandable GUI for multiple sequence analysis. Computer Applications in the Biosciences 10, 671–675. Stebbins, T. D., and Eernisse, D. J. (2009). Chitons (Mollusca: Polyplacophora) known from benthic monitoring programs in the Southern California Bight. The Festivus 41, 53–100. Strugnell, J. M., Rogers, A. D., Prodöhl, P. A., Collins, M. A., and Allcock, A. L. (2008). The thermohaline expressway: the Southern Ocean as a centre of origin for deep-sea octopuses. Cladistics 24, 853–860. doi:10.1111/j.1096-0031.2008.00234.x Todt, C., Cárdenas, P., and Rapp, H. T. (2009). The chiton Hanleya nagelfar (Polyplacophora, Mollusca) and its association with sponges in the European Northern Atlantic. Marine Biology Research 5, 408–411. doi:10.1080/17451000802572394 Varón, A., Sy Vinh, L., and Wheeler, W. C. (2010). POY version 4: phylogenetic analysis using dynamic homologies. Cladistics 26, 72–85. doi:10.1111/j.1096-0031.2009.00282.x Warén, A., and Klitgaard, A. (1991). Hanleya nagelfar, a sponge-feeding ecotype of H. hanleyi or a distinct species of chiton? Ophelia 34, 51–70. Wheeler, W. C. (1995). Sequence alignment, parameter sensitivity, and the phylogenetic analysis of molecular data. Systematic Biology 44, 321–331. Wheeler, W. C. (1996). Optimization alignment: the end of multiple sequence alignment in phylogenetics? Cladistics 12, 1–9. doi:10.1111/j.10960031.1996.tb00189.x Wilson, N. G., Rouse, G. W., and Giribet, G. (2010). Assessing the molluscan hypothesis Serialia (Monoplacophora + Polyplacophora) using novel molecular data. Molecular Phylogenetics and Evolution 54, 187–193. doi:10.1016/j.ympev.2009.07.028 Wu, S.-K., and Okutani, T. (1984). The deepsea chitons (Mollusca: Polyplacophora) collected by the R/V Soyo-Maru from Japan. I, Lepidopleuridae. Venus 44, 1–31. Manuscript received 23 September 2010, accepted 11 February 2011 http://www.publish.csiro.au/journals/is