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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Molecular Phylogenetics and Evolution 61 (2011) 392–399 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Molecular phylogeny of the order Euryalida (Echinodermata: Ophiuroidea), based on mitochondrial and nuclear ribosomal genes Masanori Okanishi a,b,⇑, Timothy D. O’Hara c, Toshihiko Fujita a,b a Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan Department of Zoology, National Museum of Nature and Science, Hyakunin-cho 3-23-1, Shinjuku-ku, Tokyo 169-0073, Japan c Museum Victoria, GPO Box 666, Melbourne 3001, Australia b a r t i c l e i n f o Article history: Received 5 January 2011 Revised 8 June 2011 Accepted 6 July 2011 Available online 21 July 2011 Keywords: Euryalida Ophiuroidea Molecular phylogeny Classification Ribosomal RNA Astrocharinae a b s t r a c t The existing taxonomy of Euryalida, one of the two orders of the Ophiuroidea (Echinodermata), is uncertain and characterized by controversial delimitation of taxonomic ranks from genus to family-level. Their phylogeny was not studied in detail until now. We investigated a dataset of sequence from a mitochondrial gene (16S rRNA) and two nucleic genes (18S rRNA and 28S rRNA) for 49 euryalid ophiuroids and four outgroup species from the order Ophiurida. The monophyly of the order Euryalida was supported as was the monophyly of Asteronychidae, Gorgonocephalidae and an Asteroschematidae + Euryalidae clade. However, the group currently known as the Asteroschematidae was paraphyletic with respect to the Euryalidae. The Asteroschematidae + Euryalidae clade, which we recognise as an enlarged Euryalidae, contains three natural groups: the Asteroschematinae (Asteroschema and Ophiocreas), a new subfamily Astrocharinae (Astrocharis) and the Euryalinae with remaining genera. These subfamilies can be distinguished by internal ossicle morphology. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction The order Euryalida Lamarck, 1816 is one of the two orders of Ophiuroidea with currently close to 190 species described (Stöhr and O’Hara, 2007). Most species of Euryalida live in waters of 100 m or greater depth, often clinging to soft corals, sponges or rocks. The Euryalida was established by Lamarck (1816) as the order Euryale (Lamarck, 1816). But since that time, the limits of the group have remained controversial. The Euryalida have been grouped into another order, Phrynophiurida Matsumoto, 1915 along with the family Ophiomyxidae Ljungman, 1867, based on the morphology of internal structures such as peristomial plates, dorsal plates, vertebrae, dental plates, oral plates, and position of gonads (Matsumoto, 1915, 1917; Fell, 1962; Murakami, 1963). However, a cladistic analysis of morphological characters of ophiuroids has supported the monophyly of the Euryalida, suggesting the Ophiomyxidae was clearly distinguished from Euryalida (Smith et al., 1995), and a recent comparative study found fundamental micro-anatomical differences between the two groups (Martynov, 2010). ⇑ Corresponding author at: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Fax: +81 3 3364 7104. E-mail address: okanishi@kahaku.go.jp (M. Okanishi). 1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.07.003 The Euryalida is currently composed of four families, Asteronychidae Müller and Troschel, 1842, Asteroschematidae Verrill, 1899, Euryalidae Gray, 1840 and Gorgonocephalidae Ljungman, 1867 (Smith et al., 1995). Two authors have attempted to subdivide the Gorgonocephalidae, based on the degree of branching of the arms (Verrill, 1899) or using a series of internal morphological features (Matsumoto, 1915, 1917), but these taxa have not been recognized by subsequent authors (Mortensen, 1933a; Spencer and Wright, 1966; Smith et al., 1995). The Asteroschematidae and Euryalidae were considered as two separate families (Verrill, 1899, 1900), as two subfamilies of the family Trichasteridae Döderlein, 1911 (Döderlein, 1911; Matsumoto, 1915, 1917), or united together in the family Trichasteridae without any subfamilial division (Döderlein, 1927, 1930). In these classifications, the diagnoses and the included genera of Asteroschematidae and Euryalidae (or Asteroschematinae and Euryalinae) have been inconsistent. Mortensen (1933a) established the current classification of these two families. He examined vertebral ossicles and classified the Asteroschematidae and Euryalidae as two separate families based on the presence/absence of an oral bridge on the arm vertebrae (Mortensen, 1933a) (Fig. 1). Currently, the Asteroschematidae is comprised of four genera, Asteroschema Örsted and Lütken, 1856 (in Lütken, 1856), Astrobrachion Döderlein, 1927, Astrocharis Koehler, 1904 and Ophiocreas Lyman, 1869, and the Euryalidae of six genera, Asteromorpha Lütken, 1869, Asterostegus Mortensen, 1933b, Astroceras Lyman, 1879, Euryale Lamarck, Author's personal copy M. Okanishi et al. / Molecular Phylogenetics and Evolution 61 (2011) 392–399 393 Fig. 1. SEM photographs of distal views of the arm vertebrae from distal portion of the arms. (A) Ophiocreas oedipus, Lyman, 1879, NSMT E-1385, without an oral bridge extending across the oral (lower) vertebral processes. (B) Astroceras annulatum Mortensen (1933), NSMT E-1534, with an oral bridge. Scale bars: 200 lm. 1816, Sthenocephalus Koehler, 1898 and Trichaster Agassiz, 1836 (Lamarck, 1816; Agassiz, 1836; Lütken, 1856, 1869; Lyman, 1869, 1879; Koehler, 1898, 1904; Döderlein, 1927; Mortensen, 1933b). All of these previous taxonomic studies were based on morphological characters alone. Molecular data of euryalid ophiuroids has been published in eight papers. In seven of the eight papers, nucleic 18S and/or 28S rRNA regions were sequenced for one or two species and a complete mitochondrial genome for another species in the context of overall echinoderm phylogeny (Smith et al., 1995; Littlewood et al., 1997; Janies, 2001; Perseke et al., 2010); mitochondrial 16S rRNA, COII and/or COI for a few more in the works on population genetic studies (Hunter and Halanych, 2008; Ward et al., 2008; Cho and Shank, 2010). Recently, Janies et al. (2011) sequenced mitochondrial 16S rRNA region, nucleic18S and 28S rRNA regions and H3 DNA region for three species (Janies et al., 2011). It is preferable to analyze multiple molecular markers across numerous taxa to reveal phylogenetic relationships (e.g. Philippe et al., 2005). To resolve the family-level phylogeny of the order Euryalida, we obtained partial mitochondrial 16S rDNA sequences and nucleic 28S and 18S rDNA sequences for 49 species covering all four families. 2. Materials and methods 2.1. Collection of samples The specimens used in this study were mainly collected by R/ Vs Tansei-Maru and Yayoi of the University of Tokyo, R/Vs SoyoMaru and Wakataka-Maru of the Fisheries Research Agency, R/V Koyo of the Tokyo Metropolitan Islands Area Research and Development Center of Agriculture, Forestry and Fisheries, M/V ShinyuMaru of the SNK Ocean Co. Ltd., T/S Toyoshio-Maru of the Hiroshima University and fishing boats, Kiyo-Maru, Taku-Maru and Yoshio-Fudo-Maru. Additional specimens were also supplied from museum collections, and provided by colleagues. Collected samples were immersed in 70–99% ethanol, or fixed in 10% formalin for one specimen of Astrobrachion constrictum (Farquhar, 1900). We analysed two species from two genera of Asteronychidae, 10 species from four genera of Asteroschematidae, 10 species from five genera of Euryalidae and 27 species from 16 genera of Gorgonocephalidae as the ingroup and selected two species from two genera of Ophiomyxidae, one species of Hemieuryalidae Verrill, 1899 and one species of Ophiactidae Matsumoto, 1915 as outgroups (Table 1). Nominotypical genera of four families and 16 type species of 27 genera were included. Accession numbers of DNA Data Bank of Japan (DDBJ), the European Molecular Biology Laboratory and GenBank, and collecting location are shown in Table 1. Voucher specimens were deposited at Auburn University, USA (AU), Florida Museum of Natural History, USA (FMNH), Museum Victoria, Australia (MV), National Museum of Nature and Science, Tokyo, Japan (NSMT), and registered numbers shown in Table 1. 2.2. DNA extraction, PCR amplification and DNA sequencing DNA was extracted from a single specimen of each species. The tip of one arm or tube feet was dissected from each ophiuroid and rinsed in ultra pure water to remove any ethanol. Extraction was performed using the DNeasy Tissue Kit (Qiagen) following the manufactur’s protocol. Regions of the mitochondrial gene, 16S rRNA, and the nuclear genes, 28S rRNA and 18S rRNA were PCRamplified from 50 region depending on the primer combination. The universal 16S primers, 16Sar (50 -CGCCTGTTTATCAAAAACAT30 ) and 16Sbr (50 -CCGGTCTGAACTCAGATCACGT-30 ) (Palumbi, 1996) were used to amplify 457–520 bp fragment of 16S. The optimum cycling parameters for those 16S primers consisted of an initial denaturation step of 95 °C/2 min followed by 41 cycles of 95 °C/30 s, 48 °C/40 s and 72 °C/1 min with final extension step at 72 °C/10 min was followed by storage at 4 °C. 28S primers LSU001 (50 -GCTAAGGAGTGTGTAACAACTCACC-30 ) and LSU002 (50 -GCTTTGTTTTAATTAGACAGTCGGA-30 ) were used to amplify 924–986 bp fragment of 28S. Both primers were newly designed modifications of primers from Palumbi (1996) and referenced to a range of Echinodermata sequences downloaded from DDBJ. For taxa that LSU002 would not amplify, an alternative reverse primer, LSU012 (50 -ACCAGTTCTGAGCCGGCTGTTT-30 ) was designed from a region approximately 40 bp internal to the 30 end of LSU002. 18S primers SSU001 (50 -GCTTGTCTTAAAGACTAAGCCATGC-30 ) and SSU002 (50 -CCGTGTTGAGTCAAATTAAGCCGC-30 ), were used to amplify a 1023–1127 bp fragment of 18S. For taxa that SSU001 and SSU002 would not amplify, alternative newly designed internal primers SSU003 (50 -GCGAAACTGCGGATGGCTCATT-30 ) and SSU004 (50 -TTCAGCTTTGCAACCATACTCC-30 ) were used, approximately 100 bp from the ends of SSU001 and SSU002, respectively. To confirm that amplifications were successful, 3 ll aliquots of PCR amplifications were visualized by 1.0% agarose gel electrophoresis. Family Asteronychidae Müller and Troschel, 1842 Asteroschematidae Verrill, 1899 Species Accession number AB605027 AB605126 AB605076 AB605028 AB605632 AB605077 Asteroschema ajax Clark, 1949 Asteroschema ferox Koehler, 1904 Asteroschema sp. Off Lord Howe Isl., Australia 34°12.180 S, 162°41.180 E. 748–772 m depth Off Takara-jima Isl., Japan 29°05.280 S, 129°09.680 E. 795 m depth Northwestern Australia 12°26.70 S, 123°36.040 E. 96 m depth Albany, Australia 35°20.100 S, 118°17.640 E. 99–100 m depth Doubtful Sound, New Zealand 45°25.080 S, 167°07.030 E. 20 m depth Off Izena Isl. Okinawa, Japan 26°51.070 N, 127°59.060 E. 260–268 m depth Off Amami-oshima Isl., Japan 28°23.930 N, 129°13.550 E. 623–635 m depth Sagami Sea, Japan. 35°02.780 N, 139°33.270 E. 1104–645 m depth Off Katsuura, Japan 34°53.N, 140°32.070 E. ca. 500 m depth South Norfork Ridge, New Zealand 33°23.410 S, 170°11.580 E. 469–526 m depth MV F99759 NSMT E-6260 MV F162658 MV F111592 NSMT E-4161 NSMT E-5632 NSMT E-6502 NSMT E-6259 NSMT E-6710 MV F99763 AB605029 – AB605078 AB605030 – AB605079 AB605031 AB605127 AB605080 AB605032 AB605128 AB605081 AB605033 Z80948 AB605082 AB605034 AB605129 AB605083 AB605035 AB605130 AB605084 AB605036 AB605131 AB605085 AB605037 AB605132 AB605086 – AB605133 AB605087 MV F111585 NSMT E-6261 NSMT E-6275 NSMT E-6268 MV F99698 NSMT E-6262 NSMT E-6291 NSMT E-6499 MV F112091 NSMT E-6263 AB605038 AB605134 AB605088 AB605039 AB605135 AB605089 AB605040 AB605136 AB605090 AB605041 AB605137 AB605091 AB605042 AB605138 AB605708 AB605043 AB605139 AB605092 Euryale aspera Lamarck, 1816⁄ Sthenocephalus anopla (Clark, 1911)⁄ Trichaster acanthifer Döderlein, 1927 Trichaster palmiferus (Lamarck, 1816)⁄ Off D’Entrecasteaux park, Australia 34°53.160 S, 115°30.420 E. 100–95 m depth Off Chichi-jima Isl., Ogasawara, Japan 27°6.70 N, 142°18.560 E. 175–176 m depth Off Chichi-jima Isl., Ogasawara, Japan 27°6.70 N, 142°18.560 E. 175–176 m depth Off Hachijo-jima Isl., Japan 33°20.830 N, 139°41.240 E. 212–177 m depth Off Lord Howe Isl., Australia 33°52.440 S, 159°14.430 E. 76–81 m depth Off Chichi-jima Isl., Ogasawara, Japan 27°04.640 N, 142°08.510 E. 175–176 m depth Mizugama, Okinawa, Japan 3–7 m depth Off Amami-oshima Isl., Japan 27°57.640 N, 129°23.490 E. 401–405 m depth Shark Bay, Australia 25°55.780 S, 112°40.080 E. 120 m depth Amami Shin-sone, Japan 28°52.610 N, 129°33.060 E. 162–153 m depth AB605044 AB605140 AB605093 AB605045 AB605141 AB605094 AB605046 AB605142 AB605095 AB605047 AB605143 AB605096 Asteroporpa hadracantha Clark, 1911 Asteroporpa australiensis H.L. Clark 1909 Asteroporpa reticulata Baker, 1980 Asteroporpa muricatopatella Sagami Sea, Japan 33°20.830 N, 139°41.240 E. 212–177 m depth Wanganella Bank, New Zealand 33°45.260 S, 167°17.070 E. 254–259 m depth North Norfork Ridge, New Zealand 28°54.390 S, 167°41.050 E. 111–113 m depth Off Yaku-shima Isl., Japan NSMT E-6264 MV F99691 MV F99693 NSMT AB605048 AB605144 AB605097 AB605049 AB605145 AB605098 – AB605146 AB605099 AB605050 AB605147 AB605100 Author's personal copy 16S NSMT E-6256 NSMT E-6257 Asteromorpha sp. 28S M. Okanishi et al. / Molecular Phylogenetics and Evolution 61 (2011) 392–399 18S Off Miyako, Iwate, Japan 39°41.N, 142°12.090 E. 216 m depth Off Onahama, Miyagi, Japan 36°57.010 N, 142°39.090 E. 4123–4094 m depth Astroceras annulatum Mortensen, 1933 Astroceras compar Koehler, 1904 Astroceras pergamenum Lyman, 1879⁄ Astroceras pleiades Baker, 1980 Astroceras sp. Gorgonocephalidae Ljungman, 1867 Museum acc. number Asteronyx loveni Müller and Troschel, 1842⁄ Astrodia tenuispina (Verrill, 1884)⁄ Astrobrachion adhaerens (Studer, 1884) Astrobrachion constrictum (Farquhar, 1900)⁄ Astrocharis monospinosa Okanishi and Fujita, 2011b Ophiocreas ambonesicus Döderlein, 1927 Ophiocreas caudatus Lyman, 1879 Ophiocreas glutinosus Döderlein, 1911 Ophiocreas sibogae Koehler, 1904 Euryalidae Gray, 1840 Locality 394 Table 1 Analysed species. Sampling locality, voucher depository and prefix of DDBJ/EMBL/genbank accession numbers of the examined specimens are shown. Type species are marked with asterisks. Institutional abbreviations: AU, Auburn University; FMNH, Florida Museum of Natural History; MV, Museum Victoria; NSMT, National Museum of Nature and Science, Tokyo. Family-level classification is followed by Smith et al. (1995). Okanishi and Fujita, 2011a Astroboa arctos Matsumoto, 1915 Astroboa globifera (Döderlein, 1902) Astroboa sp. Asterogymnotes irimurai Baker et al., 2001 Ophiomyxa anisacantha Clark, 1911 Seseko Beach, Okinawa, Japan 24.9 m depth Sagami Sea, Japan 35°03.560 N, 139°37.410 E. 212–177 m depth Hemieuryalidae Verrill. 1899 Ophiomoeris obstricta (Lyman, 1878) Ophiactidae Matsumoto, 1915 Ophiopholis aculeata (Linnaeus, 1767) Astrochele pacifica Mortensen, 1933 Astrocladus coniferus (Döderlein, 1902) Astrocladus exiguus (Lamarck, 1816) Astrocrius sp. Astroclon propugnatoris Lyman, 1879 Astrodendrum sagaminum (Döderlein, 1902)⁄ Astrodendrum sp. Astroglymma sculptum (Döderlein, 1896)⁄ Astrohamma tuberculatum (Koehler, 1923)⁄ Astrophyton muricatum (Lamarck, 1816)⁄ Astrosierra amblyconus (H.L. Clark, 1909)⁄ Astrothamnus echinaceus Matsumoto, 1912⁄ Astrothorax misakiensis Döderlein, 1911⁄ Astrothorax waitei (Benham, 1909) Astrothrombus chrysanthi Matsumoto, 1918 Astrothrombus rugosus H.L. Clark, 1909⁄ Astrothrombus vecors (Koehelr, 1904) Gorgonocephalus eucnemis Müller and Troschel, 1842 Gorgonocephalus pustulatus (H.L. Clark, 1916) Ophiocrene sp. Ophiomyxidae Ljungman, 1867 AB605051 AB605148 AB605101 AB605052 AB605149 AB605102 AB605053 AB605150 AB605103 AB605054 AB605151 AB605104 AB605055 AB605152 AB605105 AB605056 AB605153 AB605106 AB605057 AB605154 AB605107 AB605058 AB605155 AB605108 AB605059 AB605156 AB605109 AB605060 AB605157 AB605110 AB605061 AB605158 AB605111 AB605062 AB605159 AB605112 AB605063 AB605160 AB605113 AB605064 AB605161 AB605114 AB605065 AB605162 AB605115 AB605066 AB605163 AB605116 AB605067 AB605164 AB605117 AB605068 AB605165 AB605118 AB605069 AB605166 AB605119 – AB605167 AB605120 – AB605168 AB605121 AB605070 AB605169 AB605122 AB605071 AB605170 AB605631 NSMT E-6716 NSMT E-6269 AB605072 AB605171 AB605123 AB605073 AB605172 AB605124 Off Hachijyo-jima Isl., Japan 33°20.830 N, 139°41.240 E. 212–177 m depth NSMT E-6293 AB605075 AB605173 AB605125 See EMBL and GenBank See EMBL and GenBank DQ273713 DQ060806 AF314589 Author's personal copy E-5619-B NSMT E-6718 NSMT E-6258 FMNH UF5053 NSMT E-6270 NSMT E-5480 NSMT E-6265 NSMT E-6272 NSMT E-6274 NSMT E-5645 NSMT E-6273 NSMT E-6370 AU 120 2C01 FMNH UF5131 MV F92996 NSMT E-6271 NSMT E-6266 MV F99703 NSMT E-6713 NSMT E-6267 MV F99707 NSMT E-5640 MV F99712 NSMT E-6505 M. Okanishi et al. / Molecular Phylogenetics and Evolution 61 (2011) 392–399 30°49.050 N, 130°48.520 E. 140 m depth Off Minabe, Wakayama, Japan ca. 30 m Off Kodakara-jima Isl., Japan 28°33.120 N, 129°08.820 E. 175–176 m depth Florida Straits, United Status. 25°57.850 N, 81°77.180 W. 10 m depth Off Katsuura, Japan 34°53.N, 140°32.070 E. ca. 330 m depth Off Minabe, Wakayama, Japan ca. 80 m Off Yaku-shima Isl., Japan 29°47.N, 130°22.060 E. 155–170 m depth Off Katsuura, Japan 34°53.N, 140°32.070 E. ca. 330 m depth Off Tarama-jima Isl., Okinawa, Japan 24°29.720 N, 124°.330 E. 284–290 m depth Sagami Sea, Japan 35°02.780 N, 139°33.260 E. 716–681 m depth Off Otsuchi, Iwate, Japan 75 m depth Off Minabe, Wakayama, Japan ca. 30 m Off Low Isl., Antarctica 63°03.990 S, 62°24.470 E. 192 m depth Florida Straits, United Status 24°51.350 N, 83°50.040 W. 85.3 m depth Howe Reef, Australia 37°18.760 S, 150°14.050 E. 117 m depth Off Katsuura, Japan 34°53.N, 140°32.070 E. ca. 330 m depth Off Toshima Isl., Japan 34°40.020 N, 139°19.E. 312–266 m depth Wanganella Bank, New Zealand 34°22.980 S, 168°25.670 E. 373–374 m depth Off Otsuchi, Iwate, Japan 39°21.860 N, 141°59.860 E. 78.2 m depth Off Toshima Isl., Japan 34°40.020 N, 139°19.E. 312–266 m depth South Norfork Ridge, New Zealand 33°23.410 S, 170°11.580 E. 469–526 m depth Off Miyako, Iwate, Japan 39°52.470 N, 142°19.830 E. 511–512 m depth South Norfolk Ridge, New Zealand 33°23.740 S, 170°13.030 E. 465–490 m depth Off Chichi-jima Isl., Ogasawa, Japan 27°01.390 N, 142°07.400 E. 139–144 m depth 395 Author's personal copy 396 M. Okanishi et al. / Molecular Phylogenetics and Evolution 61 (2011) 392–399 The PCR products were separated from excess primers and oligonucleotides using Exo-SAP-IT (GE Healthcare) following the manufacturer’s protocol. Sequencing reactions used a BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) and 0.5–3 ll (the volume was determined depending on quantities of PCR production) of purified PCR product. Cycling conditions were according to the manufacture’s protocol. All samples were sequenced bidirectionally. Sequencing products were run on an ABI PRISM 3130 DNA sequencer. The original primers were used for the sequencing reaction. 2.3. Phylogenetic analysis All sequences were manually aligned using the Clustal W algorithm in MEGA 4.1 (Thompson et al., 1994; Tamura et al., 2007). Regions where the alignment was ambiguous, including ribosomal loops, were excluded by eye. All missing sequences (i.e. 18S of Asteroschema ajax Clark, 1949) were scored as gaps. The general time reversible model (GTR; Yang, 1994a) with a proportion of 16S rRNA and 28S rRNA sites estimated to be invariable (I; Hasegawa et al., 1985) and with 16S rRNA, 28S rRNA and 18S rRNA assumed to follow a C distribution approximated by discrete categories (C; Yang, 1994b) was consistently selected as the best-fit model of nucleotide substitution by MrModeltest v. 2.3 (Posada and Crandall, 1998). BioEdit ver. 7.0.5.3. (Hall, 1999) and Clustal W in DDBJ (http://clustalw.ddbj.nig.ac.jp/top-j.html) were used in preparing the data matrices in NEXUS format and PHYLIP format, respectively. Tree View for Win 16 was used in exploring tree files, and exploring alternative tree topologies. The phylogenetic tree was constructed with MrBayes v. 3.1.2 (Ronquist and Huelsenbeck, 2003) to obtain Bayesian posterior probabilities (BPP) and RAxMLGUI v. 0.8 beta 2 (Stamatakis, 2006; Stamatakis et al., 2005) for maximum likelihood analysis (ML) to obtain bootstrap support values (bootstrap). The three genes were placed in separate partitions. We set the maximum likelihood parameters in MrBayes as follows; the maximum likelihood model employed six substitution types (nst = 6; GTR); rate variation across sites was modelled using a gamma distribution, with a proportion of the sites being invariant (rate = invgamma); the shape, proportion of invariable sites, state frequency, and substitution rate parameters were estimated for each partition separately. The Markov-chain Monte-Carlo (MCMC) process was run with four chains for 3,000,000 generations, with trees being sampled every 100 generations and the first 5000 trees were discarded as burnin. Data sets were partitioned by gene region for the maximum likelihood analysis to allow for separate optimization of persite substitution rates. The best-known likelihood tree was found by performing 1000 replications. Nodes in the phylogenetic trees were considered as supported if BPP values were larger than 0.98 and bootstrap larger than 80%. BPP values lower than 0.90 and bootstrap lower than 60% for each node were not shown and considered as not significant. 3. Results After alignment, and the removal of ambiguously aligned sites, we obtained 438 bp of 16S, 943 bp of 18S and 876 bp of 28S. Both Bayesian and ML analyses of our dataset of partitioned nuclear and mitochondrial ribosomal genes supported the monophyly of the order Euryalida (Fig. 2, node 1, BPP 1.00, bootstrap 100%). Within the Euryalida, three major clades (Fig. 2, nodes 2, 3 and 4) were recovered. They were composed of species of Asteronychidae (Fig. 2, node 2, BPP 1.00 bootstrap 100%), Gorgonocephalidae (Fig. 2, node 3, BPP 1.00, bootstrap 96%) and Asteroschematidae + Euryalidae (Fig. 2, node 4, BPP 1.00, bootstrap 97%), respectively. Within the major clade of Gorgonocephalidae, Matsumoto’s (1915) subfamilies Astrotominae (Fig. 2, node 5, BPP 1.00) and Gorgonocephalinae (Fig. 2, node 6, BPP 0.98; see also Section 4.3) were supported by the Bayesian but not the ML analysis. There was no support for Verrill’s (1899) subdivision into groups based on the degree of branching of the arms. Within the major clade of Asteroschematidae + Euryalidae, three subclades could be recognised (Fig. 2), based on: two species of Asteroschema and four species of Ophiocreas (node 7, BPP 1.00, bootstrap 95%); one species of Astrocharis and an undescribed species of Asteroschema (node 8, BPP 1.00, bootstrap 100%); and 12 species belonging to the traditional Euryalidae and Astrobrachion (node 9, BPP 1.00, bootstrap 97%). Within the later subclade, Astrobrachion was sister to the traditional Euryalidae (node 10, BPP 1.00, bootstrap 100%). 4. Discussion 4.1. Order Euryalida Our tree supports the monophyly of Euryalida (Fig. 2, node 1). It is clearly distinguished from two species of Ophiomyxidae, Ophiodera anisacantha (Clark, 1911) and Astrogymnotes irimurai Baker et al., 2001. Astrogymnotes Clark, 1914 was once classified as a member of the Asteroschematidae, sharing stout teeth, no oral papillae and no madreporite, and was considered closer to euryalid ophiuroids than the other ophiomyxids (Clark, 1914; Matsumoto, 1917; Döderlein, 1930; Baker et al., 2001). Our tree indicates that those characters were possibly gained independently by Astrogymnotes and euryalid ophiuroids and Astrogymnotes should be retained in the Ophiomyxidae. Therefore our tree supports the classification of Ophiuroidea by Smith et al. (1995) which distinguishes the Euryalida and Ophiurida Müller and Troschel, 1842 as separate orders, the latter including Ophiomyxidae. The taxa within the Euryalida shared the following synapomorphies: the articulation surface on both the adradial genital plate and radial shield forming a large, moderately elevated condyle; stout teeth and plate-like oral papillae or disorganized spiniform teeth; lateral arm plates situated on oral side of the arms; and no tentacle scales (Mortensen, 1933a; Baker, 1980; Smith et al., 1995; Martynov, 2010). 4.2. Family Asteronychidae Although Asteronychidae has been considered as a separate taxon since its original description, its taxonomic rank has shifted between family (Verrill, 1899, 1900; Mortensen, 1933a; Fell, 1960; Murakami, 1963; Spencer and Wright, 1966; Smith et al., 1995) and subfamily levels (Döderlein, 1911, 1927, 1930; Matsumoto, 1915, 1917). The monophyly of the Asteronychidae (Fig. 2, node 2) is supported by our molecular tree and can be distinguished from the family Euryalidae morphologically by following characters: acute and spiniform teeth; gonads restricted to the disc (see also Section 4.4), and can be distinguished from the family Gorgonocephalidae by a following character: no hooklets on the aboral surface of the body (Müller and Troschel, 1842; Verrill, 1899, 1900; Mortensen, 1933a; Smith et al., 1995). 4.3. Family Gorgonocephalidae The monophyly of Gorgonocephalidae is supported by our molecular phylogeny (Fig. 2, node 3) and can be distinguished from the family Euryalidae morphologically by following characters: Author's personal copy M. Okanishi et al. / Molecular Phylogenetics and Evolution 61 (2011) 392–399 397 Fig. 2. Bayesian consensus tree based on a partitioned analysis of three nuclear and mitochondrial genes (2269 bp). Four species of Ophiurida were used as outgroup. Traditional family-level classification is indicated by dashed lines and black family names. Support values for each node are shown by Bayesian posterior probabilities and maximum likelihood bootstrap values. Numerals in circles above branches refer to the node number discussed in the text. Species with branched arms are shown by an asterisk (⁄). acute and spiniform teeth; gonads restricted to the disc (see also Section 4.4), and can be distinguished from the family Asteronychidae by a following character: hooklets on aboral surface of arms (Ljungman, 1867; Verrill, 1899, 1900; Döderlein, 1911, 1927, 1930; Mortensen, 1933a). There had been two alternative hypotheses for subdividing the Gorgonocephalidae. Verrill (1899) and Döderlein (1911, 1927, 1930) divided the Gorgonocephalidae into the Gorgonocephalinae and Astrochelinae (or Gorgonocephalidae and Astrochelidae) based on whether the arms branch or not. In contrast, Matsumoto (1915, 1917) divided Gorgonocephalidae into the Gorgonocephalinae, having spiniform oral papillae, the first vertebrae not attached by muscles, and genital slits on oral side of the disc, and the Astrotominae, having rudimentary (or absent) oral papillae, the first vertebrae attached by muscles, and genital slits on the aboral side of the disc. However, Mortensen (1933a) did not recognise any subfamilial classification within the Gorgonocephalidae and the subfamilies have not been used subsequently. In our tree (Fig. 2), there was no support for groups based on the branching of arms. Although the Bayesian analysis highly supported Matsumoto’s classification (Fig. 2, Astrotominae; node 5, 1.00 and Gorgonocephalinae; node 6, 0.98) the ML analysis did not support it. With only 16 of the 38 known genera sequenced for our study, more taxonsampling is required to fully test subfamilial classifications within Gorgonocephalidae. Within the Gorgonocephalidae, the monophyly of at least two genera, Astroboa Döderlein, 1911 and Astrodendrum Döderlein, 1911, were not supported and were shown to be polyphyletic (Fig. 2). Astroboa is distinguished from the other related genera mainly in having the following diagnostic characters: an absence of arm spines before the basal fourth branch and having uniform size dermal ossicles on the disc, and Astrodendrum by having small, uniform size tubercles on the disc and an absence of a girdle of calcareous plates on the interradial edge of the disc (Fell, 1960; Baker, Author's personal copy 398 M. Okanishi et al. / Molecular Phylogenetics and Evolution 61 (2011) 392–399 1980). Our analysis indicates that these characters may not be useful for defining these two genera. However, our dataset included only two of the nine known and one undetermined species of Astroboa and one of five known and one undetermined species of Astrodendrum. Therefore, more extensive taxon-sampling is required to reconstruct the correct classification of these two genera. 4.4. Families Asteroschematidae and Euryalidae Our tree did not support the monophyly of traditional Asteroschematidae (Fig. 2). Instead, monophyly of the major clade Asteroschematidae + Euryalidae was supported (Fig. 2, node 4). We use the family-level name Euryalidae for this major clade as the oldest available name with the nominotypical Euryale as the type-genus (Gray, 1840). This clade is well characterized morphologically by following characters: no hooklets on the aboral surface of the body; stout and large teeth forming a single ventral row on the dental plate; pavement-like domed oral papillae on the sides of the jaws; a widened basal portion of the arms due to extension of gonads in mature specimens (Verrill, 1899, 1900; Döderlein, 1911; Matsumoto, 1915, 1917; Mortensen, 1933a). The Asteroschematidae and Euryalidae have been distinguished by the following morphological characters: the presence/absence of an oral bridge on the vertebrae (Fig. 1); and the prominent lateral arm plates and long shaft of the vertebrae in the distal portion of the arms (Mortensen, 1933a; Spencer and Wright, 1966; Baker, 1980; Smith et al., 1995). However, our phylogenetic analysis found that the Asteroschematidae is paraphyletic with respect to the traditional Euryalidae. Instead of the traditional classification, our phylogeny suggests that the Asteroschematidae and Euryalidae should be divided into the following three subclades: Asteroschema and Ophiocreas; Astrocharis monospinosa and Asteroschema sp.; and Astrobrachion and Euryalidae. Out of the three subclades, the Asteroschema and Ophiocreas subclade and the A. monospinosa and Asteroschema sp. subclade were differentiated by a key morphological feature. Okanishi and Fujita, 2009, 2011) examined the radial shields of all described species of Asteroschema and Astrocharis, either by directly examining specimens or reviewing the literature. They showed that the radial shields of these species fall into two types, having either single or multiple layers of plate-shaped ossicles. Single-layered radial shields were possessed by all species of Astrocharis and Asteroschema amamiense Okanishi and Fujita, 2009 and multi-layered radial shields by the other Asteroschema species (Okanishi and Fujita, 2009, 2011). In the current study, we observed that single-layered radial shields were present in the undescribed Asteroschema sp. (sequenced here) and multi-layered radial shields in all species of Ophiocreas. Thus, the two subclades can be characterized as follows: the subclade of A. monospinosa and Asteroschema sp., has single-layered radial shields, and the subclade of Asteroschema and Ophiocreas, has multi-layered radial shields. The third subclade grouped the genus Astrobrachion with the traditional Euryalidae. Astrobrachion has been previously classified as a member of Asteroschematidae because it has no oral bridge on its vertebrae (Fig. 1; Döderlein, 1927; Mortensen, 1933a). The vertebral oral bridge of the traditional Euryalidae separates the lateral arm plates on the oral side of the arms (Fig. 1). The ventral arm plates of Asteroschema and Ophiocreas species are usually absent, at least in the distal portion of the arms, and if they do exist in the basal portion of the arms, they do not separate the lateral arm plates. However, Astrobrachion species have ventral arm plates throughout the arms, which cover the oral groove of the vertebrae, like the oral bridge of the other Euryalidae, and separate the lateral arm plates. Therefore, the lateral arm plates are separated on the oral side of the arms both in Astrobrachion and the traditional Euryalidae. Moreover, both Astrobrachion and species of traditional Euryalidae increase their number of arm spines from one to two from the fourth, or rarely fifth, arm segments, whereas the other species of the traditional Asteroschematidae increase their number on more distal segments. Thus Astrobrachion and the traditional Euryalidae share important diagnostic characters. Morphological characters support the three subclades in our tree. We recommend that the three subclades should be classified as subfamilies: the Asteroschematinae Verrill, 1899, composed of Asteroschema (except A. amamiense and Asteroschema sp.) and Ophiocreas; the Astrocharinae new subfamily, composed of A. amamiense, Asteroschema sp. and Astrocharis; and the Euryalinae Gray, 1840, composed of Asteromorpha, Asterostegus, Astrobrachion, Astroceras, Euryale, Sthenocephalus and Trichaster (see Appendix for a detailed taxonomic account). We did not include Asterostegus in this study, however, morphological characters described in the original description of the type species, A. tuberosus (Mortensen, 1933b), support its retention in the Euryalinae. 5. Conclusion The present analysis of nuclear and mitochondrial DNA sequence data is the first comprehensive molecular phylogenetic study for the order Euryalida. The phylogenetic tree supports monophyly of the Euryalida. Within the Euryalida, the monophyly of the Asteronychidae and Gorgonocephalidae respectively are supported. The monophyly of a combination of two previously recognised families (Asteroschematidae + Euryalidae) is also supported, and these are now recognised as expanded Euryalidae. Within the Euryalidae, three subclades are assigned to subfamilies, Asteroschematinae, Euryalinae and a new subfamily Astrocharinae. This classification is supported by new morphological observations. Acknowledgements Many people helped us to collect the specimens: Yuji Aoki, Ken Fujimoto, Rebecca L. Hunter, Nozomu Iwasaki, Tetsuya Kato, Hironori Komatsu, Asako K. Matsumoto, François Michonneau, Masayuki Minakawa, Takami Morita, Hiroshi Namikawa, Mark Norman, Masami Obuchi, Mark O’Louglin, Susumu Otsuka, Gustav Paulay, Brian Stewart, Hiroyuki Tachikawa, Kunihisa Yamaguchi. The materials for this study were collected by R/Vs Koyo, ShinyuMaru, Tansei-Maru, Wakataka-Maru, Yayoi, Soyo-Maru, T/S Toyoshio-Maru and fishing boats, Kiyo-Maru, Taku-Maru, YoshioFudo-Maru. Thanks are also extended to Toshiaki Kuramochi for his assistance in molecular work and Takuma Haga, Tsuneo Kakuda, Kaoru Kuriiwa, Tomoyuki Nakano, Takashi Sato and Kaori Wakabayashi for their helpful comments on molecular analysis. We wish to express our sincere gratitude to two anonymous reviewers for their critical reading of the manuscript and constructive comments. This work was partly supported by grants from the Research Institute of Marine Invertebrates (Tokyo, Japan), the Showa Seitoku Memorial Foundation, the Japanese Society for the Promotion of Science (Scientific Research [B] No. 20310144, [C] No. 22570104 and JSPS Fellows No. 22506). This is also a contribution of the project ‘‘Studies on the Origin of Biodiversity in the Sagami Sea: Fossa Magna Element and the Izu-Ogasawara Arc’’ conducted by the NSMT. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ympev.2011.07.003. Author's personal copy M. Okanishi et al. / Molecular Phylogenetics and Evolution 61 (2011) 392–399 References Agassiz, L., 1836. Prodrome d’une monographi des radiaires ou echinodermes. Mém. Soc. Sci. Nat. Neuc. 1, 168–199. Baker, A.N., 1980. Euryalinid Ophiuroidea (Echinodermata) from Australia, New Zealand, and the south-west Pacific Ocean. N. Z. J. Zool. 7, 11–83. 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