Accepted on 7 October 2008 2009 Blackwell Verlag GmbH J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2008.00509.x

1Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China; 2Beijing Museum of Natural History, Beijing, China; 3Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng Teachers College, Yancheng, China

Mitochondrial sequence data reveals the phylogeny of the Asian group of (: Brachyura: )

H. Sun1,Y.Jin1,D.Zhang1,S.Yang2,3,Q.Li1,D.Song1 and K. Zhou1

Abstract The of the Helice s. l. crabs has been recently revised, subdividing the Asian in three genera, Helice, Helicana and Pseudohelice. The last one is widespread throughout the Indo-West Pacific Ocean and a single species, Pseudohelice subquadrata, is found in Asia. The other two genera appear to be endemic to Asia. However, phylogenetic affinities among the Asian representatives of Helice s. l. are poorly understood, and different morphology-based hypotheses of relationships remain untested. In this study, we used sequence data from a mitochondrial gene-block spanning three different genes [16S rDNA-trnL1-IGS-nad1] to perform phylogenetic analyses. The results provide independent molecular evidence supporting the splitting of the Asian species into Helice and Helicana. Helice is suggested to be paraphyletic with respect to P. subquadrata.In addition, the heterogeneity of the genus Pseudohelice is shown when including the East Asian and East African counterparts. We propose that the two genera, Helice and Pseudohelice, are not monophyletic in their current composition.

Key words: Mitochondrial genes – phylogeny – Helice – Helicana – Pseudohelice

Introduction and Yatsuzuka 1980 recorded from western Japan. Sakai et al. Until recently, the Asian species of the Helice s. l. group (2006) revised the taxonomy and systematics of the Helice were assigned to three genera (Helice, Helicana and Pseudohe- group, and raised the two subgenera, Helice and Helicana to lice) according to the taxonomic arrangement proposed by genus level, and a new species, Helicana doerjesi Sakai et al. Sakai et al. (2006). These are typical semiterrestrial crabs 2006, was recorded from Taiwan, Zhejiang to Guangxi of distributed in the Indo-West Pacific Ocean, inhabiting crevices China and Vietnam. Helice leachii was considered a junior or burrows under boulders in intertidal areas of estuaries, synonym of Pseudohelice subquadrata (Dana, 1851), and especially in muddy flats and other swampy grounds in Helice crassa (Dana, 1851) from New Zealand was transferred supralittoral areas. Despite their ecological importance in to the new genus Austrohelice Sakai et al. 2006. intertidal and supertidal habitats, several features of their Apart from different hypotheses of interspecific relation- biology and evolutionary relationships are still poorly under- ships deriving from morphology-based taxonomic analyses, stood. And there is still ambiguity regarding their systematic the phylogenetic affinities within the Helice s. l. group are arrangement (Sakai 1976; Dai and Song 1977; Sakai and poorly understood. This study is the first attempt to elucidate Yatsuzuka 1980; Dai and Yang 1991; Sakai et al. 2006). phylogenetic relationships among the Asian taxa using molec- The genus Helice de Haan 1835 was originally recognized as ular characters. Previous molecular studies on this group are monotypic with the type-species de Haan 1835 scarce (Sun et al. 2002a). Sequences from fragments of large recorded from Japan, until two other species, Helice crassa and small subunit of mitochondrial ribosomal genes (rrnL or (Dana, 1851) and Helice leachii (Hess 1865) were recorded 16S rDNA, and rrnS or 12S rDNA), and cytochrome c oxidase from New Zealand and Sydney, Australia, respectively. The subunit I gene (cox1) seem to be useful to infer phylogenetic latter species was subsequently recorded in Asia and other relationships within Grapsidae and other brachyuran crabs Indo-West Pacific regions. Helice tridens has been subdivided (Sturmbauer et al. 1996; Stillman and Reeb 2001; Daniels into several subspecies. In addition to the nominal subspecies et al. 2002; Schubart et al. 2002, 2006; Sun et al. 2002a,b, H. tridens tridens, the following have been recognized: 2003; Marijnissen et al. 2006). However, the mitochondrial H. tridens latimera Parisi 1918 [=H. t. pingi Rathbun 1935; specific single-gene fragments amplified by universal PCR cf. Sakai and Yatsuzuka 1980 (from China)], H. t. formosensis primers should be used with caution, as multiple mitochon- Rathbun 1931, H. t. wuana Rathbun 1931 (=sheni Sakai 1939; drial nuclear copies arisen from the recombination or trans- Dai and Yang 1991) and H. t. tientsinensis Rathbun 1929. Dai location from mitochondrial to nuclear genome have been and Song (1977) and Sakai and Yatsuzuka (1980) proposed demonstrated in stone crabs (Schneider-Broussard and Neigel that most or all of the subspecies within the polymorphic 1997), snapping shrimps (Williams and Knowlton 2001) and H. tridens should be ranked at the species level within the various other metazoan taxa, constituting a serious potential genus Helice, considering their different features with respect risk in confounding phylogenetic reconstructions (reviewed in to ecological and geographical aspects. Zhang and Hewitt 1996; DeWoody et al. 1999). Fragments The subspecies wuana was also raised by Sakai and from multi-gene blocks of mitochondrial genome may be less Yatsuzuka (1980) to species level, but was also transferred to likely to exhibit these characters (but see Lopez et al. 1994). a new subgenus Helicana, characterized by the adult male In this study, we use as molecular markers sequences of a morphology of the first pleopods and the suborbital crest, mitochondrial gene block spanning three different genes, together with a new species, Helice (Helicana) japonica Sakai which include partial sequences of 16S rDNA and NADH 2 Sun,Jin,Zhang,Yang,Li,Song and Zhou dehydrogenase subunit 1 (nad1) genes, as well as the complete varunid crab Hemigrapsus penicillatus (De Haan 1835) (HPE278835), sequence of leucine tRNA gene with CUN recognized as was used as outgoup taxon. The unalignable sites among the 16S codon (trnL or trnL ). The main focus of our study is to alignment were initially excluded by comparing with a reference CUN 1 alignment set consisting of 24 genera of American grapsoid crabs examine: (1) the monophyly of the Asian genera within the (Schubart et al. 2000) and then applying Gblocks 0.91b (Castresana Helice s. l. group; (2) the phylogenetic relationships among the 2000). The conserved regions were concatenated in a single alignment Asian representatives of the same group. spanning 408 bp (with a total of 56 excluded sites; hereafter H16S). The phylogenetic signal present in each dataset was detected using Tree-Puzzle 5.2 (Schmidt et al. 2002) according to the maximum Materials and Methods likelihood mapping method (Strimmer and von Haeseler 1997). The Taxon sampling, PCR and DNA sequencing amount of phylogenetic signal present in a multiple alignment is mirrored by the percentage of the fully resolved quartets. A chi-square We examined specimens of eight ingroup taxa of the Helice s. l. group, test of homogeneity of base frequencies across taxa was carried out as including tridens, formosensis, tientsinensis, latimera, japonica, doerjesi, implemented in paup (Swofford 2002), including or excluding the wuana and a putative variant of wuana, and P. subquadrata (=H. outgroup taxa from our analysis. Phylogenetic trees were constructed leachii). These species sampled represent the main representatives of using three different methods based on the three datasets above. We Helice s. l. crabs in Asia. Two grapsid crabs, Hemigrapsus sanguineus used two methods from the paup 4.0b10 package (Sinauer Associates, (de Haan, 1835) (Table S1) and Eriocheir japonica sinensis H. Milne Sunderland, MA, USA) of phylogenetic programs (Swofford 2002): Edwards, 1853 (NC_006992, Sun et al. 2005) were selected as outgroup the equally weighted maximum parsimony (MP) and maximum taxa based on current understanding of the phylogenetic relationships likelihood (ML) methods, as well as Bayesian inference of phylogeny within Varunidae H. Milne Edwards, 1853 (Schubart et al. 2000, 2002, (BI) was achieved through the use of Mr Bayes, version 3.0b4 program 2006; Sun et al. 2002a). Data on the specimens used in this study are (Huelsenbeck and Ronquist 2001). Modeltest 3.7 (Posada and Cran- given in Table S1. Voucher specimens are held in Beijing Museum of dall 1998; Posada and Buckley 2004) was used to determine the best- Natural History and College of Life Sciences, Nanjing Normal fitting model for sequence evolution under the Akaike information University. criterion (AIC, Akaike 1974). The MP analyses were performed under DNA extractions were performed from fresh-frozen or alcohol- the heuristic search setting with the random addition of sequences and preserved tissue samples, using the standard proteinase K and tree-bisection-reconnection algorithm. The ML analyses were achieved Phenol chloroform techniques. The PCR amplification was performed ⁄ under heuristic search with random addition of sequences using the using the primers ES-J-10981 (Sun et al. 2005) and IHX01-H (Sun selected model. Bootstrapping was performed to assess the confidence et al. 2002b). All reactions were carried out in 30-ll volumes, level at each branch (Felsenstein 1985), with 100 iterations for ML and containing 1.0 U of Taq DNA polymerase (Promega, Shanghai, 1000 iterations for MP. Bayesian inference was performed on the basis China), 0.2 mM each dNTP, 2.0 mM MgCl , 3.0 ll10 Buffer, 2 · of the suggested model of evolution. A Markov chain Monte Carlo 0.6 lM each of primers, and 5–10 ng of template DNA. Amplification search was run with four chains for 2 · 106 generations, saving every was performed under standard PCR conditions on a MJ Research 100th tree to yield a posterior probability distribution of 20 000 trees. PTC-200 thermal cycler, with an initial 2-min predenaturation at 95 C, After eliminating the first 2500 trees as burn-in, we constructed a 50% followed by 30 cycles of 95 C denaturation for 30 s, 55 C annealing for majority-rule consensus tree, with nodal values representing the 30 s, 72 C extension for 40 s, and followed by a final 7 min extension posterior probability. at 72C. Unincorporated nucleotides and primers were removed by passing the PCR products over Sephadex columns (TaKaRa, Dalian, China) following the manufactureÕs protocol. Purified PCR products Results were sequenced directly using the ABI Prism Dye Terminator Cycle sequencing kit (Perkin-Elmer, Waltham, MA, USA). The sequences Sequence properties and variation were resolved on an ABI 310 Genetic Analyzer or an ABI 377 The length of the sequenced region in all species sampled automated sequencer (Applied Biosystems Inc., Norwalk, CT, USA). ranged from 736 to 742 bp and can be retrieved under the Both strands were sequenced. accession numbers EU367386–EU367395 (Table S1). The gene The portions of 16S rDNA and nad1 genes were identified using the organization of each sequence contains 5¢-16S rDNA-trnL - NCBI Internet Nucleotide BLAST (blastn), and the trnL1 gene was 1 identified and folded to confirm the secondary structure using the web- IGS-nad1-3¢, which are encoded on the minority strand based program tRNAscan-SE (Lowe and Eddy 1997). Residues (N-strand). Gene order and orientation are identical to those between encoding genes were recognized as intergenic spacer (IGS). described in the mitochondrial genome of Eriocheir (Sun et al. 2005). The putative start codon of nad1 of all sampled taxa was identical (ATA) with the exception of ATG in the outgroup Alignment and phylogenetic analyses taxon, Hemigrapsus. For all Helice sampled, full nucleotide Every single gene and gene fragment was aligned to the homologous sites of trnL gene are exactly the same and folded to the sequences of ingroup and outgroup taxa via CLUSTAL X (Jeann- 1 mougin et al. 1998), then carefully revised manually to maximize typical clover-leaf structure (Fig. 1a). But one to two point sequence similarity. For phylogenetic analysis, blocks of poorly mutations could be found in its TYC loop compared with the aligned sites within the alignment of 16S and IGS respectively were two outgroup taxa, Eriocheir (one C to T transition) and identified using Gblocks 0.91b (Castresana 2000). As a result, 14-bp Hemigrapsus (one deletion of T, and one G to A transition) and 7-bp sites were respectively identified as ambiguous or problematic (Fig. 1b, c). Otherwise, a 38-nucleotide IGS between trnL1 and in 16S and IGS, and were not included in later analyses. The total nad1 gene is observed in all Helice taxa sampled. A similar IGS alignment of concatenated 16S and IGS, respectively, consisted of 535- is also present at the same gene junction in Eriocheir and bp and 31-bp. The datasets of 535-bp 16S (hereafter HA16S) and the 566-bp combined 16S and IGS (hereafter HA16IGS) were finally used Hemigrapsus, of 35 and 36 nucleotides respectively. Three in the analysis. characteristic IGS sequences could be identified. One shared For investigating the phylogeny of the Asian species of Helice, two by P. subquadrata and the four Helice species sampled, could currently available 16S rDNA sequences of two Helice s. l. species in be folded to a large loop structure with short-stem (Fig. 2a). GenBank were included in our 16S dataset, of which H. leachii The other two could be also folded to a similar stem-loop (currently recognized as Pseudohelice balssi Sakai et al. 2006) collected structure, but with a long-stem, possessed by japonica ⁄ doerjesi from Mida Creek, Kenya, East Africa (AM180690; Schubart et al. 2006), and Helice crassa (now recognized as Austrohelice crassa) from and wuana ⁄ a putative variant of wuana respectively (Fig. 2b); Christchurch, New Zealand (AJ308416; Schubart et al. 2006). Another for the latter, only one C-T transitional substitution occurred doi: 10.1111/j.1439-0469.2008.00509.x 2009 Blackwell Verlag GmbH Mitochondrial sequence data reveals phylogeny of the Asian Helice crabs 3

(a)T (b)Leu T (c) Leu T Leu A–T AT A–T (CUN) C–G (CUN) C–G (CUN) C–G T–A T–A T–A A–T A–T A–T T–A T–A T–A T–A T–A T–A T–A T–A T T–A T TATCTTA T TATCT A T TATCTTA A G T A G G A G G T G A CG A T G A CGG A G A CGG A TAG A TAGA C A TAG T A A AGT A A G A A AGT GCC . T G C C . G C C . T T G T AA T A G T A A T A T G T AA A T–A T–A T–A T . T T . T T . T G–C G–C G–C G–C G–C G–C A–T A–T A–T T A T A T A TG TG TG T A G T A G T A G

Fig. 1. Deduced secondary structure of trnLCUN gene of mitochondrial DNA. (a) shared by Helice s. l. species sampled, (b) exhibit in Eriocheir and (c) in Hemigrapsus. The arrow indicates the site of point mutation

A+T), trnL1 (74.6%A+T) and 16S rDNA (546-bp, 74.7% A+T) (Table S2).

Phylogenetic analysis The phylogenetic signal presented in all analyzed datasets is shown in Fig. 3. The percentage of fully resolved quartets in the HA16S (Fig. 3a) and HA16IGS (Fig. 3b) datasets is consistent (93.9–94.5%). The H16S dataset shows an amount of phylogenetic signal with 92.0% of the quartets fully resolved (Fig. 3c). Phylogenetic congruence between tree topologies of 16S rDNA and IGS was assessed using the incongruence length difference test (Farris et al. 1995) implemented in paup Fig. 2. Comparisons of the putative stem-loop structure inferred from as the partition-homogeneity test (Swofford 2002) (p = 1.00). the 38-nt intergenic spacer between trnL1 and nad1 gene of the Helice The nucleotide composition in each datasets, HA16S, s. l. species sampled. (a) shared by the four definite Helice species, HA16IGS and H16S, was homogeneous among all ingroup H. tridens, H. formosensis, H. tientsinensis and H. latimera and and outgroup taxa (Table S3). Pseudohelice subquadrata sampled; (b) shared by all of the Helicana species, japonica, doerjesi, wuana and a putative variant of wuana. And The phylogenetic relationships among the Asian species of the latter two possess a shared single transition (C fi T) at the elev- the Helice s. l. group were reconstructed based on two different enth location resulted in G ⁄ T bond. Lines indicate Watson–Crick datasets, HA16S and HA16IGS. For both data sets, the bonds, and dots indicate G-T bonds. The number indicates the loca- TrN+I model (Tamura–Nei model with correction for the tion of each site which varied among three different groups in IGS proportion of invariable sites) was found by the Akaike information criteria to be the best-fit model of substitution. in the stem region, forming a G-T bond instead of a Watson– But for H16S dataset, the HKY+I model (Hasegawa–Kish- Crick bond at the same site. In all Helice s. l. and related taxa ino–Yano with correction for the proportion of invariable sampled there is a strong A-T bias in nucleotide composition in sites) was selected. The likelihood of the ML tree, and the all segments, with overall A+T content of 75%. The average number of parsimony informative sites (PI), the consistency A+T content of IGS (79.6%) is the largest, compared with the and retention indices (Ci, Ri), and the shortest tree length of value found in the three encoding sequences, nad1 (87-bp, 74% the MP trees are summarized for each dataset in Table S3.

(a) (b) (c)

Fig. 3. Likelihood mapping of the AH16S (a), AH16IGS (b), and H16S (c) datasets. All possible quartets were evaluated. According to the likelihood mapping method (Strimmer and von Haeseler 1997), the amount of phylogenetic signal present in the multiple alignment considered is reflected by the percentage of fully resolved quartets i.e. to the sum of the values in the corner regions of the triangle and to the length of the alignment. Conversely, the partly unresolved quartets shown in lateral rectangles, and the fully unresolved quartets shown in central triangle indicated a loss of the signal, particularly the latter doi: 10.1111/j.1439-0469.2008.00509.x 2009 Blackwell Verlag GmbH 4 Sun,Jin,Zhang,Yang,Li,Song and Zhou

(a) Eriocheir japonica sinensis

Hemigrapsus sanguineus

P. subquadrata S-China 100/100 59/56 96/96 H. tridens b 58/56 H. formosensis Helice clade -/- f -/- 86/85 96/95 -/- H. latimera 57/60 -/- 61/59 g a H. tientsinensis

AH16S/AH16IGS 100/100 98/98 H. japonica BI 98/99 ML 100/100 d MP 93/97 H. doerjesi 91/95 Helicana c clade H. wuana 99/99 e 78/73 H. wuana pv 91/89

(b)

Hemigrapsus penicillatus

Hemigrapsus sanguineus

100 P. subquadrata S-China Fig. 4. Phylogenetic relationships 69 recovered by BI, ML and MP 99 94 H. tridens treatments based on AH16S and b 43 56 AH16IGS (a), and H16S (b) data- 100 H. formosensis Helice f 56 clade sets. H. wuana pv = a putative 69 variant of Helicana wuana, P. sub- H. latimera g' quadrata S-China = Pseudohelice subquadrata sampled from South 97 H. tientsinensis 68 100 East Asian clades Coast of China, P. balssi E-Afri- 51 97 H. japonica ca = cited Pseudohelice balssi 99 a' 100 (=Helice leachii) from East Africa, 84 d H. doerjesi A. crassa New Zealand = cited H16S 91 93 c Helicana clade Austrohelice crassa (=Helice cras- BI 77 H. wuana 83 sa) from New Zealand. The values ML e MP H. wuana pv on the branches of the tree indicate 63 Bayesian posterior probability, 40 P. balssi E-Africa bootstrap in ML and MP from - AH16S, AH16IGS and H16S h A. crassa New Zealand datasets respectively

Parsimony, ML and Bayesian analyses resulted in similar tree subclade of Helice s. str. (clade f, g and gÕ) were not significant topologies (Fig. 4a, b). A number of features become apparent (see Fig. 4a, b). from the phylogenetic trees based on our three different (4) Strong support for the monophyly of Helicana + Helice datasets (AH16S, AH16IGS and H16S): (including P. subquadrata S-China) (clade a) was only found in (1) Two well supported clades are recovered within the Asian Bayesian analyses (BPP = 95 and 96%), but weakly sup- Helice s. l. group, that correspond to the two genera Helicana ported in the ML and MP analyses (BS = 57 and 60% in ML, [clade c, BPP (Bayesian posterior probability) = 100%, BS 59 and 61 in MP; Fig. 4a). (Bootstrap in ML and MP) ‡ 84%], and Helice (plus (5) Based on H16S dataset, P. balssi (=H. leachii) from P. subquadrata S-China) (clade b, BPP = 100%, BS in E-Africa was found outside of both of the East Asian clades, MP ‡ 96 and 99%; see Fig. 4a, b). and appeared as sister clade to them. Identical position was (2) The Helicana-clade (clade c) was further subdivided true for the cited A. crassa NZ (=H. crassa) from New into two subclades (d and e) with moderate to strong Zealand (Fig. 4b). support (BPP = 93, 99 and 100%; BS in ML and MP ‡ 73%; Fig. 4a, b). Discussion (3) In clade b, P. subquadrata S-China, collected from Hainan Island, China, clustered together with H. tridens, H. formo- Monophyly of the genus Helicana within the Asian Helice s. l. sensis, H. tientsinensis and H. latimera, and held a basal crabs position within this clade with high support in both BI and The Asian crabs of the Helice s. l. group were initially classified MP, whereas the supports for internal relationships within the as two species, H. tridens and H. leachii. The former species is doi: 10.1111/j.1439-0469.2008.00509.x 2009 Blackwell Verlag GmbH Mitochondrial sequence data reveals phylogeny of the Asian Helice crabs 5 an Asian endemic distributed in China, Japan, West Coast of the remarkable molecular divergence between the two species Korea and Southeast Asia, and has been split into five of Pseudohelice are not congruent from the current taxonomic subspecies, four of which are currently regarded as distinct treatment. Thus, the two genera Helice and Pseudohelice are species, i.e. H. tridens, H. formosensis, H. tientsinensis and not monophyletic in their current composition. In particular, H. latimera (Sakai et al. 2006). The other species, characterized the taxonomic position of the East Asian P. subquadrata needs by the distinct structures of the first pleopods of adult males be reconsidered. and tooth plates of the gastric mill, was segregated into a distinct subgenus Helicana, later regarded as an independent genus, together with two species described subsequently, Acknowledgements H. japonica and H. doerjesi (cf. Sakai and Yatsuzuka 1980; This research was supported by the National Natural Science Sakai et al. 2006). The remaining species, H. leachii was Foundation of China (NSFC) Project No. 30470236 (to SHY) and transferred to a new genus Pseudohelice (cf. Sakai et al. 2006). Key Project No. 30630010 (to SDX). The authors are grateful to Lu Xiu-Lin for kind help with the laboratory work, and Dr C. D. This molecular results provide strong independent evidence Schubart for translation of the abstract into German, and also for the splitting of the Asian Helicana–Helice species into two sincerely thank Dr C. D. Schubart and two anonymous reviewers for genera or subgenera (Dai and Yang 1991). However, the helpful comments on an earlier version of the manuscript. current results do not support a further distinct genus Pseudohelice. In all our analyses, H. japonica, H. doerjesi, H. wuana and a putative variant of H. wuana form a well- Zusammenfassung supported monophyletic group. It is congruent with the recent Mitochondriale Sequenzdaten rekonstruieren die Phylogenese der Asia- taxonomic treatments of the genus Helicana (Sakai and tischen Taxa der Helice-Gruppe (Decapoda: Brachyura: Varunidae) Yatsuzuka 1980; Sakai et al. 2006). This suggests that the morphological characters present in the first pleopods of Die taxonomische Systematik der Helice s.l.-Gruppe wurde ku¨ rzlich uberarbeitet. Danach lassen sich die asiatischen Vertreter von Helice adult males of Helicana should be considered phylogeneti- ¨ s.l. in drei Gattungen klassifizieren, Helice, Helicana und die neu cally informative. The specimens (1#;3$) collected from definierte Pseudohelice, von denen die letztere im indo-westpazifischen Lianyungang, Jiangsu and Chongming, Shanghai, China Raum weit verbreitet ist, aber nur mit einer Art, Pseudohelice were recognized as a putative variant of wuana, identified subquadrata (fru¨ her Helice leachii), in Ostasien auftritt. Die anderen by morphological characters of chelipeds and ambulatory beiden Gattungen scheinen in Ostasien endemisch zu sein. Phyloge- legs, and subtle features of the infraorbital crest in male netische Affinita¨ ten unter diesen ostasiatischen Vertretern sind kaum adult. erforscht, und die gegenwa¨ rtige taxonomische Behandlung und mor- phologisch basierte Systematik bleiben ungetestet. In der vorliegenden Untersuchung verwenden wir Sequenzdaten von einem mitochondri- alen DNA-Abschnitt, der Bereiche dreier unterschiedlicher Gene [16S Phylogenetic implications of a ÔHeliceÕ ingroup position of rDNA-trnL1-IGS-nad1] einschließt, um phylogenetische Analysen Pseudohelice subquadrata from southern China durchzufu¨ hren. Die Ergebnisse geben unabha¨ ngige molekulare Un- Within the Helice s. l. group, P. subquadrata appears to be the terstu¨ tzung fu¨ r die Aufspaltung der ostasiatischen Arten in die beiden most widespread species of the Indo-West Pacific fauna. It has Gattungen Helicana und Helice. Es wird postuliert, dass die derzeitige Gattung Helice paraphyletisch bezu¨ glich der Vertreter von P. sub- been recorded from the Red Sea throughout the Indian Ocean quadrata (=H. leachii) von Su¨ dchina ist. Zusa¨ tzlich, und kombiniert to Australia, north to southern China (Hainan and Taiwan) mit den 16S-Daten, konnte die Heterogeneita¨ t der asiatischen und and southern Japan (reviewed in Sakai et al. 2006). Hess ostafrikanischen Vetreter von Pseudohelice ermittelt werden. Wir (1865) described the species H. leachii from Sydney, Australia. folgern, dass die Morphospezies P. subquadrata ho¨ chstwahrscheinlich This taxon was subsequently reported from a wide Asian range einen Artenkomplex darstellt und dass die Gattungen Helice und including Japan, Korea to Taiwan and Hainan in China, south Pseudohelice in ihrer derzeitigen Umfassung nicht monophyletisch sind. to Indonesia etc. (see Sakai 1976; Dai and Yang 1991; Sakai et al. 2006). Sakai et al. 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E-mails: [email protected], [email protected], zdhla@ Schmidt HA, Strimmer K, Vingron M, von Haeseler A (2002) TREE- yahoo.com.cn, [email protected], [email protected]; Siliang Yang, PUZZLE: maximum likelihood phylogenetic analysis using quartets Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and parallel computing. Bioinformatics 18:502–504. and Environmental Protection, Yancheng Teachers College, Yancheng Schneider-Broussard R, Neigel JE (1997) A large-subunit mitochon- 224002, China. drial ribosomal DNA sequence translocated to the nuclear genome of two stone crabs (Menippe). Mol Biol Evol 14:156–165. Schubart CD, Cuesta JA, Diesel R, Felder DL (2000) Molecular Supporting Information phylogeny, taxonomy, and evolution of nonmarine lineages within Additional Supporting Information may be found in the online the American grapsoid crabs (Crustacea: Brachyura). Mol Phylo- version of this article: genet Evol 15:179–190. Table S1. 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doi: 10.1111/j.1439-0469.2008.00509.x 2009 Blackwell Verlag GmbH