Copeia, 2001(4), pp. 997±1009 Relationships of the Salamandrid Genera Paramesotriton, Pachytriton, and Cynops Based on Mitochondrial DNA Sequences LAUREN M. CHAN,KELLY R. ZAMUDIO, AND DAVID B. WAKE We compared 786 base pairs of cytochrome b mitochondrial DNA sequence to examine the evolutionary relationships among seven species belonging to three gen- era of Asian newts: Paramesotriton, Pachytriton, and Cynops. We ®nd strong evidence supporting recognition of a clade for these genera. Although bootstrap support values are relatively low, both parsimony and likelihood analyses suggest that the species of Paramesotriton sampled form a monophyletic group with Paramesotriton caudopunctatus basal to the other three species. Cynops appears to be paraphyletic, with Pachytriton and Paramesotriton being more closely related to Cynops pyrrhogaster than to Cynops cyanurus. Pachytriton and Paramesotriton exhibit some morphological similarities and have more specialized breeding habits and environmental require- ments than Cynops, suggesting that they shared an evolutionary history before di- verging. Our morphological investigations corroborate previous studies that sug- gested Cynops is the most generalized representative of the clade and that it retains several ancestral character states. SIAN newts of the genera Paramesotriton, the molecular analyses, thereby offering limited A Pachytriton, and Cynops comprise 15 cur- resolution on relationships among species. rently recognized species and several undescri- Nonetheless, in the combined molecular and bed species that are widely distributed in south- morphological analyses Cynops, Paramesotriton, eastern Asia, including Japan, China, and north- and Pachytriton form a well-supported polytomy ern Vietnam (Zhao, 1999; Thorn and RaffaeÈlli, establishing the monophyly of this group. 2001). Phylogenetic studies of morphology Studies including multiple species from each (Wake and OÈ zeti, 1969) and molecular charac- genus have also had dif®culty resolving relation- ters (Titus and Larson, 1995) for the family Sal- ships within this clade of Asian newts. An allo- amandridae have linked these three genera in zyme study of salamandrids including two spe- a trichotomy with little or no resolution. Initial cies of Cynops (pyrrhogaster and ensicauda) and surveys focusing on variation in behavior, repro- one species of Paramesotriton (hongkongensis) but ductive pattern, external morphology, and no species of Pachytriton suggested that Cynops skull/hyobranchial characters found that the re- may be paraphyletic with respect to Paramesotri- lationships among the three genera differed de- ton (Hayashi and Matsui, 1989). Finally, a phe- pending on the characters used for analyses netic investigation of the relationships within (Wake and OÈ zeti, 1969). These authors con- Paramesotriton found evident differences in hy- cluded that Pachytriton is most closely related to oid apparatus and skull characters among ®ve Cynops wolterstorf® (at the time placed in the species in this genus (Pang et al., 1992). Be- monotypic genus Hypselotriton) based on overall cause no outgroups from other genera were similarity in morphology and aquatic feeding used for comparison, it is dif®cult to determine mechanisms and argued that these two might how these results extend to further relation- have arisen from an ancestral stock close to that ships among all Asian newts. The general lack which gave rise to Cynops (only Cynops pyrrhogas- of resolution in these systematic studies stems ter was available to them). However, they were from the conservative nature of morphology in uncertain concerning the phylogenetic relation- this lineage, with numerous plesiomorphic ships of Paramesotriton, which had been consid- characters retained in species having an overall ered a close relative of Cynops and Pachytriton ``generalized'' form (OÈ zeti and Wake, 1969). (Freytag and Petzold, 1961; Freytag, 1962). Here we compare mitochondrial DNA A more recent investigation combining mo- (mtDNA) gene sequences from representatives lecular (mitochondrial DNA) and morphologi- of these three genera to examine the phyloge- cal characters was also unable to resolve these netic relationships among them. Speci®cally, we relationships (Titus and Larson, 1995). Howev- use molecular data to address three questions er, this study focused on higher level relation- about relationships within this group using mo- ships among salamandrids and included only lecular data. First, we examine higher level re- one species of each of our three focal genera in lationships among the genera Paramesotriton, q 2001 by the American Society of Ichthyologists and Herpetologists 998 COPEIA, 2001, NO. 4 Pachytriton, and Cynops. Second, we focus within eye and was straightforward because no inser- each genus to look at relationships among spe- tions or deletions were present. Amino acid cies. And third, we address the possibility of the translations of our sequences were compared paraphyly of Cynops. In addition, we compare with that of Xenopus (Roe et al., 1985) to ensure morphological data for 14 of the 15 described that there were no nonsense mutations or species and one undescribed species to identify frameshifts. We sequenced 19 individuals of diagnostic characters that may further clarify re- which 15 were unique haplotypes used for phy- lationships within this clade. logenetic analysis (submitted to GenBank under accession numbers AF295671±AF295685). In MATERIALS AND METHODS addition, we included in our analyses the partial cytochrome b sequences for two species of Tri- Laboratory protocols.ÐIncluded in our study were turus (vulgaris and carnifex) obtained from 16 individuals representing four of the six spe- GenBank. cies of Paramesotriton, one of the two species of All phylogenetic analyses of the cytochrome b Pachytriton, and two of the seven species of Cy- sequences were conducted using the program nops. Two species of Triturus (vulgaris and car- PAUP*4.0beta2 (D. L. Swofford, Sinaner Assoc., nifex) from Europe, Taricha granulosa from the Inc., Sunderland, MA, 1999, unpubl.). We as- United States, and two species of Tylototriton (tal- signed the two species of Tylototriton as out- iangensis and verrucosus) from southeast Asia groups for all analyses. Pairwise sequence diver- were selected as sequential outgroups to our gences and Kimura two-parameter (K2p) cor- clade (Titus and Larson, 1995; Appendix 1). rected divergences were estimated among all Partial cytochrome b mtDNA sequences for Tri- pairs of sequences. We assessed levels of satu- turus vulgaris and Triturus carnifex were obtained ration for base substitutions by plotting percent from GenBank (Caccone et al., 1997; accession sequence divergences against K2p distances for numbers U55498 and U55499). For all other in- transitions and transversions at each codon po- dividuals, genomic DNA was isolated from fro- sition. Kimura two-parameter values higher than zen tissues or from samples preserved in EtOH corresponding uncorrected percent sequence by standard proteinase K digestion followed by divergence suggest that transitions and transver- either salt or phenol-chloroform puri®cation. sions at the third codon position may be satu- We used the polymerase chain reaction (PCR) rated (Fig. 1). Therefore, to determine the ef- to amplify approximately 690 base pairs of the fect that saturation may have on topology, we cytochrome b region of the mtDNA with the primers MVZ16 (59-AAA TAG GAA RTA TCA analyzed our data using both equal weighting YTC TGG TTT RAT-39) and either MVZ15 (59- and with third position changes downweighted GAA CTA ATG GCC CAC ACW WTA CGN AA- to both 25% and 50% of ®rst and second posi- 39) or Triton-cytb-Fl (59-CAA CGC CAT CAA tion changes. Other than the weighting option, ACA TCT CA-39). PCR ampli®cation reactions all other assumptions and parameters were were performed in total volumes of 25 ml with identical in phylogenetic reconstruction. containing 100 ng of DNA template, 1X Taq Maximum parsimony (MP) analyses consisted buffer, 1.0 mM of each primer, 0.75 mM dNTPs, of branch-and-bound searches using initial up- per bound computed via stepwise addition, 1.5 mM MgCl2, and 0.625 units Taq polymerase. Ampli®cation consisted of initial denaturation ``furthest'' addition sequence, and ``MulTrees'' at 94 C for 5 min followed by 35 cycles of de- options in effect. We also performed MP boot- naturation for 1 min at 94 C, annealing for 1 strap analysis, with 1000 replicates, as a measure min at 45±47 C, and extension for 1.25 min at of internal support; the settings for bootstrap 72 C. PCR ampli®cations were terminated with analyses were the same as those for the original a ®nal extension period of 5 min at 72 C. We branch-and-bound search. used ABI ¯uorescent dye terminator chemistry Maximum likelihood (ML) analyses included to cycle sequence fragments in both directions heuristic searches with 100 replicates of random with the same primers used in ampli®cation. addition of sequences and one tree held at each Products were electrophoresed on a 4.75% ac- step. For ML analyses, we selected TBR branch rylammide gel on an ABI 377 automated se- swapping, the ``MulTrees'' option in effect, and quencer (Applied Biosystems, Costa Mesa, CA). ``steepest descent'' option not in effect. We chose the HKY model (Hasegawa et al., 1985), Phylogenetic analyses.ÐMtDNA sequences were with starting branch lengths obtained using aligned to each other and to the cytochrome b Rogers-Swofford approximation and no en-