Marine Genomics 22 (2015) 37–44

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Hynobiidae origin in middle Cretaceous corroborated by the new mitochondrial genome of Hynobius chinensis Da Tang, Tianjun Xu ⁎, Yuena Sun ⁎ Laboratory of Fish Biogenetics and Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan 316022, China

a r t i c l e

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Article history: Received 10 December 2014 Received in revised form 12 March 2015 Accepted 12 March 2015 Available online 24 March 2015 Keywords: Hynobiidae Hynobius chinensis Molecular clock Origin Phylogeny Zhoushan Island

a b s t r a c t Hynobius chinensis was first described by Günther in the nineteenth century. At present, the origins of the extinct Hynobius chinensis on the Zhoushan Island (Hynobius chinensis-ZI) remain a mystery. It is the only species of family Hynobiidae on the Zhoushan Island. However, there is very little empirical evidence regarding Hynobius chinensis-ZI phylogenetic relationship, and when or how did its ancestors colonized the island. Here, we used mitochondrial genome data to recover the phylogeny of family Hynobiidae. Results suggested that the origin of Hynobiidae was most likely in Middle Cretaceous (~ 112.9 Mya), and some Hynobius species of Taiwan and Japan diverged earlier than that of the mainland of China. Hynobius chinensis-ZI diverged from its closest living relative (Hynobius yiwuensis) around 6.5 Mya, and Hynobius chinensis-ZI was isolated on Zhoushan Island since the postglacial transgression in Holocene period. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The most primitive family of terrestrial salamanders, the Hynobiidae (hynobiid salamanders), comprises 59 species belonging to 10 genera (MacLeod et al., 1997; Robertson et al., 2004) Now, most species native to East Asia are found to be extinct, endangered or vulnerable (AmphibiaWeb, http://amphibiaweb.org/). Of these, Hynobius chinensis (Chinese salamander) is endangered (assigned by IUCN Red List of Threatened Species, http://www.iucnredlist.org/ and China Red Data Book of Endangered Animals (Zhao, 1998)). Hynobius chinensis was first described by Günther (1889) from two type specimens in Yichang (Hubei Province, China), and re-described by Adler and Zhao in 1990 (Adler and Zhao, 1990). However, this type locality has been doubted by morphologists, because there is no living individuals had been found for more than a century. Recently, some morphologists have reported living individuals in Yichang (Wang et al., 2007) and revealed the Hynobius populations in the Chong'an and Wenling area become extirpated (Fu et al., 2003). At present, the populations occurred in southeastern China (Fig. 1), including Yichang (Hubei Province) (Adler and Zhao, 1990), Chong'an (Fujian Province) (Pope, 1931), Wenling (Zhejiang Province) (Boring and Chang, 1933; Chang, 1933) and Zhoushan (Zhejiang Province) (Ma and Gu, 1999) were described under the same name – Hynobius chinensis. In addition, the samples

⁎ Corresponding authors. E-mail addresses: [email protected] (T. Xu), [email protected] (Y. Sun).

http://dx.doi.org/10.1016/j.margen.2015.03.006 1874-7787/© 2015 Elsevier B.V. All rights reserved.

from Qiyang (Hunan Province, China) were also assigned to Hynobius chinensis, because this locality is geographically close to Yichang (Zhang et al., 2006). Zhoushan, also known as the fourth largest island of Zhejiang Provincei, is located in East China Sea (N 30°, E 122°). This island is about 20 km away from Mainland China, leading to a geographical isolation for many animals. Currently, Hynobius chinensis is the only terrestrial salamanders on the island (Hynobius chinensis-ZI), and as many as 4000 living individuals of this geographical population in the lentic ponds. Hynobius chinensis-ZI was thought morphologically different from other mainland species (Ma and Gu, 1999). Recently, research suggested that Hynobius chinensis-ZI was also genetically different from the other species in southeastern China (Fu et al., 2003). Here, we reported the new mitochondrial genome of Hynobius chinensis from Zhoushan Island to discuss its main features, and combined with published mitochondrial DNA to investigate the phylogenetic relationship of Hynobiidae, because few Hynobiidae phylogeny has collected Hynobius chinensis DNA samples from Zhoushan Island (Weisrock et al., 2013). It is important to note at the outset that some geographic barriers appear to have a significant influence on speciation and diversification, so we are trying to find new perspectives to help in understanding the Hynobiidae origin by collecting paleogeographic information for past 150 million years (from Colorado Plateau Geosystems, http://jan.ucc.nau.edu/~rcb7/, and Goal of the PALEOMAP Project, http://www.scotese.com/). A geochronologic time point related to the formation of the arc-shaped Japanese archipelago was used as the internal calibration point in divergence estimating. We hope that the

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Fig. 1. The distribution of Hynobius populations in southeastern China. Circles indicated localities in Hubei, Hunan, Fujian and Zhejiang, colored according to the assigned name (yellow: H. amjiensis; red: H. chinensis; green: H. yiwuensis; blue: H. guabangshanensis). Cai (1985) assigned all hynobiids in Zhejiang Province to H. yiwuensis. Specimens for the studies of Zhang et al. (2006) were collected from Qiyang, Hunan Province, where is the habitat of H. guabangshanensis. Recent investigations of the Wenling and Chong'an area did not reveal any trace of Hynobius species.

phylogenetic reconstruction and molecular timescale described in this study could be beneficial in the explore and advance of hynobiid salamanders evolution. 2. Methods 2.1. Sampling, sequencing and annotating the mitochondrial genome The material used for sequencing was collected from Zhoushan Island, Zhejiang Province. Total genomic DNA isolation was conducted as detailed in standard phenol-chloroform extraction method (Sambrook and Rusell, 2001). PCR reactions were performed on the PTC-200 (MJ) and eleven pairs of primers were designed for amplification (Supplemental Table S1). The mixture included 0.2 μM of primers, 0.2 mM of dNTPS, 1 μl of DNA template, 2 unit of Taq Plus DNA polymerase, and 5.0 μl of 10× Taq Plus polymerase buffer. The quality of fragments was assessed by agarose gel electrophoresis and then purified using the Gel Extraction Kit (Takara). After conversion of clones into plasmids, we selected clones and sequenced them. The clones were sequenced using a ABI 3730 automated sequencer with M13 forward and/or M13 reverse primers. All determined sequence were compared with all sequences available in DDBJ/EMBL/GenBank on the basis of BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi/). tRNAs were annotated by first screening by using tRNAscan-SE (Lowe and Eddy, 1997) and then manual identification of missing tRNA genes by comparing with that of other related organism. Nucleotide composition was calculated using the EditSeq program of DNAStar package (DNASTAR Inc., USA). 2.2. Taxonomic coverage and sequence alignment In current update of AmphibiaWeb (AW, http://amphibiaweb.org/), there are ten genera (59 species) in Hynobiidae. Pseudohynobius and Protohynobius were recognized as two separate genera (Frost, 2011), but in Amphibian Species of the World (ASW, http://www.amnh.org/), Protohynobius was abolished. Total 27 mitochondrial sequences of family Hynobiidae are available from GenBank searches (http://www. ncbi.nlm.nih.gov/taxonomy). Sequences used in this study were given in Table 1, and Cryptobranchidae was used as the out group. The multiple alignments of mitogenome sequences were subjected to G-INS-I, FFT-NS-1 or FFT-NS-2 strategies in MAFFT software (Katoh et al., 2005). The ambiguously aligned or highly diverged alignment of data

set were excluded to make the phylogenetic analyses be more reliable (Gatesy et al., 1993). The gaps retained in aligned sequences are recognized as missing data (Nei and Kumar, 2000). The final data consisted of 11,370 bp from 13 protein-coding genes, 2537 bp from two rRNA genes, 1534 bp from 22 tRNA genes, and 786 bp from the control region. Two species, Hynobius tokyoensis and Hynobius retardatus are lack of 12S rRNA genes in the published genomes, and the control region of former was incomplete. 2.3. Phylogenetic reconstruction Phylogenetic relationships were reconstructed by the Minimum Evolution (ME), Maximum Likelihood (ML), Maximum Parsimony (MP), and Neighbor Joining (NJ) using MEGA 5 and RAxML ver. 7.2.8 (Stamatakis, 2006; Tamura et al., 2011). In NJ and ME analyses, phylogenies were performed under the Maximum Composite Likelihood method and node reliability was estimated with 1000 bootstraps. Both MP and ML analyses were derived by using heuristic searches with 500 bootstraps, and all characters were treated as being equallyweighted in MP searches. Best-fit evolutionary models were selected by jModeltest 3.7 using Bayesian Information Criteria strategy (Posada, 2008). Partitioned Bayesian analysis was constructed with MrBayes 3.2 (Ronquist and Huelsenbeck, 2003). We partitioned the combined dataset into mixed models based on gene fragment types (proteincoding genes, rRNAs, tRNAs, and codon positions), shown in Supplementary Table S2 and S3. Random starting tree was used, and the Markov Chain Monte Carlo run over 1,000,000 generations sampling every 100 generations, finally ‘burn-in’ the first 2500 trees. Selection of best partition strategy was based on Bayes Factors calculations, using the harmonic mean approximation of the marginal model likelihood. The harmonic mean approximation of the marginal model likelihood was used for calculating Bayes factors (Kass and Raferty, 1995; Nylander et al., 2004). 2.4. Tracing ancestral divergence time The Bayesian molecular dating was performed with MCMCtree program in PAML 4.6 software (Yang and Rannala, 2006). The inferred ML tree was used as the required topology. An independent rates model (IR model) was used to specify the rates among internal nodes (Zhong et al., 2009). A conservative minimum bound of the

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Table 1 Taxonomy of species used in this study. Taxonomy/Species name

English name

Accession No.

Full Length

Genetic Distance

Distribution

Order Caudata Family Hynobiidae Batrachuperus londongensis Batrachuperus pinchonii Batrachuperus tibetanus Batrachuperus yenyuanensis Hynobius amjiensis Hynobius arisanensis Hynobius chinensis-ZI Hynobius chinensis-Q Hynobius formosanus Hynobius guabangshanensis Hynobius leechii Hynobius nebulosus Hynobius quelpaertensis Hynobius retardatus Hynobius tokyoensis Hynobius yangi Hynobius yiwuensis Liua shihi Liua tsinpaensis Onychodactylus fischeri Pachyhynobius shangchengensis Paradactylodon gorganensis Paradactylodon mustersi Pseudohynobius flavomaculatus Pseudohynobius shuichengensis Protohynobius puxiongensis Ranodon sibiricus Salamandrella keyserlingii

Longdong Stream Salamander Stream Salamander Alpine Stream Salamander Yenyuan Stream Salamander Anji Salamander Arisan Salamander Chinese Salamander Chinese Salamander Formosan Salamander Guabangshan Salamander Northeastern China Hynobiid Salamander Clouded Salamander Cheju Salamander Noboribetsu Salamander Tokyo Salamander Kori Salamander Yiwu Hynobiid Wushan Salamander Tsinpa Salamander Fischer's Clawed Salamander Shangcheng Stout Salamander Gorgan Mountain Salamander Paghman Mountain Salamander Yellow-spotted Salamander – Puxiong's Protohynobiid Siberian Salamander Siberian Salamander

NC008077 NC008083 NC008085 NC012430 DQ333808 NC009335 JQ710885 NC008088 NC008084 NC013762 NC008079 HM036356 NC010224 HM036351 HM036357 NC013825 HM036354 NC008078 NC008081 NC008089 NC008080 NC008091 NC008090 FJ532059 FJ532060 FJ532058 NC004021 NC008082

16,379 16,390 16,379 16,394 16,401 16,401 16,495 16,408 16,394 16,408 16,428 16,447 16,407 16,336 16,238 16,424 16,494 16,376 16,380 16,456 16,394 16,374 16,383 16,389 16,394 16,398 16,418 16,338

0.157 0.156 0.156 0.156 0.091 0.119 – 0.093 0.120 0.091 0.102 0.102 0.112 0.123 0.126 0.104 0.023 0.170 0.169 0.226 0.172 0.177 0.175 0.179 0.180 0.186 0.159 0.172

Sichuan, China Sichuan, Guangxi, Yunnan, China Sichuan, Xiaxi, Qinghai, Xizang, Gansu, China Sichuan, China Zhejiang, China Taiwan, China (Zhoushan) Zhejiang, China (Qiyang) Huban, China Taiwan, China Hunan, China Korea; Liaoning, Jilin, Heilongjiang, China Honshu, Shikoku, Kyushu, Ikishima, Japan South Korea Hokkaido, Japan Kanto, Honshu, Japan South Korea Zhejiang, China Sichuan, Hubei, Henan, Shanxi, Hunan, China Henan, Shanxi, Sichuan, China Southern Russian; northeastern China; South Korea Henan, Hubei, Anhui, China Gorgan, Golestan, Iran Kabul, Maidan, Afghanistan Hubei, Guizhou, Hunan, Sichuan, China Guizhou, China Sichuan, China Xinjiang, China; Kazakhstan Hokkaido, Japan; Sakhalin, Kurile, Russia; Mongolia; northeastern China; Korea

Order Caudata Family Cryptobranchidae Andrias davidianus Andrias japonicus Cryptobranchus alleganiensis

Chinese Giant Salamander Japanese Giant Salamander Eastern Hellbender

NC004926 NC007446 GQ368662

16,503 16,298 16,309

0.301 0.302 0.299

China Japan America

Note: -- and – represent unknown data or inexistence, respectively, along with accession numbers, locality and genetic distance versus Hynobius chinensis-ZI.

Cryptobranchidae–Hynobiidae split was constrained to be 145 Mya based on fossil record of Chunerpeton tianyiense (Gao and Shubin, 2003). The maximum age of ingroup root was fixed with a safe calibration date, 282 Ma, based on the molecular dating of Cryptobranchoidea– Salamandroidea split (Roelants et al., 2007). A geochronologic point (50 ± 5 Myr) related to the formation time of the arc-shaped Japanese archipelago was used as the internal calibration point, based on Ron Blakey's interpretation (Colorado Plateau Geosystems, http:// jan.ucc.nau.edu/~rcb7/; Goal of the PALEOMAP Project, http://www. scotese.com/). This calibration point was constrained to be the first appearance of native Hynobius species of Japan. The stationarity of Markov chains were diagnosed by at least two runs to reach stationarity. Each analysis was burn-in of 5000 cycles, and created a total of 10,000 samplings. We considered twice independent Bayesian analysis converged on similar likelihood as the proof of a model was desirable (Rannala, 2002; Castoe et al., 2004). 3. Results 3.1. Main features of mitochondrial genome Here, we identified the entire mitochondrial genome of Hynobius chinensis from Zhoushan Island (GenBank: JQ710885). Some bioinformatics traits in this new sequence deserve to be mentioned: (1) This complete mitochondrial DNA is 16,495 bp in length, larger than all other counterparts of Hynobiidae, which has a range from 16,238 bp (Hynobius tokyoensis) to 16,494 bp (Hynobius yiwuensis). (2) This is a compact gene arrangement without any loss or shifting of genes, only

few non-coding nucleotides, but it has lots of overlapping regions. These features suggested that it has experienced a steady mitochondrial evolution in a relatively slow speed in contrast to the accelerated trend in Demospongiae (Wang and Lavrov, 2008). Gene arrangement is identical to that of other typical vertebrates, and conform to the model that most genes are transcribed on the heavy strand, while NADH dehydrogenase subunit (NADH) 6, and 8 tRNA genes (tRNAGln, tRNAAla, tRNAAsn, tRNACys, tRNATyr, tRNASer(UCN), tRNAGlu, and tRNAPro) are transcribed on the light strand. (3) 22 tRNA genes ranging from 66 to 75 bp are interspersed between the rRNAs and protein-coding genes. For example, the small and large subunits of ribosomal RNAs (12S and 16S) are located in close proximity of each other, but separated by tRNAVal. (4) The assumed boundaries of beginning or end of rRNAs, tRNAs and proteincoding genes are adjacent or overlapping, and overlaps occurred over variable of nucleotides. Intergenic non-coding regions are negligible or nearly nonexistent, but an exception was found between tRNAPro and tRNAThr with 204 nucleotides. (5) Codon usage of protein-coding genes is consistent with that of vertebrates. All individual genes begin with the ATG initiation codon except for cytochrome c oxidase subunit (COX) 1 which begins with GTG. Complete translation stop codons (TAA, TAG, and AGA) are used for all coding genes, but two abbreviated stops (TA and T) are used by cytochrome b (Cytb), NADH1, NADH4 and COX3. (6) TTA is the most frequent codon, followed by ATT, TTT, ATA, and TCA, while AGA or AGG are missing. Leucine is the most frequent amino acid, and Cystine is the least frequent. (7) Previous study suggested that genetic differences within torrent salamanders are significant during the speciation durations (Good and Wake, 1992), Genetic distance between Hynobius chinensis-ZI and other species is

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range from 0.023 to 0.180 using complete mitochondrial DNA, with an average of 0.146. (8) The nucleotide compositions are as follows: A, 33.5%; G, 13.7%; C, 22.3%, T, 32.6%.

3.2. Phylogenetic reconstruction of family Hynobiidae A total of 31 mitochondrial sequences are used for reconstructing the phylogeny of family Hynobiidae. Data from comparative analysis showed a substantial informative and constant/variable nucleotide sites in this concatenated data set. The final data set has 16,227 sites, of which 6754 sites are parsimony informative and 8280 are variable. These data provided essential information for phylogenetic analyses. A well-resolved tree is produced by employing ME, ML, MP, NJ and Partitioned Bayesian analyses (Fig. 2). The phylogenetic relationships of this study is slightly different from that of previous researches (reviewed in Larson et al., 2003; Peng et al., 2010; Pyron and Wiens, 2011), and nearly all the internal branches supported by high bootstrap values (BPs) or Bayesian posterior probabilities (BPPs). The tree topologies of partition Bayesian was plotted using Figtree v1.3.1 (Rambaut, 2006). The major phylogenetic relationships were labeled from A to N in Fig. 2: (1) Hynobiidae is confidently recovered as a monophyletic group that supported by 1.0 B.P. and 100%. (2) Onychodactylus fischeri is placed in the basal position within family Hynobiidae, and is sister to all remaining species. It suggested that Onychodactylusi is a relatively ancient genus within this family. (3) Batrachuperus, Liua, Pseudohynobius and Protohynobius are confidently recovered as monophyletic group (Clade K; BPs = 100%, BPPs = 1). In ME and NJ trees, Liua and Batrachuperus are the closest sister groups with b60% BPs. (4) In previous studies, Pachyhynobius was recovered as the sister group to all other genera excepted Onychodactylus (Zheng et al., 2011), Pachyhynobius in this study is recovered as the more distant sister group to clade M excepted Onychodactylus, Paradactylodon and

Ranodon. (5) Ranodon is strongly supported as the sister group to Paradactylodon in all phylogenetic trees. (6) Twelve Hynobius species are nested placement as the biggest genus (Clade N; BPs = 100%, BPPs = 1). Phylogenetic reconstruction suggested that Hynobius chinensis-ZI is the closest living relative of Hynobius yiwuensis, and then sister to Hynobius amjiensis. Hynobius chinensis-Q (Qiyang) is recovered as the sister group of Hynobius guabangshanensis. However, Hynobius amjiensis + Hynobius chinensis-Q + Hynobius guabangshanensis formed a group in MP tree, and then paralleled to the clade of Hynobius chinensis-ZI + Hynobius yiwuensis with only 51% supports.

3.3. Divergence time estimation With the powerful statistical approaches, the rapid accumulation of DNA sequence data is challenging our understanding about the evolution and history of life (Pagel, 1999). Here, a total of 26 divergence events in family Hynobiidae were estimated in MCMCtree program using mitogenomic DNA data set including 13 protein-coding genes, 22 tRNAs, 2 rRNAs and one control region, successively. The estimated dates of divergence for nodes are shown in Fig. 3. Bayesian relaxed clock analyses indicated that the common ancestor of 28 hynobiid salamanders was established at least 112.9 Mya, and the divergence events did not occurred in a fixed rate (Venczel, 1999; Ho et al., 2005; Welch and Bromham, 2005). This is much earlier than previous estimate about 139 Mya (Zhang et al., 2005), but is close to that proposed by Zhang et al. (2006) and Zheng et al. (2011), who suggested that the occurrence of common ancestor of hynobiid salamanders to be the Cretaceous Period. Our results suggested most splits occurred at 77.1 Mya. In deep nodes, the estimated ages are slightly older than that of previous studies (Venczel, 1999; Zheng et al., 2011). Hynobius has the most members among this family and its splits occurred since 52.0 Mya. In addition, there is no splits since 19.0 Mya but four

Fig. 2. The phylogenetic relationships among Hynobiidea. Support values are illustrated in the circles filled with different color, first number represents the Bayesian posterior probabilities and right numbers represent bootstrap proportions derived from ML, ME, NJ analysis. Branch lengths are estimated by Bayesian approach and drawn to scale, with the bar indicating 0.1 expected changes per site.

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Fig. 3. Phylogenetic tree and origin dating of Hynobiidea. (a) Molecular timescale of Hynobiidea. (b) The map of Eurasian plate is adapted from Colorado Plateau Geosystems Website (http://jan.ucc.nau.edu/~rcb7/). (c) The current distribution of Hynobiidae is on the basis of Amphibian Species of the World (http://www.amnh.org/), and the rough distribution indicated in the corresponding letters.

potentially sympatric speciation events have happened between Hynobius chinensis-ZI + Hynobius yiwuensis (6.5 Mya), Hynobius leechii + Hynobius yangi (4.7 Mya), Hynobius arisanensis + Hynobius formosanus (3.2 Mya), and Hynobius chinensis-Q + Hynobius guabangshanensis (0.3 Mya). It is worthwhile to pay attention to some Hynobius species. These species, such as Hynobius retardatus, Hynobius tokyoensis (two native species of Japan), Hynobius arisanensis, Hynobius formosanus (two native species of Taiwan, China) lived on the isolated islands, who were found diverged earlier than others of Mainland China. It might suggest the splitting time of island-adapted ones were more close to the occurrence of their common ancestor. This study has

added a new calibration constraint, and the estimated intervals of most node ages are considerably overlapped with previous studies. 4. Discussion Hynobiid salamanders have always attracted biologists across conservation, diversification, and systematics, and extensive efforts have been made to their phylogenetic consideration. Lots of morphological studies about taxonomic identification have been done in family Hynobiidae (Sato, 1941; Fei et al., 2006; Frost, 2011), and a series of methods were used to verify cryptic species (Lee et al., 1998; Kim

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et al., 2003; Tominaga et al., 2003; Matsui et al., 2006; Nishikawa et al., 2001, 2007). Molecular studies suggested that the subtle morphological differentiation might connect to genetic variation (Fu et al., 2003). It is worthy note that some hynobiid salamanders in hilly mountains as stream-adapted or pond-adapted types caused researchers to question about their distribution records (Boring and Chang, 1933; Ma and Gu, 1999; Wang et al., 2007). Yet, their highly specialized morphological characters make it difficult to find unambiguous clues to their phylogeny. For example, Cai (1985) proposed the Hynobius population in Yiwu (Zhejiang, China) to be a new species (called Hynobius yiwuuensis), Fu et al. (2003) named all Hynobius populations in southeastern China to Hynobius yiwuuensis according to the distribution pattern and morphology. While other morphologists hold an opposite opinion. Zhao and Adler (1989) prefer to restrict Hynobius chinensis and Hynobius yiwuensis to be synonym by re-described two type holotype specimens collected from Yichang (Zhao and Adler, 1989). Further morphological studies hold same opinion as Zhao and Adler (1989) (Pope, 1931; Boring and Chang, 1933; Xu, 2002). Although some morphological variations were thought in Hynobius population of Zhoushan Island, of which was shown in body length, head-tail length ratio or egg capsules shape, etc (Ma and Gu, 1999). However, based on records from Fauna Sinica and AmphibiaWeb, the Hynobius population in Zhoushan Island is still assigned to Hynobius chinensis. Recent molecular phylogenetic studies have provided different phylogeny on hynobiid salamanders, and have estimated the divergence times (Zhang et al., 2006; Nishikawa et al., 2010; Pyron and Wiens, 2011; Zheng et al., 2011). Zhang et al. (2006) recovered the phylogenetic relationships of Asiatic salamanders, and their result suggested that Asiatic salamanders might have originated from North China,

~110 Mya. As Zhang et al. (2006) hypothesized, the hynobiid salamanders might have originated from an ancestral stream-adapted form, and proposed that geographic variation played a significant role in their evolutionary radiations (Zhang et al., 2006). While in their study, data for mitochondrial genomes of Pseudohynobius and Protohynobius were lacking and some important species lived on isolated islands were not collected. Nishikawa et al. (2010) compared the complete Cytb gene sequences of Hynobius species of Mainland China and some congeners from Japan and South Korea. The result suggested that the Hynobius species of Mainland China were separated into two lineages: Hynobius chinensis (Yichang) was most close to Hynobius maoershanensis (Xingan), and then sister to Hynobius guabangshanensis (Qiyang), while Hynobius amjiensis (Anji) and Hynobius yiwuensis (Ningbo) formed a monophyletic group (Nishikawa et al., 2010). A study, by using nuclear exons or mitochondrial data, had compared the results of divergence times of Hynobiidae origin (Zheng et al., 2011). The result suggested that the estimates using nuclear exons were more recent at most node ages, and the origin time was consistent with the suggestion of Zhang et al. (2006). A supermatrix approach provides a revised phylogeny of large-scale amphibians at family levels (Pyron and Wiens, 2011). They used 46 species of Hynobiidae excepted Protohynobius to recover the phylogeny. However, the gene lengths in their study are very different, which might introduce some degree of systematic bias on long-branch in phylogeny reconstruction (Felsenstein, 1978; Nikaido, 1999). In addition, Peng et al. (2010) conclude that the separate subfamily Protohynobius should not be valid, and proposed that it is likely nested in Hynobiidae (Peng et al., 2010). Our inferred topology is consistent with previous recover (Zhang et al., 2006), but slightly different from the findings of Larson et al. (2003) and Pyron and

Fig. 4. Schematic map showing the water depth and regional circulation pattern in the East China Sea. Map showing the mountain ranges of Tiantai and distribution of muddy sediment areas in the East China Sea. The regional circulation pattern of the Zhoushan Islands and the adjacent marine area during winter times included Yangtze River Diluted Water (YRDW), East China Sea Coastal Current (ESCC) and Taiwan Warm Current (TWC), modified after (Su, 1986; Liu et al., 2007).

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Wiens (2011). The placement of Pachyhynobius in our study was different from that of Peng et al. (2010). In partitioned Bayesian analyses, we compared posterior distributions and parameter estimates in different partition strategies, and confirmed that all partition strategies generated the same topology. The Hynobiidae was confidently reproduced as a monophyly, and at genus level several genera including Batrachuperus, Hynobius, Liua, Onychodactylus, Pachyhynobius and Salamandrella were recovered as monophyletic with high support values. We agree with the hypothesis that hynobiid salamanders originated in North China in Middle Cretaceous (Zhang et al., 2006; Zheng et al., 2011). In this study, we proposed a similar origin time of Hynobiidae around 112.9 Mya, and estimated the primary splits were 77.1 Mya, in Tertiary period. Here, we try to demonstrate the potential location for Hynobiidae origin using geologic literatures and plotting data from Colorado Plateau Geosystems, reviewed in Fig. 3. These paleogeographic evidences show that during late Jurassic and early Cretaceous, North China drifting into equatorial latitudes farther north, and it has remained ancient shallow sea 150 Mya. About 120 Mya, the land began to rise and the freshwater living area appeared in this period. At the same time (about 110 to 135 Mya), the emergence of flowering plants (Angiosperms) is thought to associate with the taxonomic richness and/or diversity changes of animal evolution. All above might be a catalyst for early explosion of organism, especially for faunal group in North China. For example, the fossils of Microraptor, Dilong, Psittacosaurus, Sinornithosaurus, Sinosauropteryx, Nemicolopterus (Sauropsida), Jeholornis, Changchengornis (Aves), and Eomaia (Tetrapoda) are successively found in Northeast China (Zhou et al., 2003; Li and Gao, 2007). Although it is a pity that there is no reliable fossil support for Hynobiidae living in this period, based on Venczel's hypotheses, during Tertiary and Quaternary the geographic distribution of Hynobiidae animals should be not only distributed in Asia, but wider than now (Venczel, 1999). It is still not clear when or how the ancestors of Hynobius chinensis colonized Zhoushan Island. It is the only Hynobius species on the island. Actually, the marine strait between Mainland China and Zhoushan Island is about 20 km, and its depth is relatively narrow and shallow, which is averaging at 20–40 m (Fig. 4). Now we has found a possible mechanism to explain how the ancestors of Hynobius chinensis via the narrow and shallow marine strait and spread to the island. Zhoushan Island belongs to Mount Tiantai (Zhejiang) in continental shelf of East China Sea, and this continental shelf has experienced at least three sea-land changes during last glacial period (Pleistocene, Quaternary). In last sea-level drop (~ 15,000 to 18,000 years ago), the continental shelf of East China Sea became 150–160 meter higher than the sea level (Wang, 1995; Ha and Wang, 1996; Li et al., 2006; Liu et al., 2007). And then the postglacial transgression in the Holocene period (11,700 calendar years BP) caused sea levels to rise again, and what is more important is that Zhoushan became isolated from the mainland China. We suppose the ancestors of Hynobius chinensis colonized the island at this particular time by attaching to some floating objects – it is not too surprising given their low vagility and the divergence time estimated in this study. Some evidences of the over-water dispersal have also proposed a potential mechanism to explain how some animals colonized islands (Censky et al., 1998; Austin et al., 2013). The regional circulation pattern of the Zhoushan Island would suggest that Yangtze River Diluted Water, East China Sea Coastal Current and Taiwan Warm Current transport a large amount of muddy sediment to adjacent marine area (Fig. 4), and then the seawater obstacle prevents the gene communications between Hynobius chinensis-ZI and other Hynobius species in mainland. In addition, the long time lashing and etching of seawater may be the reason why other small islands around Zhoushan Island have no Hynobius chinensis. Evidence of biological evolution is always rare, only a very small proportion of fossils are lucky enough to be finely preserved. It is an intractable problem in biological evolution – how we can find as much molecular information as to compensate for the lack of fossil records.

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