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Asian Herpetological Research 2013, 4(3): 155–165 DOI: 10.3724/SP.J.1245.2013.00155

Relationship of Old World and New World , with Comments on Underestimation of Diversity of Chinese Pseudoxenodon

Baolin ZHANG1, 2, 3 and Song HUANG1, 3, 4*

1 College of Life and Environment Sciences, Huangshan University, Huangshan 245021, Anhui, 2 School of Life Sciences, University, Kunming 650091, Yunnan, China 3 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China 4 Institute of Biodiversity and Geobiology, Department of Life Sciences, Tibet University, Lhasa 850000, Tibet, China

Abstract Assessment of the relationship between Pseudoxenodon and Dipsadinae has been hampered by lack of adequate samples. In this paper, we conducted phylogenetic analyses using two mitochondrial genes (12S and 16S rRNA) and one nuclear gene (c-mos) from thirteen specimens representing two species of Pseudoxenodon, together with 84 sequences of caenophidians and an outgroup sequence of Boa constrictor. Our study suggests that the Southeast Asian , Pseudoxenodon forms a robust genetic subclade within South American xenodontines, indicating that at least one lineage within this genus entered or returned to the Old World (OW) from the New World (NW) across the Beringian Land Bridge during the early Tertiary and the warm mid-Miocene. It also reveals the high intraspecific genomic variation within the populations of , indicating that species diversity of Pseudoxenodon in China is likely underestimated.

Keywords phylogenetic position, 12S rRNA, 16S rRNA, Beringian Land Bridge, cryptic species

1. Introduction includes approximately 3000 species of extant (Lawson et al., 2005; Vidal et al., 2007). Currently, With the rapid emergence of new sequencing technologies a total of 205 species belonging to 66 genera in 9 since the 1970s, molecular markers have been widely families occur in China (Zhao, 2006), with many of their used in modern biology, stimulating rapid progress across classifications proposed in current literature based only on the field of biological systematics (Ride et al., 2000; morphological data sets. Few molecular studies focus on Thomson et al., 2010). Previous systematic hypotheses examining the phylogeny and phylogeographic history of of snakes relied heavily on analyses of morphological snakes in , especially for the same or closely related data. However, for many species of snakes, finding species with a transcontinental range where cryptic distinct morphological synapomorphies was not viable diversity tends to be highest (Creer et al., 2001; Giannasi or intuitive, leading to cryptic phylogeographic lineages et al., 2001; Malhotra and Thorpe, 2004; Huang et al., masquerading as a single species (Utiger et al., 2002; 2007). Pseudoxenodon is one of these poorly studied Lawson et al., 2005; Burbrink et al., 2008; Huang et al., genera, with few studies on the group’s inter- and intra- 2009; Ling et al., 2010). specific relationships. The suborder (advanced snakes) An important diagnostic feature of Pseudoxenodon

* Corresponding author: Prof. Song HUANG, from Huangshan (Chinese common name “Oblique-scaled snakes”) is University, Anhui, China, with his research focusing on ophiology, the obliquely arranged scales on anterior part of the phylogenetics and biogeography of . body. The first species of oblique-scaled snakes, namely, E-mail: [email protected] Received: 26 January 2013 Accepted: 1 May 2013 Tropidonotus macrops was described by Blyth (1855) 156 Asian Herpetological Research Vol. 4 from Darjeeling, . Boulenger (1890) subsequently Lawson et al. (2005) and previous studies. Much of the distinguished this species from Tropidonotus (= Natrix) classification proposed by Lawson et al. (2005) has been by its oblique scales and placed it in the new genus adopted and corroborated by many contemporary workers Pseudoxenodon. McDowell (1987) found hemipenal in the current literature (e.g., He et al., 2009; Huang et al., synapomorphies between Pseudoxenodon and 2009; Ling et al., 2010). , and placed both the genera in the new subfamily . Presently, the genus 2. Materials and Methods Pseudoxenodon is comprised of six species, four of which are found in China: P. bambusicola, P. karlschmidti, P. 2.1 Downloaded sequences and supraspecific terminals macrops, and P. stejnegeri (Uetz, 2013). Species in this Table 1 shows 109 terminal taxa and sequences for group are widely distributed throughout southern and three genes (12S, 16S and c-mos), together with their southeastern Asia. They are known to inhabit areas near GenBank accession numbers and references. Ninety- water across plains, mountains, hills, scrublands, lawns, eight sequences were downloaded from GenBank (77 forests, cultivated lands and stream banks. Genetic studies sequences for 12S, 67 for 16S, and 75 for c-mos) and into these species’ taxonomic status are desperately 11 sequences (haplotypes, from 13 individuals) were needed for further ecological, biological and behavioral generated by us. In addition, 19 genera are represented research (http://www.arkive.org). by different species with different sequences as terminal Previous studies revealed that Pseudoxenodontinae is nodes. In order to maximize our dataset of combined the closest relative of Dipsadinae. Zaher (1999) examined genes, different sequences from congeneric species were hemipenal variation of these two groups, finding that combined to form supraspecific terminals at the generic the hemipenes of both have a bifurcated sulcus and level according to Bininda-Emonds et al. (1998). retain calyces. Lawson et al. (2005) first conducted the 2.2 DNA extraction, amplification and sequencing In molecular (cyt b and c-mos) study of Pseudoxenodon this study, we adhered to the Wild Protection with one specimen of P. karlschmidti, and reported Law of the People’s Republic of China. All experiments Pseudoxenodon as sister of Dipsadinae. Vidal’s (2007) involving live snakes were approved by the molecular analysis based on seven nuclear protein Ethics Committee at Huangshan University. A total coding genes from one specimen of P. bambusicola also of 13 individuals (2 of P. karlschmidti and 11 of shows the same sister relationship. Several subsequent P. macrops) were collected from 9 locations in 5 studies (e.g., He et al., 2009; Zaher et al., 2009; Pyron provinces of mainland China (Figure 1) during 2009. We et al., 2011) also obtained the same results using the have to euthanize some specimens in the field because sequences from these two individuals. Support for the of a long travel process. And the others were reared at Pseudoxenodontinae-Dipsadinae clade in their reported our laboratory for further morphological examination trees was always not robust, indicating the need for until they died. Fresh liver tissue or skeletal muscle was further analysis. removed and immediately preserved in 95% ethanol for Over the last two decades, the most frequently later sequencing of mitochondrial (12S and 16S rRNA) analyzed genetic fragments for Dipsadinae are two and nuclear (c-mos) genes. Unfortunately, the specimen mitochondrial genes (12S and 16S rRNA) and one nuclear of Pseudoxenodon sp. (considered as P. macrops at first) gene (c-mos) (Vidal et al., 2000, 2010; Kelly et al., 2003; from Pailong, Nyingtri, Xizang (Tibet), China was lost Pinou et al., 2004; Hedges et al., 2009). To address this during a long arduous travel, limiting the morphological question of uncertain phylogenetic relationships of the identity during our analysis. two subfamilies, we attempted to include all the three Total genomic DNA was extracted according to the gene sequences of caenophidian available from GenBank, phenol/chloroform extraction procedure (Sambrook et al., as well as one henophidian (Boa constrictor) 1989). Sequences were amplified from total DNA extracts as an outgroup, and obtained new sequences from 13 using polymerase chain reaction (PCR). Primer pairs individuals of Pseudoxenodon. for each gene used in this study were: L1091/H1557 The systematic nomenclature used in this paper is (Knight and Mindell, 1994) for 12S rRNA; 16Sar/16Sbr primarily based on the classification proposed by Lawson (Palumbi et al., 1991) for 16S rRNA; and S77/S78 for et al. (2005). However, we simultaneously followed c-mos (Lawson et al., 2005). The PCR reaction contained the recommendation by Pyron et al. (2011) in using the approximately 100 ng of template DNA, 1 μl of each name Dipsadinae to refer to Xenodontine that presents in primer, 5 μl of 10× reaction buffer, 2 μl dNTPs (each 2.5 No. 3 Baolin ZHANG and Song HUANG Old World Pseudoxenodon and New World Dipsadinae 157

Figure 1 Map showing the Chinese populations of Pseudoxenodon analyzed. In this study, two species of Pseudoxenodon were collected. For convenience, we use Roman numerals to represent the number of specimens in parenthesis. P. macrops: 1) Pailong, Nyingtri, Xizang (I); 2) Ya’an, (III); 3) Liangshan, Sichuan (I); 4) Mengzi, Honghe, Yunnan (II); 5) Mianyang, Sichuan (I); 6) Guangyuan, Sichuan (I); 7) Mt. Funiu, Luoyang, Henan (II); and P. karlschmidti: 8) Hunan (II). mM), and 2.0 units of Taq DNA in a 50 μl reaction. The constitute a serious limitation to phylogenetic inference reactions were cycled as follows: initial denaturation at as long as the number of variable and parsimony- 94 °C for 3 min, 30 cycles of denaturation at 94 °C for informative characters is sufficient (Kelly et al., 2003; 1 min, annealing at 45–57 °C for 1 min, extension at Philippe et al., 2004; Pyron et al., 2011). As expected, 72 °C for 1 min, and final extension at 72 °C for 5 min. in the present study the phylogeny results inferred from Genes were purified from 0.8% low-melting agarose gels Bayesian Inference (BI) and Maximum-Likelihood (ML) using a BioStar Glassmilk DNA purification Kit (BioStar trees show strong statistical support for most of the nodes International, Canada) and following the manufacturer’s (see below). instructions. The purified DNA was sequenced with Separate and combined analyses for each of the three a BigDye Terminator Cycle Sequencing Kit (Applied gene regions were performed using ML in Garli v0.96 Biosystems, USA) according to the manufacturer’s (Zwickl, 2006) and MrBayes v3.0 (Huelsenbeck and protocol with the primers used in PCR. Ronquist, 2001). Modeltest 3.7 (Posada and Crandall, 2.3 Phylogenetic reconstruction Sequences of 12S, 1998) was used to determine the model of nucleotide 16S, and c-mos, and their combinations were aligned, substitution under the Akaike Information Criterion respectively with other sequences retrieved from (AIC) for ML and Bayesian Information Criterion GenBank using ClustalX (Thompson et al., 1997), (BIC) for BI. The best-fit model GTR+I+G was and proofread by eye. Nucleotide composition and selected for the 12S and 16S sequences. The following the uncorrected P-distances were calculated in Mega models were selected for analysis of the protein coding 4.0 (Tamura et al., 2007). The 16S sequences of some c-mos gene: K80+G for the first codon position; taxa used in the present study are not available from HKY+G for the second codon position; and GTR for GenBank (e.g., , Waglerophis, , , the third codon position. For the Bayesian analysis, Thamnophis, Storeria, Malpolon, and Pareas). Although a starting tree was obtained using neighbor-joining the problem of missing data is often considered to methods. Posterior probabilities (PP) were obtained be a serious obstacle for phylogenetic reconstruction by Markov Chain Monte Carlo (MCMC) analysis (Anderson, 2001; Lemmon et al., 2009), simulation and with one cold chain and three heated chains. Bayesian empirical studies have shown that even extensive missing support for the nodes was inferred through a MCMC data (alignments containing 90% of missing data) do not model as implemented in MrBayes, using 1 000 000 158 Asian Herpetological Research Vol. 4

Table 1 Samples and sequence information.

Accession No. Species Sources 12S c-mos 16S Acrochordus granulatus AF544706 Vidal and Hedges (2002) Acrochordus javanicus AF512745 AF512745 Wilcox et al. (2002) Aipysurus laevis EU547132 EU546945 EU547181 Sanders et al. (2008) assimilis GQ457843 GQ457724 Zaher et al. (2009) Apostolepis dimidiate GQ457782 Zaher et al. (2009) Aspidelaps scutatus U96790 AY187968 AY188046 Slowinski and Keogh (unpublished); Nagy et al. (2003) Atheris ceratophora DQ305410 DQ305433 Castoe and Parkinson (2006) Atheris squamigera AF544734 Vidal and Hedges (2002) trihedrurus GQ457784 GQ457846 GQ457727 Zaher et al. (2009) Azemiops feae AY352774 AF544695 AY352713 Malhotra and Thorpe (2004); Vidal and Hedges (2002) Bitis_nasicornis DQ305411 AY187970 Castoe and Parkinson (2006); Nagy et al. (2003) Bitis peringueyi DQ305435 Castoe and Parkinson (2006) Boa constrictor AF512744 AF544676 AY188080 Wilcox et al. (2002); Nagy et al. (2003) Boiruna maculate GQ457785 GQ457847 Zaher et al. (2009) Bothrolycus ater FJ404144 FJ404249 AY611859 Vidal et al. (2008) Bothrops atrox DQ469788 Noonan and Sites (unpublished) Bothrops diporus DQ305431 DQ305454 Castoe and Parkinson (2006) Bungarus fasciatus EU547135 EU366447 EU547184 Sanders et al. (2008) Cacophis squamulosus EU547101 EU366451 EU547150 Sanders et al. (2008) Cantoria violacea EF395873 EF395922 EF395848 Alfaro et al. (2008) amoenus AY577013 DQ112082 AY577022 Pinou et al. (2004); Lawson et al. (2005) Causus resimus AF544696 Vidal and Hedges (2002) Causus rhombeatus DQ305409 DQ305432 Castoe and Parkinson (2006) Clelia bicolor GQ457787 GQ457849 Zaher et al. (2009) Clelia clelia AF158472 Vidal et al. (2000) Coronella austriaca AY122836 Utiger et al. (2002) Coronella girondica AF471113 EU022641 Lawson et al. (2005); Santos et al. (2008) Daboia russellii DQ305413 AF471156 DQ305436 Castoe and Parkinson (2006); Lawson et al. (2005) Diadophis punctatus AY577015 AF544705 AY577024 Pinou et al. (2004); Vidal and Hedges (2002) catesbyi Z46496 Heise et al. (1995) Dipsas indica GQ457789 GQ457850 Zaher et al. (2009) anomalus GQ457791 GQ457852 GQ457732 Zaher et al. (2009) mastersii EU547125 EU546938 EU547174 Sanders et al. (2008) Echiopsis curta EU547121 EU546934 EU547170 Sanders et al. (2008) Eirenis modestus AY039160 AY486957 Schaetti and Utiger (2001) Eirenis persicus AY376786 Nagy et al. (2003) Elaphe anomala AY122803 Utiger et al. (2002) Elaphe schrenckii AY122804 DQ902082 Utiger et al. (2002); Burbrink and Lawson (2007) Elapognathus coronata EU547118 EU546931 EU547167 Sanders et al. (2008) Erpeton tentaculatum EF395888 EF395936 EF395864 Alfaro et al. (2008) abacura AY577016 AF471141 AY577025 Pinou et al. (2004); Lawson et al. (2005) Fordonia leucobalia EF395890 EF395938 EF395866 Alfaro et al. (2008) Gerarda prevostiana EF395891 EF395939 EF395867 Alfaro et al. (2008) Gloydius shedaoensis AF057194 AF435019 AF057241 Parkinson, (1999); Jing et al. (unpublished) Gloydius ussuriensis AF057193 AF057240 Parkinson (1999) gomesi GQ457739 Zaher et al. (2009) Helicops pictiventris GQ457800 GQ457860 Zaher et al. (2009) Hemiaspis signata EU547123 EU546936 EU547172 Sanders et al. (2008) simus AY577020 AF471142 AY577029 Pinou et al. (2004); Lawson et al. (2005) Hierophis jugularis AY486941 Nagy et al. (2004) Hierophis schmidti AY376772 Nagy et al. (2003) Hierophis viridiflavus AY541507 Utiger and Schaetti (2004) Homalopsis buccata EF395892 EF395940 EF395868 Alfaro et al. (2008) Hormonotus modestus FJ404159 FJ404261 FJ404195 Vidal et al. (2008) gigas GQ457803 GQ457863 GQ457743 Zaher et al. (2009) triangularis GQ457804 GQ457864 GQ457744 Zaher et al. (2009) Hypsiglena slevini EU728584 Mulcahy et al. (2009) No. 3 Baolin ZHANG and Song HUANG Old World Pseudoxenodon and New World Dipsadinae 159

(Continued Table 1)

Accession No. Species Sources 12S c-mos 16S Hypsiglena torquata AF471159 Lawson et al. (2005) cenchoa EU728586 GQ457865 EU728586 Mulcahy et al. (2009); Zaher et al. (2009) Lapemis curtus EU547134 EU366453 EU547183 Sanders et al. (2008) Liophis amarali GQ457807 GQ457867 Zaher et al. (2009) Liophis jaegeri GQ457749 Zaher et al. (2009) Lycophidion capense FJ404178 FJ404279 AY611893 Vidal et al. (2008) Malpolon moilensis AY643313 DQ486157 Carranza et al. (2004); Kelly et al. (2009) Micropechis ikaheka EU547091 EU366449 EU547140 Sanders et al. (2008) richardsonii EF395893 EF395941 EF395869 Alfaro et al. (2008) Natrix maura AF402623 Alfaro and Arnold (2001) Natrix natrix AF544697 Vidal and Hedges (2002) Neelaps calonotus EU547109 EU546923 EU547158 Sanders et al. (2008) Ninia atrata GQ457814 GQ457874 Zaher et al. (2009) Ophiophagus Hannah NC011394 AY058940 NC011394 Slowinski and Lawson (unpublished) clathratus GQ457815 Zaher et al. (2009) AF158482 Vidal et al. (2000) Oxyrhopus rhombifer GQ457876 Zaher et al. (2009) Oxyuranus scutellatus EU547100 EU546916 EU547149 Sanders et al. (2008) Pareas carinatus AF544773 AF544692 Vidal and Hedges (2002) nasutus GQ457818 GQ457878 GQ457757 Zaher et al. (2009) aestiva GQ457819 GQ457879 GQ457758 Zaher et al. (2009) guerini GQ457822 GQ457882 GQ457761 Zaher et al. (2009) Pseudablabes agassizi GQ457823 GQ457883 GQ457762 Zaher et al. (2009) Pseudaspis cana FJ404187 FJ404288 AY611898 Vidal et al. (2008) Pseudechis australis EU547095 EU546912 EU547144 Sanders et al. (2008) neuwiedii AF158423 AF158490 Vidal et al. (2000) Pseudoboa nigra GQ457885 Zaher et al. (2009) plicatilis GQ457826 GQ457886 GQ457765 Zaher et al. (2009) Pseudonaja modesta EU547098 EU546915 EU547147 Sanders et al. (2008) JF697342 JF697330 Pseudoxenodon karlschmidti (HuN) JF697319 This study JF697340 JF697332 Pseudoxenodon macrops (HN1) JF697321 JF697343 JF697333 This study Pseudoxenodon macrops (HN2) JF697322 JF697343 JF697336 This Study Pseudoxenodon macrops (SC1) JF697325 JF697343 JF697339 This Study Pseudoxenodon macrops (SC2) JF697328 JF697343 JF697335 This Study Pseudoxenodon macrops (SC3) JF697324 JF697343 JF697338 This Study Pseudoxenodon macrops (SC4) JF697327 JF697344 JF697334 This Study Pseudoxenodon macrops (SC5) JF697323 JF697345 JF697337 This Study Pseudoxenodon macrops (SC6) JF697326 JF697341 JF697329 This Study Pseudoxenodon sp. (XZ) JF697318 JF697345 JF697331 This Study Pseudoxenodon macrops (YN) JF697320 GQ457889 GQ457768 This Study joberti GQ457829 AF471151 FJ907950 Zaher et al. (2009) Ptyas mucosus AY122828 AF544736 EU728583 Utiger et al. (2002); Lawson et al. (2005) nebulatus EU728583 GQ457770 Mulcahy et al. (2009); Vidal and Hedges (2002) Sibynomorphus garmani GQ457892 Zaher et al. (2009) Sibynomorphus mikanii GQ457832 EU546925 EU547160 Zaher et al. (2009) bertholdi EU547111 GQ457894 GQ457773 Sanders et al. (2008) pulcher GQ457834 AF471154 Zaher et al. (2009) Storeria dekayi AF402639 GQ457895 GU018167 Alfaro and Arnold (2001); Lawson et al. (2005) peruviana GU018147 GQ457853 GQ457733 Vidal et al. (2010); Zaher et al. (2009) affinis GQ457792 Zaher et al. (2009) Thamnophis couchii AF402653 AF471165 Alfaro and Arnold (2001) Thamnophis godmani AF433658 Lawson et al. (2005) Vipera ursinii EF012817 GQ457898 Jing et al. (unpublished) Waglerophis merremi GQ457840 EF395920 EF395846 Zaher et al. (2009) Xenochrophis vittatus EF395871 GQ457899 GQ457780 Alfaro et al. (2008) argenteus GQ457842 Zaher et al. (2009) 160 Asian Herpetological Research Vol. 4 generations after discarding a “burn-in” of 100 000 12S, 16S and c-mos genes of Caenophidia, Dipsadinae generations (representing the log-likelihood scores had not and Pseudoxenodon used in the present study are reached stationarity). Sampling was performed every 100 listed in Table 2. Sequences of uncorrected P-distance generations. We ran three additional analyses starting with were calculated between ingroup taxa (excluding random trees. Consensus of all post burn-in generations Pseudoxenodon), and specimens from 11 sites for 12S, (3 600 000 generations resulting in 36 000 trees) was 16S and c-mos genes are listed in Table 3. computed from all four runs. In addition, ML analyses 3.2 Topological results In this study, separate analyses were performed with RAxML-HPC BlackBox through of unlinked genes always obtained weakly supported the CIPRES Portal 2.0 at the San Diego Supercomputing phylogenetic relationships for internal nodes (trees Center (http://www.phylo.org/portal2). for individual genes not shown), whereas support for congruent relationships in the combined dataset is 3. Results generally high. Patterns of cladogenesis derived from BI and ML based on the combined dataset were nearly 3.1 Sequence characteristics The aligned sequences identical (Figure 2). resulted in a total of 1344 nucleotides (423 for 12S, 428 The BI and ML analyses resulted in five major groups, for 16SrRNA and 493 for c-mos). The alignment of the Pareatidae, Viperidae, , , c-mos sequences revealed two internal codon deletions and (Figure 2), which were similar to those for Pseudoeryx at positions 270–275. There is one codon presented in Kelly et al. (2003), Lawson et al. (2005), and deletion at positions 271–273 for Acrochordus, Bitis, Huang et al. (2009). Furthermore, we found natricines Hormonotus, Pseudoxenodon, , , and were imbedded in Dipsadinae, which are consistent with Dipsadinae. The number and position of deletions were the Pinou et al. (2004) hypotheses. Our results provide in agreement with those reported by Lawson et al. (2005) strong posterior probability support (PP, 99%) and and Zaher et al. (2009). moderate bootstrap support (BS, 65%) for the monophyly Sequencing of the 13 specimens of Pseudoxenodon of Dipsadinae. revealed 11 haplotypes. The 12S and 16S genes each In the BI/ML tree (Figure 2), the genus Pseudoxenodon produced 11 haplotypes whereas the c-mos gene yielded is nested within a New World clade composed mostly of 5 haplotypes. For each newly sequenced gene, two South American xenodontines, with both high PP (100%) specimens of P. karlschmidti from Hunan share one and BS values (91%). All the specimens collected cluster haplotype and two specimens of P. macrops from Mengzi, into a monophyletic group and have very high (100%) BS Yunnan share one haplotype. Each of the remaining and PP support. Within this group, three subclades were specimens has its own haplotype. identified: one for Pseudoxenodon sp. (Xizang), another The average nucleotide composition (%), numbers for P. karlschmidti (Hunan) and the last contaning the of variable (V) and parsimony informative (PI) sites for other 11 sequences of P. macrops. In addition, both ML

Table 2 Average nucleotide composition, numbers of variable (V) and parsimony informative (PI) sites for 12S, 16S and c-mos genes. n: Numbers of sequences of each group; %: PI/aligned sites.

12S (423 aligned sites) c-mos (493 aligned sites) 16S (448 aligned sites) Taxa V PI V PI V PI Caenophidia n = 109 229 189 (44.70%) 226 131 (26.60%) 220 171 (38.20%) Dipsadinae n = 48 167 127 (30.00%) 129 55 (11.20%) 176 120 (26.80%) Pseudoxenodon n = 11 23 9 (2.10%) 11 1 (0.20%) 21 9 (2.00%)

Table 3 Sequence divergence information. a: The smallest distance of 0.005 between E. anomala and E. schrenckii that indicates they may be conspecific. (Utiger et al., 2002; Ling et al., 2010); n: Numbers of sequences of each group; XZ: The specimen (considered to be Pseudoxenodon macrops at first) from Pailong, Nyingtri, Xizang (Tibet).

Uncorrected P-distance 12S 16S Taxa Max Min Mean Max Min Mean Overall ingroup taxa 0.225 0.025 0.124 (n = 84) 0.145 0.013 0.082 (n = 75) Dipsadinae 0.196 0.029a 0.112 (n = 37) 0.125 0.022 0.076 (n = 35) XZ vs. Pseudoxenodon 0.048 0.039 0.045 (n = 11) 0.038 0.026 0.031 (n = 11) No. 3 Baolin ZHANG and Song HUANG Old World Pseudoxenodon and New World Dipsadinae 161

Figure 2 The ML tree based on all combined genes of 96 species of . Supraspecific terminals are labeled with generic names only. The abbreviations in parentheses after Pseudoxenodon refer to the locations of specimens obtained. XZ: Xizang; HuN: Hunan; HN: Henan; SC: Sichuan; YN: Yunnan. NA: North America; SA: South America; and CA: Central America. Numbers above the line are bootstrap supports of ML, and numbers below are posterior probabilities (PP) of the 50% majority-rule consensus tree from BI (%). 162 Asian Herpetological Research Vol. 4 and BI combined data analyses place the Pseudoxenodon the evolutionary history of Pseudoxenodon in Tibet. sp. (Xizang) in an unresolved position outside of the In this study, the sample from Tibet falls into the remaining P. macrops and P. karlschmidti (Hunan). distribution area of P. macrops (Figure 3). However, our molecular evidence suggests that it likely represents a new 4. Discussion species of Pseudoxenodon rather than being a member of P. macrops, a testament to the underestimation of species 4.1 Phylogenetic position of Pseudoxenodon The same diversity of Chinese Pseudoxenodon. More specimens of gene fragments (12S and 16S) and an additional gene Pseudoxenodon across southern and southeastern Asia (c-mos) of dipsadids and natricines yield a topological should be represented in the future phylogenetic analyses pattern similar to that of Pinou et al. (2004). Some based on large molecular and morphological data sets. previous molecular studies assigned Pseudoxenodontinae 4.3 Biogeography of snake fauna exchange between and Dipsadinae as sister taxa (Lawson et al., 2005; OW and NW Biogeographic hypotheses concerning the Vidal et al., 2007; Zaher et al., 2009; Pyron et al., 2011) origin of Caenophidians have in the past suggested that with a weak support. Fortunately, the addition of taxa in Caenophidians have an Asian origin. This is demonstrated this study yielded a better resolution of the relationship by the fact that most of the basal lineages have an between the two groups. Our phylogenetic analyses clearly Asian range. For example, Acrochordus, Pareatidae, indicate that the OW genus Pseudoxenodon, formerly Viperidae (partly Asian) and Homalopsidae always show recognized as the subfamily Pseudoxenodontinae, is a successive basal branching pattern to the monophyletic internested within a NW clade composed mostly of South Caenophidians (Vidal et al., 2007). A generally accepted American xenodontines. Our results suggest the type perception about the dates and routes of dispersal has genus (Pseudoxenodon) of Pseudoxenodontinae appears revealed that the Colubridae entered the New World to be a member of Dipsadinae; quite different from the from Asia with the formation of the Beringian Bridge previous concept of this group. Our molecular data is by the early Oligocene (Pinou et al., 2004; Burbrink consistent with Zaher’s (1999) morphological evidence, and Lawson, 2007). As one of the largest subfamilies of primarily from the hemipenes, who found both of them Colubridae, the Dipsadinae could have also entered at the have a bifurcated sulcus and retain calyces. same time (Pinou et al., 2004). In fact, the suitable habitat, 4.2 Underestimation of species diversity of warm climate and the exposed continental bridge allowed Pseudoxenodon Our phylogenetic trees strongly support multiple opportunities for snakes to disperse from the OW the monophyly of Pseudoxenodon (PP, 100%; BS, 100%). to NW during the Paleocene and mid-Miocene (Burbrink However, the internal nodes within Pseudoxenodon and Lawson, 2007). However, the same opportunities also are unclear. The specimen from Tibet does not appear have facilitated migration along the opposite route for a to be a member of P. macrops or P. karlschmidti. complete process of two-way faunal exchange (Simpson, Moreover, the minimum pairwise sequence divergences 1943). Unfortunately, few studies focus on the dispersal of (uncorrected P-distance) reveal that the populations of P. organisms from the NW to the OW. macrops have high intraspecific variation (Table 3). The Dipsadinae is one of the largest subfamilies of minimum distances of Pseudoxenodon sp. (Xizang) vs. Colubridae with about 92 genera and more than 700 Pseudoxenodon (0.039 for 12S, 0.026 for 16S) were more species (Vidal et al. 2010). Based on the analysis of 12S than those within Dipsadinae and overall ingroup taxa, rRNA and 16S rRNA sequence data, Vidal et al. (2000) respectively. A possible explanation for the high level of found snakes in this assemblage have an Asian–North sequence divergence is attributed to geographic barriers. American origin, separating into the Central and the The Hengduan Mountains that make up the division South American xenodontines in the early Tertiary. Our between the Tibetan Plateau and the Central Chinese results are consistent with these inferences. Moreover, Plain have been documented as the major barrier to gene in the present study, Pseudoxenodon forms a robust flow among the terrestrial organisms occurring at high genetic subclade within South American xenodontines, and low elevations (Fu et al., 2005; Peng et al., 2006). indicating at least one lineage was separated from this The topography of this eco-region is extremely variable clade and then dispersed from the NW to the OW across and complex. Most of the parallel mountain ranges run the Beringian Land Bridge during the early Tertiary and in a north-south direction and are separated by deep, the warm mid-Miocene. We provide evidence suggesting narrowly incised river valleys (Huang et al., 2009). These that the existing Beringian link also facilitated the two- geological barriers may have played an important role in way migration. If all the members of Pseudoxenodon No. 3 Baolin ZHANG and Song HUANG Old World Pseudoxenodon and New World Dipsadinae 163

Figure 3 Maps showing the distribution of 6 species of Pseudoxenodon. A: P. macrops occurs in 13 provinces from in the east to Xizang in the west of China, and southwestwards to , India, Burma, , , and West ; B: P. karlschmidti in 6 provinces of southern China; C: P. bambusicola in 8 provinces of southern and southeastern China, and northern Vietnam; D: P. stejnegeri in 9 provinces of central and southeastern China; E: P. baramensis mainly in East Malaysia (Borneo, Sarawak); F: P. inornatus mainly in (, Java, Kalimantan) (Zhao, 2006; Uetz, 2013). are clustered into a monophyletic group and placed at References the same position in this study, then a NW origin of Pseudoxenodon can be inferred, but our sampling is not Alfaro M. E., Arnold S. J. 2001. Molecular systematics and adequate enough to provide strong evidence suggesting evolution of Regina and the thamnophiine snakes. Mol Phylogenet Evol, 21: 408–423 that the OW Pseudoxenodon diverged from Dipsadinae. Alfaro M. E., Karns D. R., Voris H. K., Brock C. D., Stuart B. Considered the ambiguity in aligning 12S and 16S rRNA L. 2008. Phylogeny, evolutionary history, and biogeography and the admittedly un-comprehensive sample of this study, of Oriental-Australian rear-fanged water snakes (: future work should be based on more genetic markers and Homalopsidae) inferred from mitochondrial and nuclear DNA more samples across the distribution range (Figure 3). In sequences. Mol Phylogenet Evol, 46: 576–593 Anderson J. S. 2001. The phylogenetic trunk: Maximal inclusion addition, multilateral cooperation is desperately needed. of taxa with missing data in an analysis of the lepospondyli Acknowledgements We extend our sincere gratitude to (Vertebrata, Tetrapoda). Syst Biol, 50: 170–193 Xin CHEN (City University of New York, USA) and Li Bininda-Emonds O. R. P., Bryant H. N., Russell A. P. 1998. Supraspecific taxa as terminals in cladistic analysis: Implicit DING (Chengdu Institute of Biology, Chinese Academy assumptions of monophyly and a comparison of methods. Biol J of Sciences, Sichuan, China) for providing tissues used Linn Soc, 64: 101–133 in this study. We appreciate Dr. Frank T. BURBRINK Blyth E. 1855. Notices and descriptions of various reptiles, new or and Alexander D. MCKELVY (College of Staten Island, little known, Part 2. J Asiat Soc Bengal, 23: 287–302 USA) for their valuable comments and extensive revisions Boulenger G. A. 1890. The Fauna of British India, Including Ceylon and Burma. Reptilia and Batrachia. London: Taylor and Francis on the early draft of this manuscript. Also, we thank Burbrink F. T., Fontanella F., Pyron R. A. 2008. Phylogeography Tao WAN, Jinmin CHEN, Chen LING and Xaoyu SUN across a continent: The evolutionary and demographic history (Huangshan University, Anhui, China) for their help in of the North American racer (Serpentes: Colubridae: Coluber data processing. We particularly thank the reviewers constrictor). Mol Phylogenet Evol, 47: 274–288 who did extensive corrections on the manuscript. This Burbrink F. T., Lawson R. 2007. How and when did Old World research was funded by the National Natural Science ratsnakes disperse into the New World? Mol Phylogenet Evol, 43: 173–189 Foundation of China (30870290, 31071891, 31060280) Cadle J. E. 1984. Molecular systematics of neotropical xenodontine and the Students Science Research Program of Huangshan snakes: III. Overview of xenodontine phylogeny and the history University (2010xdkj019). of New World snakes. Copeia, 1984: 641–652 164 Asian Herpetological Research Vol. 4

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