Zoologica Scripta

Multilocus phylogeny reveals unexpected diversification patterns in Asian wolf ( )

CAMERON D. SILER,CARL H. OLIVEROS,ANSSI SANTANEN &RAFE M. BROWN

Submitted: 6 September 2012 Siler, C. D., Oliveros, C. H., Santanen, A., Brown, R. M. (2013). Multilocus phylogeny Accepted: 8 December 2012 reveals unexpected diversification patterns in Asian wolf snakes (genus Lycodon). —Zoologica doi:10.1111/zsc.12007 Scripta, 42, 262–277. The diverse group of Asian wolf snakes of the genus Lycodon represents one of many poorly understood radiations of advanced snakes in the superfamily Colubroidea. Outside of three having previously been represented in higher-level phylogenetic analyses, nothing is known of the relationships among species in this unique, moderately diverse, group. The genus occurs widely from central to Southeast , and contains both widespread species to forms that are endemic to small islands. One-third of the diversity is found in the Philippine archipelago. Both morphological similarity and highly variable diagnostic characters have contributed to confusion over species-level diversity. Additionally, the placement of the genus among genera in the subfamily remains uncertain, although previous studies have supported a close relationship with the genus Dinodon. In this study, we provide the first estimate of phylogenetic relationships within the genus Lycodon using a new multi- locus data set. We provide statistical tests of monophyly based on biogeographic, morpho- logical and taxonomic hypotheses. With few exceptions, we are able to reject many of these hypotheses, indicating a need for taxonomic revisions and a reconsideration of the group's biogeography. Mapping of color patterns on our preferred phylogenetic tree suggests that banded and blotched types have evolved on multiple occasions in the history of the genus, whereas the solid-color (and possibly speckled) morphotype color patterns evolved only once. Our results reveal that the colubrid genus Dinodon is nested within Lycodon—a clear finding that necessitates the placing of the former genus in synonymy with the latter. Corresponding author: Cameron D. Siler, Department of Biology, University of South Dakota, Vermillion, SD 57069, USA. E-mail: [email protected] Carl H. Oliveros, Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS, 66045-7561, USA. E-mail: [email protected] Anssi Santanen, Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS, 66045-7561, USA. E-mail: [email protected] Rafe M. Brown, Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS, 66045-7561, USA. E-mail: [email protected]

Introduction be monophyletic based on both morphological (Rieppel The superfamily Colubroidea (sensu Pyron et al. 2011), or 1988; Zaher 1999; Lee & Scanlon 2002) and molecular advanced snakes, represents one of the most strikingly data (Cadle 1988; Heise et al. 1995; Kraus & Brown 1998; diverse terrestrial radiations of living vertebrates (Lawson Gravlund 2001; Slowinski & Lawson 2002, 2005; Wilcox et al. 2005; Burbrink & Pyron 2008; Pyron et al. 2011). et al. 2002; Lawson et al. 2005; Pyron et al. 2011). Over Currently, more than 2500 species are recognized (Lawson the last decade, numerous studies have investigated rela- et al. 2005; Pyron et al. 2011) in this relatively young clade tionships among advanced snakes within the superfamily dating only to the Cenozoic (Burbrink & Pyron 2008; Colubroidea with widely varying degrees of taxonomic Vidal et al. 2009). Seven families of snakes are considered inclusion (Lawson et al. 2005; Burbrink & Pyron 2008; members of the clade (, Elapidae, Homalopsi- Wiens et al. 2008; Kelly et al. 2009; Vidal et al. 2009; dae, Lamprophiidae, Pareatidae, Viperidae and Xenoder- Zaher et al. 2009; Pyron et al. 2011). To date, the superm- matidae; Pyron et al. 2011), which has been determined to atrix of Pyron et al. (2011) has been by far the most

262 ª 2013 The Norwegian Academy of Science and Letters, 42, 3, May 2012, pp 262–277 C. D. Siler et al.  Multilocus phylogeny of Asian wolf snakes extensive of these studies, including 761 species represent- morphological data, sufficient genetic sampling has recently ing 299 genera (29% of the global species diversity of been amassed to allow for an initial molecular appraisal of advanced snakes). the genus’ phylogenetic relationships and affinities to other The computational obstacles involved in estimating phy- SE Asian genera. The geographic ranges of species in the logenies for this and other highly diverse radiations are genus Lycodon fall along a divergent spectrum, from substantial; moreover, when combined with the absence of widespread species whose range spans nearly the entire dis- available genetic samples for a large number of recognized tribution of the genus (i.e., L. aulicus) to a number of species, we likely are far from a complete understanding range-restricted, microendemic lineages (i.e., L. alcalai, of species-level relationships within advanced snakes. L. bibonius, L. chrysoprateros, L. fausti, L. ferroni, L. solivagus, Despite the relative success of new approaches and meth- L. tesselatus). Coloration varies greatly across this unique odologies to employing supermatrices in phylogenetic group of snakes; however, most species can be grouped studies of species-rich groups (Sanderson et al. 2003; into one of four distinct colour pattern categories: banded, Driskell et al. 2004; Philippe et al. 2004; Wiens et al. blotched, solid and speckled (Fig. 2). Variation in colour 2005; de Queiroz & Gatesy 2007; Thomson & Shaffer patterns among species representing each of these 2010; Pyron et al. 2011), there is still a clear need for con- morphotypes has consistently led to confusion over species tinued research aimed at filling many of the gaps in taxo- boundaries (Lanza 1999). nomic coverage and resolving more fine-scale relationships Nearly one-third of the diversity within the genus occurs among the many poorly understood genera within Colu- in the , with nine of the 11 species now consid- broidea. A characteristic example of one of these enig- ered endemic to this island archipelago (Lanza 1999; Gau- matic genera is the Wolf Snakes of the genus Lycodon lke 2002). Faunal demarcations in the archipelago have within the subfamily Colubrinae (family Colubridae). traditionally been explained by the geography of Pleisto- Although this radiation of non-venomous Asian snakes is cene aggregate island complexes (PAICs: Brown & Dies- only moderately diverse (36 currently recognized species; mos 2002, 2009; Heaney 1985; Heaney et al. 1998, 2005; The Database 2012), only three species have ever Siler et al. 2010, 2012b). During glacial periods, adjacent been included in phylogenetic studies (L. capucinus, L. lao- islands separated by shallow waters experienced greater ensis, and L. zawi; Heise et al. 1995; Kraus & Brown 1998; connectivity as decreased sea levels (100–140 m below cur- Lawson et al. 2005; Kelly et al. 2003, 2009; Pyron et al. rent levels) resulted in increased land-positive regions. The 2011). Among the studies that have included representa- cyclical nature of this process has provided an explanatory tives of Lycodon, the genus has been consistently recovered tool for explaining the distribution of biotic diversity in the as the sister species to the genus Dinodon with moderate Philippines. Although recent studies have resulted in mixed support (Kelly et al. 2003, 2009; Lawson et al. 2005; Pyron support for the PAIC model of diversification (review: Siler et al. 2011). When wider taxonomic sampling has been et al. 2010, 2012b), this model remains a heuristic tool for included in phylogenetic analyses, the genera Lycodon and exploring and understanding many of the evolutionary pro- Dinodon are recovered as members of a larger clade that cesses underlying the accumulation of biodiversity in the includes the colubrid genera Boiga, , Dipsadoboa, Philippines. Crotaphopeltis, and Telescopus, albeit with weak support In this study, we investigate the patterns of diversifica- (Kelly et al. 2003, 2009; Lawson et al. 2005; Pyron et al. tion among species of Lycodon from a phylogenetic perspec- 2011). tive, providing the first estimate of genealogical In just the last two decades, 14 new species of snakes of relationships for this unique radiation of Asian snakes. the genus Lycodon have been described, increasing the First, we attempt to estimate the phylogenetic position of known diversity of this Indian and Southeast Asian colubrid Lycodon among recognized, closely related colubrid snakes genus by nearly 40% (Ota & Ross 1994; Lanza 1999; to provide insight into patterns of morphological evolution Slowinski et al. 2001; Daltry & Wüster 2002; Gaulke 2002; and regional diversification. Second, we provide a first Mukherjee & Bhupathy 2007; Vogel et al. 2009; Vogel & assessment of the evolution of major colour patterns in Lyc- David 2010; Vogel & Luo 2011; Zhang et al. 2011; Vogel odon. Finally, we test the following suite of biogeographic, et al. 2012). The 36 recognized species of Lycodon occur morphological and taxonomic hypotheses aimed at better throughout central to , from regions east of understanding how diversity is partitioned among Wolf the Caspian Sea, eastern Iran and to southern , Snakes. Here, we address three general questions: (i) Are the Indo-Australian Archipelago, the Ryukyu Islands of currently recognized species boundaries supported by phy- and the Philippines (Lanza 1999; Fig. 1). Although logeny? (ii) Do previously defined species groups with many of these newly described species have been named common color pattern types form monophyletic clades? from just a few specimens, and solely on the basis of And (iii) do biogeographic patterns observed in other Asian

ª 2013 The Norwegian Academy of Science and Letters, 42, 3, May 2012, pp 262–277 263 Multilocus phylogeny of Asian wolf snakes  C. D. Siler et al.

Fig. 1 Distribution of Lycodon samples used for this study. Species-specific locality markers correspond to the internal figure key. Philippine samples are shown on the enlarged inset map. The recognized distribution of Dinodon species included in this study is shaded for reference. squamates also apply to Wolf Snakes and do these explain edge, tissues are unavailable for more than half of the species boundaries? recognized species diversity in the genus. To assess Our data reveal patterns of lineage diversification at odds monophyly of the genus, a broad sampling (17 taxa with currently recognized ; we conclude that representing 11 genera) from the family Colubridae was although there is some support for a few instances that included (Appendix 1) based on recent higher-level may result in the eventual recognition of additional cryptic phylogenetic studies of Colubroidea (Kraus & Brown species, diversity within some portions of the genus actually 1998; Lawson et al. 2005; Kelly et al. 2009; Pyron et al. may be overestimated as a result of taxonomic decisions 2011). based on colour pattern and untested biogeographic expec- Genomic DNA was extracted from liver tissues stored in tations. This article represents a first step towards an 95% ethanol. We sequenced the mitochondrial gene Cyto- understanding of the major evolutionary trends in colour chrome b (cyt-b) in 50 vouchered specimens using pub- patterns, biogeographic relationships and phylogeny- lished primers and protocols (Burbrink et al. 2000; Lawson informed taxonomy of Asian wolf snakes. et al. 2005; Pyron et al. 2011). For 39 and 41 of these sam- ples (Appendix 1), we also sequenced a 569 base pair Materials and methods region of the nuclear oocyte maturation factor Mos (c- Taxon sampling and data collection mos) gene and a 1018 base pair region of the fifth intron Ingroup sampling included 44 individuals representing at of the nuclear Vimentin (vim) gene, respectively, using least 16 of the 37 currently recognized species in Lycodon published primers and protocols (Pyron and Burbrink (Figs 1 and 2; Appendix 1). To the best of our knowl- 2009; Pyron et al. 2011). Amplified products were

264 ª 2013 The Norwegian Academy of Science and Letters, 42, 3, May 2012, pp 262–277 C. D. Siler et al.  Multilocus phylogeny of Asian wolf snakes

Fig. 2 Hypothesized relationships among species of Lycodon for the three genes sampled in this study (mitochondrial cyt-b; nuclear c-mos and vim), illustrated by ML bootstrap topologies. Nodes supported by  95% Bayesian PP and  70% MLBP are indicated by dots on nodes. Terminals are labeled with taxonomic names, followed by sampling localities when required for clarity. The phylogenetic position of the genus Dinodon is highlighted for reference. visualized on 1.5% agarose gels, and PCR products were (NC9406038, Amersham Biosciences) in Centri-Sep 96 purified with 1 lL of a 20% dilution of ExoSAP-IT spin plates (CS-961, Princeton Separations, Princeton, NJ, (US78201, Amersham Biosciences, Piscataway, NJ, USA). USA). Purified products were analysed with an ABI Prism Cycle sequencing reactions were run using ABI Prism Big- 3130xl Genetic Analyzer (Applied Biosystems). Continuous Dye Terminator chemistry (Ver. 3.1; Applied Biosystems, gene sequences were assembled and edited using SEQUEN- Foster City, CA, USA) and purified with Sephadex CHER 4.8 (Gene Codes Corp., Ann Arbor, MI, USA). To

ª 2013 The Norwegian Academy of Science and Letters, 42, 3, May 2012, pp 262–277 265 Multilocus phylogeny of Asian wolf snakes  C. D. Siler et al. this data set, we added 11 sequences of cyt-b and one every 5000 generations. We assessed stationarity and con- sequence of c-mos of outgroup taxa available on genbank vergence of parameters with TRACER v1.4 (Rambaut & (Appendix 1). All novel sequences were deposited in Gen- Drummond 2007) and confirmed convergence of tree splits Bank (Appendix 1). with AWTY (Wilgenbusch et al. 2004). Stationarity was achieved after three million generations (i.e., the first Sequence alignment and phylogenetic analyses 20%), and we conservatively discarded the first 25% of Initial alignments were produced in Muscle (Edgar 2004) samples as burn-in. with minimal subsequent manual adjustments. To assess Partitioned maximum likelihood (ML) analyses were phylogenetic congruence between the mitochondrial and conducted in RAXMLHPC v7.0 (Stamatakis 2006) on the con- nuclear data, we inferred the phylogeny for each gene catenated data set using the same partitioning strategy as independently using likelihood and Bayesian analyses. Fol- for Bayesian analysis. The more complex model (GTR + lowing the observation of no moderate to highly sup- Γ) was applied for all subsets (Table 1), and 100 replicate ported incongruence between data sets (Fig. 2), we felt ML inferences were performed for each analysis. Each justified in using the combined, concatenated, data for inference was initiated with a random starting tree, and subsequent analyses. Exploratory analyses of the combined nodal support was assessed with 100 bootstrap pseudorepli- data set of 61 individuals (including all taxa, some of cates (Stamatakis et al. 2008). which were missing data for c-mos and vim) and a To compare the results of concatenated analyses to a reduced data set of individuals with no missing data exhib- multilocus, coalescent analysis, we conducted partitioned, ited identical relationships; we therefore chose to include coalescent-based analyses in the program BEAST v1.7.4 all available data (61 individuals) for subsequent analyses (Drummond & Rambaut 2007). Four independent BEAST of the concatenated data set. Alignments and resulting runs of 80 million generations were completed under the topologies are deposited in Dryad (doi:10.5061/dryad. same partitioning strategy as for Bayesian analyses, impos- cp6gg). ing an uncorrelated lognormal relaxed clock prior on sub- Concatenated, partitioned Bayesian analyses were con- stitution rate (Drummond et al. 2006) and Yule speciation ducted in MRBAYES v3.2.1 (Ronquist & Huelsenbeck 2003). prior. Parameters were sampled every 20 000 generations, The mitochondrial data set was partitioned by codon posi- and the initial 50% of each run was discarded as burn-in. tion for the protein-coding region of cyt-b, c-mos and vim Convergence was evaluated under the same strategy as for were analysed as single subsets. The Akaike Information Bayesian analyses. Criterion (AIC), as implemented in JMODELTEST v0.1.1 (Posada 2008), was used to select the best model of nucleo- Hypotheses testing, biogeography, colour patterns and tide substitution for each partition (Table 1). A rate multi- taxonomy plier model was used to allow substitution rates to vary We tested a series of hypotheses based on taxonomic, among subsets, and default priors were used for all model morphological and biogeographic expectations to address parameters. We ran four independent MCMC analyses, the following three major questions concerning the pat- each with four Metropolis-coupled chains, an incremental terns of Lycodon diversification (Table 2). (i) Are currently heating temperature of 0.02, and an exponential distribu- recognized species boundaries among widespread and mic- tion with a rate parameter of 25 as the prior on branch roendemic species supported within a phylogenetic con- lengths (Marshall 2010). All analyses were run for 15 mil- text? (ii) Are the predominant color patterns exhibited by lion generations, with parameters and topologies sampled species of the genus Lycodon monophyletic groups? And (iii) do monophyletic species assemblages conform to regional biogeographic expectations? More specifically, we Table 1 Models of evolution selected by AIC (as implemented in were interested in addressing several questions concerning fi JMODELTEST) and those applied in partitioned, model-based, analy- biogeographic patterns of Lycodon diversi cation: (A) Are ses of mitochondrial (cyt-b) and nuclear (c-mos, vim) data the radiations of species of the genus Lycodon in the Phil- ippines and in Mainland Asia monophyletic? And (B) Number of within the Philippines, are recognized island groups Partition AIC model characters (Babuyan and Batanes Island Groups) regional centers of Cytb, 1st codon position GTR + Γ 370 diversity for monophyletic radiations of microendemic spe- Cytb, 2nd codon position GTR + Γ 370 cies? To statistically evaluate the probability of each Cytb, 3rd codon position GTR 370 experimentally constrained topology, we estimated the Cmos HKY + Γ 569 probability of each hypothesis within a Bayesian frame- Vimentin HKY + Γ 1018 work using the proportion of 3004 post-burn-in trees

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Table 2 Description of topology tests conducted using Bayesian methods

Hypothesis Description of constraint Posterior probability

Biogeographic tests

H1 Monophyly of Philippine Lycodon All samples of Philippine Lycodon 0.0

H2 Monophyly of Mainland Asia Lycodon All samples of Lycodon from Mainland 0.0 Asia (including Peninsular )

H3 Monophyly of Babuyan Island Group Lycodon All samples of Lycodon from the Babuyan 0.0 Island Group, Philippines

H4 Monophyly of Batanes Island Group Lycodon All samples of Lycodon from the Batanes 0.17 Island Group, Philippines Tests of hypotheses based on morphology

H5 Monophyly of solid morphotype All samples of L. alcalai, L. chrysoprateros, 1.0 L. sp. (Babuyan Claro Island), and L. sp. (Calayan Island)1

H6 Monophyly of banded morphotype All samples of Lycodon bibonius, L. dumerilii, L. effraensis, 0.0 L. cf. effraensis, L. fasciatus, L. laoensis, L. ruhstrati, L. stormi, and L. subcinctus

H7 Monophyly of blotched morphotype All samples of , L. butleri, L. muelleri, and 0.0 L. zawi Tests of hypotheses based on current taxonomy Interspecific

H8 Monophyly of Lycodon All samples of Lycodon 0.0

H9 Reciprocal Monophyly of Lycodon and Dinodon All samples of Lycodon constrained to be reciprocally monophyletic 0.0 to a clade containing all samples of Dinodon Intraspecific

H10 Monophyly of Lycodon alcalai All samples of Lycodon alcalai 0.18

H11 Monophyly of Philippine L. aulicus All samples of Lycodon aulicus from the Philippines 0.0

H12 Monophyly of Mainland Asia L. aulicus All samples of Lycodon aulicus from Mainland Asia 0.0

1The population of Lycodon on Babuyan Claro Island possesses a solid to lightly banded color pattern. For the purposes of this study we consider the population generally to possess a solid color pattern.

consistent with each topology as an estimate of the poster- Phylogenetic analyses ior probability of that hypothesis. This was accomplished With few exceptions, analyses of the combined data result by filtering the pool of post-burn-in trees for each con- in topologies with high ML bootstrap support and poster- straint topology using the program PAUP* v4.0b10 (Swof- ior probabilities among our ingroup taxa. Although analy- ford 1999). ses resulted in poor to moderate support for several relationships among outgroup taxa, general topological pat- Results terns are congruent across these analyses (Figs 2 and 3; Taxon sampling, data collection and sequence alignment Fig. S1). As has been observed in recent higher-level stud- The complete, aligned matrix contains 44 samples of Lyc- ies of the family Colubridae, poor basal support in the odon, representing 43% of the currently recognized species. clade containing Wolf Snakes results in uncertainty regard- Seventeen additional samples are included from the family ing the close relatives of the genus Lycodon (Pyron et al. Colubridae, including representative taxa of the following 2011). Our analyses do recover a clade of colubrid snakes genera: , Boiga, Dasypeltis, Dinodon, Dipsadoboa, (Boiga + Dasypeltis + Dipsadoboa + Crotaphopeltis + Telescopus) Ptyas, Gonyosoma, Lampropeltis, , Pantherophis and as sister to a clade of Lycodon + Dinodon as was similarly . Following initial unrooted analyses, and in the reported in Pyron et al. (2011), but with weak support light of recent estimates of colubrid phylogeny (Pyron et al. (Fig. 3, Clade 1). Previous studies that had limited sam- 2011), we rooted the tree using Ahaetulla prasina. A 364 pling of Lycodon have found high support for a sister rela- base pair insertion was observed in the middle of the vim tionship between the genera Lycodon and Dinodon (Kelly gene for Lycodon effraensis. Exploratory analyses excluding et al. 2003, 2009; Lawson et al. 2005; Pyron et al. 2011). this region from the data set had no observable impact on However, all concatenated and coalescent-based analyses the results. The number of variable and parsimony-infor- based on denser sampling of the genus recover Dinodon mative characters are as follows : 584 and 518 of 1110 (cyt- taxa nested within Lycodon [Figs 2 and 3 (Clades 2, 7); Fig. b); 67 and 45 of 569 (c-mos); and 137 and 99 of 1018 (vim). S1].

ª 2013 The Norwegian Academy of Science and Letters, 42, 3, May 2012, pp 262–277 267 Multilocus phylogeny of Asian wolf snakes  C. D. Siler et al.

Fig. 3 Hypothesized relationships among species of Lycodon included in this study, illustrated by the maximum clade credibility tree resulting from Bayesian analyses of the concatenated molecular data set (mtDNA + nuDNA). Nodes supported by  95% Bayesian PP and  70% MLBP are indicated by dots on nodes. Terminals are labeled with taxonomic names and sampling localities, with representative photographs showing the diversity of color patterns across the phylogeny. The phylogenetic position of the genus Dinodon is highlighted in red for reference. Numerical labels correspond to clades referred to in the Results and Discussion.

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Four major clades were recovered among the focal taxa near-solid morphotype is present only in taxa in the islands with strong support in all analyses (Fig. 3, Clades 3, 6, 10, north of Luzon, among which there is little genetic diver- and Lycodon stormi). All analyses recovered five Philippine gence. The majority of taxonomy-based hypotheses endemic species in a single monophyletic clade (Fig. 3, received no support; however, the results weakly supported Clade 3). The microendemic species from the Babuyan and the monophyly of island populations of L. alcalai Batanes Island Groups in the northern extreme of the Phil- (pp = 0.18; Table 2). ippines (L. alcalai, L. bibonius, L. chrysoprateros and L. sp. Babuyan Claro) were recovered as part of a single clade Discussion (Fig. 2, Clade 4); however, suprisingly members of this Affinities of Lycodon clade showed very low interspecific genetic diversity. This This study represents the first phylogenetic analysis of the clade of microendemics was recovered sister to L. muelleri, genus Lycodon, and although genetic samples were available which occurs in northern Philippines. Sister to L. muelleri only for roughly half of the recognized species, our analy- and the microendemics of the Babuyans and Batanes is ses resolved relationships among many of the included spe- L. dumerilii, a widespread Philippine endemic. Two major cies with strong support (Figs 2 and 3). Although previous clades recovered in all analyses consisted primarily of studies have implied that Lycodon and Dinodon are recipro- Mainland Asia and Peninsular Malaysia taxa, although pop- cally monophyletic (Kelly et al. 2003, 2009; Lawson et al. ulations of L. subcinctus and L. capucinus from the Philip- 2005; Pyron et al. 2011), this finding is an artifact of sparse pines were nested within Clade 6 and 10, respectively sampling from Lycodon. Our results do not support the (Fig. 3). The two species of Dinodon included in this study monophyly of the genus Lycodon (Figs 2 and 3; Table 2); (D. semicarinatum and D. rufozonatum) are supported to be rather, we find that Lycodon is paraphyletic with respect to monophyletic and are recovered as part of a polytomy with Dinodon. This result is not surprising considering that L. butleri, L. fasciatus and the sampled populations of members of these two genera are morphologically very L. subcinctus (Fig. 3, Clade 6). All analyses recover the two similar, being distinguished only by a single, somewhat sampled populations of L. effraensis from and ambiguously defined character, the degree of curvature of Peninsular Malaysia as deeply divergent (Fig. 2, Clade 11). the maxillary bone (Smith 1943). Genetic samples are Although both ML and Bayesian analyses resulted in con- needed for 21 species of Lycodon and six species of Dinodon sistent topological relationships, one exception was unsampled in this study to estimate relationships among all observed. In ML analyses, L. stormi is recovered as the sis- species in these two genera of colubrid snakes. ter species to the clade composed of L. effraensis, L. laoensis, Previous studies that have included samples of Lycodon L. jara, L. zawi and the widespread L. aulicus Complex have not been able to confidently resolve many of the sister (results not shown), while Bayesian analyses recovered relationships to the genus within the subfamily Colubrinae L. stormi as the sister species to Clade 6 + 10 (Fig. 3, Clade (Heise et al. 1995; Kraus & Brown 1998; Kelly et al. 2003, 5). Interestingly, several widespread species show little 2009; Lawson et al. 2005; Pyron et al. 2011). Although our genetic divergence among sampled populations (Lycodon au- attempts to sample widely from outgroup taxa results in licus Complex, L. dumerilii, L. laoensis), while other species some well-supported relationships, the most closely related show considerable intraspecific genetic divergence (L. muel- genera to the clade Lycodon + Dinodon remains weakly leri, L. subcinctus). supported.

Evaluating biogeographic, morphological and taxonomic Phylogeny and evaluation of species diversity hypotheses The discovery of species new to science cryptically masquer- Among the biogeography-based hypotheses, the Bayesian ading among widespread species complexes has been exten- method provided no support [posterior probability (pp) sively documented in Asia and beyond (Stuart et al. 2006; approaching zero] for the monophyly of species of Lycodon Bickford et al. 2007; Pfenninger & Schwenk 2007; Brown & from the Philippines, Mainland Asia, or the Babuyan and Stuart 2012). In the archipelagos of Southeast Asia, the phe- Batanes Island Groups, respectively (Table 2). However, nomenon has been shown to characterize many vertebrate the tests recovered weak support for the monophyly of groups (for reviews, see Stuart et al. 2006; Siler et al. 2012a) island populations of Lycodon from the Batanes Island and is consistent with several findings supported in this Group (pp = 0.17; Table 2). Among the morphology-based study. Our results support cases of deeply divergent lineages hypotheses, the results did not support the monophyly of within some taxa (L. effraensis, L. subcinctus; Fig. 3) that banded or blotched morphotypes, but strongly supported likely represent unrecognized and/or undescribed species. the monophyly of the solid or near-solid morphotype Some of the lineage diversity we uncovered appears to corre- (pp = 1.0; Table 2). It should be noted that the solid or spond to taxonomic entities previously identified (currently

ª 2013 The Norwegian Academy of Science and Letters, 42, 3, May 2012, pp 262–277 269 Multilocus phylogeny of Asian wolf snakes  C. D. Siler et al. recognized subspecies or synonyms) and some do not. For Philippines, few diagnostic, morphological characters have example, three subspecies currently are recognized within been presented in the literature that (i) are not recognized the widespread (L. s. subcinctus, L. s. sealei, to be highly variable characters in the genus (e.g., colour and L. s. maculatus; Lanza 1999; The pattern, relative sizes of scales), (ii) unambiguously distin- 2012). Our analyses revealed deep genetic divergences guish these recognized species from each other and (iii) are among at three populations of this species sampled from non-overlapping in their variable states with the ranges Thailand, Peninsular Malaysia, and the Philippines (Fig. 3, observed in the other island populations (Ota & Ross Clade 8). Additionally, the three divergent lineages recov- 1994). The absence of genetic divergence observed in this ered in all analyses not only appear to be morphologically study among the extreme northern Philippine lineages, distinct (Fig. 3; Lanza 1999; L. Grismer, B. Stuart, personal coupled with few diagnostic morphological characters (but communication), but previously have been described as three see Ota & Ross 1994), may indicate a need for future taxo- distinct taxonomic entities in the literature. nomic revisions of this clade of Philippine endemic Wolf Reinhardt described the nominal species in the early Snakes (Fig. 3, Clade 4), with a possible concomitant 1800s (in F. Boie 1827), and the subspecies L. s. subcinctus reduction in recognized species diversity. This anticipated is currently recognized to occur in , , Cambo- action would have important conservation implications; dia, Thailand, Peninsular Malaysia, (Bali, borneo, currently, L. chrysoprateros, a Dalupiri Island endemic, is Java, Lombok, Mentaway Archipelago, Nias, Simeulue, considered ‘Critically Endangered’ (IUCN 2012). Sumatra, Sumbawa) and possibly (Lanza 1999). Between these two extremes lie species with moderate Nutphand (1986) described L. suratensis without designat- genetic structure observed among populations (L. muelleri, ing type specimens in the description, and the species was L. aulicus Complex). Populations of L. muelleri from the subsequently synonymized with L. subcinctus subcinctus Bicol Faunal Region of Luzon Island and nearby Catandu- (Pauwels et al. 2006). Leviton (1955) described a unique anes Island are 4.4–4.9% divergent from populations in subspecies of L. subcinctus (L. s. sealei), which occurs in east-central Luzon (Fig. 3). It remains to be determined Indonesia (Borneo), Malaysia (Borneo), and the whether this genetic split also corresponds to the Mid- Philippines (Palawan Island) (Lanza 1999; Gaulke 2002). Sierra Filter Zone, a recently recognized biogeographic Whether the three lineages we reveal correspond to each barrier on Luzon Island (Welton et al. 2010; Balete et al. of these previously recognized names (requiring the 2011). Many more population-level samples are needed to elevation of the three currently recognized subspecies) is a properly evaluate whether this species has a continuous dis- subject for future study, involving careful examination of tribution across its range. It is interesting to note the large freshly collected material, use of genetic samples from the number of squamate species endemic to the Luzon faunal type localities, and examination of the name-bearing types. region that are restricted either to the Bicol Faunal Region, On the other hand, a few patterns observed in this study including Polillo and/or Catanduanes Island and nearby contradict the commonly observed phenomenon of wide- Quezon and Bulacan provinces of Luzon ( bicol- spread cryptic diversity in Southeast Asia and, in fact, indi- andia, B. brevidactylus, B. cobos, B. lukbani, B. makusog, cate that species diversity within several clades in the genus B. minimus, Cerberus microlepis, Luperosaurus cumingii, Parvo- Lycodon may be overestimated, rather than underestimated. scincus laterimaculatus, Pseudogekko smaragdinus, Varanus oli- For example, the entire clade of microendemic species vaceus) or to Luzon proper to the exclusion of the Bicol from the Babuyan and Batanes Island Groups are separated Peninsula (Brachymeles bicolor, B. elerae, B. muntingkamay, by an average of 1.3% (uncorrected p-distance for cyt-b) B. wright, Eutropis bontocensis, E. multicarinata borealis, Gekko sequence divergence among island populations (Fig. 3, carusadensis, Lipinia pulchella levitoni, Luperosaurus angliit, Clade 4), and if we exclude the one apparently divergent L. kubli, Lycodon solivagus, L. tessellatus, Parvoscincus beyeri, lineage (L. bibonius), the average genetic divergence among P. boyingi, P. hadros, P. igoratorum, P. lawtoni, P. leucospilos, these putative species is reduced to a mere 0.3%. These P. luzonensis, P. tagapayo, Varanus bitatawa), with few spe- genetically similar island populations of the Babuyan and cies still recognized to have distributions spanning the tran- Batanes Island Groups also share similar colour patterns, sition between faunal regions (Boiga dendrophila divergens, and with the exception of the banded colour pattern of B. philippina, Brachymeles boulengeri, B. kadwa, Calamaria bi- L. bibonius, the remaining island populations possess quite torques, Otosaurus cumingi, Pinoyscincus abdictus aquilonius, similar non-banded to weakly banded color patterns (Ota V. marmoratus; PhilBREO 2012 LifeDesk,). & Ross 1994; CHO and RMB, personal observation). With The morphologically variable colour patterns within and the exception of L. chrysoprateros that appears to have fewer between populations of L. aulicus have led to long-standing ventral scales (186–194 vs. >198; Ota & Ross 1994) than confusion over species boundaries. Many authors have long the remaining microendemic species of the northern recognized two subspecies, L. a. aulicus and L. a. capucinus

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(Ota & Ross 1994; Lanza 1999; Ferner et al. 2000; Slowin- ranging from to the Philippines may actually ski et al. 2001; Gaulke 2002; Das 2003; Jackson & Fritts represent a single subspecies: L. a. capucinus. Until addi- 2004), while others have recognized L. capucinus Boie, 1827 tional samples from southern Asia (i.e., Andaman Islands, as a full species, distinct from L. aulicus (Linnaeus 1758; India, , , China) become available, we are Taylor 1965; David & Vogel 1996; Daltry & Wüster 2002; unable to evaluate this possibility. Finally (iii), it remains Pauwels et al. 2005; Mukherjee & Bhupathy 2007; Vogel possible that L. aulicus is actually a complex of unique, et al. 2009; Reza 2010; Vogel & David 2010; McLeod et al. cryptic species, and future fine-scale studies of genetic and 2011; Siler et al. 2011, 2012c; Zhang et al. 2011; Brown morphological patterns across the species’ entire range may et al. 2012a,b; The Reptile Database 2012). Previous sys- result in the recognition of additional taxa. Given the low tematic studies of Lycodon have noted that both taxa differ genetic diversity observed between populations on Main- primarily in coloration alone, with single populations docu- land Asia and the Philippines, particularly for the nuclear mented to have a full spectrum of colour forms, from indi- loci sequenced in this study (Fig. 2), it seems improbable viduals with no dorsal markings to individuals to complete that many undescribed cryptic lineages are masquerading and well-defined series of dorsal crossbars (Wall 1921; among populations of L. aulicus. Lanza 1999; CDS, RMB, unpublished data). The majority of studies have consistently divided the geographic ranges Biogeographic patterns among Wolf snakes of L. a. aulicus and L. a. capucinus in Indochina (Smith With few exceptions, the results observed in this study are 1943; Leviton 1965; Fritts 1993; Zhao & Adler 1993; Das consistent with many of the biogeographic expectations for 1994; David & Vogel 1996; Lanza 1999), with populations vertebrates in Asia and the Philippines. Phylogenetic analy- of both forms reported to be sympatric in Myanmar (Lanza ses recover Philippine endemic species and Philippine pop- 1999). Lycodon aulicus aulicus is recognized to primarily ulations of widespread species as part of three distinct occur in southern Asia (Pakistan, India, Nepal, Myanmar clades (Fig. 3, Clades 3, 8, 12), suggesting that wolf snakes and ; Smith 1943; David & Vogel 1996; Lanza have colonized the archipelago on at least three, and possi- 1999), and L. a. capucinus in eastern Asia (Cook Islands bly four occasions. All endemic and microendemic Philip- [Australia], Myanmar, , Thailand, Vietnam, Sin- pine species included in this study show a southeast to gapore, Laos, China, Indonesia, Malaysia, , Masca- north progression via the eastern Philippine island arc renes, and the Philippines; Smith 1943; Leviton 1965; within a single clade of the phylogeny (Fig. 3, Clade 3), Fritts 1993; Zhao & Adler 1993; Das 1994; David & Vogel while the endemic Palawan subspecies L. subcinctus sealei 1996; Lanza 1999; Whitaker & Captain 2004), although appears to have arrived in the Philippines by means of the L. a. capucinus is also recognized to occur on the Nicobar western Philippine island arc (Fig. 3, Clade 8). These pat- and Andaman Islands of India (Smith 1943; Lanza 1999; terns are consistent with many terrestrial vertebrates in the Whitaker & Captain 2004). Philippines, with these southern routes (eastern and wes- Several possibilities remain: (i) L. aulicus may represent a tern Philippine island arcs) recognized to be the two domi- single, widespread and morphologically variable species. nant dispersal pathways into the archipelago (e.g., Our results do not show a clear split between two recipro- Diamond & Gilpin 1983; Heaney 1985, 1986; Brown & cally monophyletic clades, nor do the few well-supported Guttman 2002; Evans et al. 2003; Jansa et al. 2006; Jones splits conform to recognized biogeographic regions (e.g., & Kennedy 2008; Brown et al. 2009; Esselstyn et al. 2009, non-monophyly of the Philippines and Indochina; Fig. 3, 2010; Esselstyn & Oliveros 2010). Clade 12). Furthermore, only moderate genetic diversity is Without more complete taxon sampling, we are left with observed across the sampled populations, with an average many unanswered questions. (i) Do the Philippine species divergence of only 3.6% (range = 0.0–5.0%). This level of recovered as members of Clade 3 (Fig. 3) replace each genetic structure among populations is consistent with the other in allopatry? (ii) Is there an explanation for the degree of intraspecific genetic variation observed within apparent disparity between the extent of sympatry within other widespread Southeast Asian squamate (Gam- the Philippines (low) vs. Sundaland (high)? Finally, given ble et al. 2012; Brown et al. 2012a,b; CDS, RMB, unpub- that one-third of the diversity of Lycodon occurs in the lished data) and yet is substantially more divergent than the Philippines, what role did the archipelago play in species recognized full species of the Babuyan and Batanes Islands diversification among wolf snakes? (Ota & Ross 1994). (ii) Lycodon a. aulicus and L. a. capucinus may represent two distinct lineages, and we have simply Affinities of unsampled taxa sampled from only one of these lineages, including the On the basis of the results of this study, it is tempting to population from Myanmar where they presumably are sym- speculate on anticipated phylogenetic affinities of patric. Our moderately robust sampling of populations unsampled species diversity. Some unsampled species

ª 2013 The Norwegian Academy of Science and Letters, 42, 3, May 2012, pp 262–277 271 Multilocus phylogeny of Asian wolf snakes  C. D. Siler et al. relationships are almost certain, given colour pattern and that unsampled species of Lycodon and Dinodon, and other biogeographic relationships. For example, the unsampled unsampled colubrid genera, may be found to be allied with Panay endemic species L. fausti and the northern Luzon this clade in future studies (A. Pyron, personal communica- Island L. solivagus are morphologically very similar to tion). L. muelleri and L. dumerilii (Ota & Ross 1994; Ota 2000; Gaulke 2002); accordingly, we expect these species to be Acknowledgements members of the Philippine Clade 3 (Fig. 3) if genetic sam- Thanks are due to several institutions, government agencies ples become available in the future. In contrast, some rela- and individuals who facilitated this study or provided logis- tionships are difficult to predict. The endemic Philippine tical or material support crucial to the results presented species from Samar Island (L. ferroni) is brightly banded here. The majority of Philippine sampling that contributed and shares morphological similarity with L. stormi, L. sub- to this work was funded by a National Science Foundation cinctus and L. dumerilii, three species that come out in dis- Biotic Surveys and Inventories finding (DEB 0743491 to tinctively different parts of our preferred tree (Fig. 3). RMB) and a NSF Doctoral Dissertation Improvement However, again, as many of the Philippine endemic and grant (DEB 0804115 to CDS). We thank the Protected microendemic species are recovered as part of a single Areas and Wildlife Bureau (PAWB) of the Philippine clade, we might expect L. ferroni to be closely related to Department of Environment and Natural Resources this same group of species as well (Fig. 3, Clade 3). (DENR) for collection and export permits necessary for The reoccurrence of the banded morphotype throughout this and related studies. The Economic Planning Unit of our tree (Fig. 3), however, renders speculation tenuous Malaysia, the Chinese central government, and the govern- concerning the numerous unsampled banded species ment of Myanmar provided research and export permits to (L. cardamomensis, L. fasciatus, L. flavomaculatus, L. futsing- L. Grismer (LSUHC), R. Murphy, RMB (ROM and KU, ensis, L. gongshan, L. liuchengchaoi, L. multifasciatus, L. ophi- respectively), and J. Vindum (CAS). We thank J. McGuire, ophagus, L. paucifasciatus, L. striatus and L. synaptor). L. Grismer, B. Stuart, R. Murphy, A. Diesmos, J. Vindum, Similarly, darkly blotched or near-solid colour patterns and D. Blackburn for access to genetic material and A. appear to have arisen numerous times (Fig. 3), suggesting Diesmos, V. Yngente, and J. Fernandez for help in the that the phylogenetic placement of unsampled taxa with field. Bryan Stuart, Alex Pyron and an anonymous reviewer similar colour patterns (L. flavicollis, L. osmanhilli, L. tiwarii provided constructive comments on earlier drafts of the and L. travancoricus) cannot be confidently predicted by manuscript. Thanks are due to B. Stuart, L. Grismer and colour pattern alone. J. McGuire for use of photographs reproduced in Fig. 2.

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Appendix 1 Summary of specimens and GenBank accession numbers corresponding to genetic samples included in the study

Genbank Accession Numbers Species Voucher Locality cyt-b c-mos vim

Ahaetulla prasina KU 302954 Philippines, Polillo Island, Quezon Province, KC010338 KC010299 KC010388 Barangay Pinaglubayan Ahaetulla prasina KU 326673 Philippines, Palawan Island, Palawan Province, KC010339 KC010300 KC010389 Barangay Estrella, Estrella Falls Park Boiga cynodon KU 324614 Philippines, Negros Island, Negros Occidental KC010340 KC010301 KC010390 Province, Barangay Patag, city of Silay, Mt. Bungol Boiga dendrophila KU 302957 Philippines, Panay Island, Antique Province, KC010341 — KC010391 Barangay Duyong Dasypeltis atra CAS 201641 , Kabale district AF 471065 —— Dinodon rufozonatum CIB 098274 Not reported JF827672 JF827695 — Dinodon semicarinatus Not reported Ryukyu Archipelago, Japan AB008539 —— Dipsadoboa unicolor CAS 201660 Uganda, Rukungiri District AF 471062 —— Gonyosoma oxycephala KU 321724 Philippines, Mindanao Island, Zamboanga City KC010343 KC010302 KC010393 “Province”, Pasonanca Natural Park, Tumaga River Lampropeltis alterna Not reported Not reported AF 337128 —— Lampropeltis triangulum Not reported Not reported AF 337134 —— Lycodon alcalai KU 327847 Philippines, Bataan Peninsula, Batanes Province, KC010344 KC010303 KC010394 Barangay San Antonio, Sitio Chadpidan Lycodon alcalai KU 327848 Philippines, Sabtang Island, Batanes Province, KC010345 KC010304 KC010395 Barangay Chavayan Lycodon aulicus CAS 205000 Myanmar, Rakhine State KC010346 KC010305 KC010396 Lycodon aulicus CDSGS 4621 Philippines, Bohol Island, Bohol Province KC010347 — KC010397 Lycodon aulicus KU 305141 Philippines, Semirara Island, Antique Province, KC010348 KC010306 KC010398 Municipality of Caluya, Barangay Tinoboc Lycodon aulicus KU 315170 Philippines, Mindanao Island, Zamboanga City, KC010359 KC010317 KC010409 Pasonanca Natural Park, Tumaga River Lycodon aulicus KU 315378 Philippines, Tablas Island, Romblon Province, KC010350 KC010308 KC010400 Barangay Balogo, Sitio Piqueno Lycodon aulicus KU 328524 Thailand, SERS, Nakhon Ratchasima KC010358 KC010316 KC010408 Lycodon aulicus LSUHC 5725 West Malaysia, Johor, Pulau Rawa, KC010355 KC010313 KC010405 Lycodon aulicus LSUHC 9277 Vietnam, Kien Giang, Nam Du Island KC010356 KC010314 KC010406 Lycodon aulicus LSUHC 9695 Cambodia KC010357 KC010315 KC010407 Lycodon aulicus PNM 7705 Philippines, Leyte Island, Leyte province, Leyte state KC010349 KC010307 KC010399 University campus, Barangay Guadalupe, Municipality of Baybay Lycodon bibonius KU 304589 Philippines, Camiguin Norte Island, Cagayan Province, KC010351 KC010309 KC010401 Barangay Balatabat, Local name “Limandok” Lycodon butleri LSUHC 8066 West Malaysia, Pahang, Fraser’s Hill KC010352 KC010310 KC010402 Lycodon butleri LSUHC 9136 West Malaysia, Perak, Bukit Larut KC010353 KC010311 KC010403 Lycodon butleri LSUHC 9421 West Malaysia, Pahang, Bukit Larut KC010354 KC010312 KC010404 Lycodon chrysoprateros KU 307720 Philippines, Dalupiri Island, Cagayan Province, KC010360 KC010318 KC010410 Manolong Creek Lycodon dumerilii KU 305168 Philippines, Dinagat Island KC010362 — KC010412 Lycodon dumerilii KU 319989 Philippines, Mindanao Island, Agusan del Sur Province, KC010361 KC010319 KC010411 Barangay Bagusan II, Mt. Magdiwata Lycodon dumerilii PNM 7751 Philippines, Leyte Island, Leyte Province, Municipality of KC010363 KC010320 KC010413 Burauen, Lake Mohagnao Lycodon cf. effraensis KU 328526 Thailand, Khao Luang NP, Nakhon Si Thammarat KC010364 KC010321 KC010414 Lycodon effraensis LSUHC 9670 West Malaysia, Kedah KC010376 KC010328 — CAS 234875 Myanmar, Chin State KC010365 —— Lycodon fasciatus CAS 234957 Myanmar, Chin State KC010366 —— CAS 235387 Myanmar, Kachin State KC010367 KC010322 —

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Appendix 1 Continued

Genbank Accession Numbers Species Voucher Locality cyt-b c-mos vim

Lycodon laoensis FMNH 258659 Lao PDR, Salavan Province KC010368 KC010323 — FMNH 262186 Vietnam, Dong Nai KC010369 KC010324 — Lycodon laoensis KU 328529 Thailand, Khao Luang NP, KC010371 — KC010416 Nakhon Si Thammarat Lycodon laoensis LSUHC 8481 Cambodia, Pursat Province, O’Lakmeas KC010370 KC010325 KC010415 Lycodon muelleri CDSGS 5256 Philippines, Catanduanes Island KC010372 — KC010417 Lycodon muelleri DLSUD 031 Philippines, Luzon Island, De La Salle University KC010373 KC010326 KC010418 reference collection, Cavite Province, Municipality of Ternate, Sitio Maloayas Lycodon muelleri KU 313891 Philippines, Luzon Island, Camarines Norte Province, KC010375 KC010327 KC010420 Barangay Tulay Na Lupa Lycodon muelleri KU 323384 Philippines, Luzon Island, Aurora Province, KC010374 — KC010419 Barangay Lipimental Lycodon ruhstrati LSUHC 4199 China, Hainan Island, Wuzhi Shan KC010381 KC010332 — Lycodon sp. KU 304827 Philippines, Baboyau Claro, Cagayan Province, KC010377 KC010329 KC010421 Barangay Babuyan Claro Lycodon sp. KU 304844 Philippines, Baboyau Claro, Cagayan Province, KC010378 KC010330 KC010422 Barangay Babuyan Claro Lycodon sp. KU 304870 Philippines, Calayan, Cagayan Province, Barangay KC010379 — KC010423 Magsidel, Local Name “Macarra” Lycodon stormi JAM 7487 Indonesia, Sulawesi, Propinsi Sulawesi Tenggara, KC010380 KC010331 KC010424 Kabupaten Konawe Selatan, Kecamatan Konda, Desa Moramo, Air Terjun Moramo Lycodon subcinctus KU 309447 Philippines, Irawan, Palawan Province, Barangay KC010385 — KC010428 Irawan, Irawan Watershed Lycodon subcinctus KU 327571 Philippines, Palawan Island, Palawan Province, KC010384 KC010335 KC010427 Barangay Estrella, Estrella Falls Park Lycodon subcinctus KU 328531 Thailand, SERS, Nakhon Ratchasima KC010383 KC010334 KC010426 Lycodon subcinctus LSUHC 5016 West Malaysia, Pahang, Sungai Lembing Logging Camp, KC010382 KC010333 KC010425 Lycodon zawi CAS 239944 Myanmar, Rakhine State KC010386 KC010336 — Lycodon zawi CAS 210323 Myanmar, Sagaing Division AF 471040 Oligdon maculatus KU 321699 Philippines, Mindanao Island KC010387 KC010337 — Pantheropis bairdi isolate RDZ183 Not reported GU 073440 —— Pantheropis obsoletus isolate RDZ264 Not reported GU 073446 —— Pituophis catenifer JR18 Pet Trade FJ 627819 —— Pituophis deppei JR12 Pet Trade FJ 627818 —— Ptyas luzonensis TNHC 63002 Philippines, Luzon Island, Zambales Province, KC010342 — Municipality of Olongapo, SBMA Naval Base, “Nav-mag” area, Ilanin Forest (Triboa Bay)

CAS = California Academy of Sciences Herpetological Collections; CDSGS = Non-vouchered genetic samples deposited at the University of Kansas Natural History Museum; CIB = Chengdu Institute of Biology; DLSUD = De La Salle University Dasmari~nas Reference Collection; FMNH = Field Museum of Natural History Herpetological Collections; JAM = Jimmy A. McGuire genetic sample, deposited in Museum Zoologicum Bogoriense (National Museum of Indonesia, Cibinong, Java); JR = Javier Rodriguez Robles; KU = University of Kansas Natural History Museum; LSUHC = La Sierra University Herpetological Collections; PNM = Philippine National Museum Herpetological Collections; RDZ = reference identification acronym presented in Vandewege et al. (2012); TNHC = Texas Natural History Collections, University of Texas at Austin.

Supporting Information Fig. S1. Maximum clade credibility chronogram among Additional Supporting Information may be found in the species of Lycodon calculated in BEAST. online version of this article:

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