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

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 genus, 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 Pseudoxenodon macrops, indicating that species diversity of Pseudoxenodon in China is likely underestimated.

Full text

Asian Herpetological Research 2013, 4(3): 155–165
DOI: 10.3724/SP.J.1245.2013.00155
1. Introduction
With the rapid emergence of new sequencing technologies
since the 1970s, molecular markers have been widely
used in modern biology, stimulating rapid progress across
the field of biological systematics (Ride et al., 2000;
Thomson et al., 2010). Previous systematic hypotheses
of snakes relied heavily on analyses of morphological
data. However, for many species of snakes, finding
distinct morphological synapomorphies was not viable
or intuitive, leading to cryptic phylogeographic lineages
masquerading as a single species (Utiger et al., 2002;
Lawson et al., 2005; Burbrink et al., 2008; Huang et al.,
2009; Ling et al., 2010).
The suborder Caenophidia (advanced snakes)
Relationship of Old World Pseudoxenodon and New World
Dipsadinae, with Comments on Underestimation of Species
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, China
2 School of Life Sciences, Yunnan 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
* Corresponding author: Prof. Song HUANG, from Huangshan
University, Anhui, China, with his research focusing on ophiology,
phylogenetics and biogeography of reptiles.
E-mail: snakeman@hus.edu.cn
Received: 26 January 2013 Accepted: 1 May 2013
Keywords phylogenetic position, 12S rRNA, 16S rRNA, Beringian Land Bridge, cryptic species
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 genus, 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
intraspecic genomic variation within the populations of Pseudoxenodon macrops, indicating that species diversity of
Pseudoxenodon in China is likely underestimated.
includes approximately 3000 species of extant snakes
(Lawson et al., 2005; Vidal et al., 2007). Currently,
a total of 205 species belonging to 66 genera in 9
families occur in China (Zhao, 2006), with many of their
classications proposed in current literature based only on
morphological data sets. Few molecular studies focus on
examining the phylogeny and phylogeographic history of
snakes in Asia, especially for the same or closely related
species with a transcontinental range where cryptic
diversity tends to be highest (Creer et al., 2001; Giannasi
et al., 2001; Malhotra and Thorpe, 2004; Huang et al.,
2007). Pseudoxenodon is one of these poorly studied
genera, with few studies on the group’s inter- and intra-
specic relationships.
An important diagnostic feature of Pseudoxenodon
(Chinese common name “Oblique-scaled snakes”) is
the obliquely arranged scales on anterior part of the
body. The rst species of oblique-scaled snakes, namely,
Tropidonotus macrops was described by Blyth (1855)

Asian Herpetological Research156 Vol. 4
from Darjeeling, India. Boulenger (1890) subsequently
distinguished this species from Tropidonotus (= Natrix)
by its oblique scales and placed it in the new genus
Pseudoxenodon. McDowell (1987) found hemipenal
synapomorphies between Pseudoxenodon an d
Plagiopholis, and placed both the genera in the new
subfamily Pseudoxenodontinae. Presently, the genus
Pseudoxenodon is comprised of six species, four of which
are found in China: P. bambusicola, P. karlschmidti, P.
macrops, and P. stejnegeri (Uetz, 2013). Species in this
group are widely distributed throughout southern and
southeastern Asia. They are known to inhabit areas near
water across plains, mountains, hills, scrublands, lawns,
forests, cultivated lands and stream banks. Genetic studies
into these species’ taxonomic status are desperately
needed for further ecological, biological and behavioral
research (http://www.arkive.org).
Previous studies revealed that Pseudoxenodontinae is
the closest relative of Dipsadinae. Zaher (1999) examined
hemipenal variation of these two groups, finding that
the hemipenes of both have a bifurcated sulcus and
retain calyces. Lawson et al. (2005) first conducted the
molecular (cyt b and c-mos) study of Pseudoxenodon
with one specimen of P. karlschmidti, and reported
Pseudoxenodon as sister of Dipsadinae. Vidal’s (2007)
molecular analysis based on seven nuclear protein
coding genes from one specimen of P. bambusicola also
shows the same sister relationship. Several subsequent
studies (e.g., He et al., 2009; Zaher et al., 2009; Pyron
et al., 2011) also obtained the same results using the
sequences from these two individuals. Support for the
Pseudoxenodontinae-Dipsadinae clade in their reported
trees was always not robust, indicating the need for
further analysis.
Over the last two decades, the most frequently
analyzed genetic fragments for Dipsadinae are two
mitochondrial genes (12S and 16S rRNA) and one nuclear
gene (c-mos) (Vidal et al., 2000, 2010; Kelly et al., 2003;
Pinou et al., 2004; Hedges et al., 2009). To address this
question of uncertain phylogenetic relationships of the
two subfamilies, we attempted to include all the three
gene sequences of caenophidian available from GenBank,
as well as one henophidian snake (Boa constrictor)
as an outgroup, and obtained new sequences from 13
individuals of Pseudoxenodon.
The systematic nomenclature used in this paper is
primarily based on the classication proposed by Lawson
et al. (2005). However, we simultaneously followed
the recommendation by Pyron et al. (2011) in using the
name Dipsadinae to refer to Xenodontine that presents in
Lawson et al. (2005) and previous studies. Much of the
classication proposed by Lawson et al. (2005) has been
adopted and corroborated by many contemporary workers
in the current literature (e.g., He et al., 2009; Huang et al.,
2009; Ling et al., 2010).
2. Materials and Methods
2.1 Downloaded sequences and supraspecic terminals
Table 1 shows 109 terminal taxa and sequences for
three genes (12S, 16S and c-mos), together with their
GenBank accession numbers and references. Ninety-
eight sequences were downloaded from GenBank (77
sequences for 12S, 67 for 16S, and 75 for c-mos) and
11 sequences (haplotypes, from 13 individuals) were
generated by us. In addition, 19 genera are represented
by different species with different sequences as terminal
nodes. In order to maximize our dataset of combined
genes, different sequences from congeneric species were
combined to form supraspecic terminals at the generic
level according to Bininda-Emonds et al. (1998).
2.2 DNA extraction, amplication and sequencing In
this study, we adhered to the Wild Animals Protection
Law of the People’s Republic of China. All experiments
involving live snakes were approved by the Animal
Ethics Comm ittee at Huangshan Uni versity. A t otal
of 13 individuals (2 of P. karlschmidti and 11 of
P. macrops) were collected from 9 locations in 5
provinces of mainland China (Figure 1) during 2009. We
have to euthanize some specimens in the field because
of a long travel process. And the others were reared at
our laboratory for further morphological examination
until they died. Fresh liver tissue or skeletal muscle was
removed and immediately preserved in 95% ethanol for
later sequencing of mitochondrial (12S and 16S rRNA)
and nuclear (c-mos) genes. Unfortunately, the specimen
of Pseudoxenodon sp. (considered as P. macrops at rst)
from Pailong, Nyingtri, Xizang (Tibet), China was lost
during a long arduous travel, limiting the morphological
identity during our analysis.
Total genomic DNA was extracted according to the
phenol/chloroform extraction procedure (Sambrook et al.,
1989). Sequences were amplied from total DNA extracts
using polymerase chain reaction (PCR). Primer pairs
for each gene used in this study were: L1091/H1557
(Knight and Mindell, 1994) for 12S rRNA; 16Sar/16Sbr
(Palumbi et al., 1991) for 16S rRNA; and S77/S78 for
c-mos (Lawson et al., 2005). The PCR reaction contained
approximately 100 ng of template DNA, 1 μl of each
primer, 5 μl of 10× reaction buffer, 2 μl dNTPs (each 2.5

Baolin ZHANG and Song HUANG Old World Pseudoxenodon and New World DipsadinaeNo. 3 157
mM), and 2.0 units of Taq DNA in a 50 μl reaction. The
reactions were cycled as follows: initial denaturation at
94 °C for 3 min, 30 cycles of denaturation at 94 °C for
1 min, annealing at 45–57 °C for 1 min, extension at
72 °C for 1 min, and nal extension at 72 °C for 5 min.
Genes were puried from 0.8% low-melting agarose gels
using a BioStar Glassmilk DNA purication Kit (BioStar
International, Canada) and following the manufacturer’s
instructions. The purified DNA was sequenced with
a BigDye Terminator Cycle Sequencing Kit (Applied
Biosystems, USA) according to the manufacturer’s
protocol with the primers used in PCR.
2.3 Phylogenetic reconstruction Sequences of 12S,
16S, and c-mos, and their combinations were aligned,
re s p e ctivel y with other s e q u ences re trieved from
GenBank using ClustalX (Thompson et al., 1997),
and proofread by eye. Nucleotide composition and
the uncorrected P-distances were calculated in Mega
4.0 (Tamura et al., 2007). The 16S sequences of some
taxa used in the present study are not available from
GenBank (e.g., Boiruna, Waglerophis, Ninia, Hypsiglena,
Thamnophis, Storeria, Malpolon, and Pareas). Although
the problem of missing data is often considered to
be a serious obstacle for phylogenetic reconstruction
(Anderson, 2001; Lemmon et al., 2009), simulation and
empirical studies have shown that even extensive missing
data (alignments containing 90% of missing data) do not
constitute a serious limitation to phylogenetic inference
as long as the number of variable and parsimony-
informative characters is sufficient (Kelly et al., 2003;
Philippe et al., 2004; Pyron et al., 2011). As expected,
in the present study the phylogeny results inferred from
Bayesian Inference (BI) and Maximum-Likelihood (ML)
trees show strong statistical support for most of the nodes
(see below).
Separate and combined analyses for each of the three
gene regions were performed using ML in Garli v0.96
(Zwickl, 2006) and MrBayes v3.0 (Huelsenbeck and
Ronquist, 2001). Modeltest 3.7 (Posada and Crandall,
1998) was used to determine the model of nucleotide
substitution under the Akaike Information Criterion
(AIC) for ML and Bayesian Information Criterion
(BIC) for BI. The best-fit model GTR+I+G was
selected for the 12S and 16S sequences. The following
models were selected for analysis of the protein coding
c-mos gene: K80+G for the first codon position;
HKY+G for the second codon position; and GTR for
the third codon position. For the Bayesian analysis,
a starting tree was obtained using neighbor-joining
methods. Posterior probabilities (PP) were obtained
by Markov Chain Monte Carlo (MCMC) analysis
with one cold chain and three heated chains. Bayesian
support for the nodes was inferred through a MCMC
model as implemented in MrBayes, using 1 000 000
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, Sichuan (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).

Asian Herpetological Research158 Vol. 4
Species Accession No. 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)
Apostolepis 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)
Atractus 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)
Carphophis 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)
Dipsas catesbyi Z46496 Heise et al. (1995)
Dipsas indica GQ457789 GQ457850 Zaher et al. (2009)
Drepanoides anomalus GQ457791 GQ457852 GQ457732 Zaher et al. (2009)
Drysdalia 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)
Farancia 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)
Helicops gomesi GQ457739 Zaher et al. (2009)
Helicops pictiventris GQ457800 GQ457860 Zaher et al. (2009)
Hemiaspis signata EU547123 EU546936 EU547172 Sanders et al. (2008)
Heterodon 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 viridiavus AY541507 Utiger and Schaetti (2004)
Homalopsis buccata EF395892 EF395940 EF395868 Alfaro et al. (2008)
Hormonotus modestus FJ404159 FJ404261 FJ404195 Vidal et al. (2008)
Hydrodynastes gigas GQ457803 GQ457863 GQ457743 Zaher et al. (2009)
Hydrops triangularis GQ457804 GQ457864 GQ457744 Zaher et al. (2009)
Hypsiglena slevini EU728584 Mulcahy et al. (2009)
Table 1 Samples and sequence information.

Baolin ZHANG and Song HUANG Old World Pseudoxenodon and New World DipsadinaeNo. 3 159
Species Accession No. Sources
12S c-mos 16S
Hypsiglena torquata AF471159 Lawson et al. (2005)
Imantodes 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)
Myron 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)
Oxyrhopus clathratus GQ457815 Zaher et al. (2009)
Oxyrhopus formosus 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)
Phalotris nasutus GQ457818 GQ457878 GQ457757 Zaher et al. (2009)
Philodryas aestiva GQ457819 GQ457879 GQ457758 Zaher et al. (2009)
Phimophis 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)
Pseudoboa neuwiedii AF158423 AF158490 Vidal et al. (2000)
Pseudoboa nigra GQ457885 Zaher et al. (2009)
Pseudoeryx plicatilis GQ457826 GQ457886 GQ457765 Zaher et al. (2009)
Pseudonaja modesta EU547098 EU546915 EU547147 Sanders et al. (2008)
Pseudoxenodon karlschmidti (HuN) JF697319 JF697342 JF697330 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
Psomophis joberti GQ457829 AF471151 FJ907950 Zaher et al. (2009)
Ptyas mucosus AY122828 AF544736 EU728583 Utiger et al. (2002); Lawson et al. (2005)
Sibon 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)
Simoselaps bertholdi EU547111 GQ457894 GQ457773 Sanders et al. (2008)
Siphlophis pulcher GQ457834 AF471154 Zaher et al. (2009)
Storeria dekayi AF402639 GQ457895 GU018167 Alfaro and Arnold (2001); Lawson et al. (2005)
Tachymenis peruviana GU018147 GQ457853 GQ457733 Vidal et al. (2010); Zaher et al. (2009)
Taeniophallus afnis 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)
Xenoxybelis argenteus GQ457842 Zaher et al. (2009)
(Continued Table 1)

Asian Herpetological Research160 Vol. 4
generations after discarding a “burn-in” of 100 000
generations (representing the log-likelihood scores had not
reached stationarity). Sampling was performed every 100
generations. We ran three additional analyses starting with
random trees. Consensus of all post burn-in generations
(3 600 000 generations resulting in 36 000 trees) was
computed from all four runs. In addition, ML analyses
were performed with RAxML-HPC BlackBox through
the CIPRES Portal 2.0 at the San Diego Supercomputing
Center (http://www.phylo.org/portal2).
3. Results
3.1 Sequence characteristics The aligned sequences
resulted in a total of 1344 nucleotides (423 for 12S, 428
for 16SrRNA and 493 for c-mos). Th e alignment of
c-mos sequences revealed two internal codon deletions
for Pseudoeryx at positions 270–275. There is one codon
deletion at positions 271–273 for Acrochordus, Bitis,
Hormonotus, Pseudoxenodon, Colubrinae, Natricinae, and
Dipsadinae. The number and position of deletions were
in agreement with those reported by Lawson et al. (2005)
and Zaher et al. (2009).
Sequencing of the 13 specimens of Pseudoxenodon
revealed 11 haplotypes. The 12S and 16S genes each
produced 11 haplotypes whereas the c-mos gene yielded
5 haplotypes. For each newly sequenced gene, two
specimens of P. karlschmidti from Hunan share one
haplotype and two specimens of P. macrops from Mengzi,
Yunnan share one haplotype. Each of the remaining
specimens has its own haplotype.
The average nucleotide composition (%), numbers
of variable (V) and parsimony informative (PI) sites for
12S, 16S and c-mos genes of Caenophidia, Dipsadinae
and Pseudoxenodon used in the present study are
listed in Table 2. Sequences of uncorrected P-distance
were calculated between ingroup taxa (excluding
Pseudoxenodon), and specimens from 11 sites for 12S,
16S and c-mos genes are listed in Table 3.
3.2 Topological results In this study, separate analyses
of unlinked genes always obtained weakly supported
phylogenetic relationships for internal nodes (trees
for individual genes not shown), whereas support for
congruent relationships in the combined dataset is
generally high. Patterns of cladogenesis derived from
BI and ML based on the combined dataset were nearly
identical (Figure 2).
The BI and ML analyses resulted in ve major groups,
the Pareatidae, Viperidae, Homalopsidae, Elapidae,
and Colubridae (Figure 2), which were similar to those
presented in Kelly et al. (2003), Lawson et al. (2005), and
Huang et al. (2009). Furthermore, we found natricines
were imbedded in Dipsadinae, which are consistent with
the Pinou et al. (2004) hypotheses. Our results provide
strong posterior probability support (PP, 99%) and
moderate bootstrap support (BS, 65%) for the monophyly
of Dipsadinae.
In the BI/ML tree (Figure 2), the genus Pseudoxenodon
is nested within a New World clade composed mostly of
South American xenodontines, with both high PP (100%)
and BS values (91%). All the specimens collected cluster
into a monophyletic group and have very high (100%) BS
and PP support. Within this group, three subclades were
identied: one for Pseudoxenodon sp. (Xizang), another
for P. karlschmidti (Hunan) and the last contaning the
other 11 sequences of P. macrops. In addition, both ML
Taxa 12S (423 aligned sites) c-mos (493 aligned sites) 16S (448 aligned sites)
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%)
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 0.029a0.112 (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)
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.
Table 3 Sequence divergence information. a: The smallest distance of 0.005 between E. anomala and E. schrenckii that indicates they
may be conspecic. (Utiger et al., 2002; Ling et al., 2010); n: Numbers of sequences of each group; XZ: The specimen (considered to be
Pseudoxenodon macrops at rst) from Pailong, Nyingtri, Xizang (Tibet).

Baolin ZHANG and Song HUANG Old World Pseudoxenodon and New World DipsadinaeNo. 3 161
Figure 2 The ML tree based on all combined genes of 96 species of Alethinophidia. Supraspecic 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 (%).

Asian Herpetological Research162 Vol. 4
and BI combined data analyses place the Pseudoxenodon
sp. (Xizang) in an unresolved position outside of the
remaining P. macrops and P. karlschmidti (Hunan).
4. Discussion
4.1 Phylogenetic position of Pseudoxenodon The same
gene fragments (12S and 16S) and an additional gene
(c-mos) of dipsadids and natricines yield a topological
pattern similar to that of Pinou et al. (2004). Some
previous molecular studies assigned Pseudoxenodontinae
and Dipsadinae as sister taxa (Lawson et al., 2005;
Vidal et al., 2007; Zaher et al., 2009; Pyron et al., 2011)
with a weak support. Fortunately, the addition of taxa in
this study yielded a better resolution of the relationship
between the two groups. Our phylogenetic analyses clearly
indicate that the OW genus Pseudoxenodon, formerly
recognized as the subfamily Pseudoxenodontin ae, is
internested within a NW clade composed mostly of South
American xenodontines. Our results suggest the type
genus (Pseudoxenodon) of Pseudoxenodontinae appears
to be a member of Dipsadinae; quite different from the
previous concept of this group. Our molecular data is
consistent with Zaher’s (1999) morphological evidence,
primarily from the hemipenes, who found both of them
have a bifurcated sulcus and retain calyces.
4.2 Underestimation of species diversity of
Pseudoxenodon Our phylogenetic trees strongly support
the monophyly of Pseudoxenodon (PP, 100%; BS, 100%).
However, the internal nodes within Pseudoxenodon
are unclear. The specimen from Tibet does not appear
to be a member of P. macrops or P. karlschmidti.
Moreover, the minimum pairwise sequence divergences
(uncorrected P-distance) reveal that the populations of P.
macrops have high intraspecic variation (Table 3). The
minimum distances of Pseudoxenodon sp. (Xizang) vs.
Pseudoxenodon (0.039 for 12S, 0.026 for 16S) were more
than those within Dipsadinae and overall ingroup taxa,
respectively. A possible explanation for the high level of
sequence divergence is attributed to geographic barriers.
The Hengduan Mountains that make up the division
between the Tibetan Plateau and the Central Chinese
Plain have been documented as the major barrier to gene
flow among the terrestrial organisms occurring at high
and low elevations (Fu et al., 2005; Peng et al., 2006).
The topography of this eco-region is extremely variable
and complex. Most of the parallel mountain ranges run
in a north-south direction and are separated by deep,
narrowly incised river valleys (Huang et al., 2009). These
geological barriers may have played an important role in
the evolutionary history of Pseudoxenodon in Tibet.
In this study, the sample from Tibet falls into the
distribution area of P. macrops (Figure 3). However, our
molecular evidence suggests that it likely represents a new
species of Pseudoxenodon rather than being a member of
P. macrops, a testament to the underestimation of species
diversity of Chinese Pseudoxenodon. More specimens of
Pseudoxenodon across southern and southeastern Asia
should be represented in the future phylogenetic analyses
based on large molecular and morphological data sets.
4.3 Biogeography of snake fauna exchange between
OW and NW Biogeographic hypotheses concerning the
origin of Caenophidians have in the past suggested that
Caenophidians have an Asian origin. This is demonstrated
by the fact that most of the basal lineages have an
Asian range. For example, Acrochordus, Pareatidae,
Viperidae (partly Asian) and Homalopsidae always show
a successive basal branching pattern to the monophyletic
Caenophidians (Vidal et al., 2007). A generally accepted
perception about the dates and routes of dispersal has
revealed that the Colubridae entered the New World
from Asia with the formation of the Beringian Bridge
by the early Oligocene (Pinou et al., 2004; Burbrink
and Lawson, 2007). As one of the largest subfamilies of
Colubridae, the Dipsadinae could have also entered at the
same time (Pinou et al., 2004). In fact, the suitable habitat,
warm climate and the exposed continental bridge allowed
multiple opportunities for snakes to disperse from the OW
to NW during the Paleocene and mid-Miocene (Burbrink
and Lawson, 2007). However, the same opportunities also
have facilitated migration along the opposite route for a
complete process of two-way faunal exchange (Simpson,
1943). Unfortunately, few studies focus on the dispersal of
organisms from the NW to the OW.
Dipsadinae is one of the largest subfamilies of
Colubrida e with about 92 genera and more than 700
species (Vidal et al. 2010). Based on the analysis of 12S
rRNA and 16S rRNA sequence data, Vidal et al. (2000)
found snakes in this assemblage have an Asian–North
American origin, separating into the Central and the
South American xenodontines in the early Tertiary. Our
results are consistent with these inferences. Moreover,
in the present study, Pseudoxenodon forms a robust
genetic subclade within South American xenodontines,
indicating at least one lineage was separated from this
clade and then dispersed from the NW to the OW across
the Beringian Land Bridge during the early Tertiary and
the warm mid-Miocene. We provide evidence suggesting
that the existing Beringian link also facilitated the two-
way migration. If all the members of Pseudoxenodon

Baolin ZHANG and Song HUANG Old World Pseudoxenodon and New World DipsadinaeNo. 3 163
are clustered into a monophyletic group and placed at
the same position in this study, then a NW origin of
Pseudoxenodon can be inferred, but our sampling is not
adequate enough to provide strong evidence suggesting
that the OW Pseudoxenodon diverged from Dipsadinae.
Considered the ambiguity in aligning 12S and 16S rRNA
and the admittedly un-comprehensive sample of this study,
future work should be based on more genetic markers and
more samples across the distribution range (Figure 3). In
addition, multilateral cooperation is desperately needed.
Acknowledgements We extend our sincere gratitude to
Xin CHEN (City University of New York, USA) and Li
DING (Chengdu Institute of Biology, Chinese Academy
of Sciences, Sichuan, China) for providing tissues used
in this study. We appreciate Dr. Frank T. BURBRINK
and Alexander D. MCKELVY (College of Staten Island,
USA) for their valuable comments and extensive revisions
on the early draft of this manuscript. Also, we thank
Tao WAN, Jinmin CHEN, Chen LING and Xaoyu SUN
(Huangshan University, Anhui, China) for their help in
data processing. We particularly thank the reviewers
who did extensive corrections on the manuscript. This
research was funded by the National Natural Science
Foundation of China (30870290, 31071891, 31060280)
and the Students Science Research Program of Huangshan
University (2010xdkj019).
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