Multilocus Phylogeny of Lycodon and the Taxonomic Revision of Oligodon multizonatum

Full text

Asian Herpetological Research 2014, 5(1): 26–37
DOI: 10.3724/SP.J.1245.2014.00026
1. Introduction
The genus Oligodon Fitzinger, 1826 is widespread
throughout central and tropical Asia, containing
approximately 70 species (Green et al., 2010). Among
them, 15 are known to occur in southern China
(Zhao et al., 1998). Previous studies aimed at classifying
the genus have been based on morphological data and
yielded conflicting results (Wall, 1923; Pope, 1935;
Smith, 1943; Leviton, 1963; Campden, 1969; Wallach
and Bauer, 1996; David et al., 2008; Tillack and Günther,
2009). However, all of these studies were limited to a
species group within this complex or a limited geographic
Multilocus Phylogeny of Lycodon and the Taxonomic Revision of
Oligodon multizonatum
Juan LEI1, 2, 4, Xiaoyu SUN1, Ke JIANG3, Gernot VOGEL5, David T. BOOTH4 and
Li DING1*
* Corresponding author: Dr. Li DING, from Chengdu Institute of Biology,
Chinese Academy of Sciences, Sichuan, China, with his research
focusing on taxonomy and systematics of snakes, molecular phylogeny
and phylogeography of reptiles, faunal survey and biodiversity,
animal behavior of reptile, and conservation and public awareness
of snakes.
E-mail: dingli@cib.ac.cn
Received: 31 December 2013 Accepted: 11 March 2014
Abstract Classification of the Asian snake genera Lycodon and Oligodon has proven challenging. We conducted a
molecular phylogenetic analysis to estimate the phylogenetic relationships in the genus of Lycodon and clarify the
taxonomic status of Oligodon multizonatum using mitochondrial (cyt b, ND4) and nuclear (c-mos) genes. Phylogenetic
trees estimated using Maximum Likelihood and Bayesian Inference indicated that O. multizonatum is actually a
species of Lycodon. Comparing morphological data from O. multizonatum and its closest relatives also supported
this conclusion. Our results imply that a thorough review of the evolutionary relationships in the genus of Lycodon is
strong suggested.
area, and no study constructed a phylogenetic tree.
Green et al. (2010) produced an updated checklist and
key to the entire genus together with a phylogentic tree.
The key and checklist were given in his thesis, and the
phylogenetic data were later published (Green et al.,
2010) and concluded that several uncertainties about the
FODVVL¿FDWLRQVWLOOH[LVW+RZHYHUQR VWXG\ KDV LQFOXGHG
molecular data from Oligodon multizonatum.
Oligodon multizonatum was described by Zhao and
Jiang (1981) from Luding County, Sichuan Province,
VRXWKZHVW&KLQD7KHVSHFLHVZDV FODVVL¿HGDVDPHPEHU
of the genus Oligodon on the basis of morphological
characteristics including a short head that is not distinct
from the neck, a large rostral scale that appears protruding
when viewed from above, a cylindrical body with
paired subcaudals and smooth dorsal scales (Zhao et al.,
1998). There have been no published attempts to explore
the taxonomic position of the species since it was first
described, and no new specimens have been reported.
Currently, O. multizonatum is considered an endemic
Keywords %D\HVLDQLQIHUHQFH&KLQDFODVVL¿FDWLRQFPRVF\Wb, Lycodon, maximum likelihood, ND4, Oligodon
1 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan 610041, China
2 College of Life and Sciences, Sichuan University, Chengdu, Sichuan 610064, China
3 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of
Sciences, Kunming 650223, Yunnan Province, China
4 School of Biological Science, The University of Queensland, Brisbane, St Lucia, QLD 4072, Australia
5 Society for Southeast Asian Herpetology, Im Sand 3, D-69115 Heidelberg, Germany

Juan LEI et al. The Taxonomic Revision of Oligodon multizonatumNo. 1 27
species of China, only occurring in Sichuan and Gansu
Provinces (Zhao, 2006). A snake specimen (specimen
number KIZ01623, Figure 1) was collected in Luding
&RXQW\ƍƎ1ƍƎ(GXULQJ
a herpetological survey on July in 2009. A detailed
comparison with the species description and the holotype
specimen (CIB9964, Figure 2) suggested that it was
FRQVSHFL¿FZLWKO. multizonatum.
Recent studies of snakes (Burbrink and Castoe, 2009;
Huang et al., 2009), have shown that molecular data are
powerful tools for identifying and understanding snake
diversity. In view of this, the purpose of the present study
was to use molecular methods to clairfy the systematic
DI¿QLWLHVRIO. multizonatum. A prior study by us based
on molecular analysis with more than three genes and
89 species of Colubridae showed that O. multizonatum
clustered within Lycodon. Recently, the genus Lycodon
was suggested to include species of the old genus
Dinodon (Siler et al., 2013; Guo et al., 2013), suggesting
that many relationship within the genus of Lycodon still
need to be resolved. For example, Siler et al. (2013)
suggested that currently recognized subspecies may
Figure 1 Photographs of a new Oligodon multizonatum specimen (specimen number KIZ01623) collected in Luding province. A–C: Whole
body; D–F: Head in dorsal, ventral and right lateral views; G: Cloacal region in ventral view. Photo by Mian HOU.

Asian Herpetological Research28 Vol . 5
need to be elevated to species in further studies. Hence,
in this study, we sampled species from both Lycodon
and Dinodon in order to resolve these issues. For the
convenience of our discussion, the historic taxonomic
genera Lycodon and Dinodon continue to be used.
Additionally, we also compared the morphological data of
O. multizonatumZLWKLWVFORVHVWUHODWLYHDVLGHQWL¿HGE\
molecular data analysis to verify this conclusion.
2. Materials and methods
2.1 Morphology Measurements, except body and tail
lengths, were taken with a slide-caliper to the nearest 0.1
mm; all body lengths were made to the nearest millimeter
using a tape measure. The number of ventral scales was
counted according to Dowling (1951). Divided ventrals
were counted as one. The first scale posterior to the
cloaca was regarded as the first subcaudal, the terminal
scute was not included in the number of subcaudals. The
dorsal scale rows were counted at one head length behind
the head, at midbody (i.e., at the level of the ventral
plate corresponding to half the total number of ventrals),
and at one head length before the vent. We considered
sublabials being those shields that were completely below
a supralabial. Values for paired head characters are given
in left/right order.
The hemipenes of O. multizonatum and L.
liuchengchaoi were compared. The method for preparing
the hemipenes of preserved specimens followed Jiang
(2010) and Pesantes (1994). Hemipenial descriptive
terminology followed Dowling and Savage (1960),
Branch (1986) and Zhang et al. (1984). Drawings were
made with the aid of a stereomicroscope.
2.2 Taxon sampling Previous studies indicated that the
systematics of the genera Oligodon, Lycodon and Dinodon
are complex and possibly intertwined (Pope, 1935;
Smith, 1943; Vogel and Brachtel, 2008; Green et al.,
2010; Guo et al., 2013). Therefore, data from seven
species in Oligodon, 16 species in Lycodon and one
species in Dinodon from GenBank were used along with
new data generated during the present study from O.
multizonatum, O. formosanus, O. chinensis, L. ruhstrati,
L. liuchengchaoi, D. rufozonatum and D. flavozonatum
(Table 1). We also selected 10 taxa representing 10 genera
of Colubrinae from GenBank. The choice of outgroup
taxa (Boa constrictor and Cylindrophis ruffus) was based
on Huang et al. (2009). Accession numbers from the
Chengdu Institute of Biology (CIB), Kunming Institution
of Biology (KIZ) and the laboratory of Ding Li (DL) for
all these specimens are provided in Table 1.
2.3 DNA extraction, amplification, and sequencing
Tissue samples were either skeletal muscle or liver
preserved in 95% ethanol at the time of collection and
Figure 2 Photographs of the holotype specimen (CIB9964) of Oligodon multizonatum. A and B: Whole body; C: Ventral views; D: Cloacal
region in ventral view and hemipenis. Photo by Juan LEI.

Juan LEI et al. The Taxonomic Revision of Oligodon multizonatumNo. 1 29
Family Genus and species Accession No.
Subfamily Cyt bND4 c-mos
Colubridae
Colubrinae
'LQRGRQÀDYR]RQDWXP (DL12612) KF732927 KF732920 KF732934
Dinodon rufozonatum (DL12611) KF732924 KF732917 KF732931
Dinodon semicarinatus AB008539 AB008539
Lycodon alcalai KC010345 KC010304
Lycodon aulicus HQ735416 HQ735418
Lycodon bibonius KC010351 KC010309
Lycodon butleri KC010359 KC010312
Lycodon capucinus KC010354 U49317 KC010313
Lycodon chrysoprateros KC010360 KC010318
Lycodon dumerilii KC010363 KC010320
Lycodon effraenis KC010376 KC010328
Lycodon fasciatus KC010366
Lycodon jara KC010367 KC010322
Lycodon laoensis KC010370 KC010325
Lycodon liuchengchaoi (DL14315) KF732928 KF732921 KF732935
Lycodon muelleri KC010375
Lycodon osmanhilli KC347524 KC347403
Lycodon ruhstrati (DL12678) KF732925 KF732918 KF732932
Lycodon stormi KC010380 KC010331
Lycodon subcinctus KC010385 KC010335
Lycodon zawi AF471040 AF471111
Oligodon arnensis KC347464 KC347504 KC347404
Oligodon calamarius KC347478 KC347511 KC347405
Oligodon chinensis (DL12672) KF732930 KF732923 KF732937
Oligodon cinereus AF471033 AF471101
Oligodon formosanus (DL12643) KF732929 KF732922 KF732936
Oligodon maculatus KC010387
Oligodon multizonatum (KIZ01623) KF732926 KF732919 KF732933
Oligodon octolineatus U49316
Oligodon sublineatus KC347465 KC347521 KC347406
Oligodon taeniolatus KC347483 KC347505 KC347407
Boiga dendrophila AF471089 U49303 AF471128
Cemophora coccinea AF471091 DQ902282 AF471132
Crotaphopeltis tornieri AF471093 AF428011 AF471112
Dasypeltis atra AF471065 AF471136
Dipsadoboa unicolor AF471062 AF428017 AF471139
Elaphe carinata DQ902133 DQ902284 DQ902063
Lytorhynchus diadema DQ112076 AY187986
Pituophis melanoleucus DQ902130 DQ902312 FJ627797
Senticolis triaspis DQ902127 AF138775
Telescopus fallax AF471043 AF471108
Outgroups:
Boidae Boa constrictor AB177354 AB177354 AF471115
Cylindrophiidae Cylindrophis ruffus AB179619 AB179619 AF471113
Table 1 The information of sequences retrieved from GenBank and sequenced in this study. New sequences from this study are in bold.

Asian Herpetological Research30 Vol . 5
subsequently stored in either ethanol or frozen at –80°C.
All specimens sampled are preserved in the collections
of CIB. All tissues were treated by the standard method
of proteinase K digestion in lysis buffer followed by a
high salt DNA extraction procedure (Sambrook et al.,
1989). The mitochondrial cytochrome b (cyt b) gene and
the NADH dehydrogenase subunit 4 (ND4) gene, and the
nuclear oocyte maturation factor Mos (c-mos) gene were
DPSOL¿HGIURPWRWDO'1$H[WUDFWVXVLQJSRO\PHUDVHFKDLQ
reaction (PCR) with the following primer pairs for cyt b:
L14910/H16064 (Burbrink et al., 2000), ND4: ND4/Leu
(Arévalo et al., 1994), and c-mos: S77/S78 (Lawson et al.,
$PSOLILFDWLRQZDVSHUIRUPHGLQDȝOYROXPH
reaction with the following settings: initial denaturation
step with 4 min at 94°C, 35 cycles of denaturation for
1 min at 94°C, annealing for 1 min at 46°C for cyt b
primers and 56°C for ND4 and c-mos, extension for 1
min at 72°C. A final extension at 72°C was conducted
for 7 min. Purified PCR products were sequenced in
both directions with an ABI automated DNA sequencer
(ABI 3700). We conducted a BLAST search of acquired
sequences by using the GenBank database to verify that
generated sequences were not of pseudogenes. All novel
sequences have been deposited in GenBank (Table 1).
2.4 Phylogenetic analyses The initial alignments
of cyt b, ND4, c-mos were aligned using ClustalX
(Thompson et al., 1997) with default parameters, and
subsequently verified manually, and translated into
amino acid sequences to check for the presence of stop
codons. We tested the saturation of 3rd codon positions
of the mitochondrial protein-coding genes. These were
highly saturated, therefore, we deleted the 3rd codon
positions of mitochondrial genes (cyt b and ND4). In
addition, we also analyzed the phylogeny for each gene
independently in order to explore the congruence between
different gene data by using likelihood and Bayesian
analyses. There was no moderate to highly supported
incongruence between cyt b, ND4 and c-mos gene and
therefore we used the concatenated and combined data
for phylogenetic analyses in this study. Because some
of taxa were missing data for cyt b, ND4 and c-mos, we
did exploratory analyses of the combined data set of 41
ingroup and two outgroup taxa and found no missing data
exhibited identical relationships. Therefore, we chose
to use all data (41 taxa) for subsequent analyses of the
combined data set.
Phylogenetic analyses were performed using
Bayesian Inference (BI) and Maximum Likelihood
(ML) methodology. Partitioned Bayesian Inference (BI)
approaches were used to reconstruct phylogeny with
combined data set of three partial gene sequences, using
MrBayes v 3.1 (Huelsenbeck and Ronquist, 2001). Both
mitochondrial and nuclear data sets were partitioned by
FRGRQSRVLWLRQ7KHEHVW¿WVXEVWLWXWLRQPRGHOVHH7DEOH
2) was assigned to each partition using AIC in Modeltest
3.7 (Posada and Crandall, 1998) and PAUP* v4b10
(Swofford, 2003). Two separate runs were performed with
four Markov chains. Each run was conducted with 15 000
000 generations and sampled every 1000 generations.
When the scores were found to stabilize, a consensus tree
ZDVFDOFXODWHGDIWHURPLWWLQJ WKH¿UVW RIWKHWUHHVDV
burn-in. Node support for the Bayesian consensus tree was
determined using posterior probabilities (Erixon et al.,
2003). Maximum Likelihood (ML) with the non-
partitioned strategy with the combined data set was
used to infer trees and assess nodal support by using
RaxML (Stamatakis et al., 2005). The complex model
*75īZDVXVHGIRUHDFKSDUWLWLRQ6XSSRUWIRU0/
trees was derived from 100 nonparametric bootstrap
replicates using RaxML. Each inference was started
with a random starting tree, and 100 nonparametric
bootstrap pseudoreplicates (Stamatakis et al., 2008)
was used to assessed the nodal support. Because of less
availability of ND4 gene and the fact that the c-mos gene
was highly conserved in this study, average divergence
estimation between species was calculated from the two
mitochondrial genes using Mega 4.0 (Tamura et al.,
2008).
2.5 Topological test The Bayesian analysis and
Maximum Likelihood produced BI and ML trees. The
topological structure of phylogenetic trees were slightly
different. Results from the Shimodaira-Hasegawa (SH
test; Shimodaira and Hasegawa, 1999) and Kishino-
Hasegawa tests (KH test; Kishinino and Hasegawa, 1989)
LQGLFDWHGWKDWWKH%,WUHHZDVWKHEHVW¿W7KHUHIRUH WKH
conclusion of our analysis are based mainly on the BI tree
topological structure.
3. Results
3.1 Morphology The newly collected specimen
(KIZ01623) and the specimen of the type series of
O. multizonatum Zhao and Jiang, 1981 (CIB9964)
were used in this study. A Comparison of the main
morphological characters between O. multizonatum
(type specimens, CIB9964–9967), O. multizonatum (new
specimen, KIZ01623), L. liuchengchaoi (description
from Zhang et al. [2011], CWNU867001, CWNU84002,
and FMNH15148), L. liuchengchaoi (new specimen,
DL14315), and O. joynsoni (description from Jiang et al.

Juan LEI et al. The Taxonomic Revision of Oligodon multizonatumNo. 1 31
[2012], BMNH1946.1.4.23, BMNH 1969.1809, BMNH
1938.8.7.40, BMNH 1969.1808, MNHN 1896.0633, and
KIZ09128) are shown in Table 3. Based on morphological
examination, our results indicated that the new specimen
of O. multizonatum is same as the type specimen of O.
multizonatum, but different from the type specimen of L.
liuchengchaoi and the new specimen of L. liuchengchaoi.
In addition, Zhao and Jiang (1981) reported O. joynsoni is
the most similar species to O. multizonatum. Our analysis
indicates there are many major differences between O.
joynsoni, L. liuchengchaoi and O. multizonatum.
The hemipenis of O. multizonatum (KIZ01623) (Figure
3) is characterized as follows: smaller base, expanding
from middle to tip; relatively short, extending to the
eighth subcaudal; unforked, sulcus single and prominent,
extending to the tips of the organ; the base to middle of
the organ covered with larger hard spines, but changing to
tiny spines after middle to the tip; no nick at the tip.
The hemipenis characteristics of O. multizonatum
(KIZ01623) are similar to the description of the type
specimen of O. multizonatum (CIB9964) provided by
Zhao and Jiang (1981). Based on hemipenis morphology,
O. multizonatum can be seperated from the species of
the genus Oligodon that lack a hard spine, such as O.
joynsoni (Smith, 1917), but is similar to most species of
the genera Oligodon, Lycodon and Dinodon, which have
a hard spine on the hemipenis (Zhao et al., 1998; Zhao,
2008; Green et al., 2010). Green et al. (2010) reported
the hind teeth of snakes of the genus Oligodon are broad
and strongly recurved. However, our analysis showed
that the hind teeth of both new and type specimens of
O. multizonatum are not strongly recurved. In addition,
a striking characteristic of Oligodon is the large rostral
scale that is clearly visible when viewed from above
(Zhao et al., 1998; Zhao, 2006). Nevertheless, such
observations are to some extent subjective and might
even be dependent on the viewing angle (Figures 1–2).
Actually the rostral scale of O. multizonatum is not as
Characters O. multizonatum O. multizonatum L. liuchengchaoi L. liuchengchaoi O. joynsoni
(CIB9964–9967) (KIZ01623) (Zhang et al. [2011]) (DL14315) (Jiang et al. [2012])
Snout-vent length 173-409 428 595-676 458 568
Tail length 45-90 92 134-152 114 79
Dorsal scale rows 17-17-15 17-17-15 17-17-15 17-17-15 17-17-15
Ventrals 190-195 194 202-206 205 186-200
Subcaudals 68-75 63 68-77 73 40-50
Loreal enters eye yes yes yes yes yes
Dorsal bands 55-73 55 40-45 43 no
Tail bands 16-19 11 10-15 12 no
Upper labials 8 8 7-8 7 7-8
Temporals 2 + 3; 1/2 + 2; 2 + 3/2 2 + 3 2 + 2; 2 + 2/1 + 2; 1 + 2 2 + 2 1 + 2
Infralabials 8 8 8 8 7-8
Maxillary teeth 10-11 10 8-9 8 11-12
Anal plate divided divided divided divided entire
Table 3 A Comparison of the main morphological characters between O. multizonatum (type specimens, CIB9964–9967), O. multizonatum
(new specimen, KIZ01623), L. liuchengchaoi (description from Zhang et al. [2011], CWNU867001, CWNU84002, and FMNH15148), L.
liuchengchaoi (new specimen, DL14315), and O. joynsoni (description from Jiang et al. [2012], BMNH1946.1.4.23, BMNH 1969.1809,
BMNH 1938.8.7.40, BMNH 1969.1808, MNHN 1896.0633, and KIZ09128). Dimensions in mm.
Partitions ACI models Number of characters
cyt b, 1st codon position GTR + G 361
cyt b, 2nd codon position GTR + I + G 361
ND4, 1st codon position K81uf + G 216
ND4, 2nd codon position TrN + G 216
c-mos, 1st codon position K80 188
c-mos, 2nd codon position T-VM 188
c-mos, 3rd codon position +.<ī 188
Table 2 Models of evolution selected by AIC and partitions of mitochondrial (cyt b, ND4) and nuclear (c-mos) data applied in model-based
analyses.

Asian Herpetological Research32 Vol . 5
large as that in members of the genus Oligodon, and is
only clearly visible from above. The placement of O.
multizonatum in the genus Oligodon was based on it
having a large rostal scale (ref to original description),
but our analysis suggests that the rostal scale of O.
multizonatum is not particularly prominent.
3.2 Phylogeny The initial aligned data set contained
1085 bp of cyt b, 648 bp of ND4 and 564 bp of c-mos
IRULQJURXSDQGWZRRXWJURXSWD[DZKHUHDVWKH¿QDO
aligned data set contained 722 bp of cyt b, 432 bp of ND4
and 564 bp of c-mos after deleting the 3rd codon positions
of mitochondrial genes.
The topologies of trees derived from each dataset and
analytical method were nearly identical (see Figures
4–5). BI and ML trees showed strong support (100%
PP and 99% BS respectively) for the monophyly of
Oligodon, adding some novel molecular sequence
data of O. chinensis, O. formosanus, but excluding O.
multizonatum. Unexpectedly, all analyses demonstrated
that O. multizonatum is not part of the genus Oligodon
but is instead nested within the genus Lycodon, where it is
most closely related to L. liuchengchaoi in our sampling.
These two species together formed a highly supported
clade (100% PP and 99% BS), which are themselves
sister to L. ruhstrati in the BI tree but to other clades
including two species of L. butleri and L. fasciatus in
ML tree. All these five species formed a monophyletic
group with support values of 100% PP and 99% BS.
However, most of the main nodes were not well solved
within Oligodon and Lycodon in both BI and ML trees.
In addition, the genetic distance (uncorrected P-distance)
between O. multizonatum and L. liuchengchaoi is 0.066
(cyt b gene) and the minimum genetic distance of valid
species between L. aulicus and L. capucinus is 0.047
(cyt b gene).
4. Discussion
4.1 Phylogenetic position and morphology of O.
mulizonatum Although our sampling was incomplete
relative to the sampling of Colubridae, multilocus
phylogenetic reconstruction has indicated that all
representatives from Oligodon except O. multizonatum
formed a strongly supported clade, and those from
Lycodon with O. multizonatum clustered into another
highly supported group (Figure 1), in which the
species within Lycodon and Dinodon were shown to be
paraphyletic or polyphyletic. Previous studies support
the conclusion that Lycodon is paraphyletic with respect
to Dinodon (Siler et al., 2013). In addition, Guo et al.
(2013) concluded that Lycodon and Dinodon are
paraphyletic based on molecular and morphological
data, and suggested synonymizing Dinodon with
Lycodon. Moreover, Pyron et al. (2013) indicated the
genus of Dryocalamus which had previously been
identified as Lycodon nested within the group Dinodon
and Lycodon, suggesting that Dinodon and Lycodon
are not monophyletic. In agreement with our molecular
phylogenetic results, previous morphological studies have
noted the difficulty of separating Dinodon and Lycodon
(Pope, 1935; Smith, 1943; Vogel and Brachtel, 2008), and
thus the validity of these two genera has triggered debate.
This is the reason why we sampled widely within these
taxa and used different methods of analysis to make our
conculsions. Our results support synonymizing the genus
Dinodon and Lycodon.
Unexpectedly, our analysis indicates O. multizonatum
as the sister species of L. liuchengchaoi which is clustered
within Lycodon based on both mitochondrial and nuclear
genes. Based on morphology, Zhao and Jiang (1981)
suggested that O. multizonatum is closely related to the
Indochinese O. joynsoni, and then assigned this species to
Figure 3 The left hemipenis of Oligodon multizonatum (specimen number KIZ01623). Drawing by Ke JIANG..

Juan LEI et al. The Taxonomic Revision of Oligodon multizonatumNo. 1 33
Figure 4 The 50% majority-rule consensus tree from Bayesian analysis based on c-mos, cyt b and ND4 combined sequences. Values at
nodes are posterior probability support values. Black bar: Lycodon; Open bar: Oligodon; Gray bar: old Dinodon.
Lycodon capucinus
Lycodon aulicus
100
Lycodon osmanhilli
Lycodon zawi
100
Lycodon jara
98
Lycodon laoensis
Lycodon stormi
Crotaphopeltis tornieri
Dipsadoboa unicolor
100
Telescopus fallax
60
Boiga constrictor
Dasypeltis atra
89
97
100
Cemophora coccinea
Elaphe carinata
77
Senticolis triaspis
79
Pituophis melanoleucu s
100
Lytorhynchus diadema
57
100
Lycodon alcalai
Lycodon chrysoprateros
100
Lycodon bibonius
100
Lycodon muelleri
Lycodon dumerilii
Dinodon rufozonatum
94
99
Lycodon subcinctus
63
85
100
Lycodon effraenis
72
60
100
100
100
100
100
76
Oligodon arnensis
56
100
100
Cylindrophis ruffus
Boa constrictor
0.1
Lycodon liuchengchaoi
Oligodon multizonatum
100
96
Lycodon ruhstrati
Lycodon butleri
Lycodon fasciatus
100
100
Dinodon flavozonatum
Dinodon semicarinatus Dinodon
Lycodon
Oligodon cinereus
Oligodon octolineatus
100
Oligodon maculatus
Oligodon formosanus
Oligodon chinensis
Oligodon calamarius
Oligodon sublineatus
100
Oligodon taeniolatus
Oligodon
(old taxon)

Asian Herpetological Research34 Vol . 5
Figure 5 Maximum likelihood inferred phylogeny of the c-mos, cyt b and ND4 combined data. Bootstrap values are shown at the
corresponding nodes. Support values below 50% were not shown in this figure. Black bar: Lycodon; Open bar: Oligodon; Gray bar:
old Dinodon.
Oligodon maculatus
Oligodon cinereus
Oligodon octolineatus
99
97
Oligodon chinensis
Oligodon formosanus
100
98
Oligodon taeniolatus
Oligodon calamarius
Oligodon sublineatus
100
62
Oligodon arnensis
99
Lycodon effraenis
Lycodon stormi
Dinodon semicarinatus
Dinodon rufozonatum
Dinodon flavozonatum
84
72
Lycodon subcinctus
Lycodon butleri
Lycodon fasciatus
98
Oligodon multizonatum
Lycodon liuchengchaoi
99
50
Lycodon ruhstrati
99
Lycodon laoensis
Lycodon zawi
Lycodon osmanhilli
Lycodon capucinus
Lycodon aulicus
95
89
Lycodon jara
98
91
Lycodon dumerilii
Lycodon muelleri
Lycodon chrysoprateros
Lycodon alcalai
100
Lycodon bibonius
100
71
99
80
Dipsadoboa unicolor
Crotaphopeltis tornieri
98
Telescopus fallax
Dasypeltis atra
Boiga constrictor
50
60
Cemophora coccinea
Senticoli s triaspis
Elaphe carinata
67
71
Pituophis melanole ucus
99
Lytorhynchus diadema
93
100
Cylindrophis ruffus
Boa constrictor
0.1
Oligodon
Dinodon
Lycodon
100
(old taxon)

Juan LEI et al. The Taxonomic Revision of Oligodon multizonatumNo. 1 35
the genus Oligodon. However, there are many differences
between these two species. The former species differs
from the latter by having: 1) more subcaudal scales, 68–
75 pairs vs 40–50 pairs; 2) a divided vs entire anal plate;
3) eight upper labial scales, the third, fourth and fifth
vs the fourth and fifth touching the eye; 4) a hemipenis
with spines vs without spines. The colour pattern and
markings of these two species are also quite different
(Zhao and Jiang, 1981). The hind teeth are also different
being broad and strongly recurved, much like the shape of
the kukri knife in Oligodon (Green et al., 2010), but not
recurved in O. multizonatum. The recurved shaped teeth
of Oligodon are used to open reptile eggs (Green et al.,
2010), upon which they mainly feed. The combined
morphological data also indicate that O. multizonatum is
neither a close relative to O. joynsoni nor a member of the
genus Oligodon.
In terms of pattern, O. multizonatum is most similar
to L. liuchengzhaoi except for the fact that the number
and color of bands of the former are greater and deeper
than those of the latter (5–73 orange rings spaced along
the black body, and 16–19 orange rings spaced along
the black tail vs±ZHOOGH¿QHG\HOORZULQJVHYHQO\
spaced along the entire length of the black body, and more
than 10–15 yellow rings evenly spaced along the black
tail), but differs by the following traits: more maxillary
teeth (10–11 vs 8–9), fewer ventrals (190–195 vs 202–
206) (Zhao and Jiang, 1981; Zhang et al., 2011).
Therefore, we suggest that the species previously
assigned to O. multizonatum needs to be transferred to
Lycodon. Zhao (2006) reported that O. multizonatum feed
on reptile eggs, but no analysis of the stomach content
of this species has been reported. Further studies on prey
types consumed by species within Oligodon and Lycodon
DUHQHHGHGWRFRQ¿UPRUSURYLGHVRPHQHZHYLGHQFHWR
support the view that most of Oligodon feed on retile
eggs whereas snakes and lizards are the major food of
Lycodon. Considering that the genus Dinodon has been
merged into Lycodon, we suggested that the scientific
name of O. multizonatum should be renamed as Lycodon
multizonatum. Consistent with this we propose a new
common English name, the Luding wolf snake, referring
to the type locality, Luding County, China.
4.2 The validity of O. multizonatum and L.
liuchengzhaoi For many species, selective or
developmental constraints either prevent morphological
divergence (Colborn et al., 2001) or promote convergence
(Wake, 1991), complicating our understanding of group
composition based on evolutionary relationships inferred
from morphology (Guo et al., 2013). On the other hand,
within species individual variations in morphology can
make species identification difficult. A good example
is Lycodon, one of the most diverse genera of Asiatic
colubrids (sensustricto, see Pyron et al., 2011). Recently,
L. futsingensis (Pope, 1928), which was subsequently
synonymized with L. ruhstrati by Pope himself (Pope,
1935) was revalidated by Vogel et al. (2009). In 2010
and 2011, two new endemic species were described
from China: L. synaptor (Vogel and David, 2010) and L.
gongshan (Vogel and Luo, 2011). Based on the specimens
collected from northern Sichuan Province, China,
Zhang et al. (2011) described L. liuchengzhaoi. They
are similar to L. fasciatus in shape and were identified
as L. fasciatus previously. By careful examination of the
specimens it was noticed that they could be distinguished
from L. fasciatus and other species of the L. fasciatus
group by several morphological characters (Vogel et al.,
2009). However, O. multizonatum was not compared
with these specimens. Thus it should be cautioned
that O. multizonatum is the closest related specie to
L. liuchengchaoi from our molecular phylogenetic
analysis (Figures 4–5). Although they shared very
similar morphlogical characters, such as same dorsal
scale rows, loreal enters eye, same infralabial, similar
temporals and similar subcaudals (Table 3), the genetic
distance between these two species reached the level of
interspecific differentiation. Our molecular data showed
that the genetic distance (uncorrected P-distance)
between O. multizonatum and L. liuchengchaoi is 0.066
(cyt b gene), which is greater than the minimum genetic
distance of the valid species difference between L.
aulicus and L. capucinus which is 0.047 of cyt b gene.
Therefore, we strongly suggest that O. multizonatum and
L. liuchengchaoi are valid as distinct species.
Currently, these two species of Lycodon are known in
Sichuan Province. The three sites where L. liuchengchaoi
was found are from the east of the Hengduan mountains,
at the eastern edge of Qinghai-tibet Plateau. O.
multizonatum is known only from Luding county,
Sichuan Province, Tianshui and Kang counties, Gansu
Province. These specimen records and published literature
suggest that O. multizonatum might be distributed in
the middle eastern and northern edge of Hengduan
mountains which is sympatric with the L. liuchengchaoi.
However, it is interesting to notice that the specimen of
O. multizonatum sourced from Gansu province had fewer
rings in its body on the photograph (Zhao, 2006) is rather
similar to L. liuchengchaoi. Therefore, we suggest the
distribution of O. multizonatum in Gansu province might
be questionable.

Asian Herpetological Research36 Vol . 5
Unfortunately, the molecular phylogeny presented
here did not resolve the relationships among Lycodon and
Dinodon. Considering the morphological and phylogenetic
results in this study, we suggest future studies need to add
more markers to resolve the relationships among Lycodon
and Dinodon.
Acknowledgements This work was supported by the
National Natural Science Foundation of China (NSFC
31071913).
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