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\begin{document}

\title{Molecular phylogeny reveals cryptic diversity in Sibynophis from China
(Serpentes: Sibynophiidae)}



\author[1]{Peng Guo}%
\author[2]{Ping Wang}%
\author[1]{Bing Lyu}%
\author[1]{Qin Liu}%
\author[1]{Jieyu Zheng}%
\author[1]{Chunmei Fu}%
\author[3]{Yayong Wu}%
\author[1]{Guocheng Shu}%
\author[4]{Shaobing Hou}%
\affil[1]{Yibin University}%
\affil[2]{Sichuan Academy of Science, Chengdu, P. R. China}%
\affil[3]{Chengdu Institute of Biology}%
\affil[4]{Kunming Institute of Zoology Chinese Academy of Sciences}%


\vspace{-1em}



  
  \date{May 17, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Illumination of species diversity and their distribution is key to
evolution, genetics and conservation. The genus of Sibynophis is a group
of rare snakes with less attentions. Based on more extensive sampling,
we use both mitochondrial fragments and nuclear gene to explore the
species diversity of the species of Sibynophis occurring in China. The
results showed that S. c. miyiensis is a synonym of S. c. grahami, and
S. c. grahami should be gave a specific rank as S. graham. Sibynophis
triangularis was uncovered to be new to China and Myanmar. On basis of
our specimens and molecular phylogeny results, the species distribution
boundaries of each Chinese species were redefined.%
\end{abstract}%



\sloppy


\textbf{Molecular phylogeny reveals cryptic diversity
in\emph{Sibynophis} from China (Serpentes: Sibynophiidae)}

Peng Guo\textsuperscript{1*} Ping Wang\textsuperscript{2} Bing
Lyu\textsuperscript{1} Qin Liu\textsuperscript{1} Jieyu
Zheng\textsuperscript{1} chunmei Fu\textsuperscript{1} Yayong
Wu\textsuperscript{1} Guocheng Shu\textsuperscript{1} Shaobing
Hou\textsuperscript{3}

\textsuperscript{1}Faculty of Agriculture, Forest and Food Engineering,
Yibin University, Yibin, P. R. China

\textsuperscript{2} Sichuan Academy of Science, Chengdu, P. R. China

\textsuperscript{3}State Key Laboratory of Genetic Resources and
Evolution \& Yunnan Key Laboratory of Biodiversity and Ecological
Security of Gaoligong Mountain, Kunming Institute of Zoology, Chinese
Academy of Sciences, Kunming, P. R. China

*Corresponding author. Email: ybguop@163.com

Running title: Phylogeny of \emph{Sibynophis} from China



\textbf{Abstract}

Illumination of species diversity and their distribution is key to
evolution, genetics and conservation. The genus of \emph{Sibynophis} is
a group of rare snakes with less attentions. Based on more extensive
sampling, we use both mitochondrial fragments and nuclear gene to
explore the species diversity of the species of
\emph{Sibynophis}occurring in China. The results showed that \emph{S. c.
miyiensis} is a synonym of \emph{S. c. grahami} , and \emph{S. c.
grahami} should be gave a specific rank as \emph{S. graham} .
\emph{Sibynophistriangularis} was uncovered to be new to China and
Myanmar. On basis of our specimens and molecular phylogeny results, the
species distribution boundaries of each Chinese species were redefined.

\textbf{KEY WORDS}

taxonomy, distribution, snakes, \emph{Sibynophis grahami} , Asia

\textbf{1 INTRODUCTION}

Species diversity is central to biodiversity. Exploration of species
diversity and identification of species distribution boundaries are
pivotal to the preservation and management of biodiversity. The
accumulation of multi-locus data and development of related analytical
technologies have greatly advanced our understanding of cryptic species
diversity. In recent decades, considerable progress has been made in the
phylogeny of Asian snakes. Notably, various systematic issues have been
addressed (Guo, Zhu, Liu, Wang, \& Nguyen, 2020; Hou et al., 2021; Liu
et al., 2021; Poyarkov et al., 2022) and an increasing number of new
taxa have been described (Hou et al., 2021; Liu, Zhong, Wang, Liu, \&
Guo, 2018; Ren, Jiang, Huang, David, \& Li, 2022). However, in
comparison to well-studied common snake groups, such as \emph{Hebius}and
\emph{Lycodon} (Guo et al., 2013; Guo et al., 2014; Hou et al., 2021;
Ren et al., 2022), little attention has been paid to rare snake species,
such as those in the genus \emph{Sibynophis} .

The family Sibynophiidae is comprised of only two genera, including the
non-venomous genus \emph{Sibynophis} . These snakes are characterized by
their small body size, loose attachment of their dentary to the
articular bone, and the presence of numerous small maxillary teeth of
uniform size (Pope, 1935). Currently, nine species are recognized
within\emph{Sibynophis} , with primary distribution in South and
Southeast Asia (Uetz, Freed, Aguilar, \& Ho\selectlanguage{polish}š\selectlanguage{english}ek, 2022; Wallach, Williams,
\& Boundy, 2014). Two species, \emph{S. collaris} and \emph{S.
chinensis} , are known to occur in China (Wang et al., 2020; Zhao,
2006).\emph{Sibynophis} \emph{chinensis} was initially described based
on a sole specimen from Yichang, Hubei, China (G\selectlanguage{ngerman}ünther, 1889), but is
now known to occur in China, Korea, and Vietnam (Wallach et al., 2014).
In China, the species exhibits broad distribution, ranging from southern
Xizang in the west to Jiangsu in the east (Zhao, 2006). Three subspecies
are currently recognized (Zhao, 2006; Zhao, Huang, \& Zong, 1998),
including \emph{S. c. chinensis} , \emph{S. c. grahami} , and \emph{S.
c. miyiensis} . In addition to the nominal species, which broadly occurs
in Southwest, Central, and East China, \emph{S. c. miyiensis} is endemic
to Southwest Sichuan and Northwest Yunnan, while \emph{S. c. grahami} is
endemic to northeastern Yunnan and Guizhou (Zhao et al., 1998; Zhao \&
Yang, 1997). This taxonomical arrangement is followed by most authors
(Uetz et al., 2022; Wallach et al., 2014; Wang et al., 2020). The type
locality of \emph{S. collaris} is in Assam, India (Gray, 1853), but the
species is restricted to Xizang and Yunnan in China (Zhao, 2006; Zhao et
al., 1998). Morphologically, the two species differ from one another by
number of supralabials and anterior temporal scales only (Zhao, 2006;
Zhao et al., 1998).

Due to their rare occurrence and elusive nature, few
\emph{Sibynophis}specimens have been collected since their initial
description and few studies have been conducted on their biology,
particularly their systematics (Li et al., 2020; Pyron, Burbrink, \&
Wiens, 2013; Zaher et al., 2019). Although \emph{Sibynophis} species
have been included in certain phylogenetic studies (Chen et al., 2013;
Li et al., 2020; Pyron et al., 2013; Zaher et al., 2019), only a limited
number of samples have been examined. For example, Zaher et al. (2019)
conducted a large-scale molecular phylogenetic study of advanced
caenophidian snakes, which included five \emph{Sibynophis} species, each
with only one individual. Thus, given the small number of species and
specimens studied, the diversity and evolution of the genus remain
poorly understood.

Here, based on more extensive sampling, we explored species diversity of
the genus \emph{Sibynophis} and revised the taxonomy and distribution
boundaries of each taxon within China.

\textbf{2 MATERIALS AND METHODS}

In total, 24 specimens previously identified as \emph{S. collaris}
and\emph{S. chinensis} were collected from China (Fig. 1), then
sequenced and analyzed. Additional samples from the two species and
their congeners, were included and their sequences were retrieved from
GenBank (Table 1). \emph{Scaphiodontophis annulatus} was chosen as the
outgroup based on previous work (Pyron et al., 2013).

Total DNA was extracted from 85\% alcohol-preserved liver or muscle
tissues using M5 HiPer Universal DNA Mini Kit (Mei5 Biotechnology Co.,
Ltd., Beijing, China) following the manufacturer's protocols. Two
mitochondrial gene fragments, cytochrome \emph{b} (cyt.\emph{b} ) and
NADH subunit 2 (ND2), and nuclear gene oocyte maturation factor mos
(c-mos), were amplified by polymerase chain reaction (PCR) using primers
L14910/H16064 (Burbrink, Lawson, \& Slowinski, 2000), ND2L49/ND2H50
(Alfaro \& Arnold, 2001), and S77/S78 (Lawson, Slowinski, Crother, \&
Burbrink, 2005). Cycling parameters were identical to those described in
the above studies. Prior to sequencing, PCR products were purified using
various commercial kits. The double-stranded product was sequenced by a
commercial company (GENEWIZ Company, Suzhou, China).

The sequences were manually edited using SeqMan in Lasergene v15.1
(DNASTAR Inc., USA), aligned using Muscle with default settings, and
quality-checked using MEGA v7.0 (Kumar, Nei, Dudley, \& Tamura, 2008;
Tamura, Stecher, Peterson, Filipski, \& Kumar, 2013). Phylogenetic
analyses were performed based on the two mitochondrial DNA (mtDNA)
fragments using Bayesian inference (BI) and maximum-likelihood (ML). BI
was executed in MrBayes v3.2.2 (Ronquist et al., 2012) using
evolutionary models selected in PartitionFinder v2.1.1 under Bayesian
information criterion (BIC) (Lanfear, Calcott, Ho, \& Guindon, 2012).
All searches were performed with three independent runs, each initiating
a random tree. Each run consisted of four Markov chains (three heated
and one cold chain), with 2 × 10\textsuperscript{6} generations,
sampling every 1\selectlanguage{english} 000 generations, and 25\% of initial samples discarded
as burn-in. Convergence was assessed by examining effective sample size
(ESS) (\textgreater{} 200) and likelihood plots through time in Tracer
v1.7 (Rambaut, Drummond, Xie, Baele, \& Suchard, 2018). The resulting
trees were combined to calculate posterior probabilities (PP) for each
node in a 50\% majority-rule consensus tree. The ML trees were
constructed using RAxML v7.2.6 (Stamatakis, 2006) with the GTMMAGTRCAT
model under the same partitioning scheme as BI analysis. Branch support
was assessed using 1\selectlanguage{english} 000 non-parametric bootstrap (BS) topological
replicates.

We constructed a haplotype network to depict interspecific/clade
relationships based on c-mos sequences. Analyses were executed using
PopART v1.7 (Leigh \& Bryant, 2015) with the parameter epsilon set to 0.

Pairwise genetic distances (\emph{p} -distances) between species or
mtDNA clades were also calculated using Mega v7.0 (Kumar et al., 2008;
Tamura et al., 2013).

\textbf{3 RESULTS}

The BI and ML analyses depicted relatively consistent topologies (Fig.
2). All species of \emph{Sibynophis} formed a highly supported lineage.
Sister species \emph{S. bistrigatus} and \emph{S. subpunctatus} were
basal to the lineage, while all other specimens formed another monophyly
with strong support. The Chinese specimens were positioned in four
distinct but highly supported clades (A-D). Clade A was composed of
specimens from eastern Yunnan and Guizhou, China; clade B consisted of
samples from China (Xizang) and Myanmar; clade C contained specimens
from China (Yunnan), Vietnam, Myanmar, and Cambodia; and clade D
contained specimens from Vietnam and China (Sichuan, Shaanxi, and
southern China). The four clades formed a highly supported monophyly
with well-resolved interclade relationships (D, (A, (B, C))). A sample
previously identified as \emph{S. collaris} (CAS 240150) is much
distinct from the other \emph{S. collaris} identified samples (B clade).

Uncorrected \emph{p} -distances between species and clades are listed in
Table 2. The interspecific/clade genetic distances ranged from 8.3\%
(clades B and C) to 21.2\% (clade B and \emph{S. bistrigatus} ) based on
cyt.\emph{b} and from 7.9\% (clades B and C) to 18.3\% (clade D and
sample CAS 240150) based on ND2 (Table 2).

The network inferred from the c-mos gene showed each species/clade
exhibited unique haplotype, except clade B (\emph{S. collaris} ), which
shared a haplotype with clade C (\emph{S. triangularis}
).\emph{Sibynophis c. grahami} , \emph{S. c. chinensis} , and \emph{S.
triangularis} contained three, two, and two unique haplotypes,
respectively (Fig. 3).

\textbf{4 DISCUSSION}

This study represents the first comprehensive investigation
of\emph{Sibynophis} systematics and diversity in China. The phylogenetic
relationships reconstructed using the two methods generated mostly
identical topologies and highly similar support indices for major
clades, with slight differences in support for several shallow nodes.
Phylogenetic analyses further revealed that Chinese species
of\emph{Sibynophis} consisted of four clades (A-D), with well-supported
interclade relationships. Network analyses showed similar results.

\textbf{4.1 Systematics of \emph{S. chinensis}}

\emph{Sibynophis chinensis} is widely distributed in China. Here, the
mtDNA-based phylogeny indicated that \emph{S. chinensis} was composed of
clades A and D. Clade D consisted of specimens from Vietnam and
Southwest, Central, and Southeast China, corresponding to the \emph{S.
c. chinensis} subspecies. Clade A consisted of specimens from Yunnan,
Guizhou, and Sichuan, which are the distribution regions of the \emph{S.
c. miyiensis} and \emph{S. c. grahami} subspecies; three specimens (YBU
15152, GP 10544, and KIZ 028322) attributed to \emph{S. c.
miyiensis}were nested within the other specimens, thereby rendering
\emph{S. c. grahami} non-monophyletic. Therefore, based on priority rule
of nomenclature we concluded that clade A should be represented by
\emph{S. c. grahami} , and \emph{S. c. miyiensis} should be considered a
synonym of \emph{S. c. grahami} .

Unexpectedly, although clades A and D were highly supported, neither
formed a monophyletic lineage (Fig. 2). The genetic distance between the
two clades was 12.6\% (cyt.\emph{b} -based), which exceeds that between
certain species (e.g., 9.4\% between \emph{S. subpunctatus} and \emph{S.
bistrigatus} ) (Table 2). In addition, network analyses revealed that
the two clades did not share any nDNA haplotype. These findings suggest
that both clades represent two distinct species, i.e., clade A
represents\emph{S. grahami} and clade D represents \emph{S. chinensis} .

Given the taxonomic revision of \emph{S. chinensis} (\emph{sensu lato}
), the distribution of \emph{S. grahami} should be redefined
accordingly. Thus, based on previous records (Zhao, 2006; Zhao et al.,
1998) and our data, we conclude that \emph{S. grahami} is present in
southwestern Sichuan, Yunnan, and Guizhou in China.

\textbf{4.2. Distribution of \emph{S. triangularis} and \emph{S.
collaris} in China}

\emph{Sibynophis triangularis} was originally described based on a
single specimen from Thailand (Taylor \& Elbel, 1958). Although
initially considered a subspecies of \emph{S. collaris} , it was
subsequently elevated to a separate species, i.e., \emph{S.
triangularis} (Taylor, 1965). At present, this species is known to occur
in Thailand and Cambodia (Stuart, Sok, \& Neang, 2006; Uetz et al.,
2022; Wallach et al., 2014). However, our molecular phylogenetic
reconstruction revealed that a particular sample (FMNH 263023,
Cambodia), previously identified as \emph{S. triangularis} (Stuart et
al., 2006), formed a well-supported clade (C) with specimens from
Vietnam, Myanmar, and China (southern and northwestern Yunnan). Hence,
we confirm that clade C represents the species \emph{S. triangularis} ,
which is newly reported in China and Myanmar. Currently, \emph{S.
triangularis} is only known to occur in Fugong and Cangyuan in Yunnan of
China.

Our results identified clade B as sister to clade C,
representing\emph{S. collaris} based on geographical origin.
Unexpectedly, sample CAS 240150 collected from Myanmar and originally
identified as \emph{S. collaris} in Chen et al. (2013) formed a distinct
mtDNA lineage from clade B, with genetic distances of 8.3\%
(cyt.\emph{b} -based) and 7.9\% (ND2-based), respectively (Table 2; Fig.
2). These findings suggest that this specimen was misidentified and may
represent an undescribed taxon. Thus, further examination of this
specimen is required. In addition, in a previous large-scale systematic
study of Chinese snakes, Li et al. (2020) identified one specimen (CHS
244) from Honghe, southern Yunnan, China, as \emph{S. collaris} , and
two specimens (CHS 879 and CHS 880) from Motuo, Xizang, China, as
\emph{S. chinensis} . Our results showed the first specimen was nested
within the clade A and the latter two positioned in the clade B. Thus we
concluded that these samples were misidentified, with the former being
\emph{S. grahami} and the latter two being \emph{S. collaris} .

Zhao and Yang (1997), Zhao et al. (1998), Zhao (2006), and Yang and Rao
(2008) proposed that \emph{S. collaris} is distributed in southern
Xizang and northwestern Yunnan in China. Based on our molecular
phylogenetic analyses, however, several specimens collected from
northwestern Yunnan, which is geographically close to Motuo, Xizang,
were identified as \emph{S. grahami} and S. \emph{triangularis} (Fig.
2). Thus, it is most like that \emph{S. collaris} is endemic to Motuo in
Xizang, with no occurrence in Yunnan.

\textbf{CONCLUSIONS}

Based on more extensive sampling, we conducted a comprehensive study of
Chinese species of \emph{Sibynophis} , providing new insights into their
systematics and distribution. Our results revealed that \emph{S. c.
miyiensis} is synonymous with \emph{S. c. graham} i, which should be
elevated to a species level. Furthermore, \emph{S. triangularis} is
reported as new to China and Myanmar. In total, four species, \emph{S.
chinensis} , \emph{S. collaris} , \emph{S. grahami} , and \emph{S.
triangularis} , are distributed in China. Consequently, we revised their
distribution boundaries in China and re-examined previously
misidentified specimens.

\textbf{AUTHOR CONTRIBUTIONS}

\textbf{Peng Guo} : Conceptualization (Lead); Data curation (Lead);
Formal analysis (Lead); Funding acquisition (Lead); Investigation
(Lead); Methodology (Lead); Project administration (Lead); Writing --
original draft (Lead); Writing -- review \& editing (Lead). \textbf{Ping
Wang} : Data curation (Supporting); Methodology (Supporting); Resources
(Supporting). \textbf{Bing Lyu:} Data curation (Supporting); Resources
(Supporting); Software (Supporting); Visualization
(Supporting).\textbf{Qin Liu:} Data curation (Supporting); Formal
analysis (Supporting); Resources (Equal). \textbf{Jieyu Zheng:} Data
curation (Supporting); Formal analysis (Supporting); Resources
(Supporting).\textbf{Chunmei Fu:} Data curation (Supporting); Formal
analysis (Supporting); Resources (Supporting). \textbf{Yayong Wu:} Data
curation (Supporting); Resources (Supporting); \textbf{Guocheng Shu:}
Data curation (Supporting); Resources (Supporting). \textbf{Shaobing
Hou:}Data curation (Supporting); Resources (Supporting).

\textbf{CONFLICT OF INTEREST STATEMENT}

The authors declare no conflict of interest.

\textbf{ACKNOWLEDGEMENTS}

We would like to thank Zhong GH, Xie YL, Li K for their assistance in
samples collection, and to Zeng YM and Li L for their help in data
analyses and map preparation.

\textbf{FUNDING INFORMATION}

This study was supported by the Second Tibetan Plateau Scientific
Expedition and Research (STEP) Program (2019QZKK05010105), National
Natural Science Foundation of China (NSFC 32000308), and Sciences and
Technology Department of Sichuan Province (2020YFSY0033).

\textbf{Permission to reproduce materials from other sources}

None.

\textbf{DATA AVAILABILITY STATEMENT}

All DNA sequences newly generated are deposited in Genbank (Accessions
No: OQ981548-OQ981602)

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\textbf{FIGURE LEGENDS}

Figure 1 Map showing localities of Chinese samples of
\emph{Sibynophis}used in this study.

Figure 2 Bayesian consensus tree of \emph{Sibynophis} inferred from
mitochondrial DNA using models detailed in text. Posterior probabilities
(\textgreater{}0.50) and bootstrap support values (\textgreater{}50\%)
are given adjacent to respective nodes for major nodes. Branch support
indices are not given for shallow nodes to preserve clarity.

Figure 3 The network based on c-mos gene (lines between the haplotypes
represent one mutational step).\selectlanguage{english}
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\verb`Table 2_genetic distance.docx`  available at \url{https://authorea.com/users/574992/articles/643917-molecular-phylogeny-reveals-cryptic-diversity-in-sibynophis-from-china-serpentes-sibynophiidae}

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