A new species of the genus Lycodon (Serpentes, Colubridae) from Guangxi, China

Abstract A new species of colubrid snake, Lycodon cathayasp. nov., is described based on two adult male specimens collected from Huaping Nature Reserve, Guangxi, southern China. In a phylogenetic analyses, the new species is shown to be a sister taxon to the clade composed of L. futsingensis and L. namdongensis with low statistical support, and can be distinguished from all known congeners by the significant genetic divergence in the mitochondrial cytochrome b gene fragment (p-distance ≥ 7.9%), and morphologically by the following combination of characters: (1) dorsal scales in 17–17–15 rows, smooth throughout; (2) supralabials eight, third to fifth in contact with eye, infralabials nine; (3) ventral scales 199–200 (plus two preventral scales), subcaudals 78; (4) loreal single, elongated, in contact with eye or not, not in contact with internasals; (5) a single preocular not in contact with frontal, supraocular in contact with prefrontal, two postoculars; (6) maxillary teeth 10 (4+2+2+2); (7) two anterior temporals, three posterior temporals; (8) precloacal plate entire; (9) ground color from head to tail brownish black, with 31–35 dusty rose bands on body trunk, 13–16 on tail; (10) bands in 1–2 vertebral scales broad in minimum width; (11) bands separate ground color into brownish black ellipse patches arranged in a row along the top of body and tail; (12) elliptical patches in 3–6 scales of the vertebral row in maximum width; (13) ventral surface of body with wide brownish black strip, margined with a pair of continuous narrow greyish white ventrolateral lines. With the description of the new species, 64 congeners are currently known in the genus Lycodon, with 16 species occurring in China.


Morphometrics
Morphological examinations were performed based on two specimens collected from Huaping Nature Reserve, Guangxi, China. All specimens were fixed in 10 % buffered formalin and later transferred to 70 % ethanol for permanent preservation, and deposited in the Museum of Biology, Sun Yat-sen University (SYS).
Morphological descriptions followed Dowling (1951), , Vogel and David (2010), and Janssen et al. (2019). Measurements were taken with digital calipers to the nearest 0.1 mm. These measurements were as follows:

ED
eye horizontal diameter; HL head length (from tip of snout to posterior margin of the mandible); HW maximum head width; SVL snout-vent length (from tip of snout to posterior margin of cloacal plate); TaL tail length (from posterior margin of cloacal plate to tip of tail); TL total length (from tip of snout to tip of tail).
Scalation features and their abbreviations are as follows: dorsal scale rows (DSR) counted at one head length behind head, at midbody, and at one head length before vent, respectively; supralabials (SPL); numbers of supralabials in contact with the eye (SPL-E); infralabials (IFL); chin shields (CS); numbers of infralabials in contact with the anterior chin shield (IFL-aCS); number of infralabials in contact with the posterior chin shield (IFL-pCS); preoculars (PrO); postoculars (PtO); loreal (LoR); loreal in contact with the eye or not (L-E); anterior temporals (aTMP); posterior temporals (pTMP); preventral scales (PrV); ventral scales (V); precloacal plate (PrC); subcaudals (SC); and body scale surface (BSC). Sex was determined by dissection or by the presence/absence of everted hemipenis. The number of maxillary teeth (MT) were counted by carefully dissecting the gums of the right maxilla under the stereo microscope. The light bands on the body and tail were counted on one side; hardly visible or incomplete bands were counted as one band; obviously fused bands were counted as two bands. The collar band on the neck was not included in counts and bands covering the cloacal plate were regarded as body bands.

Phylogenetic analyses
For molecular analysis, a total of 20 samples was used, encompassing 18 samples from eight known Lycodon species (one sample of L. fasciatus, two samples of L. flavozonatus, four samples of L. futsingensis, two samples of L. liuchengchaoi, one sample of L. multizonatus, two samples of L. rufozonatus, four samples of L. ruhstrati, and two samples of L. subcinctus) and two samples of the unnamed species. Tissue samples were taken prior to fixation, and preserved in 99 % alcohol and stored at -40 °C.
Genomic DNA was extracted from muscle or liver tissue samples, using a DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. A fragment of the mitochondrial cytochrome b (CYTB) gene was amplified using the primer pair L14910 (5'-GACCT-GTGATMTGAAAACCAYCGTTGT-3') and H16064 (5'-CTTTGGTTTACAA-GAACAATGCTTTA-3') following Burbrink et al. (2000). PCR amplification was run using the following cycling conditions: initial denaturing step at 94 °C for 5 min; followed by 35 cycles of 94 °C for 30 s, 48 °C for 1 min and 72 °C for 70 s; and final extension step at 72 °C for 10 min. PCR products were purified with spin columns and then sequenced with forward primers using BigDye Terminator Cycle Sequencing Kit as per the guidelines on an ABI Prism 3730 automated DNA sequencer by Guangzhou Tianyi Huiyuan Bio-tech Co., Ltd.
Twenty sequences from 12 known Lycodon species and two out-group sequences Boiga cynodon (Boie, 1872) and Dasypeltis atra Sternfeld, 1912, following Janssen et al. (2019 were obtained from GenBank and incorporated into our dataset (Table 2). DNA sequences were aligned by the Clustal W algorithm with default parameters (Thompson et al. 1997) and trimmed with gaps partially deleted in MEGA 6 (Tamura et al. 2013). The aligned dataset was tested in jmodeltest v2.1.2 (Darriba et al. 2012) with Akaike and Bayesian information criteria, all resulting the best-fitting nucleotide substitution models of GTR+I+G. Sequence data was analyzed using Bayesian inference (BI) in MrBayes 3.2.4 (Ronquist et al. 2012), and maximum likelihood (ML) in RaxmlGUI 1.3 (Silvestro and Michalak 2012). In the BI analysis, three independent runs were conducted, each being run for 2 million generations and sampled every 1000 generations with the first 25% samples were discarded as burn-in. In the ML analysis, the bootstrap consensus tree was inferred from 1000 replicates. Pairwise distances (pdistance) were calculated in MEGA6 using the uncorrected p-distance model. ?

results
The CYTB nucleotide sequence matrix contained 1050 characters without insertion deletions. The MP and BI analyses produced essentially identical topologies, which were integrated in Fig. 2. Major nodes of the tree were supported with the Bayesian posterior probabilities (BPP) > 0.95 and the bootstrap supports (BS) for Maximum Likelihood analysis > 75. Uncorrected p-distances among Lycodon species based on the CYTB gene are shown in Table 3. The phylogenetic topologies are very similar to those recovered by previous study (Janssen et al. 2019). The unnamed Lycodon samples from Guangxi, southern China clustered in a monophyletic lineage with high nodal supports (BPP = 1.00 and = BS 100). This lineage are genetically differentiated from all congeners with the uncorrected p-distance ≥ 7.9%, which is significant when compared with that between other recognized species (e.g., p-distance = 2.7 % between L. flavozonatus and L. meridionalis, p-distance 6.8 % between L. futsingensis and L. namdongensis, and pdistance = 6.7-7.1 % between L. liuchengchaoi and L. multizonatus). The phylogenetic placement of the new lineage is largely unresolved, even though it forms the sister taxon to the clade composed of L. futsingensis and L. namdongensis while the nodal support is insignificant. Moreover, it is noteworthy that the unnamed Lycodon possesses significant morphological differences that can be easily distinguished from all other congeners (see below). Therefore, based on the combination of molecular and morphological data, we describe the unnamed population from Huaping Nature Reserve, Guangxi, southern China as a new species, Lycodon cathaya sp. nov.

Taxonomic account
Lycodon cathaya sp. nov. http://zoobank.org/BA36B7DE-36BD-4B3C-A317-BF4B8E451A26 Figures 3A, 4, 5A Etymology. The specific name cathaya is a noun referring to the monotypic botanic genus Cathaya Chun & Kuang, 1958. The single species C. argyrophylla Chun & Kuang, 1958 is an endangered relict plant, and was firstly discovered from Huaping Nature Reserve by the investigation team of Sun Yat-sen University. In memory of the predecessors and their contributions on the taxonomy of Chinese flora and fauna, we denominate this new snake species from Huaping Nature Reserve as Lycodon cathaya sp. nov. Its common name is suggested as "Huaping wolf snake" in English and "Hua Ping Bai Huan She (花坪白环蛇)" in Chinese.
Comparisons. The detailed comparisons among all Lycodon congeners are given in Table 1, with distinguishing characters marked in bold.
Description of holotype. Adult male. Body slender, TL 562.5 mm (SVL 451.4 mm,TaL 111.1 mm,TaL/TL ratio 0.198); dorsal scales in 17-17-15 rows, smooth throughout, the vertebral scales not enlarged; head elongate, moderately distinct from neck, rather flattened, longer than wide, and narrow anteriorly, HL 17.2 mm, HW 11.1 mm (HW/HL ratio 0.643); eye large, ED 2.2 mm, pupil vertically elliptic; rostral triangular, much broader than high, barely visible from above; nostril lateral, located in the middle of nasal; nasal divided into two scales by nostril; two internasals, anteriorly rounded, almost as wide as high, bordered by two large, pentagonal prefrontals posteriorly; a single enlarged hexagonal frontal, narrowed posteriorly; parietals paired, longer than wide, in contact with each other medially, with upper anterior and posterior temporals, paraparietal laterally and four nuchal scales posteriorly; paraparietal slightly elongate, nearly rectangular; one elongated loreal on each side, in contact with eye, not in contact with internasals; one preocular located above loreal, in contact with eye and supraocular posteriorly, with prefrontal anteriorly, and not in contact with frontal; two postoculars, almost equal in length, upper one in contact with eye anteriorly, with supraocular and parietal, and with upper temporal posteriorly, lower one in contact with eye anteriorly, with anterior temporals posteriorly, and with fifth and sixth supralabials below; eight supralabials on each side, first and second in contact with nasal, third to fifth entering orbit; nine infralabials on each side, first pair in broad contact with each other, first to fourth in contact with anterior pair of chin shields, fourth to fifth in contact with posterior chin shields; two pairs of chin shields, elongate, anterior pair larger, second pair meeting in midline; two anterior temporals, almost equal in size, three posterior temporals, upper one smallest, lower one largest; 199 ventrals plus two preventrals; 78 pairs of subcaudals, excluding tail tip; precloacal plate entire.
Hemipenis. Hemipenis elongated, apex not fully everted after injection of formalin. Truncus bulbous, lower 1/3 smooth without spines, spine ornamentation starting at upper part with somewhat enlarged, medium sized spines. Apex with dense microspines. Sulcus spermaticus stretches to base of apex. Apex not fully everted, ending somewhat widened with an oblique opening, with microspines inside.
Coloration of holotype. In life (Figs 3A, 4), dorsal surface of head brownish black, a distinctly dusty rose collar band that crosses over the head and nape of the neck; ventral surface of head almost white, mental, the 1 st -3 rd supralabials and the anterior pair of chin shields with brownish black patches, the 4 th and 5 th and the posterior pair of chin shields with brownish black mottles. Ground color of dorsal surface brownish black, with 35 transverse dusty rose bands on body trunk and 16 similarly colored bands on tail, including two incomplete bands between collar band and the first complete transverse band; each band in 1-2 scales of the vertebral row in minimum width and widen laterally to a width of 3-4 scales; bands link with each other in ventrolateral body and tail, and separate the ground color into brownish black ellipse patches: such patches in 3-6 scales of the vertebral row in maximum width, and arranged in a row along the top of body and tail; a brownish black ventrolateral blotch on each ventrolateral side of bands. Middle of each ventral with irregular brownish black blotches forming a relatively continuous venter strip, and greyish white on both sides, forming a pair of continuous ventrolateral lines, which run in parallel along the venter strip. Subcaudals almost entirely light brown. In preservative (Fig. 5A), the collar band faded to beige, bands become darker, and the ventral surface faded to beige.
Variations. Measurements, body proportions and scale counts of the two specimens are listed in Table 4. The paratype has a relatively small and faint collar band, just crossing over the nape of the neck; dorsal bands are faint and there are more dark brown speckles than in the holotype. It appears that this specimen represents an older age group than the holotype, and differences in coloration may indicate an ontogenetic development. The loreal is in contact with eye in the holotype, while the loreal is separated from the eye by the preocular and the third supralabial.
Distribution and habits. Currently, Lycodon cathaya sp. nov. is only known from its type locality, Huaping Nature Reserve ( Fig. 1; ca 850-1000 m a.s.l.), and is sympatric with L. meridionalis and L. ruhstrati. All of them are nocturnal species. The holotype was observed climbing on a wilted bush by the roadside, approximately half a meter above the ground (Fig. 4). The paratype and an individual of its sympatric species L. ruhstratiis  3C) were found on the ground on the same night. The surrounding environment consisted of well-preserved montane evergreen broad-leaved forest or mixed forest.

Discussion
The description of Lycodon cathaya brings the total species number of this genus to 64, 16 of which occur in China. The new discovery further emphasizes the very high diversity level of the genus Lycodon (Zhao 1981;Zhao et al. 1998;Zhao 2006;Luo et al. 2010). The Huaping Nature Reserve is located in the hilly region among Guangxi, Hunan, and Guizhou. Thus, the new species is expected to occur in southwestern Hunan and southeastern Guizhou. The area within the jurisdiction of Huaping Nature Reserve has been well valued and protected by relevant local departments, with a considerable amount of research and investigation efforts having been conducted. However, further research on the true distribution, population sizes and trends, habitat conditions and conservation actions are urgently needed in the potential distribution areas outside the jurisdiction of Huaping Nature Reserve. Moreover, since the rapid and notable developments on the knowledge about the Chinese herpetofauna, the hilly regions in southern China have received more attention and a number of new species have been discovered in the recent years (Chen et al. 2018;Li et al. 2018;Lyu et al. 2018;Peng et al. 2018;Sung et al. 2018;Wang et al. 2018ab;Chen et al. 2019;Lyu et al. 2019ab;Wang et al. 2019abc;Wang et al. 2020ab); this in turn strengthens appeals for more powerful and targeted conservation actions in these regions.