A new species of Lycodon Boie, 1826 (Serpentes, Colubridae) from northern Vietnam

Abstract A new species of the genus Lycodon is described from Cao Bang Province, Vietnam, based on three individuals with distinct differences in morphology and molecular data. The new species is differentiated from its congeners by a combination of the following characters: dorsal scales in 17-17-15 rows, smooth throughout; supralabials usually eight (rarely nine); infralabials ten; one elongated loreal on each side, in contact with the eye; precloacal plate single; ventral scales 212–218 (plus one or two preventral scales); subcaudals 90 or 91; maxillary teeth 13 or 14; dorsal surface of body with 28 or 29 light body bands; dorsal surface of tail with 13 cream bands, forming a distinct blotch in the vertebral region. Based on phylogenetic analyses of mitochondrial cytochrome b sequence data, the new species is recovered as the sister species to a clade containing L. multizonatus and L. liuchengchaoi with strong support from the Bayesian analysis. The new species is at least 7.5% divergent from other species within this clade in uncorrected pairwise distance calculated using a fragment of more than 1000 bp of the mitochondrial cytochrome b. This discovery increases the number of Lycodon species known from Vietnam to 16.


Introduction
The genus Lycodon Boie, 1827 is one of the most diverse genera of colubrid snakes, with 61 currently recognised species (Uetz et al. 2019, Luu et al. 2019. Recent phylogenetic studies showed that the genera Dinodon, Dryocalamus and Lepturophis nested within Lycodon and suggested to place them into the genus Lycodon sensu lato (Guo et al. 2013;Siler et al. 2013;Figueroa et al. 2016). The members of this genus have a broad distribution from eastern Iran to southern China and Japan, southward to the

Sampling
The field surveys were led by TQN in October 2011 and from April to May 2012. The collected specimens were euthanised with ethyl-acetate, fixed in approximately 85% ethanol for 10 hours, and subsequently transferred to 70% ethanol for permanent storage. Liver tissue samples were preserved separately in 95% ethanol. The specimens were deposited in the collections of the Institute of Ecology and Biological Resources (IEBR), Hanoi, Vietnam and of the Zoologisches Forschungsmuseum Alexander Koenig (ZFMK), Bonn, Germany.

Morphological analysis
Identification of sex was performed by dissection (inspection of gonads and presence of hemipenes). Maxillary teeth were counted by dissecting the right maxilla for teeth / sockets. Scalation and maxillary teeth number were examined with a binocular dissecting microscope. Measurements were taken following Ziegler et al. (2018) with a measuring tape to the nearest 1 mm.
Abbreviations of morphological characters are as follows: SVL Snout-vent length (from tip of snout to vent); TaL tail length; TaL / TL ratio of tail length / total length; TL total length; DSR dorsal scale rows number at one head length posterior to the head -number of dorsal scale rows at midbody -number of dorsal scale rows at one head length anterior to the vent; SL supralabials (counted on upper lips); SL / orbit number of supralabials entering orbit; IL infralabials (counted on lower lips); Lor loreals; Lor / eye loreal scale touching the eye (yes or no); PreOc preoculars; PostOc postoculars; Atem number of anterior temporals; PTem number of posterior temporals; BodySc scalation of the body (keeled or smooth); PreVen number of preventral scales; Ven number of ventral scales; SubC number of subcaudal scales; Prec precloacal (or cloacal) plate (single or divided); Teeth max number of maxillary teeth / alveoli. Scale counts were taken following Vogel et al. (2009). Ventral scales (Ven) were counted according to Dowling (1951). Bilateral scale counts were given as left / right.

Molecular data and phylogenetic analyses
Representative taxa of the genus Lycodon were included in the study. Sequences of the species were downloaded from GenBank. Two samples of the population from Cao Bang Province (ZFMK 93746,ZFMK 93747) were incorporated in the analysis. Boiga cynodon (Boie 1827) and Dipsadoboa flavida broadleyi (Broadley & Stevens, 1971) were used as outgroups based on Figueroa et al. (2016).
DNA was extracted using DNeasy Blood and Tissue kit (Qiagen, Germany) following the manufacturer's instructions. A fragment of the mitochondrial cytochrome b gene was amplified using the primer pair L14910 (5'-GACCTGTGATMTGAAAACCAY-CGTTGT-3') and H16064 (5'-CTTTGGTTTACAAGAACAATGCTTTA-3'; Burbrink et al. 2000). Extracted DNA was amplified using HotStarTaq Mastermix (Qiagen, Germany) with 21 µl volume consisting of 10 µl of Mastermix, 5 µl of water, 2 µl of each primer at 10 pmol/ml and 2 µl of DNA. PCR conditions were 95 °C for 15 minutes to activate the Taq, with 40 cycles of 95 °C for 30 s, 45 °C for 45 s, 72 °C for 60 s, and a final extension at 72 °C for six minutes. The mitochondrial cytochrome b gene was utilised in this study because it has been widely used in previous molecular analyses of Lycodon (e.g., Guo et al. 2013, Siler et al. 2013, and has been shown to be informative in revealing new species of Lycodon (e.g., Grismer et al. 2014, Luu et al. 2018. PCR products were visualised using gel electrophoresis through a 2% low melting-point agarose gel stained with ethidium bromide. Successful amplifications were purified to eliminate PCR components using GeneJET TM PCR Purification kit (Ther-moFisher Scientific, Lithuania). Purified PCR products were sent to FirstBase (Malaysia) for sequencing.
The obtained sequences were aligned in ClustalX 1.8.3 (Thompson et al. 1997) using the default settings. Data were analysed using maximum parsimony (MP) as implemented in PAUP*4.0b10 (Swofford 2001) and Bayesian inference (BI) as implemented in MrBayes v3.2 (Ronquist et al. 2012). Settings for these analyses followed Le et al. (2006), except that the number of generations in the Bayesian analysis was increased to 1×10 7 . For the maximum likelihood (ML) analysis, we used IQ-TREE v.1.6.7.1 ) with a single model and 10,000 ultrafast bootstrap replications. For ML and BI, the optimal model for nucleotide evolution was set to TrN+I+G by Modeltest v3.7 (Posada and Crandall 1998). For BI, the analysis was conducted with a random starting tree and run for 10 7 generations. Four Markov chains, one cold and three heated (utilising default heating values), were sampled every 1000 generations. Log-likelihood scores of sample points were plotted against generation time to detect stationarity of the Markov chains. The burn-in value was set to 26 in the BI analysis, as -lnL scores reached stationarity after 26,000 generations in both runs. Two independent analyses were run simultaneously. Nodal support was evaluated using Bootstrap replication (BP) as estimated in PAUP*4.0b10 and IQ-TREE v1.6.7.1 and posterior probability (PP) in MrBayes v3.2. BP ≥ 70 and PP ≥ 95% are regarded as strong support for a clade. Uncorrected pairwise distances (p) were calculated in PAUP*4.0b10.

Molecular data and phylogenetic analyses
The final matrix consisted of 1011 bp aligned characters and the alignment contained no gaps. In total, 404 characters were found to be parsimony informative. MP analysis resulted in five most parsimonious trees having 1662 steps (CI = 0.41, RI = 0.72). Our tree topologies are very similar to those recovered by Guo et al. (2015) and Luu et al. (2018). The new species was recovered to be the sister species to a clade containing L. multizonatus + L. liuchengchaoi, with strong support in BI (PP = 96), but weak support in MP and ML (BP MP = 56, BP ML = 69) (Fig. 1). The new species has an uncorrected p-distance of at least 7.5% and 8.1% from Lycodon liuchengchaoi Zhang, Jang, Vogel &Rao, 2011 andL. multizonatus Zhao &Jiang, 1981 Diagnosis. Lycodon pictus sp. nov. can be differentiated from its congeners by the following morphological characters: dorsal scales in 17-17-15 rows, all smooth; supralabials usually eight (rarely nine); infralabials ten; one elongated loreal on each side, in contact with the eye; precloacal plate single; ventral scales 212-218 (plus one or two preventral scales); subcaudals 90 or 91; a total length of 597+ mm in males and 543 mm in females; tail / total length ratio 0.211-0.215; maxillary teeth 13 or 14; dorsal surface of body with 28 or 29 light body bands; dorsal surface of tail with 13 cream bands forming a distinct blotch in the vertebral region; ventral surface of body and tail mostly cream with the dark body bands in part extending towards the venter, sometimes forming complete dark bands around the body.
Description of the holotype. Head elongate, moderately distinct from the neck, rather flattened, longer than wide, narrow anteriorly; nostril lateral, located in the middle of the nasal; eye large, pupils vertically elliptic; rostral triangular, much broader than high, hardly visible from above; nasal divided into two scales by a vertical ridge along posterior edge of nostril; two internasals, anteriorly rounded, slightly wider than high, bordered by two large, pentagonal prefrontals posteriorly; frontal single, enlarged, pentagonal to hexagonal, narrowed posteriorly; parietals longer than wide, in contact with each other medially, with upper anterior and posterior temporals, paraparietal laterally and four nuchal scales posteriorly; paraparietals elongated, anterior part widened; loreal 1/1, elongate, not entering orbit; supralabials 8/8, first and second in contact with nasal, third to fifth entering orbit, sixth largest; infralabials 10/10, first pair in broad contact with each other, first to fifth in contact with anterior pair of chin shields; anterior and posterior pairs of chin shields elongate, of the same size and shape, second pair not meeting in midline; preocular 1/1; postoculars 2/2, lowermost smaller, bordering anterior temporals; anterior temporals 2/2, posterior temporals 3/3, upper ones thinner than lower ones. Left maxilla arched, with an angular apex, distinctly bent inwards anteriorly. A total of 13 maxillary teeth or teeth alveola, with the following formula: five small anterior teeth, with the last two ones being somewhat enlarged + two strongly enlarged teeth, thick, and not much curved + a wide gap, somewhat wider than the length of the largest teeth + four small teeth + a small gap + two enlarged posterior teeth.
Coloration in preservative. Head, neck, and dorsal surface of body brownish black; light body bands beginning after 1.5 times the head length behind the head, in total 29 transverse light bands on body and at least nine light bands on tail; the first four body bands yellowish cream, and distinctly widened towards the venter, increased in size posteriorly; a dark mottling in the vertebrate region more prominent posteriorly; the subsequent light body bands with two distinct indentations on each side, fused in the middle in the last third of the body. In dorsal view the light bands forming a distinct blotch in the vertebral region, with a dark centre and a lighter frame; laterally, the middle part of the light bands forming blotches, but wider and with an extended dark centre, fused laterally in the last third part of the body; the lower and widest part of the light body bands with a dark small blotch in the centre in the anterior part of body; the light bands on the tail with a blotch like pattern in the vertebral region, but  less pronounced than that on body, and one light blotch at the lateral side of tail, widened towards the venter, with a dark centre; ventral surface of head and neck yellowish cream, belly cream and greyish cream in the last third part of body and on lower tail surface; the dark dorsal bands (28 on body and at least nine on tail) in part extending towards the venter (most prominent in the anterior five dark body bands), not forming complete dark bands around the body, but complete on the tail; lateral side of the head dark above and light below, with the lighter pattern beginning in the supralabial region; tip of lower jaw and infralabial region in part greyish; dorsal surface of the head and upper head sides a bit paler than the remaining head dorsum.
Hemipenis. Hemipenes elongated, not fully everted, not turgid. Truncus without spines. Spine ornamentation starting at truncus region with somewhat enlarged, medium sized spines. Apex with microspines. Sulcus stretches in the middle to apex. Apex not fully everted, ending somewhat widened with an oblique opening, with microspines inside, pointing to the not fully everted condition of the outer genital organ.
Variations. In the juvenile ZFMK 93747, the number of supralabials on the left side is nine, with fourth to sixth entering the orbit. The loreal does not touch the eye on the right side. The lower anterior temporal scale is not touching the postocular scale on the left side. In general, the coloration is more intense in the juvenile. The creamy pattern on the posterior third of the body sides is connected by a horizontal creamcolored stripe. It has a yellowish cream band on the head that reaches from SL 5 behind the jaws and distinctly lightens the posterior half of the head but does not touch the frontal. In the juvenile, the banded pattern is more simple, consisting of dark bands which narrow towards the venter and light bands which widen towards the venter and bear a dark pattern and a more or less distinct dark blotch at the lower side (see Fig. 5).
In the female ZFMK 93746, the lower anterior temporal scale is not touching the postocular scale on the right side. For measurements and scalation data of the examined specimens see Table 1.
Dentition. Female ZFMK 93746 and juvenile ZFMK 93747: Left maxilla arched, with an angular apex, distinctly bent inwards anteriorly. A total of 13 (in female) or 14 (in juvenile) maxillary teeth or teeth alveola, with the following formula: five small anterior teeth, with the last two ones being somewhat enlarged + two strongly enlarged teeth, thick, and not much curved + a wide gap, somewhat wider than the length of the largest teeth + four small teeth + a small gap + two enlarged posterior teeth in the female and three posterior teeth in the juvenile, with the anterior two ones enlarged.
Comparisons. In our phylogenetic analysis, Lycodon pictus sp. nov. is most closely related to L. liuchengchaoi and L. multizonatus. From L. liuchengchaoi, the new species differs in terms of body scalation (all smooth in the new species vs. feebly keeled in several median rows in L. liuchengchaoi), head scalation (ten infralabials vs. 7-9) and dentition (13 or 14 maxillary teeth vs. 8 or 9). In addition, the new species differs from the latter in having 28 or 29 cream body bands (vs. 40 yellow rings on the body in L. liuchengchaoi) (Zhang et al. 2015).  The new species differs from L. multizonatus by having more maxillary teeth (13 or 14 vs. 10 or 11 in L. multizonatus), more infralabials (10 vs. 8) and a single precloacal plate (vs. divided). In addition, the new species differs from the latter in terms of body scalation (minimum 212 ventrals and minimum 90 subcaudals vs. 190-195 ventrals and 68-75 subcaudals in L. multizonatus). Furthermore, L. pictus sp. nov. has fewer light body bands (28 or 29 vs. 55-73 in L. multizonatus) (Lei et al. 2014).
From its Vietnamese congeners, the new species can be differentiated as follows: Lycodon pictus sp. nov. differs from L. capucinus in having a single precloacal plate (vs. divided), a loreal touching the eye (vs. not in contact with the eye), in having more ventrals (minimum 212 vs. 182-211) and more subcaudals (90 or 91 vs. 59-74), and in terms of dorsal pattern (banded vs. reticulated) (Luu et al. 2019).
Lycodon pictus sp. nov. differs from L. fasciatus in having smooth dorsal scales (vs. keeled) and more maxillary teeth (13 or 14 vs. 11). Additionally, the colour pattern of Lycodon pictus sp. nov. differs in being dark brownish black with light body bands turning into a marbling posteriorly, whereas L. fasciatus is black or purplish black above with yellowish cross-bars of irregular outline and has a dark median stippling , Smith 1943. Werner (1922) described Dinodon yunnanensis from Yunnan Fu, now Kunming, Yunnan Province, southwestern China. This species was synonymized with Lycodon fasciatus by Pope (1935: 188), but according to Vogel and David (2010), this taxon might be a distinct species (see also Vogel and David 2019). Lycodon pictus sp. nov. differs from Dinodon yunnanensis Werner, 1922 in having more ventrals (minimum 212 vs. 193), more subcaudals (90 or 91 vs. 66), more infralabials (10 vs. 9) and more light body bands (28 or 29 vs. 23) (Werner 1922, Vogel and David 2010, Vogel and David 2019. Lycodon pictus sp. nov. differs from L. flavozonatus in terms of dorsal scalation (smooth vs. keeled), in having more subcaudals (90 or 91 vs. 80-88), the loreal in contact with the eye in Lycodon pictus sp. nov. (vs. separated in L. flavozonatus) and in coloration pattern (brownish black with 28 or 29 cream body bands and 9-13 light bands on the tail vs. black with 68 yellow body bands and 21 on the tail) , Vogt in Pope 1928.
Lycodon pictus sp. nov. differs from L. namdongensis in having more subcaudals (90 or 91 vs. 85) and the loreal in contact with the eye (vs. separated from the eye in L. namdongensis). The new species also differs in coloration pattern (brownish black with 28 or 29 light bands on the body vs. grey with 23 cream cross rings on the body in L. namdongensis), and in having irregular bands turning into a marbling posteriorly (vs. clearly demarcated cross bands on the body) (Luu et al. 2019).
Lycodon pictus sp. nov. differs from L. paucifasciatus in terms of dorsal scalation -15 DSR, the upper one or two plus vertebral row distinctly keeled) and fewer ventral scales (maximum 218 vs. 219-222). In addition, the new species has a loreal entering the eye (vs. separated) and 28 or 29 light body bands (vs. 14-25 beige or dirty cream body bands) (Vogel et al. 2009).
Lycodon pictus sp. nov. differs from L. rufozonatus in having a loreal in contact with the eye (vs. separated), smooth dorsal scales (vs. feebly keeled in the posterior body part), and in coloration pattern (28 or 29 cream body bands vs. 44-52 light red body bands) (Zhao 2006, Luu et al 2018. Lycodon pictus sp. nov. differs from L. ruhstrati abditus in having smooth dorsals (vs. 7-8 dorsal scale (including vertebral) rows keeled), an elongated loreal in contact with the eye (vs. separated), and in having irregular bands turning into a marbling posteriorly (vs. clearly demarcated cross bands on the body) (Vogel et al. 2009).
Lycodon pictus sp. nov. differs from L. septentrionalis by its smooth dorsal scales (vs. 7-9 median rows feebly keeled), 10 infralabials (vs. 7 or 8), and the loreal entering the orbit (vs. separated in L. septentrionalis). In addition, the new species differs in having cream irregular bands on a brown body (vs. white narrow bands on a black body forming complete annuli) (Günther 1875, Boulenger 1893, Neang et al. 2014.
Lycodon pictus sp. nov. differs from L. subcinctus in having 10 infralabials (vs. 8 or 9), one preocular (vs. preocular absent), smooth dorsal scales (vs. feebly keeled) and 28 or 29 cream bands on the body and 9-13 on the tail (vs. 9-15 bands on the body and none on the tail) (Boulenger 1893, Neang et al. 2014. Lycodon pictus sp. nov. differs from L. ophiophagus, a species from southern Thailand but with similar scalation, in having a loreal entering the eye (vs. separated) and in dorsal colour pattern (28 or 29 light bands on a brown body vs. 20 or 21 white bands on a dark body) as well as and in having irregular bands turning into a marbling posteriorly (vs. clearly demarcated cross bands on the body) (Vogel et al 2009).
For additional measurements, dentition, and scalation data see Tables 2-8. Distribution. Lycodon pictus sp. nov. is currently known only from Ha Lang and Trung Khanh districts, Cao Bang Province, northern Vietnam (Fig. 7).
Etymology. The name of the species pictus means painted or decorated in Latin and refers to its unique dorsal colour pattern.