﻿Rediscovery of Lycodongammiei (Blanford, 1878) (Serpentes, Colubridae) in Xizang, China, with comments on its systematic position

﻿Abstract Lycodongammiei (Blanford, 1878), a rarely encountered species of Asian snake, is characterized by ambiguous systematics and biology. Based on a sole specimen of L.gammiei rediscovered in southeastern Xizang, China, we conduct a detailed morphological examination and description, and investigate the systematic position of this species. Morphologically, the newly collected specimen is closely aligned with specimens previously described. Mitochondrial DNA-based phylogenetic analyses reveal that L.gammiei constitutes an independent evolutionary lineage, forming a clade with L.fasciatus (Anderson, 1879), L.gongshan Vogel & Luo, 2011, L.butleri Boulenger, 1900, and L.cavernicolus Grismer, Quah, Anuar, Muin, Wood & Nor, 2014. The closest genetic distance between L.gammiei and its congeners was 10.2%. The discovery of L.gammiei in Medog County, China, signifies an eastward expansion of its known geographical distribution.


Introduction
Exploring the boundaries of geographic distribution and systematic position of species is crucial for understanding their evolutionary origins and diversification and for devising appropriate conservation strategies.Despite considerable progress in recent years, many species remain poorly known and explored.This is particularly evident for some snake species due to their rarity and cryptic habitats.
Lycodon gammiei (Blanford, 1878), a rare non-venomous snake species within the family Colubridae, was initially described as Ophites gammiei based on a single specimen collected from Darjeeling, West Bengal, India (Blanford 1878).Subsequently, it was reclassified into the genus Lycodon (Boulenger 1890) or Dinodon (Wall 1923;Smith 1943), identifying it as Lycodon gammiei.Wall (1911) compared L. gammiei and L. fasciatus (Anderson, 1879), and he synonymized L. fasciatus with L. gammiei.However, Wall (1923) later revised this view, recognizing its distinctiveness and validity of L. fasciatus.Mahendra (1984) proposed that L. gammiei was a color variety of L. septentrionalis (Gunther, 1875), while this synonymy was not accepted by all authors.Since its initial description, L. gammiei has been found in southeastern Xizang, China (Agarwal et al. 2010) and in Bhutan (Wangyal 2013).To date, however, few specimens of the species have been collected, and no genetic data have been reported.
In 2023, we collected a living specimen of L. gammiei in Medog County, southeastern Xizang, China.The rediscovery of this species in Xizang not only extends this species' geographic distribution but also allows the exploration of its systematic position through molecular data.

Morphological examination
The specimen deposited at Yibin University (YBU 230088) was collected in Beibeng Town, Medog County, southeastern Xizang, China (29°14′02″N, 95°10′38″E) (Fig. 1) on 14 August 2023 at an elevation of 1,431 m by Xiaoqi Mi.The snake was found on a tree near a road at 23:30 hours.Characters relating to scalation, color pattern, and body proportions were recorded from the preserved specimen in laboratory.Snout-vent length (SVL) and tail length (TL) were measured using a meter ruler to the nearest 0.5 centimeter, while all remaining measurements were taken using digital calipers to the nearest millimeter.Symmetric mensural head characters were taken on the right side unless unavailable (e.g.damaged), while meristic characters were recorded on both sides and reported in left/right order.
Comparative data of other specimens of this species were taken from the literature (Blanford 1878;Mistry et al. 2007;Chettri and Bhupathy 2009;Wangyal 2013).

Molecular phylogeny
Genomic DNA was extracted from the liver tissue of the newly collected specimen using an Animal Genomic DNA Purification Kit (TIANGEN Bio-tech Co., Ltd, Beijing, China).Subsequently, a fragment of the mitochondrial gene cytochrome b (cyt b) was amplified using primers H14919 (5′-AACCACCGTTGT-TATTCAACT-3′) and L16064 (5′-CTTTGGTTTACAAGAACAATGCTTTA-3′) (Burbrink et al. 2000).The polymerase chain reaction (PCR) products were purified and sequenced in both directions by Sangon Biotech Co., Ltd (Chengdu, China).The obtained sequences were manually edited using SeqMan in Lasergene v. 7.1 (DNASTAR, USA), and aligned using the ClustalW algorithm with default parameters in MEGA v. 7.0 (Kumar et al. 2016), followed by a visual inspection for minor manual adjustments.The DNA sequences were translated into amino acid sequences using MEGA v. 7.0 to verify sequence quality and detect any unexpected stop codons (Kumar et al. 2016).Furthermore, 80 additional sequences were downloaded from GenBank (Table 1).
Both Bayesian-inference (BI) and maximum-likelihood (ML) analyses were executed for the final dataset.Prior to analyses, the best-fit model of nucleotide substitution was selected for each partition (codon position) using Akaike In- formation Criterion (AIC) implemented in PartitionFinder (Lanfear et al. 2012).
The BI analyses were conducted using MrBayes v. 3.2.2(Ronquist et al. 2012).Searches consisted of three independent runs, each involving four Markov chains (three heated chains and one cold chain), with 10 million generations, sampling every 2,000 generations and with 25% of initial samples discarded as burn-in.
Convergence was determined via effective sample size (ESS > 200) and likelihood plots against time using Tracer v. 1.7 (Rambaut et al. 2018).The resulting trees were combined to determine the posterior probabilities (PP) for each node based on a 50% majority-rule consensus tree.The ML trees were constructed in IQ-tree (Lam-Tung et al. 2015) using the GTRCAT model and the same partitioning scheme.In total, 1,000 Ultrafast bootstraps (UFB) topological replicates were performed for branch support assessment.Boiga cynodon (Boie, 1827) was selected as the outgroup following previous research (Guo et al. 2013).
Head black, with yellow spots or short lines on some shields.Large, yellow spots on each side of posterior part of head.Conspicuous yellow collar on neck.Supralabials and anterior infralabials light yellow with dusky margins.Body surrounded by alternating dusky and light-yellow rings with very irregular, crooked margins.Yellow rings on body totaling 43, first pale ring clear above, anterior dark patch not continuous across throat, remaining rings encircling body.Lower part of head and neck light yellow.On belly, across anterior part of body, dark rings only about half as broad as light-yellow rings, less difference above, dark rings near head much broader above than white rings.Yellow rings on tail totaling 21 (Fig. 2).Preserved specimen somewhat faded, with no yellow visible (Fig. 3).

Molecular phylogeny
In total, 1,047 bp of sequence data from 84 samples were aligned, with the generated novel sequence deposited in GenBank (Table 1).No deletions, insertions, or stop codons were detected, indicating that unintentional amplification of pseudogenes was unlikely (Zhang and Hewitt 1996).The best-fit evolutionary models of the data were: GTR+I+G for the first codon position, HKY+I+G for the second codon position, and GTR+G for the third codon position.
The mtDNA-based BI and ML analyses depicted relatively consistent topologies, with slight disagreement in several shallow nodes (Fig. 4).Both analyses indicated that all putative species of Lycodon formed a highly supported lineage (100 PP and 84% UFB).The newly collected specimen formed a clade with L. fasciatus, L. gongshan Vogel & Luo, 2011, L. butleri Boulenger, 1900, and L. cavernicolus Grismer, Quah, Anuar, Muin, Wood & Nor, 2014 with high support (100 PP and 97% UFB).Nevertheless, it occupied a basal position in relation to this clade and did not exhibit monophyly with any individual member.Uncorrected p-distances among the species within this clade ranged from 7.2% (L.gongshan and L. fasciatus) to 12.9% (L.gammiei and L. cavernicolus), while genetic distances between L. gammiei and its congeners within this clade ranged from 10.2% to 12.9% (data not shown).

Discussion
Lycodon gammiei is an exceedingly rare species, with a global record of only approximately 10 specimens.The majority of these are from Sikkim and West Bengal, India (Mistry et al. 2007;Chettri and Bhupathy 2009), with only two specimens reported in Cona County, Xizang, China (originally recorded in Eaglenest Wildlife Sanctuary, India) (Mistry et al. 2007) and Bhutan (Wangyal 2013), respectively.Based on the record by Mistry et al. (2007), Luo et al. (2010) recognized the existence of this species in China, although this recognition has been overlooked in subsequent publications (Wallach et al. 2014;Wang et al. 2020;Uetz et al. 2024).The discovery of this species in Medog County, Xizang, China, not only confirms its presence in China but also indicates a further eastward extension of its distribution.
The taxonomic status of L. gammiei has a controversial history.Although previously misidentified as both L. fasciatus (Wall 1911) and L. septentrionalis (Mahendra 1984), Mistry et al. (2007) later clarified its distinct status and validity based on morphological comparisons.In the current study, we present the first genetic data pertaining to this species.Notably, mtDNA-based phylogenetic analyses indicated that L. gammiei formed a highly supported monophyly with a clade containing L. fasciatus but was not the closest congener to L. fasciatus within this assemblage (Fig. 4).Lycodon gammiei shows a greater genetic distance from L. septentrionalis than from L. fasciatus, further affirming its validity and unique taxonomic position.The closer genetic affinity of L. gammiei with the clade encompassing L. fasciatus aligns with their geographical closeness along the southern slopes of the Himalayas.

Figure 2 .
Figure 2. General view of the studied specimen (YBU 230088) in life and its microhabitat a big tree trunk (by XQ Mi).

Figure 3 .
Figure 3. Views of the studied specimen (YBU 230088) in preservation.General dorsal (A) and ventral (B) views of specimen, dorsal (C), ventral (D) and lateral (E) views of head (by P Guo).

Figure 4 .
Figure4.Bayesian 50% majority-rule consensus tree of Lycodon inferred from cyt b sequences analyzed using models detailed in the text.Posterior probabilities from BI analysis (>0.50) and Ultrafast bootstraps from ML analysis (>50%) are given adjacent to respective nodes for major nodes.Branch support indices are not given for most nodes to preserve clarity.

Table 1 .
Detail information for the samples used in this study.