Sampling. The specimen was collected in the field using visual surveys. Liver and muscle tissues were extracted and immediately preserved in 95% ethanol. The specimen was fixed in 10% formaldehyde for one day, then transferred to 75% ethanol for permanent preservation and deposited in the Tibet Plateau Institute of Biology (collection number: HSR23050, voucher number TBR2023045). The animal study protocol was approved by the Laboratory Animal Care and Animal Ethics Committee of Tibet Plateau Institute of Biology. Furthermore, with the explicit permission and approval of the Tibet Autonomous Region Forestry and Grassland Administration, all sampling activities were conducted. We not only obtained the necessary permits, but also adhered to strict ethical guidelines, ensuring full compliance with local regulations and minimising any potential impact on the natural habitat.

Morphological examination. The number of ventral scales was counted according to Dowling 1951a. Dorsal scale row reduction formulae were based on Dowling 1951b. Morphometrics measurements and other scale counts were carried out following Zhao 2006 and Huang 2021. Body and tail length measurements were taken with a ruler to the nearest 1 mm. All other measurements were made using digital calipers to the nearest 0.1 mm.

Abbreviations for measurements and scale characters are following Darko et al. 2022. The measurements and their abbreviations are as follows: snout-vent length (SVL): measured from the tip of the snout to the anterior edge of the vent; tail length (TAL): measured from the anterior edge of the vent to the tip of the tail; total length (TL) defined as the sum of SVL and TAL; head length (HL): taken from the tip of snout to the posterior margin of mandible; head width (HW): measured from the widest part of the head in dorsal view; eye diameter (ED): taken from the most anterior corner of the eye to the most posterior corner. The scale characters and their abbreviations are: maxillary teeth (MT), supralabials (SL), infralabials (IL), loreals (LOR), preoculars (PRO), postoculars (PO), Chin, infralabials touching the first pair of chin shields (IL-1st Chin), temporals (TEMP), supraoculars (SPO), three dorsal scale row (DSR) counts: 1) counting from one head length behind the head, 2) at mid-body and 3) at one head length before the vent; ventral scales (VS), cloacal plate (CP) and subcaudal (SC). The specimen was compared with other species of the genus Boiga, based on descriptions in the available literature (Zhao 2006, Köhler et al. 2023).

Molecular phylogeny. Genomic DNA was extracted from liver tissue using a Qiagen DNEasy blood and tissue extraction kit (Qiagen Inc., Valencia, CA). The partial mitochondrial DNA gene encoding cytochrome b (cyt b) was obtained by polymerase chain reaction (PCR) using primer pairs L14910 (5’-GAC CTG TGA TMT GAA AAC CAY CGT TGT-3’) and H16064 (5’-CTT TGG TTT ACA AGA ACA ATG CTT TA-3’) (Burbrink et al. 2000). PCR products were sequenced by Shanghai Map Biotech Co. Ltd. and raw sequences were assembled using seqman in the DNAStar software package (Burland 2000). The newly-generated sequence was deposited in GenBank (Accession number: PP431561).

All sequences were aligned and compared with each other separately on the same gene loci by MEGA X software (Tamura et al. 2021). ModelFinder was used to select the best-fit model using BIC criterion (Kalyaanamoorthy et al. 2017). The best-fit model according to BIC is : GTR +FO +G4m. ML phylogenetic analyses were inferred, based on these alignments by RAxML-NG 1.0.0 with the GTR +FO +G4m model. Each inference was initiated with a random starting tree and a majority rule consensus tree was calculated with 1000 bootstrap replicates.

All the cyt b sequences in this study were retrieved from GenBank and uncorrected p-distances data are shown in Suppl. material 1.

In order to find the molecular systematic position of the snake in this study, we retrieved cyt b sequences from GenBank for 27 taxa of Boiga and used Telescopus beetzi as the outgroup. The alignment for cyt b was 1015 bp in length.

Maximum Likelihood (ML) methods were used to reconstruct phylogenetic trees (Fig. 5). The phylogenetic analysis suggested that this specimen was nested in the genus Boiga and formed a monophyletic clade with B. stoliczkae obtained from Tibet, China.

Figure 5.  

Maximum Likelihood (ML) tree inferred from cyt b. Numbers indicate bootstrap support estimated using Maximum Likelihood (1000 pseudoreplicates) analyses. Maximum Likelihood bootstrap supports (BS) are shown at each internal branch. Posterior probability values < 0.70 are not shown.The new record species in the present study are shown in bold.

Genetic distance on cyt b between the specimen of the B. stoliczkae collected from Tibet, China and from GenBank sequence was 1.7%, which is substantially lower than interspecific genetic distances of others (11.5-19.5%) (Suppl. material 1).

In the genus Boiga, intraspecific colouration and pattern polymorphism are known in B. forsteni (Duméril, Bibron & Duméril, 1854), B. multomaculata, B. irregularis (Bechstein, 1802), B. drapiezii (Boie, 1827) and B. dightoni (Boulenger, 1894) (Mohapatra et al. 2009, Weinell et al. 2021, Köhler et al. 2023, Narayanan et al. 2023). Previously, only a unicoloured morph was known in B. stoliczkae. The dorsal ground colour may vary from cinnamon or cinnamon-rufous to tawny. In this study, we collected the specimen of B. stoliczkae, the dorsal ground colour of which is dark brown, with a black stripe distributed almost evenly across the tail. Our results demonstrate that B. stoliczkae is polymorphic in colouration and pattern. The research indicates that colour polymorphism, defined as the co-existence of two or more distinct colour variants (morphs) within a population, constitutes a crucial aspect of phenotypic diversity and plays a significant role in enhancing survival and reproductive success (Tang et al. 2023).

In China, five species of the genus Boiga have been recorded. Our study reveals that the specimen we collected is the first record of this species in China, thereby broadening its known distribution. Over the years, new reptile species have continually been discovered in the Himalayan Region (Kai et al. 2020, Jiang et al. 2023). The Himalayan Region encompasses multiple countries and poses many access challenges, making comprehensive reptile surveys across the entire region difficult. These challenges have hindered the discovery of new species, further proving that the biodiversity of the Himalayan Region has been underestimated（Xu et al. 2020）.

The Himalaya Region is considered a biodiversity hotspot due to its unique geographic and climatic conditions, which have led to the evolution of a diverse range of plant and animal species (Xu et al. 2020). As fieldwork continues, more and more new species and records of species are being reported, which indirectly proves that there are still species awaiting discovery in the Himalayan Region. Continued biodiversity monitoring and increased attention are still needed.