Molecular phylogenetic analysis of the genus Gloydius (Squamata, Viperidae, Crotalinae), with description of two new alpine species from Qinghai-Tibet Plateau, China

Abstract We provide a molecular phylogeny of Asian pit vipers (the genus Gloydius) based on four mitochondrial genes (12S, 16S, ND4, and cytb). Sequences of Gloydiushimalayanus, the only member of the genus that occurs south of the Himalayan range, are included for the first time. In addition, two new species of the genus Gloydius are described based on specimens collected from Zayu, Tibet, west of the Nujiang River and Heishui, Sichuan, east of the Qinghai-Tibet Plateau. The new species, Gloydiuslipipengi sp. nov., can be differentiated from its congeners by the combination of the following characters: the third supralabial not reaching the orbit (separated from it by a suborbital scale); wide, black-bordered greyish postorbital stripe extending from the posterior margin of the orbit (not separated by the postoculars, covering most of the anterior temporal scale) to the ventral surface of the neck; irregular black annular crossbands on the mid-body; 23-21-15 dorsal scales; 165 ventral scales, and 46 subcaudal scales. Gloydiusswildsp. nov. can be differentiated from its congeners by the narrower postorbital stripe (only half the width of the anterior temporal scale, the lower edge is approximately straight and bordered with white); a pair of arched stripes on the occiput; lateral body lakes black spots; a pair of round spots on the parietal scales; 21 rows of mid-body dorsal scales; zigzag dark brown stripes on the dorsum; 168–170 ventral scales, and 43–46 subcaudal scales. The molecular phylogeny in this study supports the sister relationship between G.lipipengisp. nov. and G.rubromaculatus, another recently described species from the Qinghai-Tibet Plateau, more than 500 km away, and indicate the basal position of G.himalayanus within the genus and relatively distant relationship to its congeners.


Introduction
Asian pit vipers of the genus Gloydius Hoge & Romano Hoge, 1978 are small-bodied venomous snakes distributed mainly in northern Asia, but extending into southern Europe in the case of G. halys. They are quite common and have radiated into various habitats. At present, more than 20 species mainly belonging to three species groups (i.e., the G. blomhoffii complex, G. intermedius-halys complex, and G. strauchi complex) are recognized Zhao 2006;Shi et al. 2017Shi et al. , 2018. Within Gloydius, most species having 21 rows of mid-body dorsal scales and three palatine teeth have been attributed to many subspecies of G. strauchi (Bedriaga, 1912). Recently, several former subspecies have been elevated to full species (e.g., G. qinlingensis (Song & Chen, 1985)), G. liupanensis (Liu, Song & Luo, 1989), and G. monticola (Werner, 1922)) and several new species have been described from across the range of the complex (e.g., G. rubromaculatus, G. angusticeps, and G. huangi; Xu et al. 2012;Shi et al. 2017;Shi et al. 2018;Wang et al. 2019).
Given that the Gloydius strauchi complex is widely distributed in western China (Zhao et al.1998;Zhao 1999Zhao , 2006, some of the specimens from previous studies are now attributable to the recently elevated species described above. The distribution of G. strauchi sensu stricto has been restricted to western Sichuan by recent molecular and morphological studies Barabanov 1999, 2000;Shi et al. 2017Shi et al. , 2018. With respect to Tibet, older records of the G. strauchi complex refer to at least two different species, G. rubromaculatus from Jiangda (Shi et al. 2017(Shi et al. , 2018 and G. huangi from Chamdo (Wang et al. 2019). However, given this wide-ranging complex spans several biogeographic barriers and distinct environments in a poorly investigated region, we hypothesize that there still might be hidden species within the G. strauchi complex.
Additionally, Gloydius himalayanus (Günther, 1864) has long been regarded as a full species within the G. strauchi complex based on its unique morphological characters (e.g., the conspicuous rostralis and the triangular head in dorsal view; Gloyd and Conant 1990). In spite of numerous recent studies focused on the molecular phylogeny of the genus Gloydius (Xu et al. 2012;Eskandar et al. 2018;Shi et al. 2016Shi et al. , 2018Asadi et al. 2019;Wang et al. 2019), the systematic and taxonomic position of G. himalayanus in relation to the G. strauchi complex is still unclear due to lack of the sequence data for this species.
In this study, we use a molecular phylogeny of Gloydius, including data of G. himalayanus for the first time, and provide a description of two new species from the Gloydius strauchi complex from Zayu, Tibet, and Heishui, Sichuan, China.

Specimen collection
We examined preserved specimens from the Chengdu Institute of Biology (CIB), Kunming Institute of Zoology (KIZ), Institute of Zoology (IOZ), and Shenyang Normal University (SYNU). Newly obtained specimens were deposited in the Institute of Vertebrate Paleontology and Paleoanthropology (IVPP), Beijing.

Morphology
Snout-vent length (SVL), tail length (TL), and total length (TTL; i.e., SVL + TL) were measured with a flexible ruler to the nearest 1 mm. Other morphological measurements were taken with 0-200 mm vernier calipers to the nearest 0.1 mm: head length (HL, from the tip of snout to the posterior margin of mandible), head width (HW, the widest part of the head in dorsal view), head depth (HD, the deepest part of the head in lateral view), snout length (SL, from the tip of snout to the anterior margin of the eye), eye diameter (ED, measured as a horizontal distance), interorbital space (IOS, the distance between the top margin of eyes), and internasal space (INS, the distance between nostrils). Numbers of supralabials (SPL), infralabials (IFL), dorsal scales (DS), ventral scales (V, excluding four preventral scales), and subcaudal scales (SC) were counted.

X-ray scanning and three-dimensional reconstructions
The scanning was carried out with 225-kV micro-computerized tomography, developed by the Institute of High Energy Physics (IHEP), CAS. A total of 720 transmission images were reconstructed into the 2048 × 2048 matrix of 1536 slices using two-dimensional reconstruction software developed by the IHEP, CAS. The final CT reconstructed skull model was exported with a minimum resolution of 26.7 μm.

DNA extraction, polymerase chain reaction (PCR) and sequencing
Tissue samples for molecular analyses were taken separately and preserved in 95% ethanol at −40 °C. Genomic DNA was extracted with Qiaprep Spin Miniprep kit (Qia-Gen). Four mitochondrial genome fragments were specifically amplified for this study: a 859 bp fragment of 12S ribosomal RNA (12S), using primers 12SFPhe and 12SRVal, described by Knight and Mindell (1993); a 465 bp fragment of 16S ribosomal RNA (16S) using primers 16sFL and 16sRH described by Palumbi et al. (1991); a 1065 bp fragment of cytochrome b (cytb) using primers L14919 and H16064 described by Burbrink et al. (2000), and a 666 bp fragment of NADH dehydrogenase subunit 4 (ND4), using the primers ND4 and Leu, described by Arevalo et al. (1994). The standard PCR protocol was performed in a 20 μl reaction with at least 20 ng of template DNA and 10 pmol of primers. PCR conditions consisted of an initial denaturation for 3 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 30 sec, 30 sec of annealing at primer-specific temperatures (56 °C for ND4, 54 °C for 16S, 48 °C for cytb), and extension at 72 °C for 60 sec, finalized with an extension step of 10 min at 72 °C. Sequencing was conducted by Beijing Tianyi Huiyuan Bio-tech Co., Ltd.
Sequence data obtained from GenBank and from this study are listed in Table 2. Sequences were aligned in MEGA6 (Tamura et al. 2013). With respect to the different evolutionary characters of each molecular marker, the dataset was initially split into eight partitions by gene and codon positions, and then combined into five partitions taking advantage of PartitionFinder 2.1.1 (Lanfear et al. 2012) to find similarly evolving partitions.
Bayesian phylogenetic analysis was performed using MrBayes 3.1.2 (Ronquist et al. 2011). All searches consist of three heated chains and a single cold chain. Three independent iterations each comprising two runs of 100,000,000 generations were performed, sampling every 10,000 generations, and parameter estimates were plotted against generation. The first 25% of the samples were discarded as burn-in, resulting in a potential scale reduction factor (PSRF) of <0.005. Maximum likelihood analysis was run with the IQtree tool in the web server CIPRES (https://www.phylo.org/index. php), with 1,000 fast bootstrap repeats.
General time reversible (GTR) model, the most probable substitution model for the corrected ND4 p-distance matrix was calculated in PAUP 4.0.

Morphology
Comparative data of specimens examined are listed in Table 1

Molecular phylogeny
Novel sequences were uploaded to GenBank and are available under accession numbers shown in Table 2, along with accession numbers for data obtained from GenBank. The final molecular dataset consisted of 3,065 bases containing 46 specimens. The evolutionary models assigned to each of the five partitions by PartitionFinder are shown in Table  3. The phylogeny from the Bayesian analysis (BI, Fig. 5) matches those given in earlier studies of the genus (Xu et al. 2012;Shi et al. 2017Shi et al. , 2018Wang et al. 2019), except for the systematic position of the G. qinlingensis-liupanensis group, which do not form a monophyletic group with other members of the G. strauchi complex. In this study, the topological structures of the maximum likelihood (ML) and Bayesian inference (BI) trees are generally consistent. The lineage of the new specimen from Zayu, Tibet (G2), constitutes a sister group to the clade of G. rubromaculatus from Sanjiangyuan, Qinghai (Y2), but is separated from it by significant branch lengths. The clade including G. lipipengi sp. nov. and G. rubromaculatus (Clade A) is sister to the clade formed by G. huangi and G. monticola (Clade B), forming a monophyletic lineage (Clade C). Clade C is sister to the monophyletic clade constituted by G. strauchi and G. angusticeps (Clade D), forming another monophyletic clade (Clade E).
The two new specimens from Heishui, Sichuan (GR1 and GR2), forming a strongly supported monophyletic group (Clade F). The clade of G. qinlingensis is sister to the clades of G. liupanensis, forming Clade G. The samples of the nine species of G. halysintermedius group constitute another monophyletic group, Clade H. Clade G is sister to Clade F, forming a monophyletic Clade (Clade I) sister to the clade constituted by the new specimens from Heishui (Clade F), forming Clade J.  The phylogenetic position of Gloydius himalayanus, the only species of the genus to be found on the southern slopes of the Himalayan ranges, is basal to, and considerably distant from other species of Gloydius (13-16.1% p-distance for ND4, Table 4), although the genus as a whole is well supported as a monophyletic group in this analysis.
The corrected p-distance between the new specimen from Zayu, Tibet and G. rubromaculatus sequences is greater than those between other recognised species (4.4% for ND4, Table 4); the corrected p-distances between the new specimens from Heishui and one of its closest related congeners, G. rubromaculatus, are greater than those between other recognised species (8.5% for ND4, Table 4). Thus, the molecular phylogeny supports these new specimens from both Zayu and Heishui as phylogenetically independent species. Gloydius lipipengi sp. nov. differs from other congeneric species in the following characteristics: i) third supralabial scale not touching the orbit; ii) a pair of prominent black markings on the occiput; iii) black-bordered greyish cheek stripe extending from the posterior margin of orbit (not separated by the postoculars) to the ventral surface of the neck; iv) black irregular annular crossbands on the mid-body; iv) two rows of black blotches on the ventral side ; v) 23-21-15 circum-body scales; vi) 165 ventral scales; and vii) 46 subcaudal scales. Table 5 provides a brief summary of the differences between G. lipipengi sp. nov., G. swild sp. nov. and other congeneric species.    Gloydius lipipengi sp. nov. and G. swild sp. nov. can be differentiated from the species in the G. blomhoffii complex by having three palatine teeth (versus four palatine teeth), from the G. halys complex by having 21 rows of mid-body dorsal scales (versus 22 or 23 rows). Gloydius lipipengi sp. nov. differs from other species in G. strauchi complex by the third supralabial scale not touching the orbit, from G. strauchi, G. huangi, and G. rubromaculatus by having large irregular black markings on the back (versus four irregular longitudinal stripes or discrete blotches in G. strauchi, complete dark brown patches in G. huangi, and large red crossbands in G. rubromaculatus (Wang et al. 2019), from G. monticola by having seven supralabials (versus always six supralabials) and more subcaudal scales (46 pairs versus always fewer than 30 pairs), from G. qinlingensis and G. liupanensis by its greyish brown body colour (versus yellowishbrown body colour) and lacking a lateral white line on each lateral side (versus possessing a lateral white line on each side). Gloydius lipipengi sp. nov. can be differentiated from G. himalayanus by possessing an indistinct canthus rostralis (versus very distinct canthus rostralis; Gloyd and Conant 1990).
Description of the holotype. IVPP OV 2720, adult male, a slender pit viper with a total length of 628.2 mm (SVL 540.6 mm and TL 87.6 mm), preserved in 75% ethanol with its left hemipenes partially extruded (Figs 1, 2).
The head is slender and triangular shaped in dorsal view, distinct from the neck. Canthus rostralis are not distinct. The head is 25.2 mm in length, 13.2 mm in width and 8.2 mm in depth.
Scalation. Rostral scale slightly up-turned, visible from dorsal view; nasal divided, anterior part larger; seven supralabials on both sides: second smallest, not reaching the pit; third highest, not touching the bottom of orbit (separated by one small subocular); fourth longest, not touching the orbit; three preoculars, two postoculars, inferior one touching the top of the third supralabials, forming the bottom margin of the orbit; two rows of temporals (2+4); infralabials 10 on left side while 11 on right, first pair in contact behind the mental; second, third and fourth pairs meet on the chin shield; chin shield is rhomboidal in shape, the posterior chin shield comprises two pairs of scales, forming the mental groove ( Fig. 3A-C). Dorsal scales in 23-21-15 rows (reducing from 19 to 18 posteriorly at ventral 94-96), keeled except for the first scale row bordering the ventral scales; ventral scales 165 (excluding four preventral scales); anal plate single; subcaudals 46, in pairs Coloration. Eye dark brown on the upper half while black on the bottom half, pupil black, vertical with light yellow margins; postorbital stripe wide, greyish brown and black bordered on the lower edge, extending from the posterior orbit to the ventral surface of the neck; supralabials and infralabials greyish brown, scattered with very small irregularly sized black blotches. One black triangular mark on the anterodorsal head, covering the caudomedial part of prefrontals. One bold black M-shaped mark on the dorsomedial head, covering the caudal part of lateral frontals, the lateral part of parietals, merged with the postorbital stripe at the largest temporal scale (but not covering the upper postorbital). The upper postorbital white while the top part of the bottom postorbital is black (covered by the postorbital stripe). bs, basisphenoid; col, columella; cp, compound bone; d, dentary; ecp, ectopterygoid; exo, exoccipital; f, frontal; na, nasal; ma, maxilla; p, parietal; pcr, prearticular crest of compound bone; pfr, prefrontal; pmx, premaxilla; po, postorbital; pp, palatine process of maxilla; pro, prootic; psp, parasphenoid rostrum; pt, pterygoid; sac, surangular crest of compound bone; spm; septomaxilla; so, supraoccipital; sp, splenial; st, supratemporal; v, vomer. Conducted by Ye-Mao Hou and Jingsong Shi.
The body coloration is dark greyish brown, with two rows of irregular black annular crossbands on the mid-body, each covering 20 or more scales, separated by a gap of two row scale vertically, extending laterally to one or two dorsal scales from the ventrals. Ventral scales light grey, with two large black blotches on each side, clustered into two ventral stripes. The tip of tail is similar to the main body in coloration (Figs 1, 2).
Skull. The description of the skull of G. lipipengi sp. nov. is based on the 3Dreconstructed model of the holotype.
Snout. The premaxilla has bifurcated transverse process on each side. The anterior margin of the premaxilla is blunt. The dorsal tip of the ascending process of premaxilla is triangular in lateral view, not reaching the anterior tip of nasals. The horizontal laminae of the nasals are scutiform in dorsal view. The septomaxillae have prominent dorsolateral processes, nearly meeting the horizontal laminae of the nasals.
Braincase. The parietal is roughly T-shaped in dorsal view. The anterolateral part of the parietal bulges prominently laterally while the dorsoposterior part tapers medially. The postorbital processes of the parietal are prominent. The frontals are squared. The lateral margin of frontals concaved obviously on each side, forming the dorsal edge of orbit. The prefrontal has an elongate blunt lateral process, posterolaterally pointed.
The lacrimal foramen perforates the medial lamina of the prefrontal. The prefrontalfrontal join surface is waved in dorsal view.
The postorbital is relatively small and cashew-shaped, the top of the postorbital does not reach the posterolateral end of frontal. The basisphenoid is spearhead in shape, narrow anteriorly and expanded laterally. The supraoccipital is longitudinally compressed, occupies almost two thirds the total width of the otic region.
Palatomaxillary apparatus. The fang is relatively short and curved, roughly the same length of the maxilla, one third the length of ectopterygoid, attached with seven or eight replacement fangs on each side. The palatine bears three teeth. The ectopterygoid is flat and widened at the anterior part. The pterygoid is slender, the dentigerous process of pterygoid is straight, bearing 12/11 teeth (left/right), occupies almost half the total width of the pterygoid, the posterior portion of pterygoid is medially expanded.
Suspensorium and mandible. The supratemporal is slender, has a lateral process, anterolaterally pointed, lies in front of the supratemporal-quadrate articulation. The quadrate is straight, slender, and enlarged on both ends. The mandible is slender and moderately curved. The prearticular crest of the compound bone is prominent while the surangular crest is slightly concaved. The dental bone bears 11/12 teeth (left/right); the dentary teeth are perpendicular to the dentary bone, decreasing in size at the third tooth. The posterior tip of ventral process of dentary extends farther posteriorly than the dorsal process.
Dentition. Palatine: 3/3, pterygoid: 12/11, dentary: 11/12. Hemipenes. The hemipenes of G. lipipengi sp. nov. are generally similar to those of G. rubromaculatus and G. huangi but differ by the possession of longer and stronger spines, seven or eight subcaudals in length, and forked for two subcaudals. Small and stubby spines range from the basal to the distal side of the organ, without any conspicuously enlarged spines (versus 3-5 enlarged spines on the base in the G. halys complex; Gloyd 1990). The spines gradually increase in length distally.
Distribution and ecology. At present, Gloydius lipipengi sp. nov. has only been reported from the type locality, Muza village, Zayu, Tibet, China (Fig. 6). The specimen was collected at 09:00 h on leaf litter in forest near the hot, dry valley on the lower reaches of the Nujiang River (Fig. 7). Gloydius lipipengi sp. nov. accepted pink mice in captivity. Diagnosis. Gloydius swild sp. nov. differs from other congeneric species in the following characteristics: i) the narrower postorbital stripe, ii) a pair of round spots on the parietal scales; iii) the absence of the black spots on the lateral body; iv) 21 rows of mid-body dorsal scales; v) a pair of arched stripes on the occiput; vi) 168-170 ventral scales, and vii) 43-46 subcaudal scales.
Morphologically, Gloydius swild sp. nov. is quite similar to G. angusticeps, but differs by the narrower, straight bordered brown postorbital stripe (versus wider postorbital stripe with dentate lower border in G. angusticeps). G. swild sp. nov. differs from G. strauchi, G. huangi, and G. rubromaculatus by the narrow triangular head from dorsal view (versus spoon-shaped head in above-mentioned species), from G. monticola by having seven supralabials (versus always six supralabials) and more subcaudal scales  Description of the holotype. Gloydius swild sp. nov., IVPP OV 2725, adult female, a slender pit viper with a total length of 529.5 mm (SVL 462 mm and TL 67.5 mm), preserved in 75% ethanol (Fig. 1).
The head is slender and narrow triangular shaped in dorsal view, distinct from the neck. Canthus rostralis not distinct. The head is 20.8 mm in length, 12.2 mm in width and 6.6 mm in depth.
Scalation. Rostral scale slightly up-turned, visible from dorsal view; nasal divided, anterior part larger; seven supralabials on both sides: second smallest, not reaching the pit; third highest, not touching the bottom of orbit on the left (separated by one small subocular) while touching the bottom of the orbit on the right; fourth longest, not touching the orbit; three preoculars, two postoculars, inferior one touching the top of the third supralabials, forming the bottom margin of the orbit; two rows of temporals: 3+5/2+4 (L/R); infralabials 10, first pair in contact behind the mental; second, third and fourth pairs meet on the chin shield; chin shield is rhomboidal in shape, the posterior chin shield comprises two pairs of scales, forming the mental groove ( Fig. 3D-F). Dorsal scales in 21-21-15 rows, keeled except for the first scale row bordering the ventral scales; ventral scales 170 (excluding four preventral scales); anal plate single; subcaudals 46, in pairs.
Coloration. Gloydius swild sp. nov., eye light greyish brown on the upper half while black on the bottom half, pupil black, vertical with light yellow margins; postorbital stripe relatively narrow, only half the width of the anterior temporals, greyish brown and white bordered on the lower edge, extending from the posterior orbit to the lateral surface of the neck; supralabials and infralabials greyish white, scattered with large irregularly sized, black blotches, rendering the lateral head granitoid. One black Ω-shaped mark on the anterodorsal head, covering the posteromedial part of prefrontals, the anterior and lateral part of the frontals and the anterior part of the parietals. The infralabials are bordered with yellow on the lower edge.
The body coloration is dark blueish-grey, with two rows of irregular black X-shaped or C-shaped crossbands on the mid-body, each covering about 10 dorsal scales (or more), separated by a gap of one or two dorsal scales vertically, sometimes in contact with the adjacent ones forming zigzag stripes, but hardly merged on the medial dorsal line, extending laterally to one or two dorsal scales from the ventrals. Ventral scales light grey, scattered with dense irregular black blotches, rendering the ventral scales granitoid. The tip of tail is bony, similar to the main body in coloration on both ventral and dorsal sides (Figs 1C, D, 3D-F).
Infraspecific morphological variation. Despite the inconspicuous variation in the coloration among the type series of G. swild sp. nov., the scalations vary considerably between the two specimens. The range of the temporal scales of the holotype (IVPP OV 2725) is 3+5 on the left side but 2+4 on the right side, while in the paratype (IVPP OV 2726), the temporal scales are displayed as 2+4 on both sides. Ventrals range from 168-170 in females (n = 2), while range from 43-46 in females (n = 3, including one shed skin from the wild). Total length ranges from 529.5-629.1 in adult females. The infralabials of the paratype lack the greyish-yellow margins seen in the holotype. Distribution and ecology. Gloydius swild sp. nov. has been found in east part of Qinghai-Tibet plateau and Hengduanshan mountains, Heishui country, north Sichuan, about 15 km away from Dagu Holy-glacier National Geological Park, from along the route of Red Army's long march (from June to August, 1935). They were found on or under the rocks (especially near the vegetations) on sunny slopes (Figs 6, 7C).
Viviparous reproduction. One adult female (Holotype) was collected when pregnant, gave birth to eight neonates (including a couple of conjoined twins) on September 20 th , 2017 in captivity. The weight of the normal neonates ranged between 3.00-3.45 g (3.01, 3.22, 3.22, 3.23, 3.28, 3.45, average = 3.235, n = 6). The weight of the conjoined twins was 2.86 g (weighed after the first shedding).

Discussion
This study reveals the phylogenetic position of G. himalayanus within Gloydius for the first time. This study also reports two new Gloydius species, increasing the number of the recognized species in Gloydius to 23. The discovery of the new species has further verified the hypothesis that the Himalayan-Tibetan Plateau and Southwest Mountain Ranges should be considered as differentiation centres of Asian pit vipers. Furthermore, the discovery of G. swild sp. nov. suggests that the glaciers might be considered as key factors to the isolation and speciation of the alpine pit vipers in the southwest China.
Lastly, the systematic and taxonomic relationship of G. qinlingensis and G. liupanensis is still controversial. Despite their morphological similarities, these clades have not consistently formed a monophyletic group in earlier studies (Xu et al. 2012;Shi et al. 2017;Wang et al. 2019). In a subsequent study, which included more mitochondrial genes (specifically 16s rRNA) in the analysis (Li et al. 2020), G. qinlingensis and G. liupanensis formed a separate monophyletic lineage (Clade G) that is sister to the G. halys complex. The systematic position of the G. qinlingensis-liupanensis group that have been reconstructed in this study is consistent with Li et al. (2020).
In our analysis, the enigmatic clade formed by Ovophis okinavensis and Trimeresurus gracilis (Clade F) is basal to the genus Gloydius. As highlighted by Malhotra and Thorpe (2000), Tu et al. (2000), and numerous analyses since (Castoe and Parkinson 2006;Pyron et al. 2013;Alencar et al. 2016), the systematic status of these two species requires resolution.
As the squamation and body coloration variation is quite conservative within pit vipers (Gloyd and Conant 1990;Shi et al. 2017Shi et al. , 2018, it is necessary to obtain more specimens of the new species to investigate intraspecific variation in the new species. These data will be very helpful in verifying the stability of the diagnostic morphological characteristics of G. lipipengi sp. nov. Further fieldwork and molecular phylogenetics, particularly using nuclear genes, are still needed to investigate the origin, evolution, and migration of Asian pit vipers on the Qinghai-Tibet Plateau.