Molecular phylogenetic analysis of the genus Gloydius (Squamata, Viperidae, Crotalinae), with description of two new alpine species from Qinghai-Tibet Plateau, China Author Shi, Jing-Song https://orcid.org/0000-0001-9168-1734 Key Laboratory of Vertebrate Evolution and Human Origins of Chinese Academy of Sciences, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing 100044, China & Institute of Herpetology, Shenyang Normal University, Shenyang 110034, China shijingsong@ivpp.ac.cn Author Liu, Jin-Cheng Institute of Herpetology, Shenyang Normal University, Shenyang 110034, China Author Giri, Rohit Department of Zoology, Prithvi Narayan Campus, Bhimkalipatan- 1, Pokhara 33700, Nepal Author Owens, John Benjamin https://orcid.org/0000-0003-0298-451X Molecular Ecology and Evolution at Bangor, School of Natural Sciences, Bangor University, Bangor, Gwynedd LL 57 2 UW, UK & Captive and Field Herpetology, Wales, UK Author Santra, Vishal Captive and Field Herpetology, Wales, UK & Society for Nature Conservation, Research and Community Engagement (CONCERN), Nalikul, Hooghly, West Bengal, India Author Kuttalam, Sourish https://orcid.org/0000-0003-1072-1699 Captive and Field Herpetology, Wales, UK & Society for Nature Conservation, Research and Community Engagement (CONCERN), Nalikul, Hooghly, West Bengal, India Author Selvan, Melvin Endangered Wildlife Trust, Dindigull, Tamil Nadu, India Author Guo, Ke-Ji Central South Inventory and Planning Institute of National Forestry and Grassland Administration, College of Life Sciences and Technology, Central South University of Forestry and Technology, Changsha, Hunan 410004, China Author Malhotra, Anita https://orcid.org/0000-0002-1738-9046 Molecular Ecology and Evolution at Bangor, School of Natural Sciences, Bangor University, Bangor, Gwynedd LL 57 2 UW, UK a.malhotra@bangor.ac.uk text ZooKeys 2021 2021-10-04 1061 87 108 http://dx.doi.org/10.3897/zookeys.1061.70420 journal article http://dx.doi.org/10.3897/zookeys.1061.70420 1313-2970-1061-87 F2701BACD2E14F97A0BDDC8B09966247 AF38CEA80126576FA4C341BB14489336 Gloydius lipipengi Shi, Liu & Malhotra sp. nov. Etymology. The specific epithet of the new species from Tibet is dedicated to the senior author's Master's supervisor, Professor Pi-Peng Li (Institute of Herpetology, Shenyang Normal University) on Li's sixtieth birthday. Prof. Li has devoted himself to the study of the herpetological diversity of the Qinghai-Tibet Plateau. The senior author became an Asian pit viper enthusiast and professional herpetological researcher under his instruction. The common name of Gloydius lipipengi sp. nov. is suggested as "Nujiang pit viper" in English, and "Nu Jiang Fu ( 怒江蝮)" in Chinese. Type specimen. Gloydius lipipengi sp. nov., holotype. IVPP OV2720 (G2, Figs 1 - 4 ), adult male, collected from Muza Village, Zayu, Nyingchi Prefecture, Tibet ( 28.54°N , 98.23°E , 2883 m), by Jin-Cheng Liu, on 8 September 2014. Diagnosis. The specimens of the new species, IVPP OV 2720, IVPP OV 2725 and IVPP OV 2726 were identified as the member of the genus Gloydius based on the small body size, bilateral pits, and divided subcaudal scales (Hoge and Romano-Hoge 1981). 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. Table 5. Brief morphological comparisons between Gloydius lipipengi sp. nov., G. swild sp. nov. and other congeneric species.

Species	Dorsal head	Spots on supralabials	Canthus rostralis	Background coloration	Dorsal color patterns
G. lipipengi sp. nov.	triangular	greyish brown discrete spots	inconspicuous	greyish brown	irregular large black interlaced patches
G. swild sp. nov.	triangular	greyish brown discrete spots	inconspicuous	greyish brown or blueish-grey	irregular zigzag dark brown markings
G. angusticeps	triangular	greyish brown, vaporous spots	inconspicuous	greyish brown or yellowish brown	dark brown small blotches, large patcher or stripes
G. himalayanus	triangular	one triangular spot between the third and fourth supralabial	very conspicuous	reddish brown or light grey	brown, reddish brown or dark grey small blotches or large black bordered patches
G. huangi	rounded	yellowish brown small spots	inconspicuous	light or pale greyish yellow	regular large greyish brown patches
G. liupanensis	triangular	none	conspicuous	light reddish brown or yellowish brown	similar with Gloydius qinlingensis , with white stripe on the body side
G. monticola	rounded	whitish borders of the labials along the mouth line	inconspicuous	greyish brown, greenish brown or reddish brown	dark grey or dark brown blotches or zigzag stripes
G. qinlingensis	triangular	none	conspicuous	light reddish brown or yellowish brown	two columns of irregular yellowish brown or dark brown
G. rubromaculatus	rounded	irregular small black spots	inconspicuous	khaki or yellowish brown	regular large (or discrete small) scarlet or brownish yellow patches or stripes
G. strauchi	rounded	large brown between the second, third and fourth or none	inconspicuous	greenish brown, yellowish brown or nut-brown	patches, transverse crossbands or four longitudinal zigzag strips

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 yellowish-brown 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 ). Figure 1. Gloydius lipipengi sp. nov. ( A, B IVPP OV 2720, holotype) and Gloydius swild sp. nov. ( C IVPP OV 2725, holotype, D IVPP OV, 2726, paratype) in life, not to scale. Figure 2. Holotype of Gloydius lipipengi sp. nov. (IVPP OV 2720) in preservative A dorsal view B ventral view. 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 ). Figure 3. Head squamation of Gloydius lipipengi sp. nov. (Holotype, IVPP OV 2720: A lateral view B dorsal view C ventral view) and G. swild sp. nov. (Holotype, IVPP OV 2725: D lateral view E dorsal view F ventral view). Scale bar: 10 mm. 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). 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 3D-reconstructed 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 prefrontal-frontal 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. Figure 4. Color rendered three-dimensional model of Gloydius lipipengi sp. nov. (holotype, IVPP OV 2720) A dorsal view B palatal view, mandibles not shown C lateral view. Abbreviations: bo, basioccipital; 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. Figure 5. Bayesian inferenced tree of the genus Gloydius , along with some relative genus of the family Viperidae , based on 12S, 16S, ND4, and cytb sequences, with the maximum likelihood bootstrap supports (left, regular) and Bayesian posterior probabilities (right, italic) displayed on the nodes (those <50% are displayed as "-" ). Holotypes are marked with asterisks.