Zoosyst. Evol. 96 (1) 2020, 123-138 | DOI! 10.3897/zse.96.48431 yee BERLIN A new species of green pit vipers of the genus 7rimeresurus Lacépede, 1804 (Reptilia, Serpentes, Viperidae) from western Arunachal Pradesh, India Zeeshan A. Mirza', Harshal S. Bhosale*, Pushkar U. Phansalkar*?, Mandar Sawant’, Gaurang G. Gowande*?, Harshil Patel® National Centre for Biological Sciences, TIFR, Bangalore, Karnataka 560065, India Bombay Natural History Society, Mumbai, Maharashtra 400001, India A/2, Ajinkyanagari, Karvenagar, Pune, Maharashtra 411052, India Annasaheb Kulkarni Department of Biodiversity, Abasaheb Garware College, Pune, Maharashtra 411004, India Department of Biotechnology, Fergusson College, Pune, Maharashtra 411004, India Nn OR W DY Department of Biosciences, Veer Narmad South Gujarat University, Surat, Gujarat 395007, India http://zoobank.org/F4D892E 1-4D68-4736-B103-F' 1662B7D344D Corresponding author: Zeeshan A. Mirza (snakeszeeshan@gmail.com) Academic editor: Peter Bartsch # Received 13 November 2019 Accepted 9 March 2020 @ Published 15 April 2020 Abstract A new species of green pit vipers of the genus 7rimeresurus Lacépéde, 1804 is described from the lowlands of western Arunachal Pradesh state of India. The new species, 7rimeresurus salazar, is a member of the subgenus 7rimeresurus, a relationship deduced contingent on two mitochondrial genes, /6S and ND4, and recovered as sister to 7rimeresurus septentrionalis Kramer, 1977. The new species differs from the latter in bearing an orange to reddish stripe running from the lower border of the eye to the posterior part of the head in males, higher number of pterygoid and dentary teeth, and a short, bilobed hemipenis. Description of the new species and T. arunachalensis Captain, Deepak, Pandit, Bhatt & Athreya, 2019 from northeastern India in a span of less than one year highlights the need for dedicated surveys to document biodiversity across northeastern India. Key Words Biodiversity hotspot, Crotalinae, cryptic species, Himalayas, molecular phylogeny, northeastern India, taxonomy Introduction The pit vipers of the genus 7rimeresurus Lacépéde, 1804 are charismatic venomous serpents, with morphological- ly as well as ecologically diverse species (Sanders et al. 2004). They are distributed across east and southeast Asia. At least 48 nominate species (Gumprecht et al. 2004; Whitaker and Captain 2004; Wallach et al. 2014; Captain et al. 2019) are known, of which at least 15 occur in India. Of the species recorded from India, seven species occur in northeastern India (Whitaker and Captain 2004; Cap- tain et al. 2019). The most recent compilations on Indian snakes, listed the following species from northeastern In- dia, namely: 7. erythrurus (Cantor, 1839), 7? gumprechti David, Vogel, Pauwels & Vidal, 2002, 7’ medoensis Zhao, 1977, T. septentrionalis Kramer, 1977, T: albolabris Gray, 1842, 7’ popeorum Smith, 1937, and T. yunnanen- sis Schmidt, 1925 (Ao et al. 2004; Gumprecht et al. 2004; Whitaker and Captain 2004; David and Mathew 2005). In addition to the these, a new species, 7) arunachalensis Captain, Deepak, Pandit, Bhatt & Athreya, 2019 was re- cently described from Arunachal Pradesh. The diversity of pit vipers is likely underestimated, as several species are morphologically cryptic making it difficult to distin- guish them in the field (Malhotra and Thorpe 2000, 2004; Zhong et al. 2015; Zhu et al. 2016; Mulcahy et al. 2017). Copyright Zeeshan A. Mirza et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 124 For the delimitation of cryptic species, multiple lines of evidence are imperative, including morphology as well asmolecular and ecological data (Sanders et al. 2006; Mirza et al. 2018). During a herpetological expedition, Arunachal Pradesh was visited between 25 June 2019 and 5 August 2019. Arunachal Pradesh belongs to the Himalayan biodiversity hotspot and shows a high degree of heterogeneity in its landscape with elevation ranging from 100 to 7000 m and distinct climatic regimes that harbour diverse flora and fauna. Most of the state is part of the undulating terrain of the Himalayas, intersected by numerous rivers, which flow from the Himalayas and form a longitudinal network of parallel flowing rivers, which ultimately meet the River Brahmaputra. The lowland area bordering Assam mostly shares a similar biotope to that of northern Assam. During the expedition, near Pakke Tiger Reserve, we collected two specimens of a green pit viper, which resembled 7ri- meresurus septentrionalis and Trimeresurus albolabris in the number of dorsal scale rows and colouration. Howev- er, these specimens differed in the colouration of the lat- eral stripe on the head and the body in males. Comparison of the specimens from near Pakke Tiger Reserve with 7. septentrionalis and T: albolabris (specimens examined = 11) housed in the collection of the Bombay Natural His- tory Society (Mumbai, India), Natural History Museum (London, UK), and the data presented in the literature (Smith 1943; Gumprecht et al. 2004) revealed that the specimens from near Pakke Tiger Reserve represent a new species. Molecular data for the specimens corrobo- rate our morphological findings and allow us to describe a new species of green pit vipers from northeastern India. Material and methods Morphology The study was conducted under permit no. CWL/ Gen/173/2018-19/Pt.V11/2421-33 and CWL/ Gen/173/2018-19/Pt.V11/2434-43 issued by the Forest Department of Arunachal Pradesh. Two specimens of the new species were collected by hand in the field, photo- graphed, and later euthanized with halothane within 24 h of capture following ethical guidelines for animal eutha- nasia (Leary et al. 2013). The specimens were fixed in 8% formaldehyde buffer and later stored in 70% ethanol. Liver tissue was collected for molecular work and stored in molecular grade ethanol prior to specimen fixation. The specimens have been deposited in the collection of the Bombay Natural History Society (BNHS), Mumbai. Measurements were taken with the help of digital calli- pers to the nearest 0.1 mm and those for snout to vent length (SVL) and tail length (TaL) were taken with a string, which was then measured using a scale. Ventral scales (V) were counted as directed by Dowling (1951). Dorsal scales at midbody were counted at midway of the SVL. Cephalic scales (CEP) number was counted on a zse.pensoft.net Mirza, Z.A. et al.: A new Trimeresurus from Arunachal Pradesh straight line between the middle of the supraoculars; lon- gitudinal cephalic scales (LCS) number of scales counted from the posterior border off the internasals to the neck (which is here defined as the dorsal scale row, which cor- responds to the first ventral scale). Abbreviations used in the description: TL = total length, HL = head length measured from snout tip to the angle of the jaw, VED = vertical extent of the eye, DEYE = diameter of the eye measured between edges of scales surrounding it, DEL = distance between lower eye margin and the edge of lip. Morphological data for the new species were compared with the types of the sister taxa based on molecular data and original descriptions of sister species were referred to, too. Morphological data from literature were largely derived from Gumprecht et al. (2004), Kramer (1977) and Regenass and Kramer (1981). Micro-CT scan were generated for the male holotype using a Bruker Skyscan 1272 (Bruker BioSpin Cor- poration, Billerica, Massachusetts, USA). Head of the specimen was scanned for 210 minutes at resolution of 5.4 um and recording data for every 0.4° rotation for 360° with (AL) 1 mm filter. The source voltage for the scan was 65 kV and source current was 153 uA. Volume rendering was performed with CT Vox (Bruker BioSpin Corporation, Billerica, Massachusetts, USA) and imag- es were edited in Adobe Photoshop CS6. Osteological description is based on volume renders retrieved from CTVox following terminology of the skull described by Heatwole (2009). Institution acronyms BNHS MNHN Bombay Natural History Society, Mumbai, India; Muséum national d’Histoire naturelle, Paris, France; Collection Facility of the National Centre for Biological Sciences, Bangalore, India; Natural History Museum, London, UK; Zoological Museum University of Copenha- gen, Denmark. NCBS NHM ZMUC Comparative material examined: Trimeresurus albolabris NHM 1946.1.19.85, lectoype, China; BNHS 2652-2653, Taungyi Myanmar; BNHS 2654, Mayntyo, Myanmar; BNHS 2655, Myanmar; BNHS 2656, Bangkok, Thailand; BNHS 2659, Mou- linein, Myanmar; BNHS 3304, Car Nicobar, India; Trimeresurus erythrurus NCBS-AG767, NCBS- AG776, NCBS-AG78 1-782, Tripura, India Trimeresurus gumprechti MNHN_ 1999.9072_ holo- type & MNHN 1999.9073 paratype, Loe, Loei Province, Thailand, paratype: Trimeresurus popeiorum NHM 1872.4.17.137, lecto- type, Khasi hills, Meghalaya, India; BNHS 2638-2640, Tindharia, Darjeeling, West Bengal, India; BNHS 2637, Tura, Garo Hills, Meghalaya: Zoosyst. Evol. 96 (1) 2020, 123-138 Trimeresurus septentrionalis NHM 1937.3.1.15 para- type, Kullu District, Himachal Pradesh, India; paratype NHM 1937.3.1.15, paratype NHM 1955.1.13.82; Trimeresurus yunnanensis BNHS 2634, Shillong, Me- ghalaya; BNHS 2635, Tura, Assam; BNHS 2636, Tindh- aria, Darjeeling, West Bengal, India. Molecular analysis Genomic DNA was isolated from the preserved tissues of the type specimens using QIAGEN DNeasy kits fol- lowing protocols directed by the manufacturer. Molecular methods largely follow Mirza et al. (2016) and Mirza and Patel (2018). A fragment of the mitochondrial 16S rRNA (J6S) and NADH-ubiquinone oxidoreductase, subunit 4 (ND4) gene were amplified using primers used by Pyron et al. (2013) and Mirza et al. (2016). A 22.4 ul reaction was set for a bi-directional Polymerase Chain Reaction (PCR), containing 10 ul of Thermo Scientific DreamTaq PCR Master Mix, 10 ul of molecular grade water, 0.2 ul of each 10 uM primer and 2 ul template DNA, carried out with an Applied Biosystems ProFlex PCR System. Thermo-cycle profile used for amplification were as fol- lows: 95 °C for 3 min, (denaturation temperature 95 °C for 30 sec, annealing temperature 60 °C for ND4, and 45 °C for /6S for 45 sec, elongation temperature 72 °C for 1 minutes) x 36 cycles, 72 °C for 10 min, hold at 4 °C. PCR product was cleaned using QJAquick PCR Purifi- cation Kit and sequenced with an Applied Biosystems 3730 DNA Analyzer. In addition to this, /6S and ND4 sequences of 7rimeresurus spp. available on GenBank were downloaded for molecular phylogenetic reconstruc- tions (Appendix I), and the sequences were concatenated using SequenceMatrix. Taxa for molecular phylogenetics were selected based on the tree topologies recovered by Figueroa et al. (2016). Sequences were aligned in MegaX (Kumar et al. 2018) using ClustalW (Thompson and Gib- son 2002) with default settings. For optimal partitioning strategy and evolutionary substitution model, aligned data was analyzed using PartitionFinder v. 1.1.1. (Lanfear et al. 2012), implementing a greedy search algorithm under the Akaike Information Criterion (AIC). Dataset was par- titioned as per-codon position for the ND4 gene, whereas the /6S region was not partitioned per-codon position. Maximum Likelihood (ML) analysis was carried out using raxmlGUI (Silvestro and Michalak 2012), 1000 non-para- metric bootstrap pseudo-replicates were performed with rapid ML search option. Bayesian Inference (BI) was 1m- plemented in MrBayes 3.2.2. (Ronquist and Huelsenbeck 2003) and was run for 10 million generations and sam- pled every 1000 generations. BI run included five paral- lel chains, three hot and tow cold chains. The standard deviation of split frequencies of the analysis reached were below 0.01, after which the analysis was not continued further. Twenty-five percent of trees generated were dis- carded as burn-in. Data were subjected to phylogenetic reconstructions with generalised time-reversible (GTR) 1S model as the sequence substitution model, based on the optimal partitioning scheme suggested by PartitionFinder for both ML and BI. Uncorrected pairwise p-distance (% sequence divergence) was calculated in MegaX (Kumar et al. 2018) with pairwise deletions of missing data and gaps. Details of sequences and GenBank accession num- bers are presented in supporting files (Appendix I). Results Trimeresurus salazar sp. nov. http://zoobank.org/6C 165D3E-D3F2-43CA-A62E-E753E6B7FC22 Figs 1-5, 7 Holotype. BNHS 3554, adult male, collected from outskirts of Pakke Tiger Reserve, 0.64 nautical miles (1.19 km) north of Seijosa, East Kameng_ district, Arunachal Pradesh (26.968790N, 93.013984E, elevation 172 m a.s.l, datum WGS84), India by Harshal Bhosale, Pushkar Phansalkar, Mandar Sawant, and Zeeshan Mirza on | July 2019 Paratype. BNHS 3555, adult female same data as for the holotype but collected on 5 July 2019. Referred material. ZMUC R69255 & ZMUC R69256, males, from Assam, India purchased by B.W. Westermann. Etymology. The specific epithet is a noun in apposition for J.K. Rowling’s fictional Hogwarts School of Witch- craft and Wizardry’s co-founder, Salazar Slytherin. He was a Parselmouth that links him to serpents. Suggested common name: Salazar’s pit viper. Diagnosis. A species of the genus 7rimeresurus with (1) 1 supralabial fused with nasal; (2) 19—21 moderate- ly keeled dorsal scale rows at midbody; (3) dorsal co- louration greenish yellow in both sexes; (4) an orange to reddish stripe extends from the posterior borders of the preocular, running through the lower margin of the eyes to the lateral side of the nape in males; (5) ventrolater- al stripe predominantly yellow with a faint orange at the base in males, yellow in females; (6) tail to total length ratio (TaL/TL) 0.18 in males, 0.14 in females; (7) short, bilobed hemipenis reaching 8" caudal scale; (8) 6 pala- tine, 15 pterygoid and 19 dentary teeth. Comparison. The new species is here compared to all species of the genus 7rimeresurus for differing and non-overlapping characters: first supralabial fused with nasal (vs separate in 7’ macrolepis Beddome, 1862, 7. trigonocephalus (Latreille, 1801), 7! malabaricus (Jerdon, 1854), 7 strigatus Gray, 1842, T. gramineus (Shaw, 1802), 7. stejnegeri Schmidt, 1925, 7: hageni (Lidth de Jeude, 1886), 77 phuketensis Sumontha, Kun- ya, Pauwels, Nitikul & Punnadee, 2011, 7: nebularis Vogel, David & Pawels, 2004, T. truongsonensis Orlov, zse.pensoft.net 126 Ryabov, Thanh & H. Cuc, 2004, 7: gunaleni Vogel, Da- vid & Sidik, 2014, 7 sabahi Regenass & Kramer, 1981, 7. popeorum, T. yingjiangensis Chen, Zhang, Shi, Tang, Guo, Song & Ding, 2019, T. sichuanensis (Guo & Wang, 2011), 7. nebularis Vogel, David & Pauwels, 2004, and 7. yunnanensis), dorsal scale rows 19-21 (vs >23 rows in 7: andersoni Theobald, 1868, T. cantori (Blyth, 1846), 7. erythrurus, T: gracilis Oshima, 1920, T: gumprechti, T. labialis (Steindachner, 1867), T. purpureomaculatus (Gray, 1832), 7: vogeli David, Vidal & Pawels, 2001, 7. stejnegeri, and 7: arunachalensis), eye sized in relation to head not large, DEYE 2.33 (DEYE 4.03-4.46 rela- tively large eyes in 7! cardomomensis Malhotra, Thrope, Mrinalini & Staurt, 2011, 7’ macrops Karmer, 1977, and T. rubeus Malhotra, Thrope, Mrinalini & Staurt, 2011), dorsum green with a yellow tinge bearing a yellowish ventrolateral stripe along the body lacking any dorsal markings (vs dorsum reddish brown to grey, black, or green with dark markings in 7: tibetanus Huang, 1982, T. flavomaculatus (Gray, 1842), T: fasciatus (Bouleng- er, 1896), 7) arunachalensis, T; malabaricus, T: strig- atus, T. kanburiensis Smith, 1943, T. puniceus (Boie, 1827), T. schultzei Griffin, 1909, 7: mutabilis Stoliczka, 1870, T. honsonensis (Grismer, Ngo & Grismer, 2008), T! malcolmi Loveridge, 1938, T: wiroti Trutnau, 1981, T. venustus, Vogel, 1991, T. mcgregori Taylor, 1919, 7: sumatranus (Raffles, 1822), 7. andersonii, T: labialis, T. andalasensis David, Vogel, Vijaykumar & Vidal, 2006, T! borneensis (Peters, 1872), T. brongersmai Hoge, 1968, 7: cantori); 167-171 ventrals (vs 136-150 in 7. brongersmai, 141-149 in T: gracilis, 133-143 in T. mac- rolepis, 143-158 in T: malabaricus, 138-149 in T: me- doensis, and 128-150 in T. strigatus). The new species is most similar to 7) septentrionalis, T. insularis Kramer, 1977, and T: albolabris in its sca- lation but differs in bearing an orange to reddish stripe from the lower margin of the eye to the posterior of the posterior border of the mouth in males (vs a white stripe from the posterior border of the nasal to posterior part of the head in 7: septentrionalis and T: albolabris), hemip- enis short and bilobed (vs long and deeply forked in 7. septentrionalis and T: albolabris), palatine with six teeth (vs five in 7. albolabris, T. insularis, and T: septentrion- alis), pterygoid with 15 teeth (vs 11 in 7: septentrionalis, 16 in T. insularis, and 12 in T albolabris),; 19 dentary Mirza, Z.A. et al.: A new Trimeresurus from Arunachal Pradesh teeth (vs 11 in 7: septentrionalis, 12 in T: albolabris, and 14 in T. insularis). A comparison of selected characters is presented in Table 1. Description of holotype male BNHS 3554. The speci- men is in a good state of preservation, set in a coil with its head placed out of the coil (Fig. 1a). The left hemipenis is everted, and the specimen bears three ventral longitudinal incisions (Fig. 1b). The skin of the specimen seems a bit loose, likely an artifact of preservation. Body long and thin, SVL 415 mm; head triangular and elongate, head length 16.2 mm (HL/SVL 0.04); head width 12.6 mm; (HW/HL 0.77) clearly distinct from neck; distance between nostrils 3.2 mm; distance between preoculars 6 mm; distance between tip of snout and anterior border of eye 6.6 mm; distance between nostril to eye 4.8 mm. Canthus rostralis distinct; four scales between internasal and supraocular. Rostral sub- triangular, slightly visible when viewed from above; nasal and first supralabial fused, with only a trace of a suture, sub-pentagonal, wider than high (2.7 mm high, 3.2 mm wide); a pair of subrectangular interna- sals aligned in a straight manner bordered by six scales on its posterior margin; second supralabial and two preoculars encompass the loreal pit; the lower preocu- lar forms the lower margin of the loreal pit (Fig. 2a, b); one elongate and narrow supraocular (0.8 mm wide, 3.9 mm long); cephalic scales (CEP) small, much irregular, subimbricate, smooth; LCS 35, more elongate near the neck; 12 CEP between supraoculars (Fig. 2d); occipi- tal scales smooth; four to five rows of scales between the internasals and anterior border of the supraoculars flat and irregular in their shape; the rows posterior to these have a slight dome shaped appearance; temporals smooth and subequal; subocular crescent shaped; 11/11 supralabials; 1‘ supralabial fused with nasal scale, 2" as high as the 1* supralabial, nearly of the same width throughout; 3" supralabial longest among the supral- abials, lower preocular, subocular and 4" supralabial; 4 supralabial small, separated from the subocular by a single row of smooth scales; 5" supralabial in contact with temporal; the remaining supralabials slightly de- creasing in size posteriorly and in contact with temporal scales; 12/13 infralabials, the first pair in contact with each other; the first three pairs in contact with anterior Table 1. Summary of selected characters for members of the clade containing 7! albolabris. Species Dorsal colouration Colour of lateral Dorsal scale TaL/TL Ventrals Subcaudals —Hemipenis stripe on head rows at midbody T. albolabris Green White 21 0.19-0.24 149-173 48-67 bilobed, long T. andersonii Brown or black - 23 or 25 0.16 171-183 53-74 bilobed, long T. cantori Green or brown White 27 or 31 0.12-0.20 170-176 44-73 bilobed, long T. erythrurus Green White 23 0.16/0.21 151-174 49-67 bilobed, long T. fasciatus Brown - 21 0.18-0.21 158-163 63-65 bilobed, long T. insularis Green or blue - 21 0.21-0.22 156-167 54-75 bilobed, long T. purpureomaculatus — Black to dark brown - Zato 7 0.16-0.19 152-183 54-79 bilobed, long T. septentrionalis Green White wh 0.19-0.24 160-181 55-83 bilobed, long T. salazar sp. nov. Green Reddish orange 19 0r 21 0.14-0.18 163-171 59-74 bilobed, short zse.pensoft.net Zoosyst. Evol. 96 (1) 2020, 123-138 LOY: Figure 2. 7rimeresurus salazar sp. nov. holotype male BNHS 3554. A. Right view of head; b. Left view of head; c. Ventral view of head; d. Dorsal view of head. Not to scale. chin shields; six pairs of chin shields, each pair in con- tact medially; separated from infalabials by 1-5 scale rows (Fig. 2c). Palatine with six teeth; pterygoid with 15 teeth; 19 dentary teeth (Fig. 4). Body scalation: 21 dorsal scales one head length behind the head; 19 dorsal scales at midbody; 15 dorsal scales one head length anterior to the vent; dorsal scales rhom- boid, moderately keeled except for the first row which is smooth; three preventrals; 167 ventral scales; 71 subcau- dal scales; paired; anal shield entire. Eye large, with VED/ DEL ratio 0.85; tail short; ventrally depressed; Tal 94 mm; TaL/TL 0.18. Tail prehensile. Hemipenis short, bilobed, not deeply forked, extending to the 8" caudal scale. Colouration in life (Figs 5a, b, 7): dorsum green with a yellow tinge; ventrum creamy white with the dorsal green colour diffusing into the ventral scales on the ven- tral scale margins. Head dorsally dark green, which fades to lighter green to yellowish green on the nasal, labials, and preoculars. A reddish orange stripe extends from the posterior borders of the preoculars, running through the lower margin of the eyes to the lateral side of the nape. Dorsal scales yellowish green except for the first dorsal scale row, which is yellowish white and bears a faint or- ange patch; the yellow and the orange appears as a bic- oloured lateral stripe running form the neck to the vent. Tail rusty red dorsally and orange ventrally. Juveniles are brightly coloured, and the lateral stripe on the head is more prominent (Fig. 7). Colouration in preservative (Figs la, b, 2a—c): green on dorsum has turned darker and the yellow has faded to white. In some patches, the green has turned light blue. The orange stripe along the head and along the body 1s not visible. The tail is reddish brown and has lost all trace of the orange tinge. zse.pensoft.net 128 Mirza, Z.A. et al.: A new Trimeresurus from Arunachal Pradesh Figure 3. 7rimeresurus salazar sp. nov. holotype male BNHS 3554 in life. a. Body habitus; b. Left lateral view of head. zse.pensoft.net Zoosyst. Evol. 96 (1) 2020, 123-138 129 Figure 4. Micro-CT volume rendered images of the skull and mandibles of the holotype male BNHS 3554. a. Dorsal view; b. Left lateral view; c. Ventral view (lower jaw clipped for representation purpose). Scale bars: 2.5 mm (a, b); 2 mm (c). zse.pensoft.net Mirza, Z.A. et al.: A new Trimeresurus from Arunachal Pradesh Figure 5. 7rimeresurus salazar sp. nov. paratype female BNHS 3555 in life. a. Body habitus; b. Left lateral view of head. _ Trimeresurus andersonii Seta Trimeresurus erythrurus 90/0.96 - Trimeresurus purpureomaculatus smn Trimeresurus cantori TE 08 --- Trimeresurus albolabris = —<—<—< Trimeresurus insularis - Trimeresurus fasciatus - Trimeresurus septentrionalis ‘imeresurus salazar paratype imeresurus salazar holotype Figure 6. ML phylogeny of selected members of the subgenus Trimeresurus based on partial sequences of mitochondrial /6S and ND4 gene generated through 1000 non-parametric bootstrap pseudoreplicates under the GTR + G model of sequence evolution. Numbers at nodes represent ML bootstrap support and BI posterior probabality. See Appendix III for a complete tree. Variation: the paratype female BNHS 3555 agrees with the description of the holotype in most aspects ex- cept for the following difference: 21 dorsal scales rows at one head length posterior to head and at midbody; 15 dor- sal scale rows one head length anterior to vent; 10 CEP; 32 scales in a longitudinal row from internasals to neck; 10/10 supralabials; 13/12 infralabials; 171 ventral scales (+3 preventral scales); 59 paired subcaudal scales; SVL 363mm; TaL 60mm; TaL/TL 0.14. The species is sexu- ally dichromatic; the male bears a reddish orange stripe running from the margin of the eye and subocular through zse.pensoft.net the temporal region, and from the neck to the vent as a lateral stripe, which the female lacks (Fig. 5a, b). See Ap- pendix III for images of the paratype. Two male speci- mens from Assam, ZMUC R69255 bore ventrals 164 (+2 preventrals) and 74 subcaudals, and ZMUC R69256 ven- trals 163 (+2 preventrals) and 71 paired subcaudals. Natural history notes: the type specimens were found during night search between 1800-2200 hours along a road. Both individuals were found coiled on shrubs along the road. A third individual was seen but escaped in the thick undergrowth. Three individuals were seen during Zoosyst. Evol. 96 (1) 2020, 123-138 131 Figure 7. 7rimeresurus salazar sp. nov. juvenile male from Pakke Tiger Reserve photographed in 2008. Photo by Aamod Zambre and Chintan Seth, Eaglenest Biodiversity Project. night searches in six nights. Other serpent species ob- served at the locality include Boiga gokool (Gray, 1834), Boiga cyanea (Dumeril, Bibron & Dumeéril, 1854), and Lycodon jara (Shaw, 1802). For now, the new species is known only from the type locality. The specimens, ZMUC R69255 & ZMUC R69256 were collected from Assam and it is likely that the new species may occur in the adjoining state sharing similar biotope. Discussion Phylogenetic relationships within the genus 7rimeresurus are resolved, and the studies by Malhotra and Thorpe (2000, 2004) and Giannasi et al. (2001) have demonstrat- ed presence of distinct clades which have been assigned subgeneric status. David et al. (2011) suggested valuable amendments in the nomenclature of the generic splitting, and in following them, we thus assign the new species to the subgenus 7rimeresurus Lacépéde, 1804. There is a broad gap in our knowledge of the species diversity in India, especially in northeastern India, as we lack molecu- lar data for samples across the northeastern Indian states. However, based on a short fragment of /6S and ND4 gene, the new species was recovered as sister to 7’ septentrion- alis (Fig. 6), with moderate support (ML bootstrap 58 and BI posterior probability 0.74), from which it differs by an uncorrected pairwise sequence divergence (p-distance) of 6% on ND4 (Appendix II). Furthermore, the new species is recorded from an elevation of 172 ma.s.l. and is distrib- uted in the eastern parts of the Himalayan range, whereas 7. septentrionalis is distributed between above 700-3050 m a.s.l. from central Nepal to Himachal Pradesh in the Western Himalayas (Kramer 1977; Regenass and Kramer 1981; Whitaker and Captain 2004; Sharma et al. 2013). This is the second species of snake discovered after 7ra- chischium apteii Bhosale, Gowande & Mirza, 2019 (Bho- sale et al. 2019) from the Arunachal Pradesh expedition zse.pensoft.net N32 led by the authors, which merely reflects the poor nature of biodiversity documentation across northeastern India. Fu- ture dedicated surveys conducted across northeastern In- dia will help document biodiversity, which is under threat from numerous development activities that include road widening, agriculture, and hydro-electric projects. Acknowledgements We thank the Forest Department of Arunachal Pradesh for issuing the necessary permits (permit no. CWL/ Gen/173/2018-19/Pt.V11/2421-33 to GG and CWL/ Gen/173/2018-19/Pt.V11/2434-43 to ZM) to conduct sur- veys across the state. Singinawa Conservation Foundation supported ZAM. HB and his team extend their most heart- felt gratitude to Shripad Halbe and Brihad Bharatiya Sam- aj for their generous support in funding the expedition. We also thank Sandesh Kadur, Buban Gogoi and Debabrata Phukon for his help with the logistics, and Deepak Apte and Rahul Khot BNHS for their constant support. GG was partially supported by the Rufford Small Grants for Na- ture Conservation. ZAM acknowledges support from the NCBS Infosys Travel Grant to visit the NHM, MNHN, and ZMUC. GG 1s indebted to the Principal, Abasaheb Garware College, to the HoD, Annasaheb Kulkarni De- partment of Biodiversity, Abasaheb Garware College, and to the Principal, Fergusson College, and to the HoD, Bio- technology, Fergusson College and his advisor Dhanashree Paranjpe, for their constant support and encouragement. ZM thanks K. VijayRaghavan for guidance and to all lab mates for their support. Visit to museums would not have been possible without the help of an Infosys Travel Grant. Special thanks go to Sunil P. (NCBS EM facility) for help with CT scan and data analysis, which benefitted the manuscript greatly and the NCBS sequencing facility. We thank Aamod Zambre for sharing and allowing us to use the image of the juvenile from Pakke Tiger Reserve. ZM acknowledges Nicolas Vidal (MNHN), Patrick Campbell (NHM), and Peter Moller (ZMUC) for providing access to the museum collections. The manuscript greatly benefit- ted from constructive comments by the reviewer. References Ao J, David P, Bordoloi S, Ohler A (2004) Notes on a collection of snakes from Nagaland, northeast India, with 19 new records for this state. Russian Journal of Herpetology 11: 155-162. Bhosale HS, Gowande G, Mirza ZA (2019) A new species of fossorial natricid snakes of the genus Trachischium Gunther, 1858 (Serpentes: Natricidae) from the Himalayas of northeast India. Comptes Rendus Biologies 342: 323-329. https://doi.org/10.1016/j.crvi.2019.10.003 Captain A, Deepak V, Pandit R, Bhatt B, Athreya R (2019) A new spe- cies of pitviper (Serpentes: Viperidae: 7rimeresurus lacepede, 1804) from west Kameng District, Arunachal Pradesh, India. Russian Journal of Herpetology 26: 111-122. https://doi.org/10.30906/1026- 2296-2019-26-2-111-122 zse.pensoft.net Mirza, Z.A. et al.: A new Trimeresurus from Arunachal Pradesh David P, Mathew R (2005) Notes on some noteworthy snake specimens deposited in the collections of Eastern Regional Station of the Zo- ological Survey of India. Records of Zoological Survey of India. Occasional Papaer 104: 83-90. David P, Vogel G, Dubois A (2011) On the need to follow rigorously the rules of the code for the subsequent designation of a nucleospecies (type species) for a nominal genus which lacked one: the case of the nominal genus 7rimeresurus Lacépede, 1804 (Reptilia: Squamata: Viperidae). Zootaxa 51: 1-51. Dowling H (1951) A proposed standard system of counting ventrals in snakes. British Journal of Herpetology 11: 97-99. Figueroa A, Mckelvy AD, Grismer LL, Bell CD (2016) A species-level phylogeny of extant snakes with description of a new colubrid sub- family and genus. PLoS ONE 11: e0161070. https://doi.org/10.1371/ journal.pone.0161070 Giannasi N, Thorpe RS, Malhotra A (2001) The use of amplified frag- ment length polymorphism in determining species trees at fine taxonomic levels: analysis of a medically important snake, 77ri- meresurus albolabris. Molecular Ecology 10: 419-426. https://doi. org/10.1046/j.1365-294X.2001.01220.x Gumprecht A, Tillack F, Orlov NL, Captain A, Raybov S (2004) Asian pitvipers. GeitjeBooks Berlin, Berlin, 368 pp. Heatwole H (2009) Biology of the Reptilia. Volume 20, morphology H, the skull of Lepidosauria. In: Gans C, Gaunt AS, Adler K (Eds) Society for the Study of Amphibians and Reptiles, Ithaca, 610-611. https://do1.org/10.1093/icb/icp063 Kramer E (1977) Zur Schlangenfauna Nepals. Revue suisse de Zoologie 84: 721-761. https://do1.org/10.5962/bhI1.part.91420 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing plat- forms. Molecular Biology and Evolution 35: 1547-1549. https://doi. org/10.1093/molbev/mst197 Lanfear R, Calcott B, Ho S, Guindon S (2012) PartitionFinder: com- bined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution 29: 1695— 1701. https://doi.org/10.1093/molbev/mss020 Leary S, Underwood W, Anthony R, Cartner S (2013) AVMA guidelines for the euthanasia of animals: 2013 edition. Schaumburg, IL: Ameri- can Veterinary Medical Association, 98 pp. Malhotra A, Thorpe RS (2000) A phylogeny of the 7rimeresurus group of pit vipers: new evidence from a mitochondrial gene tree. Mo- lecular Marine Biology and Biotechnology 16: 199-211. https://doi. org/10.1006/mpev.2000.0779 Malhotra A, Thorpe RS (2004) A phylogeny of four mitochondrial gene regions suggests a revised taxonomy for Asian pitvipers (7rimere- surus and Ovophis). Molecular Phylogenetics and Evolution 32: 83-100. https://doi.org/10.1016/j.ympev.2004.02.008 Mirza ZA, Gowande G, Patil R, Ambekar M, Patel H (2018) First ap- pearance deceives many: disentangling the Hemidactylus triedrus species complex using an integrated approach. PeerJ: 6:e5341. https://doi.org/10.7717/peerj.5341 Mirza ZA, Patel H (2018) Back from the dead! Resurrection and reval- idation of the Indian endemic snake genus Wallophis Werner, 1929 (Squamata: Colubridae) insights from molecular data. Mitochon- drial DNA Part A: DNA Mapping, Sequencing, and Analysis 29: 331-334. https://doi.org/10.1080/24701394.2016.1278536 Mirza ZA, Vyas R, Patel H, Maheta J, Sanap RV (2016) A new Mio- cene-divergent lineage of Old World racer snake from India. 133 Zoosyst. Evol. 96 (1) 2020, 123-138 PloS ONE 11 (3): pone.0148380 Mulcahy DG, Lee JL, Miller AH, Zug GR (2017) Troublesome tri- mes: potential cryptic speciation of the 7rimeresurus (Popeia) po- e0148380. https://doi.org/10.1371/journal. peiorum complex (Serpentes: Crotalidae) around the isthmus of Kra (Myanmar and Thailand). Zootaxa 4347: 301-315. https://do1. org/10.11646/zootaxa.4347.2.6 Pyron RA, Kandambi HKD, Hendry CR, Pushpamal V, Burbrink FT, So- maweera R (2013) Genus-level phylogeny of snakes reveals the or- igins of species richness in Sri Lanka. Molecular phylogenetics and Evolution 66: 969-978. https://doi.org/10.1016/j.ympev.2012.12.004 Regenass U, Kramer E (1981) Zur Systematik der griinen Grubenottern der Gattung 7rimeresurus (Serpentes, Crotalidae). Revue Suisse de Zoologie 88: 163-205. https://doi.org/10.5962/bhl1.part.82363 Ronquist F, Huelsenbeck J (2003) MrBayes 3: Bayesian phylogenet- ic inference under mixed models. Bioinformatics 19: 1572-1574. https://do1.org/10.1093/bioinformatics/btg 1 80 Sanders KL, Malhotra A, Thorpe RS (2004) Ecological diversification in a group of Indomalayan pitvipers (7rimeresurus): convergence in taxonomically important traits has implications for species identi- fication. Journal of Evolutionary Biology 17: 721-731. https://doi. org/10.1111/j.1420-9101.2004.00735.x Sanders KL, Malhotra A, Thorpe RS (2006) Combining molecular, morphological and ecological data to infer species boundaries in a cryptic tropical pitviper. Biological Journal of the Linnean Society 87: 343-364. https://doi.org/10.1111/j.1095-8312.2006.00568.x Appendix I Sharma SK, Pandey DP, Shah KB, Tillack F, Chappuis F, Thapa CL, Alirol E, Kuch U (2013) Venomous snakes of Nepal. Lalitpur, 75 pp. Silvestro D, Michalak I (2012) RaxmlGUI: a graphical front-end for RAXxML. Organisms Diversity & Evolution 12: 335-337. https:// doi.org/10.1007/s13127-011-0056-0 Smith MA (1943) Fauna of British India, Ceylon and Burma, including the whole of the Indo-Chinese sub-region. Reptilia and Amphibia (Vol. 3). Serpentes. Taylor and Francis, London, 583 pp. Thompson J, Gibson T (2002) Multiple sequence alignment using ClustalW and ClustalX. Current Protocols in Bioinformatics 00(1): 2.3.1—2.3.22. https://doi.org/10.1002/0471250953.bi0203s00 Wallach V, Williams K, Boundy J (2014) Snakes of the world: a cata- logue of living and extinct species. Taylor & Francis Group, Lon- don, 1257 pp. https://doi.org/10.1201/b16901 Whitaker R, Captain A (2004) Snakes of India. The Field Guide. Draco Books, Chennai, 481 pp. Zhong G, Zhu F, Yan F, Fu X, Fang M, Tang T, Xiao R, Wang P, Guo P, Liu Q (2015) Cryptic diversity of green pitvipers in Yunnan, south- west China (Squamata, Viperidae). Amphibia Reptilia 36: 265-276. https://doi.org/10.1163/1568538 1 -00003004 Zhu F, Liu Q, Che J, Zhang L, Chen X, Yan F, Murphy R, Guo C, Guo P (2016) Molecular phylogeography of white-lipped tree viper (77i- meresurus, Viperidae). Zoologica Scripta 45: 252-262. https://doi. org/10.1111/zsc.12156 Genbank accession numbers for sequences used in the current study. Name ND4 Trimeresurus albolabris KF311102 AY352837 Trimeresurus andersonii AY352740 AY352835 Trimeresurus borneensis AY352722 AY352817 Trimeresurus cantori AFO57243 AY352836 Trimeresurus Cardamomensis KRO21137 KRO21070 Trimeresurus erythrurus AF517174 AY352834 Trimeresurus fasciatus GQ428466 GQ428482 Trimeresurus flavomaculatus AY059551 AY059584 Trimeresurus gracilis AY352728 AY352823 Trimeresurus gramineus AY352731 AY352827 Trimeresurus gumprechti AY352736 AY352832 Trimeresurus hageni AY059552 AY059585 Trimeresurus insularis AF517172 AY059586 Trimeresurus kanburiensis AY352737 - Trimeresurus macrops AF517176 AF517219 Trimeresurus malabaricus AY059564 AY059587 Trimeresurus malcolmi AY371793 AY371861 Trimeresurus mcgregori AY371795 - Trimeresurus medoensis AY352735 AY352831 Trimeresurus popeiorum AY059554 AY059590 Trimeresurus puniceus AF517177 AF517220 Trimeresurus purpureomaculatus AY352745 AY352840 Trimeresurus rubeus KRO21141 KRO21075 Trimeresurus Salazar BNHS 3554 MN684366 NA Trimeresurus Salazar BNHS 3555 MN684365 MN686204 Trimeresurus schultzei AY352725 AY352819 Trimeresurus septentrionalis AY352724 AY352818 Trimeresurus sichuanensis HQ850446 HQ850450 Trimeresurus stejnegeri FJ752492 AY059595 Trimeresurus sumatranus AY371788 AY371866 Trimeresurus tibetanus AY352715 AY352810 Trimeresurus trigonocephalus KC347374 AY059597 Trimeresurus truongsonensis EU443818 EU443816 Trimeresurus venustus AY352723 AY 289228 Trimeresurus vogeli AF517183 AF517225 Trimeresurus yunnanensis EU443812 EF597527 zse.pensoft.net 134 Mirza, Z.A. et al.: A new Trimeresurus from Arunachal Pradesh (a0) rap) oy 2 o) = aie By oo LO ome me) won st Ql Se CO0O°0 TON AM me a ee ee CO0O0°0 NN TtTAOON i aa Ove COOC0CO tTMOMUNO MMS = ee ee eee ee ee OOo 02-000 5+ToaUoMARTtTOWOSFS ai aoe TH OTD OOH O00 C0000 OMA MNTtTOOSF at At et SS est et COCOCO0OCCO Ke) MtTMNMNNOMNM LC ai ce a ee re ee [ee aa >) Ooooo0o00co0°o WO ™M Norns TmoOM +t a —| oHOn nA Aaa ae ome) Oooocoodco NtM™M NTMNO MONA ST zy aoe aaa aA OW Wi Qo °o Oooooo0o 000 (0.0 el ee A) NTN WT TW MR o ee ee em ee | oS Soo Ss oS ot tO 2:2 C' © Qooooooeod0 ouonnn ly NMOUOMAMOWMO WM Wo OD eSsaaa st «i Ss so st os Ss Ss Ss et ooo0o°0o Qoooo°coc7oe°o OMNMM + NMOTMNNDO NA ST 00 esos st st st es Sos sos St os st et he COO0O0C0O Ooooo0o odo NOMNNANMNN & ONAMNAMNARM®™M is ee ee ee ee ee | Onn nA OTD OO Oo 0 0000 Qoooqoco coo NatMONMOMNA HOTA NN THOM = Ce BE ce se | Ce A ee OO ee Oe Oe Ee QD | COCCOCO0OCO COCOCO0OCCO 5tTMONOOoOWWNMNWoOSTt oOtrtroOmMaRMNAO 1 Ss Soe Ss ies eS est et lS Ce A ce A ee I es GO se Oe (| Oo 00 00000 Ooooo00 00 0 nvyrurstvytrnuNoMWNn NONONMDMOoMNWoO TM wt Sool St ot Ot ed Hh St dt Our rs COOCO0OCOCO COCOCCO0OCCO onastyMNMARaATTRW tToOtTONOTMNOA © Faq Seti tal tl Gn aa Oni Tred ot) de AL tl eA COOCOOOOOOCO O00 00 0 000 Lost st © bop? a st 1 aN Sha OA TANT OD AST NRDMDODMDOUOTRORHAWOWORNNMONRRR OO nNnOnRMOnRODARMO OO 3 ~ a Seanad Pare le rl) Srl rat ele lard or eather te le te ad art ol aS Qt@- @) ©: .©O..O ©.-O" 2: OfO7@: ©7 O10 (OxO-O: OO" OO OCs ©. 1O.@: ©, ©: 2 ort tMNO*nwMOotTMntTATONMNMNMNARTANNWOST MOMNMMNMNOMM LC D N aAOTFTOO TATA TA A THO TDA ATA TAT THOTT OO aaa aA IAAI OO 5 COCDOCOOOOOOOOOOOOOOOOCO OO Q'O-O.:O: OEO+O: OF@: 3 MoHNoNFTORNONMN KR NOMNMNMNNR OMMN WO + MAMtTMNO NO tM LC —N —t O7AODOT OO FAA TATA TBHO DATA ATH TWTOW TWO 4 rr! et tet ot ©) pg OOO. O! O--O.OV'O"O-O-.©: @LOLO: O.1.O4O' (O72 OO“@ OO OOO OvO-O: Oo: (S) n S 2 g — a @ Ge x 2 w = —_ vA nA 5 2 aS 2 ror _— ao! bong os & ro) oa oe 2 “eS x a. 2 eS os yn 8 5 = iS = 2) g qo ao os ¥ o 235 QS Oy fA re -— 2 FR eo os ary — uo] eS 0 SS Sok os te BE eS et Sc SSEaolFsesgog ge oY ne) ® Se Sf Ske Sk eh oes SS OS en SG PS 8 tee Bos s B | SSSEZELESSREESSS SLSR SESS SRSSEESSSSRES Sy EO a: & S05" 22 oF SS a3 AD yeah ES os oO t Se acogiemmot SEER RASHES EAE RES SE LSS oF 3 ee a RO ROR RS RR OO > oO. 1 S OFA NMTANOORWADOANWNMST OWOORARDOHANM = ANMTNOR ODA HA FAA aaa ANNNANN NNNANNMMM MY zse.pensoft.net Zoosyst. Evol. 96 (1) 2020, 123-138 Appendix II 135 ML phylogeny of 7rimeresurus based on partial sequences of mitochondrial 16S rRNA and ND4 gene generated through 1000 non-parametric bootstrap pseudoreplicates under the GTR + G model of sequence evolution. Numbers at nodes represent ML bootstrap support.Appendix I'V 22 11 39 oy) 35 56 81 22 42 13 74 72 26 0.05 98 86 100 100 82 72 35 46 99 Trimeresurus erythrurus Trimeresurus purpureomaculatus Trimeresurus andersonii Trimeresurus cantori Trimeresurus albolabris Trimeresurus fasciatus Trimeresurus insularis Trimeresurus Salazar sp. nov. Trimeresurus septentrionalis Trimeresurus popeiorum Trimeresurus tibetanus 88 91 Trimeresurus cardamomensis Trimeresurus venustus Trimeresurus macrops Trimeresurus rubeus Trimeresurus sichuanensis Trimeresurus gumprechti Trimeresurus stejnegeri Trimeresurus vogeli Trimeresurus yunnanensis Trimeresurus malcolmi 53 Trimeresurus gramineus Trimeresurus medoensis Trimeresurus truongsonensis Trimeresurus gracilis Trimeresurus hageni Trimeresurus flavomaculatus Trimeresurus schultzei Trimeresurus Sumatranus Trimeresurus malabaricus Trimeresurus borneensis Trimeresurus puniceus Trimeresurus trigonocephalus zse.pensoft.net 136 Images of female paratype of 7: salazar sp. nov. zse.pensoft.net Mirza, Z.A. et al.: A new Trimeresurus from Arunachal Pradesh Zoosyst. Evol. 96 (1) 2020, 123-138 L374 zse.pensoft.net 138 Mirza, Z.A. et al.: A new Trimeresurus from Arunachal Pradesh zse.pensoft.net