First record of two species of venomous snakes Bungarus suzhenae and Ovophis zayuensis (Serpentes: Elapidae, Viperidae) from India

: We report Bungarus suzhenae Chen, Shi, Vogel, Ding & Shi, 2021 and Ovophis zayuensis (Jiang, 1977) for the first time from India. Specimens of B. suzhenae and O. zayuensis were collected during our field surveys in north (Arunachal Pradesh) and south (Nagaland-Manipur border) of the river Brahmaputra. Species identity was supported by partial cytochrome b (cyt b ), and 16s mitochondrial gene. We provide a detailed morphological description and a key to the two genera of this region. This report extends the westernmost distribution of B. suzhenae by ca. 300 km from Myanmar, and the southernmost range of O. zayuensis by 170 km from Tibet. Until now eight species of Bungarus and only one Ovophis species have been reported from India. Ovophis species are recently reported to be medically important venomous snakes whose venom properties have not been investigated in depth.


MATERIALS AND METHODS
We carried out field surveys during October 2012 in the Nagaland-Manipur border, between June and July 2022-2023 in Arunachal Pradesh, northeastern India (Figure 1).Collected specimens were fixed in formalin, washed, stored in 70% ethanol, and housed in the repository at the Wildlife Institute of India, Dehradun (WII-ADR1241, WII-ADR3316 and WII-ADR3491).Before fixation, live snakes were photographed, and liver tissues were collected and stored in absolute ethanol at -20°C.

Molecular phylogenetic analysis
Genomic DNA was extracted from liver tissue samples using DNeasy Blood and Tissue Kit (Qiagen™, Germany).For the Bungarus specimen (WII-ADR1241), we amplified and sequenced fragments of one mitochondrial gene, cytochrome b (cyt b) using the primers L14910 (5'-GACCTGTGATMTGAAAACCAYCGTTGT-3') and H16064 (5'-CTTTGGTTTACAAGAACAATGCTTTA) (Burbrink et al. 2000).Polymerase Chain Reaction (PCR) conditions followed was initial denaturation at 95°C for five minutes, followed by 35 cycles of denaturation at 95°C for 45 sec, annealing at 54°C for 45 sec, and extension at 72°C for 55 sec.The final extension was at 72°C for 10 min.For the Ovophis sample (WII-ADR3491) we amplified and sequenced fragments of 16s gene, using the primers 16Sar (5'-CGCCTGTTTATCAAAAACAT-3') + 16Sbr (5'-CCGGTCTGAACTCAGATCACGT-3') (Palumbi et al. 1991).Polymerase chain reaction (PCR) condition followed was initial denaturation at 95°C for five minutes, followed by 35 cycles of denaturation at 95°C for 35 sec, annealing at 55°C for 45 sec, and extension at 72°C for one minute.The final extension was at 72°C for 10 min.Amplified PCR products were run on a 2% agarose gel and viewed under a UV transilluminator.The purified PCR product was sequenced directly in an Applied Biosystems Genetic Analyzer 3500 XL in both directions using BigDye v3.1 kit.
We manually checked bidirectional sequences using the CHROMAS v2.6.6 software (http://technelysium. com.au/wp/chromas/) and aligned using ClustalW (Thompson et al. 199) with default prior settings implemented in MEGA v7. 1 (Kumar et al. 2016).For the protein-coding gene (cyt b), we checked for unexpected stop codons by translating the sequence to amino acids in MEGA v7. 1 (Kumar et al. 2016).We used Naja atra and Protobothrops mucrosquamatus as outgroup for phylogenetic analyses of Bungarus and Ovophis, respectively.The newly generated sequences were aligned with the sequences downloaded from GenBank J TT (Benson et al. 2009) (Appendix 1).
We performed maximum likelihood (ML) analyses using IQ-TREE (Nguyen et al. 2015), implemented in the web server version (http://iqtree.cibiv.univie.ac.at) (Trifinopoulos et al. 2016).For cyt b, the dataset was partitioned by codon positions using Modelfinder (Kalyaanamoorthy et al. 2017), to find the best-fit partitions and models of sequence evolution for each partition (Appendix 2).Support for internal branches was quantified using the bootstrap approximation (UFB 1000 pseudoreplicates) (Minh et al. 2013).Uncorrected P-distance was calculated in MEGA v7.1 (Kumar et al. 2016) with pairwise deletions of missing data and gaps.

Morphological character scoring and identification
Total length, snout-vent length, and tail length were measured with a thread and a metre tape in millimetre (mm).Morphometric measurements were taken with the help of a digital calliper (Mitutoyo TM) to the nearest 0.1 mm.Length and width of head scales were measured at the longest and the widest points of the respective scales.Morphological comparisons follow Das et al. (2021).All measurements were taken on the specimen's right side and scales were counted on both sides for bilateral scales.Morphometric and meristic characters were examined with the help of the Olympus SZX10 microscope.Dorsal scale rows (DSR) were counted at one head length behind the head, at midbody, and at one head length anterior to the anal scute.Ventral scales were counted following Dowling (1951).Bilateral scale counts separated by a comma are given in left, and right order.Abbreviations used are WII-ADR: Wildlife Institute of India Abhijit Das Reptile collection.

Molecular phylogeny
Based on mitochondrial gene cyt b, the specimen of Bungarus from Northeastern India clustered with the type sequences of B. suzhenae with strong bootstrap value (100) (Figure 2).Uncorrected P-distance between   3) along with two other samples from Yunnan province, China and Chin state, Myanmar.These samples altogether form a sister relationship with the samples of O. zayuensis from Tongmai and Xizang provinces, China with low node support (Figure 3).Uncorrected P-distance between our newly collected samples of Ovophis and samples of O. zayuensis from China and Myanmar was minimal (Table 2).

Morphology
We confirmed the new krait specimen as Bungarus suzhenae based on the following set of diagnostic morphological characteristics (Chen et al. 2021): enlarged hexagonal vertebral scale rows present, dorsal scales in 15:15:15 rows, anterior chin shields are slightly larger than the posterior chin shields, dorsal body black with 34 white bands, ventral scales connected with the black bands of the dorsal body by small dark patches, the ventral surface uniform white, underside of tail white with tiny brown dots in the middle.
The two viper specimens we collected were confirmed as Ovophis zayuensis based on the following set of diagnostic morphological characteristics (Che et al. 2020): the third supralabial larger is than the fourth, the second supralabial is fused with the loreal, internasal scales are separated by two scales; the ventral scale count is 169-172, dorsal scale count is 23 or 25:23:19 rows, the subcaudal count of 34-48, mostly single and J TT some are paired, anal scale is single.
The hemipenes of the newly reported specimen of B. suzhenae from India agree with the description provided by Chen et al. (2021).Hemipenis can be divided into three distal calyculate region, middle spinose region, and basal region.The top of the hemipenis is slightly bilobed.The calyculate region is covered with welldeveloped small calyces.The spinose zone is covered with fang-shaped large keratinized spines and the basal region is covered with tiny spines; the basal region is nearly smooth towards the proximal end.
Dorsally head, body, and tail are uniformly dark brown.The rostral upper half is dark brown, the lower half (below the level of the lower edge of external nares) white; the nasals are also white below the level of the lower edge of external nares; supralabials are partly dark brown on the upper part and lower part (more than half of the scales) white; 34 narrow white cross bands on the dorsal body; bands widening on the flank before meeting This newly reported specimen was encountered along Jessami-Meluri road (25.6393 0 N, 94.5791 0 E, elevation 599 m), at the Nagaland-Manipur border ca.6 km from Meluri, Nagaland towards Jessami, Manipur.This individual was found while it was crossing the road at around 2100 h on 20 October 2012.The Tiji River flows ca.20 m away from the collection locality.A roadkill Bungarus niger was also recorded 100 m away from the location of B. suzhenae indicating their sympatric occurrence.The surrounding landscape was mostly with secondary vegetation with extensive jhum cultivation areas.While photographing, the snake showed headhiding behaviour and bit the snake bag in defence.

Description of Ovophis zayuensis (WII-ADR3316 and WII-ADR3491) (Image 3,4)
Both the specimens are adult females.Specimens are in good condition, latero-ventral incision between ventral scale 99-102 in WII-ADR3316 and between ventral scale 66-70 in WII-ADR3491.Head triangular, body stout and the dorsal scales are strongly keeled, ventrally plain pale yellow or orangish in color with no patterns.There are faint black blotches on the anterior dorsal region of the body and the blotches are dark on the posterior dorsal region.After preservation, the specimen's colour changed into smokey grey with black blotches on the dorsal side.Dorsally head is completely covered with small scales.The head length ranges 34.2-37.9mm, the head width is 23.1-25 mm, the eye diameter is 2.5-2.6 mm, the eye-to-nostril distance is 5.5-7.3mm, and the snout length is 10.1-11.3mm.No supralabials touching the eye, one preocular and three postocular present.Nasal single and undivided in both specimens.There is no distinguishable temporal, parietal, and frontal scales present.The second supralabial is fused with the loreal pit.The total length of the specimens is 826-900 mm, SVL 699-746 mm, tail length 127-154 mm, ventral scales 166-167 mm, anal scale single and undivided, subcaudals 46-49 with 5-6 paired.WII-ADR3491 has nine supralabials on the left, third is the

J TT
largest measuring 4.3 mm; eight supralabials on the right, third is the largest measuring 3.6 mm.Infralabials 10 on the left, the fourth is the largest measuring 3.2 mm; 11 infralabials on the right and the sixth is the largest measuring 2.8 mm, dorsal scale rows 25:23:19.WII-ADR3316 has nine supralabials on both sides, third is the largest measuring 3.7 mm.Infralabials nine on both sides, on the right sixth is the largest measuring 3.5 mm, and on the left, fifth is the largest measuring 3.3 mm, dorsal scales rows 23:23:19.
The individual WII-ADR3491 was recorded at Dri River bank (28.8056 0 N, 95.9321 0 E, elevation 1,288 m), near Etabe village (~2.5 km north-east from Anini), Dibang Valley district, Arunachal Pradesh.This individual was found while it was moving on the ground along the edge of a first-order stream on 16 August 2022 at 2000 h.The stream edge was covered with small to moderatesized boulders covered with moss and leaf litter, fern, and other vegetation.The recorded locality was ~100 m from the Dri river near Etabe village.Along the stream, we observed a few anuran species such as Xenophrys sp. and Amolops beibengensis on the vegetation and also an unidentified rodent species.The second individual WII-ADR3316 was collected from the newly constructed Chigu Pani road, Anini, Dibang Valley (29.0182 0 N, 95.9755 0 E, elevation 1,800 m).The habitat in this region was disturbed due to the recent construction of roads, small streams were blocked by retaining walls.It was found perched in a bush 50 cm above the ground around 1900 h between the walls, blocking the stream.There was a mild yet constant drizzle throughout the night.Many Amolops froglets were found near the blocked stream and on the road.This individual was a bit aggressive while trying to bag, it puffed up, flattened   its body, and struck the snake hook.The next day around 2000 h on the same road, close to a small stream two other individuals of O. zayuensis male and female were spotted crossing the road from both sides.The blocked streams were seen to be harbouring tadpoles of multiple species of frogs including Amolops sp. and Megophrys sp.These two snakes were removed from the roadside and released by the stream.

DISCUSSION
There are over 400 known species of snakes in India (Uetz et al. 2024), where 1.2 million snakebite deaths were recorded during the years 2000-2019 (Suraweera et al. 2020).The Mountain Pit Viper Ovophis monticola Günther, 1864 has recently shown to cause medically significant envenoming (Ralph et al. 2023) while the Bungarus species have always been among the big four snakes of medical importance from India (Simpson & Norris 2007;Suraweera et al. 2020;Sunagar et al. 2021).Bungarus suzhenae and Ovophis zayuensis are two venomous snakes being reported for the first time from India, from the border of Nagaland-Manipur and Arunachal Pradesh, respectively.
Among the 18 known species of Bungarus, the maximum diversity is congregated in southeastern Asia.Until now eight species of Bungarus have been reported from India and are known for frequently causing fatal envenoming in the region (Suraweera et al. 2020;Sunagar et al. 2021).Despite several fatalities by other Bungarus species, the commercial antivenom is manufactured only against the most widespread congener B. caeruleus (Sunagar et al. 2021).This new finding reported here also indicates the lack of systematic studies on this group of snakes especially in northeastern India.Identification of Bungarus species is The genus Ovophis has eight known species globally (Malhotra et al. 2011), and now two species from India, namely: O. monticola and O. zayuensis.Though not commonly found in conflict with humans some species of Ovophis are capable of delivering a severely painful or life-threatening bite (Ralph et al. 2023).The venom composition of these less medically significant snakes and their clinical effects on humans still need to be studied (Tan et al. 2021).The current distribution range of O. zayuensis extends into the upper region of Dibang Valley, Arunachal Pradesh, Northeast India.However, it is important to carefully look into the distribution of O. zayuensis from the rest of Arunachal Pradesh.Being in the transition zone, the geographical range of these and other such species is likely to overlap in northeastern India.Therefore, proper identification of such venomous species and understanding of geographic distribution is crucial for general awareness, venom research as well as development of lifesaving antivenin.
Northeastern India is known to have Tibeto-Yunanese, Indo-Malayan, and its own unique faunal elements (Das 1996;Giri et al. 2019;Lalronunga et al. 2020;Vogel et al. 2020;Deepak et al. 2021;Das et al. 2021).The   (Ao et al. 2004;Dasgupta & Raha 2006;Das 2018).Singh (1995) and Sinate et al. 2021 also reported B. fasciatus and B. niger from Manipur respectively.Since the present specimen of B. suzhenae was encountered at the Nagaland-Manipur border, there is a possibility of the occurrence of the species in other parts of Manipur.In a herpetofaunal biodiversity report of Nagaland, Grewal et al. ( 2011) provided a photograph of a road-killed unknown Bungarus sp. from Pungro-Shatuza road (nearest distance ca. 13 km from the present locality) which is similar to B. suzhenae (based on the lower number (<40) of unpaired white cross bands on body).Therefore, the distribution range of this medically important snake is likely to be wider than hitherto known in the region.Despite several reports of fatal snake bites by other species, commercial Indian antivenoms are only manufactured against Daboia russelii, Naja naja, Echis carinatus, and Bungarus caeruleus.Hence, it is important to recognise other medically important Indian snake species as it also has great significance in the field of snake antivenom design and manufacture (Simpson & Norris 2007).

Figure 1 .First
Figure 1.Map showing the locality of Indian records of Bungarus suzhenae (green) and Ovophis zayuensis (yellow).Type locality denoted by star, known locality shown as circles and new records denoted by circles with a black centre.
shields; first chin shield is in contact with four infralabials on the right side and three infralabials on the left side; second chin shields contact with fourth infralabial on both sides, posteriorly bordered by three scales on the right side and fourth scales on the left side.Dorsal scales on body and tail smooth, no keel and apical pit; vertebral scale row enlarged and hexagonal; dorsal scale rows 15:15:15; ventrals 219; anal plate single; subcaudals 57, undivided; terminal scute conical; snoutvent length 664 mm and tail length 109 mm.Details of the morphometric and meristic characters of the specimen (WII-ADR1241) are given in (Table

Figure 2 .
Figure 2. Maximum likelihood phylogeny of Bungarus based on cyt b.The newly generated sequences is shown in blue text.Bootstrap values <50 are not shown in the tree.

Figure 3 .
Figure 3. Maximum likelihood phylogeny of Ovophis based on 16s gene.The newly generated sequence is shown in blue text.Bootstrap values <50 are not shown in the tree.
of the GenBank sequences used in this study.The sequence of Bungarus suzhenae and Ovophis zayuensis generated in this study is given in bold.

Table 3 . Morphometric and meristic data of newly collected specimens of Bungarus suzhenae. All the measurements are given in millimetre. Bilateral characters are given in left, and right order separated by comma. "N" denotes sample size, "-" indicates data not provided.
triangular; mental smaller than rostral, bordered by first pair of infralabials; infralabial seven on both sides, fourth largest; first pair of chin shields slightly larger than the

Table 4 . Morphometric and meristic data of newly collected specimens of Ovophis zayuensis. All the measurements are given in millimetre.
..... O. zayuensis new record of B. suzhenae from India indicates the possibility of the occurrence of other Bungarus species reported from Myanmar such as B. magnimaculatus, B. wanghaotingi, and B. flaviceps.Therefore, a systematic study is necessary across northeastern India to document the diversity of medically important snakes.With the present report of B. suzhenae, the number of Bungarus species in India increases to nine, and the distribution range of B. suzhenae is extended by ca.321 km north-west from the nearest known locality, Yingjiang County, Yunnan Province, China.The geographical area of occurrence of B. suzhenae is currently ca.38,000 km 2 within China, Myanmar, and northeastern India.In earlier documentation, other Bungarus species such as B. bungaroides, B. fasciatus, and B. niger were reported from Nagaland