Addition of four new records of pit vipers (Squamata: Crotalinae) to Manipur, India

: In spite of the potential richness in biodiversity, Manipur is still one of the least explored areas in northeastern India. In this work, we report on our finding of four pit viper species, namely, Trimeresurus mayaae , T. salazar , T. erythrurus , and Protobothrops jerdonii in Manipur during our herpetological surveys in 2022. We affirm the identity of these specimens based on the combination of standard morphological characters and mitochondrial cytochrome b gene. Our study not only reports four new records of pit vipers for the first time in Manipur, but also extends their respective known geographical ranges. This information calls for further dissemination especially to health care sector, as it is on venomous snakes that could be of medical importance as regards human snakebites.


INTRODUCTION
Pit vipers are a group of front-fanged venomous snakes that comprise various genera of vipers having heat sensitive pit between each eye and nostril. Asian pit vipers that are distributed across southern and southeastern Asia are known to inhabit several types of habitats like wet, humid and montane forests, and also found in a few dry forest habitats (Gumprecht et al. 2004). Recent phylogeographic studies have largely addressed the taxonomic status of certain groups of pit vipers like Trimeresurus complex (Malhotra & Thorpe 1997, 2004David et al. 2011;Mallik et al. 2021) and Protobothrops (see Guo et al. 2009). Geographical diversity as well as sexual dimorphism often led to inaccurate taxonomic assignment among the closely similar pit viper species (Malhotra & Thorpe 1997, 2004. Consequently, pit vipers are still regarded to contain several taxonomically complex species in India. Recently, many such species complexes were revised including those from the Craspedocephalus malabaricus and C. macrolepis complexes (Mallik et al. 2021), the T. medoensis complex (Rathee et al. 2022), and the T. albolabris complex (Chandramouli et al. 2020;Mirza et al. 2020;Vogel et al. 2022).
Manipur is a hilly Indian state that lies at the extreme east of the northeastern region of India bordering Nagaland in the north, Mizoram in the south-east, Assam in the west, and Myanmar towards the east and southeast. In spite of the potential richness in biodiversity, zoological exploration in Manipur is rather limited (Roonwal 1948;Singh 1995;Pawar & Birand 2001) and there exist only a handful of snake faunal reports from the area till date (e.g., Mathew 2005;Sinate et al. 2021Sinate et al. , 2022Elangbam et al. 2022). Although Wallach et al. (2014) listed Manipur as the potential range of T. erythrurus, there is no confirmatory records from the area, and none of the aforementioned literature listed pit vipers from the snake fauna of Manipur except Ovophis monticola (see Whitaker & Captain 2008). In this paper, we provide notes on our new addition of pit vipers from Manipur based on our field documentations during our herpetological surveys in 2022.

Sampling and morphology
We conducted our field surveys after obtaining herpetological specimen collection permit from the Forest Department, Government of Manipur (permit no. #3/22/2018-WL [Vol-II]). Morphological characters of the newly collected specimens were taken. We followed Dowling's (1951) method for counting ventrals (Ve), and we excluded the terminal scute in counting the subcaudals (Sc). All the measurements are given in millimeter, and other acronym used for the morphological characters are: snout-vent length, SVL; tail length, TaL; inter-orbital distance (measured on cephalic scale which was counted on a straight line between the middle of the supraoculars), IOD; supralabials, SL; infralabials, IL; and the bilateral characters are given as left/right. We euthanized the live specimens using MS-222 according to the standardized protocol (Conroy et al. 2009), and the procedure was approved by the Institutional Animal Ethics Committee (IAEC) (Permission No. MZU-IAEC/2018/12). All the specimens collected during this survey were deposited in the Departmental Museum of Zoology, Mizoram University, India (MZMU). For molecular analyses, we dissected the liver tissue from the freshly euthanized specimens and stored in 95% ethanol at -20°C refrigerator. Distribution map was prepared using QGIS v3.16.2.

DNA extraction and amplification
We employed mitochondrial cytochrome b (cytb) gene for molecular based identification. For this purpose, genomic DNA was extracted from the liver tissue using QIAamp DNA Mini Kit following the standard protocol provided. We amplified the fragment of cytb gene using a published primer pairs (Snk) (Dubey et al. 2009) under the thermal conditions of 94˚C for 3 min for initial denaturation; 35 cycles for denaturation at 94˚C for 30 sec, annealing at 49˚C for 40 sec, and extension at 72˚C for 30 sec; with the final extension of 72˚C for 5 sec, and cooling at 4°C for 15 min. The amplified products were purified using ThermoFisher ExoSAP-IT PCR product clean-up reagent and sequenced at Barcode BioSciences, Bangalore, India using Sanger's dideoxy method. The newly generated sequences were submitted to NCBI GenBank database (Benson et al. 2018) and obtained the accession numbers (OQ968475-77).

Molecular analyses
We assembled the dataset mainly based on the published datasets of Mirza et al. (2023) for Trimeresurus and Guo et al. (2009) for Protobothrops, and we obtained the published nucleotide sequences from the NCBI GenBank database (Benson et al. 2018). We used the default parameter settings of MUSCLE (Edgar 2004 with shorter nucleotide sequences are treated as missing data. We estimated the uncorrected p-distance using complete deletion for gaps/missing data in MEGA 11 (Tamura et al. 2021). We partitioned the assembled datasets (1,088 bp in length for Trimeresurus; 847 bp in length for Protobothrops) by codon position. The best partitioning schemes and nucleotide evolutionary models selected by PartitionFinder v2 (Lanfear et al. 2017) under the Bayesian Information Criterion are HKY+I for cytb pos1, TRN+G for cytb pos2, HKY+G for cytb pos3 in the dataset of Trimeresurus; HKY+I+G for cytb pos1 and pos3, and TIM+I+G for cytb pos2 in the dataset of Protobothrops. By implementing the selected models, we performed the partitioned Bayesian inference (BI) phylogeny separately for the two datasets in MrBayes v3.2.5 (Ronquist et al. 2012). We conducted four independent runs with one cold and three hot chains for 10 million generations and sampling every 5000 generations. We discarded 25% of the trees as burn-in cut off after examining the trace plots generated by the MCMC runs in Tracer v1.7 (Rambaut et al. 2018).

RESULTS AND DISCUSSION
In this report, we documented a total of four pit viper species from Manipur (Image 1), and we established their identity based on the combination of mitochondrial cyt b gene and their respective morphological characters, including key diagnostic features from relevant published literature (e.g., Guo et al. 2009;Zambre et al. 2009;Yang et al. 2011;Rathee et al. 2022;Mirza et al. 2020Mirza et al. , 2023.

Trimeresurus mayaae Rathee et al., 2022 (Image 2)
The recently discovered Maya's Pit Viper, T. mayaae is a species genetically close to T. medoensis as well as morphologically similar to T. gumprechti of Indochina Image 1. Map showing the type localities and new collection sites of the four pit vipers in Manipur, India: Trimeresurus mayaae are shown in red star (type locality) and red triangle (this study); T. salazar in green star (type locality) and green circle (this study); T. erythrurus in blue diamonds (this study); Protobothrops jerdonii in purple star (type locality) and purple square (this study). Taxa  Despite the fact that we are utilizing a single gene (cyt b) in this study, our reconstructed BI phylogram of Trimeresurus species considerably concorded to the tree topology, particularly on the clades of the subgenus "Trimeresurus" and "Viridovipera" fide Mirza et al. (2023) where they combined four mitochondrial genes (cyt b, 16S rRNA, 12S rRNA, and ND4). Accordingly, our cyt b gene tree depicted the new T. mayaae specimen (MZMU2970; Accession No. OQ968476) clustering alongside the type series of the species by a wellsupported Bayesian posterior probability support (PP = 0.99), and is nested with the holotype from Mizoram (PP=1.00) (Figure 1). Moreover, the specimen (MZMU2970) is showing 0.0-0.7% intraspecific genetic distance with respect to the type series, and the least genetic distance was seen with the holotype (0.0%) from Mizoram, India (see Table S1).

Trimeresurus salazar Mirza et al., 2020 (Image 3)
The Salazar's Pit Viper, T. salazar is one of the recently discovered Trimeresurus in the past three years. The species has been confirmed so far from centralsouthern Nepal, Southern Bhutan (Vogel et al. 2022), Northeast Indian States of Arunachal Pradesh (type locality), Assam (Mirza et al. 2020), Meghalaya (Rathee (Rathee et al. 2022 and references therein). Not much is known on the biology of this cryptic species, and its description is based on specimens originating from the Northeast Indian States of Mizoram and Meghalaya. This species, in fact represents the third new species of the genus from India, to be described in the past three years (Rathee et al. 2022).
On 17 Mirza et al. (2020) is not evident in our specimen, we considered the particular character as a phenotypically plastic trait as seen in T. mayaae (Rathee et al. 2022) and T. popeiorum (Mirza et al. 2023) rather than sexually dimorphic trait, and also in consideration of the low sampling size in the original description (three

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males and one female) (See Mirza et al. 2020). Even so, with respect to T. erythrurus, the species that usually lacks the red postocular stripe in males, the specimen (MZMU3027) is distinct in having 19 mid-dorsal scale rows vs. 23 in T. erythrurus (Mirza et al. 2020). Our cyt b based BI phylogram also depicted a subclade of salazar + septentrionalis +caudornatus that was conceived by Mirza et al. (2023); and the new T. salazar specimen (MZMU3027; Accession No. OQ968477) clustered with the conspecies from Meghalaya, India (CESS331) and Nepal (AM A100) with a well-supported branch (PP = 1.00) (Figure 1). We uncovered 0.2-0.5% intraspecific genetic distance among T. salazar samples, and our sample shows 0.2% genetic distance with respect to both of them (see Table S1).

Trimeresurus erythrurus (Cantor, 1839) (Image 4)
The Red-tailed Pit Viper, T. erythrurus was originally described from the "Delta Gangeticum" which is apparently from the Indian Sunderbans of West Bengal

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state in the present time fide Deuti et al. (2021). The species is widely distributed in parts of Southern Asia including Bhutan (Wangyal 2014;Deuti et al. 2021 and references therein), Nepal, Bangladesh, Myanmar (Mahony et al. 2009;Wallach et al. 2014;Chan et al. 2022), and the Indian states of Andhra Pradesh (Kakinada), West Bengal (Deuti et al. 2021), and Northeast India viz. Arunachal Pradesh, Assam, Mizoram, Nagaland, and Sikkim (Whitaker & Captain 2008;Wallach et al. 2014); possibly in Meghalaya and Manipur (see Wallach et al. 2014). On 23 July 2022, at ca. 0700 h, we documented a live individual of T. erythrurus from a forest trail at Bishnupur District, Manipur (24.632°N, 93.746°E; 920 m; Image 1) ca. 500 km aerial distance northeast from the type locality in Sunderbans (Deuti et al. 2021). Seeing the bulged abdomen, it was evident that the snake had recently fed, possibly upon small mammal or a bird (Image 4a). Morphologically, we identified the snake as female T. erythrurus in having golden-yellow colored eye; greenish dorsum; white lateral stripe on the head and the ventrolateral body; total length of 595 mm; Ve 168; Sc 51; and 23 mid-dorsal scale which are in agreement with the published diagnostic characters of the species (Chen et al. 2020;Mirza et al. 2020;Deuti et al. 2021). We subsequently released back the snake into the wild after taking measurements and scalation data. On 4 November 2022, at ca. 1100 h, we encountered additional uncollected road killed male individual at Noney town, Noney District, Manipur (24.851°N, 93.617°E; 510 m asl.) ca. 28 km aerial distance northwest from the point of our observation of the live individual. As the snake was badly damaged and large amount of the scales were eaten off by ants, we could barely examine the snake but nevertheless, obtained the key features like white lateral stripe on the head and ventrolateral body, 23 mid-dorsal scale rows, and Ve 158 (Image 4b). Given that we could not obtain genetic data for this species in Manipur, we advocate DNA studies on the Manipur population to further corroborate the present report.

Protobothrops jerdonii (Günther, 1875) (Image 5)
The widely distributed P. jerdonii is presently found in southwestern China, northeastern India, Nepal, northern Myanmar as well as northern Vietnam (see Guo  et al. 2009). The high level of variation in their ecology as well as morphology, particularly in the scale counts and coloration has led to a controversial intraspecific taxonomic treatment within P. jerdonii (Guo et al. 2009).
This has led some authors to recognise the species as monotypic without any subspecies, while certain other authors treat the taxon to be polytypic (see Guo et al. 2009) with a total of five species or subspecies

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proposed so far within the P. jerdonii complex. They are, apart from the nominotypical form jerdonii Günther, 1875;xanthomelas Günther, 1889;melli Vogt, 1922;meridionalis Bourret, 1935;and bourreti Klemmer, 1963. Among these, only jerdonii, bourreti and xanthomelas were considered as valid subspecies by several authors like Hoge & Romano-Hoge (1983), Golay et al. (1993), Orlov et al. (2001), and Gumprecht et al. (2004 In considering the subspecies recognized by Gumprecht et al. (2004), the Ve of our specimen (171) falls within the range of P. jerdonii jerdonii (160-170) fide Yang et al. (2011), the Sc of our specimen (66) enter the Sc range of both P. jerdonii xanthomelas (54-67) and P. jerdonii bourreti (65-72) fide Yang et al. (2011). Moreover, the basal body color of our specimen is yellowish brown, and a series of rhomboidal or irregularly shaped reddishbrown patches with black margins are present along the dorsal body. Based on the morphological data alone, we cannot certainly assign the new specimen (MZMU3011) to any of the three nominal subspecies following the recent most diagnostic keys fide Yang et al. (2011). Thus, we for now refrain from making subspecies level taxonomic identification for the specimen.
Our reconstructed cyt b gene tree also depicted paraphyly amongst P. jerdonii as conceived by Guo et al. (2009) where they sequenced four mitochondrial genes (cyt b, 12S rRNA, 16S rRNA, and ND4). Our sample (MZMU3011; Accession No. OQ968475) nested alongside the conspecific sequences from Northeast India (V14) with a high branch support (PP = 1.00), and these two formed a subclade with the other samples from China (GP87 and KIZ012737) with high support (PP = 1.00) (Figure 2). Our estimated genetic distance also showed 0.0-3.3% intraspecific distance between ours and the other P. jerdonii samples; the least genetic distance (0.0%) is seen with the Northeast Indian sample while the highest (3.3%) is seen with respect to the samples from Huili, Sichuan, China (GP11-GP14) (see Table S2).

CONCLUSION
As per the current knowledge, only O. monticola has been listed as present in Manipur (Whitaker & Captain 2008). In this work, we report the occurrence of four new records of pit vipers in Manipur for the first time, and simultaneously provide new information on their respective geographical distribution ranges. Considering the topology in our phylogenetic reconstruction and our P. jerdonii sample (MZMU3011) that disclosed the overlapping of the conventional taxonomic keys for the subspecies fide Yang et al. (2011), we are convinced that our study corroborated the taxonomic statement on the species by Guo et al. (2009) where they considered P. jerdonii as a monotypic and paraphyletic species. In the scenario of T. mayaae, both ours and the published phylogenies (Rathee et al. 2022;Mirza et al. 2023) depicting the nesting of T. mayaae next to T. medoensis. Nonetheless, combining the meristic data of the type series (Rathee et al. 2022) and the new specimens, T. mayaae is morphologically distinct in terms of the Ve counts of male i.e., 154-162 vs. 138-149 in T. medoensis, and female i.e., 152-153 vs. 141-143 in T. medoensis; also in the mid-dorsal scale row i.e., 19-21 in T. mayaae vs. 17 in T. medoensis.
The present work, however, provides evidence for the occurrence of four additional species of pit vipers in Manipur for the first time, and simultaneously provides new information on their respective geographical distribution ranges. Thus, currently, five species of pit vipers including P. jerdoni, T. salazar, T. mayaae, T. erythrurus, and O. monticola are known from Manipur (Whitaker & Captain 2004;this work). We emphasise that this new data on the occurrence of four more species of pit vipers in Manipur may be disseminated well in health care sectors. This is so because these venomous snakes may be of medical importance in terms of potential snakebites on humans (e.g., Kanwar 2018;Rai et al. 2021;Ravikar et al. 2023), more so in forest fringe areas.