PLATINUM The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservaton globally by publishing peer-reviewed artcles online OPEN ACCESS every month at a reasonably rapid rate at www.threatenedtaxa.org. All artcles published in JoTT are registered under Creatve Commons Atributon 4.0 Internatonal License unless otherwise mentoned. JoTT allows allows unrestricted use, reproducton, and distributon of artcles in any medium by providing adequate credit to the author(s) and the source of publicaton. Journal of Threatened Taxa Building evidence for conservaton globally www.threatenedtaxa.org ISSN 0974-7907 (Online) | ISSN 0974-7893 (Print) Communication Revisiting genetic structure of Wild Buffaloes Bubalus arnee Kerr, 1792 (Mammalia: Artiodactyla: Bovidae) in Koshi Tappu Wildlife Reserve, Nepal: an assessment for translocation programs Ram C. Kandel, Ram C. Poudel, Amir Sadaula, Prakrit Kandel, Kamal P. Gairhe, Chiranjibi P. Pokheral, Siddhartha B. Bajracharya, Mukesh K. Chalise & Ghan Shyam Solanki 26 December 2019 | Vol. 11 | No. 15 | Pages: 14942–14954 DOI: 10.11609/jot.4940.11.15.14942-14954 For Focus, Scope, Aims, Policies, and Guidelines visit htps://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-0 For Artcle Submission Guidelines, visit htps://threatenedtaxa.org/index.php/JoTT/about/submissions#onlineSubmissions For Policies against Scientfc Misconduct, visit htps://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-2 For reprints, contact <[email protected]> The opinions expressed by the authors do not refect the views of the Journal of Threatened Taxa, Wildlife Informaton Liaison Development Society, Zoo Outreach Organizaton, or any of the partners. The journal, the publisher, the host, and the part- Publisher & Host ners are not responsible for the accuracy of the politcal boundaries shown in the maps by the authors. Partner Member Threatened Taxa Journal of Threatened Taxa | www.threatenedtaxa.org | 26 December 2019 | 11(15): 14942–14954 Revisiting genetic structure of Wild Buffaloes Communication Bubalus arnee Kerr, 1792 (Mammalia: Artiodactyla: Bovidae) ISSN 0974-7907 (Online) in Koshi Tappu Wildlife Reserve, Nepal: an assessment for ISSN 0974-7893 (Print) translocation programs PLATINUM OPEN ACCESS Ram C. Kandel 1 , Ram C. Poudel 2 , Amir Sadaula 3 , Prakrit Kandel 4 , Kamal P. Gairhe 5 , Chiranjibi P. Pokheral 6 , Siddhartha B. Bajracharya 7 , Mukesh K. Chalise 8 & Ghan Shyam Solanki 9 1,5 Ministry of Forests and Environment, Department of Natonal Parks and Wildlife Conservaton, PO 860, Babarmahal, Nepal. 2 Nepal Academy of Science and Technology, Molecular Biotechnology Unit, PO 3323, Khumaltar, Nepal. 3,6,7 Natonal Trust for Nature Conservaton, PO 3712, Khumaltar, Nepal. 4 Kathmandu University, Department of Biotechnology, PO 6250, Dhulikhel, Nepal. 5 Chitwan Natonal Park, Headquarter, Kasara, Chitwan, Nepal. 8 Tribhuvan University, Central Department of Zoology, Kirtpur, Nepal. 9 Mizoram University, Department of Zoology, Mizoram, Aizawl, Mizoram 796009, India. 1 [email protected], 2 [email protected] (corresponding author), [email protected], 4 [email protected], 5 [email protected], [email protected], 7 [email protected], 8 [email protected], [email protected] Abstract: Koshi Tappu Wildlife Reserve (KTWR) has the last remaining Nepalese populaton of the Endangered Asiatc Wild Bufalo (Bubalus arnee Kerr, 1792). Individual animals protected inside KTWR may be of purely wild, domestc or hybrid origin, and the wild populaton is under potental threat due to habitat loss and genetc introgression from feral backcrosses. Identfcaton of genetcally pure wild individuals is important for identfying animals for translocaton to other areas within their former range. In this study we have sequenced a highly variable 422bp region of the Cytochrome b gene of 36 animals, and added 61 published sequences of both River and Swamp Bufalo from Italy and some southern Asian countries including India. The haplotype diversites ranged from 0.286-0.589 with slightly higher diversites in domestcated individuals. The AMOVA analysis revealed that 97.217% of the genetc variaton was contained within groups and 2.782% occurred among groups. An overall fxaton index (FST) was found to be 0.02782 (p>0.05). Phylogenetc relatonships derived through a reduced median network and maximum parsimony analyses reconfrmed the ancestral nature of the Wild Water Bufalo. Moreover, this study has reviewed recent achievements of molecular research in wild bufalo, assessed the technical capacites of research insttutes in Nepal to conduct molecular research required for identfying pure wild individual in KTWR and more importantly initated DNA bank and DNA sequence library of bufalos, which will enable an internatonal collaboraton for advanced molecular research in the future. Keywords: Asian Wild Bufalo, conservaton, Cytochrome b, phylogenetcs. DOI: htps://doi.org/10.11609/jot.4940.11.15.14942-14954 Editor: David Mallon, Manchester Metropolitan University, Derbyshire, UK. Date of publicaton: 26 December 2019 (online & print) Manuscript details: #4940 | Received 01 March 2019 | Final received 16 November 2019 | Finally accepted 27 November 2019 Citaton: Kandel, R.C., R.C. Poudel, A. Sadaula, P. Kandel, K.P. Gairhe, C.P. Pokheral, S.B. Bajracharya, M.K. Chalise & G.S. Solanki (2019). Revisitng genetc structure of Wild Bufaloes Bubalus arnee Kerr, 1792 (Mammalia: Artodactyla: Bovidae) in Koshi Tappu Wildlife Reserve, Nepal: an assessment for translocaton programs. Journal of Threatened Taxa 11(15): 14942–14954. htps://doi.org/10.11609/jot.4940.11.15.14942-14954 Copyright: © Kandel et al. 2019. Creatve Commons Atributon 4.0 Internatonal License. JoTT allows unrestricted use, reproducton, and distributon of this artcle in any medium by adequate credit to the author(s) and the source of publicaton. Funding: The blood and faecal sample collecton for this study was supported by Harioban Program. DNA sequencing was carried out in Nepal Academy of Science and Technology (NAST) under DNA Barcoding Program funded by Natonal Trust for Nature Conservaton (NTNC). Competng interests: The authors declare no competng interests. For Author details, Author contributon, Acknowledgements and Nepali abstract see end of this artcle. 14942 Revisitng phylogenetcs of bufaloes in KTWR Kandel et al. INTRODUCTION and widely accepted techniques. Therefore, selectng individuals for translocaton programs, identfcaton The domestcated Water Bufalo Bubalus bubalis Kerr, of wild individuals through detailed molecular study 1792 is one of the most important dairy and draf animals of the bufalo populaton protected in the reserve is a in southern Asia. Bufaloes are broadly categorized high priority for the Nepal government. In additon, into two general ‘breeds’ sometmes characterized as understanding the genetc makeup of Wild Bufalo subspecies: river (Bubalus bubalis bubalis) and swamp could be used as the basis for genetc improvement of bufalo (Bubalis bubalis carabanesis). Despite having domestc stock. Natonal capacity building to conduct distnct morphological and behavioural traits and advanced molecular studies should be initated from diferent karyotypes between these two categories of collectng blood and faecal samples, creatng a DNA bufalo, they interbreed easily and produce progeny reference library and carrying out genetc research on with intermediate chromosomes (Mishra et al. 2015). various aspects such as populaton genetcs, breeding Swamp and river bufalo have diferent purposes and are behaviours among diferent bufalo types, disease found in diferent geographical areas. The swamp type, dynamics, and food habits of bufalo populaton in the with wide-spreading horns and some white markings, is reserve. We present results of DNA sequence variaton more similar to Wild Bufalo Bubalus arnee (Kumar et al. in the partal but variable cytochrome b gene among 2007; Mishra et al. 2015). purely wild, feral and domestcated individuals and The Government of Nepal (GoN) established Koshi future prospects for advancing genetc research on Wild Tappu Wildlife Reserve (KTWR), an IUCN Category IV Bufaloes inhabitng KTWR in eastern Nepal. protected area (Heinen 1995), in 1976 primarily to conserve the last Nepalese populaton of Wild Bufalo (Heinen 1993). Wild Bufalo cohabit the reserve with MATERIAL AND METHODS highly backcrossed feral bufalo thought to have been released in the area in the 1950s (Dahmer 1978). Wild Identfcaton of organizatons Water Bufalo Bubalus arnee (Kerr, 1792) is considered Nepal Government, Ministry of Forest and Endangered globally (Kaul et al. 2019), with isolated Environment is working together with local communites populatons in KTWR and selected areas of Bhutan, India, and natonal and internatonal conservaton partners to Thailand and possibly Myanmar and Cambodia (Groves protect wild animals and restore their natural habitats. 1996; Heinen & Srikosamatara 2003; Choudhury & With the aim of establishing viable populatons of Barker 2014). In 2016, 433 Wild Bufaloes were counted endangered species in diferent areas and safeguarding in KTWR (2016). them from poaching and natural calamites such as Despite the possibility of interbreeding between wild food, fre and epidemics, the Ministry of Forests and and domestcated bufalo, it is important to assign extant Environment, Department of Natonal Parks and Wildlife individuals to wild and other types where possible to Reserve (DNPWC)