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This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. AbstractAim: We explore the phylogeography of Himalayan wolves using multiple genetic markers applied on a landscape-scale dataset and relate our findings to the biogeographic history of the region. Location: Himalayas of Nepal, the Tibetan Plateau of China and mountain ranges of Central Asia. Taxon: Himalayan wolf (also called the Tibetan wolf), Canis lupus chanco. Methods: We present a large-scale, non-invasive study of Himalayan wolves from across their estimated range. We analysed 280 wolf scat samples from western China, Kyrgyzstan and Tajikistan at two mtDNA loci, 17 microsatellite loci, four nonsynonymous SNPs in three nuclear genes related to the hypoxia pathway, and ZF genes on both sex chromosomes. | 1273 WERHAHN Et Al.

Recent molecular systematic studies of arvicoline voles of the genera Neodon , Lasiopodomys , Phaiomys , and Microtus from Central Asia suggest the inclusion of Phaiomys leucurus , Microtus clarkei , and Lasiopodomys fuscus into Neodon and moving Neodon juldaschi into Microtus ( Blanfordimys ). In addition, three new species of Neodon ( N . linzhiensis , N . medogensis , and N . nyalamensis ) have recently been described from Tibet. Analyses of concatenated mitochondrial ( Cytb , COI ) and nuclear ( Ghr , Rbp3 ) genes recovered Neodon as a well-supported monophyletic clade including all the recently described and relocated species. Kimura-2-parameter distance between Neodon from western Nepal compared to N . sikimensis (K2P = 13.1) and N . irene (K2P = 13.4) was equivalent to genetic distances observed between recognized species of this genus. The specimens sampled from western Nepal were recovered sister to N . sikimensis in the concatenated analysis. However, analyses conducted exclusively with mitochondrial loci did not support this relationship. The occlusal patterns of the first lower (m1) and third upper (M3) molars were simpler in specimens from western Nepal in comparison to N . sikimensis from eastern Nepal and India. Twelve craniodental characters and four external field measurements were examined from specimens of N . sikimensis from eastern Nepal and India, N . irene , and Neodon from western Nepal. Neodon from western Nepal were significantly different from N . sikimensis from eastern Nepal and India in ten out of 16 characters measured and from N . irene for all characters except ear height. Specimens from western Nepal were smaller in size than N . sikimensis from Eastern Nepal and India and larger than N . irene . Together the results of the molecular and morphological analyses indicate that Neodon from western Nepal are distinct under the phylogenetic, genetic and morpho species concepts.

Tiger (Panthera tigris) populations are in danger across their entire range due to habitat loss, poaching and the demand for tiger parts. The Bengal tiger (Panthera tigris tigris) is an endangered apex predator with a population size estimated to be less than 200 in Nepal. In spite of strict wildlife protection laws, illegal trade of tiger parts is increasing; and Nepal has become one of the major sources and transit routes for poached wildlife parts. Identification of wildlife parts is often challenging for law enforcement officials due to inadequate training and lack of available tools. Here, we describe a molecular forensic approach to gain insight into illegally trafficked tiger parts seized across Nepal. We created Nepal’s first comprehensive reference genetic database of wild tigers through the Nepal Tiger Genome Project (2011–2013). This database has nuclear DNA microsatellite genotype and sex profiles, including geo-spatial information, of over 60% (n = 120) of the wild tigers of Nepal. We analyzed 15 putative cases of confiscated poached tiger parts and all were confirmed to be of tiger. Ten samples were identified as male and five were female. We determined probable geo-source location for 9 of the 14 samples with 6–8 nuclear DNA microsatellite loci using inferences from four different statistical assignment methods. Six samples were assigned to Bardia National Park and one of these was an exact match to a female tiger previously profiled in our fecal DNA reference database. Two tiger samples were assigned to Shuklaphanta Wildlife Reserve and one to Chitwan National Park. We are unable to definitively assign five tiger samples which could be offspring dispersers or might have come from tiger population outside of Nepal. Our study revealed that the western region, particularly Bardia National Park, is a poaching hotspot for illegal tiger trade in Nepal. We present feasibility of using molecular forensic based evidence to incriminate criminals in a court of law in the fight against wildlife crime.

Nepal boarders India and China and all three countries lie within the Central Asian Flyway for migratory birds. Novel influenza A H7N9 caused human fatalities in China in 2013. Subclinical infections of influenza A H7N9 in birds and the potential for virus dispersal by migratory birds prompted this study to assess avian H7N9 viral intrusion into Nepal. Surveillance of influenza A virus in migratory birds was implemented in early 2014 with assistance from the Food and Agricultural Organization (FAO). Of 1811 environmental fecal samples collected from seven wetland migratory bird roosting areas, influenza A H9N2 was found in one sample from a ruddy shelduck in Koshi Tappu Wildlife Reserve located in southern Nepal. Avian H7N9 and other highly pathogenic avian influenza viruses were not detected. This study provides baseline data on the status of avian influenza virus in migratory bird populations in Nepal.

Bengal tigers ( Panthera tigris tigris ) serve a pivotal role as an apex predator in forest ecosystems. To increase our knowledge on factors impacting the viability and health of this endangered species, we studied the gut microbiota in 32 individual Bengal tigers from three geographically separated areas (Chitwan National Park (CNP), Bardia National Park (BNP) and Suklaphanta Wildlife Reserve (SWR)) in Nepal, using noninvasive genetic sampling methods. Gut microbiota influence the immune system, impact various physiological functions, and modulates metabolic reactions, that ultimately impact the host health, behavior and development. Across the tiger populations in Nepal, we found significant differences in the composition of microbial communities based on their geographic locations. Specifically, we detected significant differences between CNP and the other two protected areas (CNP vs BNP: pseudo t = 1.944, P = 0.006; CNP vs SWR: pseudo t = 1.9942, P = 0.0071), but no differences between BNP and SWR. This mirrors what has been found for tiger gene flow in the same populations, suggesting gut microbiota composition and host gene flow may be linked. Furthermore, predictive metagenome functional content analysis (PICRUSt) revealed a higher functional enrichment and diversity for significant gut microbiota in the Chitwan tiger population and the lowest enrichment and diversity in Suklaphanta. The CNP tiger population contained higher proportions of microbiota that are associated with predicted functions relevant for metabolism of amino acid, lipid, xenobiotics biodegradation, terpenoides and polyketides than the SWR population. We conclude the tiger population structure, gut microbiota profile and associated functional metabolic categories are correlated, with geographically most separated CNP and SWR tiger population having the most distinct and different host genotype and microbiota profiles. Our work dramatically expands the understanding of tiger microbiota in wild populations and provides a valuable case study on how to investigate genetic diversity at different hierarchical levels, including hosts as well as their microbial communities.

Although over 180 freshwater fish species have been reported from Nepal, little is known of their ecology and distribution. This information is needed because their diversity may be threatened by developments like hydropower constructions. We conducted Nepal’s first environmental DNA (eDNA) based fish biodiversity assessment in two major river systems- Karnali River (KR), which is still pristine and Trishuli River (TR) with numerous hydropower plants. The eDNA was concentrated by filtering (0.45 μm pore size) two liters of water collected at different sampling points in each study site. A total of 224 eDNA samples (KR = 162 and TR = 62) were collected, from which fish species were identified by 12S rRNA metabarcording approach utilizing Illumina sequencing platform. Alpha and beta diversity of species between two river basins were compared. Also, in KR site, fish (N = 795) were caught, and identified using COI gene based DNA barcoding method- building Nepal’s first fish DNA reference database. Field sampling identified 21 species through morphology and DNA barcoding, where Barilius spp. and Schizothorax spp. were the most abundant. From 244 eDNA samples, 24 Operational Taxonomic Units (OTUs) were identified in TR and 46 in KR with 19 being common to both sites, 27 being unique in KR, and five in TR only. Most fishes were of Cypriniformes and Siluriformes orders, with Barilius spp. and Schizothorax spp. being the most abundant. Long distance migratory fish (Tor spp, Neolissochilus hexagonolepis) and non-native fishes were identified in eDNA samples as well. Alpha diversity in TR was significantly lower than in KR. High beta diversity between the two sites indicated low similarity in fish diversity between the TR and KR. This study demonstrated the utility of eDNA as a non-invasive technique for biodiversity assessment which is particularly useful in areas like Nepal with scarce data on fish species distribution.

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