Molecular approaches to the conservation and management of Bengal tiger in Nepal

Bengal tiger (Panthera tigris) is an endangered species found in the lowland areas of Nepal. Much needs to be understood about this species, including its population size, genetic health, habitat and overall ecosystem dynamics. Although there have been efforts to estimate tiger population numbers using camera trapping methods, the information obtained through such efforts has been limited and there is a need to find a better method and technology to understand wild tigers in Nepal. Use of genetic sampling holds significant promise in execution of landscape-level management plans as it may allow managers to measure genetic health and even gene flow with greater certainty. Habitat connectivity and the degree of movement from one habitat patch to another can be inferred through understanding of species behavior, but genetic data can give us landscape level information by directly identifying patterns of distinctiveness, endemism and the degree to which gene flow exists between subpopulations. Non-invasive genetic studies on populations, such as those that use scat samples, have increased in recent years, and have been used to estimate population size of many elusive and endangered species. I have used this technique to understand tiger and its habitat. In my study I have uncovered prevalent sympatric biodiversity in the tiger habitat which aids in understanding the species conservation from an ecological perspective. Out of total collected scat samples (n=420), only 56% (n=237) were of tiger. The remaining non-tiger samples (n=183) included non-focal carnivores; leopard (Panthera pardus, n=83), leopard cat (Prionailurus bengalensis, n=10), jungle cat (Felis chaus, n=2), fishing cat (Prionailurus viverrinus, n=2) and fox (Vulpes spp., n=10). The spatial distribution of three small felids: leopard cat, jungle cat and fishing cat in sub-tropical deciduous forest of Terai has provided insights on sympatric occurrences of small cats with each other and with two other large felids, leopard and tiger, in CNP. Overall landscape level of information on tigers on such important aspects like genetic health, sub-population structure and gene flow will help in designing broader conservation strategies for the species. Of the 770 scat samples collected opportunistically from four protected areas and five presumed corridors, 412 were tiger (57%). Using eight microsatellite markers, I identified 78 individual tigers. I used this dataset to examine population structure, genetic variation, contemporary gene flow, and potential population bottlenecks of tigers in Nepal. I detected three genetic clusters consistent with three demographic sub-populations and found moderate levels of genetic variation (He = 0.61, AR = 3.51) and genetic differentiation (FST = 0.14) across the landscape. I detected 3-7 migrants, confirming the potential for dispersal-mediated gene flow across the landscape. I found evidence of a bottleneck signature likely caused by large-scale land-use change documented in the last two centuries in the Terai forest. Securing tiger habitat including functional forest corridors is essential to enhance gene flow across the landscape and ensure long-term tiger survival. The molecular forensic tools that we have developed have been helping the law enforcement officials in Nepal in the fight against wildlife poaching. I 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. I analyzed 15 cases of confiscated poached tiger parts. Ten were identified as males and five were females, and I determined probable geo-source location for 14 of those samples by combining inferences from two different analytical methods of population genetic structure and phylogenetic analysis. Conclusively, eleven of the fourteen samples were assigned to the western region of Nepal, while the remaining three were determined to be distantly related to the others and were identified as outliers. Among these, one sample was an exact match to a female tiger in our reference database previously detected in Bardia National Park. My study revealed the western region, particularly Bardia, is a poaching hotspot for illegal tiger trade across the Tera Arc Landscape in Nepal. I present scientific evidence to incriminate criminals in a court of law and advocate for the increased use of molecular forensics in wildlife conservation efforts. And finally, I have mapped the inter-relationship between tiger and its gut microbiome. This study is the first of its kind done on wild tigers; and our results show an association between the species with its gut microbiota and habitat. The scat microbiome of the Bengal tiger contained the phyla Proteobacteria (~37%), Firmicutes (~27.7%), Bacteroidetes (~18.6%), Fusobacteria (~13.4%) and Actinobacteria (~2.7%), but in varying proportions across the habitats. Predictive metagenome functional content analysis (PICRUST) of the gut microbiome, restricted to three significantly different bacterial genera (Comomonas, Collinsella and Fusobacteria), identified 36 significantly (p value < 0.05) differential functional content systems. Chitwan samples contained a lower proportion of sequences associated with immune system and infectious diseases, but higher proportion that are associated with xenobiotic biodegradation /metabolism, cellular processing and signaling, and neurodegenerative diseases compared to the other two. These findings underscore the importance of a conservation design that specifically seeks to maximize genetic and gut microbial diversity amongst wild tigers in Nepal. My Study, through the Nepal Tiger Genome Project, has looked into a range of molecular approaches to comprehend the tiger population from conservation genetics perspective and uncovered wealth of information which is now being utilized to formulate effective conservation strategies. Most of this work was done for the first time in Nepal; and some work, especially molecular forensics and gut microbiome study, were done for the first time. My utility based study has also been able to create necessary capacity to do similar work in other species in developing countries like Nepal, and beyond this I am looking at creating viral and dietary profiles on some of these tiger fecal samples as part of my commitment to understand this species further.