Sujeet Kumar Singh

Tigers of Sundarbans in India: Is the Population a Separate Conservation Unit?

The Sundarbans tiger inhabits a unique mangrove habitat and are morphologically distinct from the recognized tiger subspecies in terms of skull morphometrics and body size. Thus, there is an urgent need to assess their ecological and genetic distinctiveness and determine if Sundarbans tigers should be defined and managed as separate conservation unit. We utilized nine microsatellites and 3 kb from four mitochondrial DNA (mtDNA) genes to estimate genetic variability, population structure, demographic parameters and visualize historic and contemporary connectivity among tiger populations from Sundarbans and mainland India. We also evaluated the traits that determine exchangeability or adaptive differences among tiger populations. Data from both markers suggest that Sundarbans tiger is not a separate tiger subspecies and should be regarded as Bengal tiger (P. t. tigris) subspecies. Maximum likelihood phylogenetic analyses of the mtDNA data revealed reciprocal monophyly. Genetic differentiation was found stronger for mtDNA than nuclear DNA. Microsatellite markers indicated low genetic variation in Sundarbans tigers (He= 0.58) as compared to other mainland populations, such as northern and Peninsular (Hebetween 0.67- 0.70). Molecular data supports migration between mainland and Sundarbans populations until very recent times. We attribute this reduction in gene flow to accelerated fragmentation and habitat alteration in the landscape over the past few centuries. Demographic analyses suggest that Sundarbans tigers have diverged recently from peninsular tiger population within last 2000 years. Sundarbans tigers are the most divergent group of Bengal tigers, and ecologically non-exchangeable with other tiger populations, and thus should be managed as a separate “evolutionarily significant unit” (ESU) following the adaptive evolutionary conservation (AEC) concept.

Fine-scale population genetic structure of the Bengal tiger (Panthera tigris tigris) in a human-dominated western Terai Arc Landscape, India

Despite massive global conservation strategies, tiger populations continued to decline until recently, mainly due to habitat loss, human-animal conflicts, and poaching. These factors are known to affect the genetic characteristics of tiger populations and decrease local effective population sizes. The Terai Arc Landscape (TAL) at the foothills of the Himalaya is one of the 42 source sites of tigers around the globe. Therefore, information on how landscape features and anthropogenic factors affect the fine-scale spatial genetic structure and variation of tigers in TAL is needed to develop proper management strategies for achieving long-term conservation goals. We document, for the first time, the genetic characteristics of this tiger population by genotyping 71 tiger samples using 13 microsatellite markers from the western region of TAL (WTAL) of 1800 km2. Specifically, we aimed to estimate the genetic variability, population structure, and gene flow. The microsatellite markers indicated that the levels of allelic diversity (MNA = 6.6) and genetic variation (Ho = 0.50, HE = 0.64) were slightly lower than those reported previously in other Bengal tiger populations. We observed moderate gene flow and significant genetic differentiation (FST= 0.060) and identified the presence of cryptic genetic structure using Bayesian and non-Bayesian approaches. There was low and significantly asymmetric migration between the two main subpopulations of the Rajaji Tiger Reserve and the Corbett Tiger Reserve in WTAL. Sibship relationships indicated that the functionality of the corridor between these subpopulations may be retained if the quality of the habitat does not deteriorate. However, we found that gene flow is not adequate in view of changing land use matrices. We discuss the need to maintain connectivity by implementing the measures that have been suggested previously to minimize the level of human disturbance, including relocation of villages and industries, prevention of encroachment, and banning sand and boulder mining in the corridors.

Estimation of Male Gene Flow: Use Caution

Because male gene flow cannot easily be estimated directly in many organisms, Hedrick et al. (2013) provided an approach to estimate male gene flow given estimates of diploid nuclear and female differentiation. This approach appears to work well when there is lower female than male gene flow. However, in a tiger data set there was less female differentiation observed as estimated by mitochondrial DNA than expected given the observed overall nuclear diploid differentiation. To analyze these data, we suggest an alternative approach which allows incorporation of sex-specific gene flow and sex-specific effective population size. We find that the pattern of differentiation observed in tigers was consistent with a lower male than female effective population size using this alternative approach. Further, this finding is consistent with observed data in tigers where the male effective population size was 33% that of the female effective population size.

Panel of polymorphic heterologous microsatellite loci to genotype critically endangered Bengal tiger: a pilot study

In India, six landscapes and source populations that are important for long-term conservation of Bengal tigers (Panthera tigris tigris) have been identified. Except for a few studies, nothing is known regarding the genetic structure and extent of gene flow among most of the tiger populations across India as the majority of them are small, fragmented and isolated. Thus, individual-based relationships are required to understand the species ecology and biology for planning effective conservation and genetics-based individual identification has been widely used. But this needs screening and describing characteristics of microsatellite loci from DNA from good-quality sources so that the required number of loci can be selected and the genotyping error rate minimized. In the studies so far conducted on the Bengal tiger, a very small number of loci (n = 35) have been tested with high-quality source of DNA, and information on locus-specific characteristics is lacking. The use of such characteristics has been strongly recommended in the literature to minimize the error rate and by the International Society for Forensic Genetics (ISFG) for forensic purposes. Therefore, we describe for the first time locus-specific genetic and genotyping profile characteristics, crucial for population genetic studies, using high-quality source of DNA of the Bengal tiger. We screened 39 heterologous microsatellite loci (Sumatran tiger, domestic cat, Asiatic lion and snow leopard) in captive individuals (n = 8), of which 21 loci are being reported for the first time in the Bengal tiger, providing an additional choice for selection. The mean relatedness coefficient (R = −0.143) indicates that the selected tigers were unrelated. Thirty-four loci were polymorphic, with the number of alleles ranging from 2 to 7 per locus, and the remaining five loci were monomorphic. Based on the PIC values (> 0.500), and other characteristics, we suggest that 16 loci (3 to 7 alleles) be used for genetic and forensic study purposes. The probabilities of matching genotypes of unrelated individuals (3.692 × 10-19) and siblings (4.003 × 10-6) are within the values needed for undertaking studies in population genetics, relatedness, sociobiology and forensics.

A comparative study of the use of tiger-specific and heterologous microsatellite markers for population genetic studies of the Bengal tiger (Panthera tigris tigris)

Comparison of genetic diversity indices of heterologous and species-specific microsatellite loci within a species may provide a panel of appropriate markers for genetic studies, but few studies have carried out such comparisons. We examined and compared the genetic characteristics of tiger-specific and heterologous loci in eight captive Bengal tigers. The mean polymorphic information content (PIC) value of the tiger-specific microsatellite loci (n = 15) was 0.447, and the number of alleles was from 2 to 4 per locus. In comparison, the heterologous microsatellite loci (n = 15) had a mean PIC value of 0.539, and the number of alleles per locus was three to five. Our findings indicate that the heterologous markers have a higher frequency (n = 11) of polymorphic microsatellite loci and number of alleles per locus compared with tiger-specific loci. We pooled the highly polymorphic (PIC > 0.5) tiger-specific loci (n = 5) and heterologous microsatellite loci (n = 11) except one and noted a higher mean observed heterozygosity and PIC values of 0.668 and 0.575, respectively, compared with the heterologous and tiger-specific loci taken alone. Using a locus selection criterion of PIC > 0.5, we recommend a combined panel of 16 highly polymorphic loci for genetic studies of the wild population of the Bengal tigers and suggest that either a combination of tiger-specific and heterologous microsatellite primers or heterologous primers be used in genetic studies related to the ecology, biology, socio-biology and behavior of Bengal tigers as >13 loci are needed in such studies.

Identification of Galliformes through Forensically Informative Nucleotide Sequencing (FINS) and its Implication in Wildlife Forensics

Galliformes are hunted for the demand of their attractive feathers and to supply a cheap animal food for the rural communities. In such cases, species identification through visual inspection of the meat or based on feather morphometrics is a challenging task for the law enforcement agencies to enforce the Wildlife Protection Act (WPA). Here, we extracted DNA from the individual feathers of unknown species encountered during field surveys and two mitochondrial genes (12S rRNA and Cytochrome b) were amplified using universal primers for species identification. Most homologous sequences were retrieved using NCBI-BLAST for each generated sequence of both the genes. Neighbor-Joining trees based on Kimura 2 parameter distance matrices in FINS analysis identified the species from the individual feather with strong bootstrap support. Nine species specific polymorphic sites were found in the partial sequence of Cytochrome b gene that differentiated Pavo cristatus to Pavo muticus imperator. Our study highlighted the importance of feathers in identifying the species and their applicability in wildlife offence cases using FINS approach.

Curtailing Human-Leopard Conflict Using Wildlife Forensics: A Case Studyfrom Himachal Pradesh, India

Recent changes in the land use pattern have severely impacted wildlife, specifically large carnivores like leopards, by reducing natural habitat and prey base. Being highly adaptable, with a distribution more outside than inside the protected areas, leopard very often attacks human and livestock. In human-leopard conflicts, once an animal is declared as man-eater, it is either translocated or killed by officials as per Wildlife (Protection) Act 1972 of India. Identification of conflicting leopard is very difficult and sometimes lead to the castigation of innocent animal. Here, we describe the individualization of a leopard from one such human-leopard conflict in Bilaspur district of Himachal Pradesh, India using modern molecular genetic techniques. The methodology suggested in this study would be of great importance in correct identification of conflicting animals.