DNA barcoding as a tool for robust identification of cervids of India and its utility in wildlife forensics.

DNA barcoding has become a popular method of choice for identification of specimen based on molecular techniques. Here, we present preliminary findings on generating robust DNA barcode library of Cervids of India. The dataset comprising the DNA barcode library of seven deer species included in the genus Cervus, Axis and Muntiacus classified under family Cervidae. Mitochondrial Cytochrome C Oxidase subunit I gene of ca. 710 bp accepted widely as DNA barcode region, was used for generating species specific signature from 31 known samples of seven Indian deer species. Expectedly, the NJ tree clustered three genera i.e. Cervus, Axis and Muntiacus of Cervids of India into three clades. Further, the intra- and interspecies distances based on Kimura 2 parameter model also supported the results. The average intra- and interspecies sequence divergence were 0.011 (±0.09) and 0.65 (±0.14), respectively. The present study has exhibited that DNA barcoding has discriminating power to delineate boundaries among the closely related species. The data generated are of high importance to the law enforcement agencies in effective identification of species in wildlife offence cases. The similar approach can be utilized for generating DNA barcodes for other Indian mammals for making effective management and conservation action decisions.

Resolving the phylogenetic status and taxonomic relationships of the Hangul (Cervus elaphus hanglu) in the family Cervidae.

The Hangul (Cervus elaphus hanglu) is a "Least Concern" deer species, and it is the only survivor of the Red Deer group in the Indian subcontinent. The phylogenetic status of the Hangul relative to the other members of the family Cervidae is not known because sequence data are not available in public databases. Therefore, this study was carried out to determine the phylogenetic status and delineate the genetic boundaries of the Hangul with respect to the other Red Deer subspecies on the basis of cytochrome b gene sequence data (ca 421 bp). There are three major monophyletic groups of the Red Deer in the phylogenetic tree, which are referred to as the western (Hap-01 to Hap-10), eastern (Hap-11 to Hap-20) and tarim (Hap-21 to Hap-25) groups. The overall haplotype diversity and per-site nucleotide diversity were 0.9771 (±0.0523) and 0.0388 (±0.00261), respectively. In the phylogenetic tree, the Hangul clustered with the tarim group (Yarkand and Bactrian Red Deer) with a strong bootstrap support (92%) and was found to be genetically closer to the Bactrian Red Deer than to the Yarkand Red Deer. Our molecular analysis supported the idea that the Hangul diverged from the Bactrian Red Deer and migrated to India from Tajikistan approximately 1.2 MYA.

Genetic diversity of the Tibetan antelope (Pantholops hodgsonii) population of Ladakh, India, its relationship with other populations and conservation implications.

BACKGROUND: The Tibetan antelope (Pantholops hodgsonii), or chiru, is an endangered antelope, distributed in China [Xinjiang, Xizang, Qinghai, Zhuolaihu Lake (Breeding habitat)], and India (Aksai Chin and Ladakh). There is a global demand for the species prized wool, which is used in weaving shahtoosh shawls. Over the years, the population of the Tibetan antelope has drastically declined from more than a million to a few thousand individuals, mainly due to poaching. Field studies undertaken in Ladakh, India also indicated winter migration of the population to Tibet. Migration between winter and calving habitats is well established to be female-biased across the Qinghai Tibetan Plateau (QTP). For effective conservation planning, genetic characterization is considered the best way to understand the likely impact of threats for ensuring the long-term viability of the population. In this regard, genetic characteristics of all Chinese populations are well-studied using mitochondrial and microsatellite markers, but information is lacking for the Indian population. Therefore, using the control region marker, we document for the first time the genetic variation of the Indian population of the Tibetan antelope, the extent of migration and its relationships with other populations of China. RESULTS: The partial fragment of control region (259 bp) marker was successfully amplified in 30 Tibetan antelope samples that were collected from the Chang Chenmo Valley in eastern Ladakh, India. We also retrieved control region sequences (n = 88) available in the public domain from GenBank of different Chinese populations. Low levels of nucleotide (π; 0.004) and haplotype (hd; 0.543) diversity were observed in the Indian population when compared to Chinese populations (π = 0.01357-0.02048 and hd = 0.889-0.986). Commonly used indices (Tajima's D and Fu's Fs) were analyzed for inferring the demographic history of the Indian populations, and all values were negative indicating population expansion or demographic equilibrium, though nonsignificant. We observed five haplotypes in the Indian population, and these were not reported in previously studied populations of QTP. Bayesian-based phylogenetic analysis indicates the presence of four clades, however, the posterior probability support for three of these clades is weak (<0.5). Of these, the Indian population formed a distinct clade, whereas the Chinese populations exhibited shared haplotypes, and no geographic structure was observed. Median-joining network analysis was conducted for 46 haplotypes in the overall population, except the samples from India which showed a star-like topology. The Indian population is separated by one median vector from the Chinese population. CONCLUSIONS: The present study revealed the presence of different sub-clades in the Bayesian phylogenetic tree and five new haplotypes only in the Indian population or sampling location. Furthermore, in the phylogenetic tree, Indian haplotypes of Tibetan antelopes were clustered with the haplotype reported in the Chinese population of the Xinjiang region. Median-joining network analysis showed shared haplotypes pattern in all populations of QTP except the samples from India which showed new haplotypes. Given the presence of low nucleotide and haplotype diversity in eastern Ladakh populations and limited information available for populations of the western side in its range, we suggest to include genetic studies of Tibetan antelope populations around Aksai Chin (Fig. 1) under the proposed transboundary agenda between India and China and assess relationships with other populations. Such understanding would enable the planning of conservation strategies for ensuring long-term survival of westernmost populations in its range, and if required, it would establish connectivity with the other populations.

Pragmatic perspective on conservation genetics and demographic history of the last surviving population of Kashmir red deer (Cervus elaphus hanglu) in India.

The hangul (Cervus elaphus hanglu) is of great conservation concern because it represents the easternmost and only hope for an Asiatic survivor of the red deer species in the Indian subcontinent. Despite the rigorous conservation efforts of the Department of Wildlife Protection in Jammu & Kashmir, the hangul population has experienced a severe decline in numbers and range contraction in the past few decades. The hangul population once abundant in the past has largely become confined to the Dachigam landscape, with a recent population estimate of 218 individuals. We investigated the genetic variability and demographic history of the hangul population and found that it has shown a relatively low diversity estimates when compared to other red deer populations of the world. Neutrality tests, which are used to evaluate demographic effects, did not support population expansion, and the multimodal pattern of mismatch distribution indicated that the hangul population is under demographic equilibrium. Furthermore, the hangul population did not exhibit any signature of bottleneck footprints in the past, and Coalescent Bayesian Skyline plot analysis revealed that the population had not experienced any dramatic changes in the effective population size over the last several thousand years. We observed a strong evidence of sub-structuring in the population, wherein the majority of individuals were assigned to different clusters in Bayesian cluster analysis. Population viability analysis demonstrated insignificant changes in the mean population size, with a positive growth rate projected for the next hundred years. We discuss the phylogenetic status of hangul for the first time among the other red deer subspecies of the world and strongly recommend to upgrade hangul conservation status under IUCN that should be discrete from the other red deer subspecies of the world to draw more conservation attention from national and international bodies.

Loss of genetic diversity and inbreeding in Kashmir red deer (Cervus elaphus hanglu) of Dachigam National Park, Jammu & Kashmir, India.

BACKGROUND: Hangul (Cervus elaphus hanglu), the eastern most subspecies of red deer, is now confined only to the mountains in the Kashmir region of Jammu & Kashmir State of India. It is of great conservation significance as this is the last and only hope for Asiatic survivor of the red deer species in India. Wild population of free ranging hangul deer inhabiting in and around Dachigam National Park was genetically assessed in order to account for constitutive genetic attributes of hangul population using microsatellite markers. RESULTS: In a pool of 36 multi-locus genotypes, 30 unique individuals were identified based on six microsatellite loci. The estimated cumulative probability of identity assuming all individuals were siblings (PID sibs) was 0.009 (9 in 1000). Altogether, 49 different alleles were observed with mean (± s.e.) allelic number of 8.17 ± 1.05, ranging from 5 to 11 per locus. The observed heterozygosity ranged between 0.08 and 0.83, with mean 0.40 ± 0.11 and the inbreeding coefficient ranged between -0.04 and 0.87 with mean 0.38 ± 0.15. Majority of loci (5/6) were found to be informative (PIC value > 0.5). All loci deviated from Hardy-Weinberg equilibrium except Ca-38 (P > 0.05) and none of the pairs of loci showed significant linkage disequilibrium except the single pair of Ca-30 and Ca-43 (P < 0.05). CONCLUSIONS: The preliminary findings revealed that hangul population is significantly inbred and exhibited a low genetic diversity in comparison to other deer populations of the world. We suggest prioritizing the potential individuals retaining high heterozygosity for ex situ conservation and genetic monitoring of the hangul population should be initiated covering the entire distribution range to ensure the long term survival of hangul. We speculate further ignoring genetics attributes may lead to a detrimental effect which can negatively influence the reproductive fitness and survivorship of the hangul population in the wild.