Mitochondrial cytochrome b indicates the presence of two paraphyletic diverged lineages of the blue sheep Pseudois nayaur across the Indian Himalaya: conservation implications.

BACKGROUND: Populations exhibit signatures of local adaptive traits due to spatial and environmental heterogeneity resulting in microevolution. The blue sheep is widely distributed across the high Asian mountains and are the snow leopard's principal prey species. These mountains differ in their evolutionary history due to differential glaciation and deglaciation periods, orography, and rainfall patterns, and such factors causes diversification in species. METHODS AND RESULTS: Therefore, we assess the phylogeographic status of blue sheep using the mitochondrial cytochrome b gene (220 bp) across the Indian Himalayan region (IHR) and its relationship with other populations. Of the observed five haplotypes, two and three were from the western Himalayas (WH) and eastern Himalayas (EH) respectively. One of the haplotypes from WH was shared with the population of Pamir plateau, suggesting historical maternal connectivity between these areas. The phylogenetic analyses split the blue sheep into two paraphyletic clades, and western and eastern populations of IHR were within the Pamir and Tibetan plateau clades, respectively. We observed a relatively higher mean sequence divergence in the EH population than in the WH. CONCLUSION: We propose five 'Evolutionary Significant Units' across the blue sheep distribution range based on observed variation in the species' ecological requirements, orography, climatic conditions, and maternal lineages, viz.; Western Himalaya-Pamir plateau (WHPP); Eastern Himalaya-Tibetan plateau (EHTP); Qilian mountains; Helan mountains and Hengduan mountains population. Despite the small sample size, population divergence was observed across the IHR, therefore, we suggest a transboundary, collaborative study on comparative morphology, anatomy, ecology, behaviour, and population genetics using harmonized different genetic markers for identifying the overall taxonomic status of the blue sheep across its range for planning effective conservation strategies.

Investigating the genetic diversity and presence of forensically informative nucleotide sequences in Indian antelope (Antilope cervicapra) using multiple genes of the mitochondrial genome.

Indian antelope or Blackbuck (Antilope cervicapra) is one of the widely distributed endemic species in India among wild bovids and a majority of preferred habitats are in human-dominated landscapes. Poaching threats and habitat degradation are major factors for the decline in Blackbuck population from its distribution range. Till date, there is no detailed study using molecular techniques in India on Blackbuck, except a few studies entailing phylogenetic scenario based on inadequate sampling and DNA sequences restricted over limited geographic areas. In view of this, the present study is aimed to screen the Blackbuck samples from a large part of its distribution range and to investigate the genetic diversity as well as to identify the forensically informative nucleotide sequences (FINS) for species identification. We relied on multi-genes approach using three genes of mtDNA genome viz. Cytochrome Oxidase I, Cytochrome b and 16S rRNA and identified the FINS in the Blackbuck population along with conspecific sequences divergence and genetic diversity indices. In all three genes, we observed 8 to 17 haplotypes with the intra-species sequence divergence of 0.004-0.016. Inter-species sequence divergence with the other closely related species of the Blackbuck was 0.0225-0.033. We report the presence of FINS across three genes from 12 to 18 and found more informative nucleotide sites using Cytochrome Oxidase I genes compared to Cytochrome b and 16S rRNA gene. We did not observe the presence of geographic-specific FINS amongst Blackbuck population that can be used to assign individuals to geographic origin. Besides, in the phylogenetic tree, samples from different locations did not cluster into geographic-specific clade and exhibited mixed homology for these sequences. We suggest exploring the feasibility of using nuclear markers for population assignment.

Species dilemma of musk deer (Moschus spp) in India: molecular data on cytochrome c oxidase I suggests distinct genetic lineage in Uttarakhand compared to other Moschus species.

Musk deer are of high conservation priority owing to poaching pressure because of its musk pod. Representation of musk deer status using genetics is poorly documented in India, and it is not confirmed as to how many species of musk deer are present. We characterize for the first time, the genetic diversity of musk deer from Uttarakhand using Cytochrome Oxidase sub-unit (COI) gene (486 bp) and compared with the data available for other species. Results revealed the presence of six haplotypes in the Uttarakhand population amongst 17 sequences. Of these, 12 sequences shared the single haplotype. The intra-species sequences divergence was 0.003-0.017, whereas divergence with other species of musk deer was 0.071-0.081. Bayesian phylogenetic tree revealed that samples from Uttarakhand formed a separate clade with respect to other species of musk deer, whereas three species distributed in China clustered in the same clade and showed low sequences divergence, i.e., 0.002-0.061. Because of different ecomorph reported, we suggest using the barcoding based approach for inter and intra-species distinction and delineating species boundaries across the range for effective conservation. Besides, systematic classification, DNA barcoding would also help in dealing wildlife offence cases for disposal of the legal report in court.

Araniella cucurbitina: the first molecular evidence of a Palearctic species of genus Araniella inhabiting India.

The present study represents first genetic record of single spider species Araniella cucurbitina, genus Araniella from Uttarakhand, India. There are 12 identified species under Araniella genus and they are distributed in Palearctic region. Here, we used known N = 47 (2 from present study and 45 from GenBank) cytochrome oxidase 1 (CO1) sequences of A. cucurbitina representing seven different geographical groups, additionally 23 sequences of eight Araniella species were used for phylogenetic relationship. The CO1 (561 bp) sequences of A. cucurbitina consisted of n = 14 haplotypes, where haplotype 14 (Hap 14) represents Indian species, while all 13 haplotypes (Hap1-13) shared between six A. cucurbitina groups. The overall 'h' and 'π' diversities among seven groups of A. cucurbitina were 0.85291 and 0.00888, respectively, while overall evolutionary divergence was 0.04. The Indian Hap 14, showed minimum sequence divergence (0.02) from Italy and Czech Republic haplotypes (Hap 6), it means it is the closest group compared to others. Evolutionary divergence among eight species of Araniella ranges from 0.003 to 0.114. The maximum likelihood (ML) topology based on 14 haplotypes of A. cucurbitina was divided into two major clades and further two subclades. Furthermore, ML topology between eight species of Araniella was divided into three major clades, where A. cucurbitina and A. proxima clustered together in clade 'A', while six others were present together in clade 'B' and 'C'. This study helps to identify the Indian species from the rest of Araniella species and other cucurbitina population across the world. This study further needs to be on a large scale to know the exact status distribution and molecular phylogeography of this single species of genus Araniella from India.

Inferring the molecular affinity of Indian pangolin with extant Manidae species based on mitochondrial genes: a wildlife forensic perspective.

Pangolins are the world`s most trafficked mammalian species classified under family Manidae and face severe threat of extinction, largely due to the illicit trade of its parts and products, especially scales, in international markets. Pangolin scales are believed to be used in Traditional Chinese Medicines (TCM) and meat is used as delicacies in restaurants. Of the eight extant species of pangolin, morphological discrimination is easy but the situation becomes precarious once the scales and meat samples are seized and it is difficult to identify species based on morphology in such cases. However, wildlife DNA forensics has played an instrumental role in the identification of species from such type of materials. The present study investigated that three mitochondrial genes (Cyt b, 16S rRNA, and 12S rRNA) clearly showed the variation among seven extant pangolin species (Manis culionensis; possibly extinct), whereas, maximum variation was obtained in cytochrome b when compared to another two mitochondrial genes. The present study revealed that obtained SNPs based on short sequence length (Intervals) within the three mitochondrial genes will be helpful to design the short molecular marker and species-specific probe that is used in wildlife forensic for identifying pangolin species from the degraded sample. We also advocate using more than one molecular marker for species discrimination so as to minimize any false identification of the mammal's species reported in the trade. Furthermore, data generated from the study would help in strengthening the DNA database of Indian pangolin species.

Wildlife forensics in battle against veneration frauds in Uttarakhand, India: identification of protected Indian monitor lizard in items available in the local market under the name of Hatha Jodi.

Certain articles of worship are commonly sold in Uttarakhand, India by the name Hatha Jodi, a root of a rare plant found only in a few parts of central India. The present work provides genetic proof that the Hatha Jodi sold in three local markets of Uttarakhand contained material from the Varanus species, species protected under the Indian Wildlife (Protection) Act, 1972. A total of eight samples were bought, two each from the local markets in Haridwar and Rishikesh, three from Dehradun and one from an online source (Amazon). The initial inspection confirmed that two of the samples were made of plastic material. Therefore only the other six samples were subjected to DNA analysis. DNA sequences were successfully obtained and matched with reference sequences available in NCBI Genbank database through BLAST search tool for species identification. All the six samples matched 100% with the Indian monitor lizard. The findings indicate how commercialization and the wildlife trade are playing a role in decline of the population of the Indian monitor lizard. If strong protection measures are not taken as soon as possible, the Indian monitor lizards will go Extinct very soon. Therefore, we suggest that the Government and Wildlife enforcement agencies take serious action against the illegal articles available in the local markets of Uttarakhand under the name Hatha Jodi. Further, the government needs to take legal action against offenders in other states in which the product is available for sale.

The preliminary molecular study of four skink species in Rajaji Tiger Reserve (RTR), Uttarakhand, using 12S rRNA mitochondrial locus.

Skinks are present under the Scincidae family, widely distributed species in Indian subcontinent. Uttarakhand is one of the hotspot where number of identified and unidentified skink species reported. Herein, we first time provided the 12S rRNA genetic reference database of four skink species, i.e. Eutropis macularia, Eutropis carinata, Asymblepharus himalayanus and Lygosoma punctata, in Rajaji Tiger Reserve (RTR), Uttarakhand (India). The identified four species belong to three different genera, where Eutropis carinata and Asymblepharus himalayanus listed Least Concern and Vulnerable in IUCN, respectively. Here, we collected tissue samples of four different skink species from Rajaji Tiger Reserve during field survey. After successful laboratory procedure, we compared obtained sequences with publically available genetic database and we observed four sequences matched with respective species. Furthermore, the evolutionary sequence divergence result revealed that the Eutropis carinata and Eutropis macularia are close to each other with 0.11 genetic distance. The present study indicates that the exact number and population distribution of skink species are unidentified; therefore, herein we suggest the proper screening of Uttarakhand population around should be investigated, further genetic study in combination with a good sampling strategy to investigate species biology and status for conservation program.

Genetic characterization of wild swamp deer populations: ex situ conservation and forensics implications.

Swamp deer (Rucervus duvaucelii) is an endemic, Scheduled I species under the Wildlife (Protection) Act 1972, India. According to variations in antler size, it has been classified into three subspecies, namely Western (R. duvaucelii duvaucelii), Central (R. duvaucelii branderi), and Eastern (R. duvaucelii ranjitsinhii). For planning effective ex situ and in situ conservation of a wide-ranging species in different bioclimatic regions and in wildlife forensic, the use of genetic characterization in defining morpho/ecotypes has been suggested because of the geographic clines and reproductive isolation. In spite of these morphotypes, very little is known about the genetic characteristics of the three subspecies, hence no strict subspecies-based breeding plan for retaining the evolutionary characteristics in captive populations for subsequent re-introduction is available except for a few studies. We describe the genetic characteristics of these three subspecies using cytochrome b of the mtDNA genome (400 bp). The DNA sequence data indicated 11 variable sites within the three subspecies. Two paraphyletic clades, namely the Central India and Western-Eastern populations were found, whereas the Western and Eastern populations are monophyletic with a bootstrap value of 69% within the clade. We suggest the need of sorting these three subspecies using different molecular mtDNA markers in zoos for captive breeding purposes so as to retain the genetic diversity of the separate geographic clines and to use a subspecies-specific fixed-state nucleotide to assess the extent of poaching to avoid any population demography stochastically in India.

Forensically informative nucleotide sequencing (FINS) for the first time authentication of Indian Varanus species: implication in wildlife forensics and conservation.

Monitor lizards are Varanus species widely distributed, endangered reptile in the IUCN red data list. In India, based on the morphological and ecological characteristic, it is divided into four species viz. Bengal monitor lizard, Yellow monitor lizard, Desert monitor lizard and Water monitor lizard. These four species listed as Schedule I species in Indian Wildlife (Protection) Act 1972. This paper first attempt to present Forensically Informative Nucleotide Sequencing (FINS) for the Indian Varanus based on three mitochondrial genes. The molecular framework will be useful for the identification of Indian Varanus species and trade products derived from monitors and as such, have important applications for wildlife management and conservation. Here, we used known 14 individual skin pieces of four species of monitor lizards; the partial fragment of three mitochondrial genes (Cyt b, 12S rRNA, and 16S rRNA) were amplified for genetic study. In Cyt b, 12S rRNA and 16s rRNA, we observed, 5, 5 and 4 Haplotypes; 71, 69, and 43 Variables sites; 90, 89, and 50 Parsimony Informative sites within four species of Indian monitor lizards, respectively. Despite it, the nucleotide composition was T 26.4, C 32.8, A 29.2 and G11.6; T 18.8, C 29.7, A 34.0 and G 17.5; T 21.7, C 27.3, A 32.5 and G 18.5 in Cyt b, 12S rRNA and 16S rRNA, respectively. The neighbor joining phylogenetic tree and maximum parsimony tree of three mitochondrial genes, showed similar results and reveal that, there are two major clades are present in Indian monitor lizards.

Pioneer identification of fake tiger claws using morphometric and DNA-based analysis in wildlife forensics in India.

The illegal trade in wildlife is a serious threat to the existence of wild animals throughout the world. The short supply and high demand for wildlife articles have caused an influx of many different forms of fake wildlife articles into this trade. The task of identifying the materials used in making such articles poses challenges in wildlife forensics as different approaches are required for species identification. Claws constitute 3.8% of the illegal animal parts (n=2899) received at the Wildlife Institute of India (WII) for species identification. We describe the identification of seized suspected tiger claws (n=18) using a combined approach of morphometric and DNA-based analysis. The differential keratin density, determined using X-ray radiographs, indicated that none of the 18 claws were of any large cat but were fake. We determined three claw measurements, viz. ac (from the external coronary dermo-epidermal interface to the epidermis of the skin fold connecting the palmar flanges of the coronary horn), bc (from the claw tip to the epidermis of the skin fold connecting the palmar flanges of the coronary horn) and the ratio bc/ac, for all the seized (n=18), tiger (n=23) and leopard (n=49) claws. Univariate and multivariate statistical analyses were performed using SPSS. A scatter plot generated using canonical discriminant function analysis revealed that of the 18 seized claws, 14 claws formed a cluster separate from the clusters of the tiger and leopard claws, whereas the remaining four claws were within the leopard cluster. Because a discrepancy was observed between the X-ray images and the measurements of these four claws, one of the claw that clustered with the leopard claws was chosen randomly and DNA analysis carried out using the cyt b (137bp) and 16S rRNA (410bp) genes. A BLAST search and comparison with the reference database at WII indicated that the keratin material of the claw was derived from Bos taurus (cattle). This is a pioneering discovery, and we suggest that a hierarchical combination of techniques be used for identifying claws involved in wildlife offences, i.e. that an X-ray, morphometric and DNA-based analysis be carried out, to ascertain whether the claws are of tigers or leopards. To identify species in the illegal wildlife trade morphometric and genetic reference database should be developed. Morphological features as well as DNA profiles need to be used for better implementation of the Wildlife (Protection) Act, 1972 of India and other laws/treaties in South-east Asia.