An effective method for extraction and polymerase chain reaction (PCR) amplification of DNA from formalin preserved tissue samples of snow leopard

Formalin-preserved biological samples obtained from endangered species are valuable in assessing genetic diversity. To make use of snow leopard samples preserved in formalin over a period of two to seven years, we optimized the method of extracting DNA from these samples. We used (a) phenol chloroform : isoamyl alcohol, (b) the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germany), (c) the Qiagen DNeasy Blood and Tissue Kit after treating the samples with NaOH for three days and (d) the Qiagen DNeasy Blood and Tissue Kit after treating the samples with phosphate buffered saline (PBS) for three days. The usefulness of the extracted DNA was assessed on the basis of mitochondrial (150 to 550 bp) and nuclear (95 to 229 bp) markers. There was no PCR amplification with the first two methods. The PCR amplification with the NaOH and PBS treatment had a success rate of 30 to 100% for both mitochondrial and nuclear markers. The PBS method is the best method for extraction of DNA from formalin-preserved samples of longer period (two to seven years) because of higher success rate in amplifying mitochondrial gene of ca. 550 bp (60%) than the NaOH method (28%). The overall amplification of microsatellite markers in such samples was also higher in samples treated with PBS (43 to 100%) than NaOH (0 to 100%). The PCR products obtained were confirmed through DNA sequencing to be of snow leopard origin. The optimized protocol will enable genetic studies to be conducted on tissue samples of other species that have been preserved in formalin. The protocol will be particularly useful for species that are elusive and from which it is difficult to collect fresh tissue samples.Keywords: Formalin, polymerase chain reaction (PCR), mtDNA, microsatellites, snow leopardAfrican Journal of Biotechnology Vol. 12(22), pp. 3399-340

Predicting the impact of climate change on range and genetic diversity patterns of the endangered endemic Nilgiri tahr (Nilgiritragus hylocrius) in the western Ghats, India

[Context] Climate change is considered an important factor affecting the distribution and genetic diversity of species. While many studies have described the influence of climate change on population structure at various scales, little is known about the genetic consequences of a changing climate on endemic species.[Objectives] To assess possible changes in the distribution and genetic structure of the endangered Nilgiri tahr (Nilgiritragus hylocrius), which is endemic to the Western Ghats in India, under climate change and human disturbances.[Methods] We integrated tahr occurrence and nuclear DNA data with environmental geo-datasets to project the response of tahr populations to future climate change with respect to its distribution, genetic diversity and population structure. We screened the environmental variables using MaxEnt to identify a manageable set of predictors to be used in an ensemble approach, based on ten species distribution modelling techniques, to quantify the current tahr distribution. We then projected the distribution and genetic structure under two climate change scenarios.[Results] We found that suitable habitat for tahr (9,605 km2) is determined predominantly by a combination of climatic, human disturbance and topographic factors that result in a highly fragmented habitat throughout its distribution range in the Western Ghats. Under the severe high emissions RCP8.5 scenario tahr populations may lose more than half of their available habitat (55.5%) by 2070. Application of spatial Bayesian clustering suggests that their current genetic structure comprise four genetic clusters, with three of them reflecting a clear geographic structure. However, under climate change, two of these clusters may be lost, and in the future a homogenization of the genetic background of the remaining populations may arise due to prevalence of one gene pool cluster in the remaining populations.[Conclusions] Our interdisciplinary approach that combines niche modelling and genetic data identified the climate refugia (i.e., the remaining stable habitats that overlap with the current suitable areas), where the tahr populations would be restricted to small, isolated and fragmented areas. Essential factors to avert local extinctions of vulnerable tahr populations are a reduction of human disturbances, dispersal of tahr between fragmented populations, and the availability of corridors.This research was supported by the Department of Biotechnology, Ministry of Science and Technology, Government of India, and by a German Research Foundation (DFG) fellowship awarded to RK (project number 273837911).Peer reviewe

DNA barcodes and ethnomedicinal use of Sharpnose guitarfish Glaucostegus granulatus by the locals at Keylong, Lahaul and Spiti, Himachal Pradesh

Illegal trade of fishes is common and has been in practice since ages for the support of livelihood and as dietary supplements. However, several species are protected in the Wildlife (Protection) Act, 1972 of India and their trade is restricted under CITES. In this article, we report trade of Sharpnose guitarfish (Glaucostegus granulatus) for the ethnomedicinal remedy, identified using DNA barcoding in the Keylong district of Lahaul and Spiti, Himachal Pradesh. This study provides the first DNA barcode of Sharpnose guitarfish. In order to handle wildlife offense cases we emphasize that a large reference database for other fishes in trade is needed

Understanding genetic diversity and population genetic structure of three Cyprinidae fishes occupying the same habitat from Uttarakhand, India

Different pattern of genetic diversity and population genetic structure among the species are reported due to their different ecological requirements, adaptability and the evolutionary histories. Understanding such patterns in a species and between the populations is important to develop the effective conservation plans. Very limited studies are available, how different factors influencing the gene flow of a species especially in fish communities. Therefore, the present study is aimed to document the genetic diversity and population genetic structure of the three species of Cyprinidae fishes (Puntius sophore, Pethia ticto, and Pethia conchonius) sharing the same kind of habitat using the mitochondrial cytochrome c oxidase subunit 1 (CO1). We used 80 samples of the three species from different river/streams. In which we observed total 4–9 haplotypes in all three species with the intra-species sequenced divergence ranges between 0.002 and 0.019. The nucleotide and haplotype diversity was ranged from 0.002040 to 0.01007 and from 0.251 to 0.822, respectively. Neutrality test values were found to be positive only in the P. ticto but statistically non-significant. The AMOVA variation among the populations was 8.89–84.30% whereas, within the populations, it was ranged from 15.70 to 91.11%. The median-joining haplotype network suggests the stable population size over the time and haplotypes were clustered with respect to their geographic locations except the P. conchonius. Similar pattern observed in the phylogenetic tree

Time-lapse sentinel surveillance of SARS-CoV-2 spread in India.

The novel coronavirus 2019 (COVID-19) global pandemic has drastically affected the world economy, raised public anxiety, and placed a substantial psychological burden on the governments and healthcare professionals by affecting over 4.7 million people worldwide. As a preventive measure to minimise the risk of community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in India, a nationwide lockdown was imposed initially for 21 days to limit the movement of 1.3 billion people. These restrictions continue in most areas, with a conditional relaxation occurring in a few Indian states. In an attempt to assess the emerging mutants of SARS-CoV-2 and determine their spread in India, we analysed 112 complete genomes of SARS-CoV-2 in a time-lapse manner. We found 72 distinct SARS-CoV-2 haplotypes, defined by 143 polymorphic sites and high haplotype diversity, suggesting that this virus possesses a high evolutionary potential. We also demonstrated that early introduction of SARS-CoV-2 into India was from China, Italy and Iran and observed signs of community spread of the virus following its rapid demographic expansion since its first outbreak in the country. Additionally, we identified 18 mutations in the SARS-CoV-2 spike glycoprotein and a few selected mutations showed to increase stability, binding affinity, and molecular flexibility in the overall tertiary structure of the protein that may facilitate interaction between the receptor binding domain (RBD) of spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor. The study provides a pragmatic view of haplotype-dependent spread of SARS-CoV-2 in India which could be important in tailoring the pharmacologic treatments to be more effective for those infected with the most common haplotypes. The findings based on the time-lapse sentinel surveillance of SARS-CoV-2 will aid in the development of a real-time practical framework to tackle the ongoing, fast-evolving epidemic challenges in the country

Living with a large predator: Assessing the root causes of Human–brown bear conflict and their spatial patterns in Lahaul valley, Himachal Pradesh

Abstract Brown bear‐mediated conflicts have caused immense economic loss to the local people living across the distribution range. In India, limited knowledge is available on the Himalayan brown bear (HBB), making human–brown bear conflict (HBC) mitigation more challenging. In this study, we studied HBC in the Lahaul valley using a semi‐structured questionnaire survey by interviewing 398 respondents from 37 villages. About 64.8% of respondents reported conflict in two major groups—crop damage (30.6%) and livestock depredations (6.2%), while 28% reported both. Conflict incidences were relatively high in summer and frequently occurred in areas closer to the forest (<500 m) and between the elevations range of 2700 m to 3000 m above sea level (asl). The dependency of locals on forest resources (70%) for their livelihood makes them vulnerable to HBC. The “upper lower” class respondents were most impacted among the various socioeconomic classes. Two of the four clusters were identified as HBC hot spots in Lahaul valley using SaTscan analysis. We also obtained high HBC in cluster II with a 14.35 km radius. We found that anthropogenic food provisioning for HBB, livestock grazing in bear habitats, and poor knowledge of animal behavior among the communities were the major causes of HBC. We suggest horticulture crop waste management, controlled and supervised grazing, ecotourism, the constitution of community watch groups, and others to mitigate HBC. We also recommend notifying a few HBB abundant sites in the valley as protected areas for the long‐term viability of the HBB in the landscape

Phylogeography of the small Indian civet and origin of introductions to western Indian Ocean islands

International audienceThe biogeographic dynamics affecting the Indian subcontinent, East and Southeast Asia during the Plio-Pleistocene has generated complex biodiversity patterns. We assessed the molecular biogeography of the small Indian civet (Viverricula indica) through mitogenome and cytochrome b + control region sequencing of 89 historical and modern samples to (1) establish a time-calibrated phylogeography across the species' native range and (2) test introduction scenarios to western Indian Ocean islands. Bayesian phylogenetic analyses identified 3 geographic lineages (East Asia, sister-group to Southeast Asia and the Indian subcontinent + northern Indochina) diverging 3.2-2.3 million years ago (Mya), with no clear signature of past demographic expansion. Within Southeast Asia, Balinese populations separated from the rest 2.6-1.3 Mya. Western Indian Ocean populations were assigned to the Indian subcontinent + northern Indochina lineage and had the lowest mitochondrial diversity. Approximate Bayesian computation did not distinguish between single versus multiple introduction scenarios. The early diversification of the small Indian civet was likely shaped by humid periods in the Late Pliocene-Early Pleistocene that created evergreen rainforest barriers, generating areas of intra-specific endemism in the Indian subcontinent, East, and Southeast Asia. Later, Pleistocene dispersals through drier conditions in South and Southeast Asia were likely, giving rise to the species' current natural distribution. Our molecular data supported the delineation of only 4 subspecies in V. indica, including an endemic Balinese lineage. Our study also highlighted the influence of prefirst millennium AD introductions to western Indian Ocean islands, with Indian and/or Arab traders probably introducing the species for its civet oil

Indian Grey Wolf and Striped Hyaena sharing from the same bowl: High niche overlap between top predators in a human-dominated landscape

The Semi-arid wildlands of the West Bengal state of India are known habitats of Indian Grey Wolf (Canis lupus pallipes) and Striped Hyaena (Hyaena hyaena) in India. However, increasing anthropogenic pressure in these regions is one major factor behind habitat loss and rising carnivore-human conflict. We mapped the suitable habitat and estimated the niche overlap of both species in the study landscape. Further, we assessed the habitat quality, identified corridors and quantified land sharing by both species. The anthropogenic disturbance (human settlements) was found to be one of the most significant contributors for both species. The higher Schoener’s D (0.612) and Hellinger’s based I value (0.858) indicates a considerable niche overlap of about 3529 km2. We observed a high level of fragmentation in suitable patches of Grey Wolf compared to the Striped Hyaena. For both Striped Hyaena and Grey Wolf, connectivity exists among the large patch of western and southern Purulia and Bankura. The Grey Wolf corridors in the Jhargram district were also found connected with other areas. We found that both the species, which are generally known rivals showing tolerance towards each other, which is an strong evidence of land sharing among the species. We recommend that the suitable patches and corridors identified in the study may be prioritized for conservation and management

Empirical Data Suggest That the Kashmir Musk Deer (<i>Moschus cupreus</i>, Grubb 1982) Is the One Musk Deer Distributed in the Western Himalayas: An Integration of Ecology, Genetics and Geospatial Modelling Approaches

Insufficient research has been conducted on musk deer species across their distribution range, primarily because of their elusive behaviour and the fact they occupy remote high-altitude habitats in the Himalayas above 2500 m. The available distribution records, primarily derived from ecological studies with limited photographic and indirect evidence, fail to provide comprehensive information on the species distribution. Consequently, uncertainties arise when attempting to determine the presence of specific taxonomic units of musk deer in the Western Himalayas. This lack of knowledge hampers species-oriented conservation efforts, as there need to be more species-specific initiatives focused on monitoring, protecting, and combatting the illegal poaching of musk deer for their valuable musk pods. We used transect surveys (220 trails), camera traps (255 cameras), non-invasive DNA sampling (40 samples), and geospatial modelling (279 occurrence records) to resolve the taxonomic ambiguity, and identify the suitable habitat of musk deer (Moschus spp.) in Uttarkashi District of Uttarakhand and the Lahaul–Pangi landscape of Himachal Pradesh. All the captured images and DNA-based identification results confirmed the presence of only Kashmir musk deer (KDM) (Moschus cupreus) in Uttarakhand and Himachal Pradesh. The results suggest that KMD inhabit a narrow range of suitable habitats (6.9%) of the entire Western Himalayas. Since all evidence indicates that only KMD are present in the Western Himalayas, we suggest that the presence of other species of musk deer (Alpine musk deer and Himalayan musk deer) was wrongly reported. Therefore, future conservation plans and management strategies must focus only on KMD in the Western Himalayas