Preliminary study on cranial measurements and sexual dimorphism in skull bones of gaur (Bos gaurus gaurus, Smith 1827).

Cranial measurements have been widely used in various studies in wildlife sciences, ranging from understanding predator ecology to wildlife forensics. However, detailed description of morphometry and sexual dimorphism of the skull of gaur Bos gaurus gaurus is lacking. The present study was undertaken to determine the sexual dimorphism based on the cranial measurements of gaur. A total of 12 individual gaur skulls of male (n = 6) and female (n = 6) were studied in the field from the naturally deceased animals between January 2018 and December 2021 in different ranges of Bandhavgarh tiger reserve (BTR), Madhya Pradesh, India. The skull measurements were analysed using univariate and multivariate statistics to determine whether cranial dimensions could be used to differentiate male and female skulls reliably. A total of 43 morphometrical parameters grouped into nine indices were calculated. Select morphometrical parameters viz PL, GFL, AKI, LBB, LFB, GBEE, GBAN, BPOP and GTCH were significantly different (p < 0.05) between sexes, whereas GBAN were significantly higher in female skulls. The measurements demonstrated that the skull of the gaur was dolichocephalic as the profile length and the otion to otion breath in both male and female were <75% of the length. Overall, 28 linear measurements of both the sexes were statistically significant (p < 0.05; <0.01). The calculated indices revealed that the foramen magnum index in the female gaur were significantly higher. In calculated cranial indices the facial index (a) was higher in female and facial index (b) were higher in males. The two important parameters, facial breadth in facial index (a) and the greatest breadth in facial index (b) were positively correlated, though facial index (a) was statistically not significant between the sexes. The greater inner length of the foramen magnum in female skull resulted in foramen being oval whereas it was circular in males. These parameters were decisive for sexual dimorphism, skull comparison and craniological studies. This study ascertained that the frontal index and skull index had no significant influence and were not good indices for discriminating skulls between male and female. Based on the Principal Component Analysis, it was found that skull of male and female gaurs exhibits differences in cranial morphology viz. cranial profile length or total length (PL) and the least inner height of the temporal groove (LIHT). The findings of the present study provide baseline information on various craniometrical measurements of skull of gaur, indices and parameters for sex identification that can be effectively used in understanding sex biased predation ecology, provide base line information to describe variation across its geographic range, and in identifying skulls recovered in wildlife offence cases.

The cost of sympatry: spatio-temporal patterns in leopard dietary and physiological responses to tiger competition gradient in Rajaji Tiger Reserve, Uttarakhand, India.

Apex predators have critical roles in maintaining the structure of ecosystem functioning by controlling intraguild subordinate populations. Such dominant-subordinate interactions involve agonistic interactions including direct or indirect impacts on the subordinates. As these indirect effects are often mediated through physiological processes, it is important to quantify such responses to better understand population parameters. We used a large carnivore intraguild system involving tiger (Panthera tigris) and leopard (Panthera pardus) to understand the dietary and physiological responses under a spatio-temporal gradient of tiger competition pressures in Rajaji Tiger Reserve (RTR) between 2015 and 2020. We conducted systematic faecal sampling in the winters of 2015 and 2020 from the park to assess diet and physiological measures. Analyses of leopard-confirmed faeces suggest a dietary-niche separation as a consequence of tiger competition. In 2020, we found an increased occurrence of large-bodied prey species without tiger competition in western-RTR. Physiological measures followed the dietary responses where leopards with large-sized prey in the diet showed higher fT3M and lower fGCM measures in western-RTR. In contrast, eastern-RTR leopards showed lower levels of fT3M and fGCM in 2020, possibly due to intense competition from tigers. Overall, these patterns strongly indicate a physiological cost of sympatry where competition with dominant tigers resulted in elevated nutritional stress. We recommend expansion of leopard monitoring and population estimation efforts to buffers, developing appropriate plans for human-leopard conflict mitigation and intensive efforts to understand leopard population dynamics patterns to ensure their persistence during the ongoing Anthropocene.

Evolutionary insights on critically endangered Kashmir red deer or hangul (Cervus hanglu hanglu) through a mitogenomic lens.

Background: The Kashmir red deer or Hangul (Cervus hanglu hanglu) is the only Tarim red deer species endemic to India. With a current estimated population size of fewer than 200 individuals, this critically endangered species is confined to the greater Dachigam landscape in Jammu and Kashmir. Poaching, habitat loss and fragmentation, resource competition with livestock, and small population size are the major conservation challenges for this species. Methods: Blood sampling was conducted from two wild Hangul individuals during radio-collaring operations at Dachigam National Park, Kashmir in 2013 and 2020, respectively. Using next-generation sequencing approach, we sequenced the 16,351 bp long mitogenome of two wild-caught Hangul individuals (1 M:1 F at ~14× and ~10× coverage, respectively) from Dachigam National Park. Results: The annotated sequences were identical with an AT-rich composition, including 13 protein-coding genes (11,354 bp), 22 tRNA genes (1,515 bp), two ribosomal genes (2,526 bp) and a non-coding control region (917 bp) in a conserved order like other red deer species. Bayesian phylogenetic reconstruction of the red deer complex revealed two major groups: the elaphoid and the wapitoid clades. Hangul formed a distinct clade with its other subspecies C. hanglu yarkandensis and is sister to the Hungarian red deer (C. elaphus hippelaphus). Divergence time analyses suggested that the Tarim deer species group separated ~1.55 Mya from their common ancestors and Hangul diverged ~0.75 Mya from closely related C. yarkandensis, corroborating with the known paleobiogeographic events related to refugia during glaciations in the Pleistocene era. This study provides baseline information on Hangul mitogenome for further research on phylogeography and other population parameters and helps in developing suitable conservation plans for this species.

Consideration of genetic variation and evolutionary history in future conservation of Indian one-horned rhinoceros (Rhinoceros unicornis).

BACKGROUND: The extant members of the Asian rhinos have experienced severe population and range declines since Pleistocene through a combination of natural and anthropogenic factors. The one-horned rhino is the only Asian species recovered from such conditions but most of the extant populations are reaching carrying capacity. India currently harbours ~ 83% of the global wild one-horned rhino populations distributed across seven protected areas. Recent assessments recommend reintroduction-based conservation approaches for the species, and implementation of such efforts would greatly benefit from detailed genetic assessments and evolutionary history of these populations. Using mitochondrial data, we investigated the phylogeography, divergence and demographic history of one-horned rhinos across its Indian range. RESULTS: We report the first complete mitogenome from all the extant Indian wild one-horned rhino populations (n = 16 individuals). Further, we identified all polymorphic sites and assessed rhino phylogeography (2531 bp mtDNA, n = 111 individuals) across India. Results showed 30 haplotypes distributed as three distinct genetic clades (Fst value 0.68-1) corresponding to the states of Assam (n = 28 haplotypes), West Bengal and Uttar Pradesh (both monomorphic). The reintroduced population of Uttar Pradesh showed maternal signatures of Chitwan National Park, Nepal. Mitochondrial phylogenomics suggests one-horned rhino diverged from its recent common ancestors ~ 950 Kya and different populations (Assam, West Bengal and Uttar Pradesh/Nepal) coalesce at ~ 190-50 Kya, corroborating with the paleobiogeography history of the Indian subcontinent. Further, the demography analyses indicated historical decline in female effective population size ~ 300-200 Kya followed by increasing trends during ~ 110-60 Kya. CONCLUSION: The phylogeography and phylogenomic outcomes suggest recognition of three 'Evolutionary Significant Units (ESUs)' in Indian rhino. With ongoing genetic isolation of the current populations, future management efforts should focus on identifying genetically variable founder animals and consider periodic supplementation events while planning future rhino reintroduction programs in India. Such well-informed, multidisciplinary approach will be the only way to ensure evolutionary, ecological and demographic stability of the species across its range.

Tracking forest loss and fragmentation between 1930 and 2020 in Asian elephant (Elephas maximus) range in Nepal.

Forest cover is the primary determinant of elephant distribution, thus, understanding forest loss and fragmentation is crucial for elephant conservation. We assessed deforestation and patterns of forest fragmentation between 1930 and 2020 in Chure Terai Madhesh Lanscape (CTML) which covers the entire elephant range in Nepal. Forest cover maps and fragmentation matrices were generated using multi-source data (Topographic maps and Landsat satellite images of 1930, 1975, 2000, and 2020) and spatiotemporal change was quantified. At present, 19,069 km2 forest cover in CTML is available as the elephant habitat in Nepal. Overall, 21.5% of elephant habitat was lost between 1930 and 2020, with a larger (12.3%) forest cover loss between 1930 and 1975. Area of the large forests (Core 3) has decreased by 43.08% whereas smaller patches (Core 2, Core 1, edge and patch forests) has increased multifold between 1930 and 2020. The continued habitat loss and fragmentation probably fragmented elephant populations during the last century and made them insular with long-term ramifications for elephant conservation and human-elephant conflict. Given the substantial loss in forest cover and high levels of fragmentation, improving the resilience of elephant populations in Nepal would urgently require habitat and corridor restoration to enable the movement of elephants.

High frequency of an otherwise rare phenotype in a small and isolated tiger population.

Most endangered species exist today in small populations, many of which are isolated. Evolution in such populations is largely governed by genetic drift. Empirical evidence for drift affecting striking phenotypes based on substantial genetic data are rare. Approximately 37% of tigers (Panthera tigris) in the Similipal Tiger Reserve (in eastern India) are pseudomelanistic, characterized by wide, merged stripes. Camera trap data across the tiger range revealed the presence of pseudomelanistic tigers only in Similipal. We investigated the genetic basis for pseudomelanism and examined the role of drift in driving this phenotype's frequency. Whole-genome data and pedigree-based association analyses from captive tigers revealed that pseudomelanism cosegregates with a conserved and functionally important coding alteration in Transmembrane Aminopeptidase Q (Taqpep), a gene responsible for similar traits in other felid species. Noninvasive sampling of tigers revealed a high frequency of the Taqpep p.H454Y mutation in Similipal (12 individuals, allele frequency = 0.58) and absence from all other tiger populations (395 individuals). Population genetic analyses confirmed few (minimal number) tigers in Similipal, and its genetic isolation, with poor geneflow. Pairwise FST (0.33) at the mutation site was high but not an outlier. Similipal tigers had low diversity at 81 single nucleotide polymorphisms (mean heterozygosity = 0.28, SD = 0.27). Simulations were consistent with founding events and drift as possible drivers for the observed stark difference of allele frequency. Our results highlight the role of stochastic processes in the evolution of rare phenotypes. We highlight an unusual evolutionary trajectory in a small and isolated population of an endangered species.

Patterns and determinants of elephant attacks on humans in Nepal.

Attacks on humans by Asian elephant (Elephas maximus) is an extreme form of human-elephant conflict. It is a serious issue in southern lowland Nepal where elephant-related human fatalities are higher than other wildlife. Detailed understanding of elephant attacks on humans in Nepal is still lacking, hindering to devising appropriate strategies for human-elephant conflict mitigation. This study documented spatiotemporal pattern of elephant attacks on humans, factors associated with the attacks, and human/elephant behavior contributing to deaths of victims when attacked. We compiled all the documented incidences of elephant attacks on humans in Nepal for last 20 years across Terai and Chure region of Nepal. We also visited and interviewed 412 victim families (274 fatalities and 138 injuries) on elephant attacks. Majority of the victims were males (87.86%) and had low level of education. One fourth of the elephant attacks occurred while chasing the elephants. Solitary bulls or group of subadult males were involved in most of the attack. We found higher number of attacks outside the protected area. People who were drunk and chasing elephants using firecrackers were more vulnerable to the fatalities. In contrast, chasing elephants using fire was negatively associated with the fatalities. Elephant attacks were concentrated in proximity of forests primarily affecting the socioeconomically marginalized communities. Integrated settlement, safe housing for marginalized community, and community grain house in the settlement should be promoted to reduce the confrontation between elephants and humans in entire landscape for their long-term survival.

Genetic analyses reveal demographic decline and population differentiation in an endangered social carnivore, Asiatic wild dog.

Deforestation and agricultural intensification have resulted in an alarming change in the global land cover over the past 300 years, posing a threat to species conservation. Dhole is a monophyletic, social canid and, being an endangered and highly forest-dependent species, is more prone to the loss of favorable habitat in the Anthropocene. We determined the genetic differentiation and demographic history of dhole across the tiger reserves of Maharashtra using the microsatellite data of 305 individuals. Simulation-based analyses revealed a 77-85% decline in the major dhole sub-populations. Protected areas have provided refuge to the historically declining dhole population resulting in clustering with strong genetic structure in the remnant dhole population. The historical population decline coincides with the extreme events in the landscape over the past 300 years. The study highlights the pattern of genetic differentiation and diversity of a highly forest-dependent species which can be associated with the loss of forest cover outside tiger reserves. It also warrants attention to develop conservation plans for the remnant surviving population of dholes in India.

Effects of faecal inorganic content variability on quantifying glucocorticoid and thyroid hormone metabolites in large felines: Implications for physiological assessments in free-ranging animals.

Faecal glucocorticoid (GC) and triiodothyronine (T3) metabolites and their interactions are increasingly used to monitor perceived stress and nutritional challenges in free-ranging animals. However, a number of extrinsic and intrinsic factors including hormone-inert dietary materials, inorganic matters etc. are known to affect reliable hormone metabolite quantifications. In this study, the impacts of inorganic matter (IOM) on faecal GC (fGCMs) and T3 (fT3Ms) metabolite measure were addressed in wild tiger (n = 193 from Terai Arc landscape, India) and captive lion (n = 120 from Sakkarbaug Zoological Garden, Gujarat, India) and possible corrective measures were evaluated. The wild tiger samples contained highly variable IOM content (9-98%, mostly with > 40% IOM) compared to captive Asiatic lion (17-57%, majority with < 40% IOM). Significant correlations were observed between IOM content and tiger fGCM (r = -0.46, p = 0.000), fT3M (r = -0.58, p = 0.000) and lion fT3M measures (r = -0.43, p = 0.003). Two corrective measures viz. removing samples with ≥ 80% IOM and subsequently expressing concentrations as per gram of organic dry matter (instead of total dry matter) reduced IOM influence on tiger fGCM, fT3M and lion fT3M, without affecting lion fGCM measures. The corrective measures changed the interpretations of fT3M data of field-collected tiger samples with no significant changes in fGCM (both tiger and lion) and fT3M (lion) data. As faecal IOM content is common in many wild species, the results emphasize the need to reduce IOM-driven hormone data variation for ecologically relevant interpretations towards species conservation.

Microsatellite analysis reveals low genetic diversity in managed populations of the critically endangered gharial (Gavialis gangeticus) in India.

The gharial (Gavialis gangeticus) is a critically endangered crocodylian, endemic to the Indian subcontinent. The species has experienced severe population decline during the twentieth century owing to habitat loss, poaching, and mortalities in passive fishing. Its extant populations have largely recovered through translocation programmes initiated in 1975. Understanding the genetic status of these populations is crucial for evaluating the effectiveness of the ongoing conservation efforts. This study assessed the genetic diversity, population structure, and evidence of genetic bottlenecks of the two managed populations inhabiting the Chambal and Girwa Rivers, which hold nearly 80% of the global gharial populations. We used seven polymorphic nuclear microsatellite loci and a 520 bp partial fragment of the mitochondrial control region (CR). The overall mean allelic richness (Ar) was 2.80 ± 0.40, and the observed (Ho) and expected (He) heterozygosities were 0.40 ± 0.05 and 0.39 ± 0.05, respectively. We observed low levels of genetic differentiation between populations (FST = 0.039, P < 0.05; G'ST = 0.058, P < 0.05 Jost's D = 0.016, P < 0.05). The bottleneck analysis using the M ratio (Chambal = 0.31 ± 0.06; Girwa = 0.41 ± 0.12) suggested the presence of a genetic bottleneck in both populations. The mitochondrial CR also showed a low level of variation, with two haplotypes observed in the Girwa population. This study highlights the low level of genetic diversity in the two largest managed gharial populations in the wild. Hence, it is recommended to assess the genetic status of extant wild and captive gharial populations for planning future translocation programmes to ensure long-term survival in the wild.

Sequencing and annotation of the endangered wild buffalo (Bubalus arnee) mitogenome for taxonomic assessment.

The wild water buffalo (Bubalus arnee) is one of the most endangered and least studied large bovid in the Indian subcontinent. India retains 90% of the estimated global population of >4000 individuals as two fragmented populations in Assam and Chhattisgarh, both threatened by habitat loss and degradation, hunting, disease from livestock, and hybridization with the domestic buffalos. Small, fragmented population size and potential hybridisation pressures from co-occurring domestic buffalos are the major conservation challenges. For the first time, we sequenced the 16,357 bp long mitogenome of three opportunistically collected wild water buffalo samples from Assam (n = 1) and Chhattishgarh (n = 2). The annotated sequence has a base composition of 26.4% T, 26.6% C, 33.1% A and 13.9% G depicting an AT-rich mitogenome composition, including 13 protein-coding genes (11,361 bp), 22 transfer RNA (tRNA) (1514 bp), two ribosomal genes (2525 bp), and a non-coding control region (928 bp). The gene order is conserved with other bovid species. Comparative mitogenome analyses showed both populations are genetically similar but significantly different from domestic buffalo. We also identified structural differences in seven tRNA secondary structures between both species. The genetic distance between wild buffalo and other bovids varied between 0.103 and 0.122. Multiple Bayesian phylogenetic trees showed that both wild and domestic water buffalo formed sister clades which were paraphyletic to other potentially sympatric species of genus Bos. This study provides baseline information on wild buffalo mitogenome for further research on phylogeny, phylogeography and hybrid assessment and help conserving this endangered species.

Optimisation and application of a forensic microsatellite panel to combat Greater-one horned rhinoceros (Rhinoceros unicornis) poaching in India.

The Greater one-horned (GoH) rhinoceros is one of the most charismatic endemic megaherbivores of the Indian subcontinent. Threatened by poaching, habitat loss and disease, the species is found only in small areas of its historical distribution. Increasing demands for rhino horns in chinese traditional medicine has put the existing population under continuing threat, and large profits and low conviction rates make poaching difficult to contain. DNA forensics such as the RhoDIS-Africa program has helped in combating illegal rhino horn trade, but the approach is yet to be optimised for Indian GoH rhinoceros. Here we followed the International Society for Forensic Genetics (ISFG) guidelines to establish a 14 dinucleotide microsatellite panel for Indian GoH rhinoceros DNA profiling. Selected from a large initial pool (n = 34), the microsatellite markers showed high polymorphism, stable peak characteristics, consistent allele calls and produced precise, reproducible genotypes from different types of rhino samples. The panel also showed low genotyping error and produced high statistical power during individual identification (PIDsibs value of 1.2*10-4). As part of the official RhoDIS-India program, we used this panel to match poached rhino carcass with seized contraband as scientific evidence in court procedure. This program now moves to generate detailed allele-frequency maps of all GoH rhinoceros populations in India and Nepal for development of a genetic database and identification of poaching hotspots and trade routes across the subcontinent and beyond.

Genetic analyses reveal population structure and recent decline in leopards (Panthera pardus fusca) across the Indian subcontinent.

BACKGROUND: Large carnivores maintain the stability and functioning of ecosystems. Currently, many carnivore species face declining population sizes due to natural and anthropogenic pressures. The leopard, Panthera pardus, is probably the most widely distributed and highly adaptable large felid globally, still persisting in most of its historic range. However, we lack subspecies-level data on country or regional scale on population trends, as ecological monitoring approaches are difficult to apply on such wide-ranging species. We used genetic data from leopards sampled across the Indian subcontinent to investigate population structure and patterns of demographic decline. METHODS: We collected faecal samples from the Terai-Arc landscape of northern India and identified 56 unique individuals using a panel of 13 microsatellite markers. We merged this data with already available 143 leopard individuals and assessed genetic structure at country scale. Subsequently, we investigated the demographic history of each identified subpopulations and compared genetic decline analyses with countrywide local extinction probabilities. RESULTS: Our genetic analyses revealed four distinct subpopulations corresponding to Western Ghats, Deccan Plateau-Semi Arid, Shivalik and Terai region of the north Indian landscape, each with high genetic variation. Coalescent simulations with microsatellite loci revealed a possibly human-induced 75-90% population decline between ∼120-200 years ago across India. Population-specific estimates of genetic decline are in concordance with ecological estimates of local extinction probabilities in these subpopulations obtained from occupancy modeling of the historic and current distribution of leopards in India. CONCLUSIONS: Our results confirm the population decline of a widely distributed, adaptable large carnivore. We re-iterate the relevance of indirect genetic methods for such species in conjunction with occupancy assessment and recommend that detailed, landscape-level ecological studies on leopard populations are critical to future conservation efforts. Our approaches and inference are relevant to other widely distributed, seemingly unaffected carnivores such as the leopard.

Effects of personality and rearing-history on the welfare of captive Asiatic lions (Panthera leo persica).

BACKGROUND: The long-term success of ex-situ conservation programmes depends on species-appropriate husbandry and enrichment practices complemented by an accurate welfare assessment protocol. Zoos and conservation breeding programmes should employ a bottom-up approach to account for intraspecific variations in measures of animal welfare. We studied 35 (14:21) captive Asiatic lions in Sakkarbaug Zoological Garden, Junagadh, India to understand the implications of individual variations on welfare measures. We categorized the subjects based on personality traits (bold or shy), rearing history (wild-rescued or captive-raised), sex, and social-grouping. We explored the association of these categorical variables on welfare indices such as behavioural diversity, latency to approach novel objects, enclosure usage and aberrant repetitive behaviours. Further, we assessed the inter-relationships between different behavioural measures of welfare. RESULTS: Our results show that intraspecific variations based on rearing-history and personality traits are significantly associated with the welfare states of captive Asiatic lions. Asiatic lions with bold personality traits (M = 0.50, SD = 0.12, N = 21) and those raised in captivity (M = 0.47, SD = 0.12, N = 16) used enclosure space more homogenously compared to shy (M = 0.71, SD = 0.15, N = 14) and wild-rescued (M = 0.67, SD = 0.15, N = 19) animals. Behaviour diversity was significantly higher in captive-raised (M = 1.26, SD = 0.3, N = 16) and bold (M = 1.23, SD = 0.26, N = 21) subjects compared to wild-rescued (M = 0.83, SD = 0.35, N = 19) and shy (M = 0.73, SD = 0.34, N = 14) individuals. Aberrant repetitive behaviours (stereotypy) were significantly lower in bold (M = 7.01, SD = 4, N = 21) and captive-raised (M = 7.74, SD = 5.3) individuals compared to wild-rescued (M = 13.12, SD = 6.25, N = 19) and shy (M = 16.13, SD = 5.4, N = 16) lions. Sex and social-grouping of subjects did not show significant associations with behavioural welfare indices. Interestingly, behaviour diversity was reliably predicted by the enclosure usage patterns and aberrant repetitive behaviours displayed by subjects. DISCUSSION: Our findings underline the importance of individual-centric, behaviour-based, and multi-dimensional welfare assessment approaches in ex-situ conservation programmes. The results suggest that behavioural welfare indices complemented with individual variations can explain inter-individual differences in behavioural welfare measure outcomes of Asiatic lions. These findings also provide zoo managers with a non-invasive tool to reliably assess and improve husbandry practices for Asiatic lions. Understanding the unique welfare requirement of individuals in captivity will be crucial for the survival of the species.

Fecal stress, nutrition and reproductive hormones for monitoring environmental impacts on tigers (Panthera tigris).

Non-invasive stress and nutritional hormone analysis in relation to ecological and other biological indices have tremendous potential to address environmental disturbance impacts on wildlife health. To this end, we examined the relation between glucocorticoid (GC) and thyroid (T3) hormone indices of disturbance and nutritional stress in response to ACTH and TSH challenges in captive tigers, as well as how reproductive hormones vary by sex and reproductive condition. Glucocorticoid, thyroid, progesterone and androgen assays conducted on high-performance liquid chromatography separated fractions of biologically relevant fecal extracts revealed high cross-reactivity of these assays for their respective biologically relevant fecal hormone metabolites. Both adrenal and thyroid hormone metabolites were elevated in response to ACTH and TSH challenges. However, the adrenal and thyroid hormone responses to ACTH challenge were concurrent, whereas the adrenal response to TSH challenge was delayed relative to thyroid hormone elevation in both males and females. The concurrently elevated T3 in response to ACTH may serve to raise metabolic rate to maximize use of GC-mobilized glucose, whereas the relatively delayed GC rise following TSH challenge may be a response to glucose depletion due to increased metabolic rate associated with elevated T3. Progesterone, testosterone and androstenedione hormone metabolites were significantly elevated during gestation compared to lactation in a female monitored from conception through early lactation. Results suggest that the glucocorticoid, thyroid and reproductive hormone assays we tested can accurately measure the stress, nutrition and reproductive response from tiger feces, providing useful non-invasive tools to assess physiological responses to environmental stressors and their reproductive consequences in the wild.

Standardization and validation of a panel of cross-species microsatellites to individually identify the Asiatic wild dog (Cuon alpinus).

BACKGROUND: The Asiatic wild dog or dhole (Cuon alpinus) is a highly elusive, monophyletic, forest dwelling, social canid distributed across south and Southeast Asia. Severe pressures from habitat loss, prey depletion, disease, human persecution and interspecific competition resulted in global population decline in dholes. Despite a declining population trend, detailed information on population size, ecology, demography and genetics is lacking. Generating reliable information at landscape level for dholes is challenging due to their secretive behaviour and monomorphic physical features. Recent advances in non-invasive DNA-based tools can be used to monitor populations and individuals across large landscapes. In this paper, we describe standardization and validation of faecal DNA-based methods for individual identification of dholes. We tested this method on 249 field-collected dhole faeces from five protected areas of the central Indian landscape in the state of Maharashtra, India. RESULTS: We tested a total of 18 cross-species markers and developed a panel of 12 markers for unambiguous individual identification of dholes. This marker panel identified 101 unique individuals from faecal samples collected across our pilot field study area. These loci showed varied level of amplification success (57-88%), polymorphism (3-9 alleles), heterozygosity (0.23-0.63) and produced a cumulative misidentification rate or PID(unbiased) and PID(sibs) value of 4.7 × 10-10 and 1.5 × 10-4, respectively, indicating a high statistical power in individual discrimination from poor quality samples. CONCLUSION: Our results demonstrated that the selected panel of 12 microsatellite loci can conclusively identify dholes from poor quality, non-invasive biological samples and help in exploring various population parameters. This genetic approach would be useful in dhole population estimation across its range and will help in assessing population trends and other genetic parameters for this elusive, social carnivore.

Rapid molecular assays for species and sex identification of swamp deer and other coexisting cervids in human-dominated landscapes of the Terai region and upper Gangetic plains, northern India: implications in understanding species distribution and population parameters.

Burgeoning pressures of habitat loss is a major cause of herbivore decline across India, forcing them to coexist with humans in non-protected areas. Their conservation in such landscapes is challenging due to paucity of ecological and demographic information. The northern subspecies of swamp deer, Rucervus duvaucelii duvaucelii, is one such herbivore that lives across human dominated landscapes in Terai region and upper Gangetic plains of north India. Here, we describe species-specific molecular markers and a cervid-specific molecular sexing assay for swamp deer and four other coexisting cervids sambar, chital, barking deer and hog deer. Our markers show species-specific band patterns and a high success rate of 88.21% in large number of field collected referencesamples for all species. Faecal pellets from pilot swamp deer survey samples from upper Ganges basin show 93.81% success rate, and only 5.5% misidentification based on morphological characteristics. Our cervid-specific molecular sexing multiplex assay accurately ascertained 81.15% samples to respective sexes. These molecular approaches provide an easy, quick and cheap option to generate critical information on herbivore population parameters and aid their conservation in this mosaic of protected and non-protected grassland habitats.

Noninvasive DNA-based species and sex identification of Asiatic wild dog (Cuon alpinus).

Asiatic wild dog (Cuon alpinus) or dhole is an endangered canid with fragmented distribution in South, East and Southeast Asia. The remaining populations of this species face severe conservation challenges from anthropogenic interventions, but only limited information is available at population and demography levels. Here, we describe the novel molecular approaches for unambiguous species and sex identification from noninvasively collected dhole samples. We successfully tested these assays on 130 field-collected dhole faecal samples from the Vidarbha part of central Indian tiger landscape that resulted in 97 and 77% successrates in species and sex identification, respectively. These accurate, fast and cheap molecular approaches prove the efficacy of such methods in gathering ecological data from this elusive, endangered canid and show their application in generating population level information from noninvasive samples.

Cross-species screening of microsatellite markers for individual identification of snow petrel Pagodroma nivea and Wilson's storm petrel Oceanites oceanicus in Antarctica.

Seabirds are important indicators of marine ecosystem health. Species within the order Procellariiformes are the most abundant seabird species group distributed from warm tropical to cold temperate regions including Antarctica. There is a paucity of information on basic biology of the pelagic seabird species nesting on the Antarctic continents, and long-term studies are required to gather data on their population demography, genetics and other ecological parameters. Under the 'Biology and Environmental Sciences' component of the Indian Antarctic programme, long-term monitoring of Antarctic biodiversity is being conducted. In this paper, we describe results of cross-species screening of a panel of 12 and 10 microsatellite markers in two relatively little studied seabird species in Antarctica, the snow petrel Pagodroma nivea and the Wilson's storm petrel Oceanites oceanicus, respectively. These loci showed high amplification success and moderate levels of polymorphism in snow petrel (mean no. of alleles 7.08 ± 3.01 and mean observed heterozygosity 0.35 ± 0.23), but low polymorphism in Wilson's storm petrel (mean no. of alleles 3.9 ± 1.3 and mean observed heterozygosity 0.28 ± 0.18). The results demonstrate that these panels can unambiguously identify individuals of both species (cumulative PIDsibs for snow petrel is 3.7 × 10-03 and Wilson's storm petrel is 1.9 × 10-02) from field-collected samples. This work forms a baseline for undertaking long-term genetic research of these Antarctic seabird species and provides critical insights into their population genetics.

New evidence for hybrid zones of forest and savanna elephants in Central and West Africa.

The African elephant consists of forest and savanna subspecies. Both subspecies are highly endangered due to severe poaching and habitat loss, and knowledge of their population structure is vital to their conservation. Previous studies have demonstrated marked genetic and morphological differences between forest and savanna elephants, and despite extensive sampling, genetic evidence of hybridization between them has been restricted largely to a few hybrids in the Garamba region of northeastern Democratic Republic of Congo (DRC). Here, we present new genetic data on hybridization from previously unsampled areas of Africa. Novel statistical methods applied to these data identify 46 hybrid samples--many more than have been previously identified--only two of which are from the Garamba region. The remaining 44 are from three other geographically distinct locations: a major hybrid zone along the border of the DRC and Uganda, a second potential hybrid zone in Central African Republic and a smaller fraction of hybrids in the Pendjari-Arli complex of West Africa. Most of the hybrids show evidence of interbreeding over more than one generation, demonstrating that hybrids are fertile. Mitochondrial and Y chromosome data demonstrate that the hybridization is bidirectional, involving males and females from both subspecies. We hypothesize that the hybrid zones may have been facilitated by poaching and habitat modification. The localized geography and rarity of hybrid zones, their possible facilitation from human pressures, and the high divergence and genetic distinctness of forest and savanna elephants throughout their ranges, are consistent with calls for separate species classification.