Genetic Evidence of Tiger Population Structure and Migration within an Isolated and Fragmented Landscape in Northwest India

Majority of the tiger habitat in Indian subcontinent lies within high human density landscapes and is highly sensitive to surrounding pressures. These forests are unable to sustain healthy tiger populations within a tiger-hostile matrix, despite considerable conservation efforts. Ranthambore Tiger Reserve (RTR) in Northwest India is one such isolated forest which is rapidly losing its links with other tiger territories in the Central Indian landscape. Non-invasive genetic sampling for individual identification is a potent technique to understand the relationships between threatened tiger populations in degraded habitats. This study is an attempt to establish tiger movement across a fragmented landscape between RTR and its neighboring forests, Kuno-Palpur Wildlife Sanctuary (KPWLS) and Madhav National Park (MNP) based on non-invasively obtained genetic data.Data from twelve microsatellite loci was used to define population structure and also to identify first generation migrants and admixed individuals in the above forests.Population structure was consistent with the Central Indian landscape and we could determine significant gene flow between RTR and MNP. We could identify individuals of admixed ancestry in both these forests, as well as first generation migrants from RTR to KPWLS and MNP.Our results indicate reproductive mixing between animals of RTR and MNP in the recent past and migration of animals even today, despite fragmentation and poaching risk, from RTR towards MNP. Substantial conservation efforts should be made to maintain connectivity between these two subpopulations and also higher protection status should be conferred on Madhav National Park

Improved Methods of Carnivore Faecal Sample Preservation, DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers

<div><h3>Background</h3><p>Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase.</p> <h3>Methods</h3><p>We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval.</p> <h3>Results</h3><p>Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p<0.001). Maximum DNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p<0.1 and p<0.001). This difference was not significant when samples were preserved by two-step method (p>0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively.</p> <h3>Conclusions</h3><p>Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols.</p> </div

PCR success rate and dropout per locus for different categories of DNA.

<p>PCR success rate and dropout per locus for different categories of DNA.</p

Non linear regression graph of DNA concentrations obtained from samples stored by three preservation methods.

<p>Non linear regression slopes for silica, ethanol and two-step methods were -2.8, -50.6 and -84.11 respectively.</p

Comparative DNA concentrations and PCR success rates of different grades of samples.

<p>Comparative DNA concentrations and PCR success rates of different grades of samples.</p

Graph showing DNA degradation rate in 10 faecal samples exposed to direct sunlight.

<p>Maximum DNA degradation occurs between Day 1 and Day 3 of exposure to direct sunlight (p<0.001).</p

Proportional membership of each tiger in the four clusters identified by STRUCTURE.

<p>Each tiger is represented by a single vertical bar. RTR – Ranthambore Tiger Reserve, KPWLS – Kuno-Palpur Wildlife Sanctuary, MNP – Madhav National Park, BTR – Bandhavgarh Tiger Reserve, PTR – Pench Tiger Reserve, Madhya Pradesh.</p

Wright's F-statistics analysis for Madhav National Park and Ranthambore Tiger reserve populations.

<p>F_IS, F_ST, and F_IT are correlations between pairs of genes, within individuals within populations, between individuals in the same population and within individuals, respectively.</p><p>Relat, an estimator of the average relatedness of individuals within samples when compared to whole <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029827#pone.0029827-Queller1" target="_blank">[59]</a>.</p><p>Relatc estimates the inbreeding corrected relatedness <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029827#pone.0029827-Pamilo1" target="_blank">[60]</a>.</p><p>R_st, estimate of relative genetic differentiation.</p>a<p>Standard errors – estimate from jackknife over loci and significance from t-test using these estimates, p<0.05.</p