Genotyping faecal samples of Bengal tiger Panthera tigris tigris for population estimation: a pilot study

Abstract

Background: Bengal tiger Panthera tigris tigris the National Animal of India, is an endangered species. Estimating populations for such species is the main objective for designing conservation measures and for evaluating those that are already in place. Due to the tiger's cryptic and secretive behaviour, it is not possible to enumerate and monitor its populations through direct observations; instead indirect methods have always been used for studying tigers in the wild. DNA methods based on non-invasive sampling have not been attempted so far for tiger population studies in India. We describe here a pilot study using DNA extracted from faecal samples of tigers for the purpose of population estimation.

Results: In this study, PCR primers were developed based on tiger-specific variations in the mitochondrial cytochrome b for reliably identifying tiger faecal samples from those of sympatric carnivores. Microsatellite markers were developed for the identification of individual tigers with a sibling Probability of Identity of 0.005 that can distinguish even closely related individuals with 99.9% certainty. The effectiveness of using field-collected tiger faecal samples for DNA analysis was evaluated by sampling, identification and subsequently genotyping samples from two protected areas in southern India.

Conclusion: Our results demonstrate the feasibility of using tiger faecal matter as a potential source of DNA for population estimation of tigers in protected areas in India in addition to the methods currently in use.

Figure 1

Sequence alignment of some prey species of tigers, the extant sub-species of tigers and some carnivore species. Arrows indicate the tiger-specific variations at position 636 and 759 of the tiger mitochondrial cytochrome b gene that were used for designing primers for tiger-specific diagnostic PCR assay.

Figure 2

A representative gel pattern showing the specific amplification of only tiger DNA with the tiger-specific cytochrome b primers (TIF/TIR). Lane 1: 100 base pair ladder (New England Biolabs); Lane 2: negative control; Lane 3:Panthera pardus; Lane 4:Cuon alpinus; Lane 5: Neofelis nebulosa; Lane 6:Cervus unicolour; Lane 7: Panthera tigris tigris; Lane 8: P.t. altaica; Lane 9: Bos gaurus; Lane 10: Cervus axis; Lane 11: Sus scrofa; Lane 12: Cervus axis. PCR amplification with universal 'mcb' primers [48] of all animals tested rules out the possibility of false negatives in PCR with the tiger-specific primer pair (TIF/TIR). Amplification with primers TIF/TIR and mcb primers was carried out in separate reaction though the PCR products were loaded onto a gel together.

Figure 3

Neighbour-Joining tree of amplicons of two faecal samples from Nagarjunasagar Srisailam Tiger Reserve (NSTR1 and 2). Sequences of amplicons of both samples, which were positive in the tiger-specific diagnostic assay, cluster with GenBank tiger sequence proving further that the samples are tiger in origin.

Figure 4

Plot of the observed and expected P(ID) values of six loci on different population scenarios with different proportions of related individuals. The greater the number of siblings in the population, the higher is the average probability of identity and therefore the higher will be the number of loci required to distinguish individuals. The relationship proportions are numbered as: 1 = all unrelated animals; 2 = 0.1 siblings, 0.2 parents, 0.5 half siblings; 3 = 0.2 siblings, 0.2 unrelated, 0.2 parents, 0.4 half-siblings, 0.2 unrelated; 4 = 0.3 siblings, 0.2 parents, 0.3 half siblings, 0.2 unrelated; 5 = 0.5 siblings, 0.2 parents, 0.1 half-siblings, 0.2 unrelated; 6 = 0.6 siblings, 0.2 parents, 0 half-siblings, 0.2 unrelated; 7 = 0.7 siblings, 0.2 parents, 0 half-siblings, 0.1 unrelated; 8 = 0.8 siblings, 0.2 parents, 0 siblings, 0 unrelated; 9 = 0.9 siblings, 0.1 parents, 0 unrelated; 10 = 1.0 siblings.

Figure 5

Decrease in the P(ID) values with the addition of loci in decreasing order of heterozygosity. Observed and Sibling P(ID)s were calculated for six microsatellite loci on 21 captive tigers. Cumulative Observed Probability of Identity becomes zero with the three most informative loci.