Traditionally, faecal matter of tigers has been used to study the food habits of tigers and their sympatric carnivores. As they are more readily obtainable than hair, faecal samples were chosen as the non-invasive source for obtaining DNA for our study. DNA quantity and quality by different extraction trials was evaluated. The DNA extracted from faecal samples originates from the colorectal epithelial mucous on the surface of the sample. Also it appears that the DNA that is extracted from the surface of the sample has less of PCR inhibitory substances and possibly less admixture with undigested prey remains [31
]. Thus, in all the five DNA extraction methods that were evaluated here, the surface of the faecal sample was washed with the initial Lysis buffers. Three methods, namely the Digest Buffer/Phenol-Chloroform method, Guanidinium thiocyanate-silica method and Qiagen Stool DNA extraction kit showed PCR amplification with no significant difference. Guanidinium thiocyante-silica was followed for all our extractions.
We used PCR amplification to evaluate the best methods for storing the faecal samples after collection. Both the storage methods tested, namely alcohol as well as silica beads, have worked well in terms of PCR amplification ability and can be used to preserve faecal samples of carnivores. This was not tested exclusively in the faecal samples of tiger as we collected samples of tiger as well as its sympatric carnivores, leopard and wild dog. The faecal samples collected at NSTR were also used in scat identification trials discussed next.
It is essential to reliably identify the faecal samples of tiger from those of their sympatric carnivores like leopards and wild dog. Most studies on diets and occupancy of the two felids, namely the tiger and the leopard, are based on faecal samples [32
], which are identified on the basis of morphological features [34
] and associated behavioral signs like scrape marks, the absence of which makes it difficult to identify the samples. Therefore, molecular analysis that makes use of tiger-specific mitochondrial variations for distinguishing the tiger faecal samples from those of sympatric carnivores was developed and used to screen out tiger samples.
According to Davison et al
], sometimes even trained trackers have misidentified the faecal samples of similar sized carnivores. For instance, in our field study at the Mudumalai and BRT Wildlife Sanctuaries, only 70% of all the samples that were collected as supposed tiger faecal samples were actually of tiger origin. False negatives may occur in the tiger-specific PCR generated as a result of interspecific polymorphisms at the variable sites that were used in primer design. We ruled this out by PCR amplifying DNA of 25 tigers and 10 leopards DNA (data not shown) and also by amplifying representative samples of some of the animals that are the prey of tigers in India.
Microsatellite Loci E21B, E7, E6, Fca43, D10 and Fca304 target fragments of less than 200 bp long and are therefore easier to amplify [36
]. Hence they were chosen for our study. The most conservative estimate of P(ID) i.e., P(ID)sib
is determined to estimate the upper bound on the number of loci required to reliably distinguish even closely related individuals. The set of primers used in this study are sufficient to distinguish a population comprising even of several siblings (Figure ). For individual identification in population estimation studies using mark-recapture models, P(ID) value is recommended to be approximately 0.01 [19
] in order to distinguish closely related individuals with 99% certainty. Using microsatellite loci with high heterozygosity reduces the number of loci required to reach a low P(ID) value. The P(ID)sibs
for the six loci used by us is 0.005 and can be used to distinguish even closely related individuals. Therefore they can also be used in studies requiring individual identification for population estimation. Further, these loci are in Hardy-Weinberg equilibrium and not in linkage disequilibrium – an essential assumption required in calculating P(ID) where loci are expected to be independent. In our study, the observed P(ID) for the three most informative loci is zero. The P(ID) value calculated in our study was with captive tigers which may be inbred. It is therefore possible that the number of loci required to distinguish individuals can stand valid even in those wild tiger populations that may comprise of many related tigers.
The Comparative Multiple Tubes [22
] approach was used to derive consensus genotypes for faecal DNA extracts. Reference genotype profiles were generated from blood DNA of known captive tigers that were compared with genotypes generated from faecal samples of the same animals (Table ). Though there is one erroneous genotype, which might be an amplification artifact at one locus ('Roger', Locus E7, Scat1), the profiles generated from the captive samples were assigned with the corresponding reference profiles on the basis of three of the most informative loci, namely D10, Fca304 and E21B.
In order to evaluate whether faecal samples collected from the jungles could be used for genotyping and to demonstrate the error rates in the genotypes generated from such samples, a preliminary study was performed on faecal samples collected from Mudumalai and BRT Wildlife Sanctuaries. It is generally believed that the length of time between deposition of samples and collection and extraction may have some influence on the DNA quantity [37
]. This is evident as the faecal samples collected from captive tigers were amplified (microsatellite loci) with 100% success as against the 60% amplification success with faecal samples from the field. Also the rate of errors generated from captive animal faecal samples is considerably lower than the errors generated from faecal samples collected from the jungle. This could be because the DNA from faecal samples of the captive animals was extracted on the day of collection. The error rate encountered by us for faecal samples collected from the field was in the same range as has been reported by others, i.e., 46–66% [20
], though others have reported a much higher amplification success rate – 87% [22
] and 93–95% [38
As faecal samples were collected wherever encountered from all over the two protected areas, the number of genotype profiles generated thus can provide an estimate of the minimum number of tigers living in that area during that particular sampling session. Partial genotype profiles were grouped together with those samples where complete genotypes could be obtained. This kind of assignment could lead to an underestimation, but not an overestimation of animal numbers.
Sex identification of faecal samples can reveal the sex ratio of tigers, as well as territory occupancy by male and female tigers in a habitat. PCR primers that have been developed for several New World felids [30
] and which target the zinc-finger region and amelogenein gene on the X and Y-chromosome were used for sexing faecal samples. We tested these primers on known captive female and male tiger DNA samples extracted from blood before using them on faecal DNA extracts.
This pilot study demonstrates the use of faecal samples of tigers collected in the field as a source of DNA and the possibility of conducting population estimation wherein matching genotypes are considered to arise from the same individual. The genotype data generated thus could be analysed by mark-recapture analysis [39
] with appropriate models.