Studies to determine presence or absence of the Indian tiger (Panthera tigris tigris) in Kawal Wildlife Sanctuary, India
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- Reddy, P.A., Kumaraguru, A., Yadav, P.R. et al. Eur J Wildl Res (2011) 57: 517. doi:10.1007/s10344-010-0460-3
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A decade back, almost 300,000 km2 of forests in India were estimated to be potential tiger habitat. But consistent degradation and unsustainable anthropogenic pressures have adversely affected tiger presence in most of the forests outside the better protected tiger reserves. Here we use Geographic Information System data to analyze the degree of vegetation loss and landscape changes over the last decade (1998–2006), and ascertain the presence of tigers in a degraded forest like the Kawal Wildlife Sanctuary, Andhra Pradesh, India, by non-invasive fecal DNA analysis. Vegetation cover maps show a clear degradation of the sanctuary within a decade. DNA analysis of scat samples reveals tiger presence in areas where closed dense forest canopy has persisted with minimal human disturbance during the last decade.
KeywordsGISTigerFecal DNADegraded forest
“Project Tiger” was launched in March 1973 to overcome the alarming rate at which the forests were vanishing and tigers were decreasing in numbers in India. Unfortunately, the trend seems to be continuing in India as well as other parts of the tiger’s range. Dinerstein et al. (2007) reported that the current tiger range is only 7% of the historic range, and within the last one decade the estimated area known to be occupied by tigers has declined 41%. In 1999, Wikramanayake et al. estimated more than 300,000 km2 of forests as potentially suitable for tigers in India. But, according to a recent survey, potential tiger habitats in the protected areas of India add up to only 100,000 km2, while the reproductively active source populations occupy less than 20,000 km2 (Thapar 2006). Tigers in most of these potential areas outside the tiger reserves either occur in very low densities or have been locally extirpated (Karanth et al. 2004).
Despite poaching and vengeful killing, or the tremendous and unsustainable anthropogenic pressures on forests which have adversely affected all national parks/sanctuaries/ reserve forests, the tiger has survived in India. However, what are the limits of its resilience and how much of the disturbed forests outside the national parks does the tiger really use? There are not many studies showing how encroachments and unregulated biotic pressures have further degraded the already fragmented landscapes in the last decade, neither have any attempts been made to ascertain tiger presence in such areas by rigorously tested methods like camera-trap or DNA studies. This study is an attempt to address the above lacuna by using Geographic Information System (GIS) data to analyze the degree of vegetation loss and landscape changes over the last decade (1998–2006), and ascertain the presence of tigers in a degraded forest like the Kawal Wildlife Sanctuary, Andhra Pradesh, India, by non-invasive fecal DNA analysis. The implications of our results may be used for the future management of Kawal and other disturbed forests of India.
The Kawal Wildlife Sanctuary (893 km2) is located between 78.558°E, 19.274°N to 79.232°E, 19.009°N and 78.833°E, 19.346°N to 79.134°E, 18.927°N in Adilabad district of Andhra Pradesh, India. There are several villages located within and around the sanctuary resulting in severe disturbances due to activities like tree felling, harvesting of forest produce, cattle grazing, encroachment into forestland, and hunting of prey species. The dominant vegetation type is tropical dry-deciduous forest with predominantly teak (Tectonia grandis) mixed with bamboo, Terminalia, Cassia, Bombax, etc. The sanctuary supports a diverse population of mammals including large carnivore species such as leopard (Panthera pardus), sloth bear (Melursus ursinus), jungle cat (Felis chaus), and dhole (Cuon alpinus). The principal wild prey species of tigers are chital (Axis axis), nilgai (Boselephas tragocamelus), bison (Bos gaurus), and wild boar (Sus scrofa), but all these are seen in very low numbers.
GIS data and sample collection
Classified vegetation cover maps (1998–2006) of Kawal Wildlife Sanctuary were collected from the GIS Cell of the Andhra Pradesh Forest Department. The vegetation was classified into five categories: (1) dense forest, (2) open forest, (3) degraded scrub forest, (4) open area, and (5) water bodies. All map data were analyzed with ArcView software version 9.3 (ESRI, USA).
In October 2008, we started an exhaustive survey to ascertain tiger presence and to obtain an estimate of the minimum number of tigers present in Kawal Wildlife Sanctuary (893 km2). Extensive questionnaire surveys were conducted in 42 villages present in the sanctuary for locations of tiger signs, prey kills, cattle kills, etc. Tiger fecal samples were collected along forest roads and pathways within the sanctuary, and based on the first sampling the second survey was conducted in December 2008. Global positioning system locations of all the samples were recorded. All samples were collected in zip-lock covers with silica beads and stored at −20°C till further analysis.
DNA extraction and species identification of the scat samples
DNA was extracted from all the scat samples using the guanidinium thiocyanate–silica method (Reed et al. 1997). All isolation sets of 10 samples each, included a control to monitor for contamination. A PCR assay with tiger-specific cytochrome b primers (TIF and TIR) developed to identify tiger scats from those of sympatric carnivores, like leopards, was used to screen all the scat samples (Bhagavatula and Singh 2006). All the PCR products were electrophoresed and only the tiger positive samples were subjected to further analysis.
Microsatellite amplification and analysis of tiger positive scat samples
Tiger positive samples were analyzed with primers targeting 10 tetranucleotide loci (Menotti-Raymond et al. 1999) and four dinucleotide loci (Bhagavatula and Singh 2006; Menotti-Raymond et al. 1999; Williamson et al. 2002) by a two-step multiplex PCR assay (Arandjelovic et al. 2009). In the initial step, all microsatellite loci were amplified in a single reaction in 50-μl reaction volumes consisting of 1× PCR buffer II (Applied Biosystems), 2.5 mM MgCl2 (Applied Biosystems), 15 pmol of each primer (unlabeled), 200 μM dNTPs, 1× BSA (New England Biolabs), 2.5 U of AmpliTaq Gold (Applied Biosystems), and 5 μl of template DNA. PCR reactions were carried out in Mastercycler ep gradient S (Eppendorf) with the following conditions: 95°C for 10 min, 30 cycles of 94°C for 15 s, 49°C for 15 s, 72°C for 20 s, followed by a final extension of 72°C for 10 min. Three replicates of singleplex PCR were carried out as above in reaction volumes of 15 μl, except that 0.2–0.5 μl of the multiplex PCR product was used as the template. The mix also contained 1.5 mM MgCl2 (Applied Biosystems), 0.75 U of AmpliTaq Gold (Applied Biosystems), and 4 pmol each of FAM or HEX fluorescently labeled forward primer and unlabeled reverse primer. The cycling conditions were also similar to that as above except for primer-specific annealing temperatures for each singleplex PCR, which varied from 50°C to 62°C. Samples with ambiguous results or with poor amplification success were further amplified three times.
All the PCR steps, except the addition of template DNA, were performed in a hood that was UV-irradiated before and after use to avoid contamination. The PCR products from the second singleplex amplification step were electrophoresed on an ABI 3730 Genetic Analyzer and the alleles were sized relative to an internal control (500 ROX™, Applied Biosystems) using GeneMapper software version 3.7 (Applied Biosystems). Only samples that amplified at a minimum of four loci were included in the final data set.
Sex identification of genotyped samples
Tiger positive samples which amplified at a minimum of four microsatellite loci were further subjected to a PCR assay for sex identification (Bhagavatula and Singh 2006; Pilgrim et al. 2005) with primer pairs targeting the zinc-finger region and amelogenin gene. The PCR products were electrophoresed on an ABI 3730 Genetic Analyzer and the alleles were sized relative to 500 ROX™ (Applied Biosystems) using GeneMapper software version 3.7 (Applied Biosystems).
GIS data analysis
Year-wise classification of the vegetation cover (area in square kilometers) of Kawal Wildlife Sanctuary, Andhra Pradesh, India
Degraded scrub forest
In October 2008, questionnaire surveys conducted in 42 villages located within Kawal Wildlife Sanctuary gave indirect evidence of carnivore presence and the prey abundance (data not shown). Forty scat samples were collected in the first survey which covered the entire sanctuary. Subsequent to the DNA-based species identification of these samples and the questionnaires, further seven samples were collected in December 2008 only from the potential tiger areas within the sanctuary. All samples were mapped on the most recently available vegetation map (2006). All carnivore scats, irrespective of species, were found in areas of dense or open canopies sometimes adjoining scrub forests (Fig. 1b).
DNA analysis of scat samples
Out of the 47 samples collected, 21 were found to be of tiger origin (Fig. 1b). The remaining 26 samples were not analyzed further as the primary objective of our study was to determine the presence or absence of tigers in Kawal. Only five of these 21 tiger scats amplified at more than four loci and yielded four distinct genotypes. Three of the four samples turned out to be from male tigers and one was from a female tiger. These results show that there were a minimum of four tigers in Kawal Wildlife Sanctuary during the period of our sample collection. The maximum number of tiger positive samples (10) were collected from the North-western part of the sanctuary (Pembi range), where a closed, dense canopy has persisted over the last decade with minimum human disturbance (Fig. 1b). The second group of nine tiger-positive samples were again found in areas of dense forest cover in Birsaipet range in the North-central part of the sanctuary. The remaining two samples were found in two different locations but again well within dense or open forest areas.
High anthropogenic pressures and systematic poaching have led to the steady and rapid decline in the tiger population across the world. Today, India appears to be the last stronghold of the tiger (Mondol et al. 2009b). However, its protection and revival from the brink of extinction will require dedicated efforts on several fronts. Ninety percent of tiger habitat in India is outside the better protected tiger reserves (Karanth and Nichols 2002). The elusive nature of the tiger, its abilities to traverse great distances in search of food and territory, produce large litters, capture and live on diverse prey species, and to adapt to diverse climatic and ecological conditions have ensured its survival in India against all odds. The survival of the tiger also depends on the conservation and proper management of potential tiger habitats across the country. With the continuing trend of unsustainable biomass removal by the local people and rampant livestock grazing, these fragmented but vital habitats will soon be lost irrevocably. Loss of corridors and dispersing grounds will adversely affect the animals trapped in tiger reserves leading to aggravated cannibalism and inbreeding depression.
The Kawal Wildlife Sanctuary is a potential tiger habitat but is subject to heavy human disturbances, which have steeply aggravated since July 2007, following the implementation of the Forest Rights Act in India. Enumeration of tigers in Kawal Wildlife Sanctuary has always been done by the traditional pugmark method and by indirect evidences like prey kills, scrape marks, etc. Numbers obtained by these methods are ambiguous and highly subjective (Karanth et al. 2003) for various reasons. It is prone to human error since the front paw could be confused for the hind one and in addition pug marks are known to differ depending on the hardness of the substratum and the season of the year. Although the photographic capture–recapture method is an efficient and robust method for estimating tigers, it has several drawbacks in low density areas with high human disturbances (Mondol et al. 2009a). In such a scenario, non-invasive DNA-based analysis of scat samples to verify the presence of tiger and to obtain a minimum number of animals present in a given area seems to be the only authentic and reliable method. We also wanted to analyze changing forest cover over the last decade through analysis of satellite imagery, as reliable records of impacts from encroachments and logging do not exist.
Further, Fig. 2 shows that at least 26 major human settlements have come up within the forest in the same period, many of them around perennial water sources. A major river, Godavari, flows along the Southern border of the sanctuary (not shown in the maps), along the banks of which lie a town, Jannaram and several big villages, totally cutting off animal movements towards the South. But the forests of Kawal are connected in the North to those in the adjoining states of Maharashtra and Chhattisgarh which might ensure easy tiger movements in these areas. This may be the reason as to why the largest number of tiger positive samples was found in the Northwestern part of the sanctuary in Pembi range (Fig. 1b). We could successfully genotype only two of the 10 tiger samples collected in this area, and both were of the same animal. However, since all these samples were collected within a radius of 15 km2, we can safely assume that most of them must have originated from the same animal. This assumption is further strengthened because we started sample collection in October after the monsoon rains which would have washed off all old samples, and also we collected samples which did not look older than approximately 10 days. Similarly, we obtained another group of nine tiger positive samples in close proximity to each other in the North-central part of the sanctuary in Birsaipet range (Fig. 1b), one of which yielded a distinct genotype. Both these groups of samples were found in areas where the forest canopies have not changed greatly between 1998 and 2006 (Fig. 1a, b). Here again we would like to reiterate that the aim of this study was not to count tigers in Kawal Wildlife Sanctuary but to verify tiger presence in this sanctuary despite mounting human pressure. Our results confirm that habitat loss is a strong indicator of population declines (Dinerstein et al. 2007).
According to Chundawat et al. (1999), there are approximately 150,000 km2 of tropical dry forests in India which could potentially support 9,000 wild tigers, making revival of tiger populations highly feasible in India (Karanth et al. 2004). However, a lot depends on how well these forests are protected and how effectively the habitat degradation is halted. This study definitely highlights the resilient nature of the tiger to inhabit even a disturbed and rapidly degrading forest like Kawal Wildlife Sanctuary. Timely detection of the tiger’s presence in the smaller wildlife sanctuaries like Kawal and other reserve forests, vigilance and protection of habitats, and proper management of their prey base will definitely go a long way in reviving the dwindling numbers of tigers in India.
We gratefully acknowledge the GIS Cell of the Andhra Pradesh Forest Department for providing detailed vegetation maps and georeferenced maps of Kawal Wildlife Sanctuary. We thank the Department of Biotechnology, India for financially supporting this study. We also thank the Chief Wildlife Warden, Andhra Pradesh, the Deputy Forest Officer of Kawal Wildlife Sanctuary, and all their field staff for facilitating this study.