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Genetic Assessment of the Statusof Tigers in Buxa tiger reserve,
west bengal
A Report submitted to West Bengal Forest Department
September 2010
Investigating agency
Aarenyak
Finantial support
Buxa tiger reserve authority
www.aaranyak.org
Genetic Assessment of the Statusof Tigers in Buxa tiger reserve,
west bengal
A Report submitted to West Bengal Forest Department
September 2010
Investigating agency
Aarenyak
Finantial support
Buxa tiger reserve authority
www.aaranyak.org
Genetic Assessment of the Statusof Tigers in Buxa tiger reserve,
west bengal
A Report submitted to West Bengal Forest Department
September 2010
Investigating agency
Aarenyak
Finantial support
Buxa tiger reserve authority
www.aaranyak.org
© Aaranyak 2010 and West Bengal Forest Department, India
Aaranyak, 50, Samanwoy Path (Survey), PO: Beltola; Guwahati-781 028, Assam,IndiaTel: +91-361-2228418/2230250; e-mail: [email protected]
www.aaranyak.org
Contact: Udayan Borthakur [email protected] Kumar Talukdar [email protected]
Suggested citation:
Borthakur U, Das C, Talukdar B K. 2010. Genetic assessment of the status oftigers in Buxa Tiger Reserve, West Bengal. Technical Report, Aaranyak, WGP:02/2010, pp. 1 – 19.
TABLE OF CONTENTS
Executive Summary 1
Acknowledgements 2
Introduction 3
Objectives 4
Study Area 4
Methodology 5
Results and Discussion 9
Conclusion 12
References 13
Appendix I 17
Appendix II 18
Photo – Udayan BorthakurRoyal Bengal Tiger Panthera tigris tigris
WGP: 02/2010, pp. 1 – 19
1
EXECUTIVE SUMMARY
With dwindling global population, estimation of the minimum number of tigers
has always been curiosity to wildlife researchers as well as to protected area
managers. In this study, we have used DNA-based techniques for identification
of tiger individuals from their fecal (scat) samples to count the minimum number
of tiger present in Buxa Tiger Reserve (BTR), West Bengal, India. This work was
undertaken upon the request and financial support from BTR Authority. Scat
samples collected by BTR authority were handed over to Wildlife Genetics
Laboratory of Aaranyak situated at Guwahati, Assam, India. We have used
genetic markers developed by other Researchers to identify tiger scats from
other sympatric carnivores such as leopard. We have used a set of highly
polymorphic microsatellite markers to identify tiger individuals and sex
chromosome linked markers to identify gender of the tiger scat samples. We
have maintained stringent laboratory conditions for assessing and minimizing
error associated with genetic identification of individuals. We hereby confirm the
presence of 15 individual tigers, including 3 male and 9 female tigers. For three
individuals, we could not determine the gender identity following the
methodology described in this study. Our study shows that DNA-based
techniques of identification of individual tigers may be considered as a practical
and low cost option for population estimation and long term monitoring of this
species in the protected areas of India.
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2
ACKNOWLEDGEMENTS
We thank the officials of West Bengal Forest Department and Buxa Tiger Reserve
Authority for entrusting upon us by handing over the work of genetic monitoring of
tigers in Buxa Tiger Reserve.
We also thank Buxa Tiger Reserve Authority for providing us the necessary financial
support to meet the costs of laboratory work during this work.
We thank our donors to the Wildlife Genetics Programme of Aaranyak, whose support
directly or indirectly helped implementation of this study. Wildlife Genetics Laboratory,
Aaranyak receives financial support for procuring equipments from International Rhino
Foundation, Asian Rhino Project – Australia and International Foundation for Science,
which directly helped us implementing the present study.
We thank Bibek Yumnam, Senior Research Fellow, WII for his help and support to our
tiger genetic work.
We thanks Dr. Firoz Ahmed, Dr. Bibhuti P. lahkar and Dr. Anjan Kumar Talukdar for
their kind support our genetic research programme.
We thank Pranjal Kumar Das and Rumi Dev Barman of Wildlife Genetics Laboratory,
Aaranyak for their help and support during the laboratory work.
We thank all the officials of Aaranyak for their help and support throughout this work.
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INTRODUCTION
The tiger (Panthera tigris) is one of the most widely distributed big cats, yet highly
endangered throughout the range countries in which it occurs (Siedensticker et. al.
1999, Sanderson et al., 2006). The Royal Bengal tiger (Panthera tigris tigris) and
therefore is listed in the Schedule I species of the Indian Wildlife Protection Act, 1972,
also categorized as Endangered by IUCN Red List (IUCN 2008). Population monitoring
of large carnivores such as tigers are difficult to conduct because they are rare and
roam over large distances and remote areas (Schipper et al., 2008). Over the years,
remotely triggered camera has been more and more in use in wildlife studies for
determining population size or other ecological aspects of elusive, secretive and
nocturnal species such as tiger (Karanth, 1995, Karanth and Nichols, 1998, Karanth and
Nichols, 2002, Jhala et al. 2008). The opportunity to gain genetic data from natural
populations has been enhanced by the development of noninvasive genetic techniques
in past two decades (Hoss et al. 1992 ; Kohn and Wayne 1997; Goossens et al. 1998;
Fernando et al. 2003). The methodology has been increasingly applied to a wide variety
of species (Taberlet et al. 1997 ; Reed et al. 1997 ; Kohn et al. 1999 ; Ernest et al.
2000 ; Garnier et al. 2001; Eggert et al. 2003; Goossens et al. 2003; Hedmark et al.
2004; Vidya and Sukumar 2005; Smith et al. 2006; Bhagavatula and Singh 2006).
Studies have demonstrated the feasibility of using the non-invasive genetic technique
for counting tiger individuals through genetic identification of individuals from field
collected faecal samples of wild tigers in India (Bhagavatula and Singh 2006) and for
species identification (Mukherjee et al. 2007) and sex identification (Sugimoto et al.
2006). Capture-recapture based genetic population estimation of wild tigers has
successfully been carried out in India (Mondol et al. 2009), thus opening a new era of
population monitoring of this species in the country.
In the present study, we have carried out genetic analysis of tiger scat samples from
Buxa Tiger Reserve, West Bengal to identify the number of individuals present in the
area. Scat samples required for this study were collected by Buxa Tiger Reserve
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Authority. The laboratory work was undertaken in Wildlife Genetics Laboratory of
Aaranyak, situated at Guwahati, Assam.
OBJECTIVES
1. Genetic identification of tiger scats from other sympatric carnivores in the study
area
2. Genetic identification of individual tigers from tiger scats in the study area
3. Genetic identification of gender of individual tigers in the study area
STUDY AREA
Buxa Tiger Reserve (BTR) with an area of 760 km2 in the state of West Bengal,
represents part of the Terai ecosystem and lies in the Indo-Malayan biogeographical
region. BTR is considered as part of Buxa-Manas Tiger population, one of the three
major tiger populations in northeast India described by Jhala et al. (2008). This Buxa-
Manas population extends from BTR in the east to the Manas Tiger Reserve in the West
with Royal Manas in Bhutan, with a contiguous forest extent of 7,200 km2 (Jhala et al.
2008). Sympatric carnivore species includes leopard (Panthera pardus) and other
smaller cat species such as fishing cat (Prionailurus viverrinus), leopard cat (Prionailurus
bengalensis) etc. The major prey species of tiger include sambar (Cervus unicolor),
spotted deer (Axis axis), barking deer (Muntiacus muntjak), wild boar (Sus scrofa), gaur
(Bos gaurus) etc.
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METHODOLOGY
Scat Sampling
Scat samples were used as a source of DNA for genetic analysis of tigers in BTR. BTR
authority undertook sampling in the study area and handed over 72 scats to Aaranyak
in February 2010. For all scat samples, Global Positioning System (GPS) coordinates
were taken and their map locations established by BTR authority. Scats were kept in air
tight plastic bags along with silica gel. In the laboratory, these scats were dried in a hot
air oven and transferred to air tight plastic containers, following proper precautionary
measures to prevent sample cross contamination.
DNA extraction
DNA extractions for all the scats collected were performed by using commercial Stool
DNA isolation kit (QIAamp DNA Stool Kit, QIAGEN Ag., Germany) with minor
modifications. Modifications over the standard kit protocol are: (i) samples in ASL buffer
were vigorously vortexed for 10 minutes; (ii) incubation with Inhibitex at room
temperature was carried out for 15 minutes; (iii) elution was performed in 80 ul AE
buffer and incubation with elution buffer was carried out for 10 minutes. All DNA
extractions will be performed in a room dedicated to low copy-number DNA work. For
every extraction, negative controls composed of reagent blanks without the scat sample
will be included to monitor contamination. For reference tissue samples from dead
tigers, Commercial Blood and Tissue DNA extraction kit (QIAGEN Ag., Germany) was
used following standard kit protocols. Extracted DNA were run on 1% agarose gel and
visualized by ethidium bromide staining on an UV transilluminator.
Genetic species identification of tiger scats
We have used genetic markers developed by Mukherjee et al. 2007 for identification of
tigers scats collected from ONP. These are Polymerase Chain Reaction (PCR) based four
mitochondrial markers and the species identity is based on the presence or absence
products of specific size, determined through agarose gel electrophoresis. PCR reactions
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were carried out using QIAGEN Multiplex PCR Kit (QIAGEN, Germany) following
standard PCR conditions described by Mukherjee et al. 2007. 10 µl volume of PCR
reaction were performed, using 2.5 µl of scat DNA.
Genetic identification of individuals from tiger scats
Selection of polymorphic microsatellite markers – We have used polymorphic
microsatellite loci selected from screening 19 loci on freshly collected tiger scat (n = 12)
and reference blood and tissue samples (n = 5) (unpublished data, results not shown in
detail). These microsatellite loci include markers originally developed from Sumatran
tiger (Williamson et al. 2002), Amur tiger (Wu et al. 2008), Asiatic lion (Singh et al.
2002) and Domestic cat (Menotti-Raymond et al. 1999). We have also taken in to
consideration some of the microsatellite loci for individual identification of tigers, as
suggested by Bhabavatula & Singh 2006 and Mondol et al. 2009. The forward primers
of each microsatellite marker used in the study will be labelled at the 5’ end with the
fluorescent dyes 6–FAM (blue), PET (red), VIC (green) or NED (yellow), while the
reverse primers are not labelled. The PCR products generated after amplification were
loaded in a capillary electrophoresis based ABI 3130 Genetic Analyzer (Applied
Biosystems, USA) and the raw data scored for allele sizes using the software
GENEMAPPER v3.7 (Applied Biosystems, USA). The set of the genotype data from these
samples were used in selecting a panel of microsatellite markers, based on following
three criteria: (i) level of polymorphism, (ii) mean PCR success from scat DNA samples
and (iii) multiplexing compatibilities.
Individual identification of tiger scats – A panel of 7 selected polymorphic microsatellite
loci were used further to genotype all the genuine tiger scats. All the PCR were carried
out in multiplex of 4 loci in a single 10 µl reaction, each loci labelled with a separate
fluorescent label. Multiplexing were carried out using QIAGEN Multiplex PCR Kit
(QIAGEN, Germany) following standard kit protocols for reagent concentration with 0.2
μM of each primer and 2.5 μl template DNA in a 10 μl PCR reaction. The thermal cycle
were performed with 95o C initial denaturation/ activation of 15 min, followed by 40
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cycles of 94o C for 45 sec, 55o for 45 sec and 72o C for 60 sec followed by a 72o C for
15 min final extension step.
Assessing and minimizing microsatellite genotyping error
Microsatellite genotyping errors principally arise due to allelic dropout (ADO) or false
alleles (FA). The former error results in the detection of only one allele in a
heterozygous individual, and the second type of error creates a new allele, which
generates spurious heterozygotes. Each sample and loci were typed at three or more
replicates depending on the genotype discrepancy (Navidi et. al. 1992) and consensus
genotypes were created from these repeat results. Per locus and per multilocus
genotype error rates were estimated, which is a measure of the reliability of the
genotypes obtained, and is meaningful for individual identification, population
assignment, kinship and census studies (Bellemain 2004, Pompanon et. Al. 2005).
To minimize electropherogram stutter patterns and prevent Allele Drop-out, dedicated
microsatellite PCR kits (Multiplex PCR Kit, QIAGEN, Germany) were employed
throughout for genotyping.
Moreover, all the work were carried out in a room dedicated for low copy number DNA
analysis and aerosol-barrier tips will be used to prevent sample to sample
contamination.
Genetic identification of gender
Tiger scat samples were sexed using primers to amplify the Y chromosome linked SRY
(sex determining region) loci as demonstrated in the domestic cat individualization
panel, MEOWPLEX (Butler 2002, Butler et. al. 2002). This SRY primers give a single
PCR product of 99 base pairs in male individuals. We have fluorescently tagged the
forward primer of this marker, which were screened along with microsatellite loci by
capillary electrophoresis. Here microsatellite loci also act as positive controls for
distinguishing between PCR failure and false female identification. We have performed
gender identification for all the identified tiger scats, irrespective of their individual
identity. Thus the gender identity acts as an additional confirmation of multiple scats
belonging to the same tiger individual.
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8
Genetic data analysis
Allele sizing was carried out by combining automated allele calling and visual inspection
of electropherogram data for each locus in each sample. This process provides a
balance between the efficiency and consistency of automated allele-calling software
(GENEMAPPER v3.7, Applied Biosystems, USA) and the accuracy provided by human
inspection in detecting novel alleles outside of the expected range of a locus, stochastic
amplifications within the size range, and potential mistypes due to stutter or large-allele
dropout (Pompanon et al. 2005; Dewoody et al. 2006). Once samples were scored at
all loci, the data was tested for scoring errors. Consensus genotypes were checked
using the program GIMLET v 1.3.3 (Valiere 2002), and also to estimate error rates. In
order to select a final set of samples for individual identification, quality index value was
assigned to each genotype as per Miquel et al. (2006). Samples that revealed the same
genotype in all three repetitions had a quality index of one, whereas, samples that
yielded two same genotypes out of the three amplifications had a quality index of
0.667. For the purpose of selection of samples for final data analysis, quality index of
0.667 was kept as the cut-off value.
For purposes of individual identification, five to seven microsatellite loci are usually
sufficient, and act as a trade-off between achieving a reasonably low Probability of
Identity (PID) value and limiting the genotyping error (the more the loci added, the
higher will be the error rates introduced into the study). From the microsatellite
genotype data, allele frequency, observed and expected heterozygosity and probability
of identity (PID) and Probability of identity among siblings (PID-sibs) were calculated
using the software GIMLET v 1.3.3 (Valiere 2002). The unique multilocus microsatellite
genotypes, i.e., individual tigers were identified using the identity analysis module of
the program CERVUS (Marshall et al. 1998). Here, incomplete multilocus genotypes
were also considered, i.e., samples with identical genotypes for at least five or more
common loci with missing data for the rest are considered as same individual.
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RESULTS AND DISCUSSION
DNA extraction
A total of 72 scat samples were handed over by BTR authority, which were used forDNA extraction. Visible DNA could not be obtained for all the samples and DNAobtained forms smears due to degradation (Agarose gel picture in Appendix II A).
Genetic species identification of tiger scats
Analysis of 72 scat samples yields 38 scats which could be confirmed as tiger scats.Other scats were either confirmed non-tiger or failed to produce any results due to lowDNA quality (Agarose gel picture in Appendix II B). The GPS locations of identified tigerscats from BTR are given in table 1.
Table 1. GPS locations of genetically identified tiger scats from Buxa Tiger Reserve
Sl.No.
LabID GPS coordinates
Sl.No.
labID GPS coordinates
Sl.No.
labID GPS coordinates
1 605 26 45 19.5 89 25 27.0 14 622 26 36 20.4 89 38 25.5 27 647 26 47 18.9 89 30 33.7
2 606 26 41 44.4 89 87 49.2 15 623 26 35 34.9 89 27 34.5 28 648 NA
3 607 26 33 33.1 89 34 53.3 16 625 26 47 18.9 89 30 33.7 29 649 NA
4 608 26 40 29.1 89 32 42.0 17 628 26 35 45.0 89 31 29.7 30 656 26 35 49.8 89 31 07.5
5 610 26 43 13.6 89 44 59.3 18 630 26 38 63.6 89 30 83.0 31 657 26 35 43.6 89 29 58.5
6 611 26 39 53.9 89 33 58.8 19 631 26 40 37.1 89 31 44.1 32 658 26 42 61.5 89 48 27.8
7 612 26 36 39.4 89 28 07.0 20 632 26 40 29.6 89 32 14.5 33 659 NA
8 615 26 35 15.8 89 43 76.5 21 636 26 45 19.5 89 25 27.0 34 660 26 37 44.7 89 36 51.4
9 617 26 42 52.6 89 36 31.4 22 638 26 42 55.3 89 35 52.6 35 661 26 40 58.2 89 30 57.5
10 618 26 42 36.5 89 35 04.3 23 640 26 36 04.5 89 31 11.9 36 666 26 42 30.7 89 35 01.6
11 619 26 40 22.1 89 34 21.6 24 641 26 48 14.3 89 23 50.7 37 667 26 38 54.6 89 46 23.2
12 620 26 42 10.6 89 31 58.2 25 645 26 45 55.5 89 24 04.3 38 670 26 33 54.8 89 34 67.5
13 621 26 40 31.1 89 31 58.4 26 646 26 34 30.8 89 51 39.5
Genetic identification of individuals from tiger scats
Selection of microsatellite loci for analysis – From the results of screening of 19 loci, we
have selected 7 highly polymorphic loci for individual identification of tigers from BTR
(details not shown here). Figure 1 shows the low Probability of Identity values obtained
for these seven loci in combination, for five reference tiger tissue samples, which
denote high power of discrimination among different individuals.
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Figure 1. Graphical representation Product PID and PID-sibs values of 7 selected
polymorphic microsatellite loci in 5 reference tiger tissue samples
Genotyping data for all the 38 tiger scats were obtained after analysis of allele sizing
data. As per the quality index criteria mentioned in the methodology, genotype data for
27 scat samples and six highly polymorphic loci were selected for identification of
unique multilocus genotypes and determining the number of individual tigers.
Genotyping error rates (ADO and FA) were calculated from the triplicate genotyping
data for these 27 samples (Table 2). Low genotyping error rate of 2% was observed
during this study. The number of alleles, allele size range, percentage PCR success rate,
Hexp and Hobs in 27 tiger scat samples from ONP are presented in table 2. The PCR
success rate from 27 scat samples varied from 73 to 100%, with a mean high PCR
success rate of 92%. The mean observed heterozygosity obtained from the dataset of
27 samples in six loci was 46%. Consensus genotypes for all the 27 selected samples
for six loci were generated and used for further data analysis (Appendix I).
0.0E+005.0E-021.0E-011.5E-012.0E-012.5E-013.0E-013.5E-014.0E-014.5E-015.0E-01
P ID
PI Pop1
PIsibs Pop1
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Table 2. Results of 6 polymorphic loci used in individual identification from 27 tiger scat
samples
LocusName
No. ofalleles
AlleleRange
% PCRsuccess ADO FA Hexp Hobs
Fca628 6 91-125 100 0.038 0 0.71 0.67HDZ170 7 202-230 96 0.022 0 0.54 0.41Pati09 5 111-129 100 0 0 0.62 0.56Ple51 6 163-175 92 0 0 0.68 0.15Fca304 7 126-198 73 0.04 0 0.68 0.19Ple55 6 149-165 92 0 0 0.8 0.81
From the consensus genotype data for 27 scat samples, 15 unique multilocus genotypes
were obtained based on available genotype data with at least five or more loci and
allowing zero locus mismatches. The product PID and PID-Sibs value of 8.21 x 10-6 and
8.32 x 10-3 were respectively obtained from the six loci data. Thus, analysis confirms
that there are minimum of 15 individual tigers in BTR. Moreover, allowing single loci
mismatch did not change the number of individuals obtained from the dataset.
Gender identification of individual tigers
Confirmed gender identification results were obtained for 24 out of 27 scats used for
final analysis. In this dataset, presence of 9 female and 3 male tigers could be
confirmed, and three individuals with unknown gender identity (Data given in Appendix
I). Electropherogram of SRY amplification (in male tigers), along with microsatellite
locus Fca304 is shown in Appendix II B.
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CONCLUSION
1. This is for the first time that DNA-based techniques of individual
identification of tigers from scat samples have been used for
estimating the number of tigers in a protected area of West
Bengal, India.
2. This study reveals the presence of minimum 15 individual tigers,
including 3 male, 9 female and 3 unknown gender identities.
3. Results obtained in this study are based on analysis of final 27 tiger
scats out of 38 confirmed tiger scat samples, in which seven
individual tigers have only one representative sample. Therefore, it
is recommended that further scat sampling may be undertaken in
Buxa Tiger Reserve, in order to obtain a robust estimate of
population size.
4. As some of the samples did not produce data useful for further
analysis, it is recommended that at least two scat DNA preservation
methodologies may be adopted during sample collection. One of
them may be silica gel and air drying (as used during this study).
Another methodology may be either ethanol (95%) preservation or
use of buffers such as DMSO-Tris-EDTA-salt solution (DETs) for
scat storage in 10 – 15 ml plastic vials.
5. It is also recommended that scats may be collected following the
norms of capture-recapture sampling for robust population
estimation.
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Wu J-H, Lei Y-L, Fang S-G. 2008. Twenty-one novel tri- and tetranucleotidemicrosatellite loci for the Amur tiger (Panthera tigris altaica). Conserv. Genet. DOI10.1007/s10592-008-9571-8
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APPENDIX I
Multilocus microsatellite genotypes of 27 tiger scats from Buxa Tiger Reserve
(matching fill colors represent different samples sharing the same unique multilocus genotype
and white represent only one sample per unique multilocus genotype)
IndividualID
LabID
GenderID
Fca628 HDZ170 Pati09 Ple51 Fca304 Ple55
A B A B A B A B A B A B
ind1 605 F 95 105 216 216 125 125 171 171 196 196 149 155ind1 620 F 95 105 216 216 125 125 171 171 ? ? 149 155ind2 606 M 91 91 ? ? 125 125 173 173 196 196 149 155ind2 607 M 91 91 216 216 125 125 173 173 196 196 149 155ind2 610 M 91 91 216 226 125 125 173 173 196 196 149 155ind2 618 M 91 91 216 216 125 125 173 173 196 196 149 155ind2 621 M 91 91 216 216 125 125 173 173 196 196 149 155ind2 656 M 91 91 216 216 125 125 173 173 196 196 ? ?ind3 608 F 91 105 202 216 117 125 171 171 196 196 149 155ind4 615 F 111 111 216 216 125 129 175 175 148 148 153 163ind5 617 F 91 91 220 220 111 111 ? ? 128 196 163 163ind5 647 F 91 91 220 220 111 111 169 169 128 196 163 163ind6 623 F 105 119 202 216 119 129 171 171 158 158 155 165ind6 631 F 105 119 202 216 119 129 171 171 ? ? 155 165ind7 628 M 91 125 202 216 117 125 173 175 ? ? 153 161ind7 657 M 91 125 202 216 117 125 173 175 ? ? 153 161ind8 630 U 105 125 216 216 125 125 171 171 126 128 155 163ind9 632 F 91 105 212 230 119 125 163 163 196 196 149 155ind10 638 F 105 111 202 216 117 125 171 175 ? ? 155 163ind10 666 F 105 111 202 216 117 125 171 175 126 126 155 163ind11 645 F 91 105 216 216 125 129 167 167 166 166 ? ?ind12 648 U 91 105 216 216 117 125 ? ? 158 196 155 161ind13 646 M 95 105 216 216 125 129 173 173 ? ? 153 161ind13 658 M 95 105 216 216 125 129 173 173 128 128 153 161ind14 660 F 105 111 216 226 117 125 173 173 ? ? 153 161ind14 670 F 105 111 216 226 117 125 173 173 126 198 153 161ind15 667 U 91 105 220 220 117 117 173 173 166 166 163 163
? – missing data; M – Male; F – Female; U - unknown
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APPENDIX II
A. Agarose gel visualization of extracted DNA from the scats samples ofBuxa Tiger Reserve
B. Species identification of scat samples using four PCR based mitochondrial
markers
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C. Electropherogram of microsatellite locus showing (i) heterozygotes
and (ii) homezygotes
D. Electropherogram profile of fluorescently labeled SRY product along
with Microsatellite locus Fca304
(i)
(ii)
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C. Electropherogram of microsatellite locus showing (i) heterozygotes
and (ii) homezygotes
D. Electropherogram profile of fluorescently labeled SRY product along
with Microsatellite locus Fca304
(i)
(ii)
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19
C. Electropherogram of microsatellite locus showing (i) heterozygotes
and (ii) homezygotes
D. Electropherogram profile of fluorescently labeled SRY product along
with Microsatellite locus Fca304
(i)
(ii)
