Tiger (Panthera tigris) scent DNA: a valuable conservation tool for individual identification and population monitoring
Genetic monitoring of tiger source populations is a conservation priority, yet due to low sample sizes and poor DNA quality, scat DNA has failed to produce the powerful studies needed to inform management decisions in humid, tropical landscapes. Here, we report the first successful extraction of DNA from tiger scent marks, a hitherto neglected genetic resource. We show that tiger scent DNA quality is equal or superior to scat DNA, and as scent marks are encountered 2–8 times more frequently in the wild than scats, they constitute an important genetic resource for monitoring populations and individuals.
KeywordsPanthera tigris Non-invasive sampling Scent sprays Individual identification
Scent DNA samples were collected from three captive tigers (2 males, 1 female) in southern Ontario in November 2013 and June 2014. Two to four samples were collected per individual and estimated time between marking and sampling varied from 10 min to 39 h. Scent marks were swabbed using sterile cotton buds, the swabs placed in a vial containing 500 μL Buffer ASL (Qiagen) and stored at room temperature. The cotton tip was excised and placed in a sterile 2.0 mL tube with the initial Buffer ASL aliquot and 15 μL Proteinase K (Qiagen). Samples were incubated at 65 °C for 2 h, after which an additional 15 μL of Proteinase K was added prior to incubation at 37 °C for 12 h. 500 μL Buffer AL (Qiagen) was added and the sample incubated at 65 °C for 10 min, after which 500 μL of cold 100 % ethanol was added and the sample incubated at 4 °C for 1 h. The remainder of the extraction process followed the suggested QIAGEN DNeasy Blood & Tissue Kit protocol, except DNA was eluted using 50 μL Buffer AE heated to 70 °C and left to incubate on the spin column membrane for 30 min prior to centrifugation. DNA was stored at −20 °C until analyzed.
Species identification was tested by amplifying a 110 bp fragment of the cytochrome oxidase b mitochondrial gene using primers H15149 (Kocher et al. 1989) and Farrel-R (Farrell et al. 2000). Sequencing followed Caragiulo et al. (2014). Five microsatellite loci in two multiplex groups (Menotti-Raymond et al. 1999) were used for individual identification (Table S1). Gender was determined using fluorescently labeled primers for the amelogenin region of the sex chromosomes (Pilgrim et al. 2005). Each gender typing reaction consisted of 3.50 μL of QIAGEN MasterMix, 0.70 μL of Q-solution, 0.20 μL of 10 μM amelogenin primers (Pilgrim et al. 2005), 0.20 μL of DNase-free water, and 2.0 μL of DNA template. Both microsatellite and gender-typing PCRs were done in triplicate using the multiple tubes approach (Taberlet et al. 1996). All PCRs were prepared and analyzed as per Caragiulo et al. (2015). Genotyping error rates were estimated using GIMLET version 1.3.2 (Valière 2002).
Percentage of successful independent PCR, and allelic dropout and false allele rates using DNA from captive tiger scent sprays
% Positive PCR
Comparison of PCR success and error rates (mean across loci) for tiger sprays versus tiger scats
% Positive PCR
Mondol et al. (2009)
Reddy et al. (2012)
Tropical dry forest
Gour et al. (2013)
Sharma et al. (2013)
Bhagavatula and Singh (2006)
We thank Peter Klose at Jungle Cat World, and Dr. Wendy Korver and Michael Hackenberger at Bowmanville Zoo for providing tiger scent marks. We thank Tambling Wildlife Nature Conservation, and the Ministry of Forestry for the Republic of Indonesia for supporting related fieldwork.
- Gour DS, Bhagavatula J, Bhavanishankar M, Reddy PA, Gupta JA, Sarkar MS, Hussain SM, Harika S, Gulia R, Shivaji S (2013) Philopatry and dispersal patterns in tiger (Panthera tigris). PLoS One 8:e66956. doi:10.1371/journal.pone.0066956
- Smith O (2012) Population genetics and structure of the Sumatran tiger. Imperial College, LondonGoogle Scholar
- Yudakov A, Nikolaev I (2012) Winter ecology of the Amur tiger based upon observations in the west-central Sikhote-Alin Mountains, 1970–1973, 1996–2010, 2nd revised edn. Wildlife Conservation Society, Dalnauka, VladivostokGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.