The use of genetic techniques to facilitate noninvasive monitoring and conservation of wildlife Mitochondrial DNA for species-level discrimination




Corey Anco

Introduction

Noninvasive wildlife monitoring methods, such as camera trapping, hair-snaring, and scat analyses, have become increasingly useful and less costly alternatives to more invasive techniques (e.g., radio-collaring or blood/tissue collection, Waits and Paetkau 2005). Scat deposited by an animal can be collected by researchers and transported with relative ease, which minimizes potential risks to the researcher and animal. Animal scat contains a significant amount of genetic information (Chaves et al. 2012), which can be used to identify species, individuals, gender, diet, and provide information regarding population dynamics (Chaves et al. 2010; Kitano et al. 2007).

Habitat fragmentation is often cited as a primary cause resulting in global losses of biodiversity (Dirzo and Raven 2003). Biological hotspots, such as tropical forests, are areas that contain disproportionately high level of species diversity in comparatively small areas (Myers et al. 2000), and are particularly sensitive to habitat fragmentation and biodiversity loss (Dirzo and Raven 2003). Neotropical felids, such as the jaguar (Panthera onca), puma (Puma concolor), and ocelot (Leopardus pardalis) are solitary carnivores, typically present in low-densities, which can make monitoring of these organisms challenging. Wildlife biologists may rely on noninvasive survey methods to assist in the monitoring of species persisting at low-densities. The goal of this project was to test the ability to discriminate among jaguar, puma, and ocelot scat using regions of the 16s rRNA and ATP6 genes present in mitochondrial DNA, isolated from felid scat.

Materials and Methods

15 scat samples representing three species were obtained from the American Museum of Natural History and the Global Felid Genetics Program. DNA was extracted from ocelot (Leopardus pardalis; n =3), puma (Puma concolor; n = 6), and jaguar (Panthera onca; n = 6) scat using the DNA Extraction FastDNA TM SPIN Kit for Feces protocol (MP Biomedicals).

Primers were obtained for the 16s rRNA (Kitano et al. 2007) and ATP6 (Chaves et al. 2012) gene regions from published literature. Isolated DNA was amplified using PCR and visualized on 1% Agarose gel. PCR products were then purified, sequenced (GeneWiz®), and confirmed using Nucleotide BLAST (NCBI). Clustal Omega was used to align and analyze forward and reverse sequences and to discriminate among species.


Results

DNA was successfully amplified for the 16s rRNA genes (Figure 1). One of the jaguar samples (J6) failed to amplify, likely due to a small amount of starting material (Figure 1).

Two concentrations (1:10 and 1:100) of DNA were successfully amplified for the ATP6 gene (Figure 2). One of the puma concentrations (P44 1:100) and one of the ocelot samples (O77) failed to amplify (Figure 2). PCR products were loaded in the following concentration order for each sample id (e.g., P22): 1:10, 1:100

PCR products were purified for all successfully amplified samples of the 16s rRNA gene. The ATP6 gene PCR products were purified for all successfully amplified samples of 1:10 dilution (first band).

Sequences were aligned for 16s rRNA and ATP6 and analyzed using Clustal Omega (Figures 3 and 4, respectively) and DNA was confirmed using NCBI Nucleotide BLAST®.

Highlighted base pairs indicate variation among puma, ocelot, and jaguar in the forward and reverse sequences for both the 16s rRNA (Figure 3) and ATP6 (Figure 4) genes.

Discussion

Regions of the 16s rRNA and ATP6 mitochondrial genes were identified in ocelot, puma, and jaguar suitable for species-level discrimination from scat. Numerous nucleotide loci were identified that indicated differences at the species-level across all sequenced samples (Figures 3 and 4). Furthermore, sequencing of the subunit 6 region of the ATP gene for ocelots (Leopardus pardalis) is novel to this study, has not previously been sequenced, and is not available on NCBI.

References

Chaves, P.B., V.G. Graeff, M.B. Lion, L.R. Oliveira, and E. Eizirik. 2012. DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples. Molecular Ecology Resources 12: 18-35.

Dirzo, R., and P. Raven. 2003. Global state of biodiversity loss. Annual review of Environment and Resources 28: 137-167.

Kitano, T., K. Umetsu, W. Tian, and M. Osawa. 2007. Two universal primer sets for species identification among vertebrates. International Journal of Legal Medicine 121: 423-427.

Myers, N., R.A., Mittermeier, C.G. Mittermeier, G.A.B. da Fonesca, and J. Kent. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853-858.

Waits, L.P., and D. Paetkau. 2005. Noninvasive genetic sampling tools for wildlife biologists: a review of applications and recommendations for accurate data collection. Journal of Wildlife Management 69(4): 1419-1433.

Figures


Figure 1-Amplification of PCR product for 16s rRNA on 1% Agarose gel


Figure 2-Amplification of PCR product for ATP6 on 1% Agarose gel.


Figure 3-Aligned forward sequence and reverse sequence for the 16s rRNA gene


Figure 4-Aligned forward sequence and reverse sequence for the ATP6 gene


Abstract
There are a wide variety of ways researchers can monitor wildlife. Some methods are more involved than others and require a certain amount of physical interaction with the animal (radio-collaring, blood collection…). These methods are considered invasive because they directly engage the researcher with the animal. This interaction can lead to unintended consequences by inducing unhealthy stress in the animal, which may lead to a condition known as capture myopathy. Monitoring techniques that enable the researcher to collect the same or very similar data whilst minimizing the risk to both the researcher and the animal are considered as favorable alternatives. Several techniques, known collectively as noninvasive monitoring techniques, such as camera trapping, hair-snaring, and scat analyses have become increasingly useful and less costly alternatives to more invasive techniques.

Scat analysis is a noninvasive technique used to facilitate wildlife monitoring efforts and has been gaining increased use given its relative efficiency, low cost, and increased accuracy. I examined the use of molecular techniques to distinguish among felid samples using mitochondrial DNA isolated from scat. Two mitochondrial genes (16s rRNA and ATP6) were found to be effective in discriminating among the scats of three Neotropical felids: jaguars (Panthera onca), pumas (Puma concolor), and ocelots (Leopardus pardalis) at the species-level. Base pair differences were identified in the 16s rRNA and ATP6 regions at multiple nucleotide loci for all three species. This study also sequenced the subunit 6 region of the ATP gene for ocelots, previously unsequenced on NCBI.

Full Paper

Acknowledgments

I would like to thank Catharina Grubaugh, Kate Reid, and Dr. Berish Rubin for their patience and guidance throughout the duration of this project. Dr. Anthony Caragiulo and Dr. George Amato of the American Museum of Natural History provided the scat samples used for this project. This project would not have been possible without their support and generosity. I would also like to thank Chelsea Butcher, Amanda Makkay, and my advisor Dr. Evon Hekkala.

All photo credits belong to Corey Anco


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