Development and testing of an optimized method for DNA-based identification of jaguar (Panthera onca) and puma (Puma concolor) faecal samples for use in ecological and genetic studies

Universidade Federal do Rio Grande do Sul Programa de Pós-Graduação em Genética e Biologia Molecular Porto Alegre Rio Grande do Sul Brazil
Genetica (Impact Factor: 1.4). 07/2009; 136(3):505-512. DOI: 10.1007/s10709-008-9347-6


The elusive nature and endangered status of most carnivore species imply that efficient approaches for their non-invasive
sampling are required to allow for genetic and ecological studies. Faecal samples are a major potential source of information,
and reliable approaches are needed to foster their application in this field, particularly in areas where few studies have
been conducted. A major obstacle to the reliable use of faecal samples is their uncertain species-level identification in
the field, an issue that can be addressed with DNA-based assays. In this study we describe a sequence-based approach that
efficiently distinguishes jaguar versus puma scats, and that presents several desirable properties: (1) considerably high
amplification and sequencing rates; (2) multiple diagnostic sites reliably differentiating the two focal species; (3) high
information content that allows for future application in other carnivores; (4) no evidence of amplification of prey DNA;
and (5) no evidence of amplification of a nuclear mitochondrial DNA insertion known to occur in the jaguar. We demonstrate
the reliability and usefulness of this approach by evaluating 55 field-collected samples from four locations in the highly
fragmented Atlantic Forest biome of Brazil and Argentina, and document the presence of one or both of these endangered felids
in each of these areas.

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    • "Comparative fecal DNA preservation studies for carnivores were conducted primarily for canids and ursids (e.g., Wasser et al. 1997, Murphy et al. 2000, 2002, Panasci et al. 2011). For felids, a wide variety of fecal DNA methods have been applied, including freezing (e.g., Ernest et al. 2002, Sugimoto et al. 2006), air drying (e.g., Farrell et al. 2000, Weckel et al. 2006), silica desiccation (e.g., Haag et al. 2009, Jane cka et al. 2011), or liquid storage using buffer solutions (e.g., 20% dimethyl sulfoxide buffer, Vynne et al. 2012) or ethanol (EtOH; e.g., Mondol et al. 2009, Michalski et al. 2011, see also online Supporting Information Table S1). Yet, only a handful of comparative fecal DNA preservation studies examined the effectiveness of different methods on amplification of fecal DNA for felids (e.g., for mtDNA for wild tigers [Panthera tigris]; Bhagavatula and Singh 2006) and nuclear DNA (nDNA) markers for captive tigers (Reddy et al. 2012). "
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    ABSTRACT: Field-sampling methods for molecular scatology studies must be optimized, especially when working on elusive species in challenging tropical environments where rates of DNA degradation are elevated because of hot and humid weather conditions. To maximize polymerase chain reaction (PCR) amplification success and genotyping accuracy rates and to minimize genotyping error rates for fecal DNA samples of jaguars (Panthera onca) and co-occurring Neotropical felids collected in Belize, Central America, we evaluated the performance of two fecal DNA storage techniques (dimethyl sulfoxide saline solution [DETs buffer] and 95% ethanol [EtOH]) suitable for long-term preservation at remote tropical sites. Additionally, we tested fecal samples collected from 4 different locations on the scat (top, side, bottom, inside) at 2 different tropical forest types (tropical broadleaf and tropical pine forests). DETs buffer was the superior fecal DNA preservation method, with 44% higher PCR amplification success (P = 0.009) and 17% higher genotyping accuracy (P = 0.021) than 95% EtOH-stored samples. Polymerase chain reaction amplification success of fecal DNA collected at the more open, pine-forest (Pinus sp.) site differed significantly across locations on the scat, with highest mean success rates obtained from the top (85% ± 6.5% SD), followed by the side (79% ± 9.4% SD), bottom (76% ± 11.9% SD), and inside (69% ± 10.3% SD) of scat samples. Scat samples collected at the more closed-canopy broadleaf site did not show any significant differences in amplification success rates across scat locations. We recommend that researchers optimize field-sampling methods, including collection and storage protocols, by conducting a pilot study prior to their molecular scatology research efforts. © 2015 The Wildlife Society.
    Full-text · Article · Apr 2015 · Wildlife Society Bulletin
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    • "The molecular tools developed in this study thus provide certain advantages over earlier approaches used in carnivore studies (Cossíos and Angers 2006; Sugimoto et al. 2006; Bidlack et al. 2007; Livia et al. 2007; Haag et al. 2009; Roques et al. 2010). The approaches proposed here facilitate rapid screening of large number of samples using fewer steps in sample processing (DNA extraction, single multiplex PCR, electrophoresis), reduced species/sex misidentifications , and are cheaper than PCR–RFLP (Nagata Table 1 Details of the speciesspecific mtDNA primers designed in this study "
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    ABSTRACT: Assessing the distribution, abundance and demographic ratios of endangered and elusive co-occurring carnivore species at a landscape level is important for their continued survival. Despite potential to determine distribution and dietary analyses, use of faecal samples has been relatively limited in the context of multiple sympatric species living at large landscapes. We developed and optimized a range of novel non-invasive molecular techniques for species, gender and individual identification of tiger (Panthera tigris) and leopard (Panthera pardus). We collected a large number of faecal samples as part of a pilot occupancy survey in the Malenad-Mysore Tiger Landscape, Western Ghats, India. We could genetically ascertain species and gender for 88 and 57 % of the field-collected samples respectively. Additionally, we also determined a panel of nine and eight polymorphic loci for tiger and leopard individual identification, resulting in 18 tigers and 39 leopards from varied quality field-collected samples. Our pilot study suggest such molecular approaches will help in future efforts to gather landscape level distribution, demographic and other ecological information on tigers and leopards across their distribution.
    Full-text · Article · Nov 2014 · Conservation Genetics Resources
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    • "This can be particularly important when the focal species of the study occurs in sympatry with related taxa [13]. Analyses based on faecal DNA have been applied to a broad range of taxa to address questions from occupancy and food habits to abundances, species distribution, and habitat use [14] [15] [16] [17] [18] [19] [13]. "
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    ABSTRACT: Abstract The Guinean Forests are renowned for their primate diversity, with nearly 30 distinct species. These forests have been identified as some of Africa's most critical primate conservation areas. However, intensive poaching pressure and habitat degradation cause severe threats to these species. The remaining primate populations have low population densities, have secretive habitats, and are wary of humans. This can make field studies and direct observation difficult. To develop a reliable, sensitive and simple molecular identification method using Polymerase Chain Reaction (PCR), we sequenced the mitochondrial D-loop Hypervariable 1 region (HVR1) of Diana monkey (Cercopithecus diana) and Campbell monkey (Cercopithecus campbelli). From these sequences and their orthologs within Eastern black-and-white colobus (Colobus guereza) obtained from GenBank, we designed species-specific primers to amplify fragments of the mitochondrial D-loop HVR1 gene from faecal samples. These primers allow us to differentiate between Diana monkey (C. diana), Campbell monkey (C. campbelli), lesser spot-nosed monkey (Cercopithecus petaurista), Red colobus (Piliocolobus badius), Olive colobus (Procolobus verus), King colobus (C. polykomos), and Geoffroy’s black-and-white colobus (C. vellerosus), demonstrating their potential for the identification of West African threatened primates.
    Full-text · Article · Sep 2014 · Tropical Conservation Science
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