DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples

Abstract

Although species assignment of scats is important to study carnivore biology, there is still no standardized assay for the identification of carnivores worldwide, which would allow large-scale routine assessments and reliable cross-comparison of results. Here, we evaluate the potential of two short mtDNA fragments [ATP6 (126 bp) and cytochrome oxidase I gene (COI) (187 bp)] to serve as standard markers for the Carnivora. Samples of 66 species were sequenced for one or both of these segments. Alignments were complemented with archival sequences and analysed with three approaches (tree-based, distance-based and character-based). Intraspecific genetic distances were generally lower than between-species distances, resulting in diagnosable clusters for 86% (ATP6) and 85% (COI) of the species. Notable exceptions were recently diverged species, most of which could still be identified using diagnostic characters and uniqueness of haplotypes or by reducing the geographic scope of the comparison. In silico analyses were also performed for a 110-bp cytochrome b (cytb) segment, whose identification success was lower (70%), possibly due to the smaller number of informative sites and/or the influence of misidentified sequences obtained from GenBank. Finally, we performed case studies with faecal samples, which supported the suitability of our two focal markers for poor-quality DNA and allowed an assessment of prey DNA co-amplification. No evidence of prey DNA contamination was found for ATP6, while some cases were observed for COI and subsequently eliminated by the design of more specific primers. Overall, our results indicate that these segments hold good potential as standard markers for accurate species-level identification in the Carnivora.

Re-discovering jaguar in remaining coastal Atlantic Forest in southeastern Brazil by non-invasive DNA analysis

“…The neighbor-joining-tree recovered these three jaguar samples within the group of Panthera onca (Figure 1), reinforcing that the method used was able to correctly identify the species who deposited the faeces collected. The ATP6 and CytB gene have been previously used for felid identification (Chaves et al 2012, Miotto et al 2014, Wultsch et al 2016, and proved to be a powerful molecular marker for this group of carnivores.…”

“…A CytB primer pair developed by Farrel et al (2001) was used to amplify a fragment of the Cytochrome B gene, and the ATP6-DF3; ATP6-DR2 primer pair (Chaves et al 2012) was used to amplify a fragment of the ATP6 gene, following the respective PCR protocols given by the already cited authors. All PCRs were carried out using a Veriti 96 Well Thermal Cycler (Applied Biosystems) and tissue sample DNA as a positive control.…”

“…In this study, we used a non-invasive DNA analysis to identify felid scats collected in the SVU, a recognized method for species identification (Farrel et al 2000, Haag et al 2009, Chaves et al 2012, Miotto et al 2014). Our results indicated at least three individuals (two females and one male) inside SVU, thus confirming the occurrence of this key top predator in this important protected area.…”

Comparison of Noninvasive Genetics and Camera Trapping for Estimating Population Density of Ocelots (Leopardus Pardalis) on Barro Colorado Island, Panama

“…PCR conditions for species identification can be found in Chaves et al (2012). PCR conditions for microsatellite amplification and sex ID were as follows.…”

“…For species identification, we amplified a 126-bp fragment of the mitochondrial gene ATP6 by polymerase chain reaction (PCR) using primers ATP6-DF3 and ATP6-DR1 following conditions from Chaves et al [36]. PCR products were sequenced on an Applied Biosystems 3730xl DNA analyzer, and resulting sequences were compared to reference sequences using the online tool DNA Surveillance Carnivora [36].…”