Species-specific mitochondrial DNA markers for identification of non-invasive samples from sympatric carnivores in the Iberian Peninsula

Abstract

Genetic species identification of non-invasively collected samples has become an important tool in ecological research, management and conservation and wildlife forensics. This is especially true for carnivores, due to their elusive nature, and is crucial when several ecologically and phylogenetically close species, with similar faeces, hairs, bones and/or pelts, occur in sympatry. This is the case of the Iberian Peninsula, a region with a carnivore community of 16 species-about two-thirds of the European carnivore fauna. Here we present a simple, efficient and reliable PCR-based protocol, using a novel set of species-specific primers, for the unambiguous identification to species of non-invasively collected samples or forensic materials from Iberian carnivores. For each species, from the consensus of all cytochrome b haplotypes, found here and previously reported, we designed speciesspecific primer pairs for short fragments, the most likely to persist in low-quantity and degraded DNA samples. The predicted specificity of each primer pair was assessed through PCR of positive DNA extracts from the carnivore species, from an exhaustive array of potential prey and from humans. The robustness of PCR amplification for non-invasively sampled DNA was tested with scat samples. The primers did not produce false positives and correctly identified all carnivore samples to the species level. In comparison with sequencing and PCR-RFLP assays, our method is, respectively, cost-and time-effective, and is especially suited for monitoring surveys targeting multiple populations/species. It also introduces an approach that works for a whole community of carnivores living sympatrically over a large geographic area.

Record and distribution of black-fur foxes in a Mediterranean natural park, Serra de Mariola, Spain

“…3). Haplotype 171T corresponds to that previously described in other Iberian Peninsula fox populations (Fernandes et al 2008).…”

“…Genetic studies are necessary for the conservation of natural populations and the study of their interactions with non-native subspecies , and impact on natural communities in many different ways, including the introduction of novel pathogens, reduction of native prey populations and competition with native species for food resources (Mack et al 2000, Manchester Figure 3. Bayesian tree produced using a dataset including cytochrome b sequences from Frati et al (1998) [Z80957-Z80997], Perrine et al (2007) [EF064207-EF064220], Palomares et al (2002) [AJ441335], Fernandes et al (2008) [EF689058, EF689062], Aubry et al (2009) [FJ830756-FJ830776] and unpublished sequence JN709944 (Dang et al 2011). This analysis also included the sequences of these study, haplotype 171T (R1, R2) and haplotype 171C (B1-B3, R3) [KM225298-KM225303].…”

Identification of Two Tentative Strains of European Subspecies of the Eurasian River Otter <i>Lutra lutra lutra </i>(Linnaeus, 1758) from the Partial Mitochondrial Cytochrome <i>b</i> Gene

“…It might therefore be useful for the management of L. lutra pedigrees EF689068 (Fernandes, et al, 2008) X94923 (Ledje and Arnason, 1996) AJ536010 (Marmi, et al, 2004) AF057124 (Koepfli, et al, 1998) A-line (present study) Asahikawa (Suzuki, et al,1996) Latvia (Suzuki, et al,1996) FJ236015(Jang and Hwang, 2008) B-line (present study) Europe (Suzuki, et al,1996) China (Suzuki, et al,1996) EF472347 (L. sumatrana, Koepfli, et al, 2008) Aonyx cinerea (Suzuki, et al,1996) Determination and comparison of complete sequence of cytochrome b genes might be more effective. But the assessment of the fragmentation patterns by using the RFLP method described here would be an easier technique than sequencing, and more suitable for on-site screening.…”

“…The data were registered in the DDBJ/EMBL/GenBank databases and assigned the accession numbers AB933268 (for A-line Tentative Eurasian River Otter Strains kept in Japanese zoos [6]. In addition, A-line cyb had 100% identity to a number of complete nucleotide sequences of European L. lutra lutra cytochrome b gene, such as X94923 [11], EF689068 [12], AJ536010 [13], and AF057124 [14]. In contrast, among these latter sequences no sequence identical to B-line cyb was found; B-line cyb had 99% identity to L. lutra (FJ236015; Jang and Hwang, 2008, direct submission from Korea) and 95% to L. sumatrana (EF472347) [15].…”

Ecology and conservation of the polecat Mustela putorius (Linnaeus, 1758) in Portugal: a review

“…Species-specific molecular markers have been developed for non-invasive studies of Iberian carnivores (e.g. Fernandes et al, 2008a;Oliveira et al, 2010). While these markers distinguish polecats from other carnivores, they are unable to discriminate polecat from ferret samples.…”