Testing mitochondrial markers for noninvasive genetic species identification in European mammals (original) (raw)

2014, Conservation Genetics Resources

While DNA-barcoding routines have largely facilitated molecular identification of animal species, there is no common strategy for species identifications based on noninvasively collected samples. We compared the performance of six commonly used mitochondrial fragments for identification success of low-quality hair and scat samples from 15 European mammals. The standard cytochrome oxidase subunit 1 barcoding fragment performed worst in the test, with only three successful species identifications out of 32 samples. In contrast, three short fragments (16S and 12S rDNA, cytochrome b) led to reliable species identifications for [80 % of all tested samples. Success rates also differed among feeding types. Keywords Noninvasive sampling Á Species discrimination Á Mammalia Á Genetic monitoring Á Wildlife forensics Genetic monitoring based on the use of noninvasively collected samples has become an important tool in monitoring programmes and field studies of endangered mammals (Schwartz et al. 2007). While most respective studies focus on single target species (e.g., Perez et al. 2009; Karamanlidis et al. 2012) or, for example, carnivore communities (Ruell and Crooks 2007; Fernandes et al. 2008; Gompper et al. 2006), suitable genetic identification systems for entire mammal communities have not gained significant interest so far. In order to fill this gap we tested six primer pairs identified from an extensive literature survey that are commonly used for species identification on hair and scat samples from various medium-sized and large European mammals. Thirty-two samples (16 scat and 16 hair samples) of 15 species were taken from our internal data base of noninvasively collected mammal samples. We explicitly preferred samples with presumably low DNA quality, for instance due to progressed sample age or small amount of available material. The samples were derived from German wildlife parks (European wildcat, Eurasian lynx, brown bear, red deer, moufflon, goat) or field samples collected across Germany and Austria (European wildcat, Eurasian otter, raccoon, red fox, Eurasian beaver, horse, wild boar, red deer, roe deer, European hare) (Table 1). Samples were extracted in a laboratory dedicated to the pre-PCR processing of noninvasively collected materials using the commonly used Qiagen Investigator Kit (hairs) or the Qiagen Stool Kit (scats) following the recommendations of the manufacturer with slight modifications regarding dilution volumes. Mitochondrial (mt) DNA fragments were amplified from all 32 samples using six different primer pairs found in the literature (for/rev): 1. (CrL, control region) Thr-L15926 5 0 CAATTCCCCGGT CTTGTAAACC/DL-H16340 5 0 CCTGAAGTAGGAACC-AGATG (Cheney 1995), (Vila et al. 1999); 2. (CrS, control region) L15995 5 0 CTCCACTATCAGCACCCAAAG/H16 498 5 0 CCTGAAGTAAGAACCAGATG (Pun et al. 2009); 3. (COI, cytochrome oxidase 1) LCO1490 5 0 GGTCAACA AATCATAAAGATATTGG/HCO2198 5 0 TAAACTTCAG GGTGACCAAAAAATCA (Folmer et al. 1994); 4. (CytB