DRP006517
Environmental DNA collected from snow tracks useful for classification of mammalian species
Noninvasive genetic samples like scat, hair, and urine are commonly used in field surveys of terrestrial mammals. However, the collection of naturally deposited DNA samples in the field is time- and labor intensive, particularly for large- and middle-sized mammals like carnivores, which exhibit low densities and elusive behaviors. As snow tracks are the most frequently encountered natural sign of terrestrial mammals in the field in winter, we investigated several methods to recover environmental DNA (eDNA) from snow tracks. We employed both metabarcoding and Sanger sequencing analyses, in combination with universal primers for mammals (MiMammal-U primers) and target-specific primers (designed for Martes in this study), to amplify mitochondrial DNA loci. Snow samples of four Martes melampus tracks (Mm1/Mm1', Mm2/Mm2', Mm3, and Mm4), one Cervus nippon track (Cn1/Cn1'), one Vulpes vulpes track (Vv1/Vv1'), and the track on an unclassified carnivore species (Csp1) were collected in a snowfall zone in Japan in February of 2018. Five footprints were used for each sample. Regarding metabarcoding analyses with MiMammal-U primers, the sequences of three carnivore species (M. melampus, V. vulpes, and C. lupus) and a deer (C. nippon) were obtained from their respective snow tracks. However, classifiable sequences were not obtained for all samples, and several sequences were contaminated with Homo sapiens and Mus musculus DNA. Snow tracks other than that of M. melampus were classified at the species level using Sanger sequencing, but analyses using MiMammal-U primers were not efficient for the classification of M. melampus tracks, possibly because of low eDNA concentrations or the presence of contaminant DNA from non-target species. Contrarily, eDNA of M. melampus snow tracks, especially those found in shaded places, were successfully sequenced using Martes-specific primers. Metabarcoding analyses with universal primers can, thus, be used to classify the snow tracks of multiple sympatric mammals. Target-specific primers could be advantageous in reducing the influences of contaminants, and they can be used to investigate genetic variations within a particular species or phylogenetic group. Snow track surveys in combination with eDNA techniques may dramatically improve the efficiency of monitoring and conservation of mammals, which requires genetic data for immediate actions and long-term strategies.