Identification of the sibling species Apodemus sylvaticus and Apodemus flavicollis (Rodentia, Muridae)—Comparison of molecular methods

The yellow-necked field mouse, Apodemus flavicollis (Melchior, 1934), and the long-tailed field mouse, Apodemus sylvaticus (Linnaeus, 1758) are morphologically similar species, with largely overlapping geographical areas and almost equal ecological requirements. This makes reliable species diagnosis based on external characters a real challenge. When advanced multivariate methods of skull and/or dental morphometrics are employed, specimens can be successfully distinguished in most cases, but not all, making single specimen identification impossible. Application of C-band karyotyping clearly distinguishes between these two species. However, it can be applied only to live animals. Several molecular methods have also allowed successful species diagnosis, but their universality has not been tested. Here we compare the diagnostic power of three molecular approaches and their applicability to populations from a species wide distributional area, as well as their simplicity of use, both for live animals and for samples stored in alcohol for a considerable period, without DNA sequencing. A total of 200 tissue samples from Morocco, France, Belgium, Serbia, Romania, Greece, Russia, Turkey and Kazakhstan, were analyzed by AP-PCR, ISSR-PCR and species-specific primers from the mitochondrial cytochrome b gene. The first two methods gave clear species-specific DNA profiles in complete agreement with previous C-band results. However, only partial agreement was observed between the third, species-specific primer method and the other two approaches and C-banding, as there were 4.5–10.5% false positive results. Hence we propose AP-PCR and ISSR-PCR techniques with particular chosen primers for quick and reliable diagnosis of these two species. A large number of samples can be assayed in a short period, with minimal cost and effort. Moreover, these techniques can be applied to a single specimen.