To identify mammalian carnivore species distributed in the Kushiro Wetland, eastern Hokkaido, Japan, we developed molecular-genetic methods for identification of the species from fecal samples collected from the field. Species-specific primers and PCR programs were established for five native and six alien species of carnivores: Martes zibellina, Mustela nivalis, Mustela erminea, Vulpes vulpes, and Nyctereutes procyonoides as native species, and Neovison vison, Martes melampus, Mustela itatsi, Canis familiaris, Felis catus, and Procyon lotor as alien species in Hokkaido. Touchdown PCR, in which the annealing temperature is decreased 1°C every cycle, was more effective for some species from which fecal DNA was not amplified species-specifically with standard PCR programs. Of 405 fecal samples collected from the Kushiro Wetland, the species of origin of 246 samples were successfully identified: 88 samples for N. vison, 140 for M. zibellina, 13 for V. vulpes, four for C. familiaris and one for F. catus. The results show the particular applicability of this method to monitoring M. zibellina and N. vison. In addition, methods to PCR-amplify DNA from two crayfish species (Pacifastacus leniusculus and Cambaroides japonicus) were developed to determine whether the carnivore fecal samples contained detectable DNA from the prey crayfishes. DNA from P. leniusculus was amplified from feces of N. vison identified in the present study, but no DNA from C. japonicus was detected. This indicates that N. vison preys on the alien species P. leniusculus.
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