DNA-Based Identification Reveals the Limitation of Fecal Size for Distinguishing Sympatric Carnivores on Sado Island, Japan

Accurate identification of sympatric mustelid species is essential for understanding their ecological roles, yet traditional identification methods based on scat morphology often lead to misclassification. In this study, we identified mustelid feces collected on Sado Island, Japan, using DNA analysis and evaluated the reliability of fecal size for discrimination. A total of 343 fecal samples were collected, and DNA was successfully amplified in 341 samples using three commercial extraction kits. Species-specific multiplex PCR targeting the mitochondrial control region identified 331 samples as Japanese marten (Martes melampus) and 10 as Japanese weasel (Mustela itatsi). Maximum fecal diameter was measured in 323 marten and 10 weasel samples. The mean diameter was significantly larger in martens (9.8 ± 2.2 mm) than in weasels (7.3 ± 1.6 mm), with overlapping prediction intervals at both 95% and 99% confidence levels. Had species identification relied only on fecal diameter, most samples classified as weasels would actually have belonged to martens. These results highlight the limitations of fecal size as a discriminatory marker and emphasize the importance of DNA methods in mustelid research. We also show that dry feces—commonly encountered in field conditions—can be reliably used for DNA analysis, offering advantages for future studies.