Verification of the breeding periods of two clawed salamander species (Onychodactylus koreanus and O. sillanus) using environmental DNA analysis and field surveys

Determining the breeding frequencies and breeding periods of species that spend part or all of their lives underground is challenging because of limited population surveys. Environmental DNA (eDNA) analysis can be performed to address this issue. eDNA analysis and population surveys were combined to verify and compare the breeding frequencies and breeding periods of Onychodactylus koreanus and O. sillanus in mountain valley streams, which are their typical habitats in the Republic of Korea, between April 2024 and May 2025. Both species externally fertilize their eggs. Adult O. koreanus appeared at the sites during two periods, from April to June and from October to early December, whereas adult O. sillanus was not detected despite similar survey efforts. eDNA concentrations detected in the upper, middle, and lower streams differed; however, the major eDNA detection patterns were largely consistent. In O. koreanus , two major eDNA detections occurred in early May and early December, whereas in O. sillanus , eDNA detections occurred between January and June. These results suggest that the two species have different breeding frequencies and breeding periods. However, whether these differences are species- or habitat-specific needs to be further explored.