Investigating the breeding activity of externally fertilizing salamanders underground based on mitochondrial and nuclear environmental DNA (eDNA) analyses

Abstract

Abstract Spawning and fertilization are critical mating events in externally fertilizing animals and provide information on life histories and the effectiveness of conservation measures. In fish, underwater spawning events are determined by analyzing the ratio of nuclear DNA (nucDNA) to mitochondrial DNA (mtDNA) in environmental DNA (eDNA). However, the feasibility of this approach, particularly over time, has not been verified in other animal taxa, such as amphibians. We sampled the eDNA of the Korean clawed salamander ( Onychodactylus koreanus ), a subterranean externally fertilizing salamander, conducted population surveys over 14 months and performed analyses using cytochrome c oxidase subunit I (COI) and internal transcribed spacer 1 (ITS1) primers with TaqMan probes. Integrated analyses revealed that individual COI and ITS1 eDNA levels and their ratios, which were detected daily and standardized using Z scores, varied over the year and were critical for identifying the breeding periods and seasons. Major O. koreanus breeding activities may occur from late spring to early summer and small-scale, minor activities may occur from late autumn to early winter in mountain habitats. Our results suggest that the integrated analysis of standardized mitochondrial and nuclear eDNA levels, not simply their ratio, is useful for tracking the dynamics of the breeding population of subterranean breeders and complement population surveys.