The conservation of an endangered horseshoe crab, Tachypleus tridentatus, has been increasingly empha-sized worldwide. Although monitoring of T. tridentatus reproduction is effective for its conservation, the conventional monitoring system for spawning has several drawbacks. Thus, our study aimed to examine the environmental DNA (i.e., eDNA) approach to assess T. tridentatus spawning events. Six survey sites were set at the sandy beaches of the Sone tidal flat, Japan. Visual observations of the spawning events, i.e., the presence/absence of adult pairs of T. triden-tatus or that of foams, occurring because of their spawning behavior, and water sampling for eDNA assay were con-ducted 10 times at each site between 2019 and 2020. The spawning events were observed at a total 16 sites during the surveys, and the estimated numbers of T. tridentatus adult pairs were 1–5 per site. T. tridentatus DNA was detected in 15 of the 16 samples at the sites where the spawning events were observed and in 13 of 44 samples where the spawning events were not observed. The estimated number of pairs and the eDNA concentration showed a significantly positive correlation. Moreover, the presence/absence of the spawning events was distinguished with excellent accuracy based on the eDNA concentration. Therefore, our results showed that the spawning events of the species and the eDNA concentration were closely related. In conclusion, we expect that the population of T. tridentatus in spawning grounds can be evaluated more complementarily by combining the conventional observation method with the eDNA approach.
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