The Ayu Plecoglossus altivelis altivelis is an amphidromous fish that is not only the most important commercial fishery species in Japanese rivers but also has a high economic value in recreational fishing. However, the degradation of its spawning grounds has caused a decrease in its abundance. In this study, we used environmental DNA (eDNA) to monitor the Ayu in the Takatsu River in Japan to (1) identify the spawning season in three known spawning grounds, (2) clarify changes in the main spawning grounds during the spawning season, and (3) discover unknown spawning grounds. We collected 1 L of the surface river water at three known spawning grounds nine times in 2018 and seven times in 2019 in the lower reaches of the Takatsu River. We also collected samples from seven unknown sites in 2018. The water samples were filtered through glass fiber filters. Total eDNA was extracted from each filtered sample and a Real-time quantitative PCR was performed with the specific primers and probe for Ayu. The results of the eDNA analyses showed that (1) the spawning season was in November in 2018 and in September in 2019. (2) One site was used as a spawning ground in both the early and the late spawning season, depending on the year. At the second site, the frequency of use changed year by year. The third site was the main spawning ground in the middle to late spawning season every year. From these results, we elucidated that some spawning grounds are used regularly every year, while the use of others varies year by year. (3) In five of the seven unknown sites, the nighttime eDNA concentrations were high at least once during the four surveys, suggesting that these sites may have functioned as spawning grounds. In particular, one site could be an important new spawning ground.

Information on α‐ (local), β‐ (between habitats), and γ‐ (regional) diversity is fundamental to understanding biodiversity as well as the function and stability of community dynamics. Methods like environmental DNA (eDNA) metabarcoding are currently considered useful to investigate biodiversity. We compared the performance of eDNA metabarcoding with visual and capture surveys for estimating α‐ and γ‐diversity of river fish communities, and nestedness and turnover in particular. In five rivers across west Japan, by comparison to visual/capture surveys, eDNA metabarcoding detected more species in the study sites (i.e. α‐diversity). Consequently, the overall number of species in the region (i.e. γ‐diversity) was higher. In particular, the species found by visual/capture surveys were encompassed by those detected by eDNA metabarcoding. Estimates of community diversity within rivers differed between survey methods. Although we found that the methods show similar levels of community nestedness and turnover within the rivers, visual/capture surveys showed more distinct community differences from upstream to downstream. Our results suggest that eDNA metabarcoding may be a suitable method for community assemblage analysis, especially for understanding regional community patterns, for fish monitoring in rivers.

Information on alpha (local), beta (between habitats), and gamma (regional) diversity is fundamental to understanding biodiversity as well as the function and stability of community dynamics. The methods like environmental DNA (eDNA) metabarcoding are currently considered useful to investigate biodiversity.We compared the performance of eDNA metabarcoding with visual and capture surveys in estimating alpha/gamma diversity and the variation of the community assemblages of river fish communities, particularly considering community nestedness and turnover.In five rivers across west Japan, with comparing to visual/capture surveys, eDNA metabarcoding detected more species in the study sites, consequently the overall number of species in the region (i.e., gamma diversity) was higher. In particular, the species found by visual/capture surveys were encompassed by those by eDNA metabarcoding.With analyzing the community assemblages between the rivers, we showed the different results between the both methods. While, in the same river, the nestedness and species turnover changing from upstream to downstream did not significantly differ between the both methods. Our results suggest that eDNA metabarcoding may be suitable method, especially for understanding regional community patterns, for fish monitoring in rivers.

The Ryukyu ayu Plecoglossus altivelis ryukyuensis is an endangered amphidromous fish that inhabits rivers in the Ryukyu Archipelago (Japan). Populations of the species have declined dramatically. Consequently, the Ryukyu ayu has been registered as a natural monument in Japan and monitoring surveys with direct catching are restricted legally. This restriction, unfortunately, makes monitoring of population abundances difficult and creates a barrier to both advancing understanding of the species’ status and the development of appropriate conservation plans. We developed a non-invasive monitoring methodology using eDNA analyses. We designed a specific quantitative PCR assay for the Ryukyu ayu using the mitochondrial ND4 region. Using this primer/probe set, we conducted eDNA analyses in three rivers on Amami-Ohshima Island. The DNA fragments were amplified from the eDNA extracted from natural water in each river. The numbers of DNA fragments detected were positively correlated with individual counts of fish obtained by visual snorkelling surveys. Our method does not contravene restrictions and facilitates abundance monitoring of this endangered fish species.