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Environmental DNA (eDNA) analysis has seen rapid development in the last decade, as a novel biodiversity monitoring method. Previous studies have evaluated optimal strategies, at several experimental steps of eDNA metabarcoding, for the simultaneous detection of fish species. However, optimal sampling strategies, especially the season and the location of water sampling, have not been evaluated thoroughly. To identify optimal sampling seasons and locations, we performed sampling monthly or at two‐monthly intervals throughout the year in three dam reservoirs. Water samples were collected from 15 and nine locations in the Miharu and Okawa dam reservoirs in Fukushima Prefecture, respectively, and five locations in the Sugo dam reservoir in Hyogo Prefecture, Japan. One liter of water was filtered with glass‐fiber filters, and eDNA was extracted. By performing MiFish metabarcoding, we successfully detected a total of 21, 24, and 22 fish species in Miharu, Okawa, and Sugo reservoirs, respectively. From these results, the eDNA metabarcoding method had a similar level of performance compared to conventional long‐term data. Furthermore, it was found to be effective in evaluating entire fish communities. The number of species detected by eDNA survey peaked in May in Miharu and Okawa reservoirs, and in March and June in Sugo reservoir, which corresponds with the breeding seasons of many of fish species inhabiting the reservoirs. In addition, the number of detected species was significantly higher in shore, compared to offshore samples in the Miharu reservoir, and a similar tendency was found in the other two reservoirs. Based on these results, we can conclude that the efficiency of species detection by eDNA metabarcoding could be maximized by collecting water from shore locations during the breeding seasons of the inhabiting fish. These results will contribute in the determination of sampling seasons and locations for fish fauna survey via eDNA metabarcoding, in the future., Number of detected species by eDNA metabarcoding for (a) Miharu dam reservoir, (b) Okawa dam reservoir, and (c) Sugo dam reservoir. The number of species detected by eDNA survey peaked in May in Miharu and Okawa reservoirs, and in March and June in Sugo reservoir, which corresponds with the breeding seasons of many of the fish species inhabiting the reservoirs.

In Japan, farm ponds retain water throughout the year while rice fields are continuously inundated for no more than 2 months, usually from spring to early summer. Although the zooplankton fauna in artificial water bodies would be expected to vary according to differences in inundation periods, scientific confirmation of this relation is largely lacking. Due to its ubiquity, the cladoceran Moina is a suitable target for studying habitat-related differences in the planktonic fauna, but its taxonomy remains unresolved, making morphological identifications potentially uncertain. We thus applied integrative taxonomy with both morphological and genetic evaluations for reliable species delimitation to Moina samples collected primarily in Japan (with smaller collections from Taiwan). This approach increased the alpha diversity of Moina species in Japan from three (in previous studies) to seven. It also revealed different habitat preferences among Moina species, with the smaller species being distributed mostly in farm ponds (followed by natural lakes), and the larger species mostly in rice fields. We argue that the phenological match/mismatch with inundation period of rice fields was a major factor for this strong trend of spatial species turnover, with differing degrees of fish predation pressure among the habitat types being another factor.

Dam‐mediated biological invasions are a serious problem all over the world. Once established in reservoirs, the invasive species have catastrophic impacts on the river ecosystems downstream, and thus, rapid monitoring of invasive species is an urgent issue for the effective removal of them and the conservation management of native ecosystems. Here, we verified the utility of environmental DNA (eDNA) analysis as a tool to effectively monitor three invasive fish species (bluegill, largemouth bass, and smallmouth bass) in reservoirs using multiplex real‐time PCR. First, to determine the optimal sampling location and season to detect eDNA from these species, we analyzed the eDNA in water samples from shore and offshore sites in three reservoirs all year around. We found that eDNA detection rates either did not differ between sampling locations or were higher for shore than offshore sites. In addition, eDNA detection rates were higher in spring (breeding season of target species) and/or summer than winter. Second, we extensively surveyed the distribution of the three species in 30 reservoirs in Japan using eDNA analysis. Consequently, a single eDNA‐based surveillance in summer allowed to match approximately 90% of the presence/absence of the invasive fish species known from 27 yr of administrative capture‐based surveillances. Given these results, we recommend collecting the replicated water samples from shore sites in summer or the breeding season for the effective detection of invasive fish eDNA in reservoirs. Our eDNA assays with multiplex real‐time PCR enable the rapid and sensitive monitoring of invasive fish distribution in reservoirs.