Determination of a target image of freshwater fish composition in a mitigation project using eDNA metabarcoding methods

Short-term target species for mitigation should be realistically and effectively selected for restoration within a few years. Current short-term target species selection is based on spatial distribution data of past and present biota in the neighborhood based on capture surveys. However, current methods have the following problems: If historical spatial distribution data are used, the risk of including large areas, where target species cannot be dispersed, exists, as well as the risk of including extinct species. If recent spatial distribution data are used, the survey area may be narrowed due to time constraints, thereby increasing the possibility of missing species that should be selected as the target species. We examined the possibility of using the environmental DNA (eDNA) metabarcoding method, which can obtain spatial distribution data quickly, sensitively, and extensively, as a new short-term target species selection method. The study site was a restoration mitigation area (Kaihotsu-Kasumi) in the Shigenobu River system, Ehime Prefecture, Japan, where freshwater fish target species were selected based on capture surveys and past literature information. Furthermore, eDNA surveys were conducted at 17 sites in the Kaihotsu-Kasumi and its inflow and outflow rivers in 2020, and metabarcoding analysis using MiFish primers was conducted to determine the current freshwater fish fauna at each site. The visualization of the fish community composition obtained from the eDNA survey using NMDS showed that the Kaihotsu-Kasumi and surrounding watersheds were significantly divided into three clusters: upper reaches, middle and lower reaches, and estuarine reaches. Kaihotsu-Kasumi was included in Cluster 2, comprising the middle and lower reaches of the inflow and outflow rivers. Twenty-six species (including five non-native species) were detected in Cluster 2. Twenty-one fish species, excluding non-native species, were considered suitable as short-term target species as taxa with high potential for dispersal into Kaihotsu-Kasumi. The selection of short-term target species using the eDNA metabarcoding method covered all short- and medium-term target species (14 species) selected based on the capture surveys, and seven additional species were also proposed. The study also showed that intraspecific lineages of Misgurnus anguillicaudatus (Clades A and B), which were missed by the capture surveys, can be identified. These results indicate that the eDNA metabarcoding method can provide more comprehensive and realistic short-term target species selection than capture surveys, providing higher resolution monitoring through intraspecific lineage detection. ### Competing Interest Statement The authors have declared no competing interest.