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Although environmental DNA (eDNA) metabarcoding is an exceptionally useful and powerful tool for monitoring biodiversity, little is known about whether the traits of organisms and their ecosystem characteristics affect eDNA metabarcoding performance. Nationwide surveys can provide more detailed insights, yet such studies have rarely been conducted.In order to evaluate eDNA metabarcoding performance in broad‐scale monitoring, we examined the effects of species ecological/biological traits and ecosystem characteristics on species detection rates and the implications for community analysis. In addition, we tested the effects of sample mixing and transportation methods, including cooling and freezing, on eDNA metabarcoding. On a nationwide scale, we conducted eDNA metabarcoding for fish communities in 18 Japanese lakes.By comparing species records, we observed that certain traits, including body size, body shape, saltwater tolerance and habitat preference, influenced eDNA detection. In addition, the proportion of species detected decreased significantly with an increase in lake surface area owing to ecosystem size effect on species detection.We conclude that species traits, including habitat preference, body size and ecosystem size, should be considered when assessing the eDNA metabarcoding performance in broad‐scale monitoring. [ABSTRACT FROM AUTHOR]

In recent years, biodiversity loss has become one of the most serious environmental issues worldwide, especially in aquatic ecosystems. To avoid diversity loss, it is necessary to monitor biological communities, and environmental DNA (eDNA) metabarcoding has been developed as a rapid, noninvasive, and cost-effective method for aquatic biodiversity monitoring. Although this method has been applied to various environments and taxa, a detailed assessment of the efficient sampling methods for monitoring is still required. In this study, we explored eDNA metabarcoding sampling methods for fish at a single site to maximize the number of detected species using realistic effort in a natural, small river. We considered the following three parameters: sample type (water or sediment), sample position at a site (right and left shore and center of the river), and water volume (10–4000 mL). The results suggested that the number of detected species from sedimentary eDNA was equivalent to that from aqueous eDNA, although the species composition was different. The number of detected species could be saturated by collecting a 1000 mL water sample, regardless of sampling position within a survey site. However, sedimentary eDNA showed a spatially heterogeneous species composition between sampling positions within a survey site despite the short distance (5 m) between positions, without apparent differences in physical properties such as velocity and sediment particle distribution. By completing eDNA biodiversity monitoring of fish with 1000 mL water samples across the whole river, we detected more fish species than in previous traditional surveys conducted at the same sites. Thus, the aqueous eDNA metabarcoding method is as efficient as traditional surveys, while sedimentary eDNA metabarcoding could complement the results of aqueous eDNA metabarcoding. [ABSTRACT FROM AUTHOR]