No abstract

Freshwater biodiversity has been severely threatened in recent years, and to conserve endangered species, their distribution and breeding habitats need to be clarified. However, identifying breeding sites in a large area is generally difficult. Here, by combining the emerging environmental DNA (eDNA) analysis with subsequent traditional collection surveys, we successfully identified a breeding habitat for the critically endangered freshwater fish Acheilognathus typus in the mainstream of Omono River in Akita Prefecture, Japan, which is one of the original habitats of this species. Based on DNA cytochrome B sequences of A. typus and closely related species, we developed species-specific primers and a probe that were used in real-time PCR for detecting A. typus eDNA. After verifying the specificity and applicability of the primers and probe on water samples from known artificial habitats, eDNA analysis was applied to water samples collected at 99 sites along Omono River. Two of the samples were positive for A. typus eDNA, and thus, small fixed nets and bottle traps were set out to capture adult fish and verify egg deposition in bivalves (the preferred breeding substrate for A. typus) in the corresponding regions. Mature female and male individuals and bivalves containing laid eggs were collected at one of the eDNA-positive sites. This was the first record of adult A. typus in Omono River in 11 years. This study highlights the value of eDNA analysis to guide conventional monitoring surveys and shows that combining both methods can provide important information on breeding sites that is essential for species' conservation.

Molecular methods, including those for environmental DNA (eDNA), provide essential information for biological and conservation sciences. Molecular measurements are often performed in the laboratory, which limits their scope, especially for rapid on-site analysis. eDNA methods for species detection provide essential information for the management and conservation of species and communities in various environments. We have developed an innovative novel method for on-site eDNA measurements using an ultrarapid mobile PCR platform. We tested the ability of our method to detect the distribution of silver carp, Hypophthalmichthys molitrix, in rivers and lakes.Our method reduced the measurement time to 30 min and provided high detectability of aquatic organisms compared to national observation surveys that use multiple fishing nets and laboratory extraction/detection using a benchtop qPCR platform. Our on-site eDNA method can be immediately applied to various taxa and environments.

Although environmental DNA (eDNA) metabarcoding is acknowledged to be an exceptionally useful and powerful tool for monitoring surveys, it has limited applicability, particularly for nationwide surveys. To evaluate the performance of eDNA metabarcoding in broad-scale monitoring, we examined the effects of species ecological/biological traits and ecosystem characteristics on species detection rates and the consequences for community analysis. We conducted eDNA metabarcoding on fish communities in 18 Japanese lakes on a country-wide scale. By comparing species records, we found that certain species traits, including body size, body shape, saltwater tolerance, and habitat preferences, influenced eDNA detection. We also found that the proportion of species detected decreased significantly with an increase in lake surface area, owing to an ecosystem-size effect on species detection. We conclude that species traits, including habitat preferences and body size, and ecosystem size should be taken into consideration when assessing the performance of eDNA metabarcoding in broad-scale monitoring.

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.