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In freshwater ecosystems, invasive salmonid fishes can have a significant impact on native fish species. Detecting the invasion and its negative effects is critical for the conservation of native fish communities. We examined the species composition and seasonal changes in the freshwater fish community, including salmonids, on the Kamikawa Plain, Hokkaido Island, Japan, using environmental DNA (eDNA) metabarcoding. We detected 23 fish species in 176 samples collected from 16 sites over 12 months (October 2018 - August 2019). Between 11 and 20 species were detected at each site, including five native salmonids (Oncorhynchus masou, Oncorhynchus keta, Parahucho perryi, Salvelinus leucomaenis leucomaenis and Salvelinus malma krascheninnikova). The invasive alien rainbow trout Oncorhynchus mykiss was detected at all 16 sites and it was the most commonly detected salmonid. Although we found no obvious competitive exclusion of native salmonids by rainbow trout in the study area, the invasive species occurred more often and at more sites than any of the natives. We also determined the occurrence and seasonal changes in the fish community, classified as native salmonids, invasive rainbow trout, Cypriniformes and other benthic fishes. There were fewer species overall in winter, but the sites with higher species richness in winter were on the lower reaches of the river. In addition, we detected domestic invaders, such as the topmouth gudgeon, Pseudorasbora parva, although they were less prevalent than rainbow trout. These results show the effectiveness of eDNA metabarcoding, which can be used for surveying species richness at an ecosystem scale. In particular, the detection of the early stages of establishment and spread of invasive species can be achieved by eDNA monitoring. [ABSTRACT FROM AUTHOR]

Dolly Varden (Salvelinus malma) and Whitespotted Char (Salvelinus leucomaenis) are representative native fish of the family Salmonidae that inhabit the upper reaches of rivers on Hokkaido Island, Japan. They are threatened by the invasive Rainbow Trout (Oncorhynchus mykiss). In this study, environmental DNA (eDNA) real‐time polymerase chain reaction (PCR) assays to detect these three salmonids were developed and used to clarify the distribution pattern of these fish. A specificity test for each assay was conducted using DNA extracted from both target and closely related fish, and the specificity of each assay was confirmed. Then, we carried out eDNA surveys in two mountainous rivers around Mt. Daisetsu in winter, when snow depth was maximized. In the winter surveys, eDNA of all three species were successfully detected from river water samples, including under‐ice water samples. The results of eDNA detection corresponded with the results of an earlier distribution survey performed with Japanese‐style fly‐fishing and lure‐fishing. These results suggested that the eDNA assays developed in this study are applicable for inter‐seasonal surveys for these species. In this study, environmental DNA (eDNA) real‐time PCR assays to detect three salmonids were developed. We carried out eDNA surveys in two mountainous rivers around Mt. Daisetsu in winter, when snow depth was maximized. eDNA of all three species were successfully detected from river water samples, including under‐ice water samples, suggesting that the eDNA assays are applicable for inter‐seasonal surveys for these species. [ABSTRACT FROM AUTHOR]

In freshwater ecosystems, invasive salmonid fishes can have a significant impact on native fish species. Detecting the invasion and its negative effects is critical for the conservation of native fish communities. We examined the species composition and seasonal changes in the freshwater fish community, including salmonids, on the Kamikawa Plain, Hokkaido Island, Japan, using environmental DNA (eDNA) metabarcoding. We detected 23 fish species in 176 samples collected from 16 sites over 12 months (October 2018 - August 2019). Between 11 and 20 species were detected at each site, including five native salmonids ( Oncorhynchus masou , Oncorhynchus keta , Parahucho perryi , Salvelinus leucomaenis leucomaenis and Salvelinus malma krascheninnikova ). The invasive alien rainbow trout Oncorhynchus mykiss was detected at all 16 sites and it was the most commonly detected salmonid. Although we found no obvious competitive exclusion of native salmonids by rainbow trout in the study area, the invasive species occurred more often and at more sites than any of the natives. We also determined the occurrence and seasonal changes in the fish community, classified as native salmonids, invasive rainbow trout, Cypriniformes and other benthic fishes. There were fewer species overall in winter, but the sites with higher species richness in winter were on the lower reaches of the river. In addition, we detected domestic invaders, such as the topmouth gudgeon, Pseudorasbora parva , although they were less prevalent than rainbow trout. These results show the effectiveness of eDNA metabarcoding, which can be used for surveying species richness at an ecosystem scale. In particular, the detection of the early stages of establishment and spread of invasive species can be achieved by eDNA monitoring., (Akio Imamura, Kana Hayami, Masayuki K. Sakata, Toshifumi Minamoto.)

Although populations of anguillid eels have declined remarkably in recent decades, monitoring data on the spatial and temporal variation in their dynamics are often limited, particularly for tropical eel species. As there are often sympatries of multiple eel species in tropical rivers, identifying eel species based solely on morphological characteristics is challenging. Basin-scale surveys were conducted in rivers of southern Japan and northern Taiwan to investigate (1) whether the spatial distribution, abundance, and biomass of the tropical eel species, the giant mottled eel ( Anguilla marmorata ), can be monitored in rivers by comparing the results obtained from environmental DNA (eDNA) analysis with data from electrofishing and (2) the riverine distribution of the sympatric A. marmorata and the temperate eel species, the Japanese eel ( Anguilla japonica ), in this region using eDNA analysis. Although we found an much lower abundance of A. marmorata in the study region, we identified the eDNA of the species from all of the study sites (21 sites) where it was collected by electrofishing, in addition to 22 further study sites where it was not collected directly. This indicates that eDNA analysis has a greater sensitivity for detecting A. marmorata , making it a powerful tool for monitoring the spatial distribution of the species in rivers. We found a significant positive relationship between eDNA concentration and both the abundance and biomass of A. marmorata , and eDNA concentration seemed to better reflect the abundance of the species than did biomass. eDNA of both A. japonica and A. marmorata was identified from almost all rivers, indicating the sympatry of these species in this region, although the degree of sympatry differed between rivers. Though the eDNA concentration of A. japonica decreased significantly with increasing distance from the river mouth, no significant relationship was found for A. marmorata . This study is the first to demonstrate the potential usefulness of eDNA analysis for estimating the spatial distribution, abundance, and biomass of tropical eels in rivers and to further apply this method to investigate sympatry among anguillid species. eDNA analysis can help in obtaining data on the population dynamics of tropical eels, providing invaluable information for managing these species.

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.