Environmental DNA unveils deep phylogeographic structure of a freshwater fish.

Phylogeography bears an important part in ecology and evolution. However, current phylogeographic studies are largely constrained by limited numbers of individual samples. Using an environmental DNA (eDNA) assay for phylogeographic analyses, this study provides detailed information regarding the history of Siberian stone loach Barbatula toni, a primary freshwater fish across the whole range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river water samples, we detected eDNA from B. toni in 189 rivers. A total of 51 samples, representing the entire island, were then selected from the B. toni eDNA-positive sample set for the subsequent analyses. To elucidate the phylogeographic structure of B. toni, newly developed eDNA metabarcoding primers (Barba-cytb-F/R) were applied to these samples, specifically targeting their haplotypic variation in cytochrome b. After a bioinformatic processing to mitigate haplotypic false positives, a total of 50 eDNA haplotypes were identified. Two regionally restricted, genetically distinct lineages of the species were revealed as a result of phylogeographic analyses on the haplotypes and tissue-derived DNA from B. toni. According to a molecular clock analysis, they have been genetically isolated for at least 1.5 million years, suggesting their ancient origin and colonisation of Hokkaido, presumably in the glacial periods. These results demonstrate how freshwater fishes can alter their distributions over evolutionary timescales and how eDNA assay can deepen our understanding of phylogeography.

Efficient PCR Amplification Protocol of Nuclear Microsatellites for Exuviae-Derived DNA of Cicada, Yezoterpnosia nigricosta.

Although insect exuviae-based genetics is challenging, it can be a valuable method for obtaining reliable DNA resources by non-invasive sampling. This approach is especially effective when the target species is endangered/endemic or when sampling the adult is difficult. One example is cicadas, which during molt leave their exoskeletons on tree trunks, making them easily collectable. While cicada exuviae-derived DNA has previously been employed for mitochondrial DNA sequencing, this study aimed to develop a reliable method for the PCR amplification of nuclear microsatellite loci from cicada exuviae derived DNA for application in molecular ecology, conservation and population genetics. Five different PCR amplification protocols were performed, and the fragment patterns compared with those obtained using DNA extracted from adult individuals. Moreover, the relationship between the freshness of the exuviae and genotyping success was evaluated. TaKaRa LA Taq provided the best performance in the PCR amplification of DNA isolated from cicada exuviae and the electropherogram showed a clear fragment pattern that was equivalent to that obtained from the DNA extracted from the adult individual. This result suggests that cicada exuviae-derived DNA can be amplified by PCR and that multiple independent loci of nuclear DNA microsatellite markers can be easily genotyped. This study demonstrates that fresh cicada exuviae provide high quality DNA, which can be used for microsatellite genotyping. The methods developed in this study are applicable not only for cicada but other insect species for which exuviae are available. Thus, this study can make a significant contribution to insect sciences.