Environmental DNA phylogeography: Successful reconstruction of phylogeographic patterns of multiple fish species from cups of water.

Phylogeography is an integrative field of science linking micro- and macro-evolutionary processes, contributing to the inference of vicariance, dispersal, speciation, and other population-level processes. Phylogeographic surveys usually require considerable effort and time to obtain numerous samples from many geographical sites covering the distribution range of target species; this associated high cost limits their application. Recently, environmental DNA (eDNA) analysis has been useful not only for detecting species but also for assessing genetic diversity; hence, there has been growing interest in its application to phylogeography. As the first step of eDNA-based phylogeography, we examined (1) data screening procedures suitable for phylogeography and (2) whether the results obtained from eDNA analysis accurately reflect known phylogeographic patterns. For these purposes, we performed quantitative eDNA metabarcoding using group-specific primer sets in five freshwater fish species belonging to two taxonomic groups from a total of 94 water samples collected from western Japan. As a result, three-step data screening based on the DNA copy number of each haplotype detected successfully eliminated suspected false positive haplotypes. Furthermore, eDNA analysis could almost perfectly reconstruct the phylogenetic and phylogeographic patterns obtained for all target species with the conventional method. Despite existing limitations and future challenges, eDNA-based phylogeography can significantly reduce survey time and effort and is applicable for simultaneous analysis of multiple species in single water samples. eDNA-based phylogeography has the potential to revolutionize phylogeography.

Quantitative environmental DNA metabarcoding shows high potential as a novel approach to quantitatively assess fish community.

The simultaneous conservation of species richness and evenness is important to effectively reduce biodiversity loss and keep ecosystem health. Environmental DNA (eDNA) metabarcoding has been used as a powerful tool for identifying community composition, but it does not necessarily provide quantitative information due to several methodological limitations. Thus, the quantification of eDNA through metabarcoding is an important frontier of eDNA-based biomonitoring. Particularly, the qMiSeq approach has recently been developed as a quantitative metabarcoding method and has attracted much attention due to its usefulness. The aim here was to evaluate the performance of the qMiSeq approach as a quantitative monitoring tool for fish communities by comparing the quantified eDNA concentrations with the results of fish capture surveys. The eDNA water sampling and the capture surveys using the electrical shocker were conducted at a total of 21 sites in four rivers in Japan. As a result, we found significant positive relationships between the eDNA concentrations of each species quantified by qMiSeq and both the abundance and biomass of each captured taxon at each site. Furthermore, for seven out of eleven taxa, a significant positive relationship was observed between quantified DNA concentrations by sample and the abundance and/or biomass. In total, our results demonstrated that eDNA metabarcoding with the qMiSeq approach is a suitable and useful tool for quantitative monitoring of fish communities. Due to the simplicity of the eDNA analysis, the eDNA metabarcoding with qMiSeq approach would promote further growth of quantitative monitoring of biodiversity.

Illumina iSeq 100 and MiSeq exhibit similar performance in freshwater fish environmental DNA metabarcoding.

Environmental DNA (eDNA) analysis is a method of detecting DNA from environmental samples and is used as a biomonitoring tool. In recent studies, Illumina MiSeq has been the most extensively used tool for eDNA metabarcoding. The Illumina iSeq 100 (hereafter, iSeq), one of the high-throughput sequencers (HTS), has a relatively simple workflow and is potentially more affordable than other HTS. However, its utility in eDNA metabarcoding has still not been investigated. In the present study, we applied fish eDNA metabarcoding to 40 water samples from river and lake ecosystems to assess the difference in species detectability and composition between iSeq and MiSeq. To check differences in sequence quality and errors, we also assessed differences in read changes between the two HTS. There were similar sequence qualities between iSeq and MiSeq. Significant difference was observed in the number of species between two HTS, but no difference was observed in species composition between the two HTS. Additionally, the species compositions in common with the conventional method were the same between the two HTS. According to the results, using the same amplicon library for sequencing, two HTS would exhibit a similar performance of fish species detection using eDNA metabarcoding.

Using environmental DNA analyses to assess the occurrence and abundance of the endangered amphidromous fish Plecoglossus altivelis ryukyuensis.

The Ryukyu ayu Plecoglossus altivelis ryukyuensis is an endangered amphidromous fish that inhabits rivers in the Ryukyu Archipelago (Japan). Populations of the species have declined dramatically. Consequently, the Ryukyu ayu has been registered as a natural monument in Japan and monitoring surveys with direct catching are restricted legally. This restriction, unfortunately, makes monitoring of population abundances difficult and creates a barrier to both advancing understanding of the species' status and the development of appropriate conservation plans. We developed a non-invasive monitoring methodology using eDNA analyses. We designed a specific quantitative PCR assay for the Ryukyu ayu using the mitochondrial ND4 region. Using this primer/probe set, we conducted eDNA analyses in three rivers on Amami-Ohshima Island. The DNA fragments were amplified from the eDNA extracted from natural water in each river. The numbers of DNA fragments detected were positively correlated with individual counts of fish obtained by visual snorkelling surveys. Our method does not contravene restrictions and facilitates abundance monitoring of this endangered fish species.

Sediment and nutrients transport in watershed and their impact on coastal environment.

Sediment and nutrients yields especially from farmlands were studied in a watershed in Ishigaki island, Okinawa, Japan. The transport processes of these materials in rivers, mangrove, lagoon and coastal zones were studied by using various observation methods including stable isotope analysis. They were simulated by using a WEPP model which was modified to be applicable to such small islands by identifying several factors from the observations. The model predicts that a proper combination of civil engineering countermeasure and change of farming method can reduce the sediment yield from the watershed by 74%. Observations of water quality and coral recruitment test in Nagura bay indicate that the water is eutrophicated and the corals cannot grow for a long time. Based on these observations, a quantitative target of the reduction of sediment and nutrients yield in watershed can be decided rationally.