1. The use of environmental DNA (eDNA) as a new method of ecological monitoring is widely applied. Although eDNA can provide important information on the distribution and biomass of particular taxa, an organism’s DNA sequences remain unaltered throughout its existence, which complicates identifying crucial events, including reproduction, with high accuracy. We thus examined DNA methylation as a novel source of information from eDNA, considering that methylation patterns of eggs and sperm released during reproduction differ from those of somatic tissues. 2. Despite its potential applications, little is known about eDNA methylation, including its stability and methods for detection and quantification. Therefore, we conducted tank experiments and performed methylation analysis targeting 18S rDNA through bisulfite amplicon sequencing. 3. Methylation of eDNA was not affected by degradation and was equivalent to the rate of genomic DNA from somatic tissues. Unmethylated DNA, which is abundant in the ovary, was detected in eDNA during reproductive activity of fish. 4. These results indicate that eDNA methylation is a stable signal reflecting genomic methylation and demonstrate that germ cell-specific methylation patterns can be used as markers for detecting reproductive activity.

Environmental DNA (eDNA) analysis allows non-invasive and cost-effective monitoring of species distribution and composition in aquatic ecosystems. Benzalkonium chloride (BAC) treatment is an inexpensive and simple method for preserving macrobial eDNA in water samples, which is suitable for maximizing both the number of sampling replicates and water volume. However, its preservation performance has been evaluated in a limited manner by species-specific assays, targeting short fragments of mitochondrial DNA in freshwater and brackish ecosystems. Here, we examined the performance of BAC in preserving eDNA in seawater samples, targeting different fragment lengths of mitochondrial and nuclear eDNA, and community information inferred by eDNA metabarcoding. First, we quantified the time-series changes of Japanese jack mackerel (Trachurus japonicus) eDNA concentrations in experimental tanks and inshore seawater to compare the yields and decay rates of eDNA between BAC treatments. As a result, BAC addition increased the eDNA yields at the start of the experiment and substantially suppressed the initial phase of rapid degradation but not the subsequent phase of slower degradation. In addition, we performed eDNA metabarcoding targeting fish community, showing that BAC addition suppressed the decrease in species richness, where the number of fish species hardly varied throughout the day. Findings of the present and previous studies indicate high versatility of BAC in preserving qualitative (species richness) and quantitative (copy number) information on aqueous eDNA under various environmental conditions. BAC should therefore be used to minimize the false-negative detection of eDNA, regardless of target genetic regions, fragment sizes, environmental conditions, and detection strategies.

Changes in the thermal structure of lake ecosystems have been documented as a precursor of climate change, but the dynamics of biomass distribution, which fundamentally determines species conservation, have been less studied. An interdisciplinary approach was used to demonstrate the influence of climate-driven changes on the biomass distribution of two species (Gymnogobius isaza and Palaemon paucidens) in Lake Biwa. In field surveys in 2016–2017 (full water circulation) and 2019 (partial water circulation), environmental DNA concentrations of these species were used as proxies for biomass to measure 43 and 47 sites sampled at the lake bottom, respectively. A structural equation model was used to estimate the correlation between species biomass and environmental parameters. The species-environment relationship was applied to species biomass distributions under existing and future environments calculated by the model. Differences between the species were found in their responses to climate change. The biomass distribution of G. isaza will benefit in the future if full water circulation occurs, although it appears to be independent of water circulation at present. Partial water circulation enlarges the distribution area of P. paucidens, but its biomass will be low in the future, regardless of the extent of water circulation. These findings advance the knowledge of how species respond to climate change and suggest special attention should be given to species such as P. paucidens, which is currently abundant but sensitive to climate change. Furthermore, they emphasize the potential application of interdisciplinary methodologies for improved species conservation.

Understanding the processes of environmental DNA (eDNA) persistence and degradation is essential to determine the spatiotemporal scale of eDNA signals and accurately estimate species distribution. The effects of environmental factors on eDNA persistence have previously been examined; however, the influence of the physiochemical and molecular states of eDNA on its persistence is not completely understood. Here, we performed meta-analyses including 26 previously published papers on the estimation of first-order eDNA decay rate constants, and assessed the effects of filter pore size, DNA fragment size, target gene, and environmental conditions on eDNA decay rates. Almost all supported models included the interactions between the filter pore size and water temperature, between the target gene and water temperature, and between the target gene and water source, implying the influence of complex interactions between the eDNA state and environmental conditions on eDNA persistence. These findings were generally consistent with the results of a re-analysis of a previous tank experiment which measured the time-series changes in marine fish eDNA concentrations in multiple size fractions after fish removal. Our results suggest that the mechanism of eDNA persistence and degradation cannot be fully understood without knowing not only environmental factors but also cellular and molecular states of eDNA in water. Further verification of the relationship between eDNA state and persistence is required by obtaining more information on eDNA persistence in various experimental and environmental conditions, which will enhance our knowledge on eDNA persistence and support our findings.

Analysis of biodiversity in natural environments based on environmental DNA (eDNA) has been applied to a wide range of ecosystems and species. The combination of high-throughput sequencing technologies and eDNA analysis is a powerful tool that enables comprehensive non-invasive monitoring of species present in the environment. Quantitative data of the eDNA from each species is essential for understanding species abundance but until recently required individual assays targeting each species. Recently developed quantitative sequencing (qSeq) allows simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. The eDNA extracted from aquaria with five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus) across 4 days was quantified by microfluidic digital PCR using a TaqMan probe and qSeq. The eDNA abundance quantified by qSeq was consistent with dPCR for each fish species at each sampling time. However, the relative abundances of sequences obtained from high throughput sequencing did not follow the same trend as the quantitative analyses, probably due to different PCR amplification efficiencies for each species. The correlation coefficients between qSeq and dPCR were 1.052, 1.074, and 1.114 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA. The application of qSeq to eDNA of other species will provide comprehensive quantitative data that could deepen our knowledge of natural ecosystems.