Biology subjects

Minamoto, T.

Publications and source records attributed to Minamoto, T..

10 recordsLinked to original sources

bioRxiv2026

A Novel eDNA-Based Approach for Hybrid Detection: Implications for Conservation Management

Hybridization between invasive and native species poses a hidden but critical threat to biodiversity. While environmental DNA (eDNA) has revolutionized species monitoring, it has lacked the resolution to detect hybrid individuals. Here, we present the first experimental demonstration of hybrid identification using eDNA. Our method isolates a single cell in the environment (hereafter, eCell) and enables cellular-level analysis using multiplex digital PCR targeting nuclear markers from both parental species. Validation with controlled tank experiments using Oncorhynchus masou masou x Salvelinus leucomaenis leucomaenis hybrid individuals confirmed the methods ability to separately detect hybrid individuals from co-habiting purebred parent individuals. This eCell analysis overcomes the limitations of traditional eDNA methods and offers a scalable, non-invasive tool for detecting cryptic hybridization. By enabling early and accurate detection of hybrid individuals, it supports timely conservation decisions, including management prioritization and the protection of purebred populations. This novel technique bridges a critical gap in conservation genetics and enhances eDNAs utility for biodiversity management in the face of global change.

ecology↗

bioRxiv2025

Feasibility of an eDNA-based educational program for high school students

O_LIEnvironmental education plays a crucial role in raising public awareness of ecosystem conservation. Environmental DNA (eDNA) analysis is a promising environmental education tool because it enables easy and comprehensive biodiversity assessment through simple water sampling. Despite several citizen-based surveys using eDNA analysis, minimal attention has been paid to its educational effectiveness. C_LIO_LIIn this study, we examined the educational feasibility of eDNA analysis by conducting an environmental education program that incorporated eDNA-based local fish surveys in three high schools in Japan. Through detailed questionnaire surveys conducted before and after the program, we verified whether our program increased student interest in biodiversity and ecosystem services. C_LIO_LIIn the program, students proactively identified diverse fish species living in the survey area by simply collecting water. The questionnaire survey results showed that student knowledge of and interest in biodiversity and ecosystem services increased after the program. Students became aware of local fish diversity and expressed their motivation to engage in conservation activities. We confirmed the educational impact of the program not only at the end but also immediately following the water sampling experience. C_LIO_LISynthesis and applications. These results provide the first empirical evidence of the positive effects of eDNA analysis on student environmental awareness and engagement. Our program provides a feasible model for eDNA-based environmental education. Because eDNA analysis can be applied to diverse target taxa that are unique to a given area, similar environmental education programs can be conducted for various citizen groups worldwide. Our research also suggested that eDNA-based environmental education was effective for younger generations with varying levels of interest and experience in nature, showing significant potential for addressing the extinction of natural experiences and motivating citizens to participate in ecosystem conservation activities. C_LI

scientific communication and education↗

bioRxiv2025

Significant improvement of environmental DNA assay by targeting retrotransposon sequences characteristic to Anguilla eels

Environmental DNA (eDNA) analysis faces challenges regarding sensitivity and quantification accuracy, particularly in areas with low target species densities or during seasons when eDNA release decreases. Multi-copy markers such as mitochondrial genomic DNA and ribosomal DNA (rDNA) have been widely used in eDNA analysis to address this issue. However, the copy number of the DNA markers per cell remains a potential bottleneck for eDNA sensitivity. In this study, we aimed to increase the sensitivity of eDNA assays by using retrotransposons, which are abundant in the genome, as novel target markers. We developed an assay targeting UnaSINE1, a short interspersed nucleotide element (SINE) characteristic of Anguilla eels, and compared its sensitivity and accuracy with that of an established mitochondrial 16S rRNA marker. Our results demonstrated that UnaSINE1 was detected at over 100 times the copy number of the mitochondrial marker in both genomic and eDNA samples. In the river surveys, the 16S marker was positive in 32 of the 81 samples, whereas the UnaSINE1 marker was positive in 62 samples, indicating that the use of SINE marker remarkably reduced false negatives. Furthermore, both biological and technical replicates exhibited improved positive consistency and reduced variability in quantification, leading to more robust presence/absence determination and quantitative results. Utilizing retrotransposon sequences as markers requires additional effort for sequence acquisition and organization and may limit taxonomic resolution to the genus level. However, this approach significantly improves sensitivity without increasing the labor or cost of sampling and PCR analysis, making it highly practical for eDNA studies.

ecology↗

bioRxiv2022

Fish environmental DNA in lake sediment overcomes the gap of reconstructing past fauna in lake ecosystems

O_LIUnderwater sediments are a natural archive of biological information. Reconstruction of past fauna has been conducted for various taxonomic groups using morphological remains and DNA derived from these remains. However, information on past occurrences of fish species, the top predator of lake ecosystems, could have been reproduced only in exceptional environments, and past quantitative information on fish, particularly in lake ecosystems, has been a knowledge gap in reconstructing past fauna. Tracking the quantitative fluctuations of fish is essential for reconstructing multiple trophic levels of organisms in lake ecosystems. C_LIO_LITo acquire past quantitative fish information from lake sediments, we collected approximately 30 cm-length of underwater sediments in Lake Biwa. We extracted sedimentary environmental DNA (eDNA) and measured temporal fluctuations in the eDNA concentration of the native and fishery target species Plecoglossus altivelis and Gymnogobius isaza. For P. altivelis, we examined the possibility of tracking quantitative fluctuations by comparing sedimentary eDNA with recorded catch per unit effort (CPUE). C_LIO_LIThe chronology of the sediments allowed us to obtain information on sediments collected in Lake Biwa over the past 100 years. The deepest depths at which sedimentary eDNA was detected were 30 and 13 cm for P. altivelis and G. isaza from the surface, corresponding to approximately 100 and 30 years ago, respectively. In the comparison of sedimentary eDNA concentrations and biomass, we found a significant correlation between the CPUE of P. altivelis and its sedimentary eDNA concentration adjusted to compensate for DNA degradation. Sedimentary eDNA fluctuations were observed in P. altivelis, possibly reflecting the abundance fluctuation due to variations in the main food resources of zooplankton. C_LIO_LIOur findings provide essential pieces for the reconstruction of past fauna of lake ecosystems. The addition of quantitative information on fish species will reach a new phase, for instance, by investigating population shifts or biological interactions in the reconstruction of past fauna in lake ecosystems. C_LI

ecology↗

bioRxiv2021

Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia

Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians ecological traits (e.g., nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding--analysis of extra-organismal DNA released into the environment--allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160-311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata, and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set "Amph16S" had the highest resolution among the tested sets. Finally, we applied Amph16S to actual metabarcoding and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.

ecology↗

bioRxiv2020

Effects of species traits and ecosystem characteristics on species detection by eDNA metabarcoding in lake fish communities

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.

ecology↗

bioRxiv2019

Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

Environmental DNA (eDNA) analysis has recently been used as a new tool for estimating intraspecific diversity. However, whether known haplotypes contained in a sample can be detected correctly using eDNA-based methods has been examined only by an aquarium experiment. Here, we tested whether the haplotypes of Ayu fish (Plecoglossus altivelis altivelis) detected in a capture survey could also be detected from an eDNA sample derived from the field that contained various haplotypes with low concentrations and foreign substances. A water sample and Ayu specimens collected from a river on the same day were analysed by eDNA analysis and Sanger sequencing, respectively. The 10 L water sample was divided into 20 filters for each of which 15 PCR replications were performed. After high-throughput sequencing, denoising was performed using two of the most widely used denoising packages, UNOISE3 and DADA2. Of the 42 haplotypes obtained from the Sanger sequencing of 96 specimens, 38 (UNOISE3) and 41 (DADA2) haplotypes were detected by eDNA analysis. When DADA2 was used, except for one haplotype, haplotypes owned by at least two specimens were detected from all the filter replications. This study showed that the eDNA analysis for evaluating intraspecific genetic diversity provides comparable results for large-scale capture-based conventional methods, suggesting that it could become a more efficient survey method for investigating intraspecific genetic diversity in the field.

molecular biology↗

bioRxiv2018

Estimating fish population abundance by integrating quantitative data on environmental DNA and hydrodynamic modelling

We propose a general framework of abundance estimation based on spatially replicated quantitative measurements of environmental DNA in which production, transport, and degradation of DNA are explicitly accounted for. Application to a Japanese jack mackerel (Trachurus japonicus) population in Maizuru Bay revealed that the method gives an estimate of population abundance comparable to that of a quantitative echo sounder method. These findings indicate the ability of environmental DNA to reliably reflect population abundance of aquatic macroorganisms and may o[ff]er a new avenue for population monitoring based on the fast, cost-e[ff]ective, and non-invasive sampling of genetic information.

ecology↗

bioRxiv2018

Evaluating intraspecific diversity of a fish population using environmental DNA: An approach to distinguish true haplotypes from erroneous sequences

Recent advances in environmental DNA (eDNA) analysis using high-throughput sequencing (HTS) provide a non-invasive way to evaluate the intraspecific genetic diversity of aquatic macroorganisms. However, erroneous sequences present in HTS data can result in false positive haplotypes; therefore, reliable strategies are necessary to eliminate such erroneous sequences when evaluating intraspecific genetic diversity using eDNA metabarcoding. In this study, we propose an approach combining denoising using amplicon sequence variant (ASV) method and the removal of haplotypes with low detection rates. A mixture of rearing water of Ayu (Plecoglossus altivelis altivelis) was used as an eDNA sample. In total, nine haplotypes of Ayu mitochondrial D-loop region were contained in the sample and amplified by two-step tailed PCR. The 15 PCR replicates indexed different tags were prepared from the eDNA sample to compare the detection rates between true haplotypes and false positive haplotypes. All PCR replications were sequenced by HTS, and the total number of detected true haplotypes and false positive haplotypes were compared with and without denoising using the two types of ASV methods, Divisive Amplicon Denoising Algorithm 2 (DADA2) and UNOISE3. The use of both ASV methods considerably reduced the number of false positive haplotypes. Moreover, all true haplotypes were detected in all 15 PCR, whereas false positive haplotypes had detection rates varying from 1/15 to 15/15. Thus, by removing haplotypes with lower detection rates than 15/15, the number of false positive haplotypes were further reduced. The approach proposed in this study successfully eliminated most of false positive haplotypes in the HTS data obtained from eDNA samples, which allowed us to improve the detection accuracy for evaluating intraspecific genetic diversity using eDNA analysis.

molecular biology↗

bioRxiv2017

Quantitative monitoring of multispecies fish environmental DNA using high-throughput sequencing

Effective ecosystem conservation and resource management require quantitative monitoring of biodiversity, including accurate descriptions of species composition and temporal variations of species abundance. Therefore, quantitative monitoring of biodiversity has been performed for many ecosystems, but it is often time- and effort-consuming and costly. Recent studies have shown that environmental DNA (eDNA), which is released to the environment from macro-organisms living in a habitat, contains information about species identity and abundance. Thus, analyzing eDNA would be a promising approach for more efficient biodiversity monitoring. In the present study, we added internal standard DNAs (i.e., known amounts of short DNA fragments from fish species that have never been observed in a sampling area) to eDNA samples, which were collected weekly from a coastal marine ecosystem in Maizuru-Bay, Kyoto, Japan (from April 2015 to March 2016), and performed metabarcoding analysis using Illumina MiSeq to simultaneously identify fish species and quantify fish eDNA copy numbers. A correction equation was obtained for each sample using the relationship between the number of sequence reads and the added amount of the standard DNAs, and this equation was used to estimate the copy numbers from the sequence reads of non-standard fish eDNA. The calculated copy numbers showed significant positive correlation with those determined by quantitative PCR, suggesting that eDNA metabarcoding with standard DNA enabled useful quantification of eDNA. Furthermore, for samples that show a high level of PCR inhibition, our method might allow more accurate quantification than qPCR because the correction equations generated using internal standard DNAs would include the effect of PCR inhibition. A single run of Illumina MiSeq produced > 70 quantitative fish eDNA time series in our study, showing that our method could contribute to more efficient and quantitative monitoring of biodiversity.

ecology↗