Environmental DNA (eDNA) analysis is a promising tool for non-disruptive and cost-efficient estimation of species abundance. However, its practical applicability in natural environments is limited owing to a potential gap between eDNA concentration and species abundance in the field. Although the importance of accounting for eDNA dynamics, such as transport and degradation, has been discussed, the influence of eDNA characteristics, including production source and cellular/molecular state, on the accuracy of eDNA-based abundance estimation was entirely overlooked. We conducted meta-analyses using 44 of previous eDNA studies and investigated the relationships between the accuracy (R2) of eDNA-based abundance estimation and eDNA characteristics. First, we found that estimated R2 values were significantly lower for crustaceans and mussels than fish. This finding suggests that less frequent eDNA production of these taxa owing to their external morphology and physiology may impede accurate estimation of their abundance via eDNA. Moreover, linear mixed modeling showed that, despite high variances, R2 values were positively correlated with filter pore size, indicating that selective collection of larger-sized eDNA, which is typically fresher, could improve the estimation accuracy of species abundance. Although our collected dataset was somewhat biased to the studies targeting specific taxa, our findings shed a new light on the importance of what characteristics of eDNA should be targeted for more accurate estimation of species abundance. Further empirical studies are required to validate our findings and fully elucidate the relationship between eDNA characteristics and eDNA-based abundance estimation.

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.

Knowledge of the persistent state of environmental DNA (eDNA) particles in water and modeling its particle size distribution (PSD) is crucial to refine the eDNA sampling strategy. Previous studies measured eDNA concentrations at different size fractions to infer eDNA PSDs, whereas such discrete PSDs greatly depend on filter pore sizes used and may potentially bias our understanding of eDNA states. Here, I evaluated the Weibull distribution model as a tool for parameterizing eDNA PSDs. First, of the selected models developed for PSDs, the Weibull model was fitted most accurately to the eDNA PSD. Second, the Weibull parameters significantly varied depending on target genetic regions (mitochondrial/nuclear), time passages, and species, which also substantially changed the eDNA capture efficiency at a given filter pore size. According to the results, the higher detectability of multi-copy nuclear eDNA than mitochondrial one could partly be accounted for differences in their PSDs, a larger pore size filter could be suitable to detect the eDNA releases associating incidental events, and the optimum filter pore size for sufficient eDNA detection could be different among the taxa. Although further consideration is needed, this study provided the groundwork for optimizing the strategy of aqueous eDNA sampling, which contributes to efficient biodiversity conservation and ecosystem management.

Reliable abundance estimation is a primary challenge in environmental DNA (eDNA) analysis, which has been addressed by considering the effects of eDNA transport and degradation. However, these eDNA spatial dynamics depend on the cellular and molecular structure of eDNA, with its persistence state (particle size and DNA fragment length) being essential for improved abundance estimation. This existing knowledge gap is bridged by utilizing datasets obtained from two types of aquarium experiments (targeting zebrafish [Danio rerio] and Japanese jack mackerel [Trachurus japonicus]) and comparing the relationships between eDNA concentration and species abundance among different eDNA size fractions and target marker lengths. We reared the fish in experimental tanks with different individual numbers or biomass densities, filtered rearing water using different pore size filters, and quantified eDNA concentrations targeting different fragment lengths or genetic regions. Consequently, both experiments showed that the accuracy and sensitivity in abundance estimation were improved (i.e., R2 values and slopes of linear regressions increased) when targeting eDNA at the 3–10-µm size fraction. On the other hand, targeting eDNA at the >10 µm size fraction yielded a lower R2 value. This result indicates that an “appropriately” larger eDNA particle is vital for improving abundance estimation accuracy and sensitivity. Conversely, the target marker length negatively affected the R2 value. This study proposes that the relationship between eDNA concentration and species abundance relies on the complex interactions between the particle size, persistence, and spatial heterogeneity of eDNA in water.

Environmental DNA (eDNA) analysis is a promising tool for non-disruptive and cost-efficient estimation of species abundance. However, its practical applicability in natural environments is limited because it is unclear whether eDNA concentrations actually represent species abundance in the field. Although the importance of accounting for eDNA dynamics, such as transport and degradation, has been discussed, the influences of eDNA characteristics, including production source and state, and methodology, including collection and quantification strategy and abundance metrics, on the accuracy of eDNA-based abundance estimation were entirely overlooked. We conducted a meta-analysis using 56 previous eDNA literature and investigated the relationships between the accuracy (R2) of eDNA-based abundance estimation and eDNA characteristics and methodology. Our meta-regression analysis found that R2 values were significantly lower for crustaceans than fish, suggesting that less frequent eDNA production owing to their external morphology and physiology may impede accurate estimation of their abundance via eDNA. Moreover, R2 values were positively associated with filter pore size, indicating that selective collection of larger-sized eDNA, which is typically fresher, could improve the estimation accuracy of species abundance. Furthermore, R2 values were significantly lower for natural than laboratory conditions, while there was no difference in the estimation accuracy among natural environments. Our findings shed a new light on the importance of what characteristics of eDNA should be targeted for more accurate estimation of species abundance. Further empirical studies are required to validate our findings and fully elucidate the relationship between eDNA characteristics and eDNA-based abundance estimation.

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.