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.

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.