Estimation accuracy and eDNA state
We showed that the use of larger pore size filters could improve the accuracy of eDNA-based abundance estimation. According to previous studies, the cellular and molecular structure of larger eDNA particles derived from intra-cellular DNA, such as cells and tissues, is degraded into smaller eDNA particles over time (Jo et al., 2019b). Apparent persistence of such smaller-sized eDNA could thus be longer than that of larger-sized eDNA, and larger eDNA particles collected using larger pore size filters are more likely to be recently released and less degraded than smaller eDNA particles. Such larger-sized ‘fresher’ eDNA is expected to reflect species presence and abundance at a spatiotemporally finer scale, consequently improving the accuracy of eDNA-based abundance estimation. Alternatively, given that filter pore size was positively correlated with the volume of water samples (see Materials and Methods), larger volume of water samples might allow to diminish the heterogeneity of target eDNA quantity across sampling replicates and improve the estimation accuracy of species abundance. Although individual eDNA studies could not clearly infer the effects of filter pore size and water volume on the estimation accuracy (Takahara et al., 2012; Eichmiller et al., 2016), further empirical studies will be required which factors substantially contribute to the relationship between eDNA quantity and species abundance. In any case, our meta-analysis supports the applicability of larger pore size filters for improved abundance estimation via eDNA analysis for the first time.
In contrast, some datasets reported high R2 values using smaller pore size filters (Figure 3), which can collect both larger-sized fresher eDNA and smaller-sized older eDNA. Thus, these studies may have collected a higher proportion of larger-sized eDNA while using smaller pore size filters; in particular, laboratory experiments with high abundances (e.g., Takahara et al., 2012; Doi et al., 2015) may have collected large quantities of large-sized fresh eDNA. Although studies using larger pore size filters (especially >3 µm) were limited in our dataset, further empirical studies targeting larger-sized eDNA particles would conceivably contribute to the robustness of our results and potentially provide a new approach to improve the accuracy of eDNA-based abundance estimation in the field.
PCR amplicon size (i.e., DNA fragment length of target eDNA) was not significantly correlated with R2 values. However, almost all the studies in our meta-analysis targeted eDNA fragments (<500 bp) much shorter than overall genome and thus the effect of PCR amplicon size on the accuracy of eDNA-based abundance estimation may be underestimated. Owing to higher decay rates, detection of longer eDNA fragments may mitigate the effect of degraded eDNA and improve species abundance estimation accuracy (Jo et al., 2017). Nonetheless, Jo et al. (2017) was conducted in a situation where false-positive inferences of target individuals could be obvious (i.e., the effect of fish markets and dead individuals); thus, the performance of longer eDNA fragments for species abundance estimation in more ordinary situations is unknown. In addition, some studies reported that there were no significant differences in eDNA decay rate or persistence time depending on amplicon size (e.g., Ma et al., 2016; Bylemans et al., 2018). Future empirical studies targeting longer eDNA fragments are needed to elucidate the importance of PCR amplicon size on eDNA-based abundance estimation.