Estimation accuracy and eDNA assay strategy
In addition to eDNA production source and state, we investigated the
effects of abundance metrics and eDNA assay strategy, including
collection and quantification strategies, on the estimation accuracy of
species abundance via eDNA. First, there was no significant
difference in the estimation accuracy between biomass and number of
individuals. This is consistent with the finding in Yates et al. (2019)
although our study meta-analyzed a larger number of studies. However,
given that wild populations are often composed of individuals with
different age classes and body sizes, selecting either metrics itself
may ultimately hamper the accurate estimation abundance to select either
of abundance metrics. A few studies recently applied allometric scaling
coefficients to the relationship between eDNA quantity and species
abundance, showing higher R2 values for allometrically
scaled mass (ASM) than biomass and individuals (Chin et al., 2021; Yates
et al., 2021a; 2021b).
Second, the estimation accuracy of species abundance was not affected by
the assay strategy (filtration/centrifugation and
species-specific/metabarcoding). In particular, although the variation
of R2 values were higher for metabarcoding than
species-specific assay, it is surprising that metabarcoding assay had
similar abundance estimation accuracy to species-specific assay
(real-time or digital PCR). However, the result must be tempered with an
acknowledgement that (i) the proportion of studies conducting laboratory
experiments were higher for metabarcoding assay, (ii) there was no
result of metabarcoding assay targeting crustaceans eDNA, and (iii) the
number of literatures performing metabarcoding assay was much fewer (9
out of 56) than that of species-specific assay (Tables 1 and S1).
Theoretically, as the number of eDNA reads is estimated from the PCR
final product, metabarcoding assay should not be suitable for the
accurate estimation of species abundance, except for the application of
internal standard DNAs (i.e., known amounts of short artificial DNA
fragments; Ushio et al., 2018). In addition, regardless of the
substantial difference in the volume of water sample, there was no
difference in the estimation accuracy between filtration and
centrifugation. This implies that water sample volume does not have a
substantial effect on the estimation accuracy, partially contradicting
the above hypothesis that larger volume of water samples might allow to
improve the estimation accuracy of species abundance. Further studies
are required how the assay strategy and sampling water volume relate to
the accuracy of eDNA-based species abundance estimation.