Aquarium experiment
The linear relationships between zebrafish eDNA concentrations and fish
abundance were observed in all filter pore sizes and DNA fragment
lengths (Figure 1). However, there were substantial variations in theR2 values and slopes in the linear regressions.
The R2 values were significantly lower for a 10
µm pore size filter than the other pore size filters and the values were
higher by targeting shorter eDNA fragments (Figure 2a). A linear model
showed a significantly positive effect of a 3 µm pore size filter
relative to a 10 µm pore size filter (P < 0.05) and a
negative effect of DNA marker length (P < 0.01) on
Fisher’s z-transformed R2 values (Table 1).
Furthermore, the slopes tended to be larger for 3 and 0.2 µm pore size
filters than for 10 and 0.8 µm pore size filters, regardless of marker
length (Figure 2b). A linear mixed model showed significant positive
effects in the interactions of 3 and 0.2 µm pore size filters with the
number of fish individuals (both P < 0.01) on the
slopes. Concurrently, no significant interaction between zebrafish
abundance and DNA fragment length was observed (Table 2). The overallR2 values and PCR efficiencies of the standard
curves in qPCR were 0.996 ± 0.002 and 93.814% ± 6.174%, respectively
(detailed information on each marker can be seen in Table S1). Although
one of blank samples showed PCR amplification, its calculated
concentration was 0.4 copies per PCR reaction (Table S1); the potential
contamination during water filtration was considered negligible.
Amplification was not observed in any of the filtration or PCR negative
controls throughout the experiment.