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.