Environmental DNA metabarcoding fish community plots for different filter types and treatments. (A) Kernel density plots showing distribution of species richness across eDNA samples collected from different ponds and with different filtration and filter preservation strategies. The dashed blue lines indicate the mean species richness of individual eDNA samples (n = 8), the dotted black lines indicate the species richness of mixed samples (pooled aliquots of individual samples) at each sampling occasion and filtration/preservation strategy. The x axes represent the fish species richness in each pond surveyed (New Lake = 12 species; Middle Lake = 9 species) (B) PCoA plot showing distances from centroids of filter types (MCE and Sterivex [STX]; ANOVA F = 0.8521, p = 0.3611) and treatments (buffers and freezing; ANOVA F = 0.6495, p = 0.6305). Distances from centroids were calculated upon a dissimilarity matrix (Bray-Curtis) of fish species read counts. (C) Species accumulation curves for filter replicates of Sterivex filters preserved with buffers (top) and MCE filters with freezing preservation (bottom). In both figures, golden curves are calculated based on the sum of species when filter replicates/treatments of the same filter type are combined. 95% confidence intervals refer to the golden curves and boxplots of these curves show distribution of species diversity as inferred from the method “random”, which add sites in random order and was used for the SACs. Asterisks represent outliers.