Spatio-temporal distribution of environmental DNA from amphibian and turtle species in a pond ecosystem

Environmental DNA (eDNA) analysis has emerged as a transformative tool for biodiversity monitoring and conservation. However, uncertainties in the ecological processes of eDNA in aquatic environments decrease the reliability of eDNA-based surveys. Understanding the distribution and persistence patterns of eDNA is essential to effectively correlate eDNA data with species occurrence across spatial and temporal scales. Here, we investigated the spatio-temporal distribution of eDNA from amphibian and turtle species in a pond ecosystem, by establishing controlled eDNA sources from American bullfrogs (Lithobates catesbeianus) and red-eared sliders (Trachemys scripta elegans), and quantifying eDNA concentrations from different water layers and sediment samples using droplet digital PCR. Our results showed that eDNA from both species was highly concentrated within 2–5 m of the biological sources, with dispersal distance independent of the duration of organism' presence in the pond and exhibiting a vertically increasing trend over time. eDNA concentration and persistence varied significantly depending on species and substrate type, with bullfrogs showing notably higher eDNA detectability. The average eDNA concentration in sediment was 1.4 × 10⁴ times higher than in water, and eDNA persisted approximately one week longer. Our findings demonstrate that the strong aggregation patterns of eDNA can provide valuable insights into the spatial distribution of amphibians and turtles in pond systems. eDNA from surface water showed greater timeliness for biodiversity monitoring and aquatic invasive species management. The specificity of target taxa and the temporal complementarity of sedimentary eDNA should be carefully considered in future eDNA sampling designs.