A higher DNA damage rate in aqueous eDNA particles suggests intra‐cellular eDNA degradation in cellular environments

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AbstractA higher DNA quality in an environmental DNA (eDNA) sample is crucial for the improved and precise assessment of biodiversity via eDNA analysis. However, the frequency of damage of DNA molecules released from macro‐organisms in aquatic environments remains unclear. As a proof of concept, the rate of DNA damage (λ; per base pair) in an eDNA sample was estimated by measuring the quantity of target DNA with different fragment lengths using quantitative real‐time PCR (qPCR). Rearing water was collected from tanks housing zebrafish (Danio rerio) using filters with different pore sizes ranging from 0.2 to 10 μm, multiple fragment lengths of their mitochondrial eDNA concentrations were quantified and estimated the DNA damage rate based on an exponential relationship between target DNA concentrations and DNA fragment lengths. The DNA damage rate in zebrafish eDNA particles was higher for larger pore size filters, implying that cell membranes do not necessarily protect intra‐cellular DNA from degradation and high activity of intra‐cellular DNases actively degrades intra‐cellular eDNA molecules. Moreover, this finding may partly account for the apparently shorter persistence of larger eDNA particles in aquatic environments and the initial rapid decay of eDNA fragments in a biphasic degradation process. Additional field studies are required to investigate the frequency of DNA damage in different eDNA size fractions and states to facilitate the efficient collection of population genetic information on aquatic macro‐organisms from environmental samples.