Can we sort states of environmental DNA (eDNA) from a single sample?

Environmental DNA (eDNA) once shed can exist in numerous states with varying behaviors including degradation rates and transport potential. In this study we consider three states of eDNA: 1) a membrane-bound state referring to DNA enveloped in a cellular or organellar membrane, 2) a dissolved state defined as the extracellular DNA molecule in the environment without any interaction with other particles, and 3) an adsorbed state defined as extracellular DNA adsorbed to a particle surface in the environment. Capturing, isolating, and analyzing a target state of eDNA provides utility for better interpretation of eDNA degradation rates and transport potential. While methods for separating different states of DNA have been developed, they remain poorly evaluated due to the lack of state-controlled experimentation. We evaluated the methods for separating states of eDNA from a single sample by spiking DNA from three different species to represent the three states of eDNA as state-specific controls. We used chicken DNA to represent the dissolved state, cultured mouse cells for the membrane-bound state, and salmon DNA adsorbed to clay particles as the adsorbed state. We performed the separation in three water matrices, two environmental and one synthetic, spiked with the three eDNA states. The membrane-bound state was the only state that was isolated with minimal contamination from non-target states. The membrane-bound state also had the highest recovery (54.11 {+/-} 19.24 %), followed by the adsorbed state (5.08 {+/-} 2.28 %), and the dissolved state had the lowest total recovery (2.21 {+/-} 2.36 %). This study highlights the potential to sort the states of eDNA from a single sample and independently analyze them for more informed biodiversity assessments. However, further method development is needed to improve recovery and reduce cross-contamination.