Particle size distribution and optimal capture of aqueous macrobial eDNA

1. Using environmental DNA (eDNA) to detect aquatic macroorganisms is a new survey method with broad applicability. However, the origin, state and fate of aqueous macrobial eDNA - which collectively determine how well eDNA can serve as a proxy for directly observing organisms and how eDNA should be captured, purified and assayed - are poorly understood. 2. The size of aquatic particles provides clues about their origin, state and fate. We used sequential filtration size fractionation to measure the particle size distribution (PSD) of macrobial eDNA, specifically Common Carp (hereafter referred to as Carp) eDNA. We compared it to the PSDs of total eDNA (from all organisms) and suspended particle matter (SPM). We quantified Carp mitochondrial eDNA using a custom qPCR assay, total eDNA with fluorometry and SPM with gravimetric analysis. 3. In a lake and a pond, we found Carp eDNA in particles from > 180 to < 0.2 mu m, but it was most abundant from 1 to 10 mu m. Total eDNA was most abundant below 0.2 mu m, and SPM was most abundant above 100 mu m. SPM consisted of <= 0.1% total eDNA, and total eDNA consisted of <= 0.0004% Carp eDNA. 0.2 mu m filtration maximized Carp eDNA capture (85% +/- 6%) while minimizing total (i.e. non-target) eDNA capture (48% +/- 3%), but filter clogging limited this pore size to a sample volume <250 mL. To mitigate this limitation, we estimated a continuous PSD model for Carp eDNA and derived an equation for calculating isoclines of pore size and water volume that yield equivalent amounts of Carp eDNA. 4. Our results suggest that aqueous macrobial eDNA predominantly exists inside mitochondria or cells, and that settling may therefore play an important role in its fate. For optimal eDNA capture, we recommend 0.2 mu m filtration or a combination of larger pore size and water volume that exceeds the 0.2 mu m isocline. In situ filtration of large volumes could maximize detection probability when surveying large habitats for rare organisms. Our method for eDNA particle size analysis enables future research to compare the PSDs of eDNA from other organisms and environments, and to easily apply them for ecological monitoring.