Particle size distribution and optimal capture of aqueous macrobial eDNA

O_LIDetecting aquatic macroorganisms with environmental DNA (eDNA) 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.\nC_LIO_LIThe size of aquatic particles provides clues about their origin, state, and fate. We used sequential filtration size fractionation to measure, for the first time, 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.\nC_LIO_LIIn a lake and a pond, we found Carp eDNA in particles from >180 to <0.2 m, but it was most abundant from 1-10 m. Total eDNA was most abundant below 0.2 m and SPM was most abundant above 100 m. SPM was [≤]0.1% total eDNA, and total eDNA was [≤]0.0004% Carp eDNA. 0.2 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 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.\nC_LIO_LIOur results suggest that aqueous macrobial eDNA predominantly exists inside mitochondria or cells, and that settling plays an important role in its fate. For optimal eDNA capture, we recommend 0.2 m filtration or a combination of larger pore size and water volume that exceeds the 0.2 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.\nC_LI