Environmental DNA (eDNA) refers to the genetic material that can be extracted from bulk environmental samples such as soil, water, and even air. The rapidly expanding study of eDNA has generated unprecedented ability to detect species and conduct genetic analyses for conservation, management, and research, particularly in scenarios where collection of whole organisms is impractical or impossible. While the number of studies demonstrating successful eDNA detection has increased rapidly in recent years, less research has explored the ''ecology'' of eDNA-myriad interactions between extraorganismal genetic material and its environment-and its influence on eDNA detection, quantification, analysis, and application to conservation and research. Here, we outline a framework for understanding the ecology of eDNA, including the origin, state, transport, and fate of extraorganismal genetic material. Using this framework, we review and synthesize the findings of eDNA studies from diverse environments, taxa, and fields of study to highlight important concepts and knowledge gaps in eDNA study and application. Additionally, we identify frontiers of conservation-focused eDNA application where we see the most potential for growth, including the use of eDNA for estimating population size, population genetic and genomic analyses via eDNA, inclusion of other indicator biomolecules such as environmental RNA or proteins, automated sample collection and analysis, and consideration of an expanded array of creative environmental samples. We discuss how a more complete understanding of the ecology of eDNA is integral to advancing these frontiers and maximizing the potential of future eDNA applications in conservation and research.

Environmental DNA (eDNA) surveillance holds great promise for improving species conservation and management. However, few studies have investigated eDNA dynamics under natural conditions, and interpretations of eDNA surveillance results are clouded by uncertainties about eDNA degradation. We conducted a literature review to assess current understanding of eDNA degradation in aquatic systems and an experiment exploring how environmental conditions can influence eDNA degradation. Previous studies have reported macrobial eDNA persistence ranging from less than 1 day to over 2 weeks, with no attempts to quantify factors affecting degradation. Using a SYBR Green quantitative PCR assay to observe Common Carp ( Cyprinus carpio ) eDNA degradation in laboratory mesocosms, our rate of Common Carp eDNA detection decreased over time. Common Carp eDNA concentration followed a pattern of exponential decay, and observed decay rates exceeded previously published values for aquatic macrobial eDNA. Contrary to our expectations, eDNA degradation rate declined as biochemical oxygen demand, chlorophyll, and total eDNA (i.e., from any organism) concentration increased. Our results help explain the widely divergent, previously published estimates for eDNA degradation. Measurements of local environmental conditions, consideration of environmental influence on eDNA detection, and quantification of local eDNA degradation rates will help interpret future eDNA surveillance results.

ABSTRACT1. Detecting aquatic macroorganisms with environmental DNA (eDNA) is a new survey method with broad applicability. However, the origin, state, and fate of aqueous macrobial eDNAwhich 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, 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. 3.In 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. 4.Our 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 . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx

Summary1. 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 <0Á2 lm, but it was most abundant from 1 to 10 lm. Total eDNA was most abundant below 0Á2 lm, and SPM was most abundant above 100 lm. SPM consisted of ≤0Á1% total eDNA, and total eDNA consisted of ≤0Á0004% Carp eDNA. 0Á2 lm filtration maximized Carp eDNA capture (85% AE 6%) while minimizing total (i.e. non-target) eDNA capture (48% AE 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 0Á2 lm filtration or a combination of larger pore size and water volume that exceeds the 0Á2 lm 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.

Three mantras often guide species and ecosystem management: (i) for preventing invasions by harmful species, ‘early detection and rapid response’; (ii) for conserving imperilled native species, ‘protection of biodiversity hotspots’; and (iii) for assessing biosecurity risk, ‘an ounce of prevention equals a pound of cure.’ However, these and other management goals are elusive when traditional sampling tools (e.g. netting, traps, electrofishing, visual surveys) have poor detection limits, are too slow or are not feasible. One visionary solution is to use an organism’s DNA in the environment (eDNA), rather than the organism itself, as the target of detection. In this issue of Molecular Ecology, Thomsen et al. (2012) provide new evidence demonstrating the feasibility of this approach, showing that eDNA is an accurate indicator of the presence of an impressively diverse set of six aquatic or amphibious taxa including invertebrates, amphibians, a fish and a mammal in a wide range of freshwater habitats. They are also the first to demonstrate that the abundance of eDNA, as measured by qPCR, correlates positively with population abundance estimated with traditional tools. Finally, Thomsen et al. (2012) demonstrate that next-generation sequencing of eDNA can quantify species richness. Overall, Thomsen et al. (2012) provide a revolutionary roadmap for using eDNA for detection of species, estimates of relative abundance and quantification of biodiversity.