Environmental DNA (eDNA) analysis is a powerful tool for remote detection of target organisms. However, obtaining quantitative and longitudinal information from eDNA data is challenging, requiring a deep understanding of eDNA ecology. Notably, if the various size components of eDNA decay at different rates, and we can separate them within a sample, their changing proportions could be used to obtain longitudinal dynamics information on species. To test this possibility, we conducted an aquatic mesocosm experiment in which we separated fish-derived eDNA components using sequential filtration to evaluate the decay rate and changing proportion of various eDNA particle sizes over time. We then fit four alternative mathematical decay models to the data, building towards a predictive framework to interpret eDNA data from various particle sizes. We found that medium-sized particles (1-10 μm) decayed more slowly than other size classes (i.e., <1 μm and >10 μm), and thus made up an increasing proportion of eDNA particles over time. We also observed distinct eDNA particle size distribution (PSD) between our Common carp and Rainbow trout samples, suggesting that target-specific assays are required to determine starting eDNA PSDs. Additionally, we found evidence that different sizes of eDNA particles do not decay independently, with particle size conversion replenishing smaller particles over time. Nonetheless, a parsimonious mathematical model where particle sizes decay independently best explained the data. Given these results, we suggest a framework to discern target distance and abundance with eDNA data by applying sequential filtration, which theoretically has both metabarcoding and single-target applications.

Filter DNA extraction and quantification

DNA was extracted from preserved filters using Phenol-Chloroform extraction (Sambrook & Russel, 2006), following the protocol available in the Supplement, modified from Turner et al., (2014). We resuspended the extracted DNA in 100 μl of TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0), and stored it at -20°C until genetic analysis. We included an extraction negative control of RO water for every 24 samples.

We quantified target eDNA using a duplex digital droplet PCR assay (ddPCR) and species-specific primer-probe pairs developed by Takahara et al. (2012) for Common carp (C. carpio) and by Duda et al. (2021) for Rainbow trout (O. mykiss) that both target the mitochondrial gene Cytb. These primers and probes had been previously applied for qPCR only and in separate reactions, so we combined them with distinct labeling to validate a duplex ddPCR assay. The reactions were composed of 11 μl of ddPCR Supermix for probes (Bio-Rad, Inc., Hercules, CA), 250 nM of each probe, 900 nM of each primer (IDT, Coralville, IA), and 4 μl of target DNA in 22 μl total reaction volume. We generated droplets using the AutoDG Automated Droplet Generator (Bio-Rad, Inc.) and performed the PCR on a C1000 Thermocycler (Bio-Rad, Inc.) with the following cycles: 4°C for 10 minutes for droplet stabilization, 95°C for 10 minutes for initial denaturing and activation, 40 cycles of 94°C for 30 seconds and 55°C for 60 seconds for amplification, and 98 °C for 10 minutes followed by 4°C hold. We performed triplicate ddPCR reactions for each sample and droplets were read on a QX200 Droplet reader (Bio-Rad, Inc.). Each plate contained a positive control for each target and six wells of no target controls; replicates with <10,000 generated droplets were not considered on downstream analysis and were repeated when possible. 

We conducted the DNA extraction and PCR preparation and reactions in two separate buildings and laboratories, and we followed procedures to avoid sample cross contamination (further described in the extraction protocol in the SI).