DOI: 10.5061/dryad.37pvmcvnw
title: Particle size influences decay rates of environmental DNA in aquatic systems

description: 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., &lt;1 μm and &gt;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.
description: <strong>Filter DNA extraction and
 quantification</strong> 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 (<em>O. mykis</em>s) 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 &lt;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).