DOI: 10.5061/dryad.2bvq83c2d
title: Warming increases environmental DNA (eDNA) removal rates in flowing waters

description: The use of environmental DNA (eDNA) for aquatic conservation is emerging,
 but its value is limited by our understanding of how environmental factors
 like temperature impact eDNA persistence. Although elevated temperatures
 are known to increase eDNA decay in lakes and ponds, no studies have
 experimentally explored the effect of temperature on eDNA fate in flowing
 waters where physical removal could obscure the effect of temperature on
 decay rates. We compared eDNA removal rates in n=12 indoor, recirculating
 mesocosms under varying water temperatures (20, 23, 26°C) and found that,
 for small eDNA particles (0.2-1.0μm), removal rates were higher at the
 warmest temperature (Tukey’s post hoc, p ≤0.03) while removal rates were
 consistent across temperatures for larger eDNA particles (&gt;1.0μm,
 Tukey’s, p&lt;0.05). Consequently, smaller eDNA particles were removed
 faster than larger particles (&gt;1.0μm) at 26 and 23°C (Tukey’s,
 p&lt;0.001) compared to 20°C (Tukey’s, p=0.01), resulting in an
 increase in the proportion of the eDNA sample made up of small particles
 with downstream transport for the two warmer temperatures (beta linear
 model, p&lt;0.001). This suggests eDNA removal in streams reflects a
 complex interplay between physical trapping and microbial degradation
 influenced by temperature. Consequently, differences in temperature
 between geographic locations, seasons, and climates could impact the fate
 and interpretation of eDNA, even in flowing waters where physical removal
 contributes substantially to eDNA fate.
description: # Warming increases environmental DNA (eDNA) removal rates in flowing
 waters Dataset DOI: [10.5061/dryad.2bvq83c2d](10.5061/dryad.2bvq83c2d) ##
 Description of the data and file structure *Principal Investigator Contact
 Information* Name: Elise D. Snyder Institution: University of Illinois at
 Urbana-Champaign Email:
 [edsnyde2@illinois.edu](mailto:edsnyde2@illinois.edu) ***Data overview:***
 This dataset contains the data required to replicate analyses in Snyder et
 al. “Warming increases environmental DNA (eDNA) removal rates in flowing
 waters” (accepted for publication), wherein we explored two questions: 1)
 Do higher water temperatures accelerate eDNA removal from the water column
 in flowing waters? and 2) Do higher water temperatures impact eDNA removal
 differently based on particle size? To assess eDNA removal under a range
 of water temperatures (20, 23, and 26°C), we used n=12 recirculating,
 stream mesocosms, which we conditioned at their respective temperatures
 for 23 days. We then conducted pulse releases of eDNA from Common Carp
 (*Cyprinus carpio*) and Steelhead Trout (*Oncorhynchus mykiss*), measured
 the decline in eDNA concentration across 24 hours, and used vacuum
 filtration through 1.0 and 0.2-μm polycarbonate membrane filters to
 partition different sizes of eDNA particles. These data include the eDNA
 concentration measurements used to calculate eDNA removal rates for each
 mesocosm and the accompanying environmental metrics measured in each
 mesocosm during the experiment. We found that eDNA removal from the water
 column in stream mesocosms was faster under higher temperatures,
 particularly for small particles. We observed that, across our temperature
 treatments over 24 hours, eDNA removal patterns differed between larger
 (1.0 μm) and smaller (0.2 μm) eDNA particle sizes. Removal rates for
 smaller eDNA particles were faster in warmer mesocosms (23 and 26°C),
 while the two sizes displayed similar removal rates in the 20°C mesocosms.
 As a result, the proportion of smaller-sized particles decreased over time
 under elevated temperatures, with larger particles persisting for longer
 in the water column. ***Data analysis methods:*** To estimate removal
 rates for total eDNA and for each size class, we calculated eDNA mass in
 each mesocosm for both Common Carp and Steelhead Trout using the eDNA
 concentrations and water volume at each sampling time, and derived the
 eDNA removal (k; hr−1) from the decline in natural-log transformed mass in
 each mesocosm. We also converted k to depositional velocity (Vdep; mm s-1)
 as Vdep = (k*d) where *v* is mesocosm velocity and d is depth. We employed
 linear mixed models (LMMs) to compare eDNA k across temperature treatments
 for both total eDNA (i.e. the sum of both size classes), and for each size
 class, using the “lmer” function in R with the following equations: with
 the following equations:` ln(eDNA Mass) ~ Time:Temperature +
 (1+Time|Mesocosm:Target) and ln(eDNA Mass) ~ Time:Temperature x Size +
 (1+Time|Mesocosm:Target)`. The first model regressed ln(total eDNA mass)
 with the interaction of time and temperature and included random
 intercepts and slopes (in time), with each eDNA release (mesocosm and
 target) as the random unit. The second model employed the same random
 structure, but included size as an additional interacting fixed effect.
 Additionally, we ran the same models a second time with average measured
 temperature during the 24-hour release period (as opposed to temperature
 as a categorical variable) to estimate the numeric relationship between
 temperature and time.  We used the “emtrends” function within the R
 package “emmeans” for post-hoc pairwise comparisons among treatments and
 eDNA sizes. To assess whether eDNA PSD changed over time at our three
 temperatures, we used a Beta family generalized linear model (BLM) within
 the R package “betareg” with the following equation: `eDNA Proportion ~
 Time`. Finally, we compared environmental metrics with each other and with
 eDNA removal rates, using pairwise Pearson correlation tests within the R
 package “Hmisc.” For strong correlations between an environmental metric
 and eDNA removal rates (p&lt;0.05), we modeled the relationship via an LMM
 containing a random effect for mesocosm to account for the lack of
 independence between measurements for the two targets within a given
 mesocosm. We conducted all statistical analyses in R version 4.3.0. ###
 Files and variables #### File:
 Data\_and\_metadata\_for\_covariables\_and\_eDNA\_removal\_rates\_for\_Warming\_increases\_environmental\_DNA\_(eDNA)\_removal\_rates\_in\_flowing\_waters.xlsx **Description:** A .xlsx file containing the data and metadata for the eDNA concentration measurements for the experiment described in the manuscript. This data was generated using digital droplet PCR and includes the droplet counts and thresholding information for each eDNA sample, as well as eDNA concentration and mass calculated from these measurements, the water volumes used to calculate the concentration/mass, and the identifying information for each sample. #### File: Data\_and\_metadata\_for\_ddPCR\_eDNA\_concentrations\_for\_Warming\_increases\_environmental\_DNA\_(eDNA)\_removal\_rates\_in\_flowing\_waters.xlsx **Description:** A .xlsx file with the data and metadata for the covariables and eDNA removal rates (calculated using the above dataset) for the experiment described in the manuscript. These environmental covariables measured in each mesocosm include temperature, community respiration (mg/m^2/h), gross primary productivity (mg/m^2/h), chlorophyll-a (μg/cm^2/h), water column ammonium concentration (μg/L), water column soluble reactive phosphorus concentration (μg/L), water column nitrate concentration (μg/L), water column turbidity (NTU), water column pH, and water velocity (m/s). This data was derived from samples and measurements collected by the authors.