Elise D. Snyder

Sampling environmental nucleic acids (eDNA and eRNA, collectively eNA) in aquatic systems facilitates the efficient exploration of the distribution of aquatic species. Since eRNA is inherently less stable than eDNA, eRNA may yield more spatiotemporally precise information about organisms, but this volatility also poses unique methodological challenges, making it unclear whether eDNA protocols are suitable for eRNA samples. To address this gap, we conducted experiments using naturally occurring eRNA and eDNA from the mitochondrial cytochrome b (CytB) gene from largemouth bass ( Micropterus salmoides ) along with DNA and RNA extracted from bluegill tissue ( Lepomis megalotis ). We first conducted a degradation experiment to test the efficacy of refrigeration for eRNA preservation (specifically mitochondrial messenger eRNA, emRNA), given its widespread use for eDNA samples. We then compared a suite of approaches for the long‐term preservation and co‐extraction of emRNA and eDNA in the laboratory. Despite the potential that the instability of eRNA might render eDNA approaches unsuitable, we found that methods developed for eDNA samples can be readily repurposed for emRNA. Contrary to our hypothesis that refrigeration of water samples might not efficiently preserve emRNA in the short term, samples in a cooler on ice showed no decrease in concentration or detectability over 25 h for largemouth bass (Tukey's, all p > 0.3). Filtration time (i.e., time spent at room temperature) also slightly reduced eRNA yields, with concentration decreasing as filtration time increased for largemouth bass emRNA (LM, p = 0.05), highlighting the importance of cold conditions throughout processing. Additionally, as we hypothesized, a low‐cost chloroform extraction protocol yielded equivalent emRNA quantities to more expensive commercial kits for both largemouth bass and bluegill (Tukey's, p between methods > 0.8), in some cases even yielding higher concentrations than these methods. Overall, these results demonstrate the possible practical feasibility of eRNA sampling and provide guidance for future work on eRNA in natural systems.

Understanding what controls eDNA removal from the water column remains a critical challenge for interpreting eDNA results in flowing waters. Allochthonous organic matter inputs represent an important seasonal change in stream ecosystems. In smaller, headwater streams, leaf litter accumulation and decomposition modify biotic and abiotic conditions that may influence eDNA removal. We conducted experimental releases of Common Carp (Cyprinus carpio) and Steelhead Trout (Oncorhynchus mykiss) eDNA in recirculating mesocosms to explore how leaf litter density and decomposition stage might influence eDNA removal. We found that leaf density had no significant effect on eDNA removal rates, but removal rates for smaller eDNA particles (0.4–1.2 μm) were 74% higher in mesocosms with biofilm-colonized leaves relative to those with “fresh” leaves. In contrast, we found no significant difference in removal rates between colonized and uncolonized leaves for larger eDNA particles (>1.2 μm). Overall, eDNA removal rates were higher for smaller eDNA particles, particularly in the presence of biofilm-colonized leaves, implying a biological mechanism for eDNA removal from the water column. These results suggest that seasonal leaf litter can affect the fate of eDNA particles and may assist in interpreting eDNA data in natural systems.

Climate change is increasing stream temperatures globally, likely impacting stream ecosystem processes. We conducted a stream warming experiment at the Notre Dame Experimental Mesocosm Facility using N = 24 recirculating mesocosms to examine the effect of warming on biofilm colonization of organic and inorganic substrata and rates of nutrient removal. We conducted an experiment with three temperature treatments (20°C, 23°C, and 26°C) in mesocosms containing either decomposing sugar maple ( Acer saccharum ) leaves or cobble substrate under shaded and unshaded conditions, respectively. We measured biofilm chlorophyll a (Chl a ) on cobble, community respiration on leaf disks, and removal of ammonium (NH ₄ ⁺ ‐N) and soluble reactive phosphorus weekly for 21 d. Warming did not significantly alter Chl a or community respiration (generalized linear mixed model, p > 0.05). For nutrients, however, warming increased NH ₄ ⁺ ‐N removal for both leaves and cobble. For cobble, NH ₄ ⁺ ‐N removal was higher at 26°C (mean = 3.5 h ⁻¹ with a 75% credible interval of [3.1, 4.0]) compared to 20°C (2.3 h ⁻¹ [1.9, 2.7]; Bayesian regression model). For leaves, NH ₄ ⁺ ‐N removal was also higher at 26°C (1.1 h ⁻¹ [1.0, 1.2]) compared to 20°C (0.5 h ⁻¹ [0.4, 0.6]). In contrast, warming only altered soluble reactive phosphorus removal for cobble; removal was higher at 26°C at 1.9 h ⁻¹ [1.7, 2.2] compared to 20°C (1.1 h ⁻¹ [0.8, 1.3]). Warming did not alter soluble reactive phosphorus removal for leaves. We show that warming‐induced changes in nutrient removal are mediated by substrate, and that NH ₄ ⁺ ‐N removal may be more sensitive to elevated temperatures than soluble reactive phosphorus removal, with implications for altered nutrient cycling under a warmer future.

The use of environmental DNA (eDNA) in routine biomonitoring is transforming our ability to detect and quantify aquatic species. However, translating these indirect molecular detections into ecological insight requires understanding how long eDNA persists and how it moves through the environment. Here, we present a systematic mapping review of the controls on eDNA fate and transport and provide the most comprehensive synthesis to date of mechanistic evidence across aquatic systems. Drawing on over 200 manuscripts, we introduce the concept of the eDNA profile, defined by state, molecular properties, and molecular origin, as a unifying framework for interpreting why eDNA signals differ across environments and taxa. Where eDNA profile has been quantified, source, state, particle size, fragment length, and molecular origin measurably alter decay and transport, confirming that different eDNA forms experience distinct fates and movement distances within aquatic systems. Using this lens, we then synthesize the evidence for the effects of abiotic factors, biotic factors, and transport on eDNA fate. For abiotic factors, we find that temperature, pH, nutrients, suspended particles, substrates, dissolved organic matter, and dissolved oxygen measurably modulate eDNA decay, whereas light exposure (UV and visible light) and salinity show weak or no direct effects across studies. For biotic factors, we conclude that microbial activity, extracellular enzymes, and biofilms are likely major drivers of eDNA degradation, although the identities, functions, and dynamics of responsible communities are poorly resolved. Finally, we show that transport patterns differ strongly among aquatic systems, with depth-structured and short-range signals common in lakes, highly variable downstream distances in rivers that depend on discharge and eDNA particle size, and longer potential transport in coastal and open-ocean systems that often still yield predominantly local signals. In these advective environments, robust interpretation commonly requires coupling eDNA observations with hydrological and oceanographic circulation models. Finally, we highlight three priorities for a predictive framework: (1) explicitly resolve eDNA profiles, (2) isolate the causal mechanisms that govern decay and removal, and (3) identify which microbes and microbial processes drive degradation across environments. Our goal is to integrate the field, highlight critical gaps, and ultimately advance the interpretation of eDNA-based detections for biodiversity monitoring.

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°C, 23°C, 26°C) and found that, for small eDNA particles (0.2–1.0 μm), 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 (> 1.0 μm, Tukey's, p > 0.05). Consequently, smaller eDNA particles were removed faster than larger particles at 26°C and 23°C (Tukey's, p < 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 < 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.

The detection of environmental DNA (eDNA) has revolutionized aquatic species monitoring, yet interpreting eDNA data remains challenging due to gaps in our understanding of eDNA ecology (i.e., origin, state, transport, and fate) and variability in how eDNA methods are applied across the literature. A crucial aspect of the complexity of eDNA ecology is that eDNA is a heterogeneous mix of components that vary in size and other properties, thereby influencing interactions with the environment in diverse ways. In this study, we explore the interplay between three eDNA particle sizes (the physical dimension of eDNA‐containing particles) and two molecule lengths (DNA size in base pairs) in flowing water systems. Specifically, we elucidated the mechanisms governing the removal of different eDNA components using a set of 24 recirculating mesocosms where we varied light and substrate conditions. Consistent with previous observations, our findings revealed substantial differences in the mechanisms of eDNA removal between small and large eDNA particles. In mesocosms with biofilm‐colonized substrate, we found higher removal rates for smaller particles, but larger eDNA particles were removed more quickly in presence of any substrate. Importantly, we also found that biofilm removes longer eDNA molecules faster, shedding light on a probable mechanism underlying the longstanding association between eDNA removal and the presence of biofilm. Despite the association between biofilm colonization and faster removal of longer molecules, the two eDNA molecule sizes we analyzed (86 and 387 base pairs) exhibited somewhat consistent behavior. In combination, our observations highlight that particle size is an important predictor of eDNA fate, and that eDNA fate shows few differences across varying molecule lengths. Furthermore, our work suggests that conclusions regarding eDNA ecology from studies utilizing short DNA markers are applicable to metabarcoding applications, which typically use longer marker lengths.

The removal of riparian shading alters stream biogeochemical cycling by stimulating algal biofilms through the release of light limitation. Few studies have examined the combined effects of shading and biofilm growth on ammonium (NH₄⁺–N) and nitrate (NO₃⁻–N) removal in the same experiment. We quantified water column NH₄⁺–N and NO₃⁻–N removal using n = 176 short‐term additions in four experimental streams over 2 years and assessed the influence of shading (shaded vs. open canopy) and biofilm colonization (early vs. late) on removal using a Bayesian three‐level hierarchical model. First, we assessed the roles of biofilm and shade on NH₄⁺–N removal. Shading lowered NH₄⁺–N uptake velocity [V_f; slope: −0.45 (−0.69, −0.22)], while the effect of biofilm was too small to detect [0.06 (−0.18, 0.29)]. The following season, we compared NH₄⁺–N and NO₃⁻–N removal, and added night releases to estimate autotrophic and heterotrophic contributions to removal. Shading reduced NH₄⁺–N and NO₃⁻–N V_f, while the effect of biofilm often depended on shading. NH₄⁺–N V_f was higher than NO₃⁻–N V_f by 51% during the day and 38% at night, confirming preferential NH₄⁺–N removal. NO₃⁻–N V_f declined more between the Late‐Open and Late‐Shade phases compared to NH₄⁺–N, suggesting a stronger decline in NO₃⁻–N demand than for NH₄⁺–N with shade during late biofilm colonization. We found no strong diel shift in NH₄⁺–N or NO₃⁻–N demand. Results demonstrate the primacy of light on NH₄⁺–N and NO₃⁻–N removal in streams. Understanding how shading and biofilm colonization alter removal is critical as streams are vulnerable to the impacts of land use change.

Understanding fate and transport within fluvial systems requires accurate modeling of breakthrough curve (BTC) tails, which often display non‐Fickian behaviors. However, it is unclear how anomalous processes relate to the physical and biological characteristics of the stream ecosystem. We use the Stochastic Mobile Immobile model (SMIM) to determine the impact of biofilm colonization among different substrate types on reach‐scale transport velocity (V) and dispersion (D), rate of delivery to the subsurface (Λ), and retention within the subsurface (reflected by power law slope; β). During the summers of 2020 and 2021, we conducted a total of n = 42 Rhodamine‐WT releases in four experimental streams lined with contrasting substrata (sand, pea gravel, cobble, and a three‐way mix) at the Notre Dame Linked Experimental Ecosystem Facility (ND‐LEEF) in Indiana (USA). To explore the effect of biofilm colonization, we conducted releases under artificially shaded, early and late biofilm development, and senescent biofilm conditions. We found that replicated releases under constant conditions consistently reproduced stream BTCs and modeled transport parameters. Biofilm abundance, biofilm status (living vs. dead), and substrate type produced significant variations in BTC shape and transport parameterizations. We found a non‐linear relationship between algal biomass and V, where increases in biomass produced decreases in V at low biomass and increases in V at high biomass. Substrate type also predicted patterns in transport, with sand producing higher V, Λ, and β than larger substrata. These results suggest that substrate type acts as the primary driver and biofilm development the secondary control on transport in these stream systems.

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 targets. 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., 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.