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USGS2016

Critical considerations for the application of environmental DNA methods to detect aquatic species

Species detection using environmental DNA (eDNA) has tremendous potential for contributing to the understanding of the ecology and conservation of aquatic species. Detecting species using eDNA methods, rather than directly sampling the organisms, can reduce impacts on sensitive species and increase the power of field surveys for rare and elusive species. The sensitivity of eDNA methods, however, requires a heightened awareness and attention to quality assurance and quality control protocols. Additionally, the interpretation of eDNA data demands careful consideration of multiple factors. As eDNA methods have grown in application, diverse approaches have been implemented to address these issues. With interest in eDNA continuing to expand, supportive guidelines for undertaking eDNA studies are greatly needed. Environmental DNA researchers from around the world have collaborated to produce this set of guidelines and considerations for implementing eDNA methods to detect aquatic macroorganisms. Critical considerations for study design include preventing contamination in the field and the laboratory, choosing appropriate sample analysis methods, validating assays, testing for sample inhibition and following minimum reporting guidelines. Critical considerations for inference include temporal and spatial processes, limits of correlation of eDNA with abundance, uncertainty of positive and negative results, and potential sources of allochthonous DNA. We present a synthesis of knowledge at this stage for application of this new and powerful detection method.

Methods in Ecology and Evolution↗
USGS2021

Strategic considerations for invasive species managers in the utilization of environmental DNA (eDNA): Steps for incorporating this powerful surveillance tool

Invasive species surveillance programs can utilize environmental DNA sampling and analysis to provide information on the presence of invasive species. Wider utilization of eDNA techniques for invasive species surveillance may be warranted. This paper covers topics directed towards invasive species managers and eDNA practitioners working at the intersection of eDNA techniques and invasive species surveillance. It provides background information on the utility of eDNA for invasive species management and points to various examples of its use across federal and international programs. It provides information on 1) why an invasive species manager should consider using eDNA, 2) deciding if eDNA can help with the manager’s surveillance needs, 3) important components to operational implementation, and 4) a high-level overview of the technical steps necessary for eDNA analysis. The goal of this paper is to assist invasive species managers in deciding if, when, and how to use eDNA for surveillance. If eDNA use is elected, the paper provides guidance on steps to ensure a clear understanding of the strengths and limitation of the methods and how results can be best utilized in the context of invasive species surveillance.

Management of Biological Invasions↗
USGS2024

How, what, and where you sample environmental DNA affects diversity estimates and species detection

Environmental DNA (eDNA) is a complex mixture of DNA, varying in particle sizes and distributed heterogeneously in aquatic systems. Optimizing eDNA sampling is crucial for maximizing species detection, particularly in high-risk scenarios like invasive species management. In this study, we compare two eDNA sampling methods - namely tow net and grab sample, where the tow nets process large volumes of water (3500–7000 L) through a 64 μm pore size and the grab samples process 1 L sample at a single point through 0.45–1.2 μm pore size membranes. We compared these methods to ascertain what most influences (1) the detection of invasive species ( Dreissena mussels and Burmese pythons) using qPCR or ddPCR and (2) total diversity monitoring of metazoan, protist, and fungi community using a COI marker and plant communities using the ITS marker. Sampling was conducted across a wide geography and diverse aquatic environments in Minnesota and Florida, USA, and Switzerland. The tow net samples had significantly higher eDNA yield compared to grab samples; however, they exhibited equal or lower alpha diversity of OTUs (Operational Taxonomic Units). The two sampling methods measured different beta diversity of communities detected with the COI marker across all three regions, highlighting the impact of the sampling method on the diversity of eDNA captured. In comparison, the beta diversity of plant eDNA was less impacted by the sampling method. We found no clear difference in detection for the invasive species targets based on the eDNA sampling method. These results underscore the need for pilot studies before conducting biodiversity inventory and monitoring, and a need for a greater understanding of not just how much, but also what, eDNA is captured depending on method choice, considering both spatial and particle size heterogeneity.

Florida, Minnesota↗
USGS2020

Environmental DNA is an effective tool to track recolonizing migratory fish following large‐scale dam removal

Environmental DNA (eDNA) has emerged as a potentially powerful tool for use in conservation and resource management, including for tracking the recolonization dynamics of fish populations. We used eDNA to assess the effectiveness of dam removal to restore fish passage on the Elwha River in Washington State (USA). Using a suite of 11 species‐specific eDNA polymerase chain reaction (PCR) assays, we showed that most targeted anadromous species (five Pacific Salmon species and Pacific Lamprey) were able to pass upstream of both former dam sites. Multiscale occupancy modeling showed that the timing and spatial extent of recolonization differed among species during the four years of post‐dam removal monitoring. More abundant species like Chinook Salmon and Coho Salmon migrated farther into the upper portions of the watershed than less abundant species like Pink Salmon and Chum Salmon. Sampling also allowed assessment of potamodromous fish species. Bull Trout and Rainbow Trout, ubiquitous species in the watershed, were detected at all sampling locations. Environmental DNA from Brook Trout, a non‐native species isolated between the dams prior to dam removal, was detected downstream of Elwha dam but rarely upstream of the Glines Canyon Dam suggested that the species has not expanded its range appreciably in the watershed following dam removal. We found that eDNA was an effective tool to assess the response of fish populations to large‐scale dam removal on the Elwha River.

Environmental DNA↗
USGS2024

Critical considerations for communicating environmental DNA science

The economic and methodological efficiencies of environmental DNA (eDNA) based survey approaches provide an unprecedented opportunity to assess and monitor aquatic environments. However, instances of inadequate communication from the scientific community about confidence levels, knowledge gaps, reliability, and appropriate parameters of eDNA-based methods have hindered their uptake in environmental monitoring programs and, in some cases, has created misperceptions or doubts in the management community. To help remedy this situation, scientists convened a session at the Second National Marine eDNA Workshop to discuss strategies for improving communications with managers. These include articulating the readiness of different eDNA applications, highlighting the strengths and limitations of eDNA tools for various applications or use cases, communicating uncertainties associated with specified uses transparently, and avoiding the exaggeration of exploratory and preliminary findings. Several key messages regarding implementation, limitations, and relationship to existing methods were prioritized. To be inclusive of the diverse managers, practitioners, and researchers, we and the other workshop participants propose the development of communication workflow plans, using RACI (Responsible, Accountable, Consulted, Informed) charts to clarify the roles of all pertinent individuals and parties and to minimize the chance for miscommunications. We also propose developing decision support tools such as Structured Decision-Making (SDM) to help balance the benefits of eDNA sampling with the inherent uncertainty, and developing an eDNA readiness scale to articulate the technological readiness of eDNA approaches for specific applications. These strategies will increase clarity and consistency regarding our understanding of the utility of eDNA-based methods, improve transparency, foster a common vision for confidently applying eDNA approaches, and enhance their benefit to the monitoring and assessment community.

Environmental DNA↗
USGS2024

Detection and transport of environmental DNA from two federally endangered mussels

Environmental DNA (eDNA) offers a novel approach to supplement traditional surveys and provide increased spatial and temporal information on species detection, and it can be especially beneficial for detecting at risk or threatened species with minimal impact on the target species. The transport of eDNA in lotic environments is an important component in providing more informed descriptions of where and when a species is present, but eDNA transport phenomena are not well understood. In this study, we used species-specific assays to detect eDNA from two federally endangered mussels in two geographically distinct rivers. Using the eDNA concentrations measured from field samples, we developed a one-dimensional (1D) hydrodynamic transport model to predict the downstream fate and transport of eDNA. We detected eDNA from both federally endangered mussels across several seasons and flow rates and up to 3.5 km downstream from the source populations, but the detection rates and eDNA concentrations were highly variable across and within rivers and study reaches. Our 1D transport models successfully integrated the variability of the eDNA field samples into the model predictions and overall model results were generally within ±1 standard error of the eDNA field concentration values. Overall, the results of this study demonstrate the importance of optimizing the spatial locations from where eDNA is collected downstream from a source population, and it highlights the need to improve understanding on the shedding mechanisms and magnitude of eDNA from source populations and biogeomorphic processes that influence eDNA transport.

Missouri, Tennessee↗
USGS2016

Detection of an enigmatic plethodontid Salamander using Environmental DNA

The isolation and identification of environmental DNA (eDNA) offers a non-invasive and efficient method for the detection of rare and secretive aquatic wildlife, and it is being widely integrated into inventory and monitoring efforts. The Patch-Nosed Salamander (Urspelerpes brucei) is a tiny, recently discovered species of plethodontid salamander known only from headwater streams in a small region of Georgia and South Carolina. Here, we present results of a quantitative PCR-based eDNA assay capable of detecting Urspelerpes in more than 75% of 33 samples from five confirmed streams. We deployed the method at 31 additional streams and located three previously undocumented populations of Urspelerpes. We compare the results of our eDNA assay with our attempt to use aquatic leaf litterbags for the rapid detection of Urspelerpes and demonstrate the relative efficacy of the eDNA assay. We suggest that eDNA offers great potential for use in detecting other aquatic and semi-aquatic plethodontid salamanders.

Copeia↗
USGS2021

Composition and distribution of fish environmental DNA in an Adirondack watershed

Background Environmental DNA (eDNA) surveys are appealing options for monitoring aquatic biodiversity. While factors affecting eDNA persistence, capture and amplification have been heavily studied, watershed-scale surveys of fish communities and our confidence in such need further exploration. Methods We characterized fish eDNA compositions using rapid, low-volume filtering with replicate and control samples scaled for a single Illumina MiSeq flow cell, using the mitochondrial 12S ribosomal RNA locus for taxonomic profiling. Our goals were to determine: (1) spatiotemporal variation in eDNA abundance, (2) the filtrate needed to achieve strong sequencing libraries, (3) the taxonomic resolution of 12S ribosomal sequences in the study environment, (4) the portion of the expected fish community detectable by 12S sequencing, (5) biases in species recovery, (6) correlations between eDNA compositions and catch per unit effort (CPUE) and (7) the extent that eDNA profiles reflect major watershed features. Our bioinformatic approach included (1) estimation of sequencing error from unambiguous mappings and simulation of taxonomic assignment error under various mapping criteria; (2) binning of species based on inferred assignment error rather than by taxonomic rank; and (3) visualization of mismatch distributions to facilitate discovery of distinct haplotypes attributed to the same reference. Our approach was implemented within the St. Regis River, NY, USA, which supports tribal and recreational fisheries and has been a target of restoration activities. We used a large record of St. Regis-specific observations to validate our assignments. Results We found that 300 mL drawn through 25-mm cellulose nitrate filters yielded greater than 5 ng/µL DNA at most sites in summer, which was an approximate threshold for generating strong sequencing libraries in our hands. Using inferred sequence error rates, we binned 12S references for 110 species on a state checklist into 85 single-species bins and seven multispecies bins. Of 48 bins observed by capture survey in the St. Regis, we detected eDNA consistent with 40, with an additional four detections flagged as potential contaminants. Sixteen unobserved species detected by eDNA ranged from plausible to implausible based on distributional data, whereas six observed species had no 12S reference sequence. Summed log-ratio compositions of eDNA-detected taxa correlated with log(CPUE) (Pearson’s R = 0.655, P < 0.001). Shifts in eDNA composition of several taxa and a genotypic shift in channel catfish ( Ictalurus punctatus ) coincided with the Hogansburg Dam, NY, USA. In summary, a simple filtering apparatus operated by field crews without prior expertise gave useful summaries of eDNA composition with minimal evidence of field contamination. 12S sequencing achieved useful taxonomic resolution despite the short marker length, and data exploration with standard bioinformatic tools clarified taxonomic uncertainty and sources of error.

New York↗
USGS2016

Environmental DNA mapping of Zebra Mussel populations

Environmental DNA (eDNA) has become a popular tool for detecting aquatic invasive species, but advancements have made it possible to potentially answer other questions like reproduction, movement, and abundance of the targeted organism. In this study we developed a Zebra Mussel (Dreissena polymorpha) eDNA protocol. We then determined if this assay could be used to help determine Zebra Mussel biomass in a lake with a well-established population of Zebra Mussels and a lake with an emerging population of mussels. Our eDNA assay detected DNA of Zebra Mussels but not DNA from more than 20 other species of fish and mussels, many commonly found in Minnesota waters. Our assay did not predict biomass. We did find that DNA from Zebra Mussels accumulated in softer substrates in both lakes, even though the mussels were predominately on the harder substrates. Therefore, we concluded that eDNA may be useful to detect the presence of Zebra Mussels in these lakes but our assay/approach could not predict biomass.

Report↗
USGS2018

Surveys of environmental DNA (eDNA): a new approach to estimate occurrence in Vulnerable manatee populations

Environmental DNA (eDNA) detection is a technique used to non-invasively detect cryptic, low density, or logistically difficult-to-study species, such as imperiled manatees. For eDNA measurement, genetic material shed into the environment is concentrated from water samples and analyzed for the presence of target species. Cytochrome b quantitative PCR and droplet digital PCR eDNA assays were developed for the 3 Vulnerable manatee species: African, Amazonian, and both subspecies of the West Indian (Florida and Antillean) manatee. Environmental DNA assays can help to delineate manatee habitat ranges, high use areas, and seasonal population changes. To validate the assay, water was analyzed from Florida’s east coast containing a high-density manatee population and produced 31564 DNA molecules l -1 on average and high occurrence (ψ) and detection (p) estimates (ψ = 0.84 [0.40-0.99]; p = 0.99 [0.95-1.00]; limit of detection 3 copies µl -1 ). Similar occupancy estimates were produced in the Florida Panhandle (ψ = 0.79 [0.54-0.97]) and Cuba (ψ = 0.89 [0.54-1.00]), while occupancy estimates in Cameroon were lower (ψ = 0.49 [0.09-0.95]). The eDNA-derived detection estimates were higher than those generated using aerial survey data on the west coast of Florida and may be effective for population monitoring. Subsequent eDNA studies could be particularly useful in locations where manatees are (1) difficult to identify visually (e.g. the Amazon River and Africa), (2) are present in patchy distributions or are on the verge of extinction (e.g. Jamaica, Haiti), and (3) where repatriation efforts are proposed (e.g. Brazil, Guadeloupe). Extension of these eDNA techniques could be applied to other imperiled marine mammal populations such as African and Asian dugongs.

Endangered Species Research↗
USGS2015

Assessment of environmental DNA for detecting presence of imperiled aquatic amphibian species in isolated wetlands

Environmental DNA (eDNA) is an emerging tool that allows low-impact sampling for aquatic species by isolating DNA from water samples and screening for DNA sequences specific to species of interest. However, researchers have not tested this method in naturally acidic wetlands that provide breeding habitat for a number of imperiled species, including the frosted salamander ( Ambystoma cingulatum ), reticulated flatwoods salamanders ( Ambystoma bishopi ), striped newt ( Notophthalmus perstriatus ), and gopher frog ( Lithobates capito ). Our objectives for this study were to develop and optimize eDNA survey protocols and assays to complement and enhance capture-based survey methods for these amphibian species. We collected three or more water samples, dipnetted or trapped larval and adult amphibians, and conducted visual encounter surveys for egg masses for target species at 40 sites on 12 different longleaf pine ( Pinus palustris ) tracts. We used quantitative PCRs to screen eDNA from each site for target species presence. We detected flatwoods salamanders at three sites with eDNA but did not detect them during physical surveys. Based on the sample location we assumed these eDNA detections to indicate the presence of frosted flatwoods salamanders. We did not detect reticulated flatwoods salamanders. We detected striped newts with physical and eDNA surveys at two wetlands. We detected gopher frogs at 12 sites total, three with eDNA alone, two with physical surveys alone, and seven with physical and eDNA surveys. We detected our target species with eDNA at 9 of 11 sites where they were present as indicated from traditional surveys and at six sites where they were not detected with traditional surveys. It was, however, critical to use at least three water samples per site for eDNA. Our results demonstrate eDNA surveys can be a useful complement to traditional survey methods for detecting imperiled pond-breeding amphibians. Environmental DNA may be particularly useful in situations where detection probability using traditional survey methods is low or access by trained personnel is limited.

Alabama, Florida, Georgia, South Carolina↗
USGS2021

Environmental DNA metabarcoding as a tool for biodiversity assessment and monitoring: Reconstructing established fish communities of north-temperate lakes and rivers

Aim To evaluate the ability of precipitation-based environmental DNA (eDNA) sample collection and mitochondrial 12S metabarcoding sequencing to reconstruct well-studied fish communities in lakes and rivers. Specific objectives were to 1) determine correlations between eDNA species detections and known community composition based on conventional field sampling, 2) compare efficiency of eDNA to detect fish biodiversity among systems with variable morphologies and trophic states, and 3) determine if species habitat preferences predict eDNA detection. Location Upper Great Lakes Region, North America. Methods Fish community composition was estimated for seven lakes and two Mississippi River navigation pools using sequence data from the mitochondrial 12S gene amplified from 10 to 50 water samples per waterbody collected in 50-mL centrifuge tubes at a single time point. Environmental DNA (eDNA) was concentrated without filtration by centrifuging samples to reduce per-sample handling time. Taxonomic detections from eDNA were compared to established community monitoring databases containing up to 40 years of sampling and a detailed habitat/substrate preference matrix to identify patterns of bias. Results Mitochondrial 12S gene metabarcoding detected 15%–47% of the known species at each waterbody and 30%–76% of known genera. Non-metric multidimensional scaling (NMDS) assessment of the community structure indicated that eDNA-detected communities grouped in a similar pattern as known communities. Discriminant analysis of principal components indicated that there was a high degree of overlap in habitat/substrate preference of eDNA-detected and eDNA-undetected species suggesting limited habitat bias for eDNA sampling. Main conclusions Large numbers of small volume samples sequenced at the mitochondrial 12S gene can describe the coarse community structure of freshwater systems. However, additional conventional sampling and environmental DNA sampling may be necessary for a complete diversity census.

Illinois, Iowa, Wisconsin↗
USGS2018

Environmental DNA (eDNA): A tool for quantifying the abundant but elusive round goby (Neogobius melanostomus)

Environmental DNA (eDNA) is revolutionizing biodiversity monitoring, occupancy estimates, and real-time detections of invasive species. In the Great Lakes, the round goby ( Neogobius melanostomus ), an invasive benthic fish from the Black Sea, has spread to encompass all five lakes and many tributaries, outcompeting or consuming native species; however, estimates of round goby abundance are confounded by behavior and habitat preference, which impact reliable methods for estimating their population. By integrating eDNA into round goby monitoring, improved estimates of biomass may be obtainable. We conducted mesocosm experiments to estimate rates of goby DNA shedding and decay. Further, we compared eDNA with several methods of traditional field sampling to compare its use as an alternative/complementary monitoring method. Environmental DNA decay was comparable to other fish species, and first-order decay was lower at 12°C (k = 0.043) than at 19°C (k = 0.058). Round goby eDNA was routinely detected in known invaded sites of Lake Michigan and its tributaries (range log 10 4.8–6.2 CN/L), but not upstream of an artificial fish barrier. Traditional techniques (mark-recapture, seining, trapping) in Lakes Michigan and Huron resulted in fewer, more variable detections than eDNA, but trapping and eDNA were correlated (Pearson R = 0.87). Additional field testing will help correlate round goby abundance with eDNA, providing insight on its role as a prey fish and its impact on food webs.

Lake Huron, Lake Michigan↗
USGS2023

Isothermal recombinant polymerase amplification and CRIPSR (CAS12A) assay detection of Renibacterium salmoninarum as an example for wildlife pathogen detection in environmental DNA samples

Improving rapid detection methods for pathogens is important for research as we collectively aim to improve the health of ecosystems globally. In the northern hemisphere, the success of salmon ( Oncorhynchus spp.) populations is vitally important to the larger marine, aquatic, and terrestrial ecosystems they inhabit. This has led to managers cultivating salmon in hatcheries and aquaculture to bolster their populations, but young salmon face many challenges, including diseases such as bacterial kidney disease (BKD). Early detection of the BKD causative agent, Renibacterium salmoninarum , is useful for managers to avoid outbreaks in hatcheries and aquaculture stocks to enable rapid treatment with targeted antibiotics. Isothermal amplification and CRIPSR-Cas12a systems may enable sensitive, relatively rapid, detection of target DNA molecules from environmental samples compared to quantitative PCR (qPCR) and culture methods. We used these technologies to develop a sensitive and specific rapid assay to detect R. salmoninarum from water samples using isothermal recombinase polymerase amplification (RPA) and an AsCas12a RNA-guided nuclease detection. The assay was specific to R. salmoninarum (0/10 co-occurring or closely related bacteria detected) and sensitive to 0.0128 pg/µL of DNA (approximately 20–40 copies/µL) within 10 min of Cas activity. This assay successfully detected R. salmoninarum environmental DNA in 14/20 water samples from hatcheries with known quantification for the pathogen via previous qPCR (70% of qPCR-positive samples). The RPA-CRISPR/AsCas12a assay had a limit of detection (LOD) of >10 copies/µL in the hatchery water samples and stochastic detection below 10 copies/µL, similar to but slightly higher than the qPCR assay. This LOD enables 37 C isothermal detection, potentially in the field, of biologically relevant levels of R. salmoninarum in water. Further research is needed to develop easy-to-use, cost-effective, sensitive RPA/CRISPR-AsCas12a assays for rapidly detecting low concentrations of wildlife pathogens in environmental samples.

Journal of Wildlife Diseases↗
USGS2023

When are environmental DNA early detections of invasive species actionable?

Environmental DNA (eDNA) sampling provides sensitive early detection capabilities for recently introduced taxa. However, natural resource managers struggle with how to integrate eDNA results into an early detection rapid response program because positive eDNA detections are not always indicative of an eventual infestation. We used a structured decision making (SDM) framework to evaluate appropriate response actions to hypothetical eDNA early detections of an introduced aquatic plant in Sebago Lake (Maine, USA). The results were juxtaposed to a recent study that used a similar SDM approach to evaluate response actions to hypothetical eDNA early detections of introduced mussels in Jordanelle Reservoir (Utah, USA). We found that eDNA early detections were not actionable in Sebago Lake because the plant's invasion potential was spatially constrained and the current management activities provided acceptable levels of mitigation. In Jordanelle Reservoir, eDNA detections were actionable due to high invasion potential and analyses supported management actions to contain the invasion. The divergent outcomes of the two case studies are related to the unique attributes of the habitats and species, highlighting the utility of the SDM approach when considering an eDNA monitoring program. We use these two case studies to present a general SDM framework and a set of heuristics that can be efficiently applied to eDNA early detection rapid response scenarios and other instances associated with indeterminant eDNA detections, especially when there is an imperative to make decisions as quickly as possible.

Maine↗
USGS2024

Environmental DNA dynamics of three species of unionid freshwater mussels

North American freshwater mussels are of special conservation concern due to their high endemism and the multiple anthropogenic stressors affecting them. Of the over 300 species in North America, nearly one third of these species are federally listed as threatened or endangered. Environmental DNA (eDNA) analysis has been successful in detecting freshwater mussels and could aid in monitoring their populations. Production and degradation rates of eDNA for the species of interest are needed to inform interpretation of eDNA detections, allow possible modeling of relative abundance and population location, and aid in mussel conservation through population identification. Here, we designed and tested qPCR assays for three freshwater mussel species, mucket ( Ortmanniana ligamentina ), fatmucket ( Lampsilis siliquoidea ), and the federally endangered spectaclecase ( Cumberlandia monodonta ). We performed laboratory experiments under controlled conditions to measure eDNA shedding and degradation rates for each species. Different biomasses, temperatures, and food regimens were tested independently to determine if these factors influence the amount of DNA produced by the mussels. Degradation rates of eDNA were measured from experimental tank water after mussels were removed. Overall, we observed low eDNA shedding rates for freshwater mussels compared to previous studies of fish eDNA shedding rates. Furthermore, temperature and feeding showed limited or no significant effects in the species studied. Environmental DNA degradation rates were consistent with those reported in the literature for other taxa. Collectively, our results will be useful for designing eDNA monitoring studies, modeling eDNA dispersal, and interpreting eDNA results to help inform freshwater mussel conservation efforts.

Environmental DNA↗
USGS2024

Species richness and distribution of Sphaeriidae surveyed with Environmental DNA metabarcoding

Freshwater bivalves of the family Sphaeriidae (fingernail, pea, and pill clams) are difficult to survey and identify due to their small size and overlapping morphological traits. Environmental DNA (eDNA) metabarcoding offers a cost-effective method for assessing species richness and distributional patterns at large scales. We evaluated sphaeriid species richness and distribution at 15 sites in the Maumee River, Ohio, USA, based on two eDNA metabarcoding assays (broad and targeted), and we compared our results with those from a traditional benthic macroinvertebrate survey. We detected seven molecular operational taxonomic units (MOTUs) in the Maumee River, including Sphaerium transversum , five MOTUs representing Euglesa spp., and one MOTU representing Odhneripisidum sp. Sphaerium transversum was widely distributed, occurring at 10 sites, but Euglesa and Odhneripisidum were restricted to one to four sites in the upper river. Distributional patterns were broadly similar between both metabarcoding assays and benthic surveys. However, eDNA metabarcoding provided species-level identifications, resulting in higher species richness. Environmental DNA sampling augments and enhances traditional benthic surveys, but greater eDNA sample replication is needed to improve detection, and additional sphaeriid reference sequences are needed to improve species-level identification.

Freshwater Mollusk Biology and Conservation↗
USGS2025

Fish composition in a complex freshwater estuary: Environmental DNA metabarcoding versus capture surveys

Objective The potential for environmental DNA (eDNA) to disperse widely from source organisms enables high detection efficiency but raises questions about eDNA's ability to differentiate fine-scale spatial patterns relative to conventional fish capture data. Methods We evaluate these questions in the St. Louis River estuary—a hydrologically and spatially complex coastal system within Lake Superior that supports a diverse assemblage of resident and migratory fish species—via comparison of eDNA metabarcoding (12S and 16S loci) to multigear capture survey data from 2 years and two seasons. Results The eDNA and capture surveys collectively yielded 68 fish species: 2 species detected only by capture, 27 detected only by eDNA, and 39 shared across both survey types but having generally higher occurrence frequencies with eDNA than capture. Six species detected only by eDNA were unexpected, having no prior records in the Lake Superior basin. Data from paired eDNA and capture stations showed little relationship between the two survey types, with capture yielding species at stations that eDNA did not, eDNA detecting species in different habitats and distant locations from any captures, and assemblage patterns homogenized in eDNA surveys relative to capture surveys. Conclusions Our study finds that eDNA is a sensitive tool for assessing species presence at the system scale but that capture surveys may better yield the fine-scale spatial distribution information of interest to fisheries and habitat managers, especially in spatially and hydrologically complex systems.

Transactions of the American Fisheries Society↗