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Environmental DNA surveys of Burmese pythons in the Greater Everglades Ecosystem

Improving the probability of detecting invasive giant snakes is vital for the management of emerging or established populations. Burmese pythons occupy thousands of square kilometers of mostly inaccessible habitats in Florida. Environmental DNA (eDNA) methods have been shown to be time and cost effective in a number of systems and may be preferable to traditional detection methods for constrictor snakes, having been shown to be effective at detecting Burmese pythons where traditional and novel detection methods have failed. The purposes of this study were (1) to estimate Burmese python eDNA occurrence in the Greater Everglades Ecosystem based on land-use type; and (2) to conduct preliminary surveys within the Greater Everglades Ecosystem for positive eDNA detections. Twenty-eight sites were sampled in the Greater Everglades Ecosystem, with 5 field replicate samples per site, for a total of 140 water samples collected. Python eDNA was detected in samples from 25 of the 28 sites by using droplet digital polymerase chain reaction amplification. Abiotic parameters were collected and explored, but we found no conclusive relationship among them and python eDNA detections. eDNA monitoring of aquatic habitats can assist in identifying newly colonized areas where pythons have not been previously detected, as well as movement corridors and pathways of dispersal. This information could be used to delimit a population boundary as it expands further to the north in peninsular Florida.

Florida

Autonomous samplers and environmental DNA metabarcoding: Sampling day and primer choice have greatest impact on fish detection probabilities

Unprecedented rates of biodiversity loss and ecosystem function necessitate the use of rapid, efficacious, and cost-effective biomonitoring tools. The combination of autonomous samplers and high throughput sequencing (i.e., “metabarcoding”) of environmental DNA ( eDNA ) samples enables characterization of entire communities at high frequency and can be an important tool for conservation and management, allowing researchers to track fluctuations in biodiversity. We deployed two autonomous samplers at two U.S. Geological Survey streamgage sites in the upper Snake River (Wyoming and Idaho, USA) to collect eDNA samples from July-September 2021 and 2022 to characterize fish diversity. We used a probabilistic approach to evaluate the effects of water temperature, water discharge, filter pore size, water volume filtered, number of samples collected, timing, and primers on the probability of detecting eDNA from fish species known to be present. We detected eDNA from 13/15 species present in these areas of the Snake River. Overall, we did not find evidence that filter pore size, water volume filtered, water discharge, and water temperature affected the probability of detecting fish species’ eDNA . By contrast, primers and sampling day affected fish detection probabilities, indicating that primer choice and sampling day can either over- or under- estimate species diversity. These results indicate that users would ideally consider sampling on non-consecutive days and which primer set will maximize species detections.

Metabarcoding & Metagenomics

Correlating sea lamprey density with environmental DNA detections in the lab

Invasive sea lamprey (Petromyzon marinus Linnaeus, 1758) are currently managed by the Great Lakes Fishery Commission in an effort to reduce pest populations below levels that cause ecological damage. One technique to improve stream population assessments could be molecular surveillance in the form of environmental DNA (eDNA) monitoring. We developed and validated four probe-based quantitative polymerase chain reaction (qPCR) assays, then used two probes (cytb, nd1) to determine whether eDNA concentration was correlated with adult and larval sea lamprey density in the lab. We found a strong positive correlation between adult sea lamprey densities of 2, 20, and 200 individuals/2000L and eDNA concentrations in tanks using both assays (cytb, nd1). For larval laboratory tank density trials, eDNA concentrations were generally near our limit of quantification and there was no significant difference in copy numbers detected between larval sea lamprey densities of 1, 5, and 25 individuals/28L. Therefore, we examined detection probability rather than concentration with laboratory tank densities. We observed a trend of increasing detection probabilities with increased larval sea lamprey density that approached significance suggesting that when DNA copy numbers are low, detection rates may be more informative in predicting varying densities of larval sea lamprey. The ability to assess sea lamprey densities from a water sample could be a powerful tool to improve traditional assessment and stream ranking techniques. Further refinement of this method in the field may make eDNA surveillance of sea lamprey a reliable part of stream assessments. Rapid eDNA analysis from many streams may help focus traditional assessment efforts, thereby improving the efficiency of invasive sea lamprey control efforts.

Management of Biological Invasions

Calibrating environmental DNA metabarcoding to conventional surveys for measuring fish species richness

The ability to properly identify species present in a landscape is foundational to ecology and essential for natural resource management and conservation. However, many species are often unaccounted for due to ineffective direct capture and visual surveys, especially in aquatic environments. Environmental DNA metabarcoding is an approach that overcomes low detection probabilities and should consequently enhance estimates of biodiversity and its proxy, species richness. Here, we synthesize 37 studies in natural aquatic systems to compare species richness estimates for bony fish between eDNA metabarcoding and conventional methods, such as nets, visual census, and electrofishing. In freshwater systems with fewer than 100 species, we found eDNA metabarcoding detected more species than conventional methods. Using multiple genetic markers further increased species richness estimates with eDNA metabarcoding. For more diverse freshwater systems and across marine systems, eDNA metabarcoding reported similar values of species richness to conventional methods; however, more studies are needed in these environments to better evaluate relative performance. In systems with greater biodiversity, eDNA metabarcoding will require more populated reference databases, increased sampling effort, and multi-marker assays to ensure robust species richness estimates to further validate the approach. eDNA metabarcoding is reliable and provides a path for broader biodiversity assessments that can outperform conventional methods for estimating species richness.

Frontiers in Ecology and Evolution

A round-robin evaluation of the repeatability and reproducibility of environmental DNA assays for dreissenid mussels

Resource managers may be hesitant to make decisions based on environmental (e)DNA results alone since eDNA is an indirect method of species detection. One way to reduce the uncertainty of eDNA is to identify laboratory‐based protocols that ensure repeatable and reproducible results. We conducted a double‐blind round‐robin analysis of probe‐based assays for DNA of dreissenid ( Dreissena spp.) mussels, which are prolific aquatic invaders that can cause significant economic and ecological impacts. DNA extract from water samples spiked with known amounts of dreissenid DNA and from water samples collected from waters with and without dreissenids were analyzed by four independent research laboratories. We used results to calculate detection repeatability within laboratories and assays, detection reproducibility among laboratories and assays, and estimated dreissenid DNA copy number precision and accuracy. Laboratory and assay repeatability and reproducibility of detection results were high, 91% and 92%, respectively. The estimated copy numbers were neither precise nor accurate for samples spiked with <773 gene copies. These results suggest that eDNA surveillance of dreissenid mussels, using the protocols evaluated herein, can generate reliable detection data for decision‐making. However, managers should be cautious about using the quantitative information often associated with eDNA detections, especially when DNA is at lower abundance. Our results provide strong support that eDNA has the potential to provide repeatable and reproducible evidence under varying laboratory conditions and for different sample water chemistries. This is reassuring since the demand for eDNA surveillance is widespread and number of laboratories that process eDNA samples is growing steadily.

California, Montana, Nevada, New York, Oregon, Was

Testing environmental DNA from wolf snow tracks for species, sex, and individual identification

Monitoring elusive, relatively low-density, large predators, such as the grey wolf (Canis lupus), has often been accomplished by live-capture and radiocollaring. Increasingly, non-invasive methods are considered best practice whenever it is possible to use them. Recently, environmental DNA (eDNA) deposited in snow tracks was demonstrated as useful for identifying lynx (Lynx canadensis), fisher (Pekania pennanti), wolverine (Gulo gulo), sika deer (Cervus nippon), red fox (Vulpes vulpes), and the Japanese marten (Martes melampus) to species level using mitochondrial DNA (mtDNA) markers. We tested whether eDNA from fresh wolf snow tracks collected in the Superior National Forest, Minnesota, USA during winter 2019 could be used to identify species, sex, and individual. Seven of the 8 snow track samples were successfully identified to “wolf-dog” species using mtDNA, with alleles amplifying in 5 of the samples at 1 or both of loci u250 and FH2096 in the allele range for wolves for this population. None yielded enough high-quality DNA to obtain genotypes to determine individual or sex. We recommend additional field trials to determine the minimum number of tracks required per individual to obtain sufficient, high-quality eDNA, as well as collecting associated urine or blood (from estrus) when possible. If individual wolves could be identified and sexed by the eDNA in their snow tracks, researchers should be able to determine population and family or group metrics with greater precision and less effort than typically required when conducting winter scat or hair-based genetic field studies of low density, elusive carnivores. Furthermore, this method would have applications to other areas of research and management, such as hunting quota determinations, validation of field methods and, in particular regards to wolves and other predators, livestock depredation issues. Overall, this technique holds significant future promise as field and laboratory methods are further refined for greater precision and optimized regarding the varying collection, filtration, and extraction protocols for different species in various environments.

Minnesota

Looking for Lazarus: Environmental DNA (eDNA) surveillance of the federally threatened Slender Chub (Erimystax cahini) in the Clinch and Powell Rivers

Erimystax cahni (Slender Chub) is a federally threatened fish native to the Powell, Clinch, and lower Holston rivers of Tennessee and Virginia. No individuals have been captured or reported for over 2 decades. We developed a species-specific environmental DNA (eDNA) assay to survey for its presence throughout its native distribution in new and historical sites. We detected Slender Chub eDNA in both the Powell and Clinch rivers in 3 of 43 sites sampled during summer 2022. The presence of live individuals could not be confirmed, and the detection of positive eDNA matches could result from pure individuals or hybrids. Note that positive eDNA matches do not preclude the presence of hybrid individuals; however, they can help guide managers to intensively sample areas with conventional methods (e.g., snorkeling and seining), which could improve the probability of capture of living individuals.

Kentucky, Tennessee

Environmental DNA monitoring of invasive Central American boas in St. Croix at Salt River Bay National Historical Park and Ecological Preserve (SARI)

Invasive Central American boas ( Boa imperator ) have established a reproducing population on the western side of St. Croix, U.S. Virgin Islands but prevalence throughout the island is largely unknown. The large snakes threaten endemic and endangered species through competition and predation, jeopardizing island biodiversity. Environmental DNA (eDNA) methods were used to investigate occurrence and focal areas for management efforts in the Salt River Bay National Historical Park and Ecological Preserve (SARI). To validate a previously developed assay, we collected tissue samples and 13 × 60 mL water samples from a captive boa enclosure in St. Croix. We implemented this assay for both a pilot and main field sampling effort. The pilot in December 2023 resulted in analysis of 7 × 60 mL water samples per site from SARI (3 sites) and from the western forests where boas are established (1 site). The main sampling event in July 2024 collected 15 × 60 mL water samples per site within SARI (11 sites) and western forests (4 sites). Pilot sample replicates were filtered individually, while main samples were consolidated into groups, resulting in seven replicates for pilot sites and five replicates for main event sites, totaling 103 environmental samples. eDNA was isolated using a modified phenol-chloroform isolation method to remove PCR inhibitors, and target eDNA was amplified using droplet digital PCR technology. Water samples from the captive boa amplified target eDNA in 12 of 13 samples, indicating assay effectiveness ex-situ. Low concentrations of eDNA (below the 95% limit of detection) were amplified in 4 of 5 sites in the western forest and in 8 of 14 sites within the National Historic Park. Overall, boa eDNA concentrations were consistently low, as expected in water samples targeting a semi-arboreal snake species with a low rate of eDNA shedding. Further optimization of methods could enable recovery of greater eDNA concentrations in future studies. Additional eDNA method testing and ground-truthing may help to improve the assessment of invasive Boa imperator in St. Croix.

Science Report

eDNAoccupancy: An R package for multi-scale occupancy modeling of environmental DNA data

In this article we describe eDNAoccupancy, an R package for fitting Bayesian, multi-scale occupancy models. These models are appropriate for occupancy surveys that include three, nested levels of sampling: primary sample units within a study area, secondary sample units collected from each primary unit, and replicates of each secondary sample unit. This design is commonly used in occupancy surveys of environmental DNA (eDNA). eDNAoccupancy allows users to specify and fit multi-scale occupancy models with or without covariates, to estimate posterior summaries of occurrence and detection probabilities, and to compare different models using Bayesian model-selection criteria. We illustrate these features by analyzing two published data sets: eDNA surveys of a fungal pathogen of amphibians and eDNA surveys of an endangered fish species.

Molecular Ecology Resources

Using redundant primer sets to detect multiple native Alaskan fish species from environmental DNA

Accurate and timely data regarding freshwater fish communities is important for informed decision-making by local, state, tribal, and federal land and resource managers; however, conducting traditional gear-based fish surveys can be an expensive and time-consuming process, particularly in remote areas, like those that characterize much of Alaska. To help address this challenge, we developed and tested five multi-species environmental DNA (eDNA) primer sets for the simultaneous detection of up to 37 target fish species in a single sample. Using these primer sets can reduce the cost and time needed to perform future studies of fish communities. Our results comparing multiple samples from multiple lakes and streams using multiple next-generation sequencing runs show the efficacy and reproducibility of these primers.

Alaska

Using environmental DNA (eDNA) to detect the endangered Spectaclecase Mussel ( Margaritifera monodonta )

Margaritifera monodonta , or the Spectaclecase Mussel, is a federally endangered freshwater mussel species that has experienced a 55% reduction in range and is currently concentrated in 3 rivers in the Midwest region of the United States (Gasconade and Meramec rivers, Missouri, and St Croix River, Wisconsin). The detection of new populations by traditional survey methods has been limited because these mussels tend to occur under large rocks and boulders. Environmental DNA (eDNA) technology has been used to detect invasive and rare species, but its use for detection of rare, benthic-dwelling species in large flowing systems has been limited. Here, we propose using eDNA to assess known populations of M. monodonta . We designed a M. monodonta -specific quantitative polymerase chain reaction (qPCR) assay and tested it using water samples from multiple M. monodonta housing tanks, water samples from 2 known mussel beds on the St Croix River, and water samples from 3 known mussel beds on the Mississippi River. We observed higher overall eDNA detection rates on the St Croix River (30.2%) compared to the upper Mississippi River (0.60%). We also observed higher eDNA detection rates (73.3–93.1%) in 2018 for samples collected during the larval release period in May compared to samples collected in August after the reproductive period had ended (55.6–70.8%) on the St Croix River. We tested samples collected at 3 distances downstream from the 2 mussel beds found in the St Croix River, but we did not observe a substantial effect of distance on our detection rates. However, we did observe greater detection rates for samples collected near the bottom compared to at the surface. Our results indicate that this novel qPCR assay can successfully detect M. monodonta eDNA and could be used to rapidly screen locations to guide intensive physical searches for populations in riverine systems.

Wisconsin, Missouri

An integrative approach to assessing bridle shiner (Notropis bifrenatus) distribution using environmental DNA and traditional techniques

The bridle shiner ( Notropis bifrenatus ) is a small cyprinid native to the eastern United States and Canada. Bridle shiner populations have declined across their range, and the species now receives concern status or legal protection in 13 states and two provinces. Bridle shiners were historically found in southern and western Maine in densely vegetated, shallow habitats along the shorelines of streams and ponds. We surveyed areas of Maine that supported historical bridle shiner populations using environmental DNA (eDNA) and traditional seine netting methods, and then used eDNA sampling to survey areas with unknown bridle shiner presence. We rediscovered bridle shiner populations at 11 of 32 historically occupied waterbodies and documented bridle shiners in four additional waterbodies. We determined that both eDNA and seine net surveys are viable options for monitoring bridle shiners in Maine and identified ways to streamline the eDNA methods used in this study to reduce the time and cost of future surveys.

Maine

Assessing grass carp (Ctenopharyngodon idella) occupancy and detection probability within Lake Erie from environmental DNA

Grass carp ( Ctenopharyngodon idella ), an invasive cyprinid within the Laurentian Great Lakes, is naturally reproducing in several Lake Erie tributaries, which has raised concerns of the species’ spread throughout Lake Erie and the other Great Lakes. Knowledge of the recent invasion extent outside of the western basin of Lake Erie, particularly in eastern tributaries and nearshore waters, is limited. Understanding the invasion extent would improve the efficacy of ongoing coordinated multi-agency control efforts. Molecular tools, such as environmental DNA (eDNA), have shown promise for early detection of aquatic invasive species. In this study, water samples (N = 476) were collected for grass carp eDNA monthly between May and November in 2018 and 2019, at three sites in the Michigan waters of Lake Erie and the Detroit River. We fit Bayesian multi-scale occupancy models to determine differences in eDNA capture and detection probability among grass carp qPCR assays, sampling sites, and across time. To determine whether grass carp were physically present, and to validate eDNA samples, we quantified recent grass carp presence in sampled areas using an existing acoustic telemetry and field sampling framework. Our results indicate that grass carp eDNA capture probability differed among sites, but there was no difference among months. Positive grass carp eDNA detections were observed across multiple months at each site, with 69% of site-specific sampling events testing positive for grass carp eDNA on at least one assay and replicate. The majority (65%) of weeks where positive eDNA sampling detections occurred also concurrently had one or more grass carp detected via acoustic telemetry 1–6 days prior. Our results highlight the potential utility of using eDNA to monitor the invasion extent of grass carp within the nearshore waters of Lake Erie. However, further evaluation of the factors that influence grass carp eDNA characteristics among sites within Lake Erie are needed to determine its efficacy for surveillance protocols by natural resource management agencies.

Lake Erie

Using environmental DNA to assess the response of steelhead/Rainbow Trout and Coastrange Sculpin populations to postfire debris flows in coastal streams of Big Sur, California

Objective Debris flows are among the most extreme disturbances to streams and are predicted to become more frequent under climate change. We assessed the response of steelhead Oncorhynchus mykiss (anadromous Rainbow Trout)/Rainbow Trout (hereafter, collectively referred to as O. mykiss ) and Coastrange Sculpin Cottus aleuticus populations to major postfire debris flows in two small coastal basins of California using noninvasive environmental DNA (eDNA) sampling. Methods We analyzed water samples from disturbed reaches for eDNA in the first two summers after debris flows. In Big Creek, all Coastrange Sculpin habitat was disturbed by debris flows, but a major tributary occupied by O. mykiss was not affected. In Mill Creek, all available habitat for both species was disturbed. We also sampled two unburned basins as undisturbed reference streams. Result In Big Creek, O. mykiss eDNA was detected in all water samples during both years, and concentrations in some samples approached the lowest concentrations in the reference streams. Coastrange Sculpin eDNA was detected only in a single water sample in the first year and was not detected in the second year. In Mill Creek, neither species was detected in the first year, but during the second year, O. mykiss eDNA was detected at very low concentrations in 40% of samples and Coastrange Sculpin eDNA was detected in a single sample. Conclusion Our results indicate that O. mykiss and Coastrange Sculpins either survived in or quickly reoccupied the disturbed reaches from within the basins. However, the detection rates for cases in which all habitat for a species in a basin was affected by debris flows indicate that abundances were very low, suggesting that persistence may be uncertain unless there is successful reproduction or immigration. Finally, eDNA appears to be effective for monitoring sensitive fish populations after a major disturbance; however, detection rates in individual samples may be low and require appropriate sampling designs to achieve the desired detection probability.

California

Use of environmental DNA to assess American Eel distribution, abundance, and barriers in a river-canal system

Objective: The American Eel Anguilla rostrata historically was one of the most common fish species in Atlantic coast watersheds, but extensive dam construction and other factors caused a widespread population decline. One of the watersheds where American Eels have declined considerably is the Mohawk River in eastern and central New York. Recent attempts to characterize the distribution and abundance of American Eels in this watershed have been ineffective, and the extent to which a series of locks and dams on the Hudson River and lower Mohawk River limits use of the watershed is unclear. Methods: We developed a model between environmental DNA (eDNA) quantity and American Eel abundance in the Hudson River watershed in which the DNA concentration in water samples explained up to 65% of the variability in eel density and 56% of the variability in eel biomass. We then used this relationship to interpret eDNA data collected twice from 36 sites across the Mohawk River watershed in 2021 and make inferences about the distribution and abundance of American Eels. Result: American Eel DNA was detected almost exclusively in the downstream-most 4 km of the Mohawk River within a series of barriers. The concentration of DNA was reduced by approximately 80% across each successive upstream barrier before becoming too low to detect consistently. Our data suggest that eel population density was high in the Hudson River estuary and declined rapidly in the lower Mohawk River, and the species was nearly absent or undetectable in the Mohawk River and its tributaries upstream of the Crescent Dam and the Waterford Flight of Locks. Conclusion: Barriers appear to be largely restricting American Eels from using over 99% of the Mohawk River watershed. Therefore, improvements in fish passage at dams and hydroelectric facilities in the region could help the American Eel to regain access to this part of its native range.

New York

Environmental DNA: An emerging tool for understanding aquatic biodiversity

Field surveys for aquatic organisms provide critical information that is important for robust resource management. However, such surveys are expensive and labor intensive, particularly in large, remote landscapes like those that characterize much of Alaska. Traditionally, characterizing aquatic biodiversity necessitated the physical capture and identification of individual organisms, which required that field crews have some level of expertise in identifying the species likely to be present. Many other limitations of surveys that rely on direct observation of aquatic organisms have been noted (Evans and Lamberti 2018). However, what if it were possible to identify all of the species present at a site without having to capture or even see them? While we are not there yet, the recent revolution in environmental DNA (eDNA) technology is bringing us closer to that goal (Thomsen and Willerslev 2014).

Alaska

Environmental DNA sampling reveals high occupancy rates of invasive Burmese pythons at wading bird breeding aggregations in the central Everglades

The Burmese python ( Python bivittatus ) is now established as a breeding population throughout south Florida, USA. However, the extent of the invasion, and the ecological impacts of this novel apex predator on animal communities are incompletely known, in large part because Burmese pythons (hereafter “pythons”) are extremely cryptic and there has been no efficient way to detect them. Pythons are recently confirmed nest predators of long-legged wading bird breeding colonies (orders Ciconiiformes and Pelecaniformes). Pythons can consume large quantities of prey and may not be recognized as predators by wading birds, therefore they could be a particular threat to colonies. To quantify python occupancy rates at tree islands where wading birds breed, we utilized environmental DNA (eDNA) analysis—a genetic tool which detects shed DNA in water samples and provides high detection probabilities. We fitted multi-scale Bayesian occupancy models to test the prediction that pythons occupy islands with wading bird colonies at higher rates compared to representative control islands containing no breeding birds. Our results suggest that pythons are widely distributed across the central Everglades in proximity to active wading bird colonies. In support of our prediction that pythons are attracted to colonies, site-level python eDNA occupancy rates were higher at wading bird colonies (ψ = 0.88, 95% credible interval [0.59–1.00]) than at the control islands (ψ = 0.42 [0.16–0.80]) in April through June (n = 15 colony-control pairs). We found our water temperature proxy (time of day) to be informative of detection probability, in accordance with other studies demonstrating an effect of temperature on eDNA degradation in occupied samples. Individual sample concentrations ranged from 0.26 to 38.29 copies/μL and we generally detected higher concentrations of python eDNA in colony sites. Continued monitoring of wading bird colonies is warranted to determine the effect pythons are having on populations and investigate putative management activities.

Florida

Environmental DNA (eDNA) surveillance of the federally threatened Slender Chub (Erimystax cahni) in the Clinch River and Powell River

The slender chub ( Erimystax cahni ) is a federally threatened fish native to and geographically restricted to eastern North America. More specifically, the Powell, Clinch, and lower Holston Rivers in Tennessee were historical collection areas. Habitat degradation from multiple sources, including surface mining, agriculture, dams, and urbanization, is associated with the decline of this species - an obligate inhabitant of gravel shoals in large rivers. As of 1964, only 15 voucher specimens were known and no living specimens were documented for decades. A federal recovery plan and Species Status Assessment were developed to determine if slender chub still exists and could be restored. Given the lack of recent observations using conventional sampling to search for its presence, we used environmental DNA sampling to determine their potential presence. Our specific objectives were to (1) develop a quantitative polymerase chain reaction (qPCR) assay aimed at species-specific detection and (2) sample historically known areas of collection. We sampled 43 sites in the Clinch and Powell Rivers. For the first time in almost two decades, we provide evidence for the continued existence of a putatively extinct species. We detected evidence of E. cahni in both the Clinch and Powell Rivers, but only at a few sites. We cannot confirm the presence of E. cahni , and positive eDNA matches could be attributed to amplification from a hybrid. Our results do indicate slender chub DNA perseveres in the Clinch and Powell Rivers and can inform resource agencies of localities to pursue on-the-ground searches for the slender chub with conventional methods (e.g., snorkeling) and potential restoration and recovery sites.

Tennessee, Virginia