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.

Genomics reveal the origins and current structure of a genetically depauperate freshwater species in its introduced Alaskan range.

Invasive species are a major threat to global biodiversity, yet also represent large-scale unplanned ecological and evolutionary experiments to address fundamental questions in nature. Here we analyzed both native and invasive populations of predatory northern pike (Esox lucius) to characterize landscape genetic variation, determine the most likely origins of introduced populations, and investigate a presumably postglacial population from Southeast Alaska of unclear provenance. Using a set of 4329 SNPs from 351 individual Alaskan northern pike representing the most widespread geographic sampling to date, our results confirm low levels of genetic diversity in native populations (average 𝝅 of 3.18 × 10-4) and even less in invasive populations (average 𝝅 of 2.68 × 10-4) consistent with bottleneck effects. Our analyses indicate that invasive northern pike likely came from multiple introductions from different native Alaskan populations and subsequently dispersed from original introduction sites. At the broadest scale, invasive populations appear to have been founded from two distinct regions of Alaska, indicative of two independent introduction events. Genetic admixture resulting from introductions from multiple source populations may have mitigated the negative effects associated with genetic bottlenecks in this species with naturally low levels of genetic diversity. Genomic signatures strongly suggest an excess of rare, population-specific alleles, pointing to a small number of founding individuals in both native and introduced populations consistent with a species' life history of limited dispersal and gene flow. Lastly, the results strongly suggest that a small isolated population of pike, located in Southeast Alaska, is native in origin rather than stemming from a contemporary introduction event. Although theory predicts that lack of genetic variation may limit colonization success of novel environments, we detected no evidence that a lack of standing variation limited the success of this genetically depauperate apex predator.

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.

A framework to integrate innovations in invasion science for proactive management.

Invasive alien species (IAS) are a rising threat to biodiversity, national security, and regional economies, with impacts in the hundreds of billions of U.S. dollars annually. Proactive or predictive approaches guided by scientific knowledge are essential to keeping pace with growing impacts of invasions under climate change. Although the rapid development of diverse technologies and approaches has produced tools with the potential to greatly accelerate invasion research and management, innovation has far outpaced implementation and coordination. Technological and methodological syntheses are urgently needed to close the growing implementation gap and facilitate interdisciplinary collaboration and synergy among evolving disciplines. A broad review is necessary to demonstrate the utility and relevance of work in diverse fields to generate actionable science for the ongoing invasion crisis. Here, we review such advances in relevant fields including remote sensing, epidemiology, big data analytics, environmental DNA (eDNA) sampling, genomics, and others, and present a generalized framework for distilling existing and emerging data into products for proactive IAS research and management. This integrated workflow provides a pathway for scientists and practitioners in diverse disciplines to contribute to applied invasion biology in a coordinated, synergistic, and scalable manner.

Time to get real with qPCR controls: The frequency of sample contamination and the informative power of negative controls in environmental DNA studies.

Environmental (e)DNA methods have enabled rapid, sensitive and specific inferences of taxa presence throughout diverse fields of ecological study. However, use of eDNA results for decision-making has been impeded by uncertainties associated with false positive tests putatively caused by sporadic or systemic contamination. Sporadic contamination is a process that is inconsistent across samples and systemic contamination occurs consistently over a group of samples. Here, we used empirical data and laboratory experiments to (i) estimate the sporadic contamination rate for each stage of a common, targeted eDNA workflow employing best practice quality control measures under simulated conditions of rare and common target DNA presence, (ii) determine the rate at which negative controls (i.e., "blanks") detect varying concentrations of systemic contamination, and (iii) estimate the effort that would be required to consistently detect sporadic and systemic contamination. Sporadic contamination rates were very low across all eDNA workflow steps, and, therefore, an intractably high number of negative controls (>100) would be required to determine occurrence of sporadic contamination with any certainty. Contrarily, detection of intentionally introduced systemic contamination was more consistent; therefore, very few negative controls (<5) would be needed to consistently alert to systemic contamination. These results have considerable implications to eDNA study design when resources for sample analyses are constrained.

Vulnerability of Pacific salmon to invasion of northern pike (Esox lucius) in Southcentral Alaska.

The relentless role of invasive species in the extinction of native biota requires predictions of ecosystem vulnerability to inform proactive management strategies. The worldwide invasion and range expansion of predatory northern pike (Esox lucius) has been linked to the decline of native fishes and tools are needed to predict the vulnerability of habitats to invasion over broad geographic scales. To address this need, we coupled an intrinsic potential habitat modelling approach with a Bayesian network to evaluate the vulnerability of five culturally and economically vital species of Pacific salmon (Oncorhynchus spp.) to invasion by northern pike. This study was conducted along 22,875 stream km in the Southcentral region of Alaska, USA. Pink salmon (O. gorbuscha) were the most vulnerable species, with 15.2% (2,458 km) of their calculated extent identified as "highly" vulnerable, followed closely by chum salmon (O. keta, 14.8%; 2,557 km) and coho salmon (O. kisutch, 14.7%; 2,536 km). Moreover, all five Pacific salmon species were highly vulnerable in 1,001 stream km of shared habitat. This simple to implement, adaptable, and cost-effective framework will allow prioritizing habitats for early detection and monitoring of invading northern pike.

Effects of supplemental feeding on the fecal bacterial communities of Rocky Mountain elk in the Greater Yellowstone Ecosystem.

Supplemental feeding of wildlife is a common practice often undertaken for recreational or management purposes, but it may have unintended consequences for animal health. Understanding cryptic effects of diet supplementation on the gut microbiomes of wild mammals is important to inform conservation and management strategies. Multiple laboratory studies have demonstrated the importance of the gut microbiome for extracting and synthesizing nutrients, modulating host immunity, and many other vital host functions, but these relationships can be disrupted by dietary perturbation. The well-described interplay between diet, the microbiome, and host health in laboratory and human systems highlights the need to understand the consequences of supplemental feeding on the microbiomes of free-ranging animal populations. This study describes changes to the gut microbiomes of wild elk under different supplemental feeding regimes. We demonstrated significant cross-sectional variation between elk at different feeding locations and identified several relatively low-abundance bacterial genera that differed between fed versus unfed groups. In addition, we followed four of these populations through mid-season changes in supplemental feeding regimes and demonstrated a significant shift in microbiome composition in a single population that changed from natural forage to supplementation with alfalfa pellets. Some of the taxonomic shifts in this population mirrored changes associated with ruminal acidosis in domestic livestock. We discerned no significant changes in the population that shifted from natural forage to hay supplementation, or in the populations that changed from one type of hay to another. Our results suggest that supplementation with alfalfa pellets alters the native gut microbiome of elk, with potential implications for population health.

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.

Robotic environmental DNA bio-surveillance of freshwater health.

Autonomous water sampling technologies may help to overcome the human resource challenges of monitoring biological threats to rivers over long time periods and across large geographic areas. The Monterey Bay Aquarium Research Institute has pioneered a robotic Environmental Sample Processor (ESP) that overcomes some of the constraints associated with traditional sampling since it can automate water sample filtration and preservation of the captured material. The ESP was originally developed for marine environment applications. Here we evaluated whether the ESP can provide reliable, timely information on environmental (e)DNA detections of human and fish pathogens and introduced fishes at U.S. Geological Survey streamgage sites in freshwater rivers. We compared eDNA collected via ESP at high frequency (e.g., every 3 h) with manual eDNA collections collected at lower frequency (e.g., weekly). We found that water samples filtered and preserved by ESPs successfully detected the DNA of human pathogens, fish pathogens and introduced fishes. Both ESP and manually collected samples provided similar information about target DNA presence. We suggest that the greatest current benefit of the ESP is the cost savings of high frequency, bio-surveillance at remote or hard to access sites. The full potential of robotic technologies like the ESP will be realized when they can more easily execute in situ analyses of water samples and rapidly transmit results to decision-makers.

Are Environmental DNA Methods Ready for Aquatic Invasive Species Management?

Multiple studies have demonstrated environmental (e)DNA detections of rare and invasive species. However, invasive species managers struggle with using eDNA results because detections might not indicate species presence. We evaluated whether eDNA methods have matured to a point where they can be widely applied to aquatic invasive species management. We have found that eDNA methods meet legal standards for being admissible as evidence in most courts, suggesting eDNA method reliability is not the problem. Rather, we suggest the interface between results and management needs attention since there are few tools for integrating uncertainty into decision-making. Solutions include decision-support trees based on molecular best practices that integrate the temporal and spatial trends in eDNA positives relative to human risk tolerance.

What do you mean by false positive.

Misunderstandings regarding the term "false positive" present a significant hurdle to broad adoption of eDNA monitoring methods. Here, we identify three challenges to clear communication of false-positive error between scientists, managers, and the public. The first arises from a failure to distinguish between false-positive eDNA detection at the sample level and false-positive inference of taxa presence at the site level. The second is based on the large proportion of false positives that may occur when true-positive detections are likely to be rare, even when rates of contamination or other error are low. And the third misunderstanding occurs when conventional species detection approaches, often based on direct capture, are used to confirm eDNA approaches without acknowledging or quantifying the conventional approach's detection probability. The solutions to these issues include careful and consistent communication of error definitions, managing expectations of error rates, and providing a balanced discussion not only of alternative sources of species DNA, but also of the detection limitations of conventional methods. We argue that the benefit of addressing these misunderstandings will be increased confidence in the utility of eDNA methods and, ultimately, improved resource management using eDNA approaches. The term false positive is often misused in eDNA research and natural resource management. There are issues of scale of inference, the base rate fallacy, and confirmation errors using conventional methods of detection. We offer a perspective to guide discussions of errors in species detection.

Wetland drying linked to variations in snowmelt runoff across Grand Teton and Yellowstone national parks.

In Grand Teton and Yellowstone national parks wetlands offer critical habitat and play a key role in supporting biological diversity. The shallow depths and small size of many palustrine wetlands in these protected areas and elsewhere make them vulnerable to changes in climate compared with larger and deeper aquatic habitats. Here, we use a simple water balance model to generate estimates of biophysical drivers of wetland change. We then examine the relationship between wetland inundation status and four principal drivers (i.e., temperature, precipitation, evapotranspiration, and runoff) spanning varying meteorological conditions over an 8-year time series from Grand Teton and Yellowstone national parks. We found that models containing snowmelt runoff outperformed models with other meteorological drivers and determined that a higher percentage of surveyed wetlands were dry in years characterized by lower runoff. Our work further shows that wetland drying was widespread across both parks, but sub-regional variations were best described at the hydrologic subbasin-level. Documenting the varying responses of wetlands to meteorological drivers is a necessary first step to identifying which subbasins are most sensitive to recent climatic change and contemplating how future change may alter the distribution of wetlands and their dependent taxa.

Improved Conventional PCR Assay for Detecting Tetracapsuloides bryosalmonae DNA in Fish Tissues.

Conventional PCR is an established method to detect Tetracapsuloides bryosalmonaeDNA in fish tissues and to confirm diagnosis of proliferative kidney disease (PKD) caused by T. bryosalmonae. However, the commonly used PKX5f-6r primers were designed with the intention of obtaining sequence information and are suboptimal for determining parasite DNA presence. A new PCR assay to detect T. bryosalmonae 18s rDNA, PKX18s1266f-1426r, is presented that demonstrates specificity, repeatability, and enhanced sensitivity over the PKX5f-6r assay. The limit of detection of the PKX18s1266f-1426r assay at 95% confidence was 100 template copies, and the new primers detected parasite DNA more consistently at template concentrations below 100 copies than did PKX5f-6r. The PKX18s1266f-1426r also achieved 100% detection at sample DNA concentrations one order of magnitude lower than PKX5f-6r. Out of 127 salmonid fish with unknown T. bryosalmonae infection status, PKX5f-6r detected 35 positive samples, while the new assay detected 43. The discrepancy in T. bryosalmonae detection between the two primer sets may be attributed to several differences between the assays, including oligonucleotide melting temperatures, the use of a touchdown PCR thermal cycle, and amplicon length.

Correction: Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study.

[This corrects the article DOI: 10.1371/journal.pone.0162277.].

Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study.

Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling is delayed long enough to allow full degradation of DNA in the water.

Genetic reconstruction of a bullfrog invasion to elucidate vectors of introduction and secondary spread.

Reconstructing historical colonization pathways of an invasive species is critical for uncovering factors that determine invasion success and for designing management strategies. The American bullfrog (Lithobates catesbeianus) is endemic to eastern North America, but now has a global distribution and is considered to be one of the worst invaders in the world. In Montana, several introduced populations have been reported, but little is known of their sources and vectors of introduction and secondary spread. We evaluated the genetic composition of introduced populations at local (Yellowstone River floodplain) and regional (Montana and Wyoming) scales in contrast to native range populations. Our objectives were to (1) estimate the number of introductions, (2) identify probable native sources, (3) evaluate genetic variation relative to sources, and (4) characterize properties of local- and regional-scale spread. We sequenced 937 bp of the mitochondrial cytochrome b locus in 395 tadpoles collected along 100 km of the Yellowstone River, from three additional sites in MT and a proximate site in WY. Pairwise ΦST revealed high divergence among nonnative populations, suggesting at least four independent introductions into MT from diverse sources. Three cyt b haplotypes were identical to native haplotypes distributed across the Midwest and Great Lakes regions, and AMOVA confirmed the western native region as a likely source. While haplotype (H d = 0.69) and nucleotide diversity (π = 0.005) were low in introduced bullfrogs, the levels of diversity did not differ significantly from source populations. In the Yellowstone, two identified haplotypes implied few introduction vectors and a significant relationship between genetic and river distance was found. Evidence for multiple invasions and lack of subsequent regional spread emphasizes the importance of enforcing legislation prohibiting bullfrog importation and the need for continuing public education to prevent transport of bullfrogs in MT. More broadly, this study demonstrates how genetic approaches can reveal key properties of a biological invasion to inform management strategies.

The Shifting Climate Portfolio of the Greater Yellowstone Area.

Knowledge of climatic variability at small spatial extents (< 50 km) is needed to assess vulnerabilities of biological reserves to climate change. We used empirical and modeled weather station data to test if climate change has increased the synchrony of surface air temperatures among 50 sites within the Greater Yellowstone Area (GYA) of the interior western United States. This important biological reserve is the largest protected area in the Lower 48 states and provides critical habitat for some of the world's most iconic wildlife. We focused our analyses on temporal shifts and shape changes in the annual distributions of seasonal minimum and maximum air temperatures among valley-bottom and higher elevation sites from 1948-2012. We documented consistent patterns of warming since 1948 at all 50 sites, with the most pronounced changes occurring during the Winter and Summer when minimum and maximum temperature distributions increased. These shifts indicate more hot temperatures and less cold temperatures would be expected across the GYA. Though the shifting statistical distributions indicate warming, little change in the shape of the temperature distributions across sites since 1948 suggest the GYA has maintained a diverse portfolio of temperatures within a year. Spatial heterogeneity in temperatures is likely maintained by the GYA's physiographic complexity and its large size, which encompasses multiple climate zones that respond differently to synoptic drivers. Having a diverse portfolio of temperatures may help biological reserves spread the extinction risk posed by climate change.

Ecological relevance of current water quality assessment unit designations in impaired rivers.

Managers often nest sections of water bodies together into assessment units (AUs) to monitor and assess water quality criteria. Ideally, AUs represent an extent of waters with similar ecological, watershed, habitat and land-use conditions and no overlapping characteristics with other waters. In the United States, AUs are typically based on political or hydrologic boundaries rather than on ecologically relevant features, so it can be difficult to detect changes in impairment status. Our goals were to evaluate if current AU designation criteria of an impaired water body in southeastern Idaho, USA that, like many U.S. waters, has three-quarters of its mainstem length divided into two AUs. We focused our evaluation in southeastern Idaho's Portneuf River, an impaired river and three-quarters of the river is divided into two AUs. We described biological and environmental conditions at multiple reaches within each AU. We used these data to (1) test if variability at the reach-scale is greater within or among AUs and, (2) to evaluate alternate AU boundaries based on multivariate analyses of reach-scale data. We found that some biological conditions had greater variability within an AU than between AUs. Multivariate analyses identified alternative, 2- and 3-group, AUs that reduced this variability. Our results suggest that the current AU designations in the mainstem Portneuf River contain ecologically distinct sections of river and that the existing AU boundaries should be reconsidered in light of the ecological conditions measured at the reach scale. Variation in biological integrity within designated AUs may complicate water quality and biological assessments, influence management decisions or affect where monitoring or mitigation resources are directed.

Coexistence in streams: do source-sink dynamics allow salamanders to persist with fish predators?

Theory suggests that source-sink dynamics can allow coexistence of intraguild predators and prey, but empirical evidence for this coexistence mechanism is limited. We used capture-mark-recapture, genetic methods, and stable isotopes to test whether source-sink dynamics promote coexistence between stream fishes, the intraguild predator, and stream salamanders (Dicamptodon aterrimus), the intraguild prey. Salamander populations from upstream reaches without fish were predicted to maintain or supplement sink populations in downstream reaches with fish. We found instead that downstream reaches with fish were not sinks even though fish consumed salamander larvae-apparent survival, recruitment, and population growth rate did not differ between upstream and downstream reaches. There was also no difference between upstream and downstream reaches in net emigration. We did find that D. aterrimus moved frequently along streams, but believe that this is a response to seasonal habitat changes rather than intraguild predation. Our study provides empirical evidence that local-scale mechanisms are more important than dispersal dynamics to coexistence of streams salamanders and fish. More broadly, it shows the value of empirical data on dispersal and gene flow for distinguishing between local and spatial mechanisms of coexistence.

Scale-dependent genetic structure of the Idaho giant salamander (Dicamptodon aterrimus) in stream networks.

The network architecture of streams and rivers constrains evolutionary, demographic and ecological processes of freshwater organisms. This consistent architecture also makes stream networks useful for testing general models of population genetic structure and the scaling of gene flow. We examined genetic structure and gene flow in the facultatively paedomorphic Idaho giant salamander, Dicamptodon aterrimus, in stream networks of Idaho and Montana, USA. We used microsatellite data to test population structure models by (i) examining hierarchical partitioning of genetic variation in stream networks; and (ii) testing for genetic isolation by distance along stream corridors vs. overland pathways. Replicated sampling of streams within catchments within three river basins revealed that hierarchical scale had strong effects on genetic structure and gene flow. amova identified significant structure at all hierarchical scales (among streams, among catchments, among basins), but divergence among catchments had the greatest structural influence. Isolation by distance was detected within catchments, and in-stream distance was a strong predictor of genetic divergence. Patterns of genetic divergence suggest that differentiation among streams within catchments was driven by limited migration, consistent with a stream hierarchy model of population structure. However, there was no evidence of migration among catchments within basins, or among basins, indicating that gene flow only counters the effects of genetic drift at smaller scales (within rather than among catchments). These results show the strong influence of stream networks on population structure and genetic divergence of a salamander, with contrasting effects at different hierarchical scales.