Integrating invasive species risk assessment into environmental DNA metabarcoding reference libraries.

Environmental DNA (eDNA) metabarcoding has shown promise as a tool for estimating biodiversity and early detection of invasive species. In aquatic systems, advantages of this method include the ability to concurrently monitor biodiversity and detect incipient invasions simply through the collection and analysis of water samples. However, depending on the molecular markers chosen for a given study, reference libraries containing target sequences from present species may limit the usefulness of eDNA metabarcoding. To explore the extent of this issue and how it may be resolved to aid biodiversity and invasive species early detection goals, we focus on fishes in the well-studied Laurentian Great Lakes region. First, we provide a synthesis of species currently known from the region and of non-indigenous species identified as threats by international, national, regional, and introduction pathway-specific fish risk assessments. With these species lists, we then evaluate 23 primer pairs commonly used in fish eDNA metabarcoding with available databases of sequence coverage and species specificity. Finally, we identify established and potentially invasive non-indigenous fish that should be prioritized for genetic sequencing to ensure robust eDNA metabarcoding for the region. Our results should increase confidence in using eDNA metabarcoding for fisheries conservation and management in the Great Lakes region and help prioritize reference sequencing efforts. The ultimate utility of eDNA metabarcoding approaches will come when conservation management of existing fish communities is integrated with early detection efforts for invasive species surveillance to assess total fish biodiversity.

Can we manage fisheries with the inherent uncertainty from eDNA?

Environmental (e)DNA, as a general approach in aquatic systems, seeks to connect the presence of species' genetic material in the water and hence to infer the species' physical presence. However, fisheries managers face making decisions with risk and uncertainty when eDNA indicates a fish is present but traditional methods fail to capture the fish. In comparison with traditional methods such as nets, electrofishing and piscicides, eDNA approaches have more sources of underlying error that could give rise to false positives. This has resulted in some managers to question whether eDNA can be used to make management decisions because there is no fish in hand. As a relatively new approach, the methods and techniques have quickly evolved to improve confidence in eDNA. By evaluating an eDNA based research programmes through the pattern of the eDNA signal, assay design, experimental design, quality assurance and quality control checks, data analyses and concurrent search for fish using traditional gears, the evidence for fish presence can be evaluated to build confidence in the eDNA approach. The benefits for fisheries management from adopting an eDNA approach are numerous but include cost effectiveness, broader geographic coverage of habitat occupancy, early detection of invasive species, non-lethal stock assessments, exploration of previously inaccessible aquatic environments and discovery of new species hidden beneath the water's surface. At a time when global freshwater and marine fisheries are facing growing threats from over-harvest, pollution and climate change, we anticipate that growing confidence in eDNA will overcome the inherent uncertainty of not having a fish in hand and will empower the informed management actions necessary to protect and restore our fisheries.

At Palmyra Atoll, the fish-community environmental DNA signal changes across habitats but not with tides.

At Palmyra Atoll, the environmental DNA (eDNA) signal on tidal sand flats was associated with fish biomass density and captured 98%-100% of the expected species diversity there. Although eDNA spilled over across habitats, species associated with reef habitat contributed more eDNA to reef sites than to sand-flat sites, and species associated with sand-flat habitat contributed more eDNA to sand-flat sites than to reef sites. Tides did not disrupt the sand-flat habitat signal. At least 25 samples give a coverage >97.5% at this diverse, tropical, marine system.

Population dynamics of threatened Lahontan cutthroat trout in Summit Lake, Nevada.

Summit Lake, Nevada (USA) is the last high-desert terminal lake to have a native self-sustaining population of threatened Lahontan cutthroat trout (Oncorhynchus clarkii henshawi). From spring 2015 to fall 2017, we quantified adult abundance and survival and the total annual spawning run. Abundance and survival were estimated with mark-recapture using PIT tags, and the annual spawning run was estimated with PIT tag detections and counts of spawners. Adult abundance fluctuated from 830 (95% CI 559-1248) to 1085 (95% CI 747-1614), with no overall temporal trend, as a decrease in male abundance was generally offset by an equal increase in female abundance. Estimated mean adult survival was 0.51 (95% CI 0.44-0.58). The spawning run increased from 645 (2015) to 868 (2016), but then decreased slightly to 824 (2017, mean = 789 ± 118). Female spawners increased in 2016 but decreased slightly in 2017, whereas male spawners decreased each year. In addition, the proportion of adults that spawned each year increased overall. Our study suggests that the adult population remained stable although most of the study period included the recent, severe regional drought in the western United States (2012-2016).

Looking where it's hard to see: a case study documenting rare Eucyclogobius newberryi presence in a California lagoon.

Environmental DNA (eDNA) analysis is increasingly used for biomonitoring and research of fish populations and communities by environmental resource managers and academic researchers. Although managers are much interested in expanding the use of eDNA as a survey technique, they are sceptical about both its utility (given that information is often limited to presence/absence of a species) and feasibility (given the need for proper laboratory facilities for sample processing). Nonetheless, under the right circumstances, eDNA analysis is cost-effective compared to many traditional aquatic survey methods and does not disturb habitat or harm the animals being surveyed. This article presents a case study in which eDNA analysis was successfully used to document the presence of a rare fish species in a waterway earmarked for restoration. The authors discuss the conditions that allowed this study to occur quickly and smoothly and speculate on how the goals of researchers and managers can be integrated for efficient and informative use of this tool.

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.

Strong Evidence for an Intraspecific Metabolic Scaling Coefficient Near 0.89 in Fish.

As an example of applying the evidential approach to statistical inference, we address one of the longest standing controversies in ecology, the evidence for, or against, a universal metabolic scaling relationship between metabolic rate and body mass. Using fish as our study taxa, we curated 25 studies with measurements of standard metabolic rate, temperature, and mass, with 55 independent trials and across 16 fish species and confronted this data with flexible random effects models. To quantify the body mass - metabolic rate relationship, we perform model selection using the Schwarz Information Criteria (ΔSIC), an established evidence function. Further, we formulate and justify the use of ΔSIC intervals to delineate the values of the metabolic scaling relationship that should be retained for further consideration. We found strong evidence for a metabolic scaling coefficient of 0.89 with a ΔSIC interval spanning 0.82 to 0.99, implying that mechanistically derived coefficients of 0.67, 0.75, and 1, are not supported by the data. Model selection supports the use of a random intercepts and random slopes by species, consistent with the idea that other factors, such as taxonomy or ecological or lifestyle characteristics, may be critical for discerning the underlying process giving rise to the data. The evidentialist framework applied here, allows for further refinement given additional data and more complex models.

Measuring global fish species richness with eDNA metabarcoding.

Despite mounting threats to global freshwater and marine biodiversity, including climate change, habitat alteration, overharvesting and pollution, we struggle to know which species are present below the water's surface that are suffering from these stressors. However, the idea that a water sample containing environmental DNA (eDNA) can be screened using high-throughput sequencing and bioinformatics to reveal the identity of aquatic species is a revolutionary advance for studying the patterns of species extirpation, invasive species establishment and the dynamics of species richness. To date, many of the critical tests of fisheries diversity using this metabarcoding approach have been conducted in lower diversity systems (<40 fish species), but in this issue of Molecular Ecology Resources, Cilleros et al. (2018) described their eDNA application in the species-rich French Guiana fishery (>200 fish species) and showed the greater potential and some limitations of using eDNA in species-rich environments.

Investigating diversity of pathogenic microbes in commercial bait trade water.

The recreational bait trade is a potential pathway for pathogen introduction and spread when anglers dump bait shop sourced water into aquatic systems. Despite this possibility, and previous recognition of the importance of the bait trade in the spread of aquatic invasive species (AIS), to date there has been no region wide survey documenting pathogens in retail bait shops. In this study, we analyzed 96 environmental DNA samples from retail bait shops around the Great Lakes region to identify pathogens, targeting the V4 hypervariable region of the 16S rRNA gene. Additionally, we used samples from one site in Lake Michigan as a comparison to pathogen diversity and abundance in natural aquatic systems. Our results identified nine different groups of pathogens in the bait shop samples, including those that pose risks to both humans and fish species. Compared to wild sourced samples, the bait shops had higher relative abundance and greater taxonomic diversity. These findings suggest that the bait trade represents a potentially important pathway that could introduce and spread pathogens throughout the Great Lakes region. Improving pathogen screening and angler outreach should be used in combination to aid in preventing the future spread of high risk pathogens.

Controls on eDNA movement in streams: Transport, Retention, and Resuspension.

Advances in detection of genetic material from species in aquatic ecosystems, including environmental DNA (eDNA), have improved species monitoring and management. eDNA from target species can readily move in streams and rivers and the goal is to measure it, and with that infer where and how abundant species are, adding great value to delimiting species invasions, monitoring and protecting rare species, and estimating biodiversity. To date, we lack an integrated framework that identifies environmental factors that control eDNA movement in realistic, complex, and heterogeneous flowing waters. To this end, using an empirical approach and a simple conceptual model, we propose a framework of how eDNA is transported, retained, and resuspended in stream systems. Such an understanding of eDNA dispersal in streams will be essential for designing optimized sampling protocols and subsequently estimating biomass or organismal abundance. We also discuss guiding principles for more effective use of eDNA methods, highlighting the necessity of understanding these parameters for use in future predictive modeling of eDNA transport.

Estimating species richness using environmental DNA.

The foundation for any ecological study and for the effective management of biodiversity in natural systems requires knowing what species are present in an ecosystem. We assessed fish communities in a stream using two methods, depletion-based electrofishing and environmental DNA metabarcoding (eDNA) from water samples, to test the hypothesis that eDNA provides an alternative means of determining species richness and species identities for a natural ecosystem. In a northern Indiana stream, electrofishing yielded a direct estimate of 12 species and a mean estimated richness (Chao II estimator) of 16.6 species with a 95% confidence interval from 12.8 to 42.2. eDNA sampling detected an additional four species, congruent with the mean Chao II estimate from electrofishing. This increased detection rate for fish species between methods suggests that eDNA sampling can enhance estimation of fish fauna in flowing waters while having minimal sampling impacts on fish and their habitat. Modern genetic approaches therefore have the potential to transform our ability to build a more complete list of species for ecological investigations and inform management of aquatic ecosystems.

Influence of Stream Bottom Substrate on Retention and Transport of Vertebrate Environmental DNA.

While environmental DNA (eDNA) is now being regularly used to detect rare and elusive species, detection in lotic environments comes with a caveat: The species being detected is likely some distance upstream from the point of sampling. Here, we conduct a series of seminatural stream experiments to test the sensitivity of new digital droplet PCR (ddPCR) to detect low concentrations of eDNA in a lotic system, measure the residence time of eDNA compared to a conservative tracer, and we model the transport of eDNA in this system. We found that while ddPCR improves our sensitivity of detection, the residence time and transport of eDNA does not follow the same dynamics as the conservative tracer and necessitates a more stochastic framework for modeling eDNA transport. There was no evidence for differences in the transport of eDNA due to substrate type. The relatively large amount of unexplained variability in eDNA transport reveals the need for uncovering mechanisms and processes by which eDNA is transported downstream leading to species detections, particularly when inferences are to be made in natural systems where eDNA is being used for conservation management.

Using Environmental DNA for Invasive Species Surveillance and Monitoring.

The method employed for environmental DNA (eDNA) surveillance for detection and monitoring of rare species in aquatic systems has evolved dramatically since its first large-scale applications. Both active (targeted) and passive (total diversity) surveillance methods provide helpful information for management groups, but each has a suite of techniques that necessitate proper equipment training and use. The protocols described in this chapter represent some of the latest iterations in eDNA surveillance being applied in aquatic and marine systems.

Confronting species distribution model predictions with species functional traits.

Species distribution models are valuable tools in studies of biogeography, ecology, and climate change and have been used to inform conservation and ecosystem management. However, species distribution models typically incorporate only climatic variables and species presence data. Model development or validation rarely considers functional components of species traits or other types of biological data. We implemented a species distribution model (Maxent) to predict global climate habitat suitability for Grass Carp (Ctenopharyngodon idella). We then tested the relationship between the degree of climate habitat suitability predicted by Maxent and the individual growth rates of both wild (N = 17) and stocked (N = 51) Grass Carp populations using correlation analysis. The Grass Carp Maxent model accurately reflected the global occurrence data (AUC = 0.904). Observations of Grass Carp growth rate covered six continents and ranged from 0.19 to 20.1 g day(-1). Species distribution model predictions were correlated (r = 0.5, 95% CI (0.03, 0.79)) with observed growth rates for wild Grass Carp populations but were not correlated (r = -0.26, 95% CI (-0.5, 0.012)) with stocked populations. Further, a review of the literature indicates that the few studies for other species that have previously assessed the relationship between the degree of predicted climate habitat suitability and species functional traits have also discovered significant relationships. Thus, species distribution models may provide inferences beyond just where a species may occur, providing a useful tool to understand the linkage between species distributions and underlying biological mechanisms.

Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding.

Freshwater fauna are particularly sensitive to environmental change and disturbance. Management agencies frequently use fish and amphibian biodiversity as indicators of ecosystem health and a way to prioritize and assess management strategies. Traditional aquatic bioassessment that relies on capture of organisms via nets, traps and electrofishing gear typically has low detection probabilities for rare species and can injure individuals of protected species. Our objective was to determine whether environmental DNA (eDNA) sampling and metabarcoding analysis can be used to accurately measure species diversity in aquatic assemblages with differing structures. We manipulated the density and relative abundance of eight fish and one amphibian species in replicated 206-L mesocosms. Environmental DNA was filtered from water samples, and six mitochondrial gene fragments were Illumina-sequenced to measure species diversity in each mesocosm. Metabarcoding detected all nine species in all treatment replicates. Additionally, we found a modest, but positive relationship between species abundance and sequencing read abundance. Our results illustrate the potential for eDNA sampling and metabarcoding approaches to improve quantification of aquatic species diversity in natural environments and point the way towards using eDNA metabarcoding as an index of macrofaunal species abundance.

Successful survival, growth, and reproductive potential of quagga mussels in low calcium lake water: is there uncertainty of establishment risk?

The risk of quagga mussel (Dreissena rostriformis bugensis Andrusov 1897) establishment into water-bodies of the western US has expanded the geographic concern regarding the ecological and economic impacts this species will have in aquatic ecosystems. Thresholds based on calcium concentrations, an element critical for mussel growth and physiology, have been used as a primary predictor of quagga mussel establishment success to aid management decisions. We evaluated the invasion potential of quagga mussels in low calcium waters using laboratory experiments to compare the survival, growth and reproductive potential of adult mussels held for 90 days at low (9 and 12 ppm), moderate (15 to 32 ppm) and high (72 ppm) calcium water concentrations. In conjunction with adult experiments, veliger stage survival, growth and settlement were evaluated under similar low, moderate, and high calcium water treatments. Adult mussels survived, grew and showed reproductive potential in low calcium water (12 ppm). Veligers were also able to survive, grow and settle in low calcium water. Higher levels of natural seston biomass appeared to improve adult mussel life history performance in low calcium water. Survival curve analysis predicted that 99% adult mortality could occur in <170 days at 9 ppm and 12 ppm, however water with >15 ppm could have adults surviving more than a year. The results from these bioassays provide further evidence that quagga mussels have higher risk of establishment in low calcium lakes if habitats exist that have slightly elevated calcium. These results should help emphasize the vulnerability of water-body in the 12 to 15 ppm calcium range that could potentially be at risk of establishing sustainable quagga mussel populations. Furthermore, these results provide insights into the uncertainty of using a single parameter in assigning establishment risk given the complexity of variables in specific water-bodies that influence life history performance of introduced species.

Improving confidence in environmental DNA species detection.

Will we catch fish today? Our grandfathers' responses were usually something along the lines of, 'Probably. I've caught them here before'. One of the foundations of ecology is identifying which species are present, and where. This informs our understanding of species richness patterns, spread of invasive species, and loss of threatened and endangered species due to environmental change. However, our understanding is often lacking, particularly in aquatic environments where biodiversity remains hidden below the water's surface. The emerging field of metagenetic species surveillance is aiding our ability to rapidly determine which aquatic species are present, and where. In this issue of Molecular Ecology Resources, Ficetola et al. () provide a framework for metagenetic environmental DNA surveillance to foster the confidence of our grandfathers' fishing prowess by more rigorously evaluating the replication levels necessary to quantify detection errors and ultimately improving our confidence in aquatic species presence.

Estimating relative risk of within-lake aquatic plant invasion using combined measures of recreational boater movement and habitat suitability.

Effective monitoring, prevention and impact mitigation of nonindigenous aquatic species relies upon the ability to predict dispersal pathways and receiving habitats with the greatest risk of establishment. To examine mechanisms affecting species establishment within a large lake, we combined observations of recreational boater movements with empirical measurements of habitat suitability represented by nearshore wave energy to assess the relative risk of Eurasian watermilfoil (Myriophyllum spicatum) establishment. The model was evaluated using information from a 17 year (1995-2012) sequence of M. spicatum presence and absence monitoring. M. spicatum presence was not specifically correlated with recreational boater movements; however its establishment appears to be limited by wave action in Lake Tahoe. Of the sites in the "High" establishment risk category (n = 37), 54% had current or historical infestations, which included 8 of the 10 sites with the highest relative risk. Of the 11 sites in the "Medium" establishment risk category, 5 had current or historical M. spicatum populations. Most (76%) of the sites in the "Low" establishment risk category were observed in locations with higher wave action. Four sites that received zero boater visits from infested locations were occupied by M. spicatum. This suggests that the boater survey either represents incomplete coverage of boater movement, or other processes, such as the movement of propagules by surface currents or introductions from external sources are important to the establishment of this species. This study showed the combination of habitat specific and dispersal data in a relative risk framework can potentially reduce uncertainty in estimates of invasion risk.

The use of environmental DNA in invasive species surveillance of the Great Lakes commercial bait trade.

Over 180 non-native species have been introduced in the Laurentian Great Lakes region, many posing threats to native species and ecosystem functioning. One potential pathway for introductions is the commercial bait trade; unknowing or unconcerned anglers commonly release unused bait into aquatic systems. Previous surveillance efforts of this pathway relied on visual inspection of bait stocks in retail shops, which can be time and cost prohibitive and requires a trained individual that can rapidly and accurately identify cryptic species. Environmental DNA (eDNA) surveillance, a molecular tool that has been used for surveillance in aquatic environments, can be used to efficiently detect species at low abundances. We collected and analyzed 576 eDNA samples from 525 retail bait shops throughout the Laurentian Great Lake states. We used eDNA techniques to screen samples for multiple aquatic invasive species (AIS) that could be transported in the bait trade, including bighead (Hypophthalmichthys nobilis) and silver carp (H. molitrix), round goby (Neogobius melanostomus), tubenose goby (Proterorhinus marmoratus), Eurasian rudd (Scardinius erythrophthalmus), and goldfish (Carassius auratus). Twenty-seven samples were positive for at least one target species (4.7% of samples), and all target species were found at least once, except bighead carp. Despite current regulations, the bait trade remains a potential pathway for invasive species introductions in the Great Lakes region. Alterations to existing management strategies regarding the collection, transportation, and use of live bait are warranted, including new and updated regulations, to prevent future introductions of invasive species in the Great Lakes via the bait trade.