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.

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.

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.

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.

Quantifying environmental DNA signals for aquatic invasive species across multiple detection platforms.

The use of molecular surveillance techniques has become popular among aquatic researchers and managers due to the improved sensitivity and efficiency compared to traditional sampling methods. Rapid expansion in the use of environmental DNA (eDNA), paired with the advancement of molecular technologies, has resulted in new detection platforms and techniques. In this study we present a comparison of three eDNA surveillance platforms: traditional polymerase chain reaction (PCR), quantitative PCR (qPCR), and digital droplet PCR (ddPCR) in which water samples were collected over a 24 h time period from mesocosm experiments containing a population gradient of invasive species densities. All platforms reliably detected the presence of DNA, even at low target organism densities within the first hour. The two quantitative platforms (qPCR and ddPCR) produced similar estimates of DNA concentrations. The analyses completed with ddPCR was faster from sample collection through analyses and cost approximately half the expenditure of qPCR. Although a new platform for eDNA surveillance of aquatic species, ddPCR was consistent with more commonly used qPCR and a cost-effective means of estimating DNA concentrations. Use of ddPCR by researchers and managers should be considered in future eDNA surveillance applications.

Environmental conditions influence eDNA persistence in aquatic systems.

Environmental DNA (eDNA) surveillance holds great promise for improving species conservation and management. However, few studies have investigated eDNA dynamics under natural conditions, and interpretations of eDNA surveillance results are clouded by uncertainties about eDNA degradation. We conducted a literature review to assess current understanding of eDNA degradation in aquatic systems and an experiment exploring how environmental conditions can influence eDNA degradation. Previous studies have reported macrobial eDNA persistence ranging from less than 1 day to over 2 weeks, with no attempts to quantify factors affecting degradation. Using a SYBR Green quantitative PCR assay to observe Common Carp ( Cyprinus carpio ) eDNA degradation in laboratory mesocosms, our rate of Common Carp eDNA detection decreased over time. Common Carp eDNA concentration followed a pattern of exponential decay, and observed decay rates exceeded previously published values for aquatic macrobial eDNA. Contrary to our expectations, eDNA degradation rate declined as biochemical oxygen demand, chlorophyll, and total eDNA (i.e., from any organism) concentration increased. Our results help explain the widely divergent, previously published estimates for eDNA degradation. Measurements of local environmental conditions, consideration of environmental influence on eDNA detection, and quantification of local eDNA degradation rates will help interpret future eDNA surveillance results.

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.

Conservation in a cup of water: estimating biodiversity and population abundance from environmental DNA.

Three mantras often guide species and ecosystem management: (i) for preventing invasions by harmful species, 'early detection and rapid response'; (ii) for conserving imperilled native species, 'protection of biodiversity hotspots'; and (iii) for assessing biosecurity risk, 'an ounce of prevention equals a pound of cure.' However, these and other management goals are elusive when traditional sampling tools (e.g. netting, traps, electrofishing, visual surveys) have poor detection limits, are too slow or are not feasible. One visionary solution is to use an organism's DNA in the environment (eDNA), rather than the organism itself, as the target of detection. In this issue of Molecular Ecology, Thomsen et al. (2012) provide new evidence demonstrating the feasibility of this approach, showing that eDNA is an accurate indicator of the presence of an impressively diverse set of six aquatic or amphibious taxa including invertebrates, amphibians, a fish and a mammal in a wide range of freshwater habitats. They are also the first to demonstrate that the abundance of eDNA, as measured by qPCR, correlates positively with population abundance estimated with traditional tools. Finally, Thomsen et al. (2012) demonstrate that next-generation sequencing of eDNA can quantify species richness. Overall, Thomsen et al. (2012) provide a revolutionary roadmap for using eDNA for detection of species, estimates of relative abundance and quantification of biodiversity.

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.

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.

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).

Chance establishment for sexual, semelparous species: overcoming the Allee effect.

We formalize the establishment process for a sexual, semelparous organism through the use of hierarchical probability modeling from parameters of survival, probability of being female, probability of being fertilized, and expected fecundity. We show how to calculate the expected per capita growth rate and probability of extinction. An Allee effect is observed if the expected population growth rate decreases as the initial population size decreases. The model can be further extended as a stochastic process to evaluate the probability of extinction in subsequent generations. One of the novel results is the formulation of an analytical probability distribution for the next generation population size. As case studies, we use the Chinese mitten crab (Eriocheir sinensis) and the apple snail (Pomacea canaliculata), both of which appear on the World Conservation Union's list of 100 worst invaders. We evaluate the strength of the Allee effect and conclude that apple snails experience a weak Allee effect and Chinese mitten crabs experience a strong Allee effect. We emphasize one scenario where the stochastic process reveals that invasion risk can be estimated by the probability of the survival of one fertilized female, because the expected fecundity for one surviving female overwhelms the system such that population persistence is almost certain.

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.

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.

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.

Waiting for invasions: a framework for the arrival of nonindigenous species.

The process of nonindigenous species (NIS) arrival has received limited theoretical consideration despite importance in predicting and preventing the establishment of NIS. We formulate a mechanistically based hierarchical model of NIS arrival and demonstrate simplifications leading to a marginal distribution of the number of surviving introduced individuals from parameters of survival probability and propagule pressure. The marginal distribution is extended as a stochastic process from which establishment emerges with a waiting time distribution. This provides a probability of NIS establishment within a specified period and may be useful for identifying patterns of successful invaders. However, estimates of both the propagule pressure and the individual survival probability are rarely available for NIS, making estimates of the probability of establishment difficult. Alternatively, researchers are able to measure proportional estimates of propagule pressure through models of NIS transport, such as gravity models, or of survival probability through habitat-matching indexes measuring the similarity between potentially occupied and native NIS ranges. Therefore, we formulate the relative waiting time between two locations and the probability of one location being invaded before the other.

Predicting invasion risk using measures of introduction effort and environmental niche models.

The Chinese mitten crab (Eriocheir sinensis) is native to east Asia, is established throughout Europe, and is introduced but geographically restricted in North America. We developed and compared two separate environmental niche models using genetic algorithm for rule set prediction (GARP) and mitten crab occurrences in Asia and Europe to predict the species' potential distribution in North America. Since mitten crabs must reproduce in water with >15% per hundred salinity, we limited the potential North American range to freshwater habitats within the highest documented dispersal distance (1260 km) and a more restricted dispersal limit (354 km) from the sea. Applying the higher dispersal distance, both models predicted the lower Great Lakes, most of the eastern seaboard, the Gulf of Mexico and southern extent of the Mississippi River watershed, and the Pacific northwest as suitable environment for mitten crabs, but environmental match for southern states (below 35 degrees N) was much lower for the European model. Use of the lower range with both models reduced the expected range, especially in the Great Lakes, Mississippi drainage, and inland areas of the Pacific Northwest. To estimate the risk of introduction of mitten crabs, the amount of reported ballast water discharge into major United States ports from regions in Asia and Europe with established mitten crab populations was used as an index of introduction effort. Relative risk of invasion was estimated based on a combination of environmental match and volume of unexchanged ballast water received (July 1999-December 2003) for major ports. The ports of Norfolk and Baltimore were most vulnerable to invasion and establishment, making Chesapeake Bay the most likely location to be invaded by mitten crabs in the United States. The next highest risk was predicted for Portland, Oregon. Interestingly, the port of Los Angeles/Long Beach, which has a large shipping volume, had a low risk of invasion. Ports such as Jacksonville, Florida, had a medium risk owing to small shipping volume but high environmental match. This study illustrates that the combination of environmental niche- and vector-based models can provide managers with more precise estimates of invasion risk than can either of these approaches alone.

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.