Conservation in a litre of air.

Since Ficetola et al. (2008) alerted ecologists and conservation biologists to the existence of environmental DNA (eDNA), the number of studies using eDNA has exploded, with a rapidly increasing diversity of research, monitoring, and management objectives. Initial applications focused on amphibians and fishes while today's taxonomic targets span the phylogenetic tree. The environmental media that are sampled have expanded from freshwater to saltwater to soils, and, most recently, to air. In this issue of Molecular Ecology Resources, Lynggaard et al. (Molecular Ecology Resources, 2023) use eDNA captured on air filters to census vertebrate biodiversity in a forest. With a three day, six sample period, 143 sample effort in a nature park in a rural area of Zealand, Denmark, their wild species detections comprised about 25% of the terrestrial vertebrates that are known to occur in the area, including about 33% of the mammal, 17% of the bird, and 60% of the amphibian species. This study demonstrates that air sampling for eDNA has the potential to become a powerful standard method for terrestrial biodiversity assessment that is complementary to traditional methods (e.g., trapping, visual and acoustic observation, collection of scat and hair).

Environmental DNA reveals the genetic diversity and population structure of an invasive species in the Laurentian Great Lakes.

Environmental DNA (eDNA) has been established as a noninvasive and efficient approach to sample genetic material from aquatic environments. Although most commonly used to determine species presence and measure biodiversity, eDNA approaches also hold great potential to obtain population-level genetic information from water samples. In this study, we sequenced a panel of multiallelic microsatellite markers from filtered water and fish tissue samples to uncover patterns of intraspecific diversity in the freshwater Round Goby (Neogobius melanostomus) across their invaded range in the Laurentian Great Lakes region. Although we found that the concentration of nuclear eDNA is lower than mitochondrial eDNA, we nonetheless detected over two-thirds of all nuclear alleles identified from genotyped tissues in our eDNA samples, with the greatest recovery of common alleles in the population. Estimates of allele frequencies and genetic variability within and between populations were detected from eDNA in patterns that were consistent with individual tissue-based estimates of genetic diversity and differentiation. The strongest genetic differentiation in both eDNA and tissues exists in an isolation by distance pattern. Our study demonstrates the potential for eDNA-based approaches to characterize key population parameters required to effectively monitor, manage, or sustain aquatic species.

Detecting and analysing intraspecific genetic variation with eDNA: From population genetics to species abundance.

Advancements in environmental DNA (eDNA) approaches have allowed for rapid and efficient species detections in diverse environments. Although most eDNA research is focused on leveraging genetic diversity to identify taxa, some recent studies have explored the potential for these approaches to detect within-species genetic variation, allowing for population genetic assessments and abundance estimates from environmental samples. However, we currently lack a framework outlining the key considerations specific to generating, analysing and applying eDNA data for these two purposes. Here, we discuss how various genetic markers differ with regard to genetic information and detectability in environmental samples and how analysis of eDNA samples differs from common tissue-based analyses. We then outline how it may be possible to obtain species absolute abundance estimates from eDNA by detecting intraspecific genetic variation in mixtures of DNA under multiple scenarios. We also identify the major causes contributing to allele detection and frequency errors in eDNA data, discuss their consequences for population-level analyses and outline bioinformatic approaches to detect and remove erroneous sequences. This review summarizes the key advances required to harness the full potential of eDNA-based intraspecific genetic variation to inform population-level questions in ecology, evolutionary biology and conservation management.

Environment and shipping drive environmental DNA beta-diversity among commercial ports.

The spread of nonindigenous species by shipping is a large and growing global problem that harms coastal ecosystems and economies and may blur coastal biogeographical patterns. This study coupled eukaryotic environmental DNA (eDNA) metabarcoding with dissimilarity regression to test the hypothesis that ship-borne species spread homogenizes port communities. We first collected and metabarcoded water samples from ports in Europe, Asia, Australia and the Americas. We then calculated community dissimilarities between port pairs and tested for effects of environmental dissimilarity, biogeographical region and four alternative measures of ship-borne species transport risk. We predicted that higher shipping between ports would decrease community dissimilarity, that the effect of shipping would be small compared to that of environment dissimilarity and shared biogeography, and that more complex shipping risk metrics (which account for ballast water and stepping-stone spread) would perform better. Consistent with our hypotheses, community dissimilarities increased significantly with environmental dissimilarity and, to a lesser extent, decreased with ship-borne species transport risks, particularly if the ports had similar environments and stepping-stone risks were considered. Unexpectedly, we found no clear effect of shared biogeography, and that risk metrics incorporating estimates of ballast discharge did not offer more explanatory power than simpler traffic-based risks. Overall, we found that shipping homogenizes eukaryotic communities between ports in predictable ways, which could inform improvements in invasive species policy and management. We demonstrated the usefulness of eDNA metabarcoding and dissimilarity regression for disentangling the drivers of large-scale biodiversity patterns. We conclude by outlining logistical considerations and recommendations for future studies using this approach.

Long-term macrophyte and snail community responses to population declines of invasive rusty crayfish (Faxonius rusticus).

A central focus of invasive species research has been on human efforts to eradicate invaders or reduce their abundance to mitigate the worst of their impacts. In some cases, however, populations of invasive species decline without human intervention, which may inform management responses to these invaders. Such is the case of the invasive rusty crayfish (Faxonius rusticus) in northern Wisconsin, USA, where systematic population monitoring since 1975 has revealed population declines in approximately half of the lakes surveyed. Population declines of invasive species without human intervention remain understudied, but there is even less research on how communities respond following such declines. Using 10 lakes in Vilas County, Wisconsin, we investigated community recovery of habitat (macrophytes) and prey (freshwater snails) of F. rusticus following up to 33 years of declines of this invader in some lakes using a dataset with a rare, long-term span over which consistent data were collected (1987, 2002, 2011, and 2020). We compared community responses in lakes where F. rusticus populations reached a peak and subsequently declined (boom-bust lakes) and lakes where our dataset only captured the decline of F. rusticus (bust lakes) to reference lakes with consistently high or low crayfish abundance over time. We found partial recovery of macrophytes and snails in the bust and boom-bust lakes where F. rusticus has declined, with recovery of macrophyte abundance and richness in the boom-bust lakes achieving levels observed in the low-crayfish reference lakes. Snail abundance and richness increased after declines of F. rusticus, though not to the level of the low-crayfish reference lakes, suggesting that snail recovery may lag macrophyte recovery because snails are dependent on macrophytes and associated periphyton for habitat. The recovery we document potentially represents long-term ecosystem resilience of lakes to biological invasions. Our results suggest that lake communities may recover without active restoration interventions after invasive crayfish population declines, although identifying which lakes experience these natural declines remains a priority for future research and management.

A Best Practices Case Study for Scientific Collaboration between Researchers and Managers.

Effective engagement among scientists, government agency staff, and policymakers is necessary for solving fisheries challenges, but remains challenging for a variety of reasons. We present seven practices learned from a collaborative project focused on invasive species in the Great Lakes region (USA-CAN). These practices were based on a researcher-manager model composed of a research team, a management advisory board, and a bridging organization. We suggest this type of system functions well when (1) the management advisory board is provided compelling rationale for engagement; (2) the process uses key individuals as communicators; (3) the research team thoughtfully selects organizations and individuals involved; (4) the funding entity provides logistical support and allows for (5) a flexible structure that prioritizes management needs; (6) a bridging organization sustains communication between in-person meetings; and (7) the project team determines and enacts a project endpoint. We predict these approaches apply equally effectively to other challenges at the research-management-policy interface, including reductions of water pollution, transitions to renewable energy, increasing food security, and addressing climate change.

Nuclear eDNA estimates population allele frequencies and abundance in experimental mesocosms and field samples.

Advances in environmental DNA (eDNA) methodologies have led to improvements in the ability to detect species and communities in aquatic environments, yet the majority of studies emphasize biological diversity at the species level by targeting variable sites within the mitochondrial genome. Here, we demonstrate that eDNA approaches also have the capacity to detect intraspecific diversity in the nuclear genome, allowing for assessments of population-level allele frequencies and estimates of the number of genetic contributors in an eDNA sample. Using a panel of microsatellite loci developed for the round goby (Neogobius melanostomus), we tested the similarity between eDNA-based and individual tissue-based estimates of allele frequencies from experimental mesocosms and in a field-based trial. Subsequently, we used a likelihood-based DNA mixture framework to estimate the number of unique genetic contributors in eDNA samples and in simulated mixtures of alleles. In both mesocosm and field samples, allele frequencies from eDNA were highly correlated with allele frequencies from genotyped round goby tissue samples, indicating nuclear markers can be reliably amplified from water samples. DNA mixture analyses were able to estimate the number of genetic contributors from mesocosm eDNA samples and simulated mixtures of DNA from up to 58 individuals, with the degree of positive or negative bias dependent on the filtering scheme of low-frequency alleles. With this study we document the application of eDNA and multiple amplicon-based methods to obtain intraspecific nuclear genetic information and estimate the absolute abundance of a species in eDNA samples. With proper validation, this approach has the potential to advance noninvasive survey methods to characterize populations and detect population-level genetic diversity.

Network analysis of ballast-mediated species transfer reveals important introduction and dispersal patterns in the Arctic.

Rapid climate change has wide-ranging implications for the Arctic region, including sea ice loss, increased geopolitical attention, and expanding economic activity resulting in a dramatic increase in shipping activity. As a result, the risk of harmful non-native marine species being introduced into this critical region will increase unless policy and management steps are implemented in response. Using data about shipping, ecoregions, and environmental conditions, we leverage network analysis and data mining techniques to assess, visualize, and project ballast water-mediated species introductions into the Arctic and dispersal of non-native species within the Arctic. We first identify high-risk connections between the Arctic and non-Arctic ports that could be sources of non-native species over 15 years (1997-2012) and observe the emergence of shipping hubs in the Arctic where the cumulative risk of non-native species introduction is increasing. We then consider how environmental conditions can constrain this Arctic introduction network for species with different physiological limits, thus providing a tool that will allow decision-makers to evaluate the relative risk of different shipping routes. Next, we focus on within-Arctic ballast-mediated species dispersal where we use higher-order network analysis to identify critical shipping routes that may facilitate species dispersal within the Arctic. The risk assessment and projection framework we propose could inform risk-based assessment and management of ship-borne invasive species in the Arctic.

Higher-order patterns of aquatic species spread through the global shipping network.

The introduction and establishment of nonindigenous species (NIS) through global ship movements poses a significant threat to marine ecosystems and economies. While ballast-vectored invasions have been partly addressed by some national policies and an international agreement regulating the concentrations of organisms in ballast water, biofouling-vectored invasions remain largely unaddressed. Development of additional efficient and cost-effective ship-borne NIS policies requires an accurate estimation of NIS spread risk from both ballast water and biofouling. We demonstrate that the first-order Markovian assumption limits accurate modeling of NIS spread risks through the global shipping network. In contrast, we show that higher-order patterns provide more accurate NIS spread risk estimates by revealing indirect pathways of NIS transfer using Species Flow Higher-Order Networks (SF-HON). Using the largest available datasets of non-indigenous species for Europe and the United States, we then compare SF-HON model predictions against those from networks that consider only first-order connections and those that consider all possible indirect connections without consideration of their significance. We show that not only SF-HONs yield more accurate NIS spread risk predictions, but there are important differences in NIS spread via the ballast and biofouling vectors. Our work provides information that policymakers can use to develop more efficient and targeted prevention strategies for ship-borne NIS spread management, especially as management of biofouling is of increasing concern.

Author Correction: Effects of sampling effort on biodiversity patterns estimated from environmental DNA metabarcoding surveys.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Habitat explains patterns of population decline for an invasive crayfish.

Invasive nonindigenous species are defined by their impacts: they substantially change native communities or ecosystems. Accordingly, invasive species might transform their habitats in ways that eventually become unfavorable to them, causing population declines or even extirpations. Here we use over 40 yr of systematically collected data on the abundance of the invasive rusty crayfish Faxonius rusticus from 17 lakes in northern Wisconsin, USA to explore whether population declines of this invader are related to the prevalence of rocky habitat, which shelters crayfish from predators and is unchanged by crayfish. We predicted that lakes with rock-dominated substrates would be resistant to F. rusticus population declines, whereas lakes lacking rock-dominated substrates would experience F. rusticus declines due to crayfish destruction of shelter-providing macrophytes. We found that in nearly one-half (47%) of the study lakes, F. rusticus experienced population declines over the study time period, and these lakes had significantly lower proportions of rock substrate than lakes that did not experience population declines. We recommend that more studies should investigate the potential for invasive species-mediated community or ecosystem feedbacks to eventually contribute to their own population declines.

eDNA metabarcoding as a new surveillance approach for coastal Arctic biodiversity.

Because significant global changes are currently underway in the Arctic, creating a large-scale standardized database for Arctic marine biodiversity is particularly pressing. This study evaluates the potential of aquatic environmental DNA (eDNA) metabarcoding to detect Arctic coastal biodiversity changes and characterizes the local spatio-temporal distribution of eDNA in two locations. We extracted and amplified eDNA using two COI primer pairs from ~80 water samples that were collected across two Canadian Arctic ports, Churchill and Iqaluit, based on optimized sampling and preservation methods for remote regions surveys. Results demonstrate that aquatic eDNA surveys have the potential to document large-scale Arctic biodiversity change by providing a rapid overview of coastal metazoan biodiversity, detecting nonindigenous species, and allowing sampling in both open water and under the ice cover by local northern-based communities. We show that DNA sequences of ~50% of known Canadian Arctic species and potential invaders are currently present in public databases. A similar proportion of operational taxonomic units was identified at the species level with eDNA metabarcoding, for a total of 181 species identified at both sites. Despite the cold and well-mixed coastal environment, species composition was vertically heterogeneous, in part due to river inflow in the estuarine ecosystem, and differed between the water column and tide pools. Thus, COI-based eDNA metabarcoding may quickly improve large-scale Arctic biomonitoring using eDNA, but we caution that aquatic eDNA sampling needs to be standardized over space and time to accurately evaluate community structure changes.

Effects of sampling effort on biodiversity patterns estimated from environmental DNA metabarcoding surveys.

Environmental DNA (eDNA) metabarcoding can greatly enhance our understanding of global biodiversity and our ability to detect rare or cryptic species. However, sampling effort must be considered when interpreting results from these surveys. We explored how sampling effort influenced biodiversity patterns and nonindigenous species (NIS) detection in an eDNA metabarcoding survey of four commercial ports. Overall, we captured sequences from 18 metazoan phyla with minimal differences in taxonomic coverage between 18 S and COI primer sets. While community dissimilarity patterns were consistent across primers and sampling effort, richness patterns were not, suggesting that richness estimates are extremely sensitive to primer choice and sampling effort. The survey detected 64 potential NIS, with COI identifying more known NIS from port checklists but 18 S identifying more operational taxonomic units shared between three or more ports that represent un-recorded potential NIS. Overall, we conclude that eDNA metabarcoding surveys can reveal global similarity patterns among ports across a broad array of taxa and can also detect potential NIS in these key habitats. However, richness estimates and species assignments require caution. Based on results of this study, we make several recommendations for port eDNA sampling design and suggest several areas for future research.

Environmental DNA metabarcoding: Transforming how we survey animal and plant communities.

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High-throughput sequencing ("HTS") platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed "environmental DNA" or "eDNA"). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called "eDNA metabarcoding" and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.

Environmental DNA (eDNA) detects the invasive rusty crayfish Orconectes rusticus at low abundances.

Early detection is invaluable for the cost-effective control and eradication of invasive species, yet many traditional sampling techniques are ineffective at the low population abundances found at the onset of the invasion process. Environmental DNA (eDNA) is a promising and sensitive tool for early detection of some invasive species, but its efficacy has not yet been evaluated for many taxonomic groups and habitat types.We evaluated the ability of eDNA to detect the invasive rusty crayfish Orconectes rusticus and to reflect patterns of its relative abundance, in upper Midwest, USA, inland lakes. We paired conventional baited trapping as a measure of crayfish relative abundance with water samples for eDNA, which were analysed in the laboratory with a qPCR assay. We modelled detection probability for O. rusticus eDNA using relative abundance and site characteristics as covariates and also tested the relationship between eDNA copy number and O. rusticus relative abundance.We detected O. rusticus eDNA in all lakes where this species was collected by trapping, down to low relative abundances, as well as in two lakes where trap catch was zero. Detection probability of O. rusticus eDNA was well predicted by relative abundance of this species and lake water clarity. However, there was poor correspondence between eDNA copy number and O. rusticus relative abundance estimated by trap catches. Synthesis and applications. Our study demonstrates a field and laboratory protocol for eDNA monitoring of crayfish invasions, with results of statistical models that provide guidance of sampling effort and detection probabilities for researchers in other regions and systems. We propose eDNA be included as a tool in surveillance for invasive or imperilled crayfishes and other benthic arthropods.

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.

Parasites alter freshwater communities in mesocosms by modifying invasive crayfish behavior.

Parasites can alter communities by reducing densities of keystone hosts, but few studies have examined how trait-mediated indirect effects of parasites can alter ecological communities. We test how trematode parasites (Microphallus spp.) that affect invasive crayfish (Orconectes rusticus) behavior alter how crayfish impact lake littoral communities. O. rusticus drive community composition in north temperate lakes, and predatory fish can reduce crayfish activity and feeding. In laboratory studies, Microphallus parasites also alter O. rusticus behavior: infected O. rusticus eat fewer macroinvertebrates and are bolder near predatory fish than uninfected individuals. We used a 2 x 2 factorial experiment to test how predatory fish and parasites affect O. rusticus impacts in large mesocosms over 4 weeks. We predicted (1) that when predators were absent, infected crayfish would have lower impacts than uninfected crayfish on macrophytes and macroinvertebrates (as well as reduced growth and higher mortality). However, (2) when predators were present but unable to consume crayfish, infected crayfish would have greater impacts (as well as greater growth and lower mortality) than uninfected crayfish because of increased boldness. Because of its effect on crayfish feeding behavior, we also predicted (3) that infection would alter macrophyte and macroinvertebrate community composition. In contrast to our first hypothesis, we found that infected and uninfected crayfish had similar impacts on lower trophic levels when predators were absent. Across all treatments, infected crayfish were more likely to be outside shelters and had greater growth than uninfected crayfish, suggesting that the reduced feeding observed in short-term experiments does not occur over longer timescales. However, in support of the second hypothesis, when predatory fish were present, infected crayfish ate more macroinvertebrates than did uninfected crayfish, likely due to increased boldness. We also observed a trend for greater macrophyte consumption associated with infection and a trend indicating infection might alter macroinvertebrate community composition. Our results suggest that parasites can alter aquatic communities in mesocosms merely by modifying host behavior.

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.