The Multiple States of Environmental DNA and What Is Known about Their Persistence in Aquatic Environments.

Increased use of environmental DNA (eDNA) analysis for indirect species detection has spurred the need to understand eDNA persistence in the environment. Understanding the persistence of eDNA is complex because it exists in a mixture of different states (e.g., dissolved, particle adsorbed, intracellular, and intraorganellar), and each state is expected to have a specific decay rate that depends on environmental parameters. Thus, improving knowledge about eDNA conversion rates between states and the reactions that degrade eDNA in different states is needed. Here, we focus on eukaryotic extraorganismal eDNA, outline how water chemistry and suspended mineral particles likely affect conversion among each eDNA state, and indicate how environmental parameters affect persistence of states in the water column. On the basis of deducing these controlling parameters, we synthesized the eDNA literature to assess whether we could already derive a general understanding of eDNA states persisting in the environment. However, we found that these parameters are often not being measured or reported when measured, and in many cases very few experimental data exist from which to draw conclusions. Therefore, further study of how environmental parameters affect eDNA state conversion and eDNA decay in aquatic environments is needed. We recommend analytic controls that can be used during the processing of water to assess potential losses of different eDNA states if all were present in a water sample, and we outline future experimental work that would help determine the dominant eDNA states in water.

Trade-offs between reducing complex terminology and producing accurate interpretations from environmental DNA: Comment on "Environmental DNA: What's behind the term?" by Pawlowski et al., (2020).

In a recent paper, "Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring," Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived.

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.

Molecular Population Genetics of the Northern Elephant Seal Mirounga angustirostris.

The northern elephant seal, Mirounga angustirostris, was heavily hunted and declared extinct in the 19th century. However, a colony remained on remote Guadalupe Island, Mexico and the species has since repopulated most of its historical distribution. Here, we present a comprehensive evaluation of genetic variation in the species. First, we assess the effect of the demographic bottleneck on microsatellite variability and compare it with that found in other pinnipeds, demonstrating levels of variation similar to that in species that continue to be threatened with extinction. Next, we use sequence data from these markers to demonstrate that some of the limited polymorphism predates the bottleneck. However, most contemporary variation appears to have arisen recently and persisted due to exponential growth. We also describe how we use the range in allele size of microsatellites to estimate ancestral effective population size before the bottleneck, demonstrating a large reduction in effective size. We then employ a classical method for bacteria to estimate the microsatellite mutation rate in the species, deriving an estimate that is extremely similar to that estimated for a similar set of loci in humans, indicating consistency of microsatellite mutation rates in mammals. Finally, we find slight significant structure between some geographically separated colonies, although its biological significance is unclear. This work demonstrates that genetic analysis can be useful for evaluating the population biology of the northern elephant seal, in spite of the bottleneck that removed most genetic variation from the species.

Fishing in the Water: Effect of Sampled Water Volume on Environmental DNA-Based Detection of Macroinvertebrates.

Accurate detection of organisms is crucial for the effective management of threatened and invasive species because false detections directly affect the implementation of management actions. The use of environmental DNA (eDNA) as a species detection tool is in a rapid development stage; however, concerns about accurate detections using eDNA have been raised. We evaluated the effect of sampled water volume (0.25 to 2 L) on the detection rate for three macroinvertebrate species. Additionally, we tested (depending on the sampled water volume) what amount of total extracted DNA should be screened to reduce uncertainty in detections. We found that all three species were detected in all volumes of water. Surprisingly, however, only one species had a positive relationship between an increased sample volume and an increase in the detection rate. We conclude that the optimal sample volume might depend on the species-habitat combination and should be tested for the system where management actions are warranted. Nevertheless, we minimally recommend sampling water volumes of 1 L and screening at least 14 µL of extracted eDNA for each sample to reduce uncertainty in detections when studying macroinvertebrates in rivers and using our molecular workflow.

An integrated spatio-temporal view of riverine biodiversity using environmental DNA metabarcoding.

Anthropogenically forced changes in global freshwater biodiversity demand more efficient monitoring approaches. Consequently, environmental DNA (eDNA) analysis is enabling ecosystem-scale biodiversity assessment, yet the appropriate spatio-temporal resolution of robust biodiversity assessment remains ambiguous. Here, using intensive, spatio-temporal eDNA sampling across space (five rivers in Europe and North America, with an upper range of 20-35 km between samples), time (19 timepoints between 2017 and 2018) and environmental conditions (river flow, pH, conductivity, temperature and rainfall), we characterise the resolution at which information on diversity across the animal kingdom can be gathered from rivers using eDNA. In space, beta diversity was mainly dictated by turnover, on a scale of tens of kilometres, highlighting that diversity measures are not confounded by eDNA from upstream. Fish communities showed nested assemblages along some rivers, coinciding with habitat use. Across time, seasonal life history events, including salmon and eel migration, were detected. Finally, effects of environmental conditions were taxon-specific, reflecting habitat filtering of communities rather than effects on DNA molecules. We conclude that riverine eDNA metabarcoding can measure biodiversity at spatio-temporal scales relevant to species and community ecology, demonstrating its utility in delivering insights into river community ecology during a time of environmental change.

Graduate student's guide to necessary skills for nonacademic conservation careers.

Graduate education programs in conservation science generally focus on disciplinary training and discipline-specific research skills. However, nonacademic conservation professionals often require an additional suite of skills. This discrepancy between academic training and professional needs can make it difficult for graduate students to identify the skills and experiences that will best prepare them for the conservation job market. We analyzed job advertisements for conservation-science positions and interviewed conservation professionals with experience hiring early-career conservation scientists to determine what skills employers of conservation professionals seek; whether the relative importance of skills varies by job sector (government, nonprofit, and private); and how graduate students interested in careers in conservation science might signal competency in key skills to potential employers. In job advertisements, disciplinary, interpersonal, and project-management skills were in the top 5 skills mentioned across all job sectors. Employers' needs for additional skills, like program leadership, conflict resolution and negotiation, and technical and information technology skills, varied across sectors. Our interview results demonstrated that some skills are best signaled to employers via experiences obtained outside thesis or dissertation work. Our findings suggest that graduate students who wish to be competitive in the conservation job market can benefit by gaining skills identified as important to the job sector in which they hope to work and should not necessarily expect to be competent in these skills simply by completing their chosen degree path.

Drone-assisted collection of environmental DNA from tree branches for biodiversity monitoring.

The protection and restoration of the biosphere is crucial for human resilience and well-being, but the scarcity of data on the status and distribution of biodiversity puts these efforts at risk. DNA released into the environment by organisms, i.e., environmental DNA (eDNA), can be used to monitor biodiversity in a scalable manner if equipped with the appropriate tool. However, the collection of eDNA in terrestrial environments remains a challenge because of the many potential surfaces and sources that need to be surveyed and their limited accessibility. Here, we propose to survey biodiversity by sampling eDNA on the outer branches of tree canopies with an aerial robot. The drone combines a force-sensing cage with a haptic-based control strategy to establish and maintain contact with the upper surface of the branches. Surface eDNA is then collected using an adhesive surface integrated in the cage of the drone. We show that the drone can autonomously land on a variety of branches with stiffnesses between 1 and 103 newton/meter without prior knowledge of their structural stiffness and with robustness to linear and angular misalignments. Validation in the natural environment demonstrates that our method is successful in detecting animal species, including arthropods and vertebrates. Combining robotics with eDNA sampling from a variety of unreachable aboveground substrates can offer a solution for broad-scale monitoring of biodiversity.

Space-time dynamics in monitoring neotropical fish communities using eDNA metabarcoding.

The biodiverse Neotropical ecoregion remains insufficiently assessed, poorly managed, and threatened by unregulated human activities. Novel, rapid and cost-effective DNA-based approaches are valuable to improve understanding of the biological communities and for biomonitoring in remote areas. Here, we evaluate the potential of environmental DNA (eDNA) metabarcoding for assessing the structure and distribution of fish communities by analysing water and sediment from 11 locations along the Jequitinhonha River catchment (Brazil). Each site was sampled twice, before and after a major rain event in a five-week period and fish diversity was estimated using high-throughput sequencing of 12S rRNA amplicons. In total, 252 Molecular Operational Taxonomic Units (MOTUs) and 34 fish species were recovered, including endemic, introduced, and previously unrecorded species for this basin. Spatio-temporal variation of eDNA from fish assemblages was observed and species richness was nearly twice as high before the major rain event compared to afterwards. Yet, peaks of diversity were primarily associated with only four of the locations. No correlation between ß-diversity and longitudinal distance or presence of dams was detected, but low species richness observed at sites located near dams might that these anthropogenic barriers may have an impact on local fish diversity. Unexpectedly high α-diversity levels recorded at the river mouth suggest that these sections should be further evaluated as putative "eDNA reservoirs" for rapid monitoring. By uncovering spatio-temporal changes, unrecorded biodiversity components, and putative anthropogenic impacts on fish assemblages, we further strengthen the potential of eDNA metabarcoding as a biomonitoring tool, especially in regions often neglected or difficult to access.

Microbial community shifts in streams receiving treated wastewater effluent.

Wastewater treatment plant (WWTP) effluents release not only chemical constituents in watersheds, but also contain microorganisms. Thus, an understanding of what microorganisms are released and how they change microbial communities within natural streams is needed. To characterize the community shifts in streams receiving WWTP effluent, we sampled water-column microorganisms from upstream, downstream, and the effluent of WWTPs located on 23 headwater streams in which no other effluent was released upstream. We characterized the bacterial community by sequencing the V3-V4 region of the 16S rRNA gene. We hypothesized that the downstream community profile would be a hydraulic mixture between the two sources (i.e., upstream and effluent). In ordination analyses, the downstream bacterial community profile was a mixture between the upstream and effluent. For 14 of the sites, the main contribution (>50%) to the downstream community originated from bacteria in the WWTP effluent and significant shifts in relative abundance of specific sequence variants were detected. These shifts in sequence variants may serve as general bioindicators of wastewater-effluent influenced streams, with a human-gut related Ruminococcus genus displaying the highest shift (30-fold higher abundances downstream). However, not all taxa composition changes were predicted based on hydraulic mixing alone. Specifically, the decrease of Cyanobacteria/Chloroplast reads was not adequately described by hydraulic mixing. The potential alteration of stream microbial communities via a high inflow of human-gut related bacteria and a decrease in autotrophic functional groups resulting from WWTP effluent creates the potential for general shifts in stream ecosystem function.

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.

Phylogeny of the avian genus Pitohui and the evolution of toxicity in birds.

Bird species in the avian genus Pitohui contain potent neurotoxic alkaloids that may be used for defense. The genus comprises multiple species that are endemic to New Guinea and were presumed to belong to the family Pachycephalidae or Colluricinclidae, within the core corvoidea, an ancient Australasian radiation of crow-like birds. In order to understand the evolution of toxicity within the genus Pitohui, we sequenced three mitochondrial and two nuclear gene segments and reconstructed a phylogeny of the genus Pitohui and its putative relatives. We show that the genus Pitohui is polyphyletic, and consists of five different lineages. Using Bayesian ancestral state reconstruction, we estimate that toxicity likely evolved multiple times within this group. Furthermore, because the morphological and behavioral similarity among these poisonous birds appears to have evolved convergently, we hypothesize that this may be a possible example of Müllerian mimicry in birds. The Morningbird of Palau, Micronesia, that has often been included in the genus Pitohui, actually belongs in the genus Pachycephala and offers an intriguing case of pronounced evolution on a remote oceanic island.

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.

Acidity promotes degradation of multi-species environmental DNA in lotic mesocosms.

Accurate quantification of biodiversity is fundamental to understanding ecosystem function and for environmental assessment. Molecular methods using environmental DNA (eDNA) offer a non-invasive, rapid, and cost-effective alternative to traditional biodiversity assessments, which require high levels of expertise. While eDNA analyses are increasingly being utilized, there remains considerable uncertainty regarding the dynamics of multispecies eDNA, especially in variable systems such as rivers. Here, we utilize four sets of upland stream mesocosms, across an acid-base gradient, to assess the temporal and environmental degradation of multispecies eDNA. Sampling included water column and biofilm sampling over time with eDNA quantified using qPCR. Our findings show that the persistence of lotic multispecies eDNA, sampled from water and biofilm, decays to non-detectable levels within 2 days and that acidic environments accelerate the degradation process. Collectively, the results provide the basis for a predictive framework for the relationship between lotic eDNA degradation dynamics in spatio-temporally dynamic river ecosystems.

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.

Range-wide phylogeographic structure of the vernal pool fairy shrimp (Branchinecta lynchi).

Wetland habitats across the world are experiencing rapid modification and loss due to accelerating habitat conversion. Impacts to wetland habitats are particularly acute in California where up to 90% of wetland habitats have been modified or lost. Vernal pool ecosystems have therefore undergone a dramatic loss in habitat and along with them an entire endemic fauna is under threat of extinction. Recent efforts to conserve vernal pool habitat and associated species have involved restoration and creation of vernal pools as well as translocations of threatened species. The vernal pool fairy shrimp, Branchinecta lynchi, is one of several endemic and federally listed species being targeted for translocations. To guide reintroduction and conservation, detailed information on range-wide population structure and diversity is needed. We collected genetic data from two mitochondrial genes throughout the known extant range of B. lynchi to elucidate population structure and diversity of the species. We found support for phylogeographic structure throughout the range of B. lynch associated with isolated watersheds and vernal pool regions previously identified in the recovery plan for the species. The underlying mechanisms responsible for this broad pattern of genetic structure have yet to be identified. However, the evidence of only a few haplotypes being shared across the species range and patterns of isolation by distance within vernal pool regions suggests dispersal limitation may play a role. These results stress that conservation programs, at a minimum, should consider using individuals from regional populations as sources for reintroductions to maintain historical patterns of genetic differentiation. Additionally, because genetic structure is associated with vernal pool regions which are based on local hydrology and geology, translocations should proceed considering the distance between donor and recipient sites.

Environmental DNA reveals that rivers are conveyer belts of biodiversity information.

DNA sampled from the environment (eDNA) is a useful way to uncover biodiversity patterns. By combining a conceptual model and empirical data, we test whether eDNA transported in river networks can be used as an integrative way to assess eukaryotic biodiversity for broad spatial scales and across the land-water interface. Using an eDNA metabarcode approach, we detect 296 families of eukaryotes, spanning 19 phyla across the catchment of a river. We show for a subset of these families that eDNA samples overcome spatial autocorrelation biases associated with the classical community assessments by integrating biodiversity information over space. In addition, we demonstrate that many terrestrial species are detected; thus suggesting eDNA in river water also incorporates biodiversity information across terrestrial and aquatic biomes. Environmental DNA transported in river networks offers a novel and spatially integrated way to assess the total biodiversity for whole landscapes and will transform biodiversity data acquisition in ecology.