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 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.

Macrobial airborne environmental DNA analysis: A review of progress, challenges, and recommendations for an emerging application.

In the context of looming global biodiversity loss, effective species detection represents a critical concern for ecological research and management. Environmental DNA (eDNA) analysis, which refers to the collection and taxonomic identification of genetic fragments that are shed from an organism into its surroundings, emerged approximately 15 years ago as a sensitive tool for species detection. Today, one of the frontiers of eDNA research concerns the collection and analysis of genetic material in dust and other airborne materials, termed airborne eDNA analysis. As the study of airborne eDNA matures, it is an appropriate time to review the foundational and emerging studies that make up the current literature, and use the reviewed literature to summarize, synthesize, and forecast the major challenges and opportunities for this advancing research front. Specifically, we use the "ecology of eDNA" framework to organize our findings across the origin, state, transport, and fate of airborne genetic materials in the environment, and summarize what is so far known of their interactions with surrounding abiotic and biotic factors, including population and community ecologies and ecosystem processes. Within this work we identify key challenges, opportunities, and future directions associated with the application of airborne eDNA development. Lastly, we discuss the development of applications, partnerships, and messaging that promote development and growth of the field. Together, the broad potential of eDNA analysis and the rate at which research is accelerating in this field suggest that the sky's the limit for airborne eDNA science.

Batrachochytrium dendrobatidis in the Arid and Thermally Extreme Sonoran Desert.

Batrachochytrium dendrobatidis (Bd), the causative agent of the devastating global amphibian disease chytridiomycosis, was not projected to threaten amphibians in hot and arid regions due to its sensitivity to heat and desiccation. However, Bd is being detected more frequently than ever in hot and arid regions of Australia and the USA, challenging our current understanding of the environmental tolerances of the pathogen under natural conditions. We surveyed for Bd in an extremely hot and arid portion of the Sonoran Desert, where the pathogen is not projected to occur, and related presence and prevalence of the pathogen to local environmental conditions. We collected eDNA samples from isolated desert water sites including six tinajas and 13 catchments in June and August of 2020 and swabbed a total of 281 anurans of three species (red-spotted toad Anaxyrus punctatus, Couch's spadefoot Scaphiopus couchii, and the Sonoran Desert toad Incillius alvarius) across five catchments and six tinajas from June to September of 2020. Overall, Bd occurred at 68.4% of sites, despite extreme heat and aridity routinely exceeding tolerances established in laboratory studies. Average summer maximum air and water temperatures were 40.7°C and 30.7°C, respectively, and sites received an average of just 16.9 mm of precipitation throughout the summer monsoon season. Prevalence was low (5.7%) across species and life stage. Our results demonstrate that Bd is capable of persisting and infecting amphibians beyond its projected range, indicating a need to account for higher thermal tolerances when quantifying risk of Bd presence and infection.

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.

Airborne environmental DNA metabarcoding detects more diversity, with less sampling effort, than a traditional plant community survey.

BACKGROUND: Airborne environmental DNA (eDNA) research is an emerging field that focuses on the detection of species from their genetic remnants in the air. The majority of studies into airborne eDNA of plants has until now either focused on single species detection, specifically only pollen, or human health impacts, with no previous studies surveying an entire plant community through metabarcoding. We therefore conducted an airborne eDNA metabarcoding survey and compared the results to a traditional plant community survey. RESULTS: Over the course of a year, we conducted two traditional transect-based visual plant surveys alongside an airborne eDNA sampling campaign on a short-grass rangeland. We found that airborne eDNA detected more species than the traditional surveying method, although the types of species detected varied based on the method used. Airborne eDNA detected more grasses and forbs with less showy flowers, while the traditional method detected fewer grasses but also detected rarer forbs with large showy flowers. Additionally, we found the airborne eDNA metabarcoding survey required less sampling effort in terms of the time needed to conduct a survey and was able to detect more invasive species than the traditional method. CONCLUSIONS: Overall, we have demonstrated that airborne eDNA can act as a sensitive and efficient plant community surveying method. Airborne eDNA surveillance has the potential to revolutionize the way plant communities are monitored in general, track changes in plant communities due to climate change and disturbances, and assist with the monitoring of invasive and endangered species.

Detection of the Amphibian Pathogens Chytrid Fungus (Batrachochytrium dendrobatidis) and Ranavirus in West Texas, USA, Using Environmental DNA.

Environmental DNA (eDNA) methods provide novel options for the detection of pathogens. The amphibian pathogens Batrachochytrium dendrobatidis (Bd) and Ranavirus have been relatively understudied in Texas, US, so we applied eDNA assays for the surveillance of these pathogens in the upper Brazos River basin near the Texas panhandle. We collected water samples from five urban playa lakes and one reservoir in and around Lubbock, Texas. Quantitative PCR detected both Bd and Ranavirus at one playa lake, representing novel detection of both pathogens in the region. Based on these results, we recommend increased monitoring for the pathogens and symptoms of amphibian disease throughout the region.

The detection of a non-anemophilous plant species using airborne eDNA.

Genetic analysis of airborne plant material has historically focused (generally implicitly rather than as a stated goal) on pollen from anemophilous (wind-pollinated) species, such as in multiple studies examining the relationship of allergens to human health. Inspired by the recent influx of literature applying environmental DNA (eDNA) approaches to targeted-species and whole-ecosystem study, we conducted a proof-of-concept experiment to determine whether airborne samples reliably detect genetic material from non-anemophilous species that may not be releasing large plumes of pollen. We collected airborne eDNA using Big Spring Number Eight dust traps and quantified the amount of eDNA present for a flowering wind-pollinated genus (Bouteloua) and insect-pollinated honey mesquite (Prosopis glandulosa) that was not flowering at the time of the study. We were able to detect airborne eDNA from both species. Since honey mesquite is insect-pollinated and was not flowering during the time of this study, our results confirm that airborne eDNA consists of and can detect species through more than just pollen. Additionally, we were able to detect temporal patterns reflecting Bouteloua reproductive ecology and suggest that airborne honey mesquite eDNA responded to weather conditions during our study. These findings suggest a need for more study of the ecology of airborne eDNA to uncover its potential for single-species and whole-community research and management in terrestrial ecosystems.

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.

Geographic selection bias of occurrence data influences transferability of invasive Hydrilla verticillata distribution models.

Due to socioeconomic differences, the accuracy and extent of reporting on the occurrence of native species differs among countries, which can impact the performance of species distribution models. We assessed the importance of geographical biases in occurrence data on model performance using Hydrilla verticillata as a case study. We used Maxent to predict potential North American distribution of the aquatic invasive macrophyte based upon training data from its native range. We produced a model using all available native range occurrence data, then explored the change in model performance produced by omitting subsets of training data based on political boundaries. We also compared those results with models trained on data from which a random sample of occurrence data was omitted from across the native range. Although most models accurately predicted the occurrence of H. verticillata in North America (AUC > 0.7600), data omissions influenced model predictions. Omitting data based on political boundaries resulted in larger shifts in model accuracy than omitting randomly selected occurrence data. For well-documented species like H. verticillata, missing records from single countries or ecoregions may minimally influence model predictions, but for species with fewer documented occurrences or poorly understood ranges, geographic biases could misguide predictions. Regardless of focal species, we recommend that future species distribution modeling efforts begin with a reflection on potential spatial biases of available occurrence data. Improved biodiversity surveillance and reporting will provide benefit not only in invaded ranges but also within under-reported and unexplored native ranges.

Molecular detection of invasive species in heterogeneous mixtures using a microfluidic carbon nanotube platform.

Screening methods to prevent introductions of invasive species are critical for the protection of environmental and economic benefits provided by native species and uninvaded ecosystems. Coastal ecosystems worldwide remain vulnerable to damage from aquatic species introductions, particularly via ballast water discharge from ships. Because current ballast management practices are not completely effective, rapid and sensitive screening methods are needed for on-site testing of ships in transit. Here, we describe a detection technology based on a microfluidic chip containing DNA oligonucleotide functionalized carbon nanotubes. We demonstrate the efficacy of the chip using three ballast-transported species either established (Dreissena bugensis) or of potential threat (Eriocheir sinensis and Limnoperna fortuneii) to the Laurentian Great Lakes. With further refinement for on-board application, the technology could lead to real-time ballast water screening to improve ship-specific management and control decisions.

Quantitative and rapid DNA detection by laser transmission spectroscopy.

Laser transmission spectroscopy (LTS) is a quantitative and rapid in vitro technique for measuring the size, shape, and number of nanoparticles in suspension. Here we report on the application of LTS as a novel detection method for species-specific DNA where the presence of one invasive species was differentiated from a closely related invasive sister species. The method employs carboxylated polystyrene nanoparticles functionalized with short DNA fragments that are complimentary to a specific target DNA sequence. In solution, the DNA strands containing targets bind to the tags resulting in a sizable increase in the nanoparticle diameter, which is rapidly and quantitatively measured using LTS. DNA strands that do not contain the target sequence do not bind and produce no size change of the carboxylated beads. The results show that LTS has the potential to become a quantitative and rapid DNA detection method suitable for many real-world applications.