Using DNA metabarcoding to characterize national scale diatom-environment relationships and to develop indicators in streams and rivers of the United States.

Recent advancements in DNA techniques, metabarcoding, and bioinformatics could help expand the use of benthic diatoms in monitoring and assessment programs by providing relatively quick and increasingly cost-effective ways to quantify diatom diversity in environmental samples. However, such applications of DNA-based approaches are relatively new, and in the United States, unknowns regarding their applications at large scales exist because only a few small-scale studies have been done. Here, we present results from the first nationwide survey to use DNA metabarcoding (rbcL) of benthic diatoms, which were collected from 1788 streams and rivers across nine ecoregions spanning the conterminous USA. At the national scale, we found that diatom assemblage structure (1) was strongly associated with total phosphorus and total nitrogen concentrations, conductivity, and pH and (2) had clear patterns that corresponded with differences in these variables among the nine ecoregions. These four variables were strong predictors of diatom assemblage structure in ecoregion-specific analyses, but our results also showed that diatom-environment relationships, the importance of environmental variables, and the ranges of these variables within which assemblage changes occurred differed among ecoregions. To further examine how assemblage data could be used for biomonitoring purposes, we used indicator species analysis to identify ecoregion-specific taxa that decreased or increased along each environmental gradient, and we used their relative abundances of gene reads in samples as metrics. These metrics were strongly correlated with their corresponding variable of interest (e.g., low phosphorus diatoms with total phosphorus concentrations), and generalized additive models showed how their relationships compared among ecoregions. These large-scale national patterns and nine sets of ecoregional results demonstrated that diatom DNA metabarcoding is a robust approach that could be useful to monitoring and assessment programs spanning the variety of conditions that exist throughout the conterminous United States.

Developing Indicators of Nutrient Pollution in Streams Using 16S rRNA Gene Metabarcoding of Periphyton-Associated Bacteria.

Indicators based on nutrient-biota relationships in streams can inform water quality restoration and protection programs. Bacterial assemblages could be particularly useful indicators of nutrient effects because they are species-rich, important contributors to ecosystem processes in streams, and responsive to rapidly changing conditions. Here, we sampled 25 streams weekly (12-14 times each) and used 16S rRNA gene metabarcoding of periphyton-associated bacteria to quantify the effects of total phosphorus (TP) and total nitrogen (TN). Threshold indicator taxa analysis identified assemblage-level changes and amplicon sequence variants (ASVs) that increased or decreased with increasing TP and TN concentrations (i.e., low P, high P, low N, and high N ASVs). Boosted regression trees confirmed that relative abundances of gene sequence reads for these four indicator groups were associated with nutrient concentrations. Gradient forest analysis complemented these results by using multiple predictors and random forest models for each ASV to identify portions of TP and TN gradients at which the greatest changes in assemblage structure occurred. Synthesized statistical results showed bacterial assemblage structure began changing at 24 µg TP/L with the greatest changes occurring from 110 to 195 µg/L. Changes in the bacterial assemblages associated with TN gradually occurred from 275 to 855 µg/L. Taxonomic and phylogenetic analyses showed that low nutrient ASVs were commonly Firmicutes, Verrucomicrobiota, Flavobacteriales, and Caulobacterales, Pseudomonadales, and Rhodobacterales of Proteobacteria, whereas other groups, such as Chitinophagales of Bacteroidota, and Burkholderiales, Rhizobiales, Sphingomonadales, and Steroidobacterales of Proteobacteria comprised the high nutrient ASVs. Overall, the responses of bacterial ASV indicators in this study highlight the utility of metabarcoding periphyton-associated bacteria for quantifying biotic responses to nutrient inputs in streams.

First evaluation of ballast water management systems on operational ships for minimizing introductions of nonindigenous zooplankton.

Ballast water is a leading pathway for the global introduction of aquatic nonindigenous species. Most international ships are expected to install ballast water management systems (BWMS) by 2024 to treat ballast water before release. This study examines if ballast water discharges managed by BWMS are meeting standards for organisms ≥50 µm in minimum dimension (i.e., <10 organisms per m3; typically zooplankton). Representative samples of ballast water were collected from 29 ships (using 14 different BWMS) arriving to Canada during 2017-2018. Fourteen samples (48 %) had zooplankton concentrations clearly exceeding the standard (ranging from 18 to 3822 organisms per m3). Nonetheless, compared to earlier management strategies, BWMS appear to reduce the frequency of high-risk introduction events. BWMS filter mesh size was an important predictor of zooplankton concentration following treatment. Greater rates of compliance may be achieved as ship crews gain experience with operation and maintenance of BWMS.

Characterizing temporal variability in streams supports nutrient indicator development using diatom and bacterial DNA metabarcoding.

Interest in developing periphytic diatom and bacterial indicators of nutrient effects continues to grow in support of the assessment and management of stream ecosystems and their watersheds. However, temporal variability could confound relationships between indicators and nutrients, subsequently affecting assessment outcomes. To document how temporal variability affects measures of diatom and bacterial assemblages obtained from DNA metabarcoding, we conducted weekly periphyton and nutrient sampling from July to October 2016 in 25 streams in a 1293 km2 mixed land use watershed. Measures of both diatom and bacterial assemblages were strongly associated with the percent agriculture in upstream watersheds and total phosphorus (TP) and total nitrogen (TN) concentrations. Temporal variability in TP and TN concentrations increased with greater amounts of agriculture in watersheds, but overall diatom and bacterial assemblage variability within sites-measured as mean distance among samples to corresponding site centroids in ordination space-remained consistent. This consistency was due in part to offsets between decreasing variability in relative abundances of taxa typical of low nutrient conditions and increasing variability in those typical of high nutrient conditions as mean concentrations of TP and TN increased within sites. Weekly low and high nutrient diatom and bacterial metrics were more strongly correlated with site mean nutrient concentrations over the sampling period than with same day measurements and more strongly correlated with TP than with TN. Correlations with TP concentrations were consistently strong throughout the study except briefly following two major precipitation events. Following these events, biotic relationships with TP reestablished within one to three weeks. Collectively, these results can strengthen interpretations of survey results and inform monitoring strategies and decision making. These findings have direct applications for improving the use of diatoms and bacteria, and the use of DNA metabarcoding, in monitoring programs and stream site assessments.

International shipping as a potent vector for spreading marine parasites.

Aim: The global shipping fleet, the primary means of transporting goods among countries, also serves as a major dispersal mechanism for marine invasive species. To date, researchers have primarily focussed on the role of ships in transferring marine macrofauna, often overlooking transfers of associated parasites, which can have larger impacts on naïve host individuals and populations. Here, we re-examine three previously published metabarcode datasets targeting zooplankton and protists in ships' ballast water to assess the diversity of parasites across life stages arriving to three major US ports. Location: Port of Hampton Roads in the Chesapeake Bay, Virginia; Ports of Texas City, Houston and Bayport in Galveston Bay, Texas; and Port of Valdez in Prince William Sound, Alaska. Methods: We selected all known parasitic taxa, using sequences generated from the small subunit gene (SSU) from ribosomal RNA (rRNA) amplified from (1) zooplankton collected from plankton tows (35 and 80 µm datasets) and (2) eukaryotes collected from samples of ships' ballast water (3 µm dataset). Results: In all three datasets, we found a broad range of parasitic taxa, including many protistan and metazoan parasites, that infect a wide range of hosts, from teleost fish to dinoflagellates. Parasite richness was highest in the 3 µm dataset and relatively uniform across arrival regions. Several parasite taxa were found in high relative abundance (based on number of sequences recovered) either in ships entering a single or across multiple regions. Main Conclusions: The ubiquity, diversity and relative abundance of parasites detected demonstrate ships are a potent vector for spreading marine parasites across the world's oceans, potentially contributing to reported increases in outbreaks of marine diseases. Future research is urgently needed to evaluate the fate of parasites upon arrival and the efficacy of ballast water treatment systems to reduce future transfers and colonization.

Assessing cyanobacterial frequency and abundance at surface waters near drinking water intakes across the United States.

This study presents the first large-scale assessment of cyanobacterial frequency and abundance of surface water near drinking water intakes across the United States. Public water systems serve drinking water to nearly 90% of the United States population. Cyanobacteria and their toxins may degrade the quality of finished drinking water and can lead to negative health consequences. Satellite imagery can serve as a cost-effective and consistent monitoring technique for surface cyanobacterial blooms in source waters and can provide drinking water treatment operators information for managing their systems. This study uses satellite imagery from the European Space Agency's Ocean and Land Colour Instrument (OLCI) spanning June 2016 through April 2020. At 300-m spatial resolution, OLCI imagery can be used to monitor cyanobacteria in 685 drinking water sources across 285 lakes in 44 states, referred to here as resolvable drinking water sources. First, a subset of satellite data was compared to a subset of responses (n = 84) submitted as part of the U.S. Environmental Protection Agency's fourth Unregulated Contaminant Monitoring Rule (UCMR 4). These UCMR 4 qualitative responses included visual observations of algal bloom presence and absence near drinking water intakes from March 2018 through November 2019. Overall agreement between satellite imagery and UCMR 4 qualitative responses was 94% with a Kappa coefficient of 0.70. Next, temporal frequency of cyanobacterial blooms at all resolvable drinking water sources was assessed. In 2019, bloom frequency averaged 2% and peaked at 100%, where 100% indicated a bloom was always present at the source waters when satellite imagery was available. Monthly cyanobacterial abundances were used to assess short-term trends across all resolvable drinking water sources and effect size was computed to provide insight on the number of years of data that must be obtained to increase confidence in an observed change. Generally, 2016 through 2020 was an insufficient time period for confidently observing changes at these source waters; on average, a decade of satellite imagery would be required for observed environmental trends to outweigh variability in the data. However, five source waters did demonstrate a sustained short-term trend, with one increasing in cyanobacterial abundance from June 2016 to April 2020 and four decreasing.

Invasion history shapes host transcriptomic response to a body-snatching parasite.

By shuffling biogeographical distributions, biological invasions can both disrupt long-standing associations between hosts and parasites and establish new ones. This creates natural experiments with which to study the ecology and evolution of host-parasite interactions. In estuaries of the Gulf of Mexico, the white-fingered mud crab (Rhithropanopeus harrisii) is infected by a native parasitic barnacle, Loxothylacus panopaei (Rhizocephala), which manipulates host physiology and behaviour. In the 1960s, L. panopaei was introduced to the Chesapeake Bay and has since expanded along the southeastern Atlantic coast, while host populations in the northeast have so far been spared. We use this system to test the host's transcriptomic response to parasitic infection and investigate how this response varies with the parasite's invasion history, comparing populations representing (i) long-term sympatry between host and parasite, (ii) new associations where the parasite has invaded during the last 60 years and (iii) naïve hosts without prior exposure. A comparison of parasitized and control crabs revealed a core response, with widespread downregulation of transcripts involved in immunity and moulting. The transcriptional response differed between hosts from the parasite's native range and where it is absent, consistent with previous observations of increased susceptibility in populations lacking exposure to the parasite. Crabs from the parasite's introduced range, where prevalence is highest, displayed the most dissimilar response, possibly reflecting immune priming. These results provide molecular evidence for parasitic manipulation of host phenotype and the role of gene regulation in mediating host-parasite interactions.

Satellite remote sensing to assess cyanobacterial bloom frequency across the United States at multiple spatial scales.

Cyanobacterial blooms can have negative effects on human health and local ecosystems. Field monitoring of cyanobacterial blooms can be costly, but satellite remote sensing has shown utility for more efficient spatial and temporal monitoring across the United States. Here, satellite imagery was used to assess the annual frequency of surface cyanobacterial blooms, defined for each satellite pixel as the percentage of images for that pixel throughout the year exhibiting detectable cyanobacteria. Cyanobacterial frequency was assessed across 2,196 large lakes in 46 states across the continental United States (CONUS) using imagery from the European Space Agency's Ocean and Land Colour Instrument for the years 2017 through 2019. In 2019, across all satellite pixels considered, annual bloom frequency had a median value of 4% and a maximum value of 100%, the latter indicating that for those satellite pixels, a cyanobacterial bloom was detected by the satellite sensor for every satellite image considered. In addition to annual pixel-scale cyanobacterial frequency, results were summarized at the lake- and state-scales by averaging annual pixel-scale results across each lake and state. For 2019, average annual lake-scale frequencies also had a maximum value of 100%, and Oregon and Ohio had the highest average annual state-scale frequencies at 65% and 52%. Pixel-scale frequency results can assist in identifying portions of a lake that are more prone to cyanobacterial blooms, while lake- and state-scale frequency results can assist in the prioritization of sampling resources and mitigation efforts. Satellite imagery is limited by the presence of snow and ice, as imagery collected in these conditions are quality flagged and discarded. Thus, annual bloom frequencies within nine climate regions were investigated to determine whether missing data biased results in climate regions more prone to snow and ice, given that their annual summaries would be weighted toward the summer months when cyanobacterial blooms tend to occur. Results were unbiased by the time period selected in most climate regions, but a large bias was observed for the Northwest Rockies and Plains climate region. Moderate biases were observed for the Ohio Valley and the Southeast climate regions. Finally, a clustering analysis was used to identify areas of high and low cyanobacterial frequency across CONUS based on average annual lake-scale cyanobacterial frequencies for 2019. Several clusters were identified that transcended state, watershed, and eco-regional boundaries. Combined with additional data, results from the clustering analysis may offer insight regarding large-scale drivers of cyanobacterial blooms.

How to learn to stop worrying and love environmental DNA monitoring.

Environmental DNA is one of the most promising new tools in the aquatic biodiversity monitoring toolkit, with particular appeal for applications requiring assessment of target taxa at very low population densities. And yet there persists considerable anxiety within the management community regarding the appropriateness of environmental DNA monitoring for certain tasks and the degree to which environmental DNA methods can deliver information relevant to management needs. This brief perspective piece is an attempt to address that anxiety by offering some advice on how end-users might best approach these new technologies. I do not here review recent developments in environmental DNA science, but rather I explore ways in which managers and decision-makers might become more comfortable adopting environmental DNA tools-or choosing not to adopt them, should circumstances so dictate. I attempt to contextualize the central challenges associated with acceptance of environmental DNA detection by contrasting them with traditional "catch-and-look" approaches to biodiversity monitoring. These considerations lead me to recommend the cultivation of four "virtues," attitudes that can be brought into engagement with environmental DNA surveillance technologies that I hope will increase the likelihood that those engagements will be positive and that the future development and application of environmental DNA tools will further the cause of wise management.

Metabarcoding quantifies differences in accumulation of ballast water borne biodiversity among three port systems in the United States.

Characterizing biodiversity conveyed in ships' ballast water (BW), a global driver of biological invasions, is critically important for understanding risks posed by this key vector and establishing baselines to evaluate changes associated with BW management. Here we employ high throughput sequence (HTS) metabarcoding of the 18S small subunit rRNA to test for and quantify differences in the accumulation of BW-borne biodiversity among three distinct recipient port systems in the United States. These systems were located on three different coasts (Pacific, Gulf, and Atlantic) and chosen to reflect distinct trade patterns and source port biogeography. Extensive sampling of BW tanks (n = 116) allowed detailed exploration of molecular diversity accumulation. Our results indicate that saturation of introduced zooplankton diversity may be achieved quickly, with fewer than 25 tanks needed to achieve 95% of the total extrapolated diversity, if source biogeography is relatively limited. However, as predicted, port systems with much broader source geographies require more extensive sampling to estimate diversity, which continues to accumulate after sampling >100 discharges. The ability to identify BW sources using molecular indicators was also found to depend on the breadth of source biogeography and the extent to which sources had been sampled. These findings have implications both for the effort required to fully understand introduced diversity and for projecting risks associated with future changes to maritime traffic that may increase source biogeography for many recipient ports. Our data also suggest that molecular diversity may not decline significantly with BW age, indicating either that some organisms survive longer than recognized in previous studies or that nucleic acids from dead organisms persist in BW tanks. We present evidence for detection of potentially invasive species in arriving BW but discuss important caveats that preclude strong inferences regarding the presence of living representatives of these species in BW tanks.

DNA metabarcoding effectively quantifies diatom responses to nutrients in streams.

Nutrient pollution from human activities remains a common problem facing stream ecosystems. Identifying ecological responses to phosphorus and nitrogen can inform decisions affecting the protection and management of streams and their watersheds. Diatoms are particularly useful because they are a highly diverse group of unicellular algae found in nearly all aquatic environments and are sensitive responders to increased nutrient concentrations. Here, we used DNA metabarcoding of stream diatoms as an approach to quantifying effects of total phosphorus (TP) and total nitrogen (TN). Threshold indicator taxa analysis (TITAN) identified operational taxonomic units (OTUs) that increased or decreased along TP and TN gradients along with nutrient concentrations at which assemblages had substantial changes in the occurrences and relative abundances of OTUs. Boosted regression trees showed that relative abundances of gene sequence reads for OTUs identified by TITAN as low P, high P, low N, or high N diatoms had strong relationships with nutrient concentrations, which provided support for potentially using these groups of diatoms as metrics in monitoring programs. Gradient forest analysis provided complementary information by characterizing multi-taxa assemblage change using multiple predictors and results from random forest models for each OTU. Collectively, these analyses showed that notable changes in diatom assemblage structure and OTUs began around 20 µg TP/L, low P diatoms decreased substantially and community change points occurred from 75 to 150 µg/L, and high P diatoms became increasingly dominant from 150 to 300 µg/L. Diatoms also responded to TN with large decreases in low N diatoms occurring from 280 to 525 µg TN/L and a transition to dominance by high N diatoms from 525-850 µg/L. These diatom responses to TP and TN could be used to inform protection efforts (i.e., anti-degradation) and management goals (i.e., nutrient reduction) in streams and watersheds. Our results add to the growing support for using diatom metabarcoding in monitoring programs.

Recent introductions reveal differential susceptibility to parasitism across an evolutionary mosaic.

Parasitism can represent a potent agent of selection, and introduced parasites have the potential to substantially alter their new hosts' ecology and evolution. While significant impacts have been reported for parasites that switch to new host species, the effects of macroparasite introduction into naïve populations of host species with which they have evolved remain poorly understood. Here, we investigate how the estuarine white-fingered mud crab (Rhithropanopeus harrisii) has adapted to parasitism by an introduced rhizocephalan parasite (Loxothylacus panopaei) that castrates its host. While the host crab is native to much of the East and Gulf Coasts of North America, its parasite is native only to the southern end of this range. Fifty years ago, the parasite invaded the mid-Atlantic, gradually expanding through previously naïve host populations. Thus, different populations of the same host species have experienced different degrees of historical interaction (and thus potential evolutionary response time) with the parasite: long term, short term, and naïve. In nine estuaries across this range, we examined whether and how parasite prevalence and host susceptibility to parasitism differs depending on the length of the host's history with the parasite. In field surveys, we found that the parasite was significantly more prevalent in its introduced range (i.e., short-term interaction) than in its native range (long-term interaction), a result that was also supported by a meta-analysis of prevalence data covering the 50 years since its introduction. In controlled laboratory experiments, host susceptibility to parasitism was significantly higher in naïve hosts than in hosts from the parasite's native range, suggesting that host resistance to parasitism is under selection. These results suggest that differences in host-parasite historical interaction can alter the consequences of parasite introductions in host populations. As anthropogenically driven range shifts continue, disruptions of host-parasite evolutionary relationships may become an increasingly important driver of ecological and evolutionary change.

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.

Quantifying national and regional cyanobacterial occurrence in US lakes using satellite remote sensing.

Cyanobacterial harmful algal blooms are the most common form of harmful algal blooms in freshwater systems throughout the world. However, in situ sampling of cyanobacteria in inland lakes is limited both spatially and temporally. Satellite data has proven to be an effective tool to monitor cyanobacteria in freshwater lakes across the United States. This study uses data from the European Space Agency Envisat MEdium Resolution Imaging Spectrometer and the Sentinel-3 Ocean and Land Color Instrument to provide a national overview of the percentage of lakes experiencing a cyanobacterial bloom on a weekly basis for 2008-2011, 2017, and 2018. A total of 2321 lakes across the contiguous United States were included in the analysis. We examined four different thresholds to define when a waterbody is classified as experiencing a bloom. Across these four thresholds, we explored variability in bloom percentage with changes in seasonality and lake size. As a validation of algorithm performance, we analyzed the agreement between satellite observations and previously established ecological patterns, although data availability in the wintertime limited these comparisons on a year-round basis. Changes in cyanobacterial bloom percentage at the national scale followed the well-known temporal pattern of freshwater blooms. The percentage of lakes experiencing a bloom increased throughout the year, reached a maximum in fall, and decreased through the winter. Wintertime data, particularly in northern regions, were consistently limited due to snow and ice cover. With the exception of the Southeast and South, regional patterns mimicked patterns found at the national scale. The Southeast and South exhibited an unexpected pattern as cyanobacterial bloom percentage reached a maximum in the winter rather than the summer. Lake Jesup in Florida was used as a case study to validate this observed pattern against field observations of chlorophyll a. Results from this research establish a baseline of annual occurrence of cyanobacterial blooms in inland lakes across the United States. In addition, methods presented in this study can be tailored to fit the specific requirements of an individual system or region.

The risks of using molecular biodiversity data for incidental detection of species of concern.

Incidental detection of species of concern (e.g., invasive species, pathogens, threatened and endangered species) during biodiversity assessments based on high-throughput DNA sequencing holds significant risks in the absence of rigorous, fit-for-purpose data quality and reporting standards. Molecular biodiversity data are predominantly collected for ecological studies and thus are generated to common quality assurance standards. However, the detection of certain species of concern in these data would likely elicit interest from end users working in biosecurity or other surveillance contexts (e.g., pathogen detection in health-related fields), for which more stringent quality control standards are essential to ensure that data are suitable for informing decision-making and can withstand legal or political challenges. We suggest here that data quality and reporting criteria are urgently needed to enable clear identification of those studies that may be appropriately applied to surveillance contexts. In the interim, more pointed disclaimers on uncertainties associated with the detection and identification of species of concern may be warranted in published studies. This is not only to ensure the utility of molecular biodiversity data for consumers, but also to protect data generators from uncritical and potentially ill-advised application of their science in decision-making.

Strong genetic structure in a widespread estuarine crab: A test of potential versus realized dispersal.

Aim: Genetic structure has proven difficult to predict for marine and estuarine species with multi-day pelagic larval durations, since many disperse far less than expected based on passive transport models. In such cases, the gap between potential and realized dispersal may result from larval behaviours that evolved to facilitate retention and settlement in favourable environments. Behaviour is predicted to play a particularly key role in structuring truly estuarine species, which often moderate their behaviour to remain within their natal estuaries. In such systems, this restricted dispersal may lead to high divergence, local adaptation and eventual speciation across their range. Here, we test whether a geographically widespread estuarine crab, known to have behaviour promoting larval retention, exhibits high population structure despite a 2- to 4-week larval duration. Location: Atlantic and Gulf Coasts of North America. Taxon: White-fingered mud crab, Rhithropanopeus harrisii. Methods: Population genomic analyses across nine estuaries from New Hampshire to Louisiana using 12,638 transcriptome-derived SNPs. Results: We found highly differentiated genetic signatures among all nine estuaries, separated by 200-5,000 km of coastline. Estimates of gene flow suggest that migration is low and largely symmetrical between sites. We also observed deep phylogenetic divides corresponding to major biogeographical breaks. Main conclusions: These results indicate substantial and longstanding constraints to dispersal in the species' native range, likely arising from the emergence of geological and oceanographic barriers and sustained by behaviour that promotes estuarine retention during larval development. This work supports the idea that larval behaviour promoting estuarine retention can be reflected in substantial genetic structure even in species with multi-week pelagic larval durations. Such behaviour-restricted dispersal has implications for predicting adaptation and spread in estuarine species, many of which have been introduced outside their native ranges.

Assessing threats of non-native species to native freshwater biodiversity: Conservation priorities for the United States.

Non-native species pose one of the greatest threats to native biodiversity, and can have severe negative impacts in freshwater ecosystems. Identifying regions of spatial overlap between high freshwater biodiversity and high invasion pressure may thus better inform the prioritization of freshwater conservation efforts. We employ geospatial analysis of species distribution data to investigate the potential threat of non-native species to aquatic animal taxa across the continental United States. We mapped non-native aquatic plant and animal species richness and cumulative invasion pressure to estimate overall negative impact associated with species introductions. These distributions were compared to distributions of native aquatic animal taxa derived from the International Union for the Conservation of Nature (IUCN) database. To identify hotspots of native biodiversity we mapped total species richness, number of threatened and endangered species, and a community index of species rarity calculated at the watershed scale. An overall priority index allowed identification of watersheds experiencing high pressure from non-native species and also exhibiting high native biodiversity conservation value. While priority regions are roughly consistent with previously reported prioritization maps for the US, we also recognize novel priority areas characterized by moderate-to-high native diversity but extremely high invasion pressure. We further compared priority areas with existing conservation protections as well as projected future threats associated with land use change. Our findings suggest that many regions of elevated freshwater biodiversity value are compromised by high invasion pressure, and are poorly safeguarded by existing conservation mechanisms and are likely to experience significant additional stresses in the future.

Nucleic acids-based tools for ballast water surveillance, monitoring, and research.

Understanding the risks of biological invasion posed by ballast water-whether in the context of compliance testing, routine monitoring, or basic research-is fundamentally an exercise in biodiversity assessment, and as such should take advantage of the best tools available for tackling that problem. The past several decades have seen growing application of genetic methods for the study of biodiversity, driven in large part by dramatic technological advances in nucleic acids analysis. Monitoring approaches based on such methods have the potential to increase dramatically sampling throughput for biodiversity assessments, and to improve on the sensitivity, specificity, and taxonomic accuracy of traditional approaches. The application of targeted detection tools (largely focused on PCR but increasingly incorporating novel probe-based methodologies) has led to a paradigm shift in rare species monitoring, and such tools have already been applied for early detection in the context of ballast water surveillance. Rapid improvements in community profiling approaches based on high throughput sequencing (HTS) could similarly impact broader efforts to catalogue biodiversity present in ballast tanks, and could provide novel opportunities to better understand the risks of biotic exchange posed by ballast water transport-and the effectiveness of attempts to mitigate those risks. These various approaches still face considerable challenges to effective implementation, depending on particular management or research needs. Compliance testing, for instance, remains dependent on accurate quantification of viable target organisms; while tools based on RNA detection show promise in this context, the demands of such testing require considerable additional investment in methods development. In general surveillance and research contexts, both targeted and community-based approaches are still limited by various factors: quantification remains a challenge (especially for taxa in larger size classes), gaps in nucleic acids reference databases are still considerable, uncertainties in taxonomic assignment methods persist, and many applications have not yet matured sufficiently to offer standardized methods capable of meeting rigorous quality assurance standards. Nevertheless, the potential value of these tools, their growing utilization in biodiversity monitoring, and the rapid methodological advances over the past decade all suggest that they should be seriously considered for inclusion in the ballast water surveillance toolkit.

Ballast Water Exchange and Invasion Risk Posed by Intracoastal Vessel Traffic: An Evaluation Using High Throughput Sequencing.

Ballast water remains a potent vector of non-native aquatic species introductions, despite increased global efforts to reduce risk of ballast water mediated invasions. This is particularly true of intracoastal vessel traffic, whose characteristics may limit the feasibility and efficacy of management through ballast water exchange (BWE). Here we utilize high throughput sequencing (HTS) to assess biological communities associated with ballast water being delivered to Valdez, Alaska from multiple source ports along the Pacific Coast of the United States. Our analyses indicate that BWE has a significant but modest effect on ballast water assemblages. Although overall richness was not reduced with exchange, we detected losses of some common benthic coastal taxa (e.g., decapods, mollusks, bryozoans, cnidaria) and gains of open ocean taxa (e.g., certain copepods, diatoms, and dinoflagellates), including some potentially toxic species. HTS-based metabarcoding identified significantly differentiated biodiversity signatures from individual source ports; this signal persisted, though weakened, in vessels undergoing BWE, indicating incomplete faunal turnover associated with management. Our analysis also enabled identification of taxa that may be of particular concern if established in Alaskan waters. While these results reveal a clear effect of BWE on diversity in intracoastal transit, they also indicate continued introduction risk of non-native and harmful taxa.

Trends in nonindigenous aquatic species richness in the United States reveal shifting spatial and temporal patterns of species introductions.

Understanding the spatial and temporal dynamics underlying the introduction and spread of nonindigenous aquatic species (NAS) can provide important insights into the historical drivers of biological invasions and aid in forecasting future patterns of nonindigenous species arrival and spread. Increasingly, public databases of species observation records are being used to quantify changes in NAS distributions across space and time, and are becoming an important resource for researchers, managers, and policy-makers. Here we use publicly available data to describe trends in NAS introduction and spread across the conterminous United States over more than two centuries of observation records. Available data on first records of NAS reveal significant shifts in dominance of particular introduction patterns over time, both in terms of recipient regions and likely sources. These spatiotemporal trends at the continental scale may be subject to biases associated with regional variation in sampling effort, reporting, and data curation. We therefore also examined two additional metrics, the number of individual records and the spatial coverage of those records, which are likely to be more closely associated with sampling effort. Our results suggest that broad-scale patterns may mask considerable variation across regions, time periods, and even entities contributing to NAS sampling. In some cases, observed temporal shifts in species discovery may be influenced by dramatic fluctuations in the number and spatial extent of individual observations, reflecting the possibility that shifts in sampling effort may obscure underlying rates of NAS introduction.