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.

High-throughput environmental DNA analysis informs a biological assessment of an urban stream.

There is growing interest in the use of DNA barcoding and metabarcoding approaches to aid biological assessments and monitoring of waterbodies. While biodiversity measured by morphology and by DNA often has been found correlated, few studies have compared DNA data to established measures of impairment such as multimetric pollution tolerance indices used by many bioassessment programs. We incorporated environmental DNA (eDNA) metabarcoding of seston into a rigorous watershed-scale biological assessment of an urban stream to examine the extent to which eDNA richness and diversity patterns were correlated with multimetric indices and ecological impairment status designations. We also evaluated different filtering approaches and taxonomic classifications to identify best practices for environmental assessments. Seston eDNA revealed a wide diversity of eukaryotic taxa but was dominated by diatoms (36%). Differentiation among sites in alpha and beta diversity was greater when operational taxonomic units (OTUs) were classified taxonomically, but coarse resolution taxonomy (kingdom) was more informative than finer resolution taxonomy (family, genus). Correlations of DNA richness and diversity with multimetric indices for fish and macroinvertebrates were generally weak, possibly because Metazoa were not highly represented in our DNA dataset. Nonetheless, sites could be differentiated based on ecological impairment status, with more impaired sites having lower eDNA diversity as measured by the Shannon index, but higher taxonomic richness. Significant environmental drivers of community structure, as inferred from constrained ordination analyses, differed among kingdoms within the eDNA dataset, as well as from fish and macrobenthos, suggesting that eDNA provides novel environmental information. These results suggest that even a simple seston eDNA filtering protocol can provide biodiversity information of value to stream bioassessment programs. The approach bears further investigation as a potentially useful rapid assessment protocol to supplement more intensive field sampling efforts.

Dreissena veligers in western Lake Superior - inference from new low-density detection.

The notion that Lake Superior proper is inhospitable to dreissenid mussel survival has been challenged by recent finds on shipwrecks and rocky reefs in the Apostle Islands region. Motivated by concerns surrounding these finds, we conducted an intensive sampling campaign of Apostle Islands waters in 2017 to understand Dreissena prevalence and distribution. The 100-site effort combined random and targeted sites and collected zooplankton, benthos, video, environmental DNA, and supporting water quality data. We did not find settled Dreissena in any video footage or benthos samples, and quantitative PCR applied to eDNA samples was negative for Dreissena. Dreissena veligers were found in almost half the zooplankton samples but at orders of magnitude lower densities than reported from other Laurentian Great Lakes. Veligers were most prevalent around the western islands and associated with shallower depths and slightly higher phosphorus and chlorophyll, but did not spatially match known (still very localized) settled Dreissena colonies. This is the first study to conduct veliger-targeted sampling in western Lake Superior and the first to report consistent detection of veligers there. We speculate that these Apostle Islands veligers are not a new locally-spawned component of the zooplankton community, but instead are transported from an established population in the St. Louis River estuary (~100 km away) by longshore currents; i.e., low-density propagule pressure that may have been present for years. Small-mesh zooplankton data collected along a gradient from the Apostle Islands to the St. Louis River estuary and enumerated with thorough veliger searching would help elucidate these alternatives.

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.

Phylogenetic relationships of North American Gomphidae and their close relatives.

Intrafamilial relationships among clubtail dragonflies (Gomphidae) have been the subject of many morphological studies, but have not yet been systematically evaluated using molecular data. Here we present the first molecular phylogeny of Gomphidae. We include six of the eight subfamilies previously suggested to be valid, and evaluate generic relationships within them. We have included examples of all genera reported from the Nearctic except Phyllocycla. This sample includes all North American species of Ophiogomphus, which has allowed us to explore intrageneric relationships in that genus. Our particular focus is on the closest relatives of the genus Gomphus, especially those North American species groups that have been commonly treated as subgenera of Gomphus. The Gomphus complex is split into additional genera, supported by molecular and morphological evidence: Phanogomphus, Stenogomphurus, Gomphurus and Hylogomphus are here considered to be valid genera. The genus Gomphus, in our restricted sense, does not occur in the western hemisphere; in addition, G. flavipes is transferred to Stylurus.

Early detection monitoring for aquatic non-indigenous species: Optimizing surveillance, incorporating advanced technologies, and identifying research needs.

Following decades of ecologic and economic impacts from a growing list of nonindigenous and invasive species, government and management entities are committing to systematic early- detection monitoring (EDM). This has reinvigorated investment in the science underpinning such monitoring, as well as the need to convey that science in practical terms to those tasked with EDM implementation. Using the context of nonindigenous species in the North American Great Lakes, this article summarizes the current scientific tools and knowledge - including limitations, research needs, and likely future developments - relevant to various aspects of planning and conducting comprehensive EDM. We begin with the scope of the effort, contrasting target-species with broad-spectrum monitoring, reviewing information to support prioritization based on species and locations, and exploring the challenge of moving beyond individual surveys towards a coordinated monitoring network. Next, we discuss survey design, including effort to expend and its allocation over space and time. A section on sample collection and analysis overviews the merits of collecting actual organisms versus shed DNA, reviews the capabilities and limitations of identification by morphology, DNA target markers, or DNA barcoding, and examines best practices for sample handling and data verification. We end with a section addressing the analysis of monitoring data, including methods to evaluate survey performance and characterize and communicate uncertainty. Although the body of science supporting EDM implementation is already substantial, research and information needs (many already actively being addressed) include: better data to support risk assessments that guide choice of taxa and locations to monitor; improved understanding of spatiotemporal scales for sample collection; further development of DNA target markers, reference barcodes, genomic workflows, and synergies between DNA-based and morphology-based taxonomy; and tools and information management systems for better evaluating and communicating survey outcomes and uncertainty.

Inferences based on diatom compositions improve estimates of nutrient concentrations in streams.

Nutrient concentrations in streams vary strongly with flow conditions, and routinely gathered field measurements of nutrients reflect this variability. Diatom assemblage composition has been used in previous studies to infer nutrient concentrations, and because diatoms integrate nutrient concentrations over longer periods of time, diatom inferences may be less susceptible to fluctuations in streamflow. We tested this hypothesis by leveraging differences in the flashiness of streams across a large continental data set. More specifically, we tested whether the variabilities of direct measurements and diatom inferences of dissolved phosphorus and nitrate were greater in flashy versus non-flashy streams. We further considered whether models linking landscape predictor variables to nutrient concentrations yielded consistent results across flashy and non-flashy streams. Our analysis indicated that measured nutrient concentrations were more variable in flashy compared to non-flashy streams and that landscape models identified different important predictors of nutrient concentrations when fit using data from flashy vs. non-flashy streams. In contrast, variabilities of diatom-inferred nutrient concentrations were similar among stream types, as were the important predictor variables (e.g., manure application rates for nitrate and number of wet days for dissolved phosphorus). These analyses indicate that use of diatom-inferred nutrient concentrations can potentially improve efforts to quantify stream nutrient concentrations.

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.

Understanding the effects of phosphorus on diatom richness in rivers and streams using taxon-environment relationships.

Changes in phosphorus concentrations affect periphytic diatom composition in streams, yet we rarely observe strong relationships between diatom richness and phosphorus. In contrast, changes in conductivity are strongly associated with differences in both diatom composition and richness. We hypothesised that we could better understand the mechanisms that control the phosphorus-richness relationship by examining relationships between phosphorus and the occurrence of individual diatom taxa, comparing these with relationships between conductivity and taxon occurrence, and documenting how niche breadths of taxa affect richness patterns. We estimated relationships between phosphorus and taxon occurrence using DNA metabarcoding data of diatoms collected from 1,811 sites distributed across the conterminous U.S.A. and contrasted patterns in these relationships with those between conductivity and taxon occurrence. The distribution of taxon optima for phosphorus was bimodal, with most optima located at either the maximum or minimum observed phosphorus concentration. The distribution of taxon optima for conductivity was unimodal. Niche breadths of taxa for phosphorus and for conductivity both generally increased with optimum values. The distribution of conductivity optima gave rise to a prominent hump-shaped relationship between richness and conductivity. The relationship between richness and phosphorus was also slightly hump-shaped, but this relationship would not be expected from the bimodal distribution of optima. Instead, we determined that broad niche breadths caused the hump-shaped relationship between richness and phosphorus. Our results highlight the nuanced effects that increased P loadings exert on diatom assemblages in rivers and streams and identify reasons that weak relationships between taxon richness and increased phosphorus have been observed. These findings allow us to better describe how excess phosphorus and subsets of taxa and their niche breadths contribute to patterns of taxa richness in diatom assemblages, and to improve the tools used to manage phosphorus pollution.

Quantifying spatial and temporal relationships between diatoms and nutrients in streams strengthens evidence of nutrient effects from monitoring data.

Observational data are frequently used to better understand the effects of changes in P and N on stream biota, but nutrient gradients in streams are usually associated with gradients in other environmental factors, a phenomenon that complicates efforts to accurately estimate the effects of nutrients. Here, we propose a new approach for analyzing observational data in which we compare the effects of changes in nutrient concentrations in time within individual sites and in space among many sites. Covarying relationships between other, potentially confounding environmental factors and nutrient concentrations are unlikely to be the same in both time and space, and, therefore, estimated effects of nutrients that are similar in time and space are more likely to be accurate. We applied this approach to diatom rbcL metabarcoding data collected from streams in the East Fork of the Little Miami River watershed, Ohio, USA. Changes in diatom assemblage composition were consistently associated with changes in the concentration of total reactive P in both time and space. In contrast, despite being associated with spatial differences in ammonia and urea concentrations, diatom assemblage composition was not associated with temporal changes in these nitrogen species. We suggest that the results of this analysis provide evidence of a causal effect of increased P on diatom assemblage composition. We further analyzed the effects of temporal variability in measurements of total reactive P and found that averaging periods greater than ~1 wk prior to sampling best represented the effects of P on the diatom assemblage. Comparisons of biological responses in space and time can sharpen insights beyond those that are based on analyses conducted on only 1 of the 2 dimensions.

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.

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.

Invasive Dreissena Mussel Coastal Transport From an Already Invaded Estuary to a Nearby Archipelago Detected in DNA and Zooplankton Surveys.

Coastal waters of Lake Superior are generally inhospitable to the establishment of invasive Dreissena spp. mussels (both Dreissena polymorpha and Dreissena bugensis). Dreissena have inhabited the Saint Louis River estuary (SLRE; largest commercial port in the Laurentian Great Lakes) for over three decades, but only in the last few years have small colonies been found in the Apostle Islands National Lakeshore (APIS, an archipelago situated 85 km to the east of SLRE) A 2017 survey determined a low abundance Dreissena spatial distribution in APIS, with the largest colonies on the north and west islands which suggested potential veliger transport from the SLRE via longshore currents. Our objective in this study was to determine if Dreissena veligers are transported by currents at low densities along the south shore of Lake Superior from the SLRE to APIS. To do so, we used both eDNA (water and passive substrate samples) and zooplankton collection methods at eight sites evenly spaced between the SLRE and APIS with three sampling times over five weeks. Dreissena veligers were consistently detected along the south shore, although at low abundances (veligers per m3 range = 0-690, median = 8), and for every 1 km increase in distance from the SLRE, both veliger counts and water eDNA copy numbers decreased on average by 5 and 7%, respectively. D. polymorpha (suited to estuary habitats) was detected two times more than D. bugensis (better suited to deep-lake habitats). There was not a trend in the veliger size distribution along the south shore, and temperature and calcium concentrations fluctuated around the threshold for Dreissena veliger and adult development, averaging 11.0°C and 14.8 ppm, respectively. Three zooplankton taxa representative of the estuary community-Daphnia retrocurva, Diaphanosoma birgei, and Mesocyclops copepodites-decreased as the distance from the SLRE increased mirroring Dreissena veliger abundance patterns. Findings represent multiple sources of evidence of a propagule "conveyor belt" for Dreissena along the south shore of Lake Superior. We conclude that veligers are functioning as a propagule, using coastal currents to spread from the point of invasion, thereby traversing coastal habitat previously reported as inhospitable to distant habitats suitable for colonization.

Evaluating the performance of DNA metabarcoding for assessment of zooplankton communities in Western Lake Superior using multiple markers.

For DNA metabarcoding to attain its potential as a community assessment tool, we need to better understand its performance versus traditional morphological identification and work to address any remaining performance gaps in incorporating DNA metabarcoding into community assessments. Using fragments of the 18S nuclear and 16S mitochondrial rRNA genes and two fragments of the mitochondrial COI marker, we examined the use of DNA metabarcoding and traditional morphological identification for understanding the diversity and composition of crustacean zooplankton at 42 sites across western Lake Superior. We identified 51 zooplankton taxa (genus or species, depending on the finest resolution of the taxon across all identification methods), of which 17 were identified using only morphological traits, 13 using only DNA and 21 using both methods. The taxa found using only DNA metabarcoding included four species and one genus-level identification not previously known to occur in Lake Superior, the presence of which still needs to be confirmed. A substantial portion of taxa that were identified to genus or species by morphological identification, but not identified using DNA metabarcoding, had zero ("no record") or ≤ 2 ("underrepresented records") reference barcodes in the BOLD or NCBI databases (63% for COI, 80% for 16S, 74% for 18S). The two COI marker fragments identified the most genus- and species-level taxa, whereas 18S was the only marker whose family-level percent sequence abundance patterns showed high correlation to composition patterns from morphological identification, based on a NMDS analysis of Bray-Curtis similarities. Multiple replicates were collected at a subset of sites and an occupancy analysis was performed, which indicated that rare taxa were more likely to be detected using DNA metabarcoding than traditional morphology. Our results support that DNA metabarcoding can augment morphological identification for estimating zooplankton diversity and composition of zooplankton over space and time, but may require use of multiple markers. Further addition of taxa to reference DNA databases will improve our ability to use DNA metabarcoding to identify zooplankton and other invertebrates in aquatic surveys.

Comparative phylogeography uncovers evolutionary past of Holarctic dragonflies.

Here, we investigate the evolutionary history of five northern dragonfly species to evaluate what role the last glaciation period may have played in their current distributions. We look at the population structure and estimate divergence times for populations of the following species: Aeshna juncea (Linnaeus), Aeshna subarctica Walker, Sympetrum danae (Sulzer), Libellula quadrimaculata Linnaeus and Somatochlora sahlbergi Trybom across their Holarctic range. Our results suggest a common phylogeographic pattern across all species except for S. sahlbergi. First, we find that North American and European populations are genetically distinct and have perhaps been separated for more than 400,000 years. Second, our data suggests that, based on genetics, populations from the Greater Beringian region (Beringia, Japan and China) have haplotypes that cluster with North America or Europe depending on the species rather than having a shared geographic affinity. This is perhaps a result of fluctuating sea levels and ice sheet coverage during the Quaternary period that influenced dispersal routes and refugia. Indeed, glacial Beringia may have been as much a transit zone as a refugia for dragonflies. Somatochlora sahlbergi shows no genetic variation across its range and therefore does not share the geographic patterns found in the other circumboreal dragonflies studied here. Lastly, we discuss the taxonomic status of Sympetrum danae, which our results indicate is a species complex comprising two species, one found in Eurasia through Beringia, and the other in North America east and south of Beringia. Through this study we present a shared history among different species from different families of dragonflies, which are influenced by the climatic fluctuations of the past.

Comparison of Larval Fish Detections Using Morphology-Based Taxonomy versus High-Throughput Sequencing for Invasive Species Early Detection.

When first introduced, invasive species typically evade detection; DNA barcoding coupled with high-throughput sequencing (HTS) may be more sensitive and accurate than morphology-based taxonomy, and thereby improve invasive (or rare) species detection. We quantified the relative error of species detection between morphology-based and HTS-based taxonomic identification of ichthyoplankton collections from the Port of Duluth, Minnesota, an aquatic non-native species introduction 'hot-spot' in the Laurentian Great Lakes. We found HTS-based taxonomy identified 28 species and morphology-based taxonomy 30 species, of which 27 were common to both. Among samples, 76% of family-level taxonomic assignments agreed; however, only 42% of species assignments agreed. Most errors were attributed to morphology-based taxonomy, whereas HTS-based taxonomy error was low. For this study system, for most non-native fishes, the detection probability by randomized survey for larvae was similar to that by a survey that is optimized for non-native species early detection of juveniles and adults. We conclude that classifying taxonomic errors by comparing HTS results against morphology-based taxonomy is an important step toward incorporating HTS-based taxonomy into biodiversity surveys.

Early detection monitoring for non-indigenous fishes; comparison of survey approaches during two species introductions in a Great Lakes port.

Assessing relative performance of different sampling methods used for early detection monitoring (EDM) is a critical step in understanding the likelihood of detecting new non-indigenous species (NIS) in an environment of interest. EDM performance metrics are typically based on the probability of detecting established NIS or rare indigenous species; however, detection probability estimates for these proxies may not accurately reflect survey effectiveness for newly introduced NIS. We used data from three different EDM survey approaches that varied by targeted life-stage (adult-juvenile versus ichthyoplankton), media (physical fish versus environmental DNA), and taxonomic method (morphology-based versus DNA-based taxonomy) to explore relative detection sensitivity for recently introduced white bass (Morone chrysops) and gizzard shad (Dorosoma cepedianum) in the Port of Duluth-Superior, a NIS introduction hot spot within the Laurentian Great Lakes. Detection efficiency, measured by the effort (number of samples) required to achieve 95% probability of detection, differed by EDM approach and species. Also, the relative sensitivity (detection rate) of each survey approach differed by species. For both species, detection in surveys using DNA-based taxonomy was generally as good or better than the adult-juvenile survey using morphology-based taxonomy. While both species appear to have been detected at early stages of invasion, white bass were likely present up to 5 years prior to initial detection, whereas gizzard shad may have been detected in the first year of introduction. We conclude that using complimentary sampling methods can help to balance the strengths and weaknesses of each approach and provide more reliable early detection of new invaders.

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.

Sensitivity and accuracy of high-throughput metabarcoding methods for early detection of invasive fish species.

High-throughput DNA metabarcoding has gained recognition as a potentially powerful tool for biomonitoring, including early detection of aquatic invasive species (AIS). DNA based techniques are advancing, but our understanding of the limits to detection for metabarcoding complex samples is inadequate. For detecting AIS at an early stage of invasion when the species is rare, accuracy at low detection limits is key. To evaluate the utility of metabarcoding in future fish community monitoring programs, we conducted several experiments to determine the sensitivity and accuracy of routine metabarcoding methods. Experimental mixes used larval fish tissue from multiple "common" species spiked with varying proportions of tissue from an additional "rare" species. Pyrosequencing of genetic marker, COI (cytochrome c oxidase subunit I) and subsequent sequence data analysis provided experimental evidence of low-level detection of the target "rare" species at biomass percentages as low as 0.02% of total sample biomass. Limits to detection varied interspecifically and were susceptible to amplification bias. Moreover, results showed some data processing methods can skew sequence-based biodiversity measurements from corresponding relative biomass abundances and increase false absences. We suggest caution in interpreting presence/absence and relative abundance in larval fish assemblages until metabarcoding methods are optimized for accuracy and precision.

Potential for DNA-based identification of Great Lakes fauna: match and mismatch between taxa inventories and DNA barcode libraries.

DNA-based identification of mixed-organism samples offers the potential to greatly reduce the need for resource-intensive morphological identification, which would be of value both to bioassessment and non-native species monitoring. The ability to assign species identities to DNA sequences found depends on the availability of comprehensive DNA reference libraries. Here, we compile inventories for aquatic metazoans extant in or threatening to invade the Laurentian Great Lakes and examine the availability of reference mitochondrial COI DNA sequences (barcodes) in the Barcode of Life Data System for them. We found barcode libraries largely complete for extant and threatening-to-invade vertebrates (100% of reptile, 99% of fish, and 92% of amphibian species had barcodes). In contrast, barcode libraries remain poorly developed for precisely those organisms where morphological identification is most challenging; 46% of extant invertebrates lacked reference barcodes with rates especially high among rotifers, oligochaetes, and mites. Lack of species-level identification for many aquatic invertebrates also is a barrier to matching DNA sequences with physical specimens. Attaining the potential for DNA-based identification of mixed-organism samples covering the breadth of aquatic fauna requires a concerted effort to build supporting barcode libraries and voucher collections.