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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Evaluating ethanol-based sample preservation to facilitate use of DNA barcoding in routine freshwater biomonitoring programs using benthic macroinvertebrates.

Molecular methods, such as DNA barcoding, have the potential to enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI). On average, we obtained successful COI sequences (i.e. either full or partial barcodes) for between 93-99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2∶1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera) producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity.