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.

Mountaintop removal coal mining impacts on structural and functional indicators in Central Appalachian streams.

Mountaintop removal coal mining (MTR) has been a major source of landscape change in the Central Appalachians of the United States (US). Changes in stream hydrology, channel geomorphology and water quality caused by MTR coal mining can lead to severe impairment of stream ecological integrity. The objective of the Clean Water Act (CWA) is to restore and maintain the ecological integrity of the Nation's waters. Sensitive, readily measured indicators of ecosystem structure and function are needed for the assessment of stream ecological integrity. Most CWA assessments rely on structural indicators; inclusion of functional indicators could make these assessments more holistic and effective. The goals of this study were: (1) test the efficacy of selected carbon (C) and nitrogen (N) cycling and microbial structural and functional indicators for assessing MTR coal mining impacts on streams; (2) determine whether indicators respond to impacts in a predictable manner; and (3) determine if functional indicators are less likely to change than are structural indicators in response to stressors associated with MTR coal mining. The structural indicators are water quality and sediment organic matter concentrations, and the functional indicators relate to microbial activity and biofilm production. Seasonal measurements were conducted over the course of a year in streams draining small MTR-impacted and forested watersheds in the Twentymile Creek watershed of West Virginia (WV). Five of the eight structural parameters measured had significant responses, with all means greater in the MTR-impacted streams than in the forested streams. These responses resulted from changes in source or augmentation of the original source of the C and N structural parameters because of MTR coal mining. Nitrate concentration and the stable carbon isotopic ratio of dissolved inorganic carbon were the most effective indicators evaluated in this study. Only three of the fourteen functional indicators measured had significant responses to MTR coal mining, with all means greater in the forested streams than in the MTR-impacted streams. These results suggest that stressors associated with MTR coal mining caused reduction in some aspects of microbial cycling, but resource subsidies may have counterbalanced some of the inhibition leading to no observable change in most of the functional indicators. The detritus base, which is thought to confer functional stability, was likely sustained in the MTR-impacted streams by channel storage and/or leaf litter inputs from their largely intact riparian zones. Overall, our results largely support the hypothesis that certain functional processes are more resistant to stress induced change than structural properties but also suggest the difficulty of identifying suitable functional indicators for ecological integrity assessment.

Identifying invertebrate indicators for streamflow duration assessments in forested headwater streams.

Streamflow-duration assessment methods (SDAMs) are rapid, indicator-based tools for classifying streamflow duration (e.g., intermittent vs perennial flow) at the reach scale. Indicators are easily assessed stream properties used as surrogates of flow duration, which is too resource intensive to measure directly for many reaches. Invertebrates are commonly used as SDAM indicators because many are not highly mobile, and different species have life stages that require flow for different durations and times of the year. The objectives of this study were to 1) identify invertebrate taxa that can be used as SDAM indicators to distinguish between stream reaches having intermittent and perennial flow, 2) to compare indicator strength across different taxonomic and numeric resolutions, and 3) to assess the relative importance of season and habitat type on the ability of invertebrates to predict streamflow-duration class. We used 2 methods, random forest models and indicator species analysis, to analyze aquatic and terrestrial invertebrate data (presence/absence, density, and biomass) at the family and genus levels from 370 samples collected from both erosional and depositional habitats during both wet and dry seasons. In total, 36 intermittent and 53 perennial reaches were sampled along 31 forested headwater streams in 4 level II ecoregions across the United States. Random forest models for family- and genus-level datasets had stream classification accuracy ranging from 88.9 to 93.2%, with slightly higher accuracy for density than for presence/absence and biomass datasets. Season (wet/dry) tended to be a stronger predictor of streamflow-duration class than habitat (erosional/depositional). Many taxa at the family (58.8%) and genus level (61.6%) were collected from both intermittent and perennial reaches, and most taxa that were exclusive to 1 streamflow-duration class were rarely collected. However, 23 family-level or higher taxa (20 aquatic and 3 terrestrial) and 44 aquatic genera were identified as potential indicators of streamflow-duration class for forested headwater streams. The utility of the potential indicators varied across level II ecoregions in part because of representation of intermittent and perennial reaches in the dataset but also because of variable ecological responses to drying among species. Aquatic invertebrates have been an important field indicator of perennial reaches in existing SDAMs, but our findings highlight how including aquatic and terrestrial invertebrates as indicators of intermittent reaches can further maximize the data collected for streamflow-duration classifications.

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.

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.

Coarse particulate organic matter dynamics in ephemeral tributaries of a Central Appalachian stream network.

Headwater ephemeral tributaries are interfaces between uplands and downstream waters. Terrestrial coarse particulate organic matter (CPOM) is important in fueling aquatic ecosystems; however, the extent to which ephemeral tributaries are functionally connected to downstream waters through fluvial transport of CPOM has been little studied. Hydrology and deposition of leaf and wood, and surrogate transport (Ginkgo biloba leaves and wood dowels) were measured over month-long intervals through the winter and spring seasons (6 months) in 10 ephemeral tributaries (1.3-5.4 ha) in eastern Kentucky. Leaf deposition and surrogate transport varied over time, reflecting the seasonality of litterfall and runoff. Leaf deposition was higher in December than February and May but did not differ from January, March, and April. Mean percent of surrogate leaf transport from the ephemeral tributaries was highest in April (3.6% per day) and lowest in February (2.5%) and May (2%). Wood deposition and transport had similar patterns. No CPOM measures were related to flow frequency. Ephemeral tributaries were estimated to annually contribute 110.6 kg AFDM·km-1·yr-1 of leaves to the downstream mainstem. Ephemeral tributaries are functionally connected to downstream waters through CPOM storage and subsequent release that is timed when CPOM is often limited in downstream waters.

Elevated major ion concentrations inhibit larval mayfly growth and development.

Anthropogenic disturbances, including those from developing energy resources, can alter stream chemistry significantly by elevating total dissolved solids. Field studies have indicated that mayflies (Order Ephemeroptera) are particularly sensitive to high total dissolved solids. In the present study, the authors measured 20-d growth and survivorship of larval Neocloeon triangulifer exposed to a gradient of brine salt (mixed NaCl and CaCl2 ) concentrations. Daily growth rates were reduced significantly in all salt concentrations above the control (363 µS cm(-1) ) and larvae in treatments with specific conductance >812 µS cm(-1) were in comparatively earlier developmental stages (instars) at the end of the experiment. Survivorship declined significantly when specific conductance was >1513 µS cm(-1) and the calculated 20-d 50% lethal concentration was 2866 µS cm(-1) . The present study's results provide strong experimental evidence that elevated ion concentrations similar to those observed in developing energy resources, such as oil and gas drilling or coal mining, can adversely affect sensitive aquatic insect species.

Part 1: Laboratory culture of Centroptilum triangulifer (Ephemeroptera: Baetidae) using a defined diet of three diatoms.

Development of methods for assessing exposure and effects of waterborne toxicants on stream invertebrate species is important to elucidate environmentally relevant information. Current protocols for freshwater invertebrate toxicity testing almost exclusively utilize cladocerans, amphipods or chironomids rather than the more typical aquatic insect taxa found in lotic systems. Centroptilum triangulifer is a parthenogenetic mayfly occurring in depositional habitats of streams and rivers of the Eastern U.S. and Canada. C. triangulifer is an ideal stream insect for toxicity testing under field and laboratory conditions because of its short life cycle, parthenogenetic mode of reproduction, and it represents a group considered sensitive to environmental stressors. In this study, a colony of C. triangulifer was reared using a defined diet of three diatoms, Mayamaea atomus var. permitis, Nitzschia cf. pusilla, and Achnanthidium minutissimum. Percent survival (⩾80%), fecundity measurements (⩾1000 eggs) and pre-egg laying weights were used as indicators of overall colony health and fitness in our laboratory water (Lab-line) and in Moderately Hard Reconstituted Water (MHRW). Lab-line reared C. triangulifer had average survival rate of 92.69% for eleven generations and 82.99% over thirteen generations. MHRW reared C. triangulifer had an average survival rate of 80.65% for four generations and three generations of fecundities greater than 1000 eggs per individual. Pre-egg laying weight and fecundity were highly correlated and a best-fit model equation was derived to estimate egg counts for future generations. Establishment of this culturing protocol provides a more ecologically relevant species for toxicity testing and aids in further stressor identification for stream bioassessments.

Part 2: Sensitivity comparisons of the mayfly Centroptilum triangulifer to Ceriodaphnia dubia and Daphnia magna using standard reference toxicants; NaCl, KCl and CuSO4.

Criteria for establishing water quality standards that are protective for 95% of the native species are generally based upon laboratory toxicity tests. These tests utilize common model organisms that have established test methods. However, for invertebrates these species represent mostly the zooplankton community and are not inclusive of all taxa. In order to examine a potential under-representation in emerging aquatic invertebrates the US Environmental Protection Agency has cultured a parthenogenetic mayfly, Centroptilum triangulifer (Ephemeroptera: Baetidae). This study established a 48h acute and a 14-day short-term chronic testing procedure for C. triangulifer and compared its sensitivity to two model invertebrates, Ceriodaphnia dubia and Daphnia magna. Toxicity tests were conducted to determine mortality and growth effects using standard reference toxicants: NaCl, KCl and CuSO4. In 48-h acute tests, the average LC50 for the mayfly was 659mgL(-1) NaCl, 1957mgL(-1) KCl, and 11µgL(-1) CuSO4. IC25 values, using dry weight as the endpoint, were 228mgL(-1) NaCl, 356mgL(-1) KCl and 5µgL(-1) CuSO4. C. triangulifer was the most sensitive species in NaCl acute and chronic growth tests. At KCl concentrations tested, C. triangulifer was less sensitive for acute tests but was equally or more sensitive than C. dubia and D. magna for growth measurements. This study determined C. triangulifer has great potential and benefits for use in ecotoxicological studies.

Influence of trophic position and spatial location on polychlorinated biphenyl (PCB) bioaccumulation in a stream food web.

We tested the ability of delta15N-derived trophic position (TP) to predict polychlorinated biphenyl (PCB) concentrations in a historically contaminated stream (Twelvemile Creek, SC). We analyzed sediment, four types of organic matter, 27 macroinvertebrate taxa, and 25 fish species from six sites spanning 25 stream km. SigmaPCBs were high across sites (mean fish = 2505 ng g(-1) wet), with little spatial variation in concentrations within a trophic level. SigmaPCBs (wet weight) were significantly positively correlated with TP (r2 = 0.56) and lipids (r2 = 0.44), and concentrations increased 1-2 orders of magnitude among trophic levels. After adjusting for lipids, we calculated a food web magnification factor of 1.6 for SigmaPCBs, which is low compared to marine and lentic food webs. The predictive power of TP for individual congeners increased with Kow (octanol-water partition coefficient), with regression slope approximately 0.48 and r2 approximately 0.70 for Kow > 6.5. The proportion of high Kow compounds increased with distance from the source and with trophic position. Spatial variation in congener patterns was high, in contrast to marine and lentic systems where variation is typically low.