Molecular identity crisis: environmental DNA metabarcoding meets traditional taxonomy-assessing biodiversity and freshwater mussel populations (Unionidae) in Alabama.

The use of environmental DNA (eDNA) to assess aquatic biodiversity is a growing field with great potential for monitoring and managing threatened species, like freshwater mussel (Unionidae) populations. Freshwater mussels are globally imperiled and serve essential roles in aquatic systems as a food source and as a natural water filter making their management essential for ecosystem health. Unfortunately, mussel populations are often understudied, and challenges exist to accurately and efficiently describe the full suite of species present. Multispecies eDNA approaches may also be more challenging where freshwater mussel populations are most diverse due to ongoing and significant taxonomic restructuring that has been further complicated by molecular phylogenies using mitochondrial genes. For this study, we developed a microfluidic metabarcoding array that targets a wide range of species, from invertebrates to fishes, with an emphasis on detecting unionid mussels known to be present in the Sipsey River, Alabama. We compared mussel species diversity across six sites with well-studied mussel assemblages using eDNA surveys and traditional quadrat surveys in 2016. We examined how factors such as mussel population density, biomass and location in the river substrate impacted our ability to detect certain species; and investigated unexpected eDNA detections through phylogenetic analysis. Our eDNA results for fish and mussel species were broadly consistent with the data from traditional electrofishing and quadrat-based field surveys, although both community eDNA and conventional sampling detected species unique to that method. Our phylogenetic analysis agreed with other studies that treat Pleurobema decisum and P. chattanoogaense as synonymous species; however, they are still listed as unique species in molecular databases which complicates their identity in a metabarcoding assay. We also found that Fusconaia flava and F. cerina are indistinguishable from one another using a portion of the NADH dehydrogenase Subunit 1 (ND1) marker, which may warrant further investigation into whether or not they are synonymous. Our results show that many factors impacted our ability to detect and correctly identify Unionidae mussel species. Here we describe the obstacles we faced, including the murky phylogeny of Unionidae mussels and turbid river conditions, and our development of a potentially impactful freshwater mussel monitoring eDNA assay.

Detection and accumulation of environmentally-relevant glyphosate concentrations delivered via pulse- or continuous-delivery on passive samplers.

Glyphosate is the most commonly used herbicide globally, which has contributed to its ubiquitous presence in the environment. Glyphosate application rates and delivery to surface water vary with land use. Streams in urban and agricultural catchments can experience continuous delivery of low concentrations of glyphosate and aminomethylphosphonic acid (AMPA), while their presence in forest streams occurs as an episodic pulse following silvicultural application. We assessed whether trace concentrations of glyphosate delivered as a 1-day pulse (mimic silvicultural applications) followed by flushing with deionized water would affect the detection of glyphosate or AMPA on novel passive samplers, Polar Organic Chemical Integrative Sampler with Molecular Imprinted Polymer (POCIS-MIP), compared with continuous delivery (mimic agricultural or urban applications). Within each delivery type, POCIS-MIP were exposed to seven treatment concentrations of Rodeo (equivalent to 0.0 to 1.84 µg glyphosate L-1). Experimental results demonstrate POCIS-MIP can detect differences in relative glyphosate concentrations above 0.115 µg L-1 (pulse-delivery) or 0.23 µg L-1 (continuous-delivery), but were unable to distinguish trace concentrations (i.e., < 0.115 or 0.23 µg L-1). Our results suggest POCIS-MIP may better retain glyphosate when delivered as a pulse than when delivered continuously, but both underestimated actual treatment concentrations by 46 to 56%. There is a need to demonstrate the field applicability of passive sampling methods to improve environmental monitoring of silvicultural herbicides, and our results demonstrate passive samplers were unable to distinguish lower concentrations, suggesting a limited utility for determining trace concentration levels such as those experienced during or immediately after silvicultural application.

Land Use Overrides Stream Order and Season in Driving Dissolved Organic Matter Dynamics Throughout the Year in a River Network.

Anthropogenic land use has increased nutrient concentrations and altered dissolved organic matter (DOM) character and its bioavailability. Despite widespread recognition that DOM character and its reactivity can vary temporally, the relative influence of land use and stream order on DOM characteristics is poorly understood across seasons and the entire flow regime. We examined DOM character and 28-day bioavailable dissolved organic carbon (BDOC) across a river network to determine the relative roles of land use and stream order in driving variability in DOM character and bioavailability throughout the year. DOM in 1st-order streams was distinct from higher stream orders with lower DOC concentrations, less aromatic (specific ultraviolet absorbance at 254 nm (SUVA254)), more autochthonous (fluorescence index), and more recently produced (ß/α) DOM. Across all months, variability in DOM character was primarily explained by land use, rather than stream order or season. Land use and stream order explained the most DOM variation in transitional and winter months and the least during dry months. BDOC was greater in watersheds with less aromatic (SUVA254) and more recent allochthonous DOM (ß/α) and more development and impervious surface. With continued development, the bioavailability of DOM in the smallest and most impacted watersheds is expected to increase.

Shifting stoichiometry: Long-term trends in stream-dissolved organic matter reveal altered C:N ratios due to history of atmospheric acid deposition.

Dissolved organic carbon (DOC) and nitrogen (DON) are important energy and nutrient sources for aquatic ecosystems. In many northern temperate, freshwater systems DOC has increased in the past 50 years. Less is known about how changes in DOC may vary across latitudes, and whether changes in DON track those of DOC. Here, we present long-term DOC and DON data from 74 streams distributed across seven sites in biomes ranging from the tropics to northern boreal forests with varying histories of atmospheric acid deposition. For each stream, we examined the temporal trends of DOC and DON concentrations and DOC:DON molar ratios. While some sites displayed consistent positive or negative trends in stream DOC and DON concentrations, changes in direction or magnitude were inconsistent at regional or local scales. DON trends did not always track those of DOC, though DOC:DON ratios increased over time for ~30% of streams. Our results indicate that the dissolved organic matter (DOM) pool is experiencing fundamental changes due to the recovery from atmospheric acid deposition. Changes in DOC:DON stoichiometry point to a shifting energy-nutrient balance in many aquatic ecosystems. Sustained changes in the character of DOM can have major implications for stream metabolism, biogeochemical processes, food webs, and drinking water quality (including disinfection by-products). Understanding regional and global variation in DOC and DON concentrations is important for developing realistic models and watershed management protocols to effectively target mitigation efforts aimed at bringing DOM flux and nutrient enrichment under control.

Long-term hydrological response to forest harvest during seasonal low flow: Potential implications for current forest practices.

Seasonal changes in the magnitude and duration of streamflow can have important implications for aquatic species, drinking water supplies, and water quality. In many regions, including the Pacific Northwest (U.S. and Canada), seasonal low flow is declining, primarily due to a changing climate, but is also influenced by urbanization, agriculture, and forestry. We review the responses of seasonal low flow, catchment storage, and tree-water relations to forest harvest over long timescales and discuss the potential implications of these responses for current forest practices and aquatic biota. We identify three distinct periods of expected low flow responses as regrowth occurs following forest harvest: in the first period an initial increase in low flow can occur as replanted stands regenerate, in the second period low flow is characterized by mixed and variable responses as forests become established, and in the third period, which follows canopy closure, low flow declines may occur over long timescales. Of 25 small catchments with ≥10 years post-harvest data, nine catchments had no change or variable low flow and 16 catchments experienced reduced low flow years after harvest. The retention of riparian buffers, limited size of harvest units, and adherence to reforestation requirements have altered the contemporary forest landscape relative to historical forest practices, but data documenting multi-decadal hydrological responses to current harvest practices is limited. Our review suggests that the magnitude of low flow responses attenuates downstream as a broader mosaic of stand ages occurs and multiple hydrological periods are represented. Declines were not observed in the seven large catchments reviewed. The consequences of low flow declines for aquatic biota are not well understood, but where data do exist aquatic biota have not been adversely affected. We identify priorities for future research that will aid in improving predictions of low flow responses to harvest as forests regenerate.

eDNA as a tool for identifying freshwater species in sustainable forestry: A critical review and potential future applications.

Environmental DNA (eDNA) is an emerging biological monitoring tool that can aid in assessing the effects of forestry and forest manufacturing activities on biota. Monitoring taxa across broad spatial and temporal scales is necessary to ensure forest management and forest manufacturing activities meet their environmental goals of maintaining biodiversity. Our objectives are to describe potential applications of eDNA across the wood products supply chain extending from regenerating forests, harvesting, and wood transport, to manufacturing facilities, and to review the current state of the science in this context. To meet our second objective, we summarize the taxa examined with targeted (PCR, qPCR or ddPCR) or metagenomic eDNA methods (eDNA metabarcoding), evaluate how estimated species richness compares between traditional field sampling and eDNA metabarcoding approaches, and compare the geographical representation of prior eDNA studies in freshwater ecosystems to global wood baskets. Potential applications of eDNA include evaluating the effects of forestry and forest manufacturing activities on aquatic biota, delineating fish-bearing versus non fish-bearing reaches, evaluating effectiveness of constructed road crossings for freshwater organism passage, and determining the presence of at-risk species. Studies using targeted eDNA approaches focused on fish, amphibians, and invertebrates, while metagenomic studies focused on fish, invertebrates, and microorganisms. Rare, threatened, or endangered species received the least attention in targeted eDNA research, but are arguably of greatest interest to sustainable forestry and forest manufacturing that seek to preserve freshwater biodiversity. Ultimately, using eDNA methods will enable forestry and forest manufacturing managers to have data-driven prioritization for conservation actions for all freshwater species.

Watershed 'Chemical Cocktails': Forming Novel Elemental Combinations in Anthropocene Fresh Waters.

In the Anthropocene1, watershed chemical transport is increasingly dominated by novel combinations elements, which are hydrologically linked together as 'chemical cocktails.' Chemical cocktails are novel because human activities greatly enhance elemental concentrations and their probability for biogeochemical interactions and shared transport along hydrologic flowpaths. A new chemical cocktail approach advances our ability to: trace contaminant mixtures in watersheds, develop chemical proxies with high-resolution sensor data, and manage multiple water quality problems. We explore the following questions: (1) Can we classify elemental transport in watersheds as chemical cocktails using a new approach? (2) What is the role of climate and land use in enhancing the formation and transport of chemical cocktails in watersheds? To address these questions, we first analyze trends in concentrations of carbon, nutrients, metals, and salts in fresh waters over 100 years. Next, we explore how climate and land use enhance the probability of formation of chemical cocktails of carbon, nutrients, metals, and salts. Ultimately, we classify transport of chemical cocktails based on solubility, mobility, reactivity, and dominant phases: (1) sieved chemical cocktails (e.g., particulate forms of nutrients, metals and organic matter); (2) filtered chemical cocktails (e.g., dissolved organic matter and associated metal complexes); (3) chromatographic chemical cocktails (e.g., ions eluted from soil exchange sites); and (4) reactive chemical cocktails (e.g., limiting nutrients and redox sensitive elements). Typically, contaminants are regulated and managed one element at a time, even though combinations of elements interact to influence many water-quality problems such as toxicity to life, eutrophication, infrastructure and water treatment. A chemical cocktail approach significantly expands evaluations of water-quality signatures and impacts beyond single elements to mixtures. High-frequency sensor data (pH, specific conductance, turbidity, etc.) can serve as proxies for chemical cocktails and improve real-time analyses of water-quality violations, identify regulatory needs, and track water quality recovery following and extreme climate events. Ultimately, a watershed chemical cocktail approach is necessary for effectively co-managing groups of contaminants and provides a more holistic approach for studying, monitoring, and managing water quality in the Anthropocene.