A test of the loose-equilibrium concept with long-lived organisms: Evaluating temporal change in freshwater mussel assemblages.

The loose-equilibrium concept (LEC) predicts that ecological assemblages change transiently but return towards an earlier or average structure. The LEC framework can help determine whether assemblages vary within expected ranges or are permanently altered following environmental change. Long-lived, slow-growing animals typically respond slowly to environmental change, and their assemblage dynamics may respond over decades, which transcends most ecological studies. Unionid mussels are valuable for studying dynamics of long-lived animals because they can live >50 years and occur in dense, species-rich assemblages (mussel beds). Mussel beds can persist for decades, but disturbance can affect species differently, resulting in variable trajectories according to differences in species composition within and among rivers. We used long-term data sets (10-40 years) from seven rivers in the eastern United States to evaluate the magnitude, pace and directionality of mussel assemblage change within the context of the LEC. Site trajectories varied within and among streams and showed patterns consistent with either the LEC or directional change. In streams that conformed to the LEC, rank abundance of dominant species remained stable over time, but directional change in other streams was driven by changes in the rank abundance and composition of dominant species. Characteristics of mussel assemblage change varied widely, ranging from those conforming to the LEC to those showing strong directional change. Conservation approaches that attempt to maintain or create a desired assemblage condition should acknowledge this wide range of possible assemblage trajectories and that the environmental factors that influence those changes remain poorly understood.

Glacial vicariance and secondary contact shape demographic histories in a freshwater mussel species complex.

Characterizing the mechanisms influencing the distribution of genetic variation in aquatic species can be difficult due to the dynamic nature of hydrological landscapes. In North America's Central Highlands, a complex history of glacial dynamics, long-term isolation, and secondary contact have shaped genetic variation in aquatic species. Although the effects of glacial history have been demonstrated in many taxa, responses are often lineage- or species-specific and driven by organismal ecology. In this study, we reconstruct the evolutionary history of a freshwater mussel species complex using a suite of mitochondrial and nuclear loci to resolve taxonomic and demographic uncertainties. Our findings do not support Pleurobema rubrum as a valid species, which is proposed for listing as threatened under the U.S. Endangered Species Act. We synonymize P. rubrum under Pleurobema sintoxia-a common and widespread species found throughout the Mississippi River Basin. Further investigation of patterns of genetic variation in P. sintoxia identified a complex demographic history, including ancestral vicariance and secondary contact, within the Eastern Highlands. We hypothesize these patterns were shaped by ancestral vicariance driven by the formation of Lake Green and subsequent secondary contact after the last glacial maximum. Our inference aligns with demographic histories observed in other aquatic taxa in the region and mirrors patterns of genetic variation of a freshwater fish species (Erimystax dissimilis) confirmed to serve as a parasitic larval host for P. sintoxia. Our findings directly link species ecology to observed patterns of genetic variation and may have significant implications for future conservation and recovery actions of freshwater mussels.

Histological evaluations of organ tissues reveal sublethal effects in a freshwater mussel (Villosa iris) exposed to chloride and potassium concentrations below benchmark estimates.

Salinization of freshwater ecosystems due to anthropogenic sources will increasingly impact biodiversity. An example of point-source industrial salinization has occurred from historical activities at a U.S. Environmental Protection Agency Superfund Site near Saltville, Virginia USA and its associated chemical waste ponds adjacent to the North Fork Holston River. These point source discharges are documented contributors to mussel declines, partially due to high concentrations of chloride (Cl-, ≤ 26,000 mg Cl-/L) and potassium (K+, ≤ 97 mg K+/L). During a chronic 61-day laboratory study, Rainbow mussels, Villosa iris, were exposed to concentrations of Cl- (0, 416, 831, and 1,663 mg/L) and K+ (0, 4, 8, and 17 mg/L) to determine effects on survival and organ tissues. All test mussels died by day-2 in the 1,663 mg Cl-/L exposure, and 50% of mussels died by day-13 in the 17 mg K+/L concentration. Significantly greater abundances of tissue abnormalities were observed in digestive glands and kidneys with exposures to the 4 and 8 mg/L concentrations of K+ versus the control, and significantly greater abundances of lesions in kidneys were observed in the 416 and 831 mg Cl-/L concentrations compared to the control. The sublethal effects to digestive glands and kidneys were below reported effect (EC50, 20, 10 and LOEC) concentrations. Significant histological differences between control and baseline (day-0 sample) mussels were observed, suggesting the need for further study on the effects of captivity during longer-term laboratory experiments.

Description of the mantle lure and novel mimicry of the endangered Cumberlandian Combshell (Epioblasma brevidens) in the Clinch River, eastern United States.

The Cumberlandian Combshell (Epioblasma brevidens) is an endangered freshwater mussel endemic to the Tennessee and Cumberland River drainages, major tributaries of the Ohio River of the eastern United States. We conducted mask and snorkel surveys in May and June of 2021 and 2022 to locate, observe, photograph, and video female E. brevidens to document their unique mantle lures at sites in the Clinch River in Tennessee and Virginia. The mantle lure is morphologically specialized mantle tissue that mimics prey items of the host fish. The mantle lure of E. brevidens appears to mimic four distinct characteristics of the reproductive anatomy of the underside (ventral) of a gravid female crayfish, to include: (1) the external apertures of the oviducts located on the base of the third pair of walking legs, (2) crayfish larvae still encased in the egg membrane, (3) pleopods or claws, and (4) postembryonic eggs. Surprisingly, we observed males of E. brevidens displaying mantle lures that were anatomically complex and closely resembled the female mantle lure. The male lure similarly mimics oviducts, eggs, and pleopods but is diminutive (2-3 mm smaller in length or diameter) to those same structures in females. We describe for the first time the mantle lure morphology and mimicry of E. brevidens, showing its close resemblance to the reproductive anatomy of a gravid female crayfish, and a novel form of mimicry in males. To our knowledge, mantle lure displays in males have not been previously documented in freshwater mussels.

Phylogenetic assessment of endangered and look-alike Pigtoe species in a freshwater mussel diversity hotspot.

The Green River in Kentucky in the eastern United States is a freshwater mussel biodiversity hotspot, with 71 known species. Among them, the endangered Pleurobema plenum coexists with other morphologically similar species in the genera Fusconaia and Pleurobema, known colloquially as "pigtoes." Identification of species in these genera is challenging even for mussel experts familiar with them. In our study, the correct identification of these species by experts ranged from 57% to 83%. We delineated taxonomic boundaries among seven species and tested for cryptic biodiversity among these look-alike mussels utilizing mitochondrial and nuclear DNA sequence variation. Phylogenetic analysis of combined (1215 bp) mitochondrial DNA cytochrome oxidase I (COI) and NADH dehydrogenase 1 (ND1) genes showed five well-diverged groups that included F. flava, F. subrotunda, P. cordatum, and P. plenum as distinct clades, with P. sintoxia and P. rubrum grouped into a single clade. While our mitochondrial DNA analyses did not distinguish P. sintoxia and P. rubrum as phylogenetically distinct species, the typical shell forms of these two nominal taxa are very distinct. Further phylogenetic analysis using nuclear ribosomal transcribed spacer region subunit I (ITS1) DNA sequences also showed that P. sintoxia and P. rubrum were not distinct lineages. No cryptic species were detected in the Fusconaia and Pleurobema samples analyzed from the Green River. The highest haplotype diversity (h), average number of nucleotide differences (k), and nucleotide diversity (π) were observed for F. subrotunda at both the COI (h = 0.896, k = 3.805, π = 0.00808) and ND1 (h = 0.984, k = 6.595, π = 0.00886) markers, with similarly high genetic diversity in the other taxa. Our results give managers confidence that cryptic taxa do not occur within or among these morphologically similar species in the Green River, and populations appear genetically diverse, indicative of large and healthy populations.

Combined effects of copper, nickel, and zinc on growth of a freshwater mussel (Villosa iris) in an environmentally relevant context.

Trace metals rarely contaminate freshwaters independently, hence regulatory limits based on single-metal toxicity may be underprotective of aquatic life. This could be especially the case for rare and sensitive fauna like freshwater mussels, such as those suppressed in the Clinch and Powell Rivers in eastern USA where trace metals are long-term contaminants but at concentrations below regulatory limits. We hypothesized metal mixtures may be exerting combined effects on mussels, resulting in greater toxicity than would be predicted based on single-metal exposures. To test that hypothesis, we conducted two experiments exposing juvenile rainbow mussels (Villosa iris) for 42 days to dissolved copper, nickel, and zinc, individually and in three-metal mixtures, in an environmentally-relevant context of water with chemistry (hardness 155 mg/L as CaCO3, dissolved organic carbon 1.7-2.3 mg/L, pH 8.4) similar to that of the Clinch River, which receives alkaline mine drainage. We used a toxic unit approach, selecting test concentrations based on literature values for the lower of 28-day survival or growth (length) effect concentrations for Villosa iris or Lampsilis siliquoidea (fatmucket). Our first experiment confirmed survival and growth effects when acute and chronic water quality criteria, respectively, are approached and/or exceeded. Our second experiment, at lower concentrations, showed no effects on survival but combined effects on growth were evident: a mixture of Cu, Ni, and Zn (7.2 ± 1.2, 65.3 ± 6.1, 183 ± 32 µg/L, respectively) inhibited growth (dry weight) by 95% versus 73%, 74%, and 83% inhibition for single-metal exposures to Cu, Ni, and Zn of similar concentration (8.0 ± 1.1, 63.5 ± 4.8, 193 ± 31 µg/L, respectively). Furthermore, a mixture of Cu, Ni, and Zn with individual concentrations 21%, 29%, and 37% of their water quality criteria (3.4 ± 1.2, 21.8 ± 1.8, and 62.1 ± 8.4 µg/L, respectively) inhibited growth (dry weight) by 61% relative to controls. Our observation of combined effects suggests that regulatory limits based on single-metal toxicity may be underprotective of freshwater mussels when multiple metals are present.

Genetic and morphological characterization of the freshwater mussel clubshell species complex (Pleurobema clava and Pleurobema oviforme) to inform conservation planning.

The shell morphologies of the freshwater mussel species Pleurobema clava (federally endangered) and Pleurobema oviforme (species of concern) are similar, causing considerable taxonomic confusion between the two species over the last 100 years. While P. clava was historically widespread throughout the Ohio River basin and tributaries to the lower Laurentian Great Lakes, P. oviforme was confined to the Tennessee and the upper Cumberland River basins. We used two mitochondrial DNA (mtDNA) genes, 13 novel nuclear DNA microsatellite markers, and shell morphometrics to help resolve this taxonomic confusion. Evidence for a single species was apparent in phylogenetic analyses of each mtDNA gene, revealing monophyletic relationships with minimal differentiation and shared haplotypes. Analyses of microsatellites showed significant genetic structuring, with four main genetic clusters detected, respectively, in the upper Ohio River basin, the lower Ohio River and Great Lakes, and upper Tennessee River basin, and a fourth genetic cluster, which included geographically intermediate populations in the Ohio and Tennessee river basins. While principal components analysis (PCA) of morphometric variables (i.e., length, height, width, and weight) showed significant differences in shell shape, only 3% of the variance in shell shape was explained by nominal species. Using Linear Discriminant and Random Forest (RF) analyses, correct classification rates for the two species' shell forms were 65.5% and 83.2%, respectively. Random Forest classification rates for some populations were higher; for example, for North Fork Holston (HOLS), it was >90%. While nuclear DNA and shell morphology indicate that the HOLS population is strongly differentiated, perhaps indicative of cryptic biodiversity, we consider the presence of a single widespread species the most likely biological scenario for many of the investigated populations based on our mtDNA dataset. However, additional sampling of P. oviforme populations at nuclear loci is needed to corroborate this finding.

Understanding the influence of multiple pollutant stressors on the decline of freshwater mussels in a biodiversity hotspot.

The Clinch River watershed of the upper Tennessee River Basin of Virginia and Tennessee, USA supports one of North America's greatest concentrations of freshwater biodiversity, including 46 extant species of native freshwater mussels (Order Unionida), 20 of which are protected as federally endangered. Despite the global biological significance of the Clinch River, mussel populations are declining in some reaches, both in species richness and abundance. The aim of this study was to evaluate the exposure of adult resident mussels to a suite of inorganic and organic contaminant stressors in distinct sections of the Clinch River that encompassed a range of mussel abundance and health. To provide insight into the potential role of pollutants in the decline of mussels, including within a previously documented "zone of mussel decline", the mainstem Clinch River (8 sites) and its tributaries (4 sites) were examined over two consecutive years. We quantified and related metals and organic contaminant concentrations in mussels to their associated habitat compartments (bed sediment, suspended particulate sediment, pore water, and surface water). We found that concentrations of organic contaminants in resident mussels, particularly the suite of 42 polycyclic aromatic hydrocarbons (PAHs) analyzed, were related to PAH concentrations in all four habitat (media) compartments. Further, PAH concentrations in mussel tissue (range 37.8-978.1 ng/g dry weight in 2012 and 194.3-1073.7 ng/g dry weight in 2013) were negatively related to the spatial pattern in mussel densities (rs = -0.64, p ≤ 0.05 in 2012 and rs = -0.83, p ≤ 0.05 in 2013) within the river, and were highest in the "zone of mussel decline". In contrast, the suite of 22 metals analyzed in resident mussels were largely unrelated to the spatial pattern of variation of metals in the four habitat compartments except for Manganese (Mn; range 3630.5-23,749.2 µg/g dry weight in 2012 and 1540.4-12,605.8 µg/g dry weight in 2013) in surface water (rs = 0.58, p < 0.1) and pore water (rs = 0.76, p ≤ 0.05). This study revealed that PAHs and Mn are important pollutant stressors to mussels in the Clinch River and that they are largely being delivered through the Guest River tributary watershed. Accordingly, future conservation and management efforts would benefit by identifying, and ideally mitigating, the sources of PAHs, Mn, and other current or legacy mining-associated pollutants to the mainstem river and its tributaries.

Assessment of growth, survival, and organ tissues of caged mussels (Bivalvia: Unionidae) in a river-scape influenced by coal mining in the southeastern USA.

The Clinch River contains one of the most diverse freshwater mussel assemblages in the United States, with 46 extant species, 20 of which are listed as federally endangered. In a 91 km section of the Clinch River, mussel densities have severely declined at key sites from 1979 to 2014 (zone of decline), compared to other river sections that support high density and stable mussel populations (zone of stability). Coal mining has occurred in tributary watersheds that drain to the zone of decline since the late nineteenth century. To determine differences in survival, growth, and organ (gills, digestive glands, kidneys, and gonads) tissue health between the river zones, laboratory-propagated mussels were deployed to cages for one year at four sites each within the zones of mussel stability and decline. Means of growth and survival of mussels in the zone of stability were significantly greater than in the zone of decline, and mean fractions of kidney lipofuscin in mussels in the zone of decline was significantly greater than in the zone of stability. Water concentrations of K+, Na+, Al, and Mn were significantly greater in the zone of decline than in the zone of stability. Statistical correlation results indicated that mussel survival was positively associated with concentrations of Mn in water, and kidney lipofuscin was negatively associated with concentrations of dissolved K+, SO42-, and Mg2+. Most concentrations of contaminants were below published benchmark criteria, yet our results suggest that conditions continue to exist in the zone of decline that promote impacts to mussels that are at least partially associated with low concentration coal-related contaminants. More research is needed to determine the additive, synergistic, or antagonistic effects of these complex ionic mixtures on freshwater mussels from river environments, such as in the Clinch River, where constituent concentrations are relatively low.

Spatial and temporal relationships among watershed mining, water quality, and freshwater mussel status in an eastern USA river.

The Powell River of southwestern Virginia and northeastern Tennessee, USA, drains a watershed with extensive coal surface mining, and it hosts exceptional biological richness, including at-risk species of freshwater mussels, downstream of mining-disturbed watershed areas. We investigated spatial and temporal patterns of watershed mining disturbance; their relationship to water quality change in the section of the river that connects mining areas to mussel habitat; and relationships of mining-related water constituents to measures of recent and past mussel status. Freshwater mussels in the Powell River have experienced significant declines over the past 3.5 decades. Over that same period, surface coal mining has influenced the watershed. Water-monitoring data collected by state and federal agencies demonstrate that dissolved solids and associated constituents that are commonly influenced by Appalachian mining (specific conductance, pH, hardness and sulfates) have experienced increasing temporal trends from the 1960s through ~2008; but, of those constituents, only dissolved solids concentrations are available widely within the Powell River since ~2008. Dissolved solids concentrations have stabilized in recent years. Dissolved solids, specific conductance, pH, and sulfates also exhibited spatial patterns that are consistent with dilution of mining influence with increasing distance from mined areas. Freshwater mussel status indicators are correlated negatively with dissolved solids concentrations, spatially and temporally, but the direct causal mechanisms responsible for mussel declines remain unknown.

Effects of environmentally relevant mixtures of major ions on a freshwater mussel.

The Clinch and Powell Rivers (Virginia, USA) support diverse mussel assemblages. Extensive coal mining occurs in both watersheds. In large reaches of both rivers, major ion concentrations are elevated and mussels have been extirpated or are declining. We conducted a laboratory study to assess major ion effects on growth and survival of juvenile Villosa iris. Mussels were exposed to pond water and diluted pond water with environmentally relevant major ion mixtures for 55 days. Two treatments were tested to mimic low-flow concentrations of Ca(2+), Mg(2+), [Formula: see text] , [Formula: see text] , K(+) and Cl(-) in the Clinch and Powell Rivers, total ion concentrations of 419 mg/L and 942 mg/L, respectively. Mussel survival (>90%) and growth in the two treatments showed little variation, and were not significantly different than in diluted pond water (control). Results suggest that major ion chronic toxicity is not the primary cause for mussel declines in the Clinch and Powell Rivers.

Interactive effects of climate change with nutrients, mercury, and freshwater acidification on key taxa in the North Atlantic Landscape Conservation Cooperative region.

The North Atlantic Landscape Conservation Cooperative LCC (NA LCC) is a public-private partnership that provides information to support conservation decisions that may be affected by global climate change (GCC) and other threats. The NA LCC region extends from southeast Virginia to the Canadian Maritime Provinces. Within this region, the US National Climate Assessment documented increases in air temperature, total precipitation, frequency of heavy precipitation events, and rising sea level, and predicted more drastic changes. Here, we synthesize literature on the effects of GCC interacting with selected contaminant, nutrient, and environmental processes to adversely affect natural resources within this region. Using a case study approach, we focused on 3 stressors with sufficient NA LCC region-specific information for an informed discussion. We describe GCC interactions with a contaminant (Hg) and 2 complex environmental phenomena-freshwater acidification and eutrophication. We also prepared taxa case studies on GCC- and GCC-contaminant/nutrient/process effects on amphibians and freshwater mussels. Several avian species of high conservation concern have blood Hg concentrations that have been associated with reduced nesting success. Freshwater acidification has adversely affected terrestrial and aquatic ecosystems in the Adirondacks and other areas of the region that are slowly recovering due to decreased emissions of N and sulfur oxides. Eutrophication in many estuaries within the region is projected to increase from greater storm runoff and less denitrification in riparian wetlands. Estuarine hypoxia may be exacerbated by increased stratification. Elevated water temperature favors algal species that produce harmful algal blooms (HABs). In several of the region's estuaries, HABs have been associated with bird die-offs. In the NA LCC region, amphibian populations appear to be declining. Some species may be adversely affected by GCC through higher temperatures and more frequent droughts. GCC may affect freshwater mussel populations via altered stream temperatures and increased sediment loading during heavy storms. Freshwater mussels are sensitive to un-ionized ammonia that more toxic at higher temperatures. We recommend studying the interactive effects of GCC on generation and bioavailability of methylmercury and how GCC-driven shifts in bird species distributions will affect avian exposure to methylmercury. Research is needed on how decreases in acid deposition concurrent with GCC will alter the structure and function of sensitive watersheds and surface waters. Studies are needed to determine how GCC will affect HABs and avian disease, and how more severe and extensive hypoxia will affect fish and shellfish populations. Regarding amphibians, we suggest research on 1) thermal tolerance and moisture requirements of species of concern, 2) effects of multiple stressors (temperature, desiccation, contaminants, nutrients), and 3) approaches to mitigate impacts of increased temperature and seasonal drought. We recommend studies to assess which mussel species and populations are vulnerable and which are resilient to rising stream temperatures, hydrological shifts, and ionic pollutants, all of which are influenced by GCC.

Acute effects of road salts and associated cyanide compounds on the early life stages of the unionid mussel Villosa iris.

The toxicity of cyanide to the early life stages of freshwater mussels (order Unionida) has remained unexplored. Cyanide is known to be acutely toxic to other aquatic organisms. Cyanide-containing compounds, such as sodium ferrocyanide and ferric ferrocyanide, are commonly added to road deicing salts as anticaking agents. The purpose of the present study was to assess the acute toxicity of three cyanide compounds (sodium cyanide, sodium ferrocyanide, and ferric ferrocyanide), two road salts containing cyanide anticaking agents (Morton and Cargill brands), a brine deicing solution (Liquidow brand), and a reference salt (sodium chloride) on glochidia (larvae) and juveniles of the freshwater mussel Villosa iris. Sodium ferrocyanide and ferric ferrocyanide were not acutely toxic to glochidia and juvenile mussels at concentrations up to 1,000 mg/L and 100 mg/L, respectively. Lowest observed effect concentrations (LOECs) for these two chemicals ranged from 10 to >1,000 mg/L. Sodium cyanide was acutely toxic to juvenile mussels, with a 96-h median effective concentration (EC50) of 1.10 mg/L, although glochidia tolerated concentrations up to 10 mg/L. The EC50s for sodium chloride, Liquidow brine, Morton road salt, and Cargill road salt were not significantly different for tests within the same life stage and test duration (range, 1.66-4.92 g/L). These results indicate that cyanide-containing anticaking agents do not exacerbate the toxicity of road salts, but that the use of road salts and brine solutions for deicing or dust control on roads may warrant further investigation.

Homogeneity at nuclear microsatellite loci masks mitochondrial haplotype diversity in the endangered fanshell pearlymussel (Cyprogenia stegaria).

We report on multiple patterns of differentiation and connectivity in the fanshell pearlymussel (Cyprogenia stegaria), based on different markers. Knowledge of genetic variation and genetic connectivity among remaining populations of this federally endangered species is needed to initiate implementation of the species recovery plan. We collected tissue samples from 96 specimens from the Green, Rolling Fork, and Licking Rivers, tributaries to the Ohio River, and the Clinch River, a tributary to the Tennessee River, providing broad coverage of the current distributional range of the species. Results from 7 nuclear DNA microsatellite markers suggested minimal population-level differentiation, whereas a mitochondrial DNA (mtDNA) marker (ND1) exhibited significant differentiation between C. stegaria in the Clinch River and the Ohio River populations. The ND1 data also confirm the existence of 2 distinct mtDNA lineages in the genus that transcends species boundaries. Further analyses suggest that the disproportionally strong signal from 2 very divergent ND1 lineages possibly masks finer-grained structure in the Ohio River population, based on one of the mtDNA lineages only. We recommend further sampling to confirm the absence of one lineage from the upper Clinch River drainage and suggest that provisional management guidelines should limit reciprocal exchanges among C. stegaria populations from the Clinch River and those in the Ohio River system.

Chlorine toxicity to early life stages of freshwater mussels (Bivalvia: Unionidae).

Chlorine (Cl) is a highly toxic, widely used halogen disinfectant that is present in point-source pollution discharges from wastewater treatment plants and industrial facilities. The U.S. Environmental Protection Agency freshwater criteria for Cl are 19 microg total residual Cl (TRC)/L as a maximum 1-h average concentration and 11 microg TRC/L as a maximum 4-d average; however, toxicological data for unionids were not used in these calculations. To address this void in the data, we conducted acute tests with glochidia from several species and 21-d bioassays with three-month-old Epioblasma capsaeformis and three-, six-, and 12-month-old Villosa iris juveniles. The 24-h lethal concentration 50 values for glochidia were between 70 and 220 [Lg TRC/L, which are 2.5 to 37 times higher than those reported in other studies for cladocerans. Significant declines in growth and survivorship were observed in the 21-d test with E. capsaeformis at 20 microg TRC/L. Lowest-observed-adverse-effects concentrations in bioassays with juvenile V. iris were higher (30-60 microg TRC/L) but showed a significant trend of declining toxicity with increased age. Although endpoints were above water quality criteria, the long life spans of unionids and potential implications of chronic exposure to endangered juvenile mussels still warrant concern.