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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. 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.

We quantified morphological variation among genetically identified specimens of Fusconaia flava, F. subrotunda, Pleurobema cordatum, P. plenum, P. sintoxia, and P. rubrum inhabiting the Green River, Kentucky, species with shells that are morphologically similar to each other and thus difficult to identify. Molecular identifications then were compared with phenotype-based identifications by experts, who on average correctly identified 70% of the specimens. Expert identification of the putative species P. rubrum and P. sintoxia resulted in them usually being identified as the latter. Multi-variable decision tree analysis was conducted to determine the best suite of morphological variables for identifying live mussels and shells to species. Cross-validation error rates for these analyses were 12.6% and 4.14% for live mussels and shells, respectively. Both random forest and decision tree analyses showed the most important variables to be the presence/absence of a sulcus and shell shape (trapezoidal, circular, oval, equilateral triangle, or isosceles triangle). Dichotomous keys for identifying shells and live mussels were developed based on key morphological characteristics readily identifiable in the field, including foot color, beak direction, and beak position relative to the anterior margin. However, a definitive identification of these species may still need to rely on molecular methods, especially for endangered species.

Freshwater mussels are one of the most endangered faunal groups in North America, declining in terms of both diversity and abundance. Many states, including Louisiana, have implemented various studies and protections to document remaining mussel resources, and defend current populations. Louisiana currently has approximately 65 mussel species. However, numerous species have experienced significant declines in both abundance and distribution and the status of many is unknown. We investigated the current distribution and status of three rare mussel species in Louisiana, Pleurobema sintoxia (rubrum), Obovoaria unicolor, and Strophitus radiatus (target species). We surveyed for mussels at four sites historically known to harbor each species (12 sites), and four new sites with similar habitat (12 sites). We then compared our survey results to historical data to determine the health and status of each species. We recorded 4,799 live mussels of 33 species in timed searches. The assemblage was dominated by Plectomerus dombeyanus, Amblema plicata, Cyclonaias pustulosa, and Megalonaias nervosa which made up > 60% of mussels. A total of 24 target species were located, consisting of 15 P sintoxia (rubrum), from two sites, and nine S. radiatus from three sites. No O. unicolor were located. Target species were found almost exclusively at historical sites and were absent from nearly all sites. Conversely, length frequencies indicated a healthy age class, and that recruitment is occurring for both species. Despite potential loses, many of Louisiana's waterbodies retain a rich mussel fauna, including the continued presence and recruitment of several rare species. Unfortunately, these rare species appear to be surviving only in relatively low abundances, within limited ranges, though additional survey efforts across the state are needed.

Freshwater mussels in the genera Fusconaia, Pleurobema, and Pleuronaia are similar in their external shell morphology, which has made the identification and classification of species within these genera difficult and led to many taxonomic revisions. Large samples (N = 464) of select mussel species in these genera were collected from 2012 through 2014, primarily in the upper Tennessee River basin of Tennessee and Virginia, USA. Mitochondrial ND1 and nuclear ITS1 DNA sequences were analyzed to assess phylogenetic relationships among taxa. Ten species were verified as phylogenetically distinct at ND1, two of which were cryptic and previously unrecognized species. Described herein as Pleurobema parmaleei and Pleuronaia estabrookianus, each species clade was diverged at this gene region by ~3.0% from the respective closest congener. The nuclear ITS1 gene region’s nucleotide-site insertion/deletion (indel) patterns were analyzed as single mutational events rather than as fifth character states or missing data. Most species, including these two, were phylogenetically distinct at the ITS1 region when incorporating indels into analyses, but some estimated interspecific pairwise distances were lower than corresponding intraspecific estimates. Among morphological traits assessed for each species, differences in foot color and gravidity characteristics illustrated differences between phylogenetically recognized species and their closest congeners. Due to the limited known geographical distributions of these two cryptic species, each may require protection under the U.S. Endangered Species Act. While this study collected large sample sizes for each species, many streams in the basin remain unsampled and could potentially contain populations of these species or additional cryptic species.

Pleurobema riddellii is a state listed unionid in Texas, proposed for potential USFWS protections. Life history information is critical to determine whether a species warrants protection and to ensure that effective conservation measures are implemented. The Neches River is the only Texas river with substantial numbers of P. riddellii. In 2014, we selected three 25 m2 locations in the Neches River with the highest P. riddellii density and excavated those areas. All P. riddellii were collected, measured, and marked. This was repeated again in 2017. We used the inverted von Bertalanffy growth equation to create growth models for each location and an overall growth model. Models were compared to determine if there were differences between locations. The Highway 294 site had a higher growth rate and smaller individuals and was apparently a younger assemblage. To validate our models, additional P. riddellii were measured and their growth rings were counted. They were compared to the number of rings and sizes calculated by our model. Our overall growth model matched growth parameters calculated for other Pleurobema species, indicating that the model is accurately predicting P. riddellii growth. This study increases our knowledge of P. riddellii life history and can help in planning effective conservation strategies.

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. Relationships between two imperiled and morphologically similar freshwater mussel species, Pleurobema clava and Pleurobema oviforme, are re‐examined using mitochondrial DNA, morphometrics, and nuclear microsatellites. While phylogenetic analysis of mitochondrial DNA suggests a single, widespread species, one population was significantly divergent based upon microsatellites and morphometrics. Sampling of additional populations and analysis with additional nuclear loci are recommended to further define management units.

ABSTRACT Aim Freshwater mussels are considered among the most at‐risk taxa in the world. As such, comprehensive monitoring assessments of what abiotic and biotic factors influence mussel occupancy will be vital for guiding effective conservation. Here, we analysed vertebrate and mussel environmental DNA (eDNA) metabarcoding data to explore the influence of biotic (i.e., host fish diversity, predator presence, and community composition) and abiotic (i.e., drainage size, forest cover, and stream order) factors on freshwater mussel populations. Location This study utilised water samples and tactile survey data collected from streams throughout Fort Johnson, Louisiana. Methods We first evaluated the effectiveness of eDNA metabarcoding for characterising freshwater communities based on previous conventional tactile surveys. Next, we used eDNA metabarcoding analysis for freshwater mussels and vertebrate species alongside GIS‐derived satellite remote sensing data to assess how various biotic and abiotic variables impact freshwater mussel eDNA occupancy. Results Our eDNA metabarcoding survey largely agreed with both historical and contemporary surveys on Fort Johnson, while uniquely detecting Louisiana pigtoe (Pleurobema riddellii), a proposed threatened species under the US Endangered Species Act. We also found that eDNA detections and occupancy had strong seasonal variation, with increased read abundance and diversity in the spring. Vertebrate, fish, and predator diversity (as a function of habitat quality) were strongly predictive of mussel occupancy, supporting the concept of land managers focusing on the entire ecosystem for mussel conservation. Lastly, we found that percent forest cover and drainage basin size influenced mussel eDNA occupancy, informing habitat associations for mussel species of interest (i.e., the mussels occupied larger drainage sizes and perennial streams). Conclusions Our results demonstrate that combining eDNA metabarcoding of target and non‐target species with occupancy modelling can provide insights into the ecology of freshwater mussels and is a useful tool to improve their conservation and management.

Wabash Pigtoe, Fusconaia flava, and the related Round Pigtoe, Pleurobema sintoxia, are freshwater mussels (Bivalvia: Unionidae: Pleurobemini) native to the Great Lakes region of North America. Fusconaia flava is considered widespread and relatively common while P. sintoxia is considered an imperiled species. These species are similar in shell shape and coloration and have confounded many freshwater malacologists, resulting in frequent misidentifications. We sought to determine if morphometric analyses could be used to reliably distinguish between these species. Two hundred and forty-six specimens were collected from rivers in Michigan and Ontario. For each specimen, a preliminary identification was made, shell measurements and foot color (orange or white) were documented, and photos of the left shell valve were taken. A genetic sample was taken from 133 specimens for cytochrome c oxidase subunit 1 (COI) barcoding. COI sequences were used for species identification by comparing to sequences on GenBank. Twenty-one digitized landmarks along the outline of the left valve were analyzed and compared to the results of the DNA barcoding. Landmark data correctly assigned 99.2% of specimens to their DNA-confirmed species identity, compared to 82.0% accuracy of field identifications and 77.0% accuracy for foot color. The creation of a DNA-confirmed morphometric database will aid freshwater malacologists across the Great Lakes region in differentiating between these species.

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.

Biodiversity hotspots can serve as protected areas that aid in species conservation. Long-term monitoring of multiple taxonomic groups within biodiversity hotspots can offer insight into factors influencing their dynamics. Mussels (Bivalvia: Unionidae) and fish are highly diverse and imperiled groups of organisms with contrasting life histories that should influence their response to ecological factors associated with local and global change. Here we use historical and contemporary fish and mussel survey data to assess fish and mussel community changes over a 33 year period (1986–2019) and relationships between mussel abundance and their host fish abundance in Bogue Chitto Creek, a tributary of the Alabama River and a biodiversity hotspot. Mussel abundance declined by ~80% and community composition shifted, with eight species previously recorded not found in 2019, and a single individual of the endangered Pleurobema decisum. Fish abundances increased and life history strategies in the community appeared stable and there was no apparent relationship between mussel declines and abundance of host fish. Temporal variation in the proportion of life history traits composing mussel assemblages was also indicative of the disturbances specifically affecting the mussel community. However, changes and declines in mussel assemblages in Bogue Chitto Creek cannot be firmly attributed to any specific factor or events because of gaps in historical environmental and biological data. We believe that mobility differences contributed to differential responses of fish and mussel communities to stressors including habitat degradation, recent droughts and invasive species. Overall, our work indicates that monitoring biodiversity hotspots using hydrological measurements, standardized survey methods and monitoring invasive species abundance would better identify the effects of multiple and interactive stressors that impact disparate taxonomic groups in freshwater ecosystems.