Sean M. Keogh

Aim: Trait‐based approaches are increasingly recognised as tools for understanding species' vulnerabilities to intensifying global change. For species with complex life histories that involve a parasitic life stage, a combination of traits associated with either the host or parasite could influence extinction risk. Location: United States of America. Taxon: Freshwater mussels are a highly threatened assemblage of bivalves with an obligate ectoparasitic larval stage requiring a host fish. Adult mussels are sedentary, with long‐distance dispersal dictated by the dispersal ability of the host fish during the parasitic larval life phase. Methods: We integrate several open‐source spatial and trait datasets for freshwater mussels and host fishes to test hypotheses about the relationship between species‐level traits such as body size, area of occupancy (AOO), host fish dispersal, habitat fragmentation and mussel extinction risk. Results: Imperilled mussels were smaller, had smaller areas of occupancy, used small‐bodied, dispersal‐limited host fishes and had higher densities of large dams in their AOO compared to non‐imperilled mussels. Conclusions: Body size, along with AOO and dispersal potential, appears to have cascading effects on imperilment where decreases in these traits potentiate population fragmentation via large dams. Thus, in contrast to many other groups, freshwater mussel body size is inversely related to extinction risk, and small‐bodied mussels should be closely monitored to prevent future extinctions.

Morphological features play a critical role in the ability of an organism to navigate its environment. Organisms living in rivers and streams must handle a spectrum of habitats that span from dynamic flows in headwater creeks to the regularly high flows of large rivers. Freshwater mussels (order Unionoida) are an interesting group of organisms that possess a variety of hypothesized shell morphology traits adapted to life across the stream size spectrum. Fusconaia flava (family Unionidae) is a species that exhibits a pattern of clinal variation, known as Ortmann's Law of Stream Position, where shell width increases with stream size. Although speculations have been made about the causes and consequences of Ortmann's Law of Stream Position, few experiments have been conducted to help explain why some freshwater mussel species exhibit this phenotype–environment association. We conducted a comparative morphometric study and a fluvial experiment to identify how intraspecific mussel morphology and behaviour vary along a stream size gradient. We used micro‐computed tomography scanning to digitize shells from hydrologically diverse habitats, then measured the width, size and median thickness of each shell. Multiple regression was used to test the association between shell width and thickness while accounting for shell size, and linear regression models were used to describe the relationships between shell traits and hydrology. In a corresponding flume experiment, we measured valve‐gaping activity and observed presumptive burrowing behaviour of wild‐collected F. flava that exhibited ‘compressed’ (small‐river morph) and ‘inflated’ (large‐river morph) shell morphologies found in the upper and lower reaches of the Gasconade River in central Missouri, USA. We scaled flume flows to mimic observed low and high streamflows on the Gasconade River and used Hall sensors to record valve movements. We found shell thickness and shell width were strongly associated with one another and increased with aspects of stream size (i.e., Strahler stream order, flow rate and catchment area). The flume experiment demonstrated the feasibility and mechanistic potential of using wild‐collected mussels in behaviour experiments. We successfully integrated flow scaling via Froude numbers and demonstrated the sensitivity of Hall sensors for continuous behavioural monitoring. Valve‐gaping activity of ‘compressed’ mussels did not appear to have an affinity to a specific flow regime, but visually they made significant spatial movements, whereas ‘inflated’ mussels exhibited pronounced changes in valve‐gaping activity under higher flow conditions but remained more or less stationary. However, our data were not capable of definitive behavioural comparisons and instead proved the value of this setup for future, larger‐scale experiments. Given the ongoing and projected increase of droughts and catastrophic flood events due to climate change, our results provide insight into how morphological trait variation and valve‐gaping activity together allow individuals to respond to streamflow fluctuations. Additionally, our integration of comparative morphometrics and flume experiments provides a promising proof of concept for testing phenotype–environment associations.

Hydrodynamic forces and their absence appear to exert differential selection pressure on aquatic biodiversity in lake and stream habitats, creating a tight fit between organismal phenotypes and their environments. Ecophenotypic variants may be the result of genetic differentiation or phenotypic plasticity, where a genotype can produce multiple phenotypes dependent on the environment. Freshwater mussels possess a wide degree of morphological variation that frequently covaries with the environment, making them a good system to understand the mechanisms of ecophenotypic variation across hydrological conditions. We designed a two‐year experiment where individuals from the same Pyganodon grandis maternal brood (half and full siblings) were reared at a controlled site and four natural sites involving one lake and three streams. At the end of the experiment, shell shape was quantified for recaptured (N = 70), wild (N = 206), and zoo‐reared (N = 305) mussels. The maternal individual and 46 recaptured mussels were sequenced for genomic single nucleotide polymorphisms to test for multiple paternity and its effect on offspring morphology. Analysis of covariance found significant differences in shell shape between rearing sites, particularly between stream and lake habitats, but no shape differences were detected across the three stream sites. At two of the four sites, the shell shape of recaptured individuals was not significantly different than that of wild populations. Genomic sequencing and parentage analysis identified 11–27 different fathers among recaptured individuals. Yet no genetic differences were present between stream and lake habitats, and there was no paternal effect on shell shape. Taken together, phenotypic plasticity, over genetic differentiation, is identified as the primary mechanism of ecophenotypic variation. Plasticity is likely ubiquitous across freshwater mussels and may be a key adaptation given their high variance in habitat use. Multiple paternity may also play a role in the evolution of phenotypic plasticity, allowing more males from greater distances opportunities for fertilization, thus increasing genetic connectivity. Lastly, phenotypic plasticity and multiple paternity are convenient properties for freshwater mussel conservation and propagation. Multiple fathers increase the genetic variation of propagated broods, while plasticity may provide resilience to the release of stocked individuals across environmental heterogeneity.

The isolated river drainages of eastern North America serve as a natural laboratory to investigate the roles of allopatry and secondary contact in the evolutionary trajectories of recently diverged lineages. Drainage divides facilitate allopatric speciation, but due to their sensitivity to climatic and geomorphological changes, neighboring rivers frequently coalesce, creating recurrent opportunities of isolation and contact throughout the history of aquatic lineages. The freshwater mussel Quadrula quadrula is widely distributed across isolated rivers of eastern North America and possesses high phenotypic and molecular variation across its range. We integrate sequence data from three genomes, including female‐ and male‐inherited mitochondrial markers and thousands of nuclear encoded SNPs with morphology and geography to illuminate the group's divergence history. Across contemporary isolated rivers, we found continuums of molecular and morphological variation, following a pattern of isolation by distance. In contact zones, hybridization was frequent with no apparent fitness consequences, as advanced hybrids were common. Accordingly, we recognize Q. quadrula as a single cohesive species with subspecific variation (Q. quadrula rumphiana). Demographic modeling and divergence dating supported a divergence history characterized by allopatric vicariance followed by secondary contact, likely driven by river rearrangements and Pleistocene glacial cycles. Despite clinal range‐wide variation and hybridization in contact zones, the process‐based species delimitation tool delimitR, which considers demographic scenarios like secondary contact, supported the delimitation of the maximum number of species tested. As such, when interpreting species delimitation results, we suggest careful consideration of spatial sampling and subsequent geographic patterns of biological variation, particularly for wide‐ranging taxa.

2. Hydrodynamic forces and their absence appears to exert differential selection pressure between lake and stream populations, creating a tight fit between organismal phenotypes and their environments. Ecophenotypic variants may be the result from isolated genetic evolution or phenotypic plasticity, where a widespread genotype can produce multiple phenotypes dependent on the environment. Freshwater mussels possess a wide degree of morphological variation that frequently covaries with the environment, making them a good system to understand the mechanisms of ecophenotypic variation across hydrological conditions. We designed a two-year experiment where individuals from the same Pyganodon grandis maternal brood (half-full siblings) were reared at a controlled site and four natural sites involving one lake and three streams. At the end of the experiment, shell shape was quantified for recaptured (N=70), wild (N=206), and zoo-reared (N=305) mussels. Analysis of covariance found significant differences in shell shape between rearing sites, particularly between stream and lake habitats, but no shape differences were detected across the three stream sites. At two of the four sites, shell shape of recaptured individuals matched the morphology of wild populations. Genomic sequencing and parentage analysis identified 27 different fathers among recaptured individuals. Yet, no genetic differences were present between stream and lake habitats and there was no effect of parentage on shell shape. Taken together, phenotypic plasticity, over genetic differentiation, is identified as the primary mechanism of shell shape ecophenotypy. Plasticity may be a key adaptation for freshwater mussels and possibly provide a buffer against their imperilment in degraded habitats. 1. Summary statement: Streamlined and obese freshwater mussel shell shapes in stream-lake environments are developed from the same maternal brood and may reflect adaptations to the presence and absence of streamflow.

Frequent and strong morphological convergence suggests that determinism tends to supersede historical contingencies in evolutionary radiations. For many lineages living within the water column of rivers and streams, hydrodynamic forces drive widespread morphological convergence. Living below the sediment-water interface may release organisms from these hydrodynamic pressures, permitting a broad array of morphologies, and thus less convergence. However, we show here that the semi-infaunal freshwater mussels have environmentally determined convergence in shell morphology. Using 3D morphometric data from 715 individuals among 164 Nearctic species, we find that species occurring in rivers with high flow rates have evolved traits that resist dislodgement from their burrowed position in the streambed: thicker shells for their body size, with the thickest sector of the shell being the most deeply buried. Species occurring in low flow environments have evolved thinner and more uniformly thickened shells, corresponding to an alternative adaptation to dislodgement: increased burrowing efficiency. Within species, individuals also show increased shell thickness for their body size at higher flow rates, suggesting that ecophenotypy may, in part, be an important mechanism for establishing populations in new environments and thus evolutionary divergence in this highly imperiledinvertebrate group.

Natural history collections are uniquely positioned to chronicle biodiversity changes across time and space and are a fundamental data source in taxon-based research and conservation. With over 90 species listed under the Endangered Species Act, freshwater mussels are one of the most imperiled animal assemblages in the United States and are the focus of considerable conservation efforts (e.g., species status assessments, listing decisions, and recovery plans). Unfortunately, natural history collections data is often underleveraged in such efforts, in part, because much of the data are decentralized and nonstandard, and thus, difficult to access and analyze. Our objective herein is to synthesize, standardize, and enrich digitized US freshwater mussel collections data to better suit the needs of conservation stakeholders. We aggregated specimen records from 45 US natural history collections and enriched these records by programmatically standardizing taxonomic information, flagging potentially problematic records, and joining records with freshwater-specific spatial frameworks (e.g., hydrological units and stream segments) and their associated metadata (e.g., area, stream order, discharge, velocity). The assembled dataset includes 410,665 records, 302 species, and 1,494 hydrological units (8 digit-level). Using these enriched records, we estimated ecological attributes for over 280 freshwater mussel species including aspects of range size (i.e., area of occupancy and change in area of occupancy) and habitat preferences (i.e., stream order size, discharge, slope, and velocity). Listed species had significantly fewer occurrences (p<0.001) and smaller area of occupancy (p<0.001) in comparison to non-listed listed species. Listed species also tended to have a higher stream order preference (p<0.001) and discharge preference than non-listed species (p<0.001). These important ecological attributes have not been incorporated into freshwater mussel conservation efforts in a quantitative way and our novel estimates can be used to make more data-driven ecological and conservation inferences.

Aim: Freshwater mussels share habitat and are parasites of freshwater fishes during the larval life stage. Therefore, models of fish biogeography may also explain the historical biogeography of freshwater mussels. We tested this assumption using predictions of three biogeographic models constructed for northern Gulf of Mexico drainages on a freshwater mussel species complex. Specifically, we tested (1) if speciation was due to vicariant events of fluctuating sea levels that separated lineages east‐west of the Mobile Basin (Central Gulf Coast speciation hypothesis), (2) if the timing of divergences occurred 8.5–3.5 MYA (Gulf Coast allopatric speciation model) and (3) if diversification in Mississippi River populations was recent and for evidence of population increase consistent with range expansion into northern deglaciated regions (Pleistocene glaciation model).Location: Eastern North America.Taxon: Freshwater mussels (Bivalvia: Unionidae), Lampsilis teres and L. floridensis.Methods: We collected 249 specimens from 73 localities across the group's distribution. We used three molecular markers (COI, NDI & ITSI) to conduct time calibrated Bayesian phylogenetic analyses, phylogeographic analyses (AMOVA & SAMOVA) and demographic analyses including Bayesian skyline plots.Results: Lampsilis teres and L. floridensis are allopatric species whose distributions meet at the eastern edge of the Mobile Basin. Speciation was estimated to occur in the late Miocene. Populations from isolated river systems surrounding the Gulf of Mexico are almost all monophyletic. Mississippi drainage samples formed a shallow clade with recent diversification and showed evidence of recent population expansion.Main conclusions: The historical biogeography of the L. teres species complex is broadly consistent with tested ichthyofaunal models. The timing of speciation and intraspecific divergences correspond to low sea‐level events suggesting that Gulf Coast sea‐level fluctuations are responsible for dispersal (sea‐level recession) and subsequent cladogenesis (sea‐level inundation). Consistent with numerous other freshwater studies, we found the Mobile Basin to be a suture zone, which may be due to the narrow, offshore continental shelf.