Freshwater unionid bivalves currently face severe anthropogenic challenges. Over 70% of species in the United States are threatened, endangered or extinct due to pollution, damming of waterways and overfishing. These species are notable for their unusual life history strategy, parasite–host co‐evolution and biparental mitochondrial inheritance. Among this clade, the washboard mussel Megalonaias nervosa is one species that remains prevalent across the Southeastern United States, with robust population sizes. We have created a reference genome for M. nervosa to determine how genome content has evolved in the face of these widespread environmental challenges. We observe dynamic changes in genome content, with a burst of recent transposable element proliferation causing a 382 Mb expansion in genome content. Birth–death models suggest rapid expansions among gene families, with a mutation rate of 1.16 × 10−8 duplications per gene per generation. Cytochrome P450 gene families have experienced exceptional recent amplification beyond expectations based on genome‐wide birth–death processes. These genes are associated with increased rates of amino acid changes, a signature of selection driving evolution of detox genes. Fitting evolutionary models of adaptation from standing genetic variation, we can compare adaptive potential across species and mutation types. The large population size in M. nervosa suggests a 4.7‐fold advantage in the ability to adapt from standing genetic variation compared with a low diversity endemic E. hopetonensis. Estimates suggest that gene family evolution may offer an exceptional substrate of genetic variation in M. nervosa, with Psgv = 0.185 compared with Psgv = 0.067 for single nucleotide changes. Hence, we suggest that gene family evolution is a source of 'hopeful monsters’ within the genome that may facilitate adaptation when selective pressures shift. These results suggest that gene family expansion is a key driver of adaptive evolution in this key species of freshwater Unionidae that is currently facing widespread environmental challenges. This work has clear implications for conservation genomics on freshwater bivalves as well as evolutionary theory. This genome represents a first step to facilitate reverse ecological genomics in Unionidae and identify the genetic underpinnings of phenotypic diversity.

Natural resource managers use data on the spatial range of species to guide management decisions. These data come from survey or monitoring efforts that use a wide variety of tools. Environmental DNA (eDNA) is a surveillance tool that uses genetic markers for detecting species and holds potential as a tool for large‐scale monitoring programs. Two challenges of eDNA‐based studies are uncertainties created by imperfect capture of eDNA in collection samples (e.g., water field samples) and imperfect detection of eDNA using molecular methods (e.g., quantitative PCR). Occurrence models can be used to address these challenges, thus we use an occurrence model to address two objectives: first, to determine how many samples were required to detect species using eDNA; second, to examine when and where to take samples. We collected water samples from three different habitat types in the Upper Mississippi River when both Bighead Carp and Silver Carp were known to be present based on telemetry detections. Each habitat type (backwater, tributary, and impoundment) was sampled during April, May, and November. Detections of eDNA for both species varied across sites and months, but were generally low, 0–19.3% of samples were positive for eDNA. Overall, we found that eDNA‐based sampling holds promise to be a powerful monitoring tool for resource managers; however, limitations of eDNA‐based sampling include different biological and ecological characteristics of target species such as seasonal habitat usage patterns as well as aspects of different physical environments that impact the implementation of these methods such as water temperature.

The Silver Carp Hypophthalmichthys molitrix and Bighead Carp H. nobilis are two species of invasive bigheaded carp currently invading North American rivers and watersheds. Bigheaded carp were accidentally introduced into the lower Mississippi River basin in the early 1970s and have since invaded many water bodies in the Midwestern United States. Evidence of bigheaded carp reproduction and recruitment in the upper Mississippi River upstream of Lock and Dam 19 (LD19) at Keokuk, Iowa, thought to be a critical constriction point to their upstream establishment, has been limited to a few isolated detections of eggs, larvae, and juvenile life stages since 2012. Therefore, a more comprehensive assessment of bigheaded carp reproduction in this critical management zone was needed. We used quadrafoil light traps (n = 1,387) deployed during May–September 2016–2018 in Pools 17–19 of the Mississippi River to monitor for advanced larval bigheaded carp in low‐velocity habitats. Throughout the sampling period, we captured 1,747 larval and 35 postlarval bigheaded carp (N = 1,782). Bigheaded carp were collected on 15 sampling events that spanned from May 31, 2016, to September 13, 2018, with associated hatch dates estimated to represent 10 unique reproductive events from May 2016 to September 2018. The individual captures and backdated hatch estimates revealed a protracted spawning period of up to seven events in 2016, one event in 2017, and two events in 2018. Bigheaded carp were only captured in Pool 19, possibly due to the drifting requirements for egg maturation and the low‐velocity downstream reach of Pool 19. This research provides confirmation that bigheaded carp spawned upstream of LD19 are capable of transitioning past the yolk sac stage upstream of this bottleneck to more advanced larval stages. Knowledge of reproduction and larval retention and the field‐based evidence of protracted spawning fill critical research gaps needed for the management of bigheaded carp.

Environmental DNA (eDNA) sampling, the detection of species‐specific genetic material in water samples, is an emerging tool for monitoring aquatic invasive species. Optimizing eDNA sampling protocols can be challenging because there is imperfect understanding of how each step of the protocol influences its sensitivity. This paper develops a probabilistic model that characterizes each step of an eDNA sampling protocol to evaluate the protocol's overall detection sensitivity for one sample. The model is then applied to analyse how changes over time made to the eDNA sampling protocol to detect bighead (BH) and silver carp (SC) eDNA have influenced its sensitivity, and hence interpretation of the results. The model shows that changes to the protocol have caused the sensitivity of the protocol to fluctuate. A more efficient extraction method in 2013, new species‐specific markers with a qPCR assay in 2014, and a more efficient capture method in 2015 have improved the sensitivity, while switching to a larger elution volume in 2013 and a smaller sample volume in 2015 have reduced the sensitivity. Overall, the sensitivity of the current protocol is higher for BH eDNA detection and SC eDNA detection compared to the original protocol used from 2009 to 2012. The paper shows how this model of eDNA sampling can be used to evaluate the effect of proposed changes in an eDNA sampling and analysis protocol on the sensitivity of that protocol to help researchers optimize their design.

The Black Carp Mylopharyngodon piceus is an increasingly widespread invasive species in North America that threatens freshwater mussel populations. We developed four qPCR assays for detecting environmental DNA (eDNA) from these Black Carp populations. Assays were designed to target four mitochondrial DNA loci and were based on 34 complete mitochondrial genome sequences, including 29 generated in this study from samples obtained in three countries. Assays were validated for taxon specificity with in silico comparisons against archived DNA sequences and with in vitro tests of 41 DNA samples from Black Carp, as well as DNA samples from 30 nontarget fish species, all from the Mississippi River basin. All four assays were able to detect the DNA of all Black Carp samples and did not exhibit any positive results with DNA from other tested species. Tests conducted in round‐robin fashion among three different laboratories found that all four assays were able to detect DNA at very low template concentrations (limits of detection = 3 copies/qPCR, limits of quantification = 16–64 copies/qPCR) and, as part of in situ validation, were successful in detecting eDNA from Black Carp in aquaculture ponds. Despite some challenges with other attempts at in situ validation, the assays were also effective in detecting Black Carp eDNA in water samples from a drainage ditch in the upper reaches of the species’ range that was known to contain juvenile Black Carp, as well as in water samples from the Mississippi River and a connected oxbow lake in the lower reaches of the species’ range.

Before eDNA surveys can be deployed, the methods used to capture and extract DNA need to be determined. Multiple widely used eDNA capture and DNA extraction methods are available, and the effectiveness of these methods has been assessed in the literature. These studies used raw estimates of target cells captured from mesocosms and tanks, or already extracted DNA, which are not reflective of environmental DNA or precise enough to accurately compare eDNA capture and DNA extraction methods. Here, using qPCR quantification, we compared two eDNA capture and extraction methods using low and high concentrations of DNA cloned into living cells. By cloning target DNA into living cells, we can control the quantity of our starting copy number to directly compare the efficiency and effectiveness of each method throughout the process of eDNA handling and data analysis. This approach is useful for determining which capture and extraction method is suitable for the target species and sampling location of interest before full‐scale deployment. Although our target species DNA is consistently and accurately detected (even at low concentrations), stochasticity is a significant factor in our ability to consistently recover all the starting material by the end of sample processing. By recognizing that stochasticity is important for eDNA recovery, more accurate sampling models and eDNA processing protocols can be developed to increase eDNA detection sensitivity.

The barnacle Chthamalus fragilis is found along the US Atlantic seaboard historically from the Chesapeake Bay southward, and in the Gulf of Mexico. It appeared in New England circa 1900 coincident with warming temperatures, and is now a conspicuous member of rocky intertidal communities extending through the northern shore of Cape Cod, Massachusetts. The origin of northern C. fragilis is debated. It may have spread to New England from the northern end of its historic range through larval transport by ocean currents, possibly mediated by the construction of piers, marinas, and other anthropogenic structures that provided new hard substrate habitat. Alternatively, it may have been introduced by fouling on ships originating farther south in its historic distribution. Here we examine mitochondrial cytochrome c oxidase I sequence diversity and the distribution of mitochondrial haplotypes of C. fragilis from 11 localities ranging from Cape Cod, to Tampa Bay, Florida. We found significant genetic structure between northern and southern populations. Phylogenetic analysis revealed three well-supported reciprocally monophyletic haplogroups, including one haplogroup that is restricted to New England and Virginia populations. While the distances between clades do not suggest cryptic speciation, selection and dispersal barriers may be driving the observed structure. Our data are consistent with an expansion of C. fragilis from the northern end of its mid-19th century range into Massachusetts.

North American freshwater mussels are critically imperiled organisms that generally require fish hosts in order to complete their life cycle. Although numerous studies have focused on the parasitic relationship between mussels and fishes, few have examined the benefits that mussels provide to other organisms. During sampling of Altamaha River, Georgia, we observed foreign eggs occurring within body cavities of native mussels across a 253‐km reach of the river basin. Eggs were recovered from 6% of the 757 mussels examined among seven sites. Foreign eggs were present in 17% and 18% of examined mussels at two sites. Using molecular techniques, eggs were identified as American Shad Alosa sapidissima. This discovery appears to be the first documented occurrence of native fish eggs in live North American mussels. Further research into the nature and mechanism of this symbiosis is warranted to assess whether this relationship is amensalistic, mutualistic, or commensalistic as American Shad and many freshwater mussels are species of conservation concern. A commensalistic or mutualistic relationship between these taxa may result in restoration activities affecting one species facilitating restoration of others.