Mitonuclear Sex Determination? Empirical Evidence from Bivalves.

Genetic elements encoded in nuclear DNA determine the sex of an individual in many animals. In certain bivalve lineages that possess doubly uniparental inheritance (DUI), mitochondrial DNA (mtDNA) has been hypothesized to contribute to sex determination. In these cases, females transmit a female mtDNA to all offspring, while male mtDNA (M mtDNA) is transmitted only from fathers to sons. Because M mtDNA is inherited in the same way as Y chromosomes, it has been hypothesized that mtDNA may be responsible for sex determination. However, the role of mitochondrial and nuclear genes in sex determination has yet to be validated in DUI bivalves. In this study, we used DNA, RNA, and mitochondrial short noncoding RNA (sncRNA) sequencing to explore the role of mitochondrial and nuclear elements in the sexual development pathway of the freshwater mussel Potamilus streckersoni (Bivalvia: Unionida). We found that the M mtDNA sheds a sncRNA partially within a male-specific mitochondrial gene that targets a pathway hypothesized to be involved in female development and mitophagy. RNA-seq confirmed the gene target was significantly upregulated in females, supporting a direct role of mitochondrial sncRNAs in gene silencing. These findings support the hypothesis that M mtDNA inhibits female development. Genome-wide patterns of genetic differentiation and heterozygosity did not support a nuclear sex-determining region, although we cannot reject that nuclear factors are involved with sex determination. Our results provide further evidence that mitochondrial loci contribute to diverse, nonrespiratory functions and additional insights into an unorthodox sex-determining system.

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.

Did doubly uniparental inheritance (DUI) of mtDNA originate as a cytoplasmic male sterility (CMS) system?

Animal and plant species exhibit an astonishing diversity of sexual systems, including environmental and genetic determinants of sex, with the latter including genetic material in the mitochondrial genome. In several hermaphroditic plants for example, sex is determined by an interaction between mitochondrial cytoplasmic male sterility (CMS) genes and nuclear restorer genes. Specifically, CMS involves aberrant mitochondrial genes that prevent pollen development and specific nuclear genes that restore it, leading to a mixture of female (male-sterile) and hermaphroditic individuals in the population (gynodioecy). Such a mitochondrial-nuclear sex determination system is thought to be rare outside plants. Here, we present one possible case of CMS in animals. We hypothesize that the only exception to the strict maternal mtDNA inheritance in animals, the doubly uniparental inheritance (DUI) system in bivalves, might have originated as a mitochondrial-nuclear sex-determination system. We document and explore similarities that exist between DUI and CMS, and we propose various ways to test our hypothesis.

A lizard is never late: Squamate genomics as a recent catalyst for understanding sex chromosome and microchromosome evolution.

In 2011, the first high-quality genome assembly of a squamate reptile (lizard or snake) was published for the green anole. Dozens of genome assemblies were subsequently published over the next decade, yet these assemblies were largely inadequate for answering fundamental questions regarding genome evolution in squamates due to their lack of contiguity or annotation. As the "genomics age" was beginning to hit its stride in many organismal study systems, progress in squamates was largely stagnant following the publication of the green anole genome. In fact, zero high-quality (chromosome-level) squamate genomes were published between the years 2012 and 2017. However, since 2018, an exponential increase in high-quality genome assemblies has materialized with 24 additional high-quality genomes published for species across the squamate tree of life. As the field of squamate genomics is rapidly evolving, we provide a systematic review from an evolutionary genomics perspective. We collated a near-complete list of publicly available squamate genome assemblies from more than half-a-dozen international and third-party repositories and systematically evaluated them with regard to their overall quality, phylogenetic breadth, and usefulness for continuing to provide accurate and efficient insights into genome evolution across squamate reptiles. This review both highlights and catalogs the currently available genomic resources in squamates and their ability to address broader questions in vertebrates, specifically sex chromosome and microchromosome evolution, while addressing why squamates may have received less historical focus and has caused their progress in genomics to lag behind peer taxa.

The revised reference genome of the leopard gecko (Eublepharis macularius) provides insight into the considerations of genome phasing and assembly.

Genomic resources across squamate reptiles (lizards and snakes) have lagged behind other vertebrate systems and high-quality reference genomes remain scarce. Of the 23 chromosome-scale reference genomes across the order, only 12 of the ~60 squamate families are represented. Within geckos (infraorder Gekkota), a species-rich clade of lizards, chromosome-level genomes are exceptionally sparse representing only two of the seven extant families. Using the latest advances in genome sequencing and assembly methods, we generated one of the highest-quality squamate genomes to date for the leopard gecko, Eublepharis macularius (Eublepharidae). We compared this assembly to the previous, short-read only, E. macularius reference genome published in 2016 and examined potential factors within the assembly influencing contiguity of genome assemblies using PacBio HiFi data. Briefly, the read N50 of the PacBio HiFi reads generated for this study was equal to the contig N50 of the previous E. macularius reference genome at 20.4 kilobases. The HiFi reads were assembled into a total of 132 contigs, which was further scaffolded using HiC data into 75 total sequences representing all 19 chromosomes. We identified 9 of the 19 chromosomal scaffolds were assembled as a near-single contig, whereas the other 10 chromosomes were each scaffolded together from multiple contigs. We qualitatively identified that the percent repeat content within a chromosome broadly affects its assembly contiguity prior to scaffolding. This genome assembly signifies a new age for squamate genomics where high-quality reference genomes rivaling some of the best vertebrate genome assemblies can be generated for a fraction of previous cost estimates. This new E. macularius reference assembly is available on NCBI at JAOPLA010000000.

A tale of two paths: The evolution of mitochondrial recombination in bivalves with doubly uniparental inheritance.

In most animals, mitochondrial DNA is strictly maternally inherited and non-recombining. One exception to this pattern is called doubly uniparental inheritance (DUI), a phenomenon involving the independent transmission of female and male mitochondrial genomes. DUI is known only from the molluskan class Bivalvia. The phylogenetic distribution of male-transmitted mitochondrial DNA (M mtDNA) in bivalves is consistent with several evolutionary scenarios, including multiple independent gains, losses, and varying degrees of recombination with female-transmitted mitochondrial DNA (F mtDNA). In this study, we use phylogenetic methods to test M mtDNA origination hypotheses and infer the prevalence of mitochondrial recombination in bivalves with DUI. Phylogenetic modeling using site concordance factors supported a single origin of M mtDNA in bivalves coupled with recombination acting over long evolutionary timescales. Ongoing mitochondrial recombination is present in Mytilida and Venerida, which results in a pattern of concerted evolution of F mtDNA and M mtDNA. Mitochondrial recombination could be favored to offset the deleterious effects of asexual inheritance and maintain mitonuclear compatibility across tissues. Cardiida and Unionida have gone without recent recombination, possibly due to an extension of the COX2 gene in male mitochondrial DNA. The loss of recombination could be connected to the role of M mtDNA in sex determination or sexual development. Our results support that recombination events may occur throughout the mitochondrial genomes of DUI species. Future investigations may reveal more complex patterns of inheritance of recombinants, which could explain the retention of signal for a single origination of M mtDNA in protein-coding genes.

Comparative phylogenomics reveal complex evolution of life history strategies in a clade of bivalves with parasitic larvae (Bivalvia: Unionoida: Ambleminae).

Freshwater mussels are a species-rich group with biodiversity patterns strongly shaped by a life history strategy that includes an obligate parasitic larval stage. In this study, we set out to reconstruct the life history evolution and systematics in a clade of freshwater mussels adapted to parasitizing a molluscivorous host fish. Anchored hybrid enrichment and ancestral character reconstruction revealed a complex pattern of life history evolution with host switching and multiple instances of convergence, including reduction in size of larvae, increased fecundity, and growth during encapsulation. Our phylogenomic analyses also recovered non-monophyly of taxa exhibiting multiple traits used as the basis for previous taxonomic hypotheses. Taxa with axe-head shaped glochidia were resolved as paraphyletic, but our results strongly suggest the complex morphology is an adaptation to reduce larval size, with reduction in size further accentuated in taxa previously assigned to Leptodea. To more accurately reflect the evolutionary history of this group, we make multiple systematic changes, including the description of a new genus, Atlanticoncha gen. nov., and the synonymy of the genus Leptodea under Potamilus. Our findings contribute to the growing body of literature showing that cladistic hypotheses based solely on morphological characters, including larval morphology, can be flawed in freshwater mussels.

Molecular and morphological data reveal non-monophyly and speciation in imperiled freshwater mussels (Anodontoides and Strophitus).

Accurate taxonomic placement is vital to conservation efforts considering many intrinsic biological characteristics of understudied species are inferred from closely related taxa. The rayed creekshell, Anodontoides radiatus (Conrad, 1834), exists in the Gulf of Mexico drainages from western Florida to Louisiana and has been petitioned for listing under the Endangered Species Act. We set out to resolve the evolutionary history of A. radiatus, primarily generic placement and species boundaries, using phylogenetic, morphometric, and geographic information. Our molecular matrix contained 3 loci: cytochrome c oxidase subunit I, NADH dehydrogenase subunit I, and the nuclear-encoded ribosomal internal transcribed spacer I. We employed maximum likelihood and Bayesian inference to estimate a phylogeny and test the monophyly of Anodontoides and Strophitus. We implemented two coalescent-based species delimitation models to test seven species models and evaluate species boundaries within A. radiatus. Concomitant to molecular data, we also employed linear morphometrics and geographic information to further evaluate species boundaries. Molecular and morphological evidence supports the inclusion of A. radiatus in the genus Strophitus, and we resurrect the binomial Strophitus radiatus to reflect their shared common ancestry. We also found strong support for polyphyly in Strophitus and advocate the resurrection of the genus Pseudodontoideus to represent 'Strophitus' connasaugaensis and 'Strophitus' subvexus. Strophitus radiatus exists in six well-supported clades that were distinguished as evolutionary independent lineages using Bayesian inference, maximum likelihood, and coalescent-based species delimitation models. Our integrative approach found evidence for as many as 4 evolutionary divergent clades within S. radiatus. Therefore, we formally describe two new species from the S. radiatus species complex (Strophitus williamsi and Strophitus pascagoulaensis) and recognize the potential for a third putative species (Strophitus sp. cf. pascagoulaensis). Our findings aid stakeholders in establishing conservation and management strategies for the members of Anodontoides, Strophitus, and Pseudodontoideus.

A lizard is never late: squamate genomics as a recent catalyst for understanding sex chromosome and microchromosome evolution.

In 2011, the first high-quality genome assembly of a squamate reptile (lizard or snake) was published for the green anole. Dozens of genome assemblies were subsequently published over the next decade, yet these assemblies were largely inadequate for answering fundamental questions regarding genome evolution in squamates due to their lack of contiguity or annotation. As the "genomics age" was beginning to hit its stride in many organismal study systems, progress in squamates was largely stagnant following the publication of the green anole genome. In fact, zero high-quality (chromosome-level) squamate genomes were published between the years 2012-2017. However, since 2018, an exponential increase in high-quality genome assemblies has materialized with 24 additional high-quality genomes published for species across the squamate tree of life. As the field of squamate genomics is rapidly evolving, we provide a systematic review from an evolutionary genomics perspective. We collated a near-complete list of publicly available squamate genome assemblies from more than half-a-dozen international and third-party repositories and systematically evaluated them with regard to their overall quality, phylogenetic breadth, and usefulness for continuing to provide accurate and efficient insights into genome evolution across squamate reptiles. This review both highlights and catalogs the currently available genomic resources in squamates and their ability to address broader questions in vertebrates, specifically sex chromosome and microchromosome evolution, while addressing why squamates may have received less historical focus and has caused their progress in genomics to lag behind peer taxa.

The revised reference genome of the leopard gecko ( Eublepharis macularius ) provides insight into the considerations of genome phasing and assembly.

Genomic resources across squamate reptiles (lizards and snakes) have lagged behind other vertebrate systems and high-quality reference genomes remain scarce. Of the 23 chromosome-scale reference genomes across the order, only 12 of the ~60 squamate families are represented. Within geckos (infraorder Gekkota), a species-rich clade of lizards, chromosome-level genomes are exceptionally sparse representing only two of the seven extant families. Using the latest advances in genome sequencing and assembly methods, we generated one of the highest quality squamate genomes to date for the leopard gecko, Eublepharis macularius (Eublepharidae). We compared this assembly to the previous, short-read only, E. macularius reference genome published in 2016 and examined potential factors within the assembly influencing contiguity of genome assemblies using PacBio HiFi data. Briefly, the read N50 of the PacBio HiFi reads generated for this study was equal to the contig N50 of the previous E. macularius reference genome at 20.4 kilobases. The HiFi reads were assembled into a total of 132 contigs, which was further scaffolded using HiC data into 75 total sequences representing all 19 chromosomes. We identified that 9 of the 19 chromosomes were assembled as single contigs, while the other 10 chromosomes were each scaffolded together from two or more contigs. We qualitatively identified that percent repeat content within a chromosome broadly affects its assembly contiguity prior to scaffolding. This genome assembly signifies a new age for squamate genomics where high-quality reference genomes rivaling some of the best vertebrate genome assemblies can be generated for a fraction previous cost estimates. This new E. macularius reference assembly is available on NCBI at JAOPLA010000000. The genome version and its associated annotations are also available via this Figshare repository https://doi.org/10.6084/m9.figshare.20069273 .

Mitonuclear sex determination? Empirical evidence from bivalves.

Genetic elements encoded in nuclear DNA determine the sex of an individual in many animals. In bivalves, however, mitochondrial DNA (mtDNA) has been hypothesized to contribute to sex determination in lineages that possess doubly uniparental inheritance (DUI). In these cases, females transmit a female mtDNA (F mtDNA) to all offspring, while male mtDNA (M mtDNA) is transmitted only from fathers to sons. Because M mtDNA is inherited in the same way as Y chromosomes, it has been hypothesized that mtDNA may be responsible for sex determination. However, the role of mitochondrial and nuclear genes in sex determination has yet to be validated in DUI bivalves. In this study, we used DNA, RNA, and mitochondrial short non-coding RNA (sncRNA) sequencing to explore the role of mitochondrial and nuclear elements in the sexual development pathway of the freshwater mussel Potamilus streckersoni (Bivalvia: Unionida). We found that the M mtDNA shed a sncRNA partially within a male-specific mitochondrial gene that targeted pathways hypothesized to be involved in female development and mitophagy. RNA-seq confirmed the gene target was significantly upregulated in females, supporting a direct role of mitochondrial sncRNAs in gene silencing. These findings support the hypothesis that M mtDNA inhibits female development. Genome-wide patterns of genetic differentiation and heterozygosity did not support a nuclear sex determining region, although we cannot reject that nuclear factors are involved with sex determination. Our results provide further evidence that mitochondrial loci contribute to diverse, non-respiratory functions and provide a first glimpse into an unorthodox sex determining system.

A novel assembly pipeline and functional annotations for targeted sequencing: A case study on the globally threatened Margaritiferidae (Bivalvia: Unionida).

The proliferation of genomic sequencing approaches has significantly impacted the field of phylogenetics. Target capture approaches provide a cost-effective, fast and easily applied strategy for phylogenetic inference of non-model organisms. However, several existing target capture processing pipelines are incapable of incorporating whole genome sequencing (WGS). Here, we develop a new pipeline for capture and de novo assembly of the targeted regions using whole genome re-sequencing reads. This new pipeline captured targeted loci accurately, and given its unbiased nature, can be used with any target capture probe set. Moreover, due to its low computational demand, this new pipeline may be ideal for users with limited resources and when high-coverage sequencing outputs are required. We demonstrate the utility of our approach by incorporating WGS data into the first comprehensive phylogenomic reconstruction of the freshwater mussel family Margaritiferidae. We also provide a catalogue of well-curated functional annotations of these previously uncharacterized freshwater mussel-specific target regions, representing a complementary tool for scrutinizing phylogenetic inferences while expanding future applications of the probe set.

The male-type mitochondrial genome of the freshwater mussel Potamilus streckersoni Smith, Johnson, Inoue, Doyle, & Randklev, 2019 (Bivalvia: Unionidae).

The global decline of freshwater mussels emphasizes the need to establish genetic resources to better understand their biology, including a unique mitochondrial biology known as doubly uniparental inheritance. In this study, we present the complete male-type (M-type) mitochondrial genome of the freshwater mussel, Potamilus streckersoni Smith, Johnson, Inoue, Doyle, & Randklev, 2019. The M-type mtDNA is approximately 16 kilobases and contains 22 tRNAs, two rRNAs, and 14 protein-coding genes, including a male-specific open reading frame. Read coverage revealed that M-type mtDNA was more abundant than female-type mtDNA in male gonadal tissue, with respect to a non-spawning male individual. Novel mitogenomes were resolved within previously described sex-specific monophyletic clades across the subfamily Ambleminae. The availability of high-quality nuclear and mitochondrial genomic data for P. streckersoni makes it a model for future research into the potential role of mtDNA in sex determination or sexual development in freshwater mussels.

Using a multi-model ensemble approach to determine biodiversity hotspots with limited occurrence data in understudied areas: An example using freshwater mussels in México.

Species distribution models (SDMs) are an increasingly important tool for conservation particularly for difficult-to-study locations and with understudied fauna. Our aims were to (1) use SDMs and ensemble SDMs to predict the distribution of freshwater mussels in the Pánuco River Basin in Central México; (2) determine habitat factors shaping freshwater mussel occurrence; and (3) use predicted occupancy across a range of taxa to identify freshwater mussel biodiversity hotspots to guide conservation and management. In the Pánuco River Basin, we modeled the distributions of 11 freshwater mussel species using an ensemble approach, wherein multiple SDM methodologies were combined to create a single ensemble map of predicted occupancy. A total of 621 species-specific observations at 87 sites were used to create species-specific ensembles. These predictive species ensembles were then combined to create local diversity hotspot maps. Precipitation during the warmest quarter, elevation, and mean temperature were consistently the most important discriminatory environmental variables among species, whereas land use had limited influence across all taxa. To the best of our knowledge, our study is the first freshwater mussel-focused research to use an ensemble approach to determine species distribution and predict biodiversity hotspots. Our study can be used to guide not only current conservation efforts but also prioritize areas for future conservation and study.

A High-Quality Reference Genome for a Parasitic Bivalve with Doubly Uniparental Inheritance (Bivalvia: Unionida).

From a genomics perspective, bivalves (Mollusca: Bivalvia) have been poorly explored with the exception for those of high economic value. The bivalve order Unionida, or freshwater mussels, has been of interest in recent genomic studies due to their unique mitochondrial biology and peculiar life cycle. However, genomic studies have been hindered by the lack of a high-quality reference genome. Here, I present a genome assembly of Potamilus streckersoni using Pacific Bioscience single-molecule real-time long reads and 10X Genomics-linked read sequencing. Further, I use RNA sequencing from multiple tissue types and life stages to annotate the reference genome. The final assembly was far superior to any previously published freshwater mussel genome and was represented by 2,368 scaffolds (2,472 contigs) and 1,776,755,624 bp, with a scaffold N50 of 2,051,244 bp. A high proportion of the assembly was comprised of repetitive elements (51.03%), aligning with genomic characteristics of other bivalves. The functional annotation returned 52,407 gene models (41,065 protein, 11,342 tRNAs), which was concordant with the estimated number of genes in other freshwater mussel species. This genetic resource, along with future studies developing high-quality genome assemblies and annotations, will be integral toward unraveling the genomic bases of ecologically and evolutionarily important traits in this hyper-diverse group.

Establishing conservation units to promote recovery of two threatened freshwater mussel species (Bivalvia: Unionida: Potamilus).

Population genomics has significantly increased our ability to make inferences about microevolutionary processes and demographic histories, which have the potential to improve protection and recovery of imperiled species. Freshwater mussels (Bivalvia: Unionida) represent one of the most imperiled groups of organisms globally. Despite systemic decline of mussel abundance and diversity, studies evaluating spatiotemporal changes in distribution, demographic histories, and ecological factors that threaten long-term persistence of imperiled species remain lacking. In this study, we use genotype-by-sequencing (GBS) and mitochondrial sequence data (mtDNA) to define conservation units (CUs) for two highly imperiled freshwater mussel species, Potamilus amphichaenus and Potamilus streckersoni. We then synthesize our molecular findings with details from field collections spanning from 1901 to 2019 to further elucidate distributional trends, contemporary status, and other factors that may be contributing to population declines for our focal species. We collected GBS and mtDNA data for individuals of P. amphichaenus and P. streckersoni from freshwater mussel collections in the Brazos, Neches, Sabine, and Trinity drainages ranging from 2012 to 2019. Molecular analyses resolved disputing number of genetic clusters within P. amphichaenus and P. streckersoni; however, we find defensible support for four CUs, each corresponding to an independent river basin. Evaluations of historical and recent occurrence data illuminated a generally increasing trend of occurrence in each of the four CUs, which were correlated with recent increases in sampling effort. Taken together, these findings suggest that P. amphichaenus and P. streckersoni are likely rare throughout their respective ranges. Because of this, the establishment of CUs will facilitate evidence-based recovery planning and ensure potential captive propagation and translocation efforts are beneficial. Our synthesis represents a case study for conservation genomic assessments in freshwater mussels and provides a model for future studies aimed at recovery planning for these highly imperiled organisms.

A new species of freshwater mussel in the genus Popenaias Frierson, 1927, from the Gulf coastal rivers of central Mexico (Bivalvia: Unionida: Unionidae) with comments on the genus.

The Gulf coastal drainages of central Mexico are a faunal transition zone between North and South America and harbor a unique assemblage of freshwater mussels (Bivalvia: Unionida). However, little information is available regarding the taxonomy, distribution, and evolutionary history of the Mexican mussel fauna due to limited sampling over the last 100 years. To address these knowledge gaps, we evaluated species-level diversity in the genus Popenaias Frierson, 1927, in Mexican Gulf coastal drainages as part of a larger effort to inform conservation efforts for members of this genus both in Mexico and the United States of America. Based on our analyses, we describe Popenaias berezai n. sp. from the Río Valles of the Río Pánuco basin, San Luis Potosí, Mexico. We also provide presumptive distributional range, phylogenetic structure, and molecular and morphological diagnoses of the new species and provide comments on the other species currently in Popenaias. Our findings highlight the high levels of endemism among freshwater mussels in Mexican Gulf coastal drainages and will help guide impending conservation actions for P. popeii, which is listed as "endangered" in the United States.

Molecular and morphological data to facilitate future research on freshwater mussels (Bivalvia: Unionidae: Anodontinae).

This data article presents the multi-locus DNA alignments, morphometric data, and details on specimens examined to resolve the evolutionary history of Anodontoides and Strophitus, primarily generic placement and species boundaries. We sequenced 3 loci to create our molecular matrix: cytochrome c oxidase subunit I, NADH dehydrogenase subunit I, and the nuclear-encoded ribosomal internal transcribed spacer I. Aligned sequences were used in phylogenetic analyses and to identify diagnostic nucleotides for Strophitus pascagoulaensis, Strophitus radiatus, Strophitus sp. cf. pascagoulaensis, and Strophitus williamsi. Linear morphometrics (i.e. maximum height, length, and width) were also implemented to further evaluate species boundaries within Strophitus. For further details and experimental findings, please refer to the article published in Molecular Phylogenetics and Evolution (Smith et al., 2018) [1].

Integrative taxonomy resolves taxonomic uncertainty for freshwater mussels being considered for protection under the U.S. Endangered Species Act.

Objectively delimiting species boundaries remains an important challenge in systematics and becomes urgent when unresolved taxonomy complicates conservation and recovery efforts. We examined species boundaries in the imperiled freshwater mussel genus Cyclonaias (Bivalvia: Unionidae) using morphometrics, molecular phylogenetics, and multispecies coalescent models to help guide pending conservation assessments and legislative decisions. Congruence across multiple lines of evidence indicated that current taxonomy overestimates diversity in the C. pustulosa species complex. The only genetically and morphologically diagnosable species in the C. pustulosa species complex were C. pustulosa and C. succissa and we consider C. aurea, C. houstonensis, C. mortoni, and C. refulgens to be synonyms of C. pustulosa. In contrast, all three species in the C. nodulata complex (C. necki, C. nodulata, and C. petrina) were genetically, geographically, and morphologically diagnosable. Our findings have important conservation and management implications, as three nominal species (C. aurea, C. houstonensis, and C. petrina) are being considered for protection under the Endangered Species Act.