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.

A comprehensive approach uncovers hidden diversity in freshwater mussels (Bivalvia: Unionidae) with the description of a novel species.

Major geological processes have shaped biogeographical patterns of riverine biota. The Edwards Plateau of central Texas, USA, exhibits unique aquatic communities and endemism, including several species of freshwater mussels. Lampsilis bracteata (Gould, 1855) is endemic to the Edwards Plateau region; however, its phylogenetic relationship with other species in the Gulf coastal rivers and Mississippi River basin is unknown. We evaluated phylogenetic relationships, shell morphologies and soft anatomy characters of L. bracteata and a closely related congener, Lampsilis hydiana (Lea, 1838) throughout their ranges. Our results showed the presence of an undescribed species: Lampsilis bergmanni sp.n. Lampsilis bracteata and L. bergmanni sp.n. share similar shell morphologies and soft anatomy characters; however, they are genetically distinct. Geological processes, such as faulting and sea-level changes during the Miocene to Pliocene, are likely to have facilitated diversification of Lampsilis species, resulting in isolation of L. bracteata on the Edwards Plateau and diversification between L. bergmanni sp.n. and L. hydiana. We conclude that L. bracteata range is restricted to the Colorado River basin, whereas L. bergmanni sp.n. occurs only in upstream reaches of the Guadalupe River basin. Conservation actions are warranted for both species due to their restricted distributions and potential anthropogenic threats.

Evaluating the utility of the female-specific mitochondrial f-orf gene for population genetic, phylogeographic and systematic studies in freshwater mussels (Bivalvia: Unionida).

Freshwater mussels (order: Unionida) represent one of the most critically imperilled groups of animals; consequently, there exists a need to establish a variety of molecular markers for population genetics and systematic studies in this group. Recently, two novel mitochondrial protein-coding genes were described in unionoids with doubly uniparental inheritance of mtDNA. These genes are the f-orf in female-transmitted mtDNA and the m-orf in male-transmitted mtDNA. In this study, whole F-type mitochondrial genome sequences of two morphologically similar Lampsilis spp. were compared to identify the most divergent protein-coding regions, including the f-orf gene, and evaluate its utility for population genetic and phylogeographic studies in the subfamily Ambleminae. We also tested whether the f-orf gene is phylogenetically informative at the species level. Our preliminary results indicated that the f-orf gene could represent a viable molecular marker for population- and species-level studies in freshwater mussels.

Incongruence between mtDNA and nuclear data in the freshwater mussel genus Cyprogenia (Bivalvia: Unionidae) and its impact on species delineation.

Accurately identifying species is a crucial step for developing conservation strategies for freshwater mussels, one of the most imperiled faunas in North America. This study uses genetic data to re-examine species delineation in the genus Cyprogenia. Historically, Cyprogenia found west of the Mississippi River have been ascribed to Cyprogenia aberti (Conrad 1850), and those east of the Mississippi River were classified as Cyprogenia stegaria (Rafinesque 1820). Previous studies using mitochondrial DNA sequences indicated that C. aberti and C. stegaria were not reciprocally monophyletic groups, suggesting the need for systematic revision. We generated a novel dataset consisting of 10 microsatellite loci and combined it with sequence data from the mitochondrial ND1 gene for 223 Cyprogenia specimens. Bayesian analysis of the ND1 nucleotide sequences identified two divergent clades that differ by 15.9%. Members of these two clades occur sympatrically across most sampling locations. In contrast, microsatellite genotypes support recognition of three allopatric clusters defined by major hydrologic basins. The divergent mitochondrial lineages are highly correlated with the color of the conglutinate lures used by mussels to attract and infest host fishes, and tests for selection at the ND1 locus were positive. We infer that the incongruence between mtDNA and microsatellite data in Cyprogenia may be the result of a combination of incomplete lineage sorting and balancing selection on lure color. Our results provide further evidence that mitochondrial markers are not always neutral with respect to selection, and highlight the potential problems of relying on a single-locus-marker for delineating species.

Phylogenetic and morphometric analyses reveal ecophenotypic plasticity in freshwater mussels Obovaria jacksoniana and Villosa arkansasensis (Bivalvia: Unionidae).

Freshwater mollusk shell morphology exhibits clinal variation along a stream continuum that has been termed the Law of Stream Distribution. We analyzed phylogenetic relationships and morphological similarity of two freshwater mussels (Bivalvia: Unionidae), Obovaria jacksoniana and Villosa arkansasensis, throughout their ranges. The objectives were to investigate phylogenetic structure and evolutionary divergence of O. jacksoniana and V. arkansasensis and morphological similarity between the two species. Our analyses were the first explicit tests of phenotypic plasticity in shell morphologies using a combination of genetics and morphometrics. We conducted phylogenetic analyses of mitochondrial DNA (1416 bp; two genes) and morphometric analyses for 135 individuals of O. jacksoniana and V. arkansasensis from 12 streams. We examined correlations among genetic, morphological, and spatial distances using Mantel tests. Molecular phylogenetic analyses revealed a monophyletic relationship between O. jacksoniana and V. arkansasensis. Within this O. jacksoniana/V. arkansasensis complex, five distinct clades corresponding to drainage patterns showed high genetic divergence. Morphometric analysis revealed relative differences in shell morphologies between the two currently recognized species. We conclude that morphological differences between the two species are caused by ecophenotypic plasticity. A series of Mantel tests showed regional and local genetic isolation by distance. We observed clear positive correlations between morphological and geographic distances within a single drainage. We did not observe correlations between genetic and morphological distances. Phylogenetic analyses suggest O. jacksoniana and V. arkansasensis are synonomous and most closely related to a clade composed of O. retusa, O. subrotunda, and O. unicolor. Therefore, the synonomous O. jacksoniana and V. arkansasensis should be recognized as Obovaria arkansasensis (Lea 1862) n. comb. Phylogenetic analyses also showed relative genetic isolation among drainages, suggesting no current gene flow. Further investigation of in-progress speciation and/or cryptic species within O. arkansasensis is warranted followed by appropriate revision of conservation management designations. In this study, we found Obovaria jacksoniana and Villosa arkansasensis are synonomous. We suggest that morphological differences between the two species are caused by ecophenotypic plasticity, where V. arkansasensis is the upstream morphotype and O. jacksoniana is the downstream morphotype of a single species.

Rapid quantitative analysis using a single molecule counting approach.

Single molecule detection of target molecules specifically bound by paired fluorescently labeled probes has shown great potential for sensitive quantitation of biomolecules. To date, no reports have rigorously evaluated the analytical capabilities of a single molecule detection platform employing this dual-probe approach or the performance of its data analysis methodology. In this paper, we describe a rapid, automated, and sensitive multicolor single molecule detection apparatus and a novel extension of coincident event counting based on detection of fluorescent probes. The approach estimates the number of dual-labeled molecules of interest from the total number of coincident fluorescent events observed by correcting for unbound probes that randomly pass through the interrogation zone simultaneously. Event counting was evaluated on three combinations of distinct fluorescence channels and was demonstrated to outperform conventional spatial cross-correlation in generating a wider linear dynamic response to target molecules. Furthermore, this approach succeeded in detecting subpicomolar concentrations of a model RNA target to which fluorescently labeled oligonucleotide probes were hybridized in a complex background of RNA. These results illustrate that the fluorescent event counting approach described represents a general tool for rapid sensitive quantitative analysis of any sample analyte, including nucleic acids and proteins, for which pairs of specific probes can be developed.