New microsatellite markers for Ellipse, Venustaconcha ellipsiformis (Bivalvia: Unionidae), with notes on optimal sample size and cross-species amplification.

Venustaconcha ellipsiformis (Unionidae) is a freshwater mussel species inhabiting small to medium streams of the Midwestern United States; however, its occurrence is rather sporadic and populations are often isolated. Due to anthropogenic habitat degradation and water pollution, this species is designated as some sort of conservation status in many states. To prioritize conservation strategies, highly variable genetic markers are necessary to assess population genetic structure and potential genetic erosion of V. ellipsiformis. Using whole genome sequence data, we developed and characterized microsatellite markers for V. ellipsiformis. Among 23 tetranucleotide loci tested, 14 loci were consistently amplified and showed polymorphism. Analyses performed on three populations in the upper Mississippi River basin showed that the number of alleles per locus ranged from 2 to 11 and the observed heterozygosity varied from 0.15 to 0.75. Based on genotypic and allelic rarefaction curves, these loci had adequate statistical power to genetically discriminate between individuals and the sample size was large enough to capture most alleles available in the populations at most loci. Finally, cross-species screening of the loci successfully amplified and showed polymorphism in six species in the tribe Lampsilini. The microsatellite loci developed in this study provide a valuable addition to extant genetic markers for freshwater mussels and can be useful to provide high-level resolution of population genetic parameters for V. ellipsiformis. Such information will be of great value for resource managers developing and prioritizing conservation strategies for imperiled mussel species.

Pyganodon (Bivalvia: Unionoida: Unionidae) phylogenetics: a male- and female-transmitted mitochondrial DNA perspective.

Species boundaries, evolutionary relationships and geographic distributions of many unionoid bivalve species, like those in the genus Pyganodon, remain unresolved in Eastern North America. Because unionoid bivalves are one of the most imperiled groups of animals in the world, understanding the genetic variation within and among populations as well as among species is crucial for effective conservation planning. Conservation of unionoid species is indispensable from a freshwater habitat perspective but also because they possess a unique mitochondrial inheritance system where distinct gender-associated mitochondrial DNA lineages coexist: a female-transmitted (F) mt genome and a male-transmitted (M) mt genome that are involved in the maintenance of separate sexes (=dioecy). In this study, 42 populations of Pyganodon sp. were sampled across a large geographical range and fragments of two mitochondrial genes (cox1 and cox2) were sequenced from both the M- and F-transmitted mtDNA genomes. Our results support the recency of the divergence between P. cataracta and P. fragilis. We also found two relatively divergent F and M lineages within P. grandis. Surprisingly, the relationships among the P. grandis specimens in the F and M sequence trees are not congruent. We found that a single haplotype in P. lacustris has recently swept throughout the M genotype space leading to an unexpectedly low diversity in the M lineage in that species. Our survey put forward some challenging results that force us to rethink hybridization and species boundaries in the genus Pyganodon. As the M and F genomes do not always display the same phylogeographic story in each species, we also discuss the importance of being careful in the interpretation of molecular data based solely on maternal transmitted mtDNA genomes. The involvement of F and M genomes in unionoid bivalve sex determination likely played a role in the genesis of the unorthodox phylogeographic patterns reported herein.

Deciphering the Link between Doubly Uniparental Inheritance of mtDNA and Sex Determination in Bivalves: Clues from Comparative Transcriptomics.

Bivalves exhibit an astonishing diversity of sexual systems and sex-determining mechanisms. They can be gonochoric, hermaphroditic or androgenetic, with both genetic and environmental factors known to determine or influence sex. One unique sex-determining system involving the mitochondrial genome has also been hypothesized to exist in bivalves with doubly uniparental inheritance (DUI) of mtDNA. However, the link between DUI and sex determination remains obscure. In this study, we performed a comparative gonad transcriptomics analysis for two DUI-possessing freshwater mussel species to better understand the mechanisms underlying sex determination and DUI in these bivalves. We used a BLAST reciprocal analysis to identify orthologs between Venustaconcha ellipsiformis and Utterbackia peninsularis and compared our results with previously published sex-specific bivalve transcriptomes to identify conserved sex-determining genes. We also compared our data with other DUI species to identify candidate genes possibly involved in the regulation of DUI. A total of ∼12,000 orthologous relationships were found, with 2,583 genes differentially expressed in both species. Among these genes, key sex-determining factors previously reported in vertebrates and in bivalves (e.g., Sry, Dmrt1, Foxl2) were identified, suggesting that some steps of the sex-determination pathway may be deeply conserved in metazoans. Our results also support the hypothesis that a modified ubiquitination mechanism could be responsible for the retention of the paternal mtDNA in male bivalves, and revealed that DNA methylation could also be involved in the regulation of DUI. Globally, our results suggest that sets of genes associated with sex determination and DUI are similar in distantly-related DUI species.

The extremely divergent maternally- and paternally-transmitted mitochondrial genomes are co-expressed in somatic tissues of two freshwater mussel species with doubly uniparental inheritance of mtDNA.

Freshwater mussel species with doubly uniparental inheritance (DUI) of mtDNA are unique because they are naturally heteroplasmic for two extremely divergent mtDNAs with ~50% amino acid differences for protein-coding genes. The paternally-transmitted mtDNA (or M mtDNA) clearly functions in sperm in these species, but it is still unknown whether it is transcribed when present in male or female soma. In the present study, we used PCR and RT-PCR to detect the presence and expression of the M mtDNA in male and female somatic and gonadal tissues of the freshwater mussel species Venustaconcha ellipsiformis and Utterbackia peninsularis (Unionidae). This is the first study demonstrating that the M mtDNA is transcribed not only in male gonads, but also in male and female soma in freshwater mussels with DUI. Because of the potentially deleterious nature of heteroplasmy, we suggest the existence of different mechanisms in DUI species to deal with this possibly harmful situation, such as silencing mechanisms for the M mtDNA at the transcriptional, post-transcriptional and/or post-translational levels. These hypotheses will necessitate additional studies in distantly-related DUI species that could possess different mechanisms of action to deal with heteroplasmy.