RAD-tag and mitochondrial DNA sequencing reveal the genetic structure of a widespread and regionally imperiled freshwater mussel, Obovaria olivaria (Bivalvia: Unionidae).

Obovaria olivaria is a species of freshwater mussel native to the Mississippi River and Laurentian Great Lakes-St. Lawrence River drainages of North America. This mussel has experienced population declines across large parts of its distribution and is imperiled in many jurisdictions. Obovaria olivaria uses the similarly imperiled Acipenser fulvescens (Lake Sturgeon) as a host for its glochidia. We employed mitochondrial DNA sequencing and restriction site-associated DNA sequencing (RAD-seq) to assess patterns of genetic diversity and population structure of O. olivaria from 19 collection locations including the St. Lawrence River drainage, the Great Lakes drainage, the Upper Mississippi River drainage, the Ohioan River drainage, and the Mississippi Embayment. Heterozygosity was highest in Upper Mississippi and Great Lakes populations, followed by a reduction in diversity and relative effective population size in the St. Lawrence populations. Pairwise F ST ranged from 0.00 to 0.20, and analyses of genetic structure revealed two major ancestral populations, one including all St. Lawrence River/Ottawa River sites and the other including remaining sites; however, significant admixture and isolation by river distance across the range were evident. The genetic diversity and structure of O. olivaria is consistent with the existing literature on Acipenser fulvescens and suggests that, although northern and southern O. olivaria populations are genetically distinct, genetic structure in O. olivaria is largely clinal rather than discrete across its range. Conservation and restoration efforts of O. olivaria should prioritize the maintenance and restoration of locations where O. olivaria remain, especially in northern rivers, and to ensure connectivity that will facilitate dispersal of Acipenser fulvescens and movement of encysted glochidia.

Boundaries and hybridization in a secondary contact zone between freshwater mussel species (Family:Unionidae).

Correct species identification and delineation are crucial for effective conservation and management. However, species delineation can be problematic in the presence of morphological ambiguities due to phenotypic plasticity, convergence, and/or interspecific hybridization. Here, we investigated the degree of hybridization between two closely related freshwater mussel species [Bivalvia: Unionidae; Lampsilis siliquoidea (Barnes) and L. radiata (Gmelin)] that present intermediate forms in areas of sympatry. Unionids have a distinct form of mitochondrial DNA (mtDNA) inheritance, termed doubly uniparental inheritance (DUI) where female mtDNA (F-type) is transmitted to all progeny but male mtDNA (M-type) is mostly inherited by the males resulting in mostly homoplasmic females and heteroplasmic males. An individual was identified as hybrid when F-type and M-type mtDNA of the two different species were found in the same individual. Twelve out of 116 sequenced males were identified as hybrids indicating that these species hybridize where their geographic range overlaps in the lower Great Lakes and St. Lawrence basins. Microsatellite analyses further support the occurrence of hybridization but at a larger spatial scale than indicated by the mitochondrial analyses. We also found that strong within-species population genetic structure affects the detection of purebred individuals overestimating the number of hybrids. Given the large geographic scale and proportion of hybrids found in this study, natural hybridization and introgression need to be considered when implementing local biodiversity inventories, identifying waterbodies as source of organisms for relocation and restoration projects and when setting appropriate conservation policies.

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.

Mesozoic mitogenome rearrangements and freshwater mussel (Bivalvia: Unionoidea) macroevolution.

Using a new fossil-calibrated mitogenome-based approach, we identified macroevolutionary shifts in mitochondrial gene order among the freshwater mussels (Unionoidea). We show that the early Mesozoic divergence of the two Unionoidea clades, Margaritiferidae and Unionidae, was accompanied by a synchronous split in the gene arrangement in the female mitogenome (i.e., gene orders MF1 and UF1). Our results suggest that this macroevolutionary jump was completed within a relatively short time interval (95% HPD 201-226 Ma) that coincided with the Triassic-Jurassic mass extinction. Both gene orders have persisted within these clades for ~200 Ma. The monophyly of the so-called "problematic" Gonideinae taxa was supported by all the inferred phylogenies in this study using, for the first time, the M- and F-type mitogenomes either singly or combined. Within Gonideinae, two additional splits in the gene order (UF1 to UF2, UF2 to UF3) occurred in the Mesozoic and have persisted for ~150 and ~100 Ma, respectively. Finally, the mitogenomic results suggest ancient connections between freshwater basins of East Asia and Europe near the Cretaceous-Paleogene boundary, probably via a continuous paleo-river system or along the Tethys coastal line, which are well supported by at least three independent but almost synchronous divergence events.

Testing the utility of DNA barcodes and a preliminary phylogenetic framework for Chinese freshwater mussels (Bivalvia: Unionidae) from the middle and lower Yangtze River.

The middle and lower portions of the Yangtze River basin is the most species-rich region for freshwater mussels in Asia. The management and conservation of the taxa in this region has been greatly hampered by the lack of a well-developed phylogeny and species-level taxonomic framework. In this study, we tested the utility of two mitochondrial genes commonly used as DNA barcodes: the first subunit of the cytochrome oxidase c gene (COI) and the first subunit of the NADH dehydrogenase gene (ND1) for 34 putative species representing 15 genera, and also generated phylogenetic hypotheses for Chinese unionids based on the combined dataset of the two mitochondrial genes. The results showed that both loci performed well as barcodes for species identification, but the ND1 sequences provided better resolution when compared to COI. Based on the two-locus dataset, Bayesian Inference (BI) and Maximum Likelihood (ML) phylogenetic analyses indicated 3 of the 15 genera of Chinese freshwater mussels examined were polyphyletic. Additionally, the analyses placed the 15 genera into 3 subfamilies: Unioninae (Aculamprotula, Cuneopsis, Nodularia and Schistodesmus), Gonideninae (Lamprotula, Solenaia and Ptychorhychus) and Anodontinae (Cristaria, Arconaia, Acuticosta, Lanceolaria, Anemina and Sinoanodonta). Our results contradict previous taxonomic classification that placed the genera Arconaia, Acuticosta and Lanceolaria in the Unioninae. This study represents one of the first attempts to develop a molecular phylogenetic framework for the Chinese members of the Unionidae and will provide a basis for future research on the evolution, ecology, and conservation of Chinese freshwater mussels.

Genetic structure and diversity of Nodularia douglasiae (Bivalvia: Unionida) from the middle and lower Yangtze River drainage.

The Yangtze River drainage in China is among the most species rich rivers for freshwater mussels (order Unionida) on Earth with at least 68 species known. The freshwater mussels of the Yangtze River face a variety of threats with indications that species are declining in abundance and area of occupancy. This study represents the first analyses of the genetic structure and diversity for the common and widespread freshwater mussel Nodularia douglasiae based on microsatellite DNA genotypes and mitochondrial DNA sequences. Phylogenetic analysis a fragment of the COI mitochondrial gene indicated that N. douglasiae collected from across the middle and lower Yangtze River drainage are monophyletic with N. douglasiae from Japan, Russia, and South Korea. The results of the analysis of both the mtDNA and microsatellite datasets indicated that the seven collection locations of N. douglasiae in the middle and lower Yangtze River drainage showed high genetic diversity, significant genetic differentiation and genetic structure, and stable population dynamics over time. Moreover, we found that the connections among tributaries rivers and lakes in the Yangtze River drainage were important in maintaining gene flow among locations that N. douglasiae inhabits. An understanding of the genetic structure and diversity of a widespread species like N. douglasiae could be used as a surrogate to better understand the populations of other freshwater mussel species that are more rare in the Yangtze River drainage. At the same time, these results could provide a basis for the protection of genetic diversity and management of unionid mussels diversity and other aquatic organisms in the system.

Detection of barriers to dispersal is masked by long lifespans and large population sizes.

Population genetic analyses of species inhabiting fragmented landscapes are essential tools for conservation. Occasionally, analyses of fragmented populations find no evidence of isolation, even though a barrier to dispersal is apparent. In some cases, not enough time may have passed to observe divergence due to genetic drift, a problem particularly relevant for long-lived species with overlapping generations. Failing to consider this quality during population structure analyses could result in incorrect conclusions about the impact of fragmentation on the species. We designed a model to explore how lifespan and population size influence perceived population structure of isolated populations over time. This iterative model tracked how simulated populations of variable lifespan and population size were affected by drift alone, using a freshwater mussel, Quadrula quadrula (mapleleaf), as a model system. In addition to exhibiting dramatic lifespan variability among species, mussels are also highly imperiled and exhibit fragmentation by dams throughout the range of many species. Results indicated that, unless population size was small (<50 individuals) or lifespan short (<22 years), observing genetic divergence among populations was unlikely. Even if wild populations are isolated, observing population structure in long-lived mussels from modern damming practices is unlikely because it takes longer for population structure to develop in these species than most North American dams have existed. Larger population sizes and longer lifespans increase the time needed for significant divergence to occur. This study helps illuminate the factors that influence genetic responses by populations to isolation and provides a useful model for conservation-oriented research.

Phylogeny of the most species-rich freshwater bivalve family (Bivalvia: Unionida: Unionidae): Defining modern subfamilies and tribes.

Freshwater mussels of the order Unionida are key elements of freshwater habitats and are responsible for important ecological functions and services. Unfortunately, these bivalves are among the most threatened freshwater taxa in the world. However, conservation planning and management are hindered by taxonomic problems and a lack of detailed ecological data. This highlights the urgent need for advances in the areas of systematics and evolutionary relationships within the Unionida. This study presents the most comprehensive phylogeny to date of the larger Unionida family, i.e., the Unionidae. The phylogeny is based on a combined dataset of 1032bp (COI+28S) of 70 species in 46 genera, with 7 of this genera being sequenced for the first time. The resulting phylogeny divided the Unionidae into 6 supported subfamilies and 18 tribes, three of which are here named for the first time (i.e., Chamberlainiini nomen novum, Cristariini nomen novum and Lanceolariini nomen novum). Molecular analyses were complemented by investigations of selected morphological, anatomical and behavioral characters used in traditional phylogenetic studies. No single morphological, anatomical or behavioral character was diagnostic at the subfamily level and few were useful at the tribe level. However, within subfamilies, many tribes can be recognized based on a subset of these characters. The geographical distribution of each of the subfamilies and tribes is also presented. The present study provides important advances in the systematics of these extraordinary taxa with implications for future ecological and conservation studies.

Competitive replacement of invasive congeners may relax impact on native species: interactions among zebra, quagga, and native unionid mussels.

Determining when and where the ecological impacts of invasive species will be most detrimental and whether the effects of multiple invaders will be superadditive, or subadditive, is critical for developing global management priorities to protect native species in advance of future invasions. Over the past century, the decline of freshwater bivalves of the family Unionidae has been greatly accelerated by the invasion of Dreissena. The purpose of this study was to evaluate the current infestation rates of unionids by zebra (Dreissena polymorpha) and quagga (D. rostriformis bugensis) mussels in the lower Great Lakes region 25 years after they nearly extirpated native unionids. In 2011-2012, we collected infestation data for over 4000 unionids from 26 species at 198 nearshore sites in lakes Erie, Ontario, and St. Clair, the Detroit River, and inland Michigan lakes and compared those results to studies from the early 1990 s. We found that the frequency of unionid infestation by Dreissena recently declined, and the number of dreissenids attached to unionids in the lower Great Lakes has fallen almost ten-fold since the early 1990s. We also found that the rate of infestation depends on the dominant Dreissena species in the lake: zebra mussels infested unionids much more often and in greater numbers. Consequently, the proportion of infested unionids, as well as the number and weight of attached dreissenids were lower in waterbodies dominated by quagga mussels. This is the first large-scale systematic study that revealed how minor differences between two taxonomically and functionally related invaders may have large consequences for native communities they invade.

Evolution of active host-attraction strategies in the freshwater mussel tribe Lampsilini (Bivalvia: Unionidae).

Most freshwater mussels (Bivalvia: Unionoida) require a host, usually a fish, to complete their life cycle. Most species of mussels show adaptations that increase the chances of glochidia larvae contacting a host. We investigated the evolutionary relationships of the freshwater mussel tribe Lampsilini including 49 of the approximately 100 extant species including 21 of the 24 recognized genera. Mitochondrial DNA sequence data (COI, 16S, and ND1) were used to create a molecular phylogeny for these species. Parsimony and Bayesian likelihood topologies revealed that the use of an active lure arose early in the evolution of the Lampsiline mussels. The mantle flap lure appears to have been the first to evolve with other lure types being derived from this condition. Apparently, lures were lost independently in several clades. Hypotheses are discussed as to how some of these lure strategies may have evolved in response to host fish prey preferences.