Nathan A. Johnson

Suggestive association of major histocompatibility IB genetic markers with resistance to bacterial cold water disease in rainbow trout (Oncorhynchus mykiss).

Genes within the major histocompatibility complex (MHC) are important for both innate and adaptive immune responses in mammals; however, much less is known regarding their contribution in teleost fishes. We examined the involvement of four major histocompatibility (MH) genomic regions in rainbow trout in resistance to the causative agent of bacterial coldwater disease (BCWD), Flavobacterium psychrophilum. Fish from the 2005 NCCCWA brood-year (71 full-sib families) were challenged with F. psychrophilum strain CSF 259–93. The overall mortality rate was 70%, with large variation in mortality between families. Disease resistance was quantified as post-challenge days to death. Phenotypic variation and additive genetic variation were estimated using mixed models of survival analysis. To examine association, eight microsatellite markers were isolated from MH gene-containing BAC clones and mapped onto the rainbow trout genetic linkage map. The parents and grandparents of the 2005 brood-year families were genotyped with these eight markers and another two markers tightly linked to the MH-IB region to assess the extent of linkage disequilibrium (LD) of MH genomic regions MH-IA, MH-IB, TAP1, and MH-II with survival post-challenge. MH-IB and MH-II markers were linked to BCWD survivability when data were analyzed by family. Tests for disease association at the population level substantiated the involvement of MH-IB, but not MH-II, with disease resistance. The impact of selective breeding for disease resistance on MH sequence variation is discussed in the context of aquaculture production.

Misidentification of freshwater mussel species (Bivalvia:Unionidae): contributing factors, management implications, and potential solutions

Abstract Surveys of freshwater mussel populations are used frequently to inform conservation decisions by providing information about the status and distribution of species. It is generally accepted that not all mussels or species are collected during surveys, and incomplete detection of individuals and species can bias data and can affect inferences. However, considerably less attention has been given to the potential effects of species misidentification. To evaluate the prevalence of and potential reasons for species misidentification, we conducted a laboratory-based identification exercise and quantified the relationships between mussel species characteristics, observer experience, and misidentification rate. We estimated that misidentification was fairly common, with rates averaging 27% across all species and ranging from 0 to 56%, and was related to mussel shell characteristics and observer experience. Most notably, species with shell texturing were 6.09× less likely than smooth-shelled species to be misidentified. Misidentification rates declined with observer experience, but for many species the risk of misidentification averaged >10% even for observers with moderate levels of experience (5–6 y). In addition, misidentification rates among observers showed substantial variability after controlling for experience. Our results suggest that species misidentification may be common in field surveys of freshwater mussels and could potentially bias estimates of population status and trends. Misidentification rates possibly could be reduced through use of regional workshops, testing and certification programs, and the availability of archived specimens and tissue samples in museum collections.

American alligator digestion rate of blue crabs and its implications for stomach contents analysis.

Stomach contents analysis (SCA) provides a snap-shot observation of a consumers diet. Interpretation of SCA data can be complicated by many factors, including variation in gastric residence times and digestion rates among prey taxa. Although some SCA methods are reported to efficiently remove all stomach contents, the effectiveness of these techniques has rarely been tested for large irregular shaped prey with hard exoskeletons. We used a controlled feeding trial to estimate gastric residency time and decomposition rate of a large crustacean prey item, the Blue Crab (Callinectes sapidus), which is consumed by American Alligators (Alligator mississippiensis), an abundant apex predator in coastal habitats of the southeastern United States. The decomposition rate of C. sapidus in the stomachs of A. mississippiensis followed a predictable pattern, and some crab pieces remained in stomachs for at least 14 days. We also found that certain portions of C. sapidus were prone to becoming caught within the stomach or esophagus, meaning not all crab parts are consistently recovered using gastric lavage techniques. However, because the state of decomposition of crabs was predictable, it is possible to estimate time since consumption for crabs recovered from wild alligators. This information, coupled with a detailed understanding of crab distributions and alligator movement tactics could help elucidate patterns of cross-ecosystem foraging by the American Alligator in coastal habitats.

Generic reclassification and species boundaries in the rediscovered freshwater mussel ‘ Quadrula’ mitchelli (Simpson in Dall, 1896)

The Central Texas endemic freshwater mussel, Quadrula mitchelli (Simpson in Dall, 1896), had been presumed extinct until relict populations were recently rediscovered. To help guide ongoing and future conservation efforts focused on Q. mitchelli we set out to resolve several uncertainties regarding its evolutionary history, specifically its unknown generic position and untested species boundaries. We designed a molecular matrix consisting of two loci (cytochrome c oxidase subunit I and internal transcribed spacer I) and 57 terminal taxa to test the generic position of Q. mitchelli using Bayesian inference and maximum likelihood phylogenetic reconstruction. We also employed two Bayesian species validation methods to test five a priori species models (i.e. hypotheses of species delimitation). Our study is the first to test the generic position of Q.mitchelli and we found robust support for its inclusion in the genus Fusconaia. Accordingly, we introduce the binomial, Fusconaia mitchelli comb. nov., to accurately represent the systematic position of the species. We resolved F. mitchelli individuals in two well supported and divergent clades that were generally distinguished as distinct species using Bayesian species validation methods, although alternative hypotheses of species delineation were also supported. Despite strong evidence of genetic isolation within F. mitchelli, we do not advocate for species-level status of the two clades as they are allopatrically distributed and no morphological, behavioral, or ecological characters are known to distinguish them. These results are discussed in the context of the systematics, distribution, and conservation of F. mitchelli.

Homogeneity at Nuclear Microsatellite Loci Masks Mitochondrial Haplotype Diversity in the Endangered Fanshell Pearlymussel (Cyprogenia stegaria)

We report on multiple patterns of differentiation and connectivity in the fanshell pearlymussel (Cyprogenia stegaria), based on different markers. Knowledge of genetic variation and genetic connectivity among remaining populations of this federally endangered species is needed to initiate implementation of the species recovery plan. We collected tissue samples from 96 specimens from the Green, Rolling Fork, and Licking Rivers, tributaries to the Ohio River, and the Clinch River, a tributary to the Tennessee River, providing broad coverage of the current distributional range of the species. Results from 7 nuclear DNA microsatellite markers suggested minimal population-level differentiation, whereas a mitochondrial DNA (mtDNA) marker (ND1) exhibited significant differentiation between C. stegaria in the Clinch River and the Ohio River populations. The ND1 data also confirm the existence of 2 distinct mtDNA lineages in the genus that transcends species boundaries. Further analyses suggest that the disproportionally strong signal from 2 very divergent ND1 lineages possibly masks finer-grained structure in the Ohio River population, based on one of the mtDNA lineages only. We recommend further sampling to confirm the absence of one lineage from the upper Clinch River drainage and suggest that provisional management guidelines should limit reciprocal exchanges among C. stegaria populations from the Clinch River and those in the Ohio River system.

Molecular phylogeny of the Nearctic and Mesoamerican freshwater mussel genus Megalonaias

Megalonaias is the most geographically widespread genus of the subfamily Ambleminae and is distributed across much of the eastern half of North America, from Minnesota to Nicaragua. Despite the large geographic distribution, the species-level diversity of Megalonaias is quite depauperate (2 spp.), suggesting the genus may not be constrained by the same physical, ecological, or physiological barriers that limit dispersal in many other amblemines. However, this hypothesis is contingent on the assumption that the current taxonomy of Megalonaias accurately reflects its evolutionary history, which remains incompletely understood due to the marginalization of Mesoamerican populations in systematic research. Using one mitochondrial (COI) and one nuclear marker (ITS1) sequenced from 41 individuals distributed across both the Nearctic and Mesoamerican ecoregions, we set out to better understand the species boundaries and genetic diversity within Megalonaias. The reconstructed molecular phylogeny and the observed genetic diversity suggests that Megalonaias is a monotypic genus and that Megalonaias nickliniana, currently considered a federally endangered species, is not a valid species. These results are discussed in the context of their systematic and conservation implications, as well as how the unusual life history strategy of Megalonaias may be influencing its molecular diversity.

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.

Molecular systematics of the critically-endangered North American spinymussels (Unionidae: Elliptio and Pleurobema ) and description of Parvaspina gen. nov.

Despite being common in numerous marine bivalve lineages, lateral spines are extremely rare among freshwater bivalves (Bivalvia: Unionidae), with only three known species characterized by the presence of spines: Elliptio spinosa, Elliptio steinstansana, and Pleurobema collina. All three taxa are endemic to the Atlantic Slope of southeastern North America, critically endangered, and protected by the US Endangered Species Act. Currently, these species are recognized in two genera and remain a source of considerable taxonomic confusion. Because spines are rare in freshwater mussels and restricted to a small region of North America, we hypothesized that spinymussels represent a monophyletic group. We sequenced two mtDNA gene fragments (COI and ND1) and a fragment of the nuclear ITS-1 locus from >70 specimens. Bayesian and maximum-likelihood phylogenetic reconstructions suggest that the spinymussels do not comprise a monophyletic group. Elliptio steinstansana is sister to P. collina, forming a monophyletic clade that was estimated to have diverged from its most recent ancestor in the late Miocene and is distinct from both Elliptio and Pleurobema; we describe a new genus (Parvaspina gen. nov.) to reflect this relationship. Additionally, E. spinosa forms a monophyletic clade that diverged from members of the core Elliptio lineage in the mid-Pliocene. Furthermore, E. spinosa is genetically divergent from the other spinymussel species, suggesting that spines, while extremely rare in freshwater mussels worldwide, may have evolved independently in two bivalve lineages. Recognizing the genetic distinctiveness and inter-generic relationships of the spinymussels is an important first step towards effectively managing these imperiled species and lays the groundwork for future conservation genetics studies.

Deciphering the link between doubly uniparental inheritance of mtDNA and sex determination in bivalves: Clues from comparative transcriptomics

Abstract 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.

Endangered Rough Pigtoe Pearlymussel: Assessment of Phylogenetic Status and Genetic Differentiation of Two Disjunct Populations

Abstract We conducted a genetic characterization of two extant populations of an endangered freshwater mussel, the rough pigtoe Pleurobema plenum, in the Clinch River, Tennessee of the Tennessee River basin, and the Green River, Kentucky, of the Ohio River basin for purposes of conservation recovery planning. First, phylogenetic status of this species within the Pleurobema cordatum species complex (P. cordatum, plenum, rubrum, sintoxia) was assessed using mitochondrial ND1 deoxyribonucleic acid (DNA) sequences, which showed that all investigated species of Pleurobema are genetically distinct. Our results indicated that P. cordatum, P. plenum, P. rubrum, and P. sintoxia each represent monophyletic clades; however, the latter two species were closely related, separated by only one to three nucleotide differences. Further, DNA sequence haplotypes from both populations of P. plenum grouped together into one monophyletic clade and did not support characterizing the populations as separate species. Thus, our ph...