Amplicon sequencing of 42 nuclear loci supports directional gene flow between South Pacific populations of a hydrothermal vent limpet.

In the past few decades, population genetics and phylogeographic studies have improved our knowledge of connectivity and population demography in marine environments. Studies of deep-sea hydrothermal vent populations have identified barriers to gene flow, hybrid zones, and demographic events, such as historical population expansions and contractions. These deep-sea studies, however, used few loci, which limit the amount of information they provided for coalescent analysis and thus our ability to confidently test complex population dynamics scenarios. In this study, we investigated population structure, demographic history, and gene flow directionality among four Western Pacific hydrothermal vent populations of the vent limpet Lepetodrilus aff. schrolli. These vent sites are located in the Manus and Lau back-arc basins, currently of great interest for deep-sea mineral extraction. A total of 42 loci were sequenced from each individual using high-throughput amplicon sequencing. Amplicon sequences were analyzed using both genetic variant clustering methods and evolutionary coalescent approaches. Like most previously investigated vent species in the South Pacific, L. aff. schrolli showed no genetic structure within basins but significant differentiation between basins. We inferred significant directional gene flow from Manus Basin to Lau Basin, with low to no gene flow in the opposite direction. This study is one of the very few marine population studies using >10 loci for coalescent analysis and serves as a guide for future marine population studies.

Effects of temperature and salinity on prevalence and intensity of infection of blue crabs, Callinectes sapidus, by Vibrio cholerae, V. parahaemolyticus, and V. vulnificus in Louisiana.

Coastal marine and estuarine environments are experiencing higher average temperatures, greater frequency of extreme temperature events, and altered salinities. These changes are expected to stress organisms and increase their susceptibility to infectious diseases. However, beyond these generalities, little is known about how environmental factors influence host-pathogen relationships in the marine realm. We investigated the prevalence and intensity of infections by Vibrio cholerae, V. parahaemolyticus, and V. vulnificus in blue crabs, Callinectes sapidus, from Louisiana saltmarshes in relation to temperature and salinity. We evaluated relationships for single measurements taken at the time of collection and for more complex measurements representing accumulated exposure to physiologically-stressful environmental conditions for up to 31 days prior to collection. We found that: (1) prevalence of infection varied across the Louisiana coast, (2) prevalence of all three Vibrio species was influenced by temperature and salinity, and (3) measurements that represent accumulated exposure to extreme conditions are useful predictors of infection prevalence and can provide insights into underlying biological mechanisms.

Differential host mortality explains the effect of high temperature on the prevalence of a marine pathogen.

Infectious diseases threaten marine populations, and the extent of their impacts is often assessed by prevalence of infection (the proportion of infected individuals). Changes in prevalence are often attributed to altered rates of transmission, although the rates of birth, recovery, and mortality also determine prevalence. The parasitic dinoflagellate Hematodinium perezi causes a severe, often fatal disease in blue crabs. It has been speculated that decreases in prevalence associated with high temperatures result from lower rates of infection. We used field collections, environmental sensor data, and high-temperature exposure experiments to investigate the factors that change prevalence of infections in blue crab megalopae (post-larvae). These megalopae migrate from offshore waters, where temperatures are moderate, to marshes where temperatures may be extremely high. Within a few days of arriving in the marsh, the megalopae metamorphose into juvenile crabs. We found a strong negative association between prevalence of Hematodinium infection in megalopae and the cumulative time water temperatures in the marsh exceeded 34°C over the preceding two days. Temperatures this high are known to be lethal for blue crabs, suggesting that higher mortality of infected megalopae could be the cause of reduced prevalence. Experimental exposure of megalopae from the marsh to a temperature of 34°C resulted in higher mortality for infected than uninfected individuals, and decreased the prevalence of infection among survivors from 18% to 3%.

Molecular detection of the parasitic dinoflagellate Hematodinium perezi from blue crabs Callinectes sapidus in Louisiana, USA.

The dinoflagellate Hematodinium perezi is a prolific pathogen of the blue crab Callinectes sapidus along the Atlantic and Gulf of Mexico coasts of North America. High prevalence, sometimes approaching 100%, and outbreaks with high mortality are associated with higher salinities. H. perezi has not been reported previously in blue crabs from Louisiana, USA, where salinities in coastal habitats are generally below the parasite's favorable range. However, the possibility that H. perezi infects blue crabs in higher salinity habitats offshore has not been investigated. A PCR-based test for H. perezi was used to screen blue crabs collected from both high and low salinity areas. These included juvenile and adult crabs from inshore marshes where salinities are relatively low and from higher salinity offshore shoals that are spawning sites for females. H. perezi was detected in blue crabs from offshore shoals (prevalence=5.6%) but not in juvenile or adult crabs from inshore habitats. Megalopae (post-larvae) were also collected from inshore locations. Although megalopae settle inshore where salinities are relatively low, the megalopal stage is preceded by a planktonic phase in higher salinity offshore waters. We detected H. perezi in 11.2% of settling megalopae tested. Although the prevalence of H. perezi was relatively low within our samples, if spawning females and megalopae are especially vulnerable, the impact on the population could be compounded. This is the first report of H. perezi from blue crabs in Louisiana and demonstrates the importance of examining all life stages in determining the prevalence of a harmful parasite.

De novo assembly of a transcriptome from juvenile blue crabs (Callinectes sapidus) following exposure to surrogate Macondo crude oil.

BACKGROUND: The blue crab, Callinectes sapidus, is economically and ecologically important in western Atlantic and Gulf of Mexico coastal estuaries. In 2010 blue crabs in the northern Gulf of Mexico were exposed to crude oil and chemical dispersants from the Deepwater Horizon oil spill. To characterize the blue crab transcriptome and identify genes that could be regulated in response to oil exposure we sequenced transcriptomes from hepatopancreas and gill tissues of juvenile blue crabs after exposing them to a water-accommodated fraction of surrogate Macondo crude oil in the laboratory and compared them to transcriptomes from an unexposed control group. RESULTS: Illumina sequencing provided 42.5 million paired-end sequencing reads for the control group and 44.9 million paired-end reads for the treatment group. From these, 73,473 transcripts and 52,663 genes were assembled. Comparison of control and treatment transcriptomes revealed about 100 genes from each tissue type that were differentially expressed. However, a much larger number of transcripts, approximately 2000 from each tissue type, were differentially expressed. Several examples of alternatively spliced transcripts were verified by qPCR, some of which showed significantly different expression patterns. The combined transcriptome from all tissues and individuals was annotated to assign putative gene products to both major gene ontology categories as well as specific roles in responses to cold and heat, metabolism of xenobiotic compounds, defence, hypoxia, osmoregulation and ecdysis. Among the annotations for upregulated and alternatively-spliced genes were candidates for the metabolism of oil-derived compounds. CONCLUSIONS: Previously, few genomic resources were available for blue crabs or related brachyuran crabs. The transcriptome sequences reported here represent a major new resource for research on the biology of blue crabs. These sequences can be used for studies of differential gene expression or as a source of genetic markers. Genes identified and annotated in this study include candidates for responses of the blue crab to xenobiotic compounds, which could serve as biomarkers for oil exposure. Changes in gene expression also suggest other physiological changes that may occur as the result of exposure to oil.

Detecting selection in the blue crab, Callinectes sapidus, using DNA sequence data from multiple nuclear protein-coding genes.

The identification of genes involved in the adaptive evolution of non-model organisms with uncharacterized genomes constitutes a major challenge. This study employed a rigorous and targeted candidate gene approach to test for positive selection on protein-coding genes of the blue crab, Callinectes sapidus. Four genes with putative roles in physiological adaptation to environmental stress were chosen as candidates. A fifth gene not expected to play a role in environmental adaptation was used as a control. Large samples (n>800) of DNA sequences from C. sapidus were used in tests of selective neutrality based on sequence polymorphisms. In combination with these, sequences from the congener C. similis were used in neutrality tests based on interspecific divergence. In multiple tests, significant departures from neutral expectations and indicative of positive selection were found for the candidate gene trehalose 6-phosphate synthase (tps). These departures could not be explained by any of the historical population expansion or bottleneck scenarios that were evaluated in coalescent simulations. Evidence was also found for balancing selection at ATP-synthase subunit 9 (atps) using a maximum likelihood version of the Hudson, Kreitmen, and Aguadé test, and positive selection favoring amino acid replacements within ATP/ADP translocase (ant) was detected using the McDonald-Kreitman test. In contrast, test statistics for the control gene, ribosomal protein L12 (rpl), which presumably has experienced the same demographic effects as the candidate loci, were not significantly different from neutral expectations and could readily be explained by demographic effects. Together, these findings demonstrate the utility of the candidate gene approach for investigating adaptation at the molecular level in a marine invertebrate for which extensive genomic resources are not available.

Where are they now? The fates of two genetic lineages in an introduced Hawaiian reef fish.

Humans, both wittingly and unwittingly, have been transporting marine organisms beyond their native ranges for centuries (Ruiz et al. 1997). A central challenge of invasion biology is to identify the factors that determine whether introduced species fail to become established, become benign members of a community, or spread so far and reach such densities as to be considered invasive. Organismal features such as physiological tolerance, niche breadth and fecundity are critical, but by themselves are inaccurate predictors of the fates of introduced species (Sakai et al. 2001). The size, age distribution, and genetic makeup of founder populations are also important, but because they are usually unknown they are most often viewed as sources of uncertainty. For marine species with planktonic larvae, the challenge is even greater because the consequences of a planktonic phase for dispersal and population viability are not well understood. In this issue, Gaither et al. (2010a) present a remarkable account of the introduction of a reef fish for which the number and genetic makeup of the founders are known. Between 1956 and 1961, the Division of Fish and Game for the Territory of Hawaii introduced 12 non-indigenous fish species into Hawaiian waters to establish commercial and sport fisheries. The introduction of Lutjanus kasmira, the bluestriped snapper, was the most successful (Fig. 1). There were two releases of fish from French Polynesia. In 1958, 2431 fish from the Marquesas Islands were released on Oahu, followed in 1961 with an additional 728 fish from the Society Islands. The blue striped snapper rapidly spread to the other Hawaiian Islands, reaching the northwestern end of the archipelago by 1992. The choice of the Marquesas as one of two sources for the introduction was fortuitous. Gaither et al. (2010b) found that the Marquesas population is genetically distinct from all other Indo-Pacific populations of L. kasmira. Mitochondrial cytochrome b sequences of fish from the Marquesas belong to a separate lineage that diverged from others in the species roughly half a Ma. Allele frequencies for several nuclear loci are also distinct. This provided Gaither et al. (2010a) with an extraordinary opportunity to examine what became of the mixed genetic heritage of Hawaiian blue striped snappers after 50 years.

Utility of arginine kinase for resolution of phylogenetic relationships among Brachyuran genera and families.

The molecular phylogenetics of decapod crustaceans has been based on sequence data from a limited number of genes. These have included rapidly evolving mitochondrial genes, which are most appropriate for studies of closely related species, and slowly evolving nuclear ribosomal RNA genes, which have been most useful for resolution of deep branches within the Decapoda. Here we examine the utility of the nuclear gene that encodes arginine kinase for phylogenetic reconstruction at intermediate levels (relationships among genera and families) within the decapod infraorder Brachyura (the true crabs). Analyses based on arginine kinase sequences were compared and combined with those for the mitochondrial cytochrome oxidase I gene. All of the genera in our taxon sample were resolved with high support with arginine kinase data alone. However, some of these genera were grouped into clades that are in conflict with recognized brachyuran families. A phylogeny based on cytochrome oxidase I was consistent with the arginine kinase phylogeny, but with weaker support. A recently proposed measure of phylogenetic informativeness indicated that arginine kinase was generally more informative than cytochrome oxidase I for relationships above the level of genus. Combined analysis of data from both genes provided strong support for clades that are in conflict with current assignments of genera to the families Epialtidae, Mithracidae, Pisidae, and Portunidae.

Isolation and characterisation of main olfactory and vomeronasal receptor gene families from the Atlantic salmon (Salmo salar).

The Atlantic salmon (Salmo salar) has been widely used as a model species in studies of olfactory signal transduction and processing. Here we report the isolation and characterisation of salmon olfactory receptor (SOR) and salmon vomeronasal receptor (SVR) partial sequences from Atlantic salmon. Six groups of SOR sequences (SORA-F) and three groups of SVR sequences (SVRA-C) were identified. All SORB, SORF, SVRB and SVRC sequences contained uninterrupted open reading frames. However, all SORA sequences and members of the SVRA sequence family contained multiple stop codons while SORC and SORE sequences were truncated in the 3' region of the sequence. Full length SORF and almost complete SORB sequences displayed amino acid residues and motifs conserved in fish olfactory receptor genes. In sequence phylogenies, SOR sequences fell into the main olfactory receptor (MOR) type I clade and were most closely related to either delta or zeta reference sequences, while all SVR sequences grouped within a clade of fish type 2 vomeronasal receptor (V2R) sequences. A family of sequences (Sasa CaSR1-6), isolated using the same degenerate primers that amplified SVR sequences, clustered within a group of calcium sensing receptor (CaSR) sequences. Analysis of tissue expression patterns of sequences by reverse transcriptase polymerase chain reaction showed that they were transcribed in olfactory epithelium (SORB, SORF, all SVR and Sasa CaSR sequences), testis (SORB, SORD and Sasa CaSR) and/or anterior kidney (SORB and Sasa CaSR). Similar analysis of expression supported the identification of SORA sequences as non-transcribed pseudogene(s). Although the level of occurrence of OR pseudogenes is within the range found for other, well-characterised vertebrate OR genomes, it does not seem to reflect the importance of olfaction in the biology of the Atlantic salmon.

Population structure at two geographic scales in the burrowing crustacean Callichirus islagrande (Decapoda, Thalassinidea): historical and contemporary barriers to planktonic dispersal.

There has been much recent interest in the extent to which marine planktonic larvae connect local populations demographically and genetically. Uncertainties about the true extent of larval dispersal have impeded our understanding of the ecology and evolution of marine species as well as our attempts to effectively manage marine populations. Because direct measurements of larval movements are difficult, genetic markers have often been used for indirect measurements of gene flow among marine populations. Here we examine data from allozymes, mitochondrial DNA sequences, and microsatellite length polymorphisms to assess the extent of gene flow among populations of the burrowing crustacean Callichirus islagrande. All three types of markers revealed a genetic break between populations separated by the Louisiana Chenier Plain. The extent of mitochondrial sequence divergence across this break indicates that the nominal species, C. islagrande, consists of at least two lineages that have been reproductively isolated for about a million years. Within the eastern lineage microsatellite allele frequencies were significantly heterogeneous among populations as little as 10 km apart. Maximum likelihood estimates of gene flow and effective population size based on a coalescent model for the microsatellite data indicated that local populations are nearly closed. A model-based clustering method identified four or five groups from the microsatellite data, although individuals sampled from each location generally consisted of mixtures of these groups. This suggests a mechanism that would lead to genetic differentiation of open populations: gene flow from different source populations that are themselves genetically distinct.

ENHANCING THE RETRDEVABILITY OF POPULATION GENETIC SURVEY DATA? AN ASSESSMENT OF ANIMAL MITOCHONDRIAL DNA STUDIES.

Surveys of genetic variation in natural populations represent a valuable and often irreplaceable resource. It may be desirable to reanalyze data as new methods are developed for comparisons with other populations or for comparisons with the same populations at different times. We evaluated existing mechanisms of data preservation in a survey of 627 published surveys of mitochondrial DNA variation in animal and found that over half of the datasets (56%) contained insufficient information for reanalysis. In many cases, publication of complete data would not have added excessively to the length of the publication. Because at present, publications represent the main archive of population genetic data, we offer recommendations for how the essential data from mtDNA surveys can be presented in a form that is complete and concise.

TESTS OF PHYLOGEOGRAPHIC MODELS WITH NUCLEAR AND MITOCHONDRIAL DNA SEQUENCE VARIATION IN THE STONE CRABS, MENIPPE ADINA AND MENIPPE MERCENARIA.

Evolutionary relationships among stone crabs (Menippe) from the Gulf of Mexico and western Atlantic were investigated by comparisons of restriction sites within anonymous nuclear DNA sequences and nucleotide sequences of both mitochondrial and a duplicated nuclear form of the mitochondrial large subunit ribosomal RNA (LSrDNA) gene. A survey of over 100 restriction sites by Southern blot analysis with 10 anonymous nuclear DNA sequence probes failed to reveal any differences between Menippe adina and M. mercenaria. Sequence comparisons of both mitochondrial and nuclear forms of the LSrDNA gene also did not distinguish these species. Although both LSrDNA gene sequences were variable, some haplotypes were shared by the two species, implying either incomplete gene lineage sorting or introgressive hybridization. Based on molecular clock calibrations, we estimate that all of the observed mitochondrial LSrDNA sequences share a common ancestor between 1.5 and 2.7 million years before present (M.Y.B.P.). However, because identical sequences are shared by the two species, these data are also compatible with a more recent common ancestry. These findings conflict with a previously proposed biogeographic scenario for North American Menippe, which featured a relict hybrid zone on the Atlantic Coast. We suggest an alternative scenario based on relatively recent events and ongoing, rather than historical, gene flow.

ESTIMATION OF SINGLE GENERATION MIGRATION DISTANCES FROM GEOGRAPHIC VARIATION IN ANIMAL MITOCHONDRIAL DNA.

A new approach is introduced for the analysis of dispersal from the geographic distributions of mtDNA lineages. The method is based on the expected spatial distributions of lineages arising under a multigeneration random walk process. Unlike previous methods based on the predicted equilibria between genetic drift and gene flow, this approach is appropriate for non-equilibrium conditions, and yields an estimate of dispersal distance rather than dispersal rate. The theoretical basis for this method is examined, and an analysis of mtDNA restriction site data for Peromyscus maniculatus is presented as an example of how this approach can be applied to empirical data.

GENE GENEALOGIES WITHIN THE ORGANISMAL PEDIGREES OF RANDOM-MATING POPULATIONS.

Using computer simulations, we generated and analyzed genetic distances among selectively neutral haplotypes transmitted through gene genealogies with random-mating organismal pedigrees. Constraints and possible biases on haplotype distances due to correlated ancestry were evaluated by comparing observed distributions of distances to those predicted from an inbreeding theory that assumes independence among haplotype pairs. Results suggest that: 1) mean time to common ancestry of neutral haplotypes can be a reasonably good predictor of evolutionary effective population size; 2) the nonindependence of haplotype paths of descent within a given gene genealogy typically produces significant departures from the theoretical probability distributions of haplotype distances; 3) frequency distributions of distances between haplotypes drawn from "replicate" organismal pedigrees or from multiple unlinked loci within an organismal pedigree exhibit very close agreement with the theory for independent haplotypes. These results are relevant to interpretations of current molecular data on genetic distances among nonrecombining haplotypes at either nuclear or cytoplasmic loci.

THE PRECISION OF HISTOCOMPATIBILITY RESPONSE IN CLONAL RECOGNITION IN TROPICAL MARINE SPONGES.

Recently discovered histocompatibility-like phenomena in sponges (Phylum Porifera) have prompted attempts to measure the precision with which allogeneic grafts are recognized and rejected. The results of these investigations have been extremely varied, ranging from suggestions that allorecognition does not occur to suggestions that every genetically distinct individual may be unique in histocompatibility type. Interpretation of these findings is complicated by the variation in methods and species used by different workers. Here we compare various measurements of allorecognition precision for several species of tropical marine sponges. From our results we conclude that 1) tissue implant grafts are more prone to artifact than grafts between intact sponges; 2) the possibility of clonal propogation should be considered when graft acceptances are observed between sponges selected from a single population; and 3) allozyme variation in Niphates erecta shows that occasional grafts between genetically different individuals may be accepted.