Seascape genetics of the stalked kelp Pterygophora californica and comparative population genetics in the Santa Barbara Channel.

We conducted a population genetic analysis of the stalked kelp, Pterygophora californica, in the Santa Barbara Channel, California, USA. The results were compared with previous work on the genetic differentiation of giant kelp, Macrocystis pyrifera, in the same region. These two sympatric kelps not only share many life history and dispersal characteristics but also differ in that dislodged P. californica does not produce floating rafts with buoyant fertile sporophytes, commonly observed for M. pyrifera. We used a comparative population genetic approach with these two species to test the hypothesis that the ability to produce floating rafts increases the genetic connectivity among kelp patches in the Santa Barbara Channel. We quantified the association of habitat continuity and oceanographic distance with the genetic differentiation observed in stalked kelp, like previously conducted for giant kelp. We compared both overall (across all patches) and pairwise (between patches) genetic differentiation. We found that oceanographic transit time, habitat continuity, and geographic distance were all associated with genetic connectivity in P. californica, supporting similar previous findings for M. pyrifera. Controlling for differences in heterozygosity between kelp species using Jost's DEST , we showed that global differentiation and pairwise differentiation were similar among patches between the two kelp species, indicating that they have similar dispersal capabilities despite their differences in rafting ability. These results suggest that rafting sporophytes do not play a significant role in effective dispersal of M. pyrifera at ecologically relevant spatial and temporal scales.

Human-mediated and natural dispersal of an invasive fish in the eastern Great Lakes.

The globally invasive Round Goby (Neogobius melanostomus) was introduced to the Great Lakes around 1990, spreading widely and becoming the dominant benthic fish in many areas. The speed and scope of this invasion is remarkable and calls into question conventional secondary spread models and scenarios. We utilized nine microsatellites to identify large-scale genetic structure in Round Goby populations in the eastern Great Lakes, and assessed the role of colonization vs. secondary transport and dispersal in developing this structure. We identified three clusters, corresponding with Lake Huron, eastern Lake Erie, and western Lake Erie plus eastern Lake Ontario, along with three highly divergent populations. Bottleneck analysis identified founder effects in two divergent populations. Regression analyses of isolation by distance and allelic richness vs. distance from the initial invasion site were consistent with limited migration. However, some populations in eastern Lake Erie and Lake Ontario showed anomalously low genetic distance from the original site of colonization, consistent with secondary transport of large numbers of individuals via ballast water. We conclude that genetic structure of Round Goby in the Great Lakes principally resulted from long-distance secondary transport via ballast water with additional movement of individual via bait buckets and natural dispersal. The success of Round Gobies represents an interesting model for colonization characterization; however, those same attributes present significant challenges for conservation and fisheries management. Current management likely prevents many new species from arriving in the Great Lakes, but fails to address the transport of species within the lakes after they arrive; this is an issue of clear and pressing importance.

Molecular Insights Into the Ctenophore Genus Beroe in Europe: New Species, Spreading Invaders.

The genus Beroe Browne, 1756 (Ctenophora, Beroidae) occurs worldwide, with 25 currently-described species. Because the genus is poorly studied, the definitive number of species is uncertain. Recently, a possible new Beroe species was suggested based on internal transcribed spacer 1 (ITS1) sequences from samples collected in Svalbard, Norway. Another species, Beroe ovata, was introduced to Europe from North America, initially in the Black Sea and subsequently (and possibly secondarily) into the Mediterranean and Baltic Seas. In areas where ctenophores have been introduced, they have often had significant detrimental ecological effects. The potential for other cryptic and/or undescribed Beroe species and history of spread of some species in the genus give reason for additional study. When alive, morphological hallmarks may be challenging to spot and photograph owing to the animals' transparency and near-constant motion. We sampled and analyzed 109 putative Beroe specimens from Europe, using morphological and molecular approaches. DNA analyses were conducted using cytochrome oxidase 1 and internal transcribed spacer sequences and, together with published sequences from GenBank, phylogenetic relationships of the genus were explored. Our study suggests the presence of at least 5 genetic lineages of Beroe in Europe, of which 3 could be assigned to known species: Beroe gracilis Künne 1939; Beroe cucumis Fabricius, 1780; and Beroe ovata sensu Mayer, 1912. The other 2 lineages (here provisionally named Beroe "norvegica" and Beroe "anatoliensis") did not clearly coincide with any known species and might therefore reflect new species, but confirmation of this requires further study.

Inbreeding effects on gene-specific DNA methylation among tissues of Chinook salmon.

Inbreeding depression is the loss of fitness resulting from the mating of genetically related individuals. Traditionally, the study of inbreeding depression focused on genetic effects, although recent research has identified DNA methylation as also having a role in inbreeding effects. Since inbreeding depression and DNA methylation change with age and environmental stress, DNA methylation is a likely candidate for the regulation of genes associated with inbreeding depression. Here, we use a targeted, multigene approach to assess methylation at 22 growth-, metabolic-, immune- and stress-related genes. We developed PCR-based DNA methylation assays to test the effects of intense inbreeding on intragenic gene-specific methylation in inbred and outbred Chinook salmon. Inbred fish had altered methylation at three genes, CK-1, GTIIBS and hsp70, suggesting that methylation changes associated with inbreeding depression are targeted to specific genes and are not whole-genome effects. While we did not find a significant inbreeding by age interaction, we found that DNA methylation generally increases with age, although methylation decreased with age in five genes, CK-1, IFN-É£, HNRNPL, hsc71 and FSHb, potentially due to environmental context and sexual maturation. As expected, we found methylation patterns differed among tissue types, highlighting the need for careful selection of target tissue for methylation studies. This study provides insight into the role of epigenetic effects on ageing, environmental response and tissue function in Chinook salmon and shows that methylation is a targeted and regulated cellular process. We provide the first evidence of epigenetically based inbreeding depression in vertebrates.

Role of genomics and transcriptomics in selection of reintroduction source populations.

The use and importance of reintroduction as a conservation tool to return a species to its historical range from which it has been extirpated will increase as climate change and human development accelerate habitat loss and population extinctions. Although the number of reintroduction attempts has increased rapidly over the past 2 decades, the success rate is generally low. As a result of population differences in fitness-related traits and divergent responses to environmental stresses, population performance upon reintroduction is highly variable, and it is generally agreed that selecting an appropriate source population is a critical component of a successful reintroduction. Conservation genomics is an emerging field that addresses long-standing challenges in conservation, and the potential for using novel molecular genetic approaches to inform and improve conservation efforts is high. Because the successful establishment and persistence of reintroduced populations is highly dependent on the functional genetic variation and environmental stress tolerance of the source population, we propose the application of conservation genomics and transcriptomics to guide reintroduction practices. Specifically, we propose using genome-wide functional loci to estimate genetic variation of source populations. This estimate can then be used to predict the potential for adaptation. We also propose using transcriptional profiling to measure the expression response of fitness-related genes to environmental stresses as a proxy for acclimation (tolerance) capacity. Appropriate application of conservation genomics and transcriptomics has the potential to dramatically enhance reintroduction success in a time of rapidly declining biodiversity and accelerating environmental change.

Seascape drivers of Macrocystis pyrifera population genetic structure in the northeast Pacific.

At small spatial and temporal scales, genetic differentiation is largely controlled by constraints on gene flow, while genetic diversity across a species' distribution is shaped on longer temporal and spatial scales. We assess the hypothesis that oceanographic transport and other seascape features explain different scales of genetic structure of giant kelp, Macrocystis pyrifera. We followed a hierarchical approach to perform a microsatellite-based analysis of genetic differentiation in Macrocystis across its distribution in the northeast Pacific. We used seascape genetic approaches to identify large-scale biogeographic population clusters and investigate whether they could be explained by oceanographic transport and other environmental drivers. We then modelled population genetic differentiation within clusters as a function of oceanographic transport and other environmental factors. Five geographic clusters were identified: Alaska/Canada, central California, continental Santa Barbara, California Channel Islands and mainland southern California/Baja California peninsula. The strongest break occurred between central and southern California, with mainland Santa Barbara sites forming a transition zone between the two. Breaks between clusters corresponded approximately to previously identified biogeographic breaks, but were not solely explained by oceanographic transport. An isolation-by-environment (IBE) pattern was observed where the northern and southern Channel Islands clustered together, but not with closer mainland sites, despite the greater distance between them. The strongest environmental association with this IBE pattern was observed with light extinction coefficient, which extends suitable habitat to deeper areas. Within clusters, we found support for previous results showing that oceanographic connectivity plays an important role in the population genetic structure of Macrocystis in the Northern hemisphere.

Looking into the black box: simulating the role of self-fertilization and mortality in the genetic structure of Macrocystis pyrifera.

Patterns of spatial genetic structure (SGS), typically estimated by genotyping adults, integrate migration over multiple generations and measure the effective gene flow of populations. SGS results can be compared with direct ecological studies of dispersal or mating system to gain additional insights. When mismatches occur, simulations can be used to illuminate the causes of these mismatches. Here, we report a SGS and simulation-based study of self-fertilization in Macrocystis pyrifera, the giant kelp. We found that SGS is weaker than expected in M. pyrifera and used computer simulations to identify selfing and early mortality rates for which the individual heterozygosity distribution fits that of the observed data. Only one (of three) population showed both elevated kinship in the smallest distance class and a significant negative slope between kinship and geographical distance. All simulations had poor fit to the observed data unless mortality due to inbreeding depression was imposed. This mortality could only be imposed for selfing, as these were the only simulations to show an excess of homozygous individuals relative to the observed data. Thus, the expected data consistently achieved nonsignificant differences from the observed data only under models of selfing with mortality, with best fits between 32% and 42% selfing. Inbreeding depression ranged from 0.70 to 0.73. The results suggest that density-dependent mortality of early life stages is a significant force in structuring Macrocystis populations, with few highly homozygous individuals surviving. The success of these results should help to validate simulation approaches even in data-poor systems, as a means to estimate otherwise difficult-to-measure life cycle parameters.

The mitochondrial genomes of Euphausia pacifica and Thysanoessa raschii sequenced using 454 next-generation sequencing, with a phylogenetic analysis of their position in the Malacostracan family tree.

Euphausiid krill play a critical role in coastal and oceanic food webs, linking primary producers to upper trophic levels. In addition, some species support commercial fisheries worldwide. Despite their ecological importance, the genetics of these important species remain poorly described. To improve our understanding of the genetics of these ecological links, we sequenced the mitochondrial genomes of two species of North Pacific krill, Euphausia pacifica and Thysanoessa raschii, using long-range PCR and 454 GS Junior next-generation sequencing technology. The E. pacifica mitogenome (14,692 + base pairs (bp)) encodes 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, and at least 22 transfer RNA (tRNA) genes. The T. raschii mitogenome (14,240 + bp) encodes 13 PCGs, two rRNA genes, and at least 19 tRNA genes. The gene order in both species is similar to that of E. superba. Comparisons between Bering Sea and Yellow Sea E. pacifica revealed a total of 644 variable sites. The most variable protein-coding gene were atp8 (7.55 %, 12 of 159 sites variable), nad4 (6.35 %, 85 variable sites) and nad6 (6.32 %, 33 variable sites). Phylogenetic analyses to assess the phylogenetic position of the Euphausiacea, using the concatenated nucleic acid sequences of E. pacifica and T. raschii along with 46 previously published malacostracan mitogenomes, support the monophyly of the order Decapoda and indicate that the Euphausiacea share a common ancestor with the Decapoda. Future research should utilize this sequence data to explore the population genetics and molecular ecology of these species.

Olfactory receptor related to class A, type 2 (V1r-like Ora2) genes are conserved between distantly related rockfishes (genus Sebastes).

V1r-like Ora genes express putative chemoreceptors that may function as pheromone receptors in fishes. We used a candidate gene approach to test whether V1r-like Ora2 genes show evidence of positive selection that could suggest a role in mate recognition and the avoidance of hybridization between closely related rockfishes. We amplified a 492-bp fragment of a single V1r-like Ora2 gene from each of 5 species of rockfish. Despite separation of up to 7.8 My, the sequence of V1r-like Ora2 is highly conserved. Genetic distances are small, and all our study species shared at least one sequence with another species. Sequence comparisons suggested that, although most amino acids were subject to purifying selection, 9 amino acids showed evidence of positive selection. Because many of these amino acids were not associated with the areas of the protein suggested to be involved in ligand binding based on structural similarity to other olfactory receptors, this signal may reflect an echo of the relaxation of selection associated with the speciation events that separate these species. Strong sequence conservation suggests that this gene is of functional significance. However, because of shared alleles among species, the V1r-like Ora2 gene, in isolation, would be unlikely to differentiate species during mating season.

Conventional versus real-time quantitative PCR for rare species detection.

Detection of species in nature at very low abundance requires innovative methods. Conventional PCR (cPCR) and real-time quantitative PCR (qPCR) are two widely used approaches employed in environmental DNA (eDNA) detection, though lack of a comprehensive comparison of them impedes method selection. Here we test detection capacity and false negative rate of both approaches using samples with different expected complexities. We compared cPCR and qPCR to detect invasive, biofouling golden mussels (Limnoperna fortunei), in samples from laboratory aquaria and irrigation channels where this mussel was known to occur in central China. Where applicable, the limit of detection (LoD), limit of quantification (LoQ), detection rate, and false negative rate of each PCR method were tested. Quantitative PCR achieved a lower LoD than cPCR (1 × 10-7 vs. 10-6 ng/µl) and had a higher detection rate for both laboratory (100% vs. 87.9%) and field (68.6% vs. 47.1%) samples. Field water samples could only be quantified at a higher concentration than laboratory aquaria and total genomic DNA, indicating inhibition with environmental samples. The false negative rate was inversely related to the number of sample replicates. Target eDNA concentration was negatively related to distance from sampling sites to the water (and animal) source. Detection capacity difference between cPCR and qPCR for genomic DNA and laboratory aquaria can be translated to field water samples, and the latter should be prioritized in rare species detection. Field environmental samples may involve more complexities-such as inhibitors-than laboratory aquaria samples, requiring more target DNA. Extensive sampling is critical in field applications using either approach to reduce false negatives.

Modeling sampling strategies for determination of zooplankton abundance in ballast water.

Ballast water has been a major source of non-indigenous species introductions. The International Maritime Organization has proposed performance standard that will establish an upper limit for viable organisms in discharged ballast. Here we test different sampling efforts for zooplankton in ballast water on a commercial vessel. We fit different probability density functions to find the most representative and evaluated sampling efforts necessary to achieve error rates (α, ß) of <0.05. Our tests encompassed four seasonal trials and five sample volumes. To estimate error rates, we performed simulations which drew from 1 to 30 replicates of each volume (0.10-3.00m3) for mean densities ranging between 1 and 20 organisms m-3. Fieldwork and simulations suggested that >0.5m3 samples had the best accuracy and precision, and that the Poisson distribution fit these communities best. This study provides the first field test of a sampling strategy to assess compliance with the future IMO standard for large vessels.

Population structure of three species of Anisakis nematodes recovered from Pacific sardines (Sardinops sagax) distributed throughout the California Current system.

Members of the Anisakidae are known to infect over 200 pelagic fish species and have been frequently used as biological tags to identify fish populations. Despite information on the global distribution of Anisakis species, there is little information on the genetic diversity and population structure of this genus, which could be useful in assessing the stock structure of their fish hosts. From 2005 through 2008, 148 larval anisakids were recovered from Pacific sardine (Sardinops sagax) in the California Current upwelling zone and were genetically sequenced. Sardines were captured off Vancouver Island, British Columbia in the north to San Diego, California in the south. Three species, Anisakis pegreffii, Anisakis simplex 'C', and Anisakis simplex s.s., were identified with the use of sequences from the internal transcribed spacers (ITS1 and ITS2) and the 5.8s subunit of the nuclear ribosomal DNA. The degree of nematode population structure was assessed with the use of the cytochrome c oxidase 2 (cox2) mitochondrial DNA gene. All 3 Anisakis species were distributed throughout the study region from 32°N to 50°N latitude. There was no association between sardine length and either nematode infection intensity or Anisakis species recovered. Larval Anisakis species and mitochondrial haplotype distributions from both parsimony networks and analyses of molecular variance revealed a panmictic distribution of these parasites, which infect sardines throughout the California Current ecosystem. Panmictic distribution of the larval Anisakis spp. populations may be a result of the presumed migratory pathways of the intermediate host (the Pacific sardine), moving into the northern portion of the California Current in summer and returning to the southern portion to overwinter and spawn in spring. However, the wider geographic range of paratenic (large piscine predators), and final hosts (cetaceans) can also explain the observed distribution pattern. As a result, the recovery of 3 Anisakis species and a panmictic distribution of their haplotypes could not be used to confirm or deny the presence of population subdivision of Pacific sardines in the California Current system.