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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.

Aim: Ballast water is a major vector of non-indigenous species introductions world-wide. Our understanding of population dynamics of organisms entrained in ballast is largely limited to studies of zooplankton and phytoplankton. Bacteria are more numerous and diverse than zooplankton or phytoplankton, yet remain comparatively understudied. We apply a metagenomics approach to characterize changes in the microbial ballast water community over the course of three voyages on one ship, and assess the effects of ballast water exchange (BWE), spring/summer sampling month and time since voyage start. Location: Quebec City and Deception Bay, Quebec, and the coastal marine region offshore of eastern Canada. Methods: We used universal primers to Ion Torrent sequence a fragment of the bacterial 16S ribosomal DNA for samples collected over three voyages of one ship between Quebec City and Deception Bay in June, July and August 2015. We compared richness (total number of species in the community) and diversity (accounts for both species abundance and evenness) using linear mixed-effects analysis and compared community composition using non-metric multidimensional scaling and permutational multivariate analysis of variance. Initial comparisons were between months. Subsequent analyses focused on each month separately. Results: Ion Torrent sequencing returned c. 2.9 million reads and revealed monthly differences in diversity and richness, and in community structure in ballast water. June had higher richness and diversity than either July or August, and showed most clearly the effect of BWE on the microbial community. Main conclusions: Our results suggest that environmental conditions associated with different spring/summer sampling months drive differences in microbial diversity in ballast water. This study showed that BWE removes some components of the freshwater starting microbial community and replaces them with other taxa. BWE also changed proportional representation of some microbes without removing them completely. It appears that some taxa are resident in ballast tanks and are not removed by BWE. © 2017 John Wiley & Sons Ltd

A developing body of theory and empirical evidence suggest that feeding behaviour as measured by the functional response (FR) can assist researchers in assessing the relative potential, ecological impacts and competitive abilities of native and introduced species. Here, we explored the FRs of two land snails that occur in south-western Ontario, one native (Mesodonthyroidus) and one non-indigenous (Cepaeanemoralis) to Canada. The non-indigenous species appears to have low ecological impact and inferior competitive abilities. Consistent with theory, while both species conformed to Type II functional responses, the native species had a significantly higher attack rate (5.30 vs 0.41, respectively) and slightly lower handling time (0.020 vs 0.023), and hence a higher maximum feeding rate (50.0 vs 43.5). The non-indigenous species exhibited a significantly longer time to contact for a variety of food types, and appeared less discriminating of paper that was offered as a non-food type. The non-indigenous species also ate significantly less food when in mixed species trials with the native snail. These feeding patterns match the known low ecological impact of the introduced snail and are consistent with the view that it is an inferior competitor relative to the native species. However, field experimentation is required to clarify whether the largely microallopatric distributions of the two species in south-western Ontario reflect competitive dominance by the native species or other factors such as habitat preference, feeding preferences or predator avoidance. The relative patterns of feeding behaviour and ecological impact are, however, fully in line with recent functional response theory and application.

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-ɣ, 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.

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.

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 version of record is embargoed until 07-24-2016. The final peer reviewed, accepted manuscript is available without an embargo. The published article is copyrighted by Inter-Research and can be found at: http://www.int-res.com/journals/meps/meps-home/.

In the US Pacific Northwest, rockfishes Sebastes spp. have recently become a focus for increased management efforts; several species are currently managed under extreme conser- vation measures due to low population levels and intense fishing pressure. Rockfish recruitment is extremely variable, and a better understanding of the factors influencing recruitment and set- tlement would assist in prioritizing management and conservation efforts. The goal of this study was to investigate natural and anthropogenic influences on the estuarine settlement process of rockfishes, with a focus on black rockfish S. melanops. Trap surveys conducted in 6 Oregon estu- aries indicate that young-of-the-year (YOY; Age-0) rockfish utilize multiple Oregon estuaries from spring through late fall. As shown by late season increases in catch rates and the capture of mul- tiple Age-1 individuals, rockfishes may be present in highly developed estuaries through their first winter. Genetic identification confirms that the majority of the YOY rockfish captured during this study were black rockfish S. melanops. Catches were higher in the more developed estuaries, suggesting that the continued development of Oregon estuaries may not adversely affect the rock- fish settlement process. This study provides strong evidence of widespread use of estuarine habi- tat by black rockfish on the Oregon coast during their first year of life, and provides additional support that structure is an important component to the settlement process.

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.

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 32u Nt o 50uN 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.

The copper rockfish is a benthic, nonmigratory, temperate rocky reef marine species with pelagic larvae and juveniles. A previous range-wide study of the population-genetic structure of copper rockfish revealed a pattern consistent with isolation-by-distance. This could arise from an intrinsically limited dispersal capability in the species or from regularly-spaced extrinsic barriers that restrict gene flow (offshore jets that advect larvae offshore and/or habitat patchiness). Tissue samples were collected along the West Coast of the contiguous USA between Neah Bay, WA and San Diego, CA, with dense sampling along Oregon. At the whole-coast scale (approximately 2200 km), significant population subdivision (F(ST) = 0.0042), and a significant correlation between genetic and geographical distance were observed based on 11 microsatellite DNA loci. Population divergence was also significant among Oregon collections (approximately 450 km, F(ST) = 0.001). Hierarchical amova identified a weak but significant 130-km habitat break as a possible barrier to gene flow within Oregon, across which we estimated that dispersal (N(e)m) is half that of the coast-wide average. However, individual-based Bayesian analyses failed to identify more than a single population along the Oregon coast. In addition, no correlation between pairwise population genetic and geographical distances was detected at this scale. The offshore jet at Cape Blanco was not a significant barrier to gene flow in this species. These findings are consistent with low larval dispersal distances calculated in previous studies on this species, support a mesoscale dispersal model, and highlight the importance of continuity of habitat and adult population size in maintaining gene flow.