Contaminants reach everywhere: Fish dietary samples should be surface decontaminated prior to molecular diet analysis.

Knowledge of trophic interaction is necessary to understand the dynamics of ecosystems and develop ecosystem-based management. The key data to measure these interactions should come from large-scale diet analyses with good taxonomic resolution. To that end, molecular methods that analyze prey DNA from guts and feces provide high-resolution dietary taxonomic data. However, molecular diet analysis may also produce unreliable results if the samples are contaminated by external sources of DNA. Employing the freshwater European whitefish (Coregonus lavaretus) as a tracer for sample contamination, we studied the possible route of whitefish in beaked redfish (Sebastes mentella) guts sampled in the Barents Sea. We used whitefish-specific COI primers for diagnostic analysis, and fish-specific 12S and metazoa-specific COI primers for metabarcoding analyses of intestine and stomach contents of fish samples that were either not cleaned, water cleaned, or bleach cleaned after being in contact with whitefish. Both the diagnostic and COI metabarcoding revealed clear positive effects of cleaning samples as whitefish were detected in significantly higher numbers of uncleaned samples compared to water or bleach-cleaned samples. Stomachs were more susceptible to contamination than intestines and bleach cleaning reduced the frequency of whitefish contamination. Also, the metabarcoding approach detected significantly more reads of whitefish in the stomach than in intestine samples. The diagnostic analysis and COI metabarcoding detected contaminants in a higher and comparable number of gut samples than the 12S-based approach. Our study underlines thus the importance of surface decontamination of aquatic samples to obtain reliable diet information from molecular data.

Benthic invertebrates in Svalbard fjords-when metabarcoding does not outperform traditional biodiversity assessment.

To protect and restore ecosystems and biodiversity is one of the 10 challenges identified by the United Nations's Decade of the Ocean Science. In this study we used eDNA from sediments collected in two fjords of the Svalbard archipelago and compared the taxonomic composition with traditional methods through metabarcoding, targeting mitochondrial CO1, to survey benthos. Clustering of 21.6 mill sequence reads with a d value of 13 in swarm, returned about 25 K OTU reads. An identification search with the BOLD database returned 12,000 taxonomy annotated sequences spanning a similarity range of 50% to 100%. Using an acceptance filter of minimum 90% similarity to the CO1 reference sequence, we found that 74% of the ca 100 taxon identified sequence reads were Polychaeta and 22% Nematoda. Relatively few other benthic invertebrate species were detected. Many of the identified sequence reads were extra-organismal DNA from terrestrial, planktonic, and photic zone sources. For the species rich Polychaeta, we found that, on average, only 20.6% of the species identified from morphology were also detected with DNA. This discrepancy was not due to missing reference sequences in the search database, because 90-100% (mean 96.7%) of the visually identified species at each station were represented with barcodes in Boldsystems. The volume of DNA samples is small compared with the volume searched in visual sorting, and the replicate DNA-samples in sum covered only about 2% of the surface area of a grab. This may considerably reduce the detection rate of species that are not uniformly distributed in the sediments. Along with PCR amplification bias and primer mismatch, this may be an important reason for the limited congruence of species identified with the two approaches. However, metabarcoding also identified 69 additional species that are usually overlooked in visual sample sorting, demonstrating how metabarcoding can complement traditional methodology by detecting additional, less conspicuous groups of organisms.

Geographic variation in gene flow from a genetically distinct migratory ecotype drives population genetic structure of coastal Atlantic cod (Gadus morhua L.).

Identifying how physical and biotic factors shape genetic connectivity among populations in time and space is essential to our understanding of the evolutionary trajectory as well as the management of marine species. Atlantic cod is a widespread and commercially important marine species displaying several ecotypes with different life history strategies. Using three sets of SNPs: neutral, informative, and genome-inversion linked, we studied population genetic structure of ~2500 coastal Atlantic cod (CC) from 40 locations along Norway's 2500 km coastline, including nine fjords. We observed: (1) a genetic cline, suggesting a mechanism of isolation by distance, characterized by a declining F ST between CC and North East Arctic Cod (NEAC-genetically distinct migratory ecotype) with increasing latitude, (2) that in the north, samples of CC from outer-fjord areas were genetically more similar to NEAC than were samples of CC from their corresponding inner-fjord areas, (3) greater population genetic differentiation among CC sampled from outer-fjord areas along the coast, than among CC sampled from their corresponding inner-fjord areas, (4) genetic differentiation among samples of CC from both within and among fjords. Collectively, these results permit us to draw two main conclusions. First, that differences in the relative presence of the genetically highly distinct, migratory ecotype NEAC, declining from north to south and from outer to inner fjord, plays the major role in driving population genetic structure of the Norwegian CC. Second, that there is limited connectivity between CC from different fjords. These results suggest that the current management units implemented for this species in Norway should be divided into smaller entities. Furthermore, the situation where introgression from one ecotype drives population genetic structure of another, as is the case here, may exist in other species and geographical regions, thus creating additional challenges for sustainable fisheries management.

Hierarchical genetic structure in an evolving species complex: Insights from genome wide ddRAD data in Sebastes mentella.

The diverse biology and ecology of marine organisms may lead to complex patterns of intraspecific diversity for both neutral and adaptive genetic variation. Sebastes mentella displays a particular life-history as livebearers, for which existence of multiple ecotypes has been suspected to complicate the genetic population structure of the species. Double digest restriction-site associated DNA was used to investigate genetic population structure in S. mentella and to scan for evidence of selection. In total, 42,288 SNPs were detected in 277 fish, and 1,943 neutral and 97 tentatively adaptive loci were selected following stringent filtration. Unprecedented levels of genetic differentiation were found among the previously defined 'shallow pelagic', 'deep pelagic' and 'demersal slope' ecotypes, with overall mean FST = 0.05 and 0.24 in neutral and outlier SNPs, respectively. Bayesian computation estimated a concurrent and historical divergence among these three ecotypes and evidence of local adaptation was found in the S. mentella genome. Overall, these findings imply that the depth-defined habitat divergence of S. mentella has led to reproductive isolation and possibly adaptive radiation among these ecotypes. Additional sub-structuring was detected within the 'shallow' and 'deep' pelagic ecotypes. Population assignment of individual fish showed more than 94% agreement between results based on SNP and previously generated microsatellite data, but the SNP data provided a lower estimate of hybridization among the ecotypes than that by microsatellite data. We identified a SNP panel with only 21 loci to discriminate populations in mixed samples based on a machine-learning algorithm. This first SNP based investigation clarifies the population structure of S. mentella, and provides novel and high-resolution genomic tools for future investigations. The insights and tools provided here can readily be incorporated into the management of S. mentella and serve as a template for other exploited marine species exhibiting similar complex life history traits.

Genomic analysis reveals neutral and adaptive patterns that challenge the current management regime for East Atlantic cod Gadus morhua L.

Challenging long-held perceptions of fish management units can help to protect vulnerable stocks. When a fishery consisting of multiple genetic stocks is managed as a single unit, overexploitation and depletion of minor genetic units can occur. Atlantic cod (Gadus morhua) is an economically and ecologically important marine species across the North Atlantic. The application of new genomic resources, including SNP arrays, allows us to detect and explore novel structure within specific cod management units. In Norwegian waters, coastal cod (i.e. those not undertaking extensive migrations) are divided into two arbitrary management units defined by ICES: one between 62° and 70°N (Norwegian coastal cod; NCC) and one between 58° and 62°N (Norwegian coastal south; NCS). Together, these capture a fishery area of >25,000 km2 containing many spawning grounds. To assess whether these geographic units correctly represent genetic stocks, we analysed spawning cod of NCC and NCS for more than 8,000 SNPs along with samples of Russian White Sea cod, north-east Arctic cod (NEAC: the largest Atlantic stock), and outgroup samples representing the Irish and Faroe Sea's. Our analyses revealed large differences in spatial patterns of genetic differentiation across the genome and revealed a complex biological structure within NCC and NCS. Haplotype maps from four chromosome sets show regional specific SNP indicating a complex genetic structure. The current management plan dividing the coastal cod into only two management units does not accurately reflect the genetic units and needs to be revised. Coastal cod in Norway, while highly heterogenous, is also genetically distinct from neighbouring stocks in the north (NEAC), west (Faroe Island) and the south. The White Sea cod are highly divergent from other cod, possibly yielding support to the earlier notion of subspecies rank.

The pantophysin gene and its relationship with survival in early life stages of Atlantic cod.

Genetic markers are widely used in fisheries management around the world. While the genetic structure and markers selected are usually based on samples from the wild, very few controlled experiments have been carried out to investigate possible differences in influence on traits between markers. Here we examine the bi-allelic gene pantophysin (Pan I), widely used in the management of Atlantic cod, in a series of in vitro crosses under a range of temperatures. It has been proposed that this gene, or another tightly linked gene, may be under strong divergent selection. Resolving this issue is essential in order to interpret results when using this gene marker for stock management. We found no evidence of departure from the expected 1 : 2 : 1 Mendelian ratio for any of the three genotypes during the egg stage, while both the 6 and 12°C temperature regimes in tank experiments favoured the survival of the Pan IAA genotype. No difference in genotype survival was, however, found in a more natural mesocosm environment. Collectively, these results suggest that for the early life stages of Atlantic cod, and under the current experimental conditions, there is no strong consistent influence of Pan I genotype on survival. The results also emphasize the importance of varied experimental studies to verify the importance of environmental factors influencing genotype selection.

Boreal marine fauna from the Barents Sea disperse to Arctic Northeast Greenland.

As a result of ocean warming, the species composition of the Arctic seas has begun to shift in a boreal direction. One ecosystem prone to fauna shifts is the Northeast Greenland shelf. The dispersal route taken by boreal fauna to this area is, however, not known. This knowledge is essential to predict to what extent boreal biota will colonise Arctic habitats. Using population genetics, we show that Atlantic cod (Gadus morhua), beaked redfish (Sebastes mentella), and deep-sea shrimp (Pandalus borealis) recently found on the Northeast Greenland shelf originate from the Barents Sea, and suggest that pelagic offspring were dispersed via advection across the Fram Strait. Our results indicate that boreal invasions of Arctic habitats can be driven by advection, and that the fauna of the Barents Sea can project into adjacent habitats with the potential to colonise putatively isolated Arctic ecosystems such as Northeast Greenland.

Analysis of coastal cod (Gadus morhua L.) sampled on spawning sites reveals a genetic gradient throughout Norway's coastline.

BACKGROUND: Atlantic cod (Gadus morhua L.) has formed the basis of many economically significant fisheries in the North Atlantic, and is one of the best studied marine fishes, but a legacy of overexploitation has depleted populations and collapsed fisheries in several regions. Previous studies have identified considerable population genetic structure for Atlantic cod. However, within Norway, which is the country with the largest remaining catch in the Atlantic, the population genetic structure of coastal cod (NCC) along the entire coastline has not yet been investigated. We sampled > 4000 cod from 55 spawning sites. All fish were genotyped with 6 microsatellite markers and Pan I (Dataset 1). A sub-set of the samples (1295 fish from 17 locations) were also genotyped with an additional 9 microsatellites (Dataset 2). Otoliths were read in order to exclude North East Arctic Cod (NEAC) from the analyses, as and where appropriate. RESULTS: We found no difference in genetic diversity, measured as number of alleles, allelic richness, heterozygosity nor effective population sizes, in the north-south gradient. In both data sets, weak but significant population genetic structure was revealed (Dataset 1: global FST = 0.008, P < 0.0001. Dataset 2: global FST = 0.004, P < 0.0001). While no clear genetic groups were identified, genetic differentiation increased among geographically-distinct samples. Although the locus Gmo132 was identified as a candidate for positive selection, possibly through linkage with a genomic region under selection, overall trends remained when this locus was excluded from the analyses. The most common allele in loci Gmo132 and Gmo34 showed a marked frequency change in the north-south gradient, increasing towards the frequency observed in NEAC in the north. CONCLUSION: We conclude that Norwegian coastal cod displays significant population genetic structure throughout its entire range, that follows a trend of isolation by distance. Furthermore, we suggest that a gradient of genetic introgression between NEAC and NCC contributes to the observed population genetic structure. The current management regime for coastal cod in Norway, dividing it into two stocks at 62°N, represents a simplification of the level of genetic connectivity among coastal cod in Norway, and needs revision.

Geographic extent of introgression in Sebastes mentella and its effect on genetic population structure.

Genetic population structure is often used to identify management units in exploited species, but the extent of genetic differentiation may be inflated by geographic variation in the level of hybridization between species. We identify the genetic population structure of Sebastes mentella and investigate possible introgression within the genus by analyzing 13 microsatellites in 2,562 redfish specimens sampled throughout the North Atlantic. The data support an historical divergence between the "shallow" and "deep" groups, beyond the Irminger Sea where they were described previously. A third group, "slope," has an extended distribution on the East Greenland Shelf, in addition to earlier findings on the Icelandic slope. Furthermore, S. mentella from the Northeast Arctic and Northwest Atlantic waters are genetically different populations. In both areas, interspecific introgression may influence allele frequency differences among populations. Evidence of introgression was found for almost all the identified Sebastes gene pools, but to a much lower extent than suggested earlier. Greenland waters appear to be a sympatric zone for many of the genetically independent Sebastes groups. This study illustrates that the identified groups maintain their genetic integrity in this region despite introgression.

Genetically distinct populations of northern shrimp, Pandalus borealis, in the North Atlantic: adaptation to different temperatures as an isolation factor.

The large-scale population genetic structure of northern shrimp, Pandalus borealis, was investigated over the species' range in the North Atlantic, identifying multiple genetically distinct groups. Genetic divergence among sample localities varied among 10 microsatellite loci (range: FST = -0.0002 to 0.0475) with a highly significant average (FST = 0.0149; P < 0.0001). In contrast, little or no genetic differences were observed among temporal replicates from the same localities (FST = 0.0004; P = 0.33). Spatial genetic patterns were compared to geographic distances, patterns of larval drift obtained through oceanographic modelling, and temperature differences, within a multiple linear regression framework. The best-fit model included all three factors and explained approximately 29% of all spatial genetic divergence. However, geographic distance and larval drift alone had only minor effects (2.5-4.7%) on large-scale genetic differentiation patterns, whereas bottom temperature differences explained most (26%). Larval drift was found to promote genetic homogeneity in parts of the study area with strong currents, but appeared ineffective across large temperature gradients. These findings highlight the breakdown of gene flow in a species with a long pelagic larval phase (up to 3 months) and indicate a role for local adaptation to temperature conditions in promoting evolutionary diversification and speciation in the marine environment.

Simultaneous analysis of six microsatellite markers in Atlantic cod ( Gadus morhua): a novel multiplex assay system for use in selective breeding studies.

A novel hexaplex assay system including Gmo8, Gmo19, Gmo35, Gmo37, Tch11, and Tch12 microsatellites from Atlantic cod, consisting of trinucleotide or tetranucleotide repeat units, is introduced. All 6 loci were coamplified in a single reaction employing dye-labeled primers. Alleles from these loci were sized using an internal standard by automated sample processing in an ABI 310 Genetic Analyser. Amplified alleles in profiles containing selected microsatellites were typed clearly, providing easily interpretable results. Sequencing data indicated that alleles at all loci consisted of simple repeat units. This may help minimize the likelihood of stuttering upon polymerase chain reaction amplification. The results suggest that the presented hexaplex assay system may be a useful tool in a selective breeding program in which genetic identification will allow different genotypes to be reared together from fertilization. This should have a great impact as it will make selective breeding more efficient.