Despite the enormous economic and ecological importance of marine organisms, the spatial scales of adaptation and biocomplexity remain largely unknown. Yet, the preservation of local stocks that possess adaptive diversity is critical to the long-term maintenance of productive stable fisheries and ecosystems. Here, we document genomic evidence of range-wide adaptive differentiation in a broadcast spawning marine fish, Atlantic cod (Gadus morhua), using a genome survey of single nucleotide polymorphisms. Of 1641 gene-associated polymorphisms examined, 70 (4.2%) tested positive for signatures of selection using a Bayesian approach. We identify a subset of these loci (n ¼ 40) for which allele frequencies show parallel temperature-associated clines (p , 0.001, r 2 ¼ 0.89) in the eastern and western north Atlantic. Temperature associations were robust to the statistical removal of geographic distance or latitude effects, and contrasted 'neutral' loci, which displayed no temperature association. Allele frequencies at temperature-associated loci were significantly correlated, spanned three linkage groups and several were successfully annotated supporting the involvement of multiple independent genes. Our results are consistent with the evolution and/or selective sweep of multiple genes in response to ocean temperature, and support the possibility of a new conservation paradigm for non-model marine organisms based on genomic approaches to resolving functional and adaptive diversity.

We address the bioinformatic issue of accurately separating amplified genes of the major histocompatibility complex (MHC) from artefacts generated during high-throughput sequencing workflows. We fit observed ultra-deep sequencing depths (hundreds to thousands of sequences per amplicon) of allelic variants to expectations from genetic models of copy number variation (CNV). We provide a simple, accurate and repeatable method for genotyping multigene families, evaluating our method via analyses of 209 b of MHC class IIb exon 2 in guppies (Poecilia reticulata). Genotype repeatability for resequenced individuals (N = 49) was high (100%) within the same sequencing run. However, repeatability dropped to 83.7% between independent runs, either because of lower mean amplicon sequencing depth in the initial run or random PCR effects. This highlights the importance of fully independent replicates. Significant improvements in genotyping accuracy were made by greatly reducing type I genotyping error (i.e. accepting an artefact as a true allele), which may occur when using low-depth allele validation thresholds used by previous methods. Only a small amount (4.9%) of type II error (i.e. rejecting a genuine allele as an artefact) was detected through fully independent sequencing runs. We observed 1-6 alleles per individual, and evidence of sharing of alleles across loci. Variation in the total number of MHC class II loci among individuals, both among and within populations was also observed, and some genotypes appeared to be partially hemizygous; total allelic dosage added up to an odd number of allelic copies. Collectively, observations provide evidence of MHC CNV and its complex basis in natural populations.

As populations diverge, genomic regions associated with adaptation display elevated differentiation. These genomic islands of adaptive divergence can inform conservation efforts in exploited species, by refining the delineation of management units, and providing genomic tools for more precise and effective population monitoring and the successful assignment of individuals and products. We explored heterogeneity in genomic divergence and its impact on the resolution of spatial population structure in exploited populations of Atlantic cod, Gadus morhua, using genome wide expressed sequence derived single nucleotide polymorphisms in 466 individuals sampled across the range. Outlier tests identified elevated divergence at 5.2% of SNPs, consistent with directional selection in onethird of linkage groups. Genomic regions of elevated divergence ranged in size from a single position to several cM. Structuring at neutral loci was associated with geographic features, whereas outlier SNPs revealed genetic discontinuities in both the eastern and western Atlantic. This fine-scale geographic differentiation enhanced assignment to region of origin, and through the identification of adaptive diversity, fundamentally changes how these populations should be conserved. This work demonstrates the utility of genome scans for adaptive divergence in the delineation of stock structure, the traceability of individuals and products, and ultimately a role for population genomics in fisheries conservation.

MEGASAT is software that enables genotyping of microsatellite loci using next-generation sequencing data. Microsatellites are amplified in large multiplexes, and then sequenced in pooled amplicons. MEGASAT reads sequence files and automatically scores microsatellite genotypes. It uses fuzzy matches to allow for sequencing errors and applies decision rules to account for amplification artefacts, including nontarget amplification products, replication slippage during PCR (amplification stutter) and differential amplification of alleles. An important feature of MEGASAT is the generation of histograms of the length-frequency distributions of amplification products for each locus and each individual. These histograms, analogous to electropherograms traditionally used to score microsatellite genotypes, enable rapid evaluation and editing of automatically scored genotypes. MEGASAT is written in Perl, runs on Windows, Mac OS X and Linux systems, and includes a simple graphical user interface. We demonstrate MEGASAT using data from guppy, Poecilia reticulata. We genotype 1024 guppies at 43 microsatellites per run on an Illumina MiSeq sequencer. We evaluated the accuracy of automatically called genotypes using two methods, based on pedigree and repeat genotyping data, and obtained estimates of mean genotyping error rates of 0.021 and 0.012. In both estimates, three loci accounted for a disproportionate fraction of genotyping errors; conversely, 26 loci were scored with 0-1 detected error (error rate ≤0.007). Our results show that with appropriate selection of loci, automated genotyping of microsatellite loci can be achieved with very high throughput, low genotyping error and very low genotyping costs.

Individual assignment and genetic mixture analysis are commonly utilized in contemporary wildlife and fisheries management. Although microsatellite loci provide unparalleled numbers of alleles per locus, their use in assignment applications is increasingly limited. However, next‐generation sequencing, in conjunction with novel bioinformatic tools, allows large numbers of microsatellite loci to be simultaneously genotyped, presenting new opportunities for individual assignment and genetic mixture analysis. Here, we scanned the published Atlantic salmon genome to identify 706 microsatellite loci, from which we developed a final panel of 101 microsatellites distributed across the genome (average 3.4 loci per chromosome). Using samples from 35 Atlantic salmon populations (n = 1,485 individuals) from coastal Labrador, Canada, a region characterized by low levels of differentiation in this species, this panel identified 844 alleles (average of 8.4 alleles per locus). Simulation‐based evaluations of assignment and mixture identification accuracy revealed unprecedented resolution, clearly identifying 26 rivers or groups of rivers spanning 500 km of coastline. This baseline was used to examine the stock composition of 696 individuals harvested in the Labrador Atlantic salmon fishery and revealed that coastal fisheries largely targeted regional groups (<300 km). This work suggests that the development and application of large sequenced microsatellite panels presents great potential for stock resolution in Atlantic salmon and more broadly in other exploited anadromous and marine species.