BackgroundAnadromous migratory fish species such as Atlantic salmon (Salmo salar) have significant economic, cultural and ecological importance, but present a complex case for management and conservation due to the range of their migration. Atlantic salmon exist in rivers across the North Atlantic, returning to their river of birth with a high degree of accuracy; however, despite continuing efforts and improvements in in-river conservation, they are in steep decline across their range. Salmon from rivers across Europe migrate along similar routes, where they have, historically, been subject to commercial netting. This mixed stock exploitation has the potential to devastate weak and declining populations where they are exploited indiscriminately. Despite various tagging and marking studies, the effect of marine exploitation and the marine element of the salmon lifecycle in general, remain the "black-box" of salmon management. In a number of Pacific salmonid species and in several regions within the range of the Atlantic salmon, genetic stock identification and mixed stock analysis have been used successfully to quantify exploitation rates and identify the natal origins of fish outside their home waters - to date this has not been attempted for Atlantic salmon in the south of their European range.ResultsTo facilitate mixed stock analysis (MSA) of Atlantic salmon, we have produced a baseline of genetic data for salmon populations originating from the largest rivers from Spain to northern Scotland, a region in which declines have been particularly marked. Using 12 microsatellites, 3,730 individual fish from 57 river catchments have been genotyped. Detailed patterns of population genetic diversity of Atlantic salmon at a sub-continent-wide level have been evaluated, demonstrating the existence of regional genetic signatures. Critically, these appear to be independent of more commonly recognised terrestrial biogeographical and political boundaries, allowing reporting regions to be defined. The implications of these results on the accuracy of MSA are evaluated and indicate that the success of MSA is not uniform across the range studied; our findings indicate large differences in the relative accuracy of stock composition estimates and MSA apportioning across the geographical range of the study, with a much higher degree of accuracy achieved when assigning and apportioning to populations in the south of the area studied. This result probably reflects the more genetically distinct nature of populations in the database from Spain, northwest France and southern England. Genetic stock identification has been undertaken and validation of the baseline microsatellite dataset with rod-and-line and estuary net fisheries of known origin has produced realistic estimates of stock composition at a regional scale.ConclusionsThis southern European database and supporting phylogeographic and mixed-stock analyses of net samples provide a unique tool for Atlantic salmon research and management, in both their natal rivers and the marine en...

Microsatellite genotyping is a common DNA characterization technique in population, ecological and evolutionary genetics research. Since different alleles are sized relative to internal size-standards, different laboratories must calibrate and standardize allelic designations when exchanging data. This interchange of microsatellite data can often prove problematic. Here, 16 microsatellite loci were calibrated and standardized for the Atlantic salmon, Salmo salar, across 12 laboratories. Although inconsistencies were observed, particularly due to differences between migration of DNA fragments and actual allelic size (‘size shifts’), inter-laboratory calibration was successful. Standardization also allowed an assessment of the degree and partitioning of genotyping error. Notably, the global allelic error rate was reduced from 0.05 ± 0.01 prior to calibration to 0.01 ± 0.002 post-calibration. Most errors were found to occur during analysis (i.e. when size-calling alleles; the mean proportion of all errors that were analytical errors across loci was 0.58 after calibration). No evidence was found of an association between the degree of error and allelic size range of a locus, number of alleles, nor repeat type, nor was there evidence that genotyping errors were more prevalent when a laboratory analyzed samples outside of the usual geographic area they encounter. The microsatellite calibration between laboratories presented here will be especially important for genetic assignment of marine-caught Atlantic salmon, enabling analysis of marine mortality, a major factor in the observed declines of this highly valued species.Electronic supplementary materialThe online version of this article (doi:10.1007/s10709-011-9554-4) contains supplementary material, which is available to authorized users.

The identification of localized discrete populations is particularly important to the management and conservation of animal species, especially in the marine environment, where potential for dispersal is high but barriers to gene flow are often not clear. We investigated population genetic structuring of bottlenose dolphins Tursiops truncatus found along the west coast of Ireland, with particular attention to the Shannon Estuary, which is the only candidate Special Area of Conservation designated for this species in Irish waters. A genetic structure analysis using 62 biopsy samples from free ranging dolphins and 23 necropsies from stranded dolphins revealed fine-scale population structure among three distinct populations. The Shannon Estuary population appears to be genetically isolated from adjacent coastal areas, with the exception of four animals sampled from a small group of six dolphins that are now resident in Cork Harbour (south coast) indicating ongoing gene flow or recent dispersal between these two areas. A second genetically distinct aggregation was identified in the Connemara-Mayo region, where recent photoidentification studies have suggested that dolphins found in this area show a degree of site fidelity. We found moderate nuclear (15 microsatellites) and low mitochondrial (544 bp of the control region) gene diversity in dolphins using the Shannon Estuary and the Connemara-Mayo region, while dolphins that stranded along the coast showed markedly higher levels of gene diversity at both classes of markers. Specifically, these stranded dolphins formed a third genetically distinct cluster, which may be part of a larger pelagic population, as also suggested by the high levels of gene diversity. These results provide new insights into population structure of bottlenose dolphins in Irish waters and will aid future management and conservation of the species in the eastern North Atlantic.

Previous genetic studies using neutral markers such as allozymes, mtDNA and minisatellite loci have demonstrated varying amounts of population structure in cod Gadus morhua throughout the Atlantic. Microsatellite loci, which are potentially the most informative of presently available neutral genetic markers, have been applied extensively within western and eastern Atlantic areas but not on a range-wide basis. In the present study, six microsatellite DNA loci were used to screen cod samples from nine locations throughout the geographic range from the Scotian Shelf in the West Atlantic to the Barents and Baltic Seas in the east. Overall F ST value was 0Á03 (P ¼ < 0Á001) across all samples. Statistically significant population differences over all loci combined were evident between more geographically distant samples, using either heterogeneity tests or F ST analysis, with at least one locus showing significant differences between all samples (prior to Bonferroni correction). A significant correlation was observed between genetic and geographical distance, suggesting a higher level of historical and contemporary gene flow between adjacent populations than more distant populations. Samples from either end of the geographic range (Scotian Shelf and Baltic Sea) were particularly distinct when analysed using the STRUCTURE programme and also showed a high level of selfassignment when individuals of either the Scotian Shelf or Baltic Sea were tested against the entire data set. The present microsatellite study demonstrates a high level of geographic population structure between the western Atlantic, middle and eastern Atlantic and Baltic Sea, and thus, the findings should be useful in devising overall management and conservation strategies for the species.

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While environmental DNA (eDNA) is becoming increasingly established in biodiversity monitoring of freshwater ecosystems, the use of eDNA surveys in the marine environment is still in its infancy. Here, we use two approaches: targeted quantitative PCR (qPCR) and whole-genome enrichment capture followed by shotgun sequencing in an effort to amplify killer whale DNA from seawater samples. Samples were collected in close proximity to killer whales in inshore and offshore waters, in varying sea conditions and from the surface and subsurface but none returned strongly positive detections of killer whale eDNA. We validated our laboratory methodologies by successfully amplifying a dilution series of a positive control of killer whale DNA. Furthermore, DNA of Atlantic mackerel, which was present at all sites during sampling, was successfully amplified from the same seawater samples, with positive detections found in ten of the eighteen eDNA extracts. We discuss the various eDNA collection and amplification methodologies used and the abiotic and biotic factors that influence eDNA detection. We discuss possible explanations for the lack of positive killer whale detections, potential pitfalls, and the apparent limitations of eDNA for genetic research on cetaceans, particularly in offshore regions. K E Y W O R D S eDNA, environmental DNA, metagenomics, Orcinus orca, PCR, Scomber scombrus, wholegenome enrichment | 317 PINFIELD Et aL. S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article: Pinfield R, Dillane E, Runge AKW, et al. False-negative detections from environmental DNA collected in the presence of large numbers of killer whales (Orcinus orca).

Several studies have documented the genetic effects of intraspecific hybridization of cultured and wild Atlantic salmon (Salmo salar L.). However, the effect of salmon aquaculture on wild congeners is not so well understood. Diseases, introduced or increased in incidence by salmon aquaculture activities, may have an impact on co-occurring wild sea trout (Salmo trutta L.), as implied by the steep decline in sea trout numbers in many Irish, Scottish, and Norwegian rivers since the late 1980s, which may be linked to sea lice infestations associated with marine salmonid farming. Our data suggest that salmon farming and ocean ranching can indirectly affect, most likely mediated by disease, the genetics of cohabiting sea trout by reducing variability at major histocompatibility class I genes. We studied samples of DNA extracted from scales of sea trout in the Burrishoole River, in the west of Ireland, before and at intervals during aquaculture activities. In these samples, allelic variation at a microsatellite marker, tightly linked to a locus critical to immune response (Satr-UBA), was compared with variation at six neutral microsatellite loci. A significant decline in allelic richness and gene diversity at the Satr-UBA marker locus, observed since aquaculture started and which may indicate a selective response, was not reflected by similar reductions at neutral loci. Subsequent recovery of variability at the Satr-UBA marker, seen among later samples, may reflect an increased contribution by resident brown trout to the remaining sea trout stock.