Genomic signatures and correlates of widespread population declines in salmon.

Global losses of biodiversity are occurring at an unprecedented rate, but causes are often unidentified. Genomic data provide an opportunity to isolate drivers of change and even predict future vulnerabilities. Atlantic salmon (Salmo salar) populations have declined range-wide, but factors responsible are poorly understood. Here, we reconstruct changes in effective population size (Ne) in recent decades for 172 range-wide populations using a linkage-based method. Across the North Atlantic, Ne has significantly declined in >60% of populations and declines are consistently temperature-associated. We identify significant polygenic associations with decline, involving genomic regions related to metabolic, developmental, and physiological processes. These regions exhibit changes in presumably adaptive diversity in declining populations consistent with contemporary shifts in body size and phenology. Genomic signatures of widespread population decline and associated risk scores allow direct and potentially predictive links between population fitness and genotype, highlighting the power of genomic resources to assess population vulnerability.

Trans-oceanic genomic divergence of Atlantic cod ecotypes is associated with large inversions.

Chromosomal rearrangements such as inversions can play a crucial role in maintaining polymorphism underlying complex traits and contribute to the process of speciation. In Atlantic cod (Gadus morhua), inversions of several megabases have been identified that dominate genomic differentiation between migratory and nonmigratory ecotypes in the Northeast Atlantic. Here, we show that the same genomic regions display elevated divergence and contribute to ecotype divergence in the Northwest Atlantic as well. The occurrence of these inversions on both sides of the Atlantic Ocean reveals a common evolutionary origin, predating the >100 000-year-old trans-Atlantic separation of Atlantic cod. The long-term persistence of these inversions indicates that they are maintained by selection, possibly facilitated by coevolution of genes underlying complex traits. Our data suggest that migratory behaviour is derived from more stationary, ancestral ecotypes. Overall, we identify several large genomic regions-each containing hundreds of genes-likely involved in the maintenance of genomic divergence in Atlantic cod on both sides of the Atlantic Ocean.

Barcoding Atlantic Canada's commonly encountered marine fishes.

Marine fishes from the northwest Atlantic Ocean were analysed to determine whether barcoding was effective at identifying species. Our data included 177 species, 136 genera, 81 families and 28 orders. Overall, 88% of nominal species formed monophyletic clusters based on >500 bp of the CO1 region, and the average bootstrap value for these species was 98%. Although clearly effective, the percentage of species that were distinguishable with barcoding based on the criterion of reciprocal monophyletic clusters was slightly lower than has been documented in other studies of marine fishes. Eelpouts, sculpins and rocklings proved to be among the most challenging groups for barcoding, although we suspect that difficult identifications based on traditional (morphology based) taxonomy played a role. Within several taxa, speciation may have occurred too recently for barcoding to be effective (e.g. within Sebastes, Thunnus and Ammodytes) or the designation of distinct species may have been erroneous (e.g. within Antimora and Macrourus). Results were consistent with previous work recognizing particularly high levels of divergence within certain taxa, some of which have been recognized as distinct species (e.g. Osmerus mordax and Osmerus dentex; and Liparis gibbus and Liparis bathyarcticus), and some of which have not (e.g. within Halargyreus johnsonii and within Mallotus villosus). The results from this study suggest that morphology-based identification and taxonomy can be challenging in marine fishes, even within a region as well characterized as Atlantic Canada. Barcoding proved to be a very useful tool for species identification that will likely find a wide range of applications, including the fisheries trade, studies of range expansion, ecological analyses and population assessments.

DNA barcoding of Canada's skates.

DNA-based identifications have been employed across broad taxonomic ranges and provide an especially useful tool in cases where external identification may be problematic. This study explored the utility of DNA barcoding in resolving skate species found in Atlantic Canadian waters. Most species were clearly resolved, expanding the utility for such identification on a taxonomically problematic group. Notably, one genus (Amblyraja) contained three of four species whose distributions do not overlap that could not be readily identified with this method. On the other hand, two common and partially sympatric species (Little and Winter skates) were readily identifiable. There were several instances of inconsistency between the voucher identification and the DNA sequence data. In some cases, these were at the intrageneric level among species acknowledged to be prone to misidentification. However, several instances of intergeneric discrepancies were also identified, suggesting either evidence of past introgressive hybridization or misidentification of vouchered specimens across broader taxonomic ranges. Such occurrences highlight the importance of retaining vouchered specimens for subsequent re-examination in the light of conflicting DNA evidence.

Structural and functional connectivity of marine fishes within a semi-enclosed Newfoundland fjord.

The interplay between structural connectivity (i.e. habitat continuity) and functional connectivity (i.e. dispersal probability) in marine fishes was examined in a coastal fjord (Holyrood Pond, Newfoundland, Canada) that is completely isolated from the North Atlantic Ocean for most of the year. Genetic differentiation was described in three species (rainbow smelt Osmerus mordax, white hake Urophycis tenuis and Atlantic cod Gadus morhua) with contrasting life histories using seven to 10 microsatellite loci and a protein-coding locus, PanI (G. morhua). Analysis of microsatellite differentiation indicated clear genetic differences between the fjord and coastal regions; however, the magnitude of difference was no more elevated than adjacent bays and was not enhanced by the fjord's isolation. Osmerus mordax was characterized by the highest structure overall with moderate differentiation between the fjord and St Mary's Bay (F(ST)c.0.047). In contrast, U. tenuis and G. morhua displayed weak differentiation (F(ST) < 0.01). Nonetheless, these populations did demonstrate high rates (< 75%) of Bayesian self-assignment. Furthermore, elevated differentiation was observed at the PanI locus in G. morhua between the fjord and other coastal locations. Interestingly, locus-specific genetic differentiation and expected heterozygosity were negatively associated in O. mordax, in contrast to the positive associations observed in U. tenuis and G. morhua. Gene flow in these species is apparently unencumbered by limited structural connectivity, yet the observed differentiation suggests that population structuring exists over small scales despite high dispersal potential.

Estimating contemporary early life-history dispersal in an estuarine fish: integrating molecular and otolith elemental approaches.

Dispersal during the early life history of the anadromous rainbow smelt, Osmerus mordax, was examined using assignment testing and mixture analysis of multilocus genotypes and otolith elemental composition. Six spawning areas and associated estuarine nurseries were sampled throughout southeastern Newfoundland. Samples of adults and juveniles isolated by > 25 km displayed moderate genetic differentiation (F(ST) ~ 0.05), whereas nearby (< 25 km) spawning and nursery samples displayed low differentiation (F(ST) < 0.01). Self-assignment and mixture analysis of adult spawning samples supported the hypothesis of independence of isolated spawning locations (> 80% self-assignment) with nearby runs self-assigning at rates between 50 % and 70%. Assignment and mixture analysis of juveniles using adult baselines indicated high local recruitment at several locations (70-90%). Nearby (< 25 km) estuaries at the head of St Mary's Bay showed mixtures of individuals (i.e. 20-40% assignment to adjacent spawning location). Laser ablation inductively coupled mass spectrometry transects across otoliths of spawning adults of unknown dispersal history were used to estimate dispersal among estuaries across the first year of life. Single-element trends and multivariate discriminant function analysis (Sr:Ca and Ba:Ca) classified the majority of samples as estuarine suggesting limited movement between estuaries (< 0.5%). The mixtures of juveniles evident in the genetic data at nearby sites and a lack of evidence of straying in the otolith data support a hypothesis of selective mortality of immigrants. If indeed selective mortality of immigrants reduces the survivorship of dispersers, estimates of dispersal in marine environments that neglect survival may significantly overestimate gene flow.

Inverse relationship between F and microsatellite polymorphism in the marine fish, walleye pollock (Theragra chalcogramma): implications for resolving weak population structure.

Microsatellites have proved to be useful for the detection of weak population structure in marine fishes and other species characterized by large populations and high gene flow. None the less, uncertainty remains about the net effects of the particular mutational properties of these markers, and the wide range of locus polymorphism they exhibit, on estimates of differentiation. We examined the effect of varying microsatellite polymorphism on the magnitude of observed differentiation in a population survey of walleye pollock, Theragra chalcogramma. Genetic differentiation at 14 microsatellite loci among six putative populations from across the North Pacific Ocean and Bering Sea was weak but significant on large geographical scales and conformed to an isolation-by-distance pattern. A negative relationship was found between locus variability and the magnitude of estimated population subdivision. Estimates of F(ST) declined with locus polymorphism, resulting in diminished power to discriminate among samples, and we attribute this loss to the effects of size homoplasy. This empirical result suggests that mutation rates of some microsatellite loci are sufficiently high to limit resolution of weak genetic structure typical of many marine fishes.

Evidence for positive selection at the pantophysin (Pan I) locus in walleye pollock, Theragra chalcogramma.

Nucleotide polymorphism at the pantophysin (Pan I) locus in walleye pollock, Theragra chalcogramma, was examined using DNA sequence data. Two distinct allelic lineages were detected in pollock, resulting from three amino acid replacement mutations in the first intravesicular domain of the protein. The common Pan I allelic group, comprising 94% of the samples, was less polymorphic (pi = 0.005) than the uncommon group (pi = 0.008), and nucleotide diversity in both was higher than for two allelic lineages in the related Atlantic cod, Gadus morhua. Phylogenetic analyses of Pan I sequences from these two species did not clearly resolve orthology among allelic groups, in part because of recombination that has occurred between the two pollock lineages. Conventional tests of neutrality comparing polymorphisms within and between homologous regions of the Pan I locus in walleye pollock and Atlantic cod did not detect the effects of selection. This result is likely attributed to low levels of synonymous divergence among allelic lineages and a lack of mutation-drift equilibrium inferred from nucleotide mismatch frequency distributions. However, the ratio of nonsynonymous to synonymous substitutions per site (dN/dS) exceeded unity in two intravesicular domains of the protein and the influence of positive selection at multiple codon sites was strongly inferred through the use of maximum-likelihood analyses. In addition, the frequency spectrum of linked neutral variation showed indirect effects of adaptive hitchhiking in pollock resulting from a selective sweep of the common allelic lineage. Recombination between the two allelic classes may have prevented complete loss of the older, more polymorphic lineage. The results suggest that recurrent sweeps driven by positive selection is the principle mode of evolution at the Pan I locus in gadid fishes.

A consolidated linkage map for rainbow trout (Oncorhynchus mykiss).

Androgenetic doubled haploid progeny produced from a cross between the Oregon State University and Arlee clonal rainbow trout (Oncorhynchus mykiss) lines, used for a previous published rainbow trout map, were used to update the map with the addition of more amplified fragment length polymorphic (AFLP) markers, microsatellites, type I and allozyme markers. We have added more than 900 markers, bringing the total number to 1359 genetic markers and the sex phenotype including 799 EcoRI AFLPs, 174 PstI AFLPs, 226 microsatellites, 72 VNTR, 38 SINE markers, 29 known genes, 12 minisatellites, five RAPDs, and four allozymes. Thirty major linkage groups were identified. Synteny of linkage groups in our map with the outcrossed microsatellite map has been established for all except one linkage group in this doubled haploid cross. Putative homeologous relationships among linkage groups, resulting from the autotetraploid nature of the salmonid genome, have been revealed based on the placement of duplicated microsatellites and type I loci.

Reconstructing recent divergence: evaluating nonequilibrium population structure in New Zealand chinook salmon.

Newly established or perturbed populations are often the focus of conservation concerns but they pose special challenges for population genetics because drift-migration equilibrium is unlikely. To advance our understanding of the evolution of such populations, we investigated structure and gene flow among populations of chinook salmon that formed via natural straying following introduction to New Zealand in the early 1900s. We examined 11 microsatellite loci from samples collected in several sites and years to address two questions: (i) what population differentiation has arisen in the approximately 30 generations since salmon were introduced to New Zealand, relative to temporal variation within populations; and (ii) what are the approximate effective population sizes and amounts of gene flow in these populations? These questions are routinely addressed in studies of indigenous populations, but less often in the case of new populations and rarely with consideration of equilibrium assumptions. We show that despite the recent introduction, continued gene flow and high temporal variability among samples, detectable population structure has arisen among the New Zealand populations, consistent with their colonization pattern and isolation by geographical distance. Furthermore, we use simple individual-based simulations and estimates of effective population sizes to estimate the effective gene flow among drainages under likely nonequilibrium conditions. Similar methodology may be broadly applicable to other studies of population structure and phenotypic evolution under similar nonequilibrium, high gene flow conditions.

Allozyme and microsatellite loci provide discordant estimates of population differentiation in the endangered dusky grouper (Epinephelus marginatus) within the Mediterranean Sea.

The dusky grouper, Epinephelus marginatus, inhabits coastal reefs in the Mediterranean Sea and Atlantic Ocean. A decline in the abundance of this long-lived protogynous hermaphrodite has led to its listing as an endangered species in the Mediterranean, and heightened management concerns regarding its genetic variability and population substructure. To address these concerns, we analysed genetic variation at seven microsatellite and 28 allozyme loci in dusky groupers sampled from seven areas (for microsatellites) and three areas (for allozymes) in the west-central Mediterranean. Levels of genetic variability were higher for microsatellites than for allozymes (mean H(E) = 0.78 and 0.07, respectively), but similar to those observed in other marine fishes with comparable markers. Both microsatellites and allozymes revealed significant genetic differentiation among all areas analysed with each class of marker, but the magnitude of differentiation revealed by allozymes over three locales (F(ST) = 0.214) was greater than that detected with microsatellites over seven areas, or over the three areas shared with the allozyme analysis (F(ST) = 0.018 and approximately 0, respectively). A large proportion of the allozyme differentiation was due to a single locus (ADA*) possibly influenced by selection, but allozyme differentiation over the three areas was still highly significant (F(ST) = 0.06, P < 0.0001), and the 95% confidence intervals for allozyme and microsatellite F(ST) did not overlap when this locus was excluded. There was no evidence of isolation by distance with either class of markers. Our results lead us to conclude that dusky groupers are not panmictic in the Mediterranean Sea and suggest that they should be managed on a local basis. However, more work is needed to elucidate genetic relationships among populations.

The aunt and uncle effect: an empirical evaluation of the confounding influence of full sibs of parents on pedigree reconstruction.

This study used simulations and a known two-generation pedigree of chinook salmon (Oncorhynchus tshawytscha) to evaluate the effect of full sibs of parents on pedigree reconstruction. Parentage analysis was conducted on 100 parent pair-offspring relationships from pedigrees with unrelated (simulation) and related (chinook salmon) candidate parents. Parentage assignment success for the chinook salmon was lower than in the simulated populations. For example, the six most variable loci (mean H(E) = 0.87) provided a mean of 97% unambiguous assignments in the simulated population and 67% unambiguous assignments for the chinook salmon. Estimates of the pairwise relatedness coefficient ((xy)) for most nonexcluded false parents and true parents of chinook salmon offspring exceeded 0.50. These results support the conclusion that closely related candidate parents decrease the power of genetic markers for pedigree reconstruction based on exclusion. Ambiguous parentage may be resolved using single parent- and parent pair-offspring likelihood analysis, however, these methods should be used with caution and they are not replacements for using more loci when many candidate parents are full sibs.

Kinship analysis of Pacific salmon: insights into mating, homing, and timing of reproduction.

Multilocus microsatellite genotypes were used to infer kinship and relatedness in two species of Pacific salmon from three populations in Washington State. Even in the absence of direct genetic data from parents, clustering of individuals according to allele sharing and reconstruction of parental genotypes allowed resolution of full- and half-sib relationships among 135 chinook salmon (Oncorhynchus tshawytscha) sampled as preemergent juveniles from 14 redds in the Dungeness River. Inferred reproductive behaviors included single-pair matings, polyandry in which females mated with two to three males at a single redd, polygyny in which males mated with two females at different redds, use of two redds by a single female, and use of one redd site by two females. Greater average relatedness (rxy) in the upper reach of the Dungeness River implied within-reach homing of returning adults. In steelhead trout (O. mykiss), the frequency of related pairs (dyads) of mature individuals that migrated up Snow Creek less than a week apart was greater than expected for randomly chosen dyads, as was the frequency of steelhead dyads that were spawned on the same day in the Forks Creek hatchery. These results imply a heritable basis for upstream migration date and maturation date in steelhead trout.

Rapid evolution of reproductive isolation in the wild: evidence from introduced salmon.

Colonization of new environments should promote rapid speciation as a by-product of adaptation to divergent selective regimes. Although this process of ecological speciation is known to have occurred over millennia or centuries, nothing is known about how quickly reproductive isolation actually evolves when new environments are first colonized. Using DNA microsatellites, population-specific natural tags, and phenotypic variation, we tested for reproductive isolation between two adjacent salmon populations of a common ancestry that colonized divergent reproductive environments (a river and a lake beach). We found evidence for the evolution of reproductive isolation after fewer than 13 generations.

Isolation and Characterization of Twelve Microsatellite Loci for Rockfish (Sebastes).

: We describe the first microsatellites for rockfishes in the diverse genus Sebastes. Clones containing microsatellites were isolated from the genomic library of a quillback rockfish, Sebastes maliger. Twelve microsatellites are characterized; six of these are polymorphic in quillback rockfish, and eight are polymorphic in at least one rockfish species on which they were tested. The number of alleles per variable locus ranged from 4 to 15 and averaged 6.8. The expected heterozygosities ranged from 0.38 to 0.79 and averaged 0.60 in these loci. These loci should prove valuable in studies examining species identification, population genetics, hybridization, paternity, kinship, and microsatellite evolution.

Tandem repeat polymorphism and heteroplasmy in the mitochondrial control region of redfishes (Sebastes: Scorpaenidae).

Three species of redfish (Sebastes) share a common pattern of mitochondrial DNA tandem repeat polymorphism and heteroplasmy in the northwest Atlantic Ocean. All three species exhibit 9-17 copies of an approximately 275 base pair (bp) tandem repeat situated within the 3' domain of the control region. Sequence analysis of cloned mtDNA from S. mentella revealed that the tandem array is adjacent to the tRNA(phe) gene, and that the repeat shares 53% identity with the tRNA(phe) gene and part of the 12S rRNA gene. These features, as well as potential secondary structure assumed by the repeat, are consistent with previously proposed models explaining tandem duplications in the 3' end of the control region. In a sample comprising 36 S. fasciatus, 52 S. mentella, and 13 S. marinus taken near Newfoundland, neither the mean number of repeats per fish (12.2-12.7) nor the frequency of heteroplasmy varied significantly among species. A total of 42% of the redfishes were heteroplasmic, bearing either two or three repeat variants (33% and 9%, respectively). The similarity of the frequency distributions of tandem repeat variants in the three species suggests either a common balance between mutation and selection in the three species, or mitochondrial gene flow between them.