Recolonization history and large-scale dispersal in the open sea: the case study of the North Atlantic cod, Gadus morhua L.

Most studies of the genetic structure of Atlantic cod have focused on small geographical scales. In the present study, the genetic structure of cod sampled on spawning grounds in the North Atlantic was examined using eight microsatellite loci and the Pan I locus. A total of 954 cod was collected from nine different regions: the Baltic Sea, the North Sea, the Celtic Sea, the Irish Sea and Icelandic waters during spring 2002 and spring 2003, from Norwegian waters and the Faroe Islands (North and West spawning grounds) in spring 2003, and from Canadian waters in 1998. Temporal stability among spawning grounds was observed in Icelandic waters and the Celtic Sea, and no significant difference was observed between the samples from the Baltic Sea and between the samples from Faroese waters. F -statistics showed significant differences between most populations and a pattern of isolation-by-distance was described with microsatellite loci. The Pan I locus revealed the presence of two genetically distinguishable basins, the North-west Atlantic composed of the Icelandic and Canadian samples and the North-east Atlantic composed of all other samples. Permutation of allele sizes at each microsatellite locus among allelic states supported a mutational component to the genetic differentiation, indicating a historical origin of the observed variation. Estimation of the time of divergence was approximately 3000 generations, which places the origin of current genetic pattern of cod in the North Atlantic in the late Weichselian (Wisconsinian period), at last glacial maximum.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 315–329.     

Intraspecific variation in expression of candidate genes for osmoregulation, heme biosynthesis and stress resistance suggests local adaptation in European flounder (Platichthys flesus)

Despite the recent discovery of significant genetic structuring in a large number of marine organisms, the evolutionary significance of these often minute genetic differences are still poorly understood. To elucidate the adaptive relevance of low genetic differentiation among marine fish populations, we studied expression differences of osmoregulatory and stress genes in genetically weakly differentiated populations of the European flounder (Platichthys flesus), distributed across a natural salinity gradient. Flounders were maintained in a long-term reciprocal transplantation experiment mimicking natural salinities in the North Sea and the Baltic Sea. Applying real-time quantitative PCR and microarray analysis we studied expression of four candidate genes (hsp70, angiotensinogen, Na/K-ATPase-alpha and 5-aminolevulinic acid synthase (ALAS)) in gill, kidney and liver tissues. Genes involved in osmoregulative processes (Na/K-ATPases-alpha and angiotensinogen) showed highly plastic but similar expression in the two populations dependent on environmental salinity. However, we observed a unique sixfold up-regulation of hsp70 in kidney tissue of flounder from the North Sea following long-term acclimation to Baltic salinities. Similarly, significant differences between North Sea and Baltic flounders in expression of ALAS in response to different salinities were found in gill and liver tissue. These findings strongly suggest that gene expression in flounders is shaped by adaptation to local environmental conditions. This identification of adaptive differences in high gene flow marine organisms adds a new dimension to our current understanding of evolutionary processes in the sea and is of paramount importance for identification, protection and sustainable management of marine biodiversity.     

Disentangling structural genomic and behavioural barriers in a sea of connectivity

Genetic divergence among populations arises through natural selection or drift and is counteracted by connectivity and gene flow. In sympatric populations, isolating mechanisms are thus needed to limit the homogenizing effects of gene flow to allow for adaptation and speciation. Chromosomal inversions act as an important mechanism maintaining isolating barriers, yet their role in sympatric populations and divergence with gene flow is not entirely understood. Here, we revisit the question of whether inversions play a role in the divergence of connected populations of the marine fish Atlantic cod (Gadus morhua), by exploring a unique data set combining whole‐genome sequencing data and behavioural data obtained with acoustic telemetry. Within a confined fjord environment, we find three genetically differentiated Atlantic cod types belonging to the oceanic North Sea population, the western Baltic population and a local fjord‐type cod. Continuous behavioural tracking over 4 year revealed temporally stable sympatry of these types within the fjord. Despite overall weak genetic differentiation consistent with high levels of gene flow, we detected significant frequency shifts of three previously identified inversions, indicating an adaptive barrier to gene flow. In addition, behavioural data indicated that North Sea cod and individuals homozygous for the LG12 inversion had lower fitness in the fjord environment. However, North Sea and fjord‐type cod also occupy different depths, possibly contributing to prezygotic reproductive isolation and representing a behavioural barrier to gene flow. Our results provide the first insights into a complex interplay of genomic and behavioural isolating barriers in Atlantic cod and establish a new model system towards an understanding of the role of genomic structural variants in adaptation and diversification.     

Environmental correlates of population differentiation in Atlantic herring

The marine environment is characterized by few physical barriers, and pelagic fishes commonly show high migratory potential and low, albeit in some cases statistically significant, levels of genetic divergence in neutral genetic marker analyses. However, it is not clear whether low levels of differentiation reflect spatially separated populations experiencing gene flow or shallow population histories coupled with limited random genetic drift in large, demographically isolated populations undergoing independent evolutionary processes. Using information for nine microsatellite loci in a total of 1951 fish, we analyzed genetic differentiation among Atlantic herring from eleven spawning locations distributed along a longitudinal gradient from the North Sea to the Western Baltic. Overall genetic differentiation was low (theta = 0.008) but statistically significant. The area is characterized by a dramatic shift in hydrography from the highly saline and temperature stable North Sea to the brackish Baltic Sea, where temperatures show high annual variation. We used two different methods, a novel computational geometric approach and partial Mantel correlation analysis coupled with detailed environmental information from spawning locations to show that patterns of reproductive isolation covaried with salinity differences among spawning locations, independent of their geographical distance. We show that reproductive isolation can be maintained in marine fish populations exhibiting substantial mixing during larval and adult life stages. Analyses incorporating genetic, spatial, and environmental parameters indicated that isolating mechanisms are associated with the specific salinity conditions on spawning locations.     

Adaptive differences in gene expression in European flounder (Platichthys flesus)

Population structure was previously believed to be very limited or absent in classical marine fishes, but recently, evidence of weakly differentiated local populations has been accumulating using noncoding microsatellite markers. However, the evolutionary significance of such minute genetic differences remains unknown. Therefore, in order to elucidate the relationship between genetic markers and adaptive divergence among populations of marine fishes, we combined cDNA microarray and microsatellite analysis in European flounders (Platichthys flesus). We demonstrate that despite extremely low levels of neutral genetic divergence, a high number of genes were significantly differentially expressed between North Sea and Baltic Sea flounders maintained in a long-term reciprocal transplantation experiment mimicking natural salinities. Several of the differentially regulated genes could be directly linked to fitness traits. These findings demonstrate that flounders, despite little neutral genetic divergence between populations, are differently adapted to local environmental conditions and imply that adaptation in gene expression could be common in other marine organisms with similar low levels of population subdivision.     

Local adaptation and oceanographic connectivity patterns explain genetic differentiation of a marine diatom across the North Sea–Baltic Sea salinity gradient

Drivers of population genetic structure are still poorly understood in marine micro‐organisms. We exploited the North Sea–Baltic Sea transition for investigating the seascape genetics of a marine diatom, Skeletonema marinoi. Eight polymorphic microsatellite loci were analysed in 354 individuals from ten locations to analyse population structure of the species along a 1500‐km‐long salinity gradient ranging from 3 to 30 psu. To test for salinity adaptation, salinity reaction norms were determined for sets of strains originating from three different salinity regimes of the gradient. Modelled oceanographic connectivity was compared to directional relative migration by correlation analyses to examine oceanographic drivers. Population genetic analyses showed distinct genetic divergence of a low‐salinity Baltic Sea population and a high‐salinity North Sea population, coinciding with the most evident physical dispersal barrier in the area, the Danish Straits. Baltic Sea populations displayed reduced genetic diversity compared to North Sea populations. Growth optima of low salinity isolates were significantly lower than those of strains from higher native salinities, indicating local salinity adaptation. Although the North Sea–Baltic Sea transition was identified as a barrier to gene flow, migration between Baltic Sea and North Sea populations occurred. However, the presence of differentiated neutral markers on each side of the transition zone suggests that migrants are maladapted. It is concluded that local salinity adaptation, supported by oceanographic connectivity patterns creating an asymmetric migration pattern between the Baltic Sea and the North Sea, determines genetic differentiation patterns in the transition zone.     

Genetic population structure of turbot (Scophthalmus maximus L.) supports the presence of multiple hybrid zones for marine fishes in the transition zone between the Baltic Sea and the North Sea

Genetic population structure of turbot (Scophthalmus maximus L.) in the Northeast Atlantic was investigated using eight highly variable microsatellite loci. In total 706 individuals from eight locations with temporal replicates were assayed, covering an area from the French Bay of Biscay to the Aaland archipelago in the Baltic Sea. In contrast to previous genetic studies of turbot, we found significant genetic differentiation among samples with a maximum pairwise FST of 0.032. Limited or no genetic differentiation was found among samples within the Atlantic/North Sea area and within the Baltic Sea, suggesting high gene flow among populations in these areas. In contrast, there was a sharp cline in genetic differentiation going from the low saline Baltic Sea to the high saline North Sea. The data were explained best by two divergent populations connected by a hybrid zone; however, a mechanical mixing model could not be ruled out. A significant part of the genetic variance could be ascribed to variation among years within locality. Nevertheless, the population structure was relatively stable over time, suggesting that the observed pattern of genetic differentiation is biologically significant. This study suggests that hybrid zones are a common phenomenon for marine fishes in the transition area between the North Sea and the Baltic Sea and highlights the importance of using interspecific comparisons for inferring population structure in high gene flow species such as most marine fishes.     

Salinity gradient shapes distance decay of similarity among parasite communities in three marine fishes

Published data were used to compare the distance decay of similarity in parasite communities of three marine fish hosts: Atlantic cod Gadus morhua, the dab Limanda limanda and the flounder Platichthys flesus in two adjacent areas that differ with respect to the strength of a salinity gradient. In the Baltic Sea, which exhibits a strong salinity gradient from its connection with the North Sea in the west to its head in the north‐east, parasite communities in all three fish hosts showed a significant decline of similarity with increasing distance. In contrast, among host populations in the North Sea, which is a fully marine environment, there was no such decline or only a weak relationship. The results suggest that environmental gradients like salinity can be strong driving forces behind patterns of distance decay in parasite communities of fishes.     

Tissue specific haemoglobin gene expression suggests adaptation to local marine conditions in North Sea flounder (Platichthys flesus L.)

Recent genetic analyses of candidate genes and gene expression in marine fishes have provided evidence of local adaptation in response to environmental differences, despite the lack of strong signals of population structure from conventional neutral genetic markers. In this study expression of the haemoglobin alpha and beta subunit genes was studied in reciprocally transplanted European flounder Platichthys flesus from the highly saline North Sea and the brackish Baltic Sea. Clear differences in expression patterns of haemoglobin alpha and beta subunit genes were found among different types of tissue in flounder. In gill tissue a plastic response to salinity treatments was observed with general up-regulation of these genes concomitant with higher salinity. For liver tissue a population specific expression differences was observed with lower expression at simulated non-native compared to native salinities. Finally, for kidney tissue a stress response was observed in one population, with gene up-regulation when North Sea flounders were transplanted to low salinity. This study underlines the importance of tissue specific gene expression and the significance of gene expression for evolution of local adaptation in high gene flow marine fishes.     

Population genomic evidence for adaptive differentiation in the Baltic Sea herring

Detecting and estimating the degree of genetic differentiation among populations of highly mobile marine fish having pelagic larval stages is challenging because their effective population sizes can be large, and thus, little genetic drift and differentiation is expected in neutral genomic sites. However, genomic sites subject to directional selection stemming from variation in local environmental conditions can still show substantial genetic differentiation, yet these signatures can be hard to detect with low‐throughput approaches. Using a pooled RAD‐seq approach, we investigated genomewide patterns of genetic variability and differentiation within and among 20 populations of Atlantic herring in the Baltic Sea (and adjacent Atlantic sites), where previous low‐throughput studies and/or studies based on few populations have found limited evidence for genetic differentiation. Stringent quality control was applied in the filtering of 1 791 254 SNPs, resulting in a final data set of 68 182 polymorphic loci. Clear differentiation was identified between Atlantic and Baltic populations in many genomic sites, while differentiation within the Baltic Sea area was weaker and geographically less structured. However, outlier analyses – whether including all populations or only those within the Baltic Sea – uncovered hundreds of directionally selected loci in which variability was associated with either salinity, temperature or both. Hence, our results support the view that although the degree of genetic differentiation among Baltic Sea herring populations is low, there are many genomic regions showing elevated divergence, apparently as a response to temperature‐ and salinity‐related natural selection. As such, the results add to the increasing evidence of local adaptation in highly mobile marine organisms, and those in the young Baltic Sea in particular.     

Microsatellite variation in cod Gadus morhua throughout its geographic range

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 0·03 ( P = < 0·001) 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 self-assignment 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.     

The effect of migratory behaviour on genetic diversity and population divergence: a comparison of anadromous and freshwater Atlantic salmon Salmo salar

The genetic diversity of anadromous and freshwater Atlantic salmon ( Salmo salar ) populations from north-west Russia and other north European locations was compared using microsatellite variation to evaluate the importance of anadromous migration, population size and population glacial history in determining population genetic diversity and divergence. In anadromous Atlantic salmon populations, the level of genetic diversity was significantly higher and the level of population divergence was significantly lower than among the freshwater Atlantic salmon populations, even after correcting for differences in stock size. The phylogeographic origin of the populations also had a significant effect on the genetic diversity characteristics of populations: anadromous populations from the basins of the Atlantic Ocean, White Sea and Barents Sea possessed higher levels of genetic diversity than anadromous populations from the Baltic Sea basin. Among the freshwater populations, the result was the opposite: the Baltic freshwater populations were more variable. The results of this study imply that differences in the level of long-term gene flow between freshwater populations and anadromous populations have led to different levels of genetic diversity, which was also evidenced by the hierarchical analysis of molecular variance. Furthermore, the results emphasize the importance of taking the life history of a population into consideration when developing conservation strategies: due to the limited possibilities for new genetic diversity to be generated via gene flow, it is expected that freshwater Atlantic salmon populations would be more vulnerable to extinction following a population crash. Hence, high conservation status is warranted in order to ensure the long-term survival of the limited number of European populations with this life-history strategy.     

Evidence of a hybrid-zone in Atlantic cod (Gadus morhua) in the Baltic and the Danish Belt Sea revealed by individual admixture analysis

The study of hybrid zones is central to our understanding of the genetic basis of reproductive isolation and speciation, yet very little is known about the extent and significance of hybrid zones in marine fishes. We examined the population structure of cod in the transition area between the North Sea and the Baltic Sea employing nine microsatellite loci. Genetic differentiation between the North Sea sample and the rest increased along a transect to the Baltic proper, with a large increase in level of differentiation occurring in the Western Baltic area. Our objective was to determine whether this pattern was caused purely by varying degrees of mechanical mixing of North Sea and Baltic Sea cod or by interbreeding and formation of a hybrid swarm. Simulation studies revealed that traditional Hardy-Weinberg analysis did not have sufficient power for detection of a Wahlund effect. However, using a model-based clustering method for individual admixture analysis, we were able to demonstrate the existence of intermediate genotypes in all samples from the transition area. Accordingly, our data were explained best by a model of a hybrid swarm flanked by pure nonadmixed populations in the North Sea and the Baltic Sea proper. Significant correlation of gene identities across loci (gametic phase disequilibrium) was found only in a sample from the Western Baltic, suggesting this area as the centre of the apparent hybrid zone. A hybrid zone for cod in the ecotone between the high-saline North Sea and the low-saline Baltic Sea is discussed in relation to its possible origin and maintenance, and in relation to a classical study of haemoglobin variation in cod from the Baltic Sea/Danish Belt Sea, suggesting mixing of two divergent populations without interbreeding.     

Evidence of microsatellite hitch-hiking selection in Atlantic cod (Gadus morhua L.): implications for inferring population structure in nonmodel organisms

Microsatellites have gained wide application for elucidating population structure in nonmodel organisms. Since they are generally noncoding, neutrality is assumed but rarely tested. In Atlantic cod (Gadus morhua L.), microsatellite studies have revealed highly heterogeneous estimates of genetic differentiation among loci. In particular one locus, Gmo 132, has demonstrated elevated genetic differentiation. We investigated possible hitch-hiking selection at this and other microsatellite loci in Atlantic cod. We employed 11 loci for analysing samples from the Baltic Sea, North Sea, Barents Sea and Newfoundland covering a large part of the species' distributional range. The 'classical' Lewontin-Krakauer test for selection based on variance in estimates of F(ST) and (standardized genetic differentiation) revealed only one significant pairwise test (North Sea-Barents Sea), and the source of the elevated variance could not be ascribed exclusively to Gmo 132. In contrast, different variants of the recently developed ln Rtheta test for selective sweeps at microsatellite loci revealed a high number of significant outcomes of pair-wise tests for Gmo 132. Further, the presence of selection was indicated in at least one other locus. The results suggest that many previous estimates of genetic differentiation in cod based on microsatellites are inflated, and in some cases relationships among populations are obscured by one or more loci being the subject to hitch-hiking selection. Likewise, temporal estimates of effective population sizes in Atlantic cod may be flawed. We recommend, generally, to use a higher number of microsatellite loci to elucidate population structure in marine fishes and other nonmodel species to allow for identification of outlier loci that are subject to selection.     

Geographical variation in Fucus vesiculosus morphology in the Baltic and North Seas

Effects of stress and disturbance on morphology, reproductive effort, size and sex ratio were studied for Fucus vesiculosus populations from the Baltic Sea at Askö and the North Sea on the west coast of Sweden at Tjäm[otilde]. High morphological variation was found between Fucus populations, with significant differences in length and weight of individuals, thallus breadth, number of branches and receptacles and receptacle weight, not only between Baltic and North Sea populations but also between populations within the same area, differing in wave exposure. With increasing disturbance, individuals in both studied populations were smaller and less branched. Differences were observed in plant size, with longer, broader and more branched plants being found in Askö compared with Tjärnö. Fucus populations at Tjämö allocated more biomass to reproduction and had longer, heavier receptacles than at Askö. Although the observed morphological changes may be partly explained by differences in wave exposure and salinity between the two sites, it is not possible to rule out genetic differences between the Baltic and North Sea populations. However, it is unlikely that the variations observed within the populations and between populations from the same area are genetically determined.     

Adaptive divergence in a high gene flow environment: Hsc70 variation in the European flounder (Platichthys flesus L.)

Little is known about local adaptations in marine fishes since population genetic surveys in these species have typically not applied genetic markers subject to selection. In this study, we used a candidate gene approach to investigate adaptive population divergence in the European flounder (Platichthys flesus L.) throughout the northeastern Atlantic. We contrasted patterns of genetic variation in a presumably neutral microsatellite baseline to patterns from a heat-shock cognate protein gene, Hsc70. Using two different neutrality tests we found that the microsatellite data set most likely represented a neutral baseline. In contrast, Hsc70 strongly deviated from neutral expectations. Importantly, when estimating standardized levels of population divergence (F(ST)'), we also found a large discrepancy in the patterns of structuring in the two data sets. Thus, samples grouped according to geographical or historical proximity with regards to microsatellites, but according to environmental similarities with regards to Hsc70. The differences between the data sets were particularly pronounced in pairwise comparisons involving populations in the western and central Baltic Sea. For instance, the genetic differentiation between geographically close Baltic Sea and North Sea populations was found to be 0.02 and 0.45 for microsatellites and Hsc70 respectively. Our results strongly suggest adaptive population divergence and indicate local adaptations at the DNA level in a background of high levels of gene flow, typically found in many marine fish species. Furthermore, this study highlights the usefulness of the candidate gene approach for demonstrating local selection in non-model organisms such as most marine fishes.     

HETEROGENEOUS GENOMIC DIFFERENTIATION IN MARINE THREESPINE STICKLEBACKS: ADAPTATION ALONG AN ENVIRONMENTAL GRADIENT

Evolutionary divergence among populations occupying ecologically distinct environments can occur even in the face of on‐going gene flow. However, the genetic underpinnings, as well as the scale and magnitude at which this differentiation occurs in marine habitats are not well understood. We investigated the patterns and degree of genomic heterogeneity in threespine sticklebacks (Gasterosteus aculeatus) by assessing genetic variability in 20 nongenic and 20 genic (associated with genes important for freshwater adaptation) microsatellite loci in samples collected from 38 locations spanning the entire Baltic Sea coast to the North Sea boundary. Population divergence (F_ST ≈ 0.026) and structuring (five genetic clusters) was significantly more pronounced in the genic as compared to nongenic markers (F_ST ≈ 0.008; no genetic clusters). Patterns of divergence in the genic markers—45% of which were identified as outliers—correlated with local differences in salinity. Yet, a strong positive correlation between divergence in genic and nongenic markers, and their association with environmental factors suggests that adaptive divergence is reducing gene flow across the genome. Apart from providing a clear demonstration of heterogeneous genomic patterns of differentiation in a marine species, the results are indicative of adaptive population structuring across the relatively young Baltic Sea in spite of ample opportunities for gene flow.     

Genetic variation within and among North Atlantic and Baltic populations of the benthic alga Phycodrys rubens (Rhodophyta)

Genetic variation was examined within and among North Atlantic, North Sea and Baltic populations of the benthic red alga Phycodrys rubens using allozymes and random amplified polymorphic DNA (RAPD) markers. On western and eastern North Atlantic coasts distinct allozyme types were found, with the exception of western Newfoundland where East and West Atlantic types co-occur. Along the European coasts, two genetic groups were distinguished by fixed allelic differences: an outer oceanic group and a North Sea/Baltic group. The two genetic types co-occur in the Skagerrak and Kattegat region. Reproductive isolation between the two types is suggested by the lack of hybrids in the overlap zones, and they may therefore represent sibling species. Unexpectedly, an analysis of RAPD variation was unable to recover the two cryptic species identified using allozymes. Within-population RAPD variation was similar to or greater than between-population variation. The lack of structure in the RAPD data cannot be attributed solely to technical artefacts of the method but appears to reflect real biological variability. Within-population genomic polymorphisms caused by frequent mutational events are discussed, as are high amounts of genetic drift and possible disruptive selection brought about by stressed habitats. Finally, Baltic and extra-Baltic salinity ecotypes are known to exist in P. rubens. However, no correlation between ecotypic variation and allozyme groups was detected.