Genetic population structure constrains local adaptation in sticklebacks.

Repeated and independent adaptation to specific environmental conditions from standing genetic variation is common. However, if genetic variation is limited, the evolution of similar locally adapted traits may be restricted to genetically different and potentially less optimal solutions or prevented from happening altogether. Using a quantitative trait locus (QTL) mapping approach, we identified the genomic regions responsible for the repeated pelvic reduction (PR) in three crosses between nine-spined stickleback populations expressing full and reduced pelvic structures. In one cross, PR mapped to linkage group 7 (LG7) containing the gene Pitx1, known to control pelvic reduction also in the three-spined stickleback. In the two other crosses, PR was polygenic and attributed to 10 novel QTL, of which 90% were unique to specific crosses. When screening the genomes from 27 different populations for deletions in the Pitx1 regulatory element, these were only found in the population in which PR mapped to LG7, even though the morphological data indicated large-effect QTL for PR in several other populations as well. Consistent with the available theory and simulations parameterized on empirical data, we hypothesize that the observed variability in genetic architecture of PR is due to heterogeneity in the spatial distribution of standing genetic variation caused by >2× stronger population structuring among freshwater populations and >10× stronger genetic isolation by distance in the sea in nine-spined sticklebacks as compared to three-spined sticklebacks.

A phylogenomic perspective on diversity, hybridization and evolutionary affinities in the stickleback genus Pungitius.

Hybridization and convergent evolution are phenomena of broad interest in evolutionary biology, but their occurrence poses challenges for reconstructing evolutionary affinities among affected taxa. Sticklebacks in the genus Pungitius are a case in point: evolutionary relationships and taxonomic validity of different species and populations in this circumpolarly distributed species complex remain contentious due to convergent evolution of traits regarded as diagnostic in their taxonomy, and possibly also due to frequent hybridization among taxa. To clarify the evolutionary relationships among different Pungitius species and populations globally, as well as to study the prevalence and extent of introgression among recognized species, genomic data sets of both reference genome-anchored single nucleotide polymorphisms and de novo assembled RAD-tag loci were constructed with RAD-seq data. Both data sets yielded topologically identical and well-supported species trees. Incongruence between nuclear and mitochondrial DNA-based trees was found and suggested possibly frequent hybridization and mitogenome capture during the evolution of Pungitius sticklebacks. Further analyses revealed evidence for frequent nuclear genetic introgression among Pungitius species, although the estimated proportions of autosomal introgression were low. Apart from providing evidence for frequent hybridization, the results challenge earlier mitochondrial and morphology-based hypotheses regarding the number of species and their affinities in this genus: at least seven extant species can be recognized on the basis of genetic data. The results also shed new light on the biogeographical history of the Pungitius-complex, including suggestion of several trans-Arctic invasions of Europe from the Northern Pacific. The well-resolved phylogeny should facilitate the utility of this genus as a model system for future comparative evolutionary studies.

A four-questions perspective on public information use in sticklebacks (Gasterosteidae).

Whether learning primarily reflects general processes or species-specific challenges is a long-standing matter of dispute. Here, we present a comprehensive analysis of public information use (PI-use) in sticklebacks (Gasterosteidae). PI-use is a form of social learning by which animals are able to assess the relative quality of resources, here prey patches, by observing the behaviour of others. PI-use was highly specific with only Pungitius and their closest relative Culaea inconstans showing evidence of PI-use. We saw no effects of ontogenetic experience upon PI-use in Pungitius pungitius. Experiments with live demonstrators and animated fish revealed that heightened activity and feeding strikes by foraging conspecifics are important cues in the transmission of PI. Finally, PI-use was the only form of learning in which P. pungitius and another stickleback, Gasterosteus aculeatus differed. PI-use in sticklebacks is species-specific and may represent an 'ecological specialization' for social foraging. Whether this reflects selection on perception, attentional or cognitive processes remains to be determined.

The evolution of sex determination associated with a chromosomal inversion.

Sex determination is a fundamentally important and highly diversified biological process, yet the mechanisms behind the origin of this diversity are mostly unknown. Here we suggest that the evolution of sex determination systems can be driven by a chromosomal inversion. We show that an XY system evolved recently in particular nine-spined stickleback (Pungitius pungitius) populations, which arose from ancient hybridization between two divergent lineages. Our phylogenetic and genetic mapping analyses indicate that the XY system is formed in a large inversion that is associated with hybrid sterility between the divergent lineages. We suggest that a new male-determining gene evolved in the inversion in response to selection against impaired male fertility in a hybridized population. Given that inversions are often associated with hybrid incompatibility in animals and plants, they might frequently contribute to the diversification of sex determination systems.

Deciphering the genomic architecture of the stickleback brain with a novel multilocus gene-mapping approach.

Quantitative traits important to organismal function and fitness, such as brain size, are presumably controlled by many small-effect loci. Deciphering the genetic architecture of such traits with traditional quantitative trait locus (QTL) mapping methods is challenging. Here, we investigated the genetic architecture of brain size (and the size of five different brain parts) in nine-spined sticklebacks (Pungitius pungitius) with the aid of novel multilocus QTL-mapping approaches based on a de-biased LASSO method. Apart from having more statistical power to detect QTL and reduced rate of false positives than conventional QTL-mapping approaches, the developed methods can handle large marker panels and provide estimates of genomic heritability. Single-locus analyses of an F2 interpopulation cross with 239 individuals and 15 198, fully informative single nucleotide polymorphisms (SNPs) uncovered 79 QTL associated with variation in stickleback brain size traits. Many of these loci were in strong linkage disequilibrium (LD) with each other, and consequently, a multilocus mapping of individual SNPs, accounting for LD structure in the data, recovered only four significant QTL. However, a multilocus mapping of SNPs grouped by linkage group (LG) identified 14 LGs (1-6 depending on the trait) that influence variation in brain traits. For instance, 17.6% of the variation in relative brain size was explainable by cumulative effects of SNPs distributed over six LGs, whereas 42% of the variation was accounted for by all 21 LGs. Hence, the results suggest that variation in stickleback brain traits is influenced by many small-effect loci. Apart from suggesting moderately heritable (h2  ≈ 0.15-0.42) multifactorial genetic architecture of brain traits, the results highlight the challenges in identifying the loci contributing to variation in quantitative traits. Nevertheless, the results demonstrate that the novel QTL-mapping approach developed here has distinctive advantages over the traditional QTL-mapping methods in analyses of dense marker panels.

Quantitative trait locus analysis of body shape divergence in nine-spined sticklebacks based on high-density SNP-panel.

Heritable phenotypic differences between populations, caused by the selective effects of distinct environmental conditions, are of commonplace occurrence in nature. However, the actual genomic targets of this kind of selection are still poorly understood. We conducted a quantitative trait locus (QTL) mapping study to identify genomic regions responsible for morphometric differentiation between genetically and phenotypically divergent marine and freshwater nine-spined stickleback (Pungitius pungitius) populations. Using a dense panel of SNP-markers obtained by restriction site associated DNA sequencing of an F2 recombinant cross, we found 22 QTL that explained 3.5-12.9% of phenotypic variance in the traits under investigation. We detected one fairly large-effect (PVE = 9.6%) QTL for caudal peduncle length-a trait with a well-established adaptive function showing clear differentiation among marine and freshwater populations. We also identified two large-effect QTL for lateral plate numbers, which are different from the lateral plate QTL reported in earlier studies of this and related species. Hence, apart from identifying several large-effect QTL in shape traits showing adaptive differentiation in response to different environmental conditions, the results suggest intra- and interspecific heterogeneity in the genomic basis of lateral plate number variation.

Complete mitochondrial genome of the Greek nine-spined stickleback Pungitius hellenicus (Gasterosteiformes, Gasterosteidae).

The complete mitochondrial genome of the Greek nine-spined stickleback Pungitius hellenicus was obtained using Illumina high-throughput sequencing of genomic DNA. The genome was 16 713 bp long, and contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a control region. The arrangement of the genes was identical to that of other Gasterosteidae fishes. However, the control region of P. hellenicus contained three copies of imperfect repeated sequences (72-78 bp in single motifs), while P. pungitius and P. tymensis have one or two copies. Nucleotide identity between P. hellenicus and three other Pungitius species across all the 37 genic regions was 93.0% to 95.5%.

Complete mitochondrial genome of the Ukrainian nine-spined stickleback Pungitius platygaster (Gasterosteiformes, Gasterosteidae).

The complete mitochondrial genome of the Ukrainian nine-spined stickleback Pungitius platygaster was obtained using massive parallel sequencing of genomic DNA. The mitogenome sequence was 16 570 bp long, and the gene order and contents were identical to those of other sequenced Pungitius mitogenomes. In a phylogenetic analysis, the mitogenome of P. platygaster clustered with other Pungitius mitogenomes, yet being clearly distinct from those of P. pungitius, P. sinensis and P. kaibarae.

Complete mitochondrial genomes of the smooth tail nine-spined sticklebacks Pungitius laevis (Gasterosteiformes, Gasterosteidae).

The complete mitochondrial genome sequences of three divergent lineages of the smooth tail nine-spined stickleback (Pungitius laevis) were obtained with massive parallel sequencing of their genomic DNA. The genome sequences were 16 574-16 580 bp long, and the gene order and contents were identical to those of other sequenced Pungitius mitogenomes. Although the mitogenome sequences of all three P. laevis lineages clustered within the genus Pungitius, they were clearly distinct and showed divergence comparable to that seen between some Pungitius species.

Complete mitochondrial genome of the Sakhalin nine-spined stickleback Pungitius tymensis (Gasterosteiformes, Gasterosteidae).

The complete mitochondrial genome of the Sakhalin nine-spined stickleback Pungitius tymensis was determined using Illumina paired-end sequencing of genomic DNA. The genome sequence was 16 481 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and a control region. The content and arrangement of the genes were identical to those of other Gasterosteidae species. P. tymensis was phylogenetically positioned with other Pungitius species (P. kaibarae, P. pungitius and P. sinensis) with a clear distinction from them. Nucleotide identity in the 37 genic regions ranged from 94.7% to 94.9% between P. tymensis and the other Pungitius species.

Construction of Ultradense Linkage Maps with Lep-MAP2: Stickleback F2 Recombinant Crosses as an Example.

High-density linkage maps are important tools for genome biology and evolutionary genetics by quantifying the extent of recombination, linkage disequilibrium, and chromosomal rearrangements across chromosomes, sexes, and populations. They provide one of the best ways to validate and refine de novo genome assemblies, with the power to identify errors in assemblies increasing with marker density. However, assembly of high-density linkage maps is still challenging due to software limitations. We describe Lep-MAP2, a software for ultradense genome-wide linkage map construction. Lep-MAP2 can handle various family structures and can account for achiasmatic meiosis to gain linkage map accuracy. Simulations show that Lep-MAP2 outperforms other available mapping software both in computational efficiency and accuracy. When applied to two large F2-generation recombinant crosses between two nine-spined stickleback (Pungitius pungitius) populations, it produced two high-density (∼6 markers/cM) linkage maps containing 18,691 and 20,054 single nucleotide polymorphisms. The two maps showed a high degree of synteny, but female maps were 1.5-2 times longer than male maps in all linkage groups, suggesting genome-wide recombination suppression in males. Comparison with the genome sequence of the three-spined stickleback (Gasterosteus aculeatus) revealed a high degree of interspecific synteny with a low frequency (<5%) of interchromosomal rearrangements. However, a fairly large (ca. 10 Mb) translocation from autosome to sex chromosome was detected in both maps. These results illustrate the utility and novel features of Lep-MAP2 in assembling high-density linkage maps, and their usefulness in revealing evolutionarily interesting properties of genomes, such as strong genome-wide sex bias in recombination rates.

Genetic Variability and Structuring of Arctic Charr (Salvelinus alpinus) Populations in Northern Fennoscandia.

Variation in presumably neutral genetic markers can inform us about evolvability, historical effective population sizes and phylogeographic history of contemporary populations. We studied genetic variability in 15 microsatellite loci in six native landlocked Arctic charr (Salvelinus alpinus) populations in northern Fennoscandia, where this species is considered near threatened. We discovered that all populations were genetically highly (mean FST ≈ 0.26) differentiated and isolated from each other. Evidence was found for historical, but not for recent population size bottlenecks. Estimates of contemporary effective population size (Ne) ranged from seven to 228 and were significantly correlated with those of historical Ne but not with lake size. A census size (NC) was estimated to be approximately 300 individuals in a pond (0.14 ha), which exhibited the smallest Ne (i.e. Ne/NC = 0.02). Genetic variability in this pond and a connected lake is severely reduced, and both genetic and empirical estimates of migration rates indicate a lack of gene flow between them. Hence, albeit currently thriving, some northern Fennoscandian populations appear to be vulnerable to further loss of genetic variability and are likely to have limited capacity to adapt if selection pressures change.

Signatures of selection in the three-spined stickleback along a small-scale brackish water - freshwater transition zone.

Local adaptation is often obvious when gene flow is impeded, such as observed at large spatial scales and across strong ecological contrasts. However, it becomes less certain at small scales such as between adjacent populations or across weak ecological contrasts, when gene flow is strong. While studies on genomic adaptation tend to focus on the former, less is known about the genomic targets of natural selection in the latter situation. In this study, we investigate genomic adaptation in populations of the three-spined stickleback Gasterosteus aculeatus L. across a small-scale ecological transition with salinities ranging from brackish to fresh. Adaptation to salinity has been repeatedly demonstrated in this species. A genome scan based on 87 microsatellite markers revealed only few signatures of selection, likely owing to the constraints that homogenizing gene flow puts on adaptive divergence. However, the detected loci appear repeatedly as targets of selection in similar studies of genomic adaptation in the three-spined stickleback. We conclude that the signature of genomic selection in the face of strong gene flow is weak, yet detectable. We argue that the range of studies of genomic divergence should be extended to include more systems characterized by limited geographical and ecological isolation, which is often a realistic setting in nature.

Genome-wide linkage disequilibrium in nine-spined stickleback populations.

Variation in the extent and magnitude of genome-wide linkage disequilibrium (LD) among populations residing in different habitats has seldom been studied in wild vertebrates. We used a total of 109 microsatellite markers to quantify the level and patterns of genome-wide LD in 13 Fennoscandian nine-spined stickleback (Pungitius pungitius) populations from four (viz. marine, lake, pond, and river) different habitat types. In general, high magnitude (D' > 0.5) of LD was found both in freshwater and marine populations, and the magnitude of LD was significantly greater in inland freshwater than in marine populations. Interestingly, three coastal freshwater populations located in close geographic proximity to the marine populations exhibited similar LD patterns and genetic diversity as their marine neighbors. The greater levels of LD in inland freshwater compared with marine and costal freshwater populations can be explained in terms of their contrasting demographic histories: founder events, long-term isolation, small effective sizes, and population bottlenecks are factors likely to have contributed to the high levels of LD in the inland freshwater populations. In general, these findings shed new light on the patterns and extent of variation in genome-wide LD, as well as the ecological and evolutionary factors driving them.

Geographic variation in age structure and longevity in the nine-spined stickleback (Pungitius pungitius).

Variation in age and size of mature nine-spined sticklebacks (Pungitius pungitius) within and among 16 Fennoscandian populations were assessed using skeletochronology. The average age of individuals in a given population varied from 1.7 to 4.7 years. Fish from pond populations were on average older than those from lake and marine populations, and females tended to be older than males. Reproduction in marine and lake populations commenced typically at an age of two years, whereas that in ponds at an age of three years. The maximum life span of the fish varied from 3 to 7 years. Mean body size within and among populations increased with increasing age, but the habitat and population differences in body size persisted even after accounting for variation in population age (and sex) structure. Hence, the population differences in mean body size are not explainable by age differences alone. As such, much of the pronounced intraspecific variation in population age structure can be attributed to delayed maturation and extended longevity of the pond fish. The results are contrasted and discussed in the context of similar data from the three-spined stickleback (Gasterosteus aculeatus) occupying the same geographic area.

Identification of major and minor QTL for ecologically important morphological traits in three-spined sticklebacks (Gasterosteus aculeatus).

Quantitative trait locus (QTL) mapping studies of Pacific three-spined sticklebacks (Gasterosteus aculeatus) have uncovered several genomic regions controlling variability in different morphological traits, but QTL studies of Atlantic sticklebacks are lacking. We mapped QTL for 40 morphological traits, including body size, body shape, and body armor, in a F2 full-sib cross between northern European marine and freshwater three-spined sticklebacks. A total of 52 significant QTL were identified at the 5% genome-wide level. One major QTL explaining 74.4% of the total variance in lateral plate number was detected on LG4, whereas several major QTL for centroid size (a proxy for body size), and the lengths of two dorsal spines, pelvic spine, and pelvic girdle were mapped on LG21 with the explained variance ranging from 27.9% to 57.6%. Major QTL for landmark coordinates defining body shape variation also were identified on LG21, with each explaining ≥15% of variance in body shape. Multiple QTL for different traits mapped on LG21 overlapped each other, implying pleiotropy and/or tight linkage. Thus, apart from providing confirmatory data to support conclusions born out of earlier QTL studies of Pacific sticklebacks, this study also describes several novel QTL of both major and smaller effect for ecologically important traits. The finding that many major QTL mapped on LG21 suggests that this linkage group might be a hotspot for genetic determinants of ecologically important morphological traits in three-spined sticklebacks.

QTL analysis of behavior in nine-spined sticklebacks (Pungitius pungitius).

The genetic architecture of behavioral traits is yet relatively poorly understood in most non-model organisms. Using an F2-intercross (n = 283 offspring) between behaviorally divergent nine-spined stickleback (Pungitius pungitius) populations, we tested for and explored the genetic basis of different behavioral traits with the aid of quantitative trait locus (QTL) analyses based on 226 microsatellite markers. The behaviors were analyzed both separately (viz. feeding activity, risk-taking and exploration) and combined in order to map composite behavioral type. Two significant QTL-explaining on average 6 % of the phenotypic variance-were detected for composite behavioral type on the experiment-wide level, located on linkage groups 3 and 8. In addition, several suggestive QTL located on six other linkage groups were detected on the chromosome-wide level. Apart from providing evidence for the genetic basis of behavioral variation, the results provide a good starting point for finer-scale analyses of genetic factors influencing behavioral variation in the nine-spined stickleback.

Quantitative trait loci for growth and body size in the nine-spined stickleback Pungitius pungitius L.

Body size is an ecologically important trait shown to be genetically variable both within and among different animal populations as revealed by quantitative genetic studies. However, few studies have looked into underlying genetic architecture of body size variability in the wild using genetic mapping methods. With the aid of quantitative trait loci (QTL) analyses based on 226 microsatellite markers, we mapped body size and growth rate traits in the nine-spined stickleback (Pungitius pungitius) using an F2 -intercross (n = 283 offspring) between size-divergent populations. In total, 17 QTL locations were detected. The proportion of phenotypic variation explained by individual body size-related QTL ranged from 3% to 12% and those related to growth parameters and increments from 3% to 10%. Several of the detected QTL affected either early or late growth. These results provide a solid starting point for more in depth investigations of structure and function of genomic regions involved in determination of body size in this popular model of ecological and evolutionary research.

Genetic architecture of parallel pelvic reduction in ninespine sticklebacks.

Teleost fish genomes are known to be evolving faster than those of other vertebrate taxa. Thus, fish are suited to address the extent to which the same vs. different genes are responsible for similar phenotypic changes in rapidly evolving genomes of evolutionary independent lineages. To gain insights into the genetic basis and evolutionary processes behind parallel phenotypic changes within and between species, we identified the genomic regions involved in pelvic reduction in Northern European ninespine sticklebacks (Pungitius pungitius) and compared them to those of North American ninespine and threespine sticklebacks (Gasterosteus aculeatus). To this end, we conducted quantitative trait locus (QTL) mapping using 283 F2 progeny from an interpopulation cross. Phenotypic analyses indicated that pelvic reduction is a recessive trait and is inherited in a simple Mendelian fashion. Significant QTL for pelvic spine and girdle lengths were identified in the region of the Pituitary homeobox transcription factor 1 (Pitx1) gene, also responsible for pelvic reduction in threespine sticklebacks. The fact that no QTL was observed in the region identified in the mapping study of North American ninespine sticklebacks suggests that an alternative QTL for pelvic reduction has emerged in this species within the past 1.6 million years after the split between Northern European and North American populations. In general, our study provides empirical support for the view that alternative genetic mechanisms that lead to similar phenotypes can evolve over short evolutionary time scales.

High degree of genetic differentiation in marine three-spined sticklebacks (Gasterosteus aculeatus).

Populations of widespread marine organisms are typically characterized by a low degree of genetic differentiation in neutral genetic markers, but much less is known about differentiation in genes whose functional roles are associated with specific selection regimes. To uncover possible adaptive population divergence and heterogeneous genomic differentiation in marine three-spined sticklebacks (Gasterosteus aculeatus), we used a candidate gene-based genome-scan approach to analyse variability in 138 microsatellite loci located within/close to (<6 kb) functionally important genes in samples collected from ten geographic locations. The degree of genetic differentiation in markers classified as neutral or under balancing selection-as determined with several outlier detection methods-was low (F(ST) = 0.033 or 0.011, respectively), whereas average FST for directionally selected markers was significantly higher (F(ST) = 0.097). Clustering analyses provided support for genomic and geographic heterogeneity in selection: six genetic clusters were identified based on allele frequency differences in the directionally selected loci, whereas four were identified with the neutral loci. Allelic variation in several loci exhibited significant associations with environmental variables, supporting the conjecture that temperature and salinity, but not optic conditions, are important drivers of adaptive divergence among populations. In general, these results suggest that in spite of the high degree of physical connectivity and gene flow as inferred from neutral marker genes, marine stickleback populations are strongly genetically structured in loci associated with functionally relevant genes.