A low-density SNP array for analyzing differential selection in freshwater and marine populations of threespine stickleback (Gasterosteus aculeatus)

Background

The threespine stickleback (Gasterosteus aculeatus) has become an important model species for studying both contemporary and parallel evolution. In particular, differential adaptation to freshwater and marine environments has led to high differentiation between freshwater and marine stickleback populations at the phenotypic trait of lateral plate morphology and the underlying candidate gene Ectodysplacin (EDA). Many studies have focused on this trait and candidate gene, although other genes involved in marine-freshwater adaptation may be equally important. In order to develop a resource for rapid and cost efficient analysis of genetic divergence between freshwater and marine sticklebacks, we generated a low-density SNP (Single Nucleotide Polymorphism) array encompassing markers of chromosome regions under putative directional selection, along with neutral markers for background.

Results

RAD (Restriction site Associated DNA) sequencing of sixty individuals representing two freshwater and one marine population led to the identification of 33,993 SNP markers. Ninety-six of these were chosen for the low-density SNP array, among which 70 represented SNPs under putatively directional selection in freshwater vs. marine environments, whereas 26 SNPs were assumed to be neutral. Annotation of these regions revealed several genes that are candidates for affecting stickleback phenotypic variation, some of which have been observed in previous studies whereas others are new.

Conclusions

We have developed a cost-efficient low-density SNP array that allows for rapid screening of polymorphisms in threespine stickleback. The array provides a valuable tool for analyzing adaptive divergence between freshwater and marine stickleback populations beyond the well-established candidate gene Ectodysplacin (EDA).

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-867) contains supplementary material, which is available to authorized users.     

Genomic signatures of the plateless phenotype in the threespine stickleback

Understanding the genetic basis of traits involved in adaptive divergence and speciation is one of the most fundamental objectives in evolutionary biology. Toward that end, we look for signatures of extreme plate loss in the genome of freshwater threespine sticklebacks (Gasterosteus aculeatus). Plateless stickleback have been found in only a few lakes and streams across the world; they represent the far extreme of a phenotypic continuum (plate number) that has been studied for years, although plateless individuals have not yet been the subject of much investigation. We use a dense single nucleotide polymorphism dataset made using RADseq to study fish from three freshwater populations containing plateless and low plated individuals, as well as fish from full plated marine populations. Analyses were performed using FastStructure, sliding windows F_ST, Bayescan and latent factor mixed models to search for genomic differences between the low plated and plateless phenotypes both within and among the three lakes. At least 18 genomic regions which may contribute to within‐morph plate number variation were detected in our low plated stickleback populations. We see no evidence of a selective sweep between low and plateless fish; rather reduction of plate number within the low plated morph seems to be polygenic.     

Partially repeatable genetic basis of benthic adaptation in threespine sticklebacks

The extent to which convergent adaptation to similar ecological niches occurs by a predictable genetic basis remains a fundamental question in biology. Threespine stickleback fish have undergone an adaptive radiation in which ancestral oceanic populations repeatedly colonized and adapted to freshwater habitats. In multiple lakes in British Columbia, two different freshwater ecotypes have evolved: a deep‐bodied benthic form adapted to forage near the lake substrate, and a narrow‐bodied limnetic form adapted to forage in open water. Here, we use genome‐wide linkage mapping in marine × benthic F2 genetic crosses to test the extent of shared genomic regions underlying benthic adaptation in three benthic populations. We identify at least 100 Quantitative Trait Loci (QTL) harboring genes influencing skeletal morphology. The majority of QTL (57%) are unique to one cross. However, four genomic regions affecting eight craniofacial and armor phenotypes are found in all three benthic populations. We find that QTL are clustered in the genome and overlapping QTL regions are enriched for genomic signatures of natural selection. These findings suggest that benthic adaptation has occurred via both parallel and nonparallel genetic changes.     

Chromosomal Fusions Facilitate Adaptation to Divergent Environments in Threespine Stickleback

Chromosomal fusions are hypothesized to facilitate adaptation to divergent environments, both by bringing together previously unlinked adaptive alleles and by creating regions of low recombination that facilitate the linkage of adaptive alleles; but, there is little empirical evidence to support this hypothesis. Here, we address this knowledge gap by studying threespine stickleback (Gasterosteus aculeatus), in which ancestral marine fish have repeatedly adapted to freshwater across the northern hemisphere. By comparing the threespine and ninespine stickleback (Pungitius pungitius) genomes to a de novo assembly of the fourspine stickleback (Apeltes quadracus) and an outgroup species, we find two chromosomal fusion events involving the same chromosomes have occurred independently in the threespine and ninespine stickleback lineages. On the fused chromosomes in threespine stickleback, we find an enrichment of quantitative trait loci underlying traits that contribute to marine versus freshwater adaptation. By comparing whole-genome sequences of freshwater and marine threespine stickleback populations, we also find an enrichment of regions under divergent selection on these two fused chromosomes. There is elevated genetic diversity within regions under selection in the freshwater population, consistent with a simulation study showing that gene flow can increase diversity in genomic regions associated with local adaptation and our demographic models showing gene flow between the marine and freshwater populations. Integrating our results with previous studies, we propose that these fusions created regions of low recombination that enabled the formation of adaptative clusters, thereby facilitating freshwater adaptation in the face of recurrent gene flow between marine and freshwater threespine sticklebacks.     

Habitat‐specific trends in ontogeny of body shape in stickleback from coastal archipelago: Potential for rapid shifts in colonizing populations

We investigated ontogenetic trends in body shape of 54 freshwater (48 lake, seven stream) and six anadromous populations of threespine stickleback (Gasterosteus aculeatus L.) from the Haida Gwaii archipelago off the west coast of Canada. Multivariate analysis of covariance on the partial warp scores generated from 12 homologous landmarks on 1,958 digital images of subadult and adult male stickleback indicated that there was considerable variability of population ontogenetic slopes. We used discriminant function analysis to quantify body shape and determined that anadromous stickleback, which are ancestral to the freshwater populations, have a strongly negative ontogenetic slope (?5.62; increased streamlining with increased size). All freshwater populations exhibit a more positive slope (91% differed significantly from the marine slope), with the differences being most accentuated in populations from ponds and streams. In pristine lakes, ontogenetic slope could be predicted by lake volume as well as multivariate measures of habitat. Evidence from field transplant experiments of one of the intact populations indicates a rapid change (5 years) from allometric to isometric growth, equivalent to about half of the total slope variation among intact populations on the archipelago. We interpret this shift as developmental plasticity and suggest this may comprise the precursor for selection of optimal body shapes in these stickleback populations. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Phenotypic divergence among threespine stickleback that differ in nuptial coloration

By studying systems in their earliest stages of differentiation, we can learn about the evolutionary forces acting within and among populations and how those forces could contribute to reproductive isolation. Such an understanding would help us to better discern and predict how selection leads to the maintenance of multiple morphs within a species, rather than speciation. The postglacial adaptive radiation of the threespine stickleback (Gasterosteus aculeatus) is one of the best‐studied cases of evolutionary diversification and rapid, repeated speciation. Following deglaciation, marine stickleback have continually invaded freshwater habitats across the northern hemisphere and established resident populations that diverged innumerable times from their oceanic ancestors. Independent freshwater colonization events have yielded broadly parallel patterns of morphological differences in freshwater and marine stickleback. However, there is also much phenotypic diversity within and among freshwater populations. We studied a lesser‐known freshwater “species pair” found in southwest Washington, where male stickleback in numerous locations have lost the ancestral red sexual signal and instead develop black nuptial coloration. We measured phenotypic variation in a suite of traits across sites where red and black stickleback do not overlap in distribution and at one site where they historically co‐occurred. We found substantial phenotypic divergence between red and black morphs in noncolor traits including shape and lateral plating, and additionally find evidence that supports the hypothesis of sensory drive as the mechanism responsible for the evolutionary switch in color from red to black. A newly described third “mixed” morph in Connor Creek, Washington, differs in head shape and size from the red and black morphs, and we suggest that their characteristics are most consistent with hybridization between anadromous and freshwater stickleback. These results lay the foundation for future investigation of the underlying genetic basis of this phenotypic divergence as well as the evolutionary processes that may drive, maintain, or limit divergence among morphs.     

Association mapping of morphological traits in wild and captive zebra finches: reliable within,but not between populations

Identifying causal genetic variants underlying heritable phenotypic variation is a long‐standing goal in evolutionary genetics. We previously identified several quantitative trait loci (QTL) for five morphological traits in a captive population of zebra finches (Taeniopygia guttata) by whole‐genome linkage mapping. We here follow up on these studies with the aim to narrow down on the quantitative trait variants (QTN) in one wild and three captive populations. First, we performed an association study using 672 single nucleotide polymorphisms (SNPs) within candidate genes located in the previously identified QTL regions in a sample of 939 wild‐caught zebra finches. Then, we validated the most promising SNP–phenotype associations (n = 25 SNPs) in 5228 birds from four populations. Genotype–phenotype associations were generally weak in the wild population, where linkage disequilibrium (LD) spans only short genomic distances. In contrast, in captive populations, where LD blocks are large, apparent SNP effects on morphological traits (i.e. associations) were highly repeatable with independent data from the same population. Most of those SNPs also showed significant associations with the same trait in other captive populations, but the direction and magnitude of these effects varied among populations. This suggests that the tested SNPs are not the causal QTN but rather physically linked to them, and that LD between SNPs and causal variants differs between populations due to founder effects. While the identification of QTN remains challenging in nonmodel organisms, we illustrate that it is indeed possible to confirm the location and magnitude of QTL in a population with stable linkage between markers and causal variants.     

Computational identification of miRNAs,their targets and functions in three‐spined stickleback (Gasterosteus aculeatus)

An intriguing question in biology is how the evolution of gene regulation is shaped by natural selection in natural populations. Among the many known regulatory mechanisms, regulation of gene expression by microRNAs (miRNAs) is of critical importance. However, our understanding of their evolution in natural populations is limited. Studying the role of miRNAs in three‐spined stickleback, an important natural model for speciation research, may provide new insights into adaptive polymorphisms. However, lack of annotation of miRNA genes in its genome is a bottleneck. To fill this research gap, we used the genome of three‐spined stickleback to predict miRNAs and their targets. We predicted 1486 mature miRNAs using the homology‐based miRNA prediction approach. We then performed functional annotation and enrichment analysis of these targets, which identified over‐represented motifs. Further, a database resource (GAmiRdb) has been developed for dynamically searching miRNAs and their targets exclusively in three‐spined stickleback. Finally, the database was used in two case studies focusing on freshwater adaptation in natural populations. In the first study, we found 44 genomic regions overlapping with predicted miRNA targets. In the second study, we identified two SNPs altering the MRE seed site of sperm‐specific glyceraldehyde‐3‐phosphate gene. These findings highlight the importance of the GAmiRdb knowledge base in understanding adaptive evolution.     

The genomic signature of parallel adaptation from shared genetic variation

Parallel adaptation is common and may often occur from shared genetic variation, but the genomic consequences of this process remain poorly understood. We first use individual‐based simulations to demonstrate that comparisons between populations adapted in parallel to similar environments from shared variation reveal a characteristic genomic signature around a selected locus: a low‐divergence valley centred at the locus and flanked by twin peaks of high divergence. This signature is initiated by the hitchhiking of haplotype tracts differing between derived populations in the broader neighbourhood of the selected locus (driving the high‐divergence twin peaks) and shared haplotype tracts in the tight neighbourhood of the locus (driving the low‐divergence valley). This initial hitchhiking signature is reinforced over time because the selected locus acts as a barrier to gene flow from the source to the derived populations, thus promoting divergence by drift in its close neighbourhood. We next empirically confirm the peak‐valley‐peak signature by combining targeted and RAD sequence data at three candidate adaptation genes in multiple marine (source) and freshwater (derived) populations of threespine stickleback. Finally, we use a genome‐wide screen for the peak‐valley‐peak signature to discover additional genome regions involved in parallel marine‐freshwater divergence. Our findings offer a new explanation for heterogeneous genomic divergence and thus challenge the standard view that peaks in population divergence harbour divergently selected loci and that low‐divergence regions result from balancing selection or localized introgression. We anticipate that genome scans for peak‐valley‐peak divergence signatures will promote the discovery of adaptation genes in other organisms.     

Gene expression plasticity evolves in response to colonization of freshwater lakes in threespine stickleback

Phenotypic plasticity is predicted to facilitate individual survival and/or evolve in response to novel environments. Plasticity that facilitates survival should both permit colonization and act as a buffer against further evolution, with contemporary and derived forms predicted to be similarly plastic for a suite of traits. On the other hand, given the importance of plasticity in maintaining internal homeostasis, derived populations that encounter greater environmental heterogeneity should evolve greater plasticity. We tested the evolutionary significance of phenotypic plasticity in coastal British Columbian postglacial populations of threespine stickleback (Gasterosteus aculeatus) that evolved under greater seasonal extremes in temperature after invading freshwater lakes from the sea. Two ancestral (contemporary marine) and two derived (contemporary freshwater) populations of stickleback were raised near their thermal tolerance extremes, 7 and 22 °C. Gene expression plasticity was estimated for more than 14 000 genes. Over five thousand genes were similarly plastic in marine and freshwater stickleback, but freshwater populations exhibited significantly more genes with plastic expression than marine populations. Furthermore, several of the loci shown to exhibit gene expression plasticity have been previously implicated in the adaptive evolution of freshwater populations, including a gene involved in mitochondrial regulation (PPARAa). Collectively, these data provide molecular evidence that highlights the importance of plasticity in colonization and adaptation to new environments.     

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 F_ST 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.     

GENETIC ARCHITECTURE OF SKELETAL EVOLUTION IN EUROPEAN LAKE AND STREAM STICKLEBACK

Advances in genomic techniques are greatly facilitating the study of molecular signatures of selection in diverging natural populations. Connecting these signatures to phenotypes under selection remains challenging, but benefits from dissections of the genetic architecture of adaptive divergence. We here perform quantitative trait locus (QTL) mapping using 488 F₂ individuals and 2011 single nucleotide polymorphisms (SNPs) to explore the genetic architecture of skeletal divergence in a lake‐stream stickleback system from Central Europe. We find QTLs for gill raker, snout, and head length, vertebral number, and the extent of lateral plating (plate number and height). Although two large‐effect loci emerge, QTL effect sizes are generally small. Examining the neighborhood of the QTL‐linked SNPs identifies several genes involved in bone formation, which emerge as strong candidate genes for skeletal evolution. Finally, we use SNP data from the natural source populations to demonstrate that some SNPs linked to QTLs in our cross also exhibit striking allele frequency differences in the wild, suggesting a causal role of these QTLs in adaptive population divergence. Our study paves the way for comparative analyses across other (lake‐stream) stickleback populations, and for functional investigations of the candidate genes.     

Genome‐wide association analyses reveal polygenic genomic architecture underlying divergent shell morphology in Spanish Littorina saxatilis ecotypes

Gene flow between diverging populations experiencing dissimilar ecological conditions can theoretically constrain adaptive evolution. To minimize the effect of gene flow, alleles underlying traits essential for local adaptation are predicted to be located in linked genome regions with reduced recombination. Local reduction in gene flow caused by selection is expected to produce elevated divergence in these regions. The highly divergent crab‐adapted and wave‐adapted ecotypes of the marine snail Littorina saxatilis present a model system to test these predictions. We used genome‐wide association (GWA) analysis of geometric morphometric shell traits associated with microgeographic divergence between the two L. saxatilis ecotypes within three separate sampling sites. A total of 477 snails that had individual geometric morphometric data and individual genotypes at 4,066 single nucleotide polymorphisms (SNPs) were analyzed using GWA methods that corrected for population structure among the three sites. This approach allowed dissection of the genomic architecture of shell shape divergence between ecotypes across a wide geographic range, spanning two glacial lineages. GWA revealed 216 quantitative trait loci (QTL) with shell size or shape differences between ecotypes, with most loci explaining a small proportion of phenotypic variation. We found that QTL were evenly distributed across 17 linkage groups, and exhibited elevated interchromosomal linkage, suggesting a genome‐wide response to divergent selection on shell shape between the two ecotypes. Shell shape trait‐associated loci showed partial overlap with previously identified outlier loci under divergent selection between the two ecotypes, supporting the hypothesis of diversifying selection on these genomic regions. These results suggest that divergence in shell shape between the crab‐adapted and wave‐adapted ecotypes is produced predominantly by a polygenic genomic architecture with positive linkage disequilibrium among loci of small effect.     

Allopatric and sympatric diversification within roach (Rutilus rutilus) of large pre‐alpine lakes

Intraspecific differentiation in response to divergent natural selection between environments is a common phenomenon in some northern freshwater fishes, especially salmonids and stickleback. Understanding why these taxa diversify and undergo adaptive radiations while most other fish species in the same environments do not, remains an open question. The possibility for intraspecific diversification has rarely been evaluated for most northern freshwater fish species. Here, we assess the potential for intraspecific differentiation between and within lake populations of roach (Rutilus rutilus)—a widespread and abundant cyprinid species—in lakes in which salmonids have evolved endemic adaptive radiations. Based on more than 3,000 polymorphic RADseq markers, we detected low but significant genetic differentiation between roach populations of two ultraoligotrophic lakes and between these and populations from other lakes. This, together with differentiation in head morphology and stable isotope signatures, suggests evolutionary and ecological differentiation among some of our studied populations. Next, we tested for intralacustrine diversification of roach within Lake Brienz, the most pristine lake surveyed in this study. We found significant phenotypic evidence for ecological intralacustrine differentiation between roach caught over a muddy substrate and those caught over a rocky substrate. However, evidence for intralacustrine genetic differentiation is at best subtle and phenotypic changes may therefore be mostly plastic. Overall, our findings suggest roach can differ between ecologically distinct lakes, but the extent of intralacustrine ecological differentiation is weak, which contrasts with the strong differentiation among endemic species of whitefish in the same lakes.     

Fast Evolution from Precast Bricks: Genomics of Young Freshwater Populations of Threespine Stickleback Gasterosteus aculeatus

Adaptation is driven by natural selection; however, many adaptations are caused by weak selection acting over large timescales, complicating its study. Therefore, it is rarely possible to study selection comprehensively in natural environments. The threespine stickleback (Gasterosteus aculeatus) is a well-studied model organism with a short generation time, small genome size, and many genetic and genomic tools available. Within this originally marine species, populations have recurrently adapted to freshwater all over its range. This evolution involved extensive parallelism: pre-existing alleles that adapt sticklebacks to freshwater habitats, but are also present at low frequencies in marine populations, have been recruited repeatedly. While a number of genomic regions responsible for this adaptation have been identified, the details of selection remain poorly understood. Using whole-genome resequencing, we compare pooled genomic samples from marine and freshwater populations of the White Sea basin, and identify 19 short genomic regions that are highly divergent between them, including three known inversions. 17 of these regions overlap protein-coding genes, including a number of genes with predicted functions that are relevant for adaptation to the freshwater environment. We then analyze four additional independently derived young freshwater populations of known ages, two natural and two artificially established, and use the observed shifts of allelic frequencies to estimate the strength of positive selection. Adaptation turns out to be quite rapid, indicating strong selection acting simultaneously at multiple regions of the genome, with selection coefficients of up to 0.27. High divergence between marine and freshwater genotypes, lack of reduction in polymorphism in regions responsible for adaptation, and high frequencies of freshwater alleles observed even in young freshwater populations are all consistent with rapid assembly of G. aculeatus freshwater genotypes from pre-existing genomic regions of adaptive variation, with strong selection that favors this assembly acting simultaneously at multiple loci.     

The easy road to genome‐wide medium density SNP screening in a non‐model species: development and application of a 10 K SNP‐chip for the house sparrow (Passer domesticus)

With the advent of next generation sequencing, new avenues have opened to study genomics in wild populations of non‐model species. Here, we describe a successful approach to a genome‐wide medium density Single Nucleotide Polymorphism (SNP) panel in a non‐model species, the house sparrow (Passer domesticus), through the development of a 10 K Illumina iSelect HD BeadChip. Genomic DNA and cDNA derived from six individuals were sequenced on a 454 GS FLX system and generated a total of 1.2 million sequences, in which SNPs were detected. As no reference genome exists for the house sparrow, we used the zebra finch (Taeniopygia guttata) reference genome to determine the most likely position of each SNP. The 10 000 SNPs on the SNP‐chip were selected to be distributed evenly across 31 chromosomes, giving on average one SNP per 100 000 bp. The SNP‐chip was screened across 1968 individual house sparrows from four island populations. Of the original 10 000 SNPs, 7413 were found to be variable, and 99% of these SNPs were successfully called in at least 93% of all individuals. We used the SNP‐chip to demonstrate the ability of such genome‐wide marker data to detect population sub‐division, and compared these results to similar analyses using microsatellites. The SNP‐chip will be used to map Quantitative Trait Loci (QTL) for fitness‐related phenotypic traits in natural populations.     

Divergence with gene flow is driven by local adaptation to temperature and soil phosphorus concentration in teosinte subspecies (Zea mays parviglumis and Zea mays mexicana)

Patterns of genomic divergence between hybridizing taxa can be heterogeneous along the genome. Both differential introgression and local adaptation may contribute to this pattern. Here, we analysed two teosinte subspecies, Zea mays ssp. parviglumis and ssp. mexicana, to test whether their divergence has occurred in the face of gene flow and to infer which environmental variables have been important drivers of their ecological differentiation. We generated 9,780 DArTseqTM SNPs for 47 populations, and used an additional data set containing 33,454 MaizeSNP50 SNPs for 49 populations. With these data, we inferred features of demographic history and performed genome wide scans to determine the number of outlier SNPs associated with climate and soil variables. The two data sets indicate that divergence has occurred or been maintained despite continuous gene flow and/or secondary contact. Most of the significant SNP associations were to temperature and to phosphorus concentration in the soil. A large proportion of these candidate SNPs were located in regions of high differentiation that had been identified previously as putative inversions. We therefore propose that genomic differentiation in teosintes has occurred by a process of adaptive divergence, with putative inversions contributing to reduced gene flow between locally adapted populations.