Brian R. Summers

The genomic basis of adaptive evolution in threespine sticklebacks

Marine stickleback fish have colonized and adapted to thousands of streams and lakes formed since the last ice age, providing an exceptional opportunity to characterize genomic mechanisms underlying repeated ecological adaptation in nature. Here we develop a high-quality reference genome assembly for threespine sticklebacks. By sequencing the genomes of twenty additional individuals from a global set of marine and freshwater populations, we identify a genome-wide set of loci that are consistently associated with marine–freshwater divergence. Our results indicate that reuse of globally shared standing genetic variation, including chromosomal inversions, has an important role in repeated evolution of distinct marine and freshwater sticklebacks, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. Both coding and regulatory changes occur in the set of loci underlying marine–freshwater evolution, but regulatory changes appear to predominate in this well known example of repeated adaptive evolution in nature.

THE GENETICS OF ADAPTIVE SHAPE SHIFT IN STICKLEBACK: PLEIOTROPY AND EFFECT SIZE

Abstract The distribution of effect sizes of genes underlying adaptation is unknown (Orr 2005). Are suites of traits that diverged under natural selection controlled by a few pleiotropic genes of large effect (major genes model), by many independently acting genes of small effect (infinitesimal model), or by a combination, with frequency inversely related to effect size (geometric model)? To address this we carried out a quantitative trait loci (QTL) study of a suite of 54 position traits describing body shapes of two threespine stickleback species: an ancestral Pacific marine form and a highly derived benthic species inhabiting a geologically young lake. About half of the 26 detected QTL affected just one coordinate and had small net effects, but several genomic regions affected multiple aspects of shape and had large net effects. The distribution of effect sizes followed the gamma distribution, as predicted by the geometric model of adaptation when detection limits are taken into account. The sex-determining chromosome region had the largest effect of any QTL. Ancestral sexual dimorphism was similar to the direction of divergence, and was largely eliminated during freshwater adaptation, suggesting that sex differences may provide variation upon which selection can act. Several shape QTL are linked to Eda, a major gene responsible for reduction of lateral body armor in freshwater. Our results are consistent with predictions of the geometric model of adaptation. Shape evolution in stickleback results from a few genes with large and possibly widespread effects and multiple genes of smaller effect.

Modular Skeletal Evolution in Sticklebacks Is Controlled by Additive and Clustered Quantitative Trait Loci

Understanding the genetic architecture of evolutionary change remains a long-standing goal in biology. In vertebrates, skeletal evolution has contributed greatly to adaptation in body form and function in response to changing ecological variables like diet and predation. Here we use genome-wide linkage mapping in threespine stickleback fish to investigate the genetic architecture of evolved changes in many armor and trophic traits. We identify >100 quantitative trait loci (QTL) controlling the pattern of serially repeating skeletal elements, including gill rakers, teeth, branchial bones, jaws, median fin spines, and vertebrae. We use this large collection of QTL to address long-standing questions about the anatomical specificity, genetic dominance, and genomic clustering of loci controlling skeletal differences in evolving populations. We find that most QTL (76%) that influence serially repeating skeletal elements have anatomically regional effects. In addition, most QTL (71%) have at least partially additive effects, regardless of whether the QTL controls evolved loss or gain of skeletal elements. Finally, many QTL with high LOD scores cluster on chromosomes 4, 20, and 21. These results identify a modular system that can control highly specific aspects of skeletal form. Because of the general additivity and genomic clustering of major QTL, concerted changes in both protective armor and trophic traits may occur when sticklebacks inherit either marine or freshwater alleles at linked or possible “supergene” regions of the stickleback genome. Further study of these regions will help identify the molecular basis of both modular and coordinated changes in the vertebrate skeleton.

GENETIC SIGNATURE OF ADAPTIVE PEAK SHIFT IN THREESPINE STICKLEBACK

Transition of an evolving population to a new adaptive optimum is predicted to leave a signature in the distribution of effect sizes of fixed mutations. If they affect many traits (are pleiotropic), large effect mutations should contribute more when a population evolves to a farther adaptive peak than to a nearer peak. We tested this prediction in wild threespine stickleback fish (Gasterosteus aculeatus) by comparing the estimated frequency of large effect genetic changes underlying evolution as the same ancestor adapted to two lake types since the end of the ice age. A higher frequency of large effect genetic changes (quantitative trait loci) contributed to adaptive evolution in populations that adapted to lakes representing a more distant optimum than to lakes in which the optimum phenotype was nearer to the ancestral state. Our results also indicate that pleiotropy, not just optimum overshoot, contributes to this difference. These results suggest that a series of adaptive improvements to a new environment leaves a detectable mark in the genome of wild populations. Although not all assumptions of the theory are likely met in natural systems, the prediction may be robust enough to the complexities of natural environments to be useful when forecasting adaptive responses to large environmental changes.

A recurrent regulatory change underlying altered expression and Wnt response of the stickleback armor plates gene EDA

Armor plate changes in sticklebacks are a classic example of repeated adaptive evolution. Previous studies identified ectodysplasin (EDA) gene as the major locus controlling recurrent plate loss in freshwater fish, though the causative DNA alterations were not known. Here we show that freshwater EDA alleles have cis-acting regulatory changes that reduce expression in developing plates and spines. An identical T → G base pair change is found in EDA enhancers of divergent low-plated fish. Recreation of the T → G change in a marine enhancer strongly reduces expression in posterior armor plates. Bead implantation and cell culture experiments show that Wnt signaling strongly activates the marine EDA enhancer, and the freshwater T → G change reduces Wnt responsiveness. Thus parallel evolution of low-plated sticklebacks has occurred through a shared DNA regulatory change, which reduces the sensitivity of an EDA enhancer to Wnt signaling, and alters expression in developing armor plates while preserving expression in other tissues. DOI: http://dx.doi.org/10.7554/eLife.05290.001

A Simple and Efficient Microinjection Protocol for Making Transgenic Sticklebacks

Gasterosteus aculeatus , the threespine stickleback, has been used for many years to study vertebrate behavior, physiology, evolution and ecology. Further studies of this organism at the molecular level would be greatly enhanced by methods to increase or decrease the activity of specific genes, or to transfer genes between fish with different morphologies. We have tested a variety of methods for microinjection of DNA into 1-2 cell stickleback embryos. Holding the embryos in place using a custom-made glass chamber made it possible to penetrate the chorion with a glass capillary needle, and inject small volumes of DNA solutions into the blastodisc region of the fertilized egg. To test for expression and maintenance of DNA following injection, we injected a DNA plasmid containing the zebrafish muscle specific (α) actin promoter fused to the coding region of a jellyfish green fluorescent protein (GFP) gene, together with the rare cutting endonuclease, ISceI . Injected sticklebacks expressed the GFP reporter gene in developing muscle, maintained expression for at least 6 months, and transmitted the GFP construct to their own progeny when raised to sexual maturity. Further use of this method should make it possible to introduce a variety of constructs into developing sticklebacks and to study the molecular basis of the interesting morphological, behavioral, and physiological differences among recently evolved stickleback forms.

Dorsal spine evolution in threespine sticklebacks via a splicing change in MSX2A

BackgroundDorsal spine reduction in threespine sticklebacks (Gasterosteus aculeatus) is a classic example of recurrent skeletal evolution in nature. Sticklebacks in marine environments typically have long spines that form part of their skeletal armor. Many derived freshwater populations have evolved shorter spines. Changes in spine length are controlled in part by a quantitative trait locus (QTL) previously mapped to chromosome 4, but the causative gene and mutations underlying the repeated evolution of this interesting skeletal trait have not been identified.ResultsRefined mapping of the spine length QTL shows that it lies near the MSX2A transcription factor gene. MSX2A is expressed in developing spines. In F1 marine × freshwater fish, the marine allele is preferentially expressed.Differences in expression can be attributed to splicing regulation. Due to the use of an alternative 5 ′ splice site within the first exon, the freshwater allele produces greater amounts of a shortened, non-functional transcript and makes less of the full-length transcript. Sequence changes in the MSX2A region are shared by many freshwater fish, suggesting that repeated evolution occurs by reuse of a spine-reduction variant.To demonstrate the effect of full-length MSX2A on spine length, we produced transgenic freshwater fish expressing a copy of marine MSX2A. The spines of the transgenic fish were significantly longer on average than those of their non-transgenic siblings, partially reversing the reduced spine lengths that have evolved in freshwater populations.ConclusionsMSX2A is a major gene underlying dorsal spine reduction in freshwater sticklebacks. The gene is linked to a separate gene controlling bony plate loss, helping explain the concerted effects of chromosome 4 on multiple armor-reduction traits. The nature of the molecular changes provides an interesting example of morphological evolution occurring not through a simple amino acid change, nor through a change only in gene expression levels, but through a change in the ratio of splice products encoding both normal and truncated proteins.