The genomic basis of adaptive evolution in threespine sticklebacks

Abstract

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.

Figure 1. Genome scans for parallel marine-freshwater divergence

a. Marine (red) and freshwater (blue) stickleback populations were surveyed from diverse locations. b. Morphometric analysis was used to select individuals for re-sequencing. The 20 chosen individuals are from multiple geographically-proximate pairs of populations with typical marine and freshwater morphology (solid symbols). Points: population mean morphologies; ellipses: 95% confidence intervals for ecotypes. c. Genomes were analysed using SOM/HMM (upper) and CSS (lower) methods to identify parallel marine-freshwater divergent regions. Across most of the genome, the dominant patterns reflect neutral divergence or geographic structure. In contrast, <0.5% of the genome show haplotype-ecotype association, a pattern characteristic of divergent marine and freshwater adaptation via parallel reuse of standing genetic variation^,.

Figure 2. Parallel divergence signals at known armour plates locus

a. Ensembl gene models around EDA. b. Visual genotypes for sequenced fish [homozygous sites for most frequent allele in marine fish (red); homozygous for alternate allele (blue); heterozygous (yellow), or nonvariable/missing/repeat-masked data (white)]. c. DDC cluster assignments for marine (red) and freshwater populations (blue). Most fish are assigned to cluster k1, except in boxed region, where freshwater fish are assigned to a distinct cluster (k2). d. SOM/HMM analysis supports patterns of divergence with a marine-freshwater-like tree topology in the centre, but not edges, of the window (trees a-d). Similar support is shown by CSS analysis (e) and its associated P-value (f). The combined analyses define a consensus 16 kb region shared in freshwater fish (vertical shaded box), matching the minimal haplotype known to control repeated low armour evolution in sticklebacks.

Figure 3. Genome-wide distribution of marine-freshwater divergence regions

Whole-genome profiles of SOM/HMM and CSS analyses reveal many loci distributed on multiple chromosomes (plus unlinked scaffolds, here grouped as "ChrUn"). Extended regions of marine-freshwater divergence on chrI, XI, and XXI correspond to inversions (red arrows). Marine-freshwater divergent regions detected by CSS are shown as grey peaks with grey points above chromosomes indicating regions of significant marine-freshwater divergence (FDR 0.05). Genomic regions with marine-freshwater-like tree topologies detected by SOM/HMM are shown as green points below chromosomes.

Figure 4. How much of local marine-freshwater adaptation occurs by reuse of global variants?

a. Classic marine and freshwater ecotypes are maintained in downstream and upstream locations of the River Tyne, despite extensive hybridization at intermediate sites. b. Pairwise sequence comparisons identify many genomic regions that show high divergence between upstream and downstream fish (X-axis). Many, but not all, of these regions also show high global marine-freshwater divergence (Y-axis; red points indicate significant CSS FDR<0.05), indicating that both global and local variants contribute to formation and reproductive isolation of a marine-freshwater species pair.

Figure 5. Chromosome inversions and marine-freshwater divergence

a. Multiple marine BAC clones have paired-end reads that place anomalously against the freshwater reference genome (grey arrows below chromosome bars; see Supplementary Methods for BAC names). b. Intrachromosomal inversions on chrI, XI, and XXI resolve orientation and size anomalies for all marine clones. c. The chrXI inversion breakpoints map inside the exons of KCNH4, a potassium transporter gene. Duplicated 3’ exons lead to different transcript orientations and gene products in marine (red gene model) and freshwater fish (blue gene model). d. The chrXXI inversion occurs in a region with separate QTLs controlling armour plate number and body shape^,, traits that differ between marine and freshwater fish.

Figure 6. Contributions of coding and regulatory changes to parallel marine-freshwater stickleback adaptation

a. A genome-wide set of marine-freshwater loci recovered by both SOM/HMM and CSS analyses includes regions with consistent amino acid substitutions between marine and freshwater ecotypes (yellow sector); regions with no predicted coding sequence (green sector); and regions with both coding and non-coding sequences, but no consistent marine-freshwater amino acid substitutions (grey). b. Genome-wide expression analysis shows that marine-freshwater regions identified by SOM/HMM or CSS analyses are enriched for genes showing significant expression differences in 6 out of 7 tissues between marine LITC and freshwater FTC fish (observed: grey bars; expected: white bars; * P<0.01, **P<0.001, ***P<0.0001, ****P≪0.00001), consistent with a role for regulatory changes in marine-freshwater evolution.