Evolving New Skeletal Traits by cis-Regulatory Changes in Bone Morphogenetic Proteins.

Changes in bone size and shape are defining features of many vertebrates. Here we use genetic crosses and comparative genomics to identify specific regulatory DNA alterations controlling skeletal evolution. Armor bone-size differences in sticklebacks map to a major effect locus overlapping BMP family member GDF6. Freshwater fish express more GDF6 due in part to a transposon insertion, and transgenic overexpression of GDF6 phenocopies evolutionary changes in armor-plate size. The human GDF6 locus also has undergone distinctive regulatory evolution, including complete loss of an enhancer that is otherwise highly conserved between chimps and other mammals. Functional tests show that the ancestral enhancer drives expression in hindlimbs but not forelimbs, in locations that have been specifically modified during the human transition to bipedalism. Both gain and loss of regulatory elements can localize BMP changes to specific anatomical locations, providing a flexible regulatory basis for evolving species-specific changes in skeletal form.

Reproductive isolation in a three-way contact zone.

Contact zones between divergent forms within a species provide insight into the role of gene flow in adaptation and speciation. Previous work has focused on contact zones involving only two divergent forms, but in nature, many more than two populations may overlap simultaneously and experience gene flow. Patterns of introgression in wild populations are, therefore, likely much more complicated than is often assumed. We begin to address this gap in current knowledge by investigating patterns of divergence and introgression across a complex natural contact zone. We use phenotypic and genomic data to confirm the existence of a three-way contact zone among divergent freshwater resident, saltwater resident and saltwater migratory three-spined stickleback (Gasterosteus aculeatus) on the island of North Uist, Scottish Western Isles. We find evidence for hybridization, mostly between saltwater resident and saltwater migratory forms. Despite hybridization, genomic analyses reveal pairwise islands of divergence between all forms that are maintained across the contact zone. Genomic cline analyses also provide evidence for selection and/or hybrid incompatibilities in divergent regions. Divergent genomic regions occur across multiple chromosomes and involve many known adaptive loci and several chromosomal inversions. We also identify distinct immune gene expression profiles between forms, but no evidence for transgressive expression in hybrids. Our results suggest that reproductive isolation is maintained in this three-way contact zone, despite some hybridization, and that reduced recombination in chromosomal inversions may play an important role in maintaining this isolation.

Longer or shorter spines: Reciprocal trait evolution in stickleback via triallelic regulatory changes in Stanniocalcin2a.

Vertebrates have repeatedly modified skeletal structures to adapt to their environments. The threespine stickleback is an excellent system for studying skeletal modifications, as different wild populations have either increased or decreased the lengths of their prominent dorsal and pelvic spines in different freshwater environments. Here we identify a regulatory locus that has a major morphological effect on the length of stickleback dorsal and pelvic spines, which we term Maser (major spine enhancer). Maser maps in a closely linked supergene complex that controls multiple armor, feeding, and behavioral traits on chromosome IV. Natural alleles in Maser are differentiated between marine and freshwater sticklebacks; however, alleles found among freshwater populations are also differentiated, with distinct alleles found in short- and long-spined freshwater populations. The distinct freshwater alleles either increase or decrease expression of the bone growth inhibitor gene Stanniocalcin2a in developing spines, providing a simple genetic mechanism for either increasing or decreasing spine lengths in natural populations. Genomic surveys suggest many recurrently differentiated loci in sticklebacks are similarly specialized into three or more distinct alleles, providing multiple ancient standing variants in particular genes that may contribute to a range of phenotypes in different environments.

Fitness maps to a large-effect locus in introduced stickleback populations.

Mutations of small effect underlie most adaptation to new environments, but beneficial variants with large fitness effects are expected to contribute under certain conditions. Genes and genomic regions having large effects on phenotypic differences between populations are known from numerous taxa, but fitness effect sizes have rarely been estimated. We mapped fitness over a generation in an F2 intercross between a marine and a lake stickleback population introduced to a freshwater pond. A quantitative trait locus map of the number of surviving offspring per F2 female detected a single, large-effect locus near Ectodysplasin (Eda), a gene having an ancient freshwater allele causing reduced bony armor and other changes. F2 females homozygous for the freshwater allele had twice the number of surviving offspring as homozygotes for the marine allele, producing a large selection coefficient, s = 0.50 ± 0.09 SE. Correspondingly, the frequency of the freshwater allele increased from 0.50 in F2 mothers to 0.58 in surviving offspring. We compare these results to allele frequency changes at the Eda gene in an Alaskan lake population colonized by marine stickleback in the 1980s. The frequency of the freshwater Eda allele rose steadily over multiple generations and reached 95% within 20 y, yielding a similar estimate of selection, s = 0.49 ± 0.05, but a different degree of dominance. These findings are consistent with other studies suggesting strong selection on this gene (and/or linked genes) in fresh water. Selection on ancient genetic variants carried by colonizing ancestors is likely to increase the prevalence of large-effect fitness variants in adaptive evolution.

Detecting differential copy number variation between groups of samples.

We present a method to detect copy number variants (CNVs) that are differentially present between two groups of sequenced samples. We use a finite-state transducer where the emitted read depth is conditioned on the mappability and GC-content of all reads that occur at a given base position. In this model, the read depth within a region is a mixture of binomials, which in simulations matches the read depth more closely than the often-used negative binomial distribution. The method analyzes all samples simultaneously, preserving uncertainty as to the breakpoints and magnitude of CNVs present in an individual when it identifies CNVs differentially present between the two groups. We apply this method to identify CNVs that are recurrently associated with postglacial adaptation of marine threespine stickleback (Gasterosteus aculeatus) to freshwater. We identify 6664 regions of the stickleback genome, totaling 1.7 Mbp, which show consistent copy number differences between marine and freshwater populations. These deletions and duplications affect both protein-coding genes and cis-regulatory elements, including a noncoding intronic telencephalon enhancer of DCHS1 The functions of the genes near or included within the 6664 CNVs are enriched for immunity and muscle development, as well as head and limb morphology. Although freshwater stickleback have repeatedly evolved from marine populations, we show that freshwater stickleback also act as reservoirs for ancient ancestral sequences that are highly conserved among distantly related teleosts, but largely missing from marine stickleback due to recent selective sweeps in marine populations.

Convergent evolution of SWS2 opsin facilitates adaptive radiation of threespine stickleback into different light environments.

Repeated adaptation to a new environment often leads to convergent phenotypic changes whose underlying genetic mechanisms are rarely known. Here, we study adaptation of color vision in threespine stickleback during the repeated postglacial colonization of clearwater and blackwater lakes in the Haida Gwaii archipelago. We use whole genomes from 16 clearwater and 12 blackwater populations, and a selection experiment, in which stickleback were transplanted from a blackwater lake into an uninhabited clearwater pond and resampled after 19 y to test for selection on cone opsin genes. Patterns of haplotype homozygosity, genetic diversity, site frequency spectra, and allele-frequency change support a selective sweep centered on the adjacent blue- and red-light sensitive opsins SWS2 and LWS. The haplotype under selection carries seven amino acid changes in SWS2, including two changes known to cause a red-shift in light absorption, and is favored in blackwater lakes but disfavored in the clearwater habitat of the transplant population. Remarkably, the same red-shifting amino acid changes occurred after the duplication of SWS2 198 million years ago, in the ancestor of most spiny-rayed fish. Two distantly related fish species, bluefin killifish and black bream, express these old paralogs divergently in black- and clearwater habitats, while sticklebacks lost one paralog. Our study thus shows that convergent adaptation to the same environment can involve the same genetic changes on very different evolutionary time scales by reevolving lost mutations and reusing them repeatedly from standing genetic variation.

Genomics and the origin of species.

Speciation is a fundamental evolutionary process, the knowledge of which is crucial for understanding the origins of biodiversity. Genomic approaches are an increasingly important aspect of this research field. We review current understanding of genome-wide effects of accumulating reproductive isolation and of genomic properties that influence the process of speciation. Building on this work, we identify emergent trends and gaps in our understanding, propose new approaches to more fully integrate genomics into speciation research, translate speciation theory into hypotheses that are testable using genomic tools and provide an integrative definition of the field of speciation genomics.

Genetics of ecological divergence during speciation.

Ecological differences often evolve early in speciation as divergent natural selection drives adaptation to distinct ecological niches, leading ultimately to reproductive isolation. Although this process is a major generator of biodiversity, its genetic basis is still poorly understood. Here we investigate the genetic architecture of niche differentiation in a sympatric species pair of threespine stickleback fish by mapping the environment-dependent effects of phenotypic traits on hybrid feeding and performance under semi-natural conditions. We show that multiple, unlinked loci act largely additively to determine position along the major niche axis separating these recently diverged species. We also find that functional mismatch between phenotypic traits reduces the growth of some stickleback hybrids beyond that expected from an intermediate phenotype, suggesting a role for epistasis between the underlying genes. This functional mismatch might lead to hybrid incompatibilities that are analogous to those underlying intrinsic reproductive isolation but depend on the ecological context.

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.

A role for a neo-sex chromosome in stickleback speciation.

Sexual antagonism, or conflict between the sexes, has been proposed as a driving force in both sex-chromosome turnover and speciation. Although closely related species often have different sex-chromosome systems, it is unknown whether sex-chromosome turnover contributes to the evolution of reproductive isolation between species. Here we show that a newly evolved sex chromosome contains genes that contribute to speciation in threespine stickleback fish (Gasterosteus aculeatus). We first identified a neo-sex chromosome system found only in one member of a sympatric species pair in Japan. We then performed genetic linkage mapping of male-specific traits important for reproductive isolation between the Japanese species pair. The neo-X chromosome contains loci for male courtship display traits that contribute to behavioural isolation, whereas the ancestral X chromosome contains loci for both behavioural isolation and hybrid male sterility. Our work not only provides strong evidence for a large X-effect on reproductive isolation in a vertebrate system, but also provides direct evidence that a young neo-X chromosome contributes to reproductive isolation between closely related species. Our data indicate that sex-chromosome turnover might have a greater role in speciation than was previously appreciated.

Fine-scale contemporary recombination variation and its fitness consequences in adaptively diverging stickleback fish.

Despite deep evolutionary conservation, recombination rates vary greatly across the genome and among individuals, sexes and populations. Yet the impact of this variation on adaptively diverging populations is not well understood. Here we characterized fine-scale recombination landscapes in an adaptively divergent pair of marine and freshwater populations of threespine stickleback from River Tyne, Scotland. Through whole-genome sequencing of large nuclear families, we identified the genomic locations of almost 50,000 crossovers and built recombination maps for marine, freshwater and hybrid individuals at a resolution of 3.8 kb. We used these maps to quantify the factors driving variation in recombination rates. We found strong heterochiasmy between sexes but also differences in recombination rates among ecotypes. Hybrids showed evidence of significant recombination suppression in overall map length and in individual loci. Recombination rates were lower not only within individual marine-freshwater-adaptive loci, but also between loci on the same chromosome, suggesting selection on linked gene 'cassettes'. Through temporal sampling along a natural hybrid zone, we found that recombinants showed traits associated with reduced fitness. Our results support predictions that divergence in cis-acting recombination modifiers, whose functions are disrupted in hybrids, may play an important role in maintaining differences among adaptively diverging populations.

Late Pleistocene stickleback environmental genomes reveal the chronology of freshwater adaptation.

Directly observing the chronology and tempo of adaptation in response to ecological change is rarely possible in natural ecosystems. Sedimentary ancient DNA (sedaDNA) has been shown to be a tractable source of genome-scale data of long-dead organisms1,2,3 and to thereby potentially provide an understanding of the evolutionary histories of past populations.4,5 To date, time series of ecosystem biodiversity have been reconstructed from sedaDNA, typically using DNA metabarcoding or shotgun sequence data generated from less than 1 g of sediment.6,7 Here, we maximize sequence coverage by extracting DNA from ∼50× more sediment per sample than the majority of previous studies1,2,3 to achieve genotype resolution. From a time series of Late Pleistocene sediments spanning from a marine to freshwater ecosystem, we compare adaptive genotypes reconstructed from the environmental genomes of three-spined stickleback at key time points of this transition. We find a staggered temporal dynamic in which freshwater alleles at known loci of large effect in marine-freshwater divergence of three-spined stickleback (e.g., EDA)8 were already established during the brackish phase of the formation of the isolation basin. However, marine alleles were still detected across the majority of marine-freshwater divergence-associated loci, even after the complete isolation of the lake from marine ingression. Our retrospective approach to studying adaptation from environmental genomes of three-spined sticklebacks at the end of the last glacial period complements contemporary experimental approaches9,10,11 and highlights the untapped potential for retrospective "evolve and resequence" natural experiments using sedaDNA.

Phylogeography and adaptation genetics of stickleback from the Haida Gwaii archipelago revealed using genome-wide single nucleotide polymorphism genotyping.

Threespine stickleback populations are model systems for studying adaptive evolution and the underlying genetics. In lakes on the Haida Gwaii archipelago (off western Canada), stickleback have undergone a remarkable local radiation and show phenotypic diversity matching that seen throughout the species distribution. To provide a historical context for this radiation, we surveyed genetic variation at >1000 single nucleotide polymorphism (SNP) loci in stickleback from over 100 populations. SNPs included markers evenly distributed throughout genome and candidate SNPs tagging adaptive genomic regions. Based on evenly distributed SNPs, the phylogeographic pattern differs substantially from the disjunct pattern previously observed between two highly divergent mtDNA lineages. The SNP tree instead shows extensive within watershed population clustering and different watersheds separated by short branches deep in the tree. These data are consistent with separate colonizations of most watersheds, despite underlying genetic connections between some independent drainages. This supports previous suppositions that morphological diversity observed between watersheds has been shaped independently, with populations exhibiting complete loss of lateral plates and giant size each occurring in several distinct clades. Throughout the archipelago, we see repeated selection of SNPs tagging candidate freshwater adaptive variants at several genomic regions differentiated between marine-freshwater populations on a global scale (e.g. EDA, Na/K ATPase). In estuarine sites, both marine and freshwater allelic variants were commonly detected. We also found typically marine alleles present in a few freshwater lakes, especially those with completely plated morphology. These results provide a general model for postglacial colonization of freshwater habitat by sticklebacks and illustrate the tremendous potential of genome-wide SNP data sets hold for resolving patterns and processes underlying recent adaptive divergences.

Population genomics of parallel phenotypic evolution in stickleback across stream-lake ecological transitions.

Understanding the genetics of adaptation is a central focus in evolutionary biology. Here, we use a population genomics approach to examine striking parallel morphological divergences of parapatric stream-lake ecotypes of threespine stickleback fish in three watersheds on the Haida Gwaii archipelago, western Canada. Genome-wide variation at greater than 1000 single nucleotide polymorphism loci indicate separate origin of giant lake and small-bodied stream fish within each watershed (mean F(ST) between watersheds = 0.244 and within = 0.114). Genome scans within watersheds identified a total of 21 genomic regions that are highly differentiated between ecotypes and are probably subject to directional selection. Most outliers were watershed-specific, but genomic regions undergoing parallel genetic changes in multiple watersheds were also identified. Interestingly, several of the stream-lake outlier regions match those previously identified in marine-freshwater and benthic-limnetic genome scans, indicating reuse of the same genetic loci in different adaptive scenarios. We also identified multiple new outlier loci, which may contribute to unique aspects of differentiation in stream-lake environments. Overall, our data emphasize the important role of ecological boundaries in driving both local and broadly occurring parallel genetic changes during adaptation.

Genomic changes underlying repeated niche shifts in an adaptive radiation.

In adaptive radiations, single lineages rapidly diversify by adapting to many new niches. Little is known yet about the genomic mechanisms involved, that is, the source of genetic variation or genomic architecture facilitating or constraining adaptive radiation. Here, we investigate genomic changes associated with repeated invasion of many different freshwater niches by threespine stickleback in the Haida Gwaii archipelago, Canada, by resequencing single genomes from one marine and 28 freshwater populations. We find 89 likely targets of parallel selection in the genome that are enriched for old standing genetic variation. In contrast to theoretical expectations, their genomic architecture is highly dispersed with little clustering. Candidate genes and genotype-environment correlations match the three major environmental axes predation regime, light environment, and ecosystem size. In a niche space with these three dimensions, we find that the more divergent a new niche from the ancestral marine habitat, the more loci show signatures of parallel selection. Our findings suggest that the genomic architecture of parallel adaptation in adaptive radiation depends on the steepness of ecological gradients and the dimensionality of the niche space.

Ancient and modern stickleback genomes reveal the demographic constraints on adaptation.

Adaptation is typically studied by comparing modern populations with contrasting environments. Individuals persisting in the ancestral habitat are typically used to represent the ancestral founding population; however, it has been questioned whether these individuals are good proxies for the actual ancestors.1 To address this, we applied a paleogenomics approach2 to directly access the ancestral genepool: partially sequencing the genomes of two 11- to 13,000-year-old stickleback recovered from the transitionary layer between marine and freshwater sediments of two Norwegian isolation lakes3 and comparing them with 30 modern stickleback genomes from the same lakes and adjacent marine fjord, in addition to a global dataset of 20 genomes.4 The ancient stickleback shared genome-wide ancestry with the modern fjord population, whereas modern lake populations have lost substantial ancestral variation following founder effects, and subsequent drift and selection. Freshwater-adaptive alleles found in one ancient stickleback genome have not risen to high frequency in the present-day population from the same lake. Comparison to the global dataset suggested incomplete adaptation to freshwater in our modern lake populations. Our findings reveal the impact of population bottlenecks in constraining adaptation due to reduced efficacy of selection on standing variation present in founder populations.

Predicting future from past: The genomic basis of recurrent and rapid stickleback evolution.

Similar forms often evolve repeatedly in nature, raising long-standing questions about the underlying mechanisms. Here, we use repeated evolution in stickleback to identify a large set of genomic loci that change recurrently during colonization of freshwater habitats by marine fish. The same loci used repeatedly in extant populations also show rapid allele frequency changes when new freshwater populations are experimentally established from marine ancestors. Marked genotypic and phenotypic changes arise within 5 years, facilitated by standing genetic variation and linkage between adaptive regions. Both the speed and location of changes can be predicted using empirical observations of recurrence in natural populations or fundamental genomic features like allelic age, recombination rates, density of divergent loci, and overlap with mapped traits. A composite model trained on these stickleback features can also predict the location of key evolutionary loci in Darwin's finches, suggesting that similar features are important for evolution across diverse taxa.

Predominance of cis-regulatory changes in parallel expression divergence of sticklebacks.

Regulation of gene expression is thought to play a major role in adaptation, but the relative importance of cis- and trans- regulatory mechanisms in the early stages of adaptive divergence is unclear. Using RNAseq of threespine stickleback fish gill tissue from four independent marine-freshwater ecotype pairs and their F1 hybrids, we show that cis-acting (allele-specific) regulation consistently predominates gene expression divergence. Genes showing parallel marine-freshwater expression divergence are found near to adaptive genomic regions, show signatures of natural selection around their transcription start sites and are enriched for cis-regulatory control. For genes with parallel increased expression among freshwater fish, the quantitative degree of cis- and trans-regulation is also highly correlated across populations, suggesting a shared genetic basis. Compared to other forms of regulation, cis-regulation tends to show greater additivity and stability across different genetic and environmental contexts, making it a fertile substrate for the early stages of adaptive evolution.

Genome-wide recombination map construction from single individuals using linked-read sequencing.

Meiotic recombination rates vary across the genome, often involving localized crossover hotspots and coldspots. Studying the molecular basis and mechanisms underlying this variation has been challenging due to the high cost and effort required to construct individualized genome-wide maps of recombination crossovers. Here we introduce a new method, called ReMIX, to detect crossovers from gamete DNA of a single individual using Illumina sequencing of 10X Genomics linked-read libraries. ReMIX reconstructs haplotypes and identifies the valuable rare molecules spanning crossover breakpoints, allowing quantification of the genomic location and intensity of meiotic recombination. Using a single mouse and stickleback fish, we demonstrate how ReMIX faithfully recovers recombination hotspots and landscapes that have previously been built using hundreds of offspring. ReMIX provides a high-resolution, high-throughput, and low-cost approach to quantify recombination variation across the genome, providing an exciting opportunity to study recombination among multiple individuals in diverse organisms.