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.

Similar forms often evolve repeatedly in nature, raising longstanding questions about the underlying mechanisms. Here we use repeated evolution in sticklebacks to identify a large set of genomic loci that change recurrently during colonization of new 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. Dramatic genotypic and phenotypic changes arise within 5-7 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 similar features are important for evolution across diverse taxa.

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.

Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human–chimpanzee divergence, we have generated human and chimpanzee autotetraploids and allotetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or autotetraploid iPSCs. Analysis of gene expression patterns in interspecific allotetraploid iPSCs shows that human–chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allotetraploid cells. We successfully generated derivative cells with nested deletions or interspecific recombination on the X chromosome. These studies confirm an important role for the X chromosome in trans regulation of expression differences between species and illustrate the potential of this system for more detailed cis and trans mapping of the molecular basis of human and chimpanzee evolution.

Understanding the mechanisms leading to new traits or additional features in organisms is a fundamental goal of evolutionary biology. We show that HOXDB regulatory changes have been used repeatedly in different fish genera to alter the length and number of the prominent dorsal spines used to classify stickleback species. In Gasterosteus aculeatus (typically ‘three-spine sticklebacks’), a variant HOXDB allele is genetically linked to shortening an existing spine and adding an additional spine. In Apeltes quadracus (typically ‘four-spine sticklebacks’), a variant HOXDB allele is associated with lengthening a spine and adding an additional spine in natural populations. The variant alleles alter the same non-coding enhancer region in the HOXDB locus but do so by diverse mechanisms, including single-nucleotide polymorphisms, deletions and transposable element insertions. The independent regulatory changes are linked to anterior expansion or contraction of HOXDB expression. We propose that associated changes in spine lengths and numbers are partial identity transformations in a repeating skeletal series that forms major defensive structures in fish. Our findings support the long-standing hypothesis that natural Hox gene variation underlies key patterning changes in wild populations and illustrate how different mutational mechanisms affecting the same region may produce opposite gene expression changes with similar phenotypic outcomes.

Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human-chimpanzee divergence, we have generated human and chimpanzee auto-tetraploids and allo-tetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or auto-tetraploid iPSCs. Analysis of gene expression patterns in inter-specific allo-tetraploid iPSCs shows that human-chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves, and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allo-tetraploid cells. We successfully generated derivative cells with nested deletions or inter-specific recombination on the X chromosome. These studies identify a long distance cis-regulatory domain of the Fragile X-associated gene (FMR1), confirm an important role for the X chromosome in trans-regulation of other expression differences, and illustrate the potential of this system for more detailed mapping of the molecular basis of human and chimpanzee evolution.

SummaryUnderstanding the genetic mechanisms leading to new traits is a fundamental goal of evolutionary biology. We show that HOXDB regulatory changes have been used repeatedly in different stickleback fish species to alter the length and number of bony dorsal spines. In Gasterosteus aculeatus, a variant HOXDB allele is genetically linked to shortening an existing spine and adding a spine. In Apeltes quadracus, a variant allele is associated with lengthening an existing spine and adding a spine. The alleles alter the same conserved non-coding HOXDB enhancer by diverse molecular mechanisms, including SNPs, deletions, and transposable element insertions. The independent cis-acting regulatory changes are linked to anterior expansion or contraction of HOXDB expression. Our findings support the long-standing hypothesis that natural Hox gene variation underlies key morphological patterning changes in wild populations and illustrate how different mutational mechanisms affecting the same region may produce opposite gene expression changes with similar phenotypic outcomes.Abstract Figure

<p>The intersection of environmental conditions with the conditions permissive for life defines habitability. Consequently, our understanding of habitability is fundamentally limited by our understanding of the multidimensional niche space for life, which up to now, is based on our one known data point: life on Earth. Terrestrial life has evolved to tolerate environmental conditions found on Earth, and as most physiological studies are limited to extant organisms, it is likely that life has potential for a far broader niche space than observed today.</p><p>Potentially habitable extraterrestrial environments present challenges not only in single environmental dimensions (temperature, pH, radiation, etc.), but also in combination. For example, Martian brines feature both low temperature and high concentrations of perchlorate, while Venusian clouds feature both desiccating conditions and extreme acidity. We do not know whether the inability of known life to reproduce under analogous conditions reflects a fundamental boundary condition or simply a lack of terrestrial selection pressure. A mixture of environmental challenges may be similarly common among exoplanets and other potentially habitable environments within our solar system.</p><p>We are addressing this key gap in our understanding of habitability by using adaptive laboratory evolution, functional metagenomics, and synthetic biology to expand the known environmental limits of life. First, we are determining and pushing the limits of pH (acidic and basic), salt (both chloride and perchlorate), and UV tolerance individually and in combination with temperature for <em>B. subtilis</em>, <em>E. coli</em>, and <em>D. radiodurans </em>through adaptive laboratory evolution. This will define a multidimensional niche-space for these organisms and assess how firm these boundaries are. Second, we are taking advantage of the rich genetic diversity present on Earth to identify genetic elements providing transferable survival benefits under extreme environmental conditions. One of the most powerful resources available to us for this endeavor to expand the boundary conditions of life is the extensive biodiversity present on Earth, particularly those capable of surviving in extreme environments. Prior work demonstrates that extremophile genes can expand an organism’s niche space, including increased resistance to desiccation, salinity, radiation, and low temperatures. However, despite all we have learned from them, at present it remains difficult and laborious to characterize their genetic mechanisms of adaptation and test their ability to facilitate an enlarged environmental niche. Through a combination of cDNA- and DNA-based libraries, we aim to establish a high throughput method of assaying novel organisms for additional mechanisms of expanding the niche-space of life. Third, we will use codon-optimized cassettes containing genes either identified in our screen or from published research to verify the synthetic acquisition of functional capabilities and to test if the same genetic constructs can expand the niche-space of multiple species.</p><p>Through these approaches, we will provide both selection pressure and genetic resources to challenge life to evolve beyond environmental conditions found naturally on Earth. Such work will improve our understanding of what environmental conditions are compatible with life as we know it and allow firm reclassification of some extraterrestrial environments from “probably habitable” to “definitely habitable.”</p>