Many familiar traits in the natural world—from lions’ manes to the longevity of bristlecone pine trees—arose in the distant past, and have long since fixed in their respective species. A key challenge in evolutionary genetics is to figure out how and why species-defining traits have come to be. We used the thermotolerance growth advantage of the yeast Saccharomyces cerevisiae over its sister species Saccharomyces paradoxus as a model for addressing these questions. Analyzing loci at which the S. cerevisiae allele promotes thermotolerance, we detected robust evidence for positive selection, including amino acid divergence between the species and conservation within S. cerevisiae populations. Since such signatures were particularly strong at the chromosome segregation gene ESP1, we used this locus as a case study for focused mechanistic follow-up. Experiments revealed that, in culture at high temperature, the S. paradoxus ESP1 allele conferred a qualitative defect in biomass accumulation and cell division relative to the S. cerevisiae allele. Only genetic divergence in the ESP1 coding region mattered phenotypically, with no functional impact detectable from the promoter. Together, these data support a model in which an ancient ancestor of S. cerevisiae, under selection to boost viability at high temperature, acquired amino acid variants at ESP1 and many other loci, which have been constrained since then. Complex adaptations of this type hold promise as a paradigm for interspecies genetics, especially in deeply diverged traits that may have taken millions of years to evolve.

Decades of successes in statistical genetics have revealed the molecular underpinnings of traits as they vary across individuals of a given species. But standard methods in the field cant be applied to divergences between reproductively isolated taxa. Genome-wide reciprocal hemizygosity mapping (RH-seq), a mutagenesis screen in an inter-species hybrid background, holds promise as a method to accelerate the progress of interspecies genetics research. Toward this end we pioneered an improvement to RH-seq in which mutants harbor barcodes for cheap and straightforward phenotyping-by-sequencing. As a proof of concept for the new tool, we carried out genetic dissection of the difference in thermotolerance between two reproductively isolated budding yeast species. Experimental screening and sequencing identified dozens of loci at which variation between the species contributed to the thermotolerance trait. These hits were enriched for mitosis genes and other housekeeping factors, and among them were multiple loci with robust sequence signatures of positive selection. Together, our results shed new light on the mechanisms by which evolution solved the problems of cell survival and division at high temperature in the yeast clade, and they illustrate the power of the barcoded RH-seq approach.

A central goal of evolutionary genetics is to understand, at the molecular level, how organisms adapt to their environments. For a given trait, the answer often involves the acquisition of variants at unlinked sites across the genome. Genomic methods have achieved landmark successes in pinpointing these adaptive loci. To figure out how a suite of adaptive alleles work together, and to what extent they can reconstitute the phenotype of interest, requires their transfer into an exogenous background. We studied the joint effect of adaptive, gain-of-function thermotolerance alleles at eight unlinked genes from Saccharomyces cerevisiae, when introduced into a thermosensitive sister species, S. paradoxus. Although the loci damped each other’s beneficial impact (that is, they were subject to negative epistasis), most boosted high-temperature growth alone and in combination, and none was deleterious. The complete set of eight genes was sufficient to confer ~15% of the S. cerevisiae thermotolerance phenotype in the S. paradoxus background. The same loci also contributed to a heretofore unknown advantage in cold growth by S. paradoxus. Together, our data establish temperature resistance in yeasts as a model case of a genetically complex evolutionary tradeoff, which can be partly reconstituted from the sequential assembly of unlinked underlying loci.

Decades of successes in statistical genetics have revealed the molecular underpinnings of traits as they vary across individuals of a given species. But standard methods in the field can’t be applied to divergences between reproductively isolated taxa. Genome-wide reciprocal hemizygosity mapping (RH-seq), a mutagenesis screen in an inter-species hybrid background, holds promise as a method to accelerate the progress of interspecies genetics research. Here we describe an improvement to RH-seq in which mutants harbor barcodes for cheap and straightforward sequencing after selection in a condition of interest. As a proof of concept for the new tool, we carried out genetic dissection of the difference in thermotolerance between two reproductively isolated budding yeast species. Experimental screening identified dozens of candidate loci at which variation between the species contributed to the thermotolerance trait. Hits were enriched for mitosis genes and other housekeeping factors, and among them were multiple loci with robust sequence signatures of positive selection. Together, these results shed new light on the mechanisms by which evolution solved the problems of cell survival and division at high temperature in the yeast clade, and they illustrate the power of the barcoded RH-seq approach.

A central goal of evolutionary genetics is to understand, at the molecular level, how organisms adapt to their environments. Landmark work has characterized steric clashes between variants arising in a given protein as it evolves a new function. For many traits, any such single gene represents only part of a complex architecture, whose genetic mechanisms remain poorly understood. We studied the joint effect of eight genes underlying thermotolerance in Saccharomyces cerevisiae, when introduced into a thermosensitive species, S. paradoxus. The data revealed no sign epistasis: most gene combinations boosted thermotolerance, and none was deleterious. And the genes also governed a heretofore unknown advantage in cold growth by S. paradoxus. These results shed light on how and why thermotolerance arose in S. cerevisiae, and they suggest a paradigm in which, if protein repacking is a difficult step in adaptation, combining whole-gene modules may be far less constrained.Author summaryThe building of new traits by evolution can be difficult and slow. A given DNA variant may be the linchpin of a beneficial trait in some individuals and, in others, have the opposite effect—torpedo fitness altogether. Such effects have been best studied among amino acids in a given protein, whose changing side chains crowd each other unless they arise in a particular order. We set out to complement this literature by studying adaptive variants that are not in the same protein, but rather scattered across unlinked genes. We used as a model system eight genes that govern the ability of the unicellular yeast Saccharomyces cerevisiae to grow at high temperature. We introduced this suite of genes stepwise into a non-thermotolerant sister species, and found that the more S. cerevisiae loci we added, the better the phenotype. We saw no evidence for toxic interactions between the variant genes as they were combined. We also used the eight-fold transgenic to dissect the mechanism and the evolutionary forces underlying the thermotolerance trait. Together, our data suggest a principle for the field in which repacking a given protein is the hard part of evolution, and assembling combinations of unlinked loci is far easier.