The Repertoire and Dynamics of Evolutionary Adaptations to Controlled Nutrient-Limited Environments in Yeast

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Abstract
The experimental evolution of laboratory populations of microbes provides an opportunity to observe the evolutionary dynamics of adaptation in real time. Until very recently, however, such studies have been limited by our inability to systematically find mutations in evolved organisms. We overcome this limitation by using a variety of DNA microarray-based techniques to characterize genetic changes—including point mutations, structural changes, and insertion variation—that resulted from the experimental adaptation of 24 haploid and diploid cultures of Saccharomyces cerevisiae to growth in either glucose, sulfate, or phosphate-limited chemostats for ∼200 generations. We identified frequent genomic amplifications and rearrangements as well as novel retrotransposition events associated with adaptation. Global nucleotide variation detection in ten clonal isolates identified 32 point mutations. On the basis of mutation frequencies, we infer that these mutations and the subsequent dynamics of adaptation are determined by the batch phase of growth prior to initiation of the continuous phase in the chemostat. We relate these genotypic changes to phenotypic outcomes, namely global patterns of gene expression, and to increases in fitness by 5–50%. We found that the spectrum of available mutations in glucose- or phosphate-limited environments combined with the batch phase population dynamics early in our experiments allowed several distinct genotypic and phenotypic evolutionary pathways in response to these nutrient limitations. By contrast, sulfate-limited populations were much more constrained in both genotypic and phenotypic outcomes. Thus, the reproducibility of evolution varies with specific selective pressures, reflecting the constraints inherent in the system-level organization of metabolic processes in the cell. We were able to relate some of the observed adaptive mutations (e.g., transporter gene amplifications) to known features of the relevant metabolic pathways, but many of the mutations pointed to genes not previously associated with the relevant physiology. Thus, in addition to answering basic mechanistic questions about evolutionary mechanisms, our work suggests that experimental evolution can also shed light on the function and regulation of individual metabolic pathways. Adaptive evolution is a central biological process that underlies diverse phenomena from the acquisition of antibiotic resistance by microbes to the evolution of niche specialization. Two unresolved questions regarding adaptive evolution are what types of genomic variation are associated with adaptation and how repeatable is the process. We evolved yeast populations for more than 200 generations in nutrient-limited chemostats. We find that the phenotype of adapted individuals, as measured using global gene expression, is much less variable in clones adapted to sulfate limitation than either glucose or phosphate limitation. We comprehensively analyzed the genomes of adapted clones and found that those adapted to sulfate limitation almost invariably carry amplifications of the gene encoding a sulfur transporter, but the mutations in individuals adapted to glucose and phosphate limitation are much more diverse. This parallelism holds true at the level of single-nucleotide mutations. Although there may be other paths to adapt to sulfate limitation, one path confers a much greater advantage than all others so it dominates. By contrast, there are a number of ways to adapt to glucose and phosphate limitation that confer similar advantages. We conclude that the reproducibility of evolution depends on the specific selective pressure experienced by the organism.