| Understanding the dynamics and mechanisms of adaptive evolution is a central question in evolutionary biology. However, realizing this goal remains challenging due to the difficulty of observing adaptive evolution in real time and deducing its molecular basis. Long-term Experimental Evolution (LTEE) using microbes and chemostats provides a means of overcoming to these limitations to address these central questions. I studied the evolution of genetic networks in Saccharomyces cerevisiae (budding yeast) populations propagated for more than 200 generations in different nitrogen-limiting conditions using chemostats. I find that rapid adaptive evolution in nitrogen-poor environments is dominated by the de novo generation and selection of copy number variants (CNVs), a large fraction of which contain genes encoding specific nitrogen transporters. The large fitness increases associated with these alleles limits the genetic heterogeneity of adapting populations even in environments with multiple nitrogen sources. Complete identification of acquired point mutations, in individual lineages and entire populations, identified heterogeneity at the level of genetic loci but common themes at the level of functional modules, including genes controlling phosphatidylinositol-3-phosphate metabolism and vacuole biogenesis. Adaptive strategies shared with other nutrient-limited environments point to selection of genetic variation in the TORC1 and Ras/PKA signaling pathways as a general mechanism underlying improved growth in nutrient-limited environments. By studying the fitness of individual alleles, and their combination, as well as the evolutionary history of the evolving population, I find that the order in which adaptive mutations are acquired is constrained by epistasis. I observed the repeated selection of non-synonymous mutations in the zinc finger DNA binding domain of the GATA transcription factor, GAT1, an activator of the nitrogen catabolite repression (NCR) regulon. The functional effects of GAT1 mutations are exerted both directly, and indirectly by rewiring of incoherent feed-forward loops comprising multiple GATA transcription factors and their common NCR regulon targets. This suggests that under strong selection the evolution of gene expression is highly repeatable and that rewiring transcriptional networks can lead to both direct and indirect effects. Studies using LTEE are potentially applicable to understanding pathogenic strategies adopted by viruses, microbes and even human cancer cells. For example, recurrent mutations in the DNA binding domain of GAT1 is reminiscent of recurrent missense mutations in the DNA binding domain of TP53 found in a variety of tumors. |
