Evolutionary outcomes depend not only on the selective forces acting upon a species, but also on the genetic background. However, large timescales and uncertain historical selection pressures can make it difficult to discern such important background differences between species. Experimental evolution is one tool to compare evolutionary potential of known genotypes in a controlled environment. Here we utilized a highly reproducible evolutionary adaptation in Saccharomyces cerevisiae to investigate whether experimental evolution of other yeast species would select for similar adaptive mutations. We evolved populations of S. cerevisiae, S. paradoxus, S. mikatae, S. uvarum, and interspecific hybrids between S. uvarum and S. cerevisiae for ~200–500 generations in sulfate-limited continuous culture. Wild-type S. cerevisiae cultures invariably amplify the high affinity sulfate transporter gene, SUL1. However, while amplification of the SUL1 locus was detected in S. paradoxus and S. mikatae populations, S. uvarum cultures instead selected for amplification of the paralog, SUL2. We measured the relative fitness of strains bearing deletions and amplifications of both SUL genes from different species, confirming that, converse to S. cerevisiae, S. uvarum SUL2 contributes more to fitness in sulfate limitation than S. uvarum SUL1. By measuring the fitness and gene expression of chimeric promoter-ORF constructs, we were able to delineate the cause of this differential fitness effect primarily to the promoter of S. uvarum SUL1. Our data show evidence of differential sub-functionalization among the sulfate transporters across Saccharomyces species through recent changes in noncoding sequence. Furthermore, these results show a clear example of how such background differences due to paralog divergence can drive changes in genome evolution.

Eukaryotic DNA replication initiates from multiple discrete sites in the genome, termed origins of replication (origins). Prior to S phase, multiple origins are poised to initiate replication by recruitment of the pre-replicative complex (pre-RC). For proper replication to occur, origin activation must be tightly regulated. At the population level, each origin has a distinct firing time and frequency of activation within S phase. Many studies have shown that chromatin can strongly influence initiation of DNA replication. However, the chromatin parameters that affect properties of origins have not been thoroughly established. We found that nucleosome occupancy in G1 varies greatly around origins across the S. cerevisiae genome, and nucleosome occupancy around origins significantly correlates with the activation time and efficiency of origins, as well as pre-RC formation. We further demonstrate that nucleosome occupancy around origins in G1 is established during transition from G2/M to G1 in a pre-RC-dependent manner. Importantly, the diminished cell-cycle changes in nucleosome occupancy around origins in the orc1-161 mutant are associated with an abnormal global origin usage profile, suggesting that proper establishment of nucleosome occupancy around origins is a critical step for regulation of global origin activities. Our work thus establishes nucleosome occupancy as a novel and key chromatin parameter for proper origin regulation.

2Evolutionary outcomes depend not only on the selective forces acting upon a species, but 3 also on the genetic background. However, large timescales and uncertain historical 4 selection pressures can make it difficult to discern such important background differences 5 between species. Experimental evolution is one tool to compare evolutionary potential of 6 known genotypes in a controlled environment. Here we utilized a highly reproducible 7 evolutionary adaptation in Saccharomyces cerevisiae to investigate whether other yeast 8 species would adopt similar evolutionary trajectories. We evolved populations of S. 9 cerevisiae, S. paradoxus, S. mikatae, S. uvarum, and interspecific hybrids between S. 1 0 uvarum and S. cerevisiae for ~200-500 generations in sulfate-limited continuous culture. 1 1 Wild-type S. cerevisiae cultures invariably amplify the high affinity sulfate transporter 1 2 gene, SUL1. However, while amplification of the SUL1 locus was detected in S. 1 3 paradoxus and S. mikatae populations, S. uvarum cultures instead selected for 1 4amplification of the paralog, SUL2. We measured the relative fitness of strains bearing 1 5 deletions and amplifications of both SUL genes from different species, confirming that, 1 6 converse to S. cerevisiae, S. uvarum SUL2 contributes more to fitness in sulfate limitation 1 7 than S. uvarum SUL1. By measuring the fitness and gene expression of chimeric 1 8 promoter-ORF constructs, we were able to delineate the cause of this differential fitness 1 9 hold at 95°C for 4 min, followed by 50 cycles of 10 sec at 95°C and 45 sec at 56°C. The