## 3 Results  
Three independent continuous culture experiments were performed for the wild-type haploid W303 strain. In each of these biological replicates, we analyzed transcriptome changes that occurred between the 40 th generation time point, i.e., the early stage of the adaptive phase, and the time point after an additional 200 generations of growth in the adaptive phase. The changes in transcriptomes during this growth period were determined by the concurrent hybridization of Cy3- or Cy5-labeled RNA samples to yeast v.2 microarrays. Microarray data were validated by qRT-PCR for 8 randomly selected genes (see Materials and Methods and Table S1 for more details). The correlation coefficient between the microarray and qRT-PCR data for the selected genes was 0.931 (see Supplementary Figure S1). Some of the transcriptome changes identified in W303 strain cultures were specific to the individual biological replicates and could be ascribed to the specific mutations found in the gene pools of the respective evolved cell populations. Briefly, in Replicate 1 we detected significant mutations in _HOG1_ and _MSY1_ genes, in Replicate 2 we found mutations in _HOG1_ and _URE2_ genes, and in Replicate 3 we identified a mutation in the _MSY1_ gene. These data are summarized in the Supplementary data file. Intriguingly, however, we also noticed changes in the transcriptome that were uniform across biological replicates even though no mutations were affecting the same genes in all three independent cultures of the W303 strain. While these changes were most likely adaptive, they could not have been the consequence of adaptive mutations. The steadiness of the continuous culture conditions ruled out the possibility that the observed transcriptome changes constituted a response to the changing environment. This striking finding encouraged us to investigate further the observed phenomenon. The criteria for the selection of genes whose expression was uniformly and significantly changed between the two time points were as follows: (|Mean Log2 Ratio|) \(\geq\) 0.9 AND \(p\)-value \(\leq\) 0.05 Standard Deviation \(\leq\) 0.8 AND (Standard Deviation)/(Mean Log2 Ratio)2 \(\leq\) 0.4, where |Mean Log2 Ratio| is the modulus of the logarithm of fold difference of expression between the two time points averaged across the biological replicates and Standard Deviation is calculated based on the values of these biological replicates. These criteria allowed us to reveal 212 genes whose expression was higher after 240 generations of adaptive growth relative to 40 generations of growth and 241 genes whose expression was lower when comparing the same time points. The analysis of Gene Ontology terms overrepresentation, performed with Cytoscape (v3.8.0) [18] and the BiNGO (v3.0.3) plugin [19] allowed us to identify several functional categories that were overrepresented within the selected gene sets. The results of this analysis are summarized in Figure 1. Figure 2 shows all the genes in our gene set sorted by their assigned functions. Some categories were merged to reduce their number and increase clarity. Below the gray bars are genes whose functional categories were not overrepresented and genes of unknown function. Genes most strongly up- or downregulated, with LogRatio above 2.33 or below -2.33, respectively i.e., whose expression changed at least 5-fold, are marked in bold face. Figure S3 shows the expanded version of Figure 2, listing genes together with their short descriptions and the LogRatio values denoting the increase or decrease of gene expression between 40 and 240 generations. The data presented allow for several general remarks. Downregulated genes dominated in our gene set. More of them have known functions and more of them belonged to overrepresented Gene Ontology categories compared to upregulated genes. Notably, in both up- and downregulated gene lists there was a 1.5-fold excess of genes qualified in SGD as Uncharacterized relative to the whole _S. cerevisiae_ genome (62 of 453 in our gene set versus 578 of 6256 _S.