Optical density, OD_{600 nm} (closed squares) and glucose (open squares), ethanol (closed circles), glycerol (closed triangles) and acetic acid (open diamonds) concentrations are shown. In each case a representative experiment is shown. (a) Batch culture of D. bruxellensis Y879 (CBS 2499) under aerobic conditions in the defined minimal medium. (b) Batch culture of D. bruxellensis Y879 under strict anaerobic conditions. (c) Batch culture of B. naardenensis Y922 (CBS 754) under aerobic conditions. B. naardenensis strains could not grow under strict anaerobic conditions.

(a) The phylogenetic relationship is based on the mitochondrial 15S rDNA sequence (for details see ref. 15). Accumulation of substitutions was used to deduce the timing of the divergence events. The split between S. cerevisiae and S. kluyveri (post- and pre-WGD lineages in the S/K complex) was taken as 100 mya24 and this distance (or the number of accumulated substitutions) was then used to calibrate the rest of the events. Each of the D/B and S/K yeast complexes can be divided into two subgroups, based on their phylogenetic relationship and the physiological traits. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. (b) The phylogenetic relationship based on the nuclear 26S rDNA sequences (for details see ref. 16). The accession numbers of the sequences, analysed using the neighbour-joining method22, can be found in the Supplementary Methods.

The S. cerevisiae (a) and D. bruxellensis (b, c) promoters of the respiration- and rapid growth-associated genes were analysed for the presence of the AATTTT cis-elements positioned at the conserved sites. For D. bruxellensis two different rapid growth-associated gene sets are shown: set 1 (containing 15 available promoters and including rRNA-processing-associated genes, black line, see b) and set 2 (including 21 available promoters and including CRP-associated genes, blue line, see c). For S. cerevisiae only set 1 is shown (and it includes only the available D. bruxellensis orthologues, black line), the set 2 data can be found elsewhere5. Sets 1 and 2 contain genes as defined by ref. 5. The 500 bp of sequence upstream was checked for the presence of the regulatory element; the percentage of the gene promoters containing the AATTTT motif (and its reverse complement) within the blocks of 50 bp was plotted against the distance from the start codon (see also ref. 5). The AATTTT motif was overrepresented in the promoters of the rapid growth-associated genes in the regions 101–150 bp upstream from the ATG codon in S. cerevisiae (a) and 101–250 bp upstream from the ATG codon in D. bruxellensis (b, c). The AATTTT motif did not exhibit any positional conservation in the respiration-associated genes (red line), and it was either missing or was spread equally along the promoter sequence. The differences in the distribution pattern of the motif in rRNA/CRP versus MRP genes in D. bruxellensis were statistically significant according to the repeated-measures analysis of variance.

Ratio of the expression levels of the rapid growth-associated genes (CRP, blue) and respiration-associated genes (MRP, red) in different yeasts species: (a) S. cerevisiae, (b) S. bayanus, (c) K. lactis, (d) D. bruxellensis Y872 (CBS1943) and (e) D. bruxellensis Y883 (CBS3025). Each yeast species was grown in a defined minimal medium with a fermentable carbon source (glucose) and a defined minimal medium with a non-fermentable carbon source (ethanol). The results were expressed as a ratio of expression levels in the glucose and ethanol experiments. The α-tubulin encoding gene (YML085C), which has the same expression level in both media (according to the microarray studies; for example, ref. 5), was used to normalize the data. The upper graphs for each species show the ratio of expression levels of a set of CRP and a set of MRP genes from a single representative experiment (each gene measurement was done in duplicates). The take-off and the amplification values, obtained from the relative quantification performed using the RotorGene 2000 software, were used to quantify the expression ratios with help of REST 2009 V2.0.13 with RG mode5. These results are presented as Whisker box plot. The lower graphs show the average of the expression ratios calculated for all CRP and MRP genes in each strain and are based on four (b–d) or two (a, e) replicates. The error bars represent the standard deviation. Note that all the orthologous gene names follow the S. cerevisiae naming system and the genes are lined along the x axis. The following genes were used: a S. cerevisiae genes: A1-YLR029C; A2-YLR264W; A3-YBR031W; A4-YCR012W; A5-YKL156W; A6-YBR282W; A7-YMR225C; A8-YMR193W; A9-YMR188C. b S. bayanus genes: B1-YOR063W; B2-YPL131W; B3-YNL178W; B4-YBR031W; B5-YDR116C; B6-YGR165W; B7-YKL167C; B8-YOR158W; B9-YGR220C; B10-YBR122C. c K. lactis genes: D1-YBR189W; D2-YPL143W; D3-YHR141C; D4-YHR021C; D5-YLR367W; D6-YGL129C; D7-YEL050C; D8-YKL138C; D9-YHR147C; D10-YDR237W; D11-YMR158W; D12-YNR037C; D13-YNL185C; D14-YGR165W; D15-YDR175C; D16-YDR322W. d D. bruxellensis Y879 genes: C1-YDL081C; C2-YPL143W; C3-YPL131W; C4-YDR405W; C5-YHR075C; C6-YGR165W; C7-YGR220C; C8-YNL005C. e D. bruxellensis Y883 genes: G1-YPL143W; G2-YDL081C; G3-YPL131W; G4-YDR025W; G5-YGL103W; G6-YDR405W; G7-YGR165W; G8-YGR220C; G9-YNL005C; G10-YGR215W; G11-YEL050C; G12-YML009C; G13-YHR075C.