Supplemental Materials and Methods Widespread Genetic Incompatibilities Between First-Step Mutations During Parallel Adaptation of Saccharomyces cerevisiae to a Common Environment

All possible haploid and diploid genotypes were created for each pair of four beneficial mutations (one in each of ERG3, ERG5, ERG6 and ERG7, main text Table 5). Each mutation was initially isolated in the BY4741 haploid background (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and given a Beneficial Mutation Nystatin (BMN) strain number [1]. Each BMN strain was mated to BY4739 (MATα leu2Δ0 lys2Δ0 ura3Δ0) (Open Biosystems) to create strains heterozygous for a single ERG mutation, and diploids were positively selected on plates lacking both histidine and lysine. Similarly, diploid non-mutant strains were created by mating BY4741 and BY4739. In each case, single colonies were then grown up on a second selection plate and frozen at -80°C in 15% glycerol. MATα single mutant strains were isolated by sporulation of the heterozygous diploids. Diploid stock grown on YPD a plate was used to inoculate 10 mL of YPD and grown overnight on a rotor at 30°C. 200 μL of culture was then washed, spread on potassium acetate plates (1% KOAc, 2% agar) and sporulated at 25°C until a sufficient number of tetrads could be observed. The resulting tetrads were dissected by micromanipulation on YPD plates. The spores were allowed to germinate and grown at 30°C for three days before replica plating to test for auxotrophies, mating type, and nystatin growth ability. Auxotrophy was assessed on SC plates lacking the appropriate amino acid. Mating type was tested by replica plating tetrads onto plates containing a lawn of MATa or MATα yeast carrying a histidine (his1123) auxotrophy, allowing them to mate, and subsequently testing for mating on a plate lacking arginine, histidine, leucine, lysine, methionine, tryptophan, adenine and uracil (i.e., a plate on which no original haploid strain could grow). Nystatin growth was assessed on YPD + 8 μM nystatin because growth of ancestral strains was not noticeably inhibited on plates with a lower concentration of nystatin. YPD + 8 μM nystatin plates were made by preparing YPD medium with agar as usual, subsequently adding the appropriate amount of 2.7 mM nystatin stock, and mixing by inversion immediately before pouring. All tetrads were verified for 2:2 segregation of auxotrophies and mating type. Once this was confirmed, the spores that showed growth on the nystatin plate and contained the desired MATα lys2Δ0 mutation were frozen at -80°C in 15% glycerol. Throughout strain construction, histidine and lysine auxotrophies were