Sporulation of the evolved strains was attempted in both liquid and solid media. For liquid culture sporulation, fresh colonies were innoculated into 0.8% glucose YNB or YPD media and grown overnight at 30 degrees C. 0.5 ml of this culture was spun down and resuspended in 2 ml SPO++ media (Guthrie and Fink 1991) and grown at 30 degrees C for 3-5 days. 0.5 ml sporulated culture was resuspended in water. 17 microliters of culture was incubated with 3 microliters of beta-glucuronidase for 20-40 minutes, and tetrads were dissected with a micromanipulator onto 0.8% glucose YNB or YPD plates.

For solid culture sporulation, 5x10^8 cells were spun down and washed twice in 5 ml water before being resuspended in 200 microliters waters. 10 microliters of this was spotted onto an agar plate with 1% acetate. Cells were grown for 3-5 days at 30 degrees C before being dissected as above.

SPO++ media (1L)
2.5 g yeast extract
15 g potassium acetate
Autoclave.
Add:
0.5 g glucose
40 mg adenine, uracil, and tyrosine
20 mg histidine, leucine, lysine, tryptophan, methionine, and arginine
100 mg phenylalanine
350 mg threonine