Ancient balancing selection maintains incompatible versions of the galactose pathway in yeast

Metabolic pathways differ across species but are expected to be similar within a species. We discovered two functional, incompatible versions of the galactose pathway in Saccharomyces cerevisiae. We identified a three-locus genetic interaction for growth in galactose, and used precisely engineered alleles to show that it arises from variation in the galactose utilization genes GAL2, GAL1/10/7, and phosphoglucomutase (PGM1), and that the reference allele of PGM1 is incompatible with the alternative alleles of the other genes. Multiloci balancing selection has maintained the two incompatible versions of the pathway for millions of years. Strains with alternative alleles are found primarily in galactose-rich dairy environments, and they grow faster in galactose but slower in glucose, revealing a trade-off on which balancing selection may have acted.