Sign epistasis between experimentally evolved adaptive mutations causes a rugged fitness landscape

The fitness landscape captures the relationship between genotype and evolutionary fitness. On a rugged fitness landscape, peaks of high fitness are separated by valleys of lower fitness. Theoretical studies have shown that these valleys are created by reciprocal sign epistasis, where negative interactions between two mutations result in a fitness that is less than each individual mutation’s fitness. However, little molecular work has been done to test this theory. Here we demonstrate reciprocal sign epistasis between two adaptive mutations in the genes mth1 and HXT6/HXT7 that arose in different lineages during experimental evolution of an asexual population of Saccharomyces cerevisiae. This epistasis causes the two mutations to be mutually exclusive. This is the first example in an experimental evolution of a rugged, two-peaked molecular fitness landscape, where the adaptive mutations in mth1 and HXT6/HXT7 are at the two fitness peaks, and the mutant carrying both the mth1 and HXT6/HXT7 mutations is in a fitness valley. The constraint caused by the reciprocal sign epistasis has far-reaching evolutionary implications, including the maintenance of biodiversity, the evolution of sex, and speciation. Our study draws some surprising parallels with cancer, where mutually exclusive mutations in the Ras pathway have been identified in colorectal cancers.