Gene loss and compensatory evolution promotes the emergence of morphological novelties in budding yeast.

Deleterious mutations are generally considered to be irrelevant for morphological evolution. However, they could be compensated by conditionally beneficial mutations, thereby providing access to new adaptive paths. Here we use high-dimensional phenotyping of laboratory-evolved budding yeast lineages to demonstrate that new cellular morphologies emerge exceptionally rapidly as a by-product of gene loss and subsequent compensatory evolution. Unexpectedly, the capacities for invasive growth, multicellular aggregation and biofilm formation also spontaneously evolve in response to gene loss. These multicellular phenotypes can be achieved by diverse mutational routes and without reactivating the canonical regulatory pathways. These ecologically and clinically relevant traits originate as pleiotropic side effects of compensatory evolution and have no obvious utility in the laboratory environment. The extent of morphological diversity in the evolved lineages is comparable to that of natural yeast isolates with diverse genetic backgrounds and lifestyles. Finally, we show that both the initial gene loss and subsequent compensatory mutations contribute to new morphologies, with their synergistic effects underlying specific morphological changes. We conclude that compensatory evolution is a previously unrecognized source of morphological diversity and phenotypic novelties.