The evolution of multicellularity is considered to be a major transition in the history of life on Earth1. In the evolution from unicellularity to obligate multicellularity, facultative clonal multicellularity may constitute an intermediate state, in which unicellular proliferation and clonal multicellular growth are switchable2–4. However, little is known about the mechanisms of switching. Here we identify the genetic and cellular basis of nutrition-responsive facultative clonal multicellularity in two black-yeast species of Dothideomycetes. Deletion of any one of ten genes in Hortaea werneckii5,6 results in near-obligate unicellularity or multicellularity. Six of these genes encode regulators of conidiation (asexual sporulation) in filamentous fungi7, despite conidiation not being observed in H. werneckii. Second-site mutations often restore or reverse the phenotype, revealing genetic flexibility underlying facultative multicellularity. A Myb protein functions as a switch-like regulator of state transitions in H. werneckii; its expression and degradation are coupled to nutrient conditions, stabilizing unicellular or multicellular growth. However, while conidiation regulators are similarly co-opted to enable facultative multicellularity, the Myb gene is dispensable in the related species Neodothiora pruni8, further highlighting molecular diversity in plasticity regulation. Ecologically, multicellular-prone H. werneckii ecotypes are isolated from sponges, and sponge-conditioned medium induces multicellularity. This study establishes a tractable model system for dissecting facultative clonal multicellularity across genetic, cellular and ecological scales, and outlines genetic and cellular strategies to gain, lose and regain multicellularity and, more broadly, phenotypic plasticity.
