Abstract
While early multicellular lineages necessarily started out as relatively simple groups of cells, little is known about how they became Darwinian entities capable of sustained multicellular evolution(1-3). Here we investigate this with a multicellularity long-term evolution experiment, selecting for larger group size in the snowflake yeast (Saccharomyces cerevisiae) model system. Given the historical importance of oxygen limitation(4), our ongoing experiment consists of three metabolic treatments(5)-anaerobic, obligately aerobic and mixotrophic yeast. After 600 rounds of selection, snowflake yeast in the anaerobic treatment group evolved to be macroscopic, becoming around 2 × 10(4) times larger (approximately mm scale) and about 10(4)-fold more biophysically tough, while retaining a clonal multicellular life cycle. This occurred through biophysical adaptation-evolution of increasingly elongate cells that initially reduced the strain of cellular packing and then facilitated branch entanglements that enabled groups of cells to stay together even after many cellular bonds fracture. By contrast, snowflake yeast competing for low oxygen(5) remained microscopic, evolving to be only around sixfold larger, underscoring the critical role of oxygen levels in the evolution of multicellular size. Together, this research provides unique insights into an ongoing evolutionary transition in individuality, showing how simple groups of cells overcome fundamental biophysical limitations through gradual, yet sustained, multicellular evolution.