Many genotypes of S. cerevisiae aggregate into biofilm-like clusters of cells, termed “flocs,” by producing adhesive glycoproteins in their cell walls (25), but we found that snowflake-phenotype yeast do not arise from floc-type aggregation. Individual cells, obtained by enzymatic digestion of snowflake clusters, were tracked via microscopy for 16 h of growth (Movie S1). During this time each cell was seen to give rise to a new snowflake-type cluster, whereas aggregation was never seen, demonstrating that clusters arise via postdivision adhesion and not by aggregation of previously separate cells. Cell adhesion sites were identified by calcofluor staining, which preferentially stains yeast bud scars, confirming that the
group of cells making up the snowflake phenotype arises via successive divisions of component cells (Fig. 2B). Snowflake yeast are also phenotypically stable: we transferred three replicate populations of snowflake yeast (drawn from replicate population 1, day 30, of our first evolution experiment) 35 times without gravitational selection and did not detect invasion by any unicellular strains.