Evolution of clustering in snowflake-phenotype yeast. (A) Clusters have greater fitness only with gravitational selection. Five replicate populations of S. cerevisiae strain Y55 were transferred 60 times either with or without selection for settling. One representative genotype was isolated from each population (multicellular cluster with centrifugation, unicellular without centrifugation). The relative fitness of these isolates was determined by competition with a common unicellular competitor and a GFP-marked Y55 isolate either with or without gravitational selection. Relative fitness during a single 24-h growth period (one transfer) was calculated as the ratio of the Malthusian growth parameters of the test strain to the common competitor. Clustering genotypes possess a large fitness advantage with gravitational selection, but appear to pay a small cost when transferred without centrifugation. Significance: *P = 0.004, ^§P = 0.06, one-sided t tests. Error bars are the SEM of five replicate populations. (B) A representative genotype (drawn from replicate population 1, day 30, of our first evolution experiment) was grown overnight in yeast peptone dextrose (YPD) media and stained with the blue-fluorescent chitin-binding fluorescent stain calcofluor. All attachments between cells occur at “bud scars” (arrow), demonstrating that the cluster is formed by incomplete separation of daughter and mother cells.

Across-population comparison of early vs. late snowflake phenotype yeast. (A) Snowflake-phenotype yeast face a trade-off between growth and settling rates. Relative growth rate is calculated as the number of doublings per isolate during a 4-h experiment, relative to the fastest growing isolate. Snowflake yeast adapted during the course of the experiment, moving the trade-off function away from the origin. Symbols indicate the replicate population (▲, replicate population 1; ■, replicate population 5; , replicate population 7; ●, replicate population 8; replicate population 9). (B) The frequency of apoptotic cells (measured by dihydrorhodamine 123 staining for reactive oxygen species) was not correlated with settling rate in the first snowflake genotypes to evolve in each population (r² = 0.005). By 60 transfers, however, settling rate and apoptosis are highly correlated (r² = 0.91).

High rates of apoptosis evolve, decreasing propagule size. (A and B) High rates of apoptosis evolve between transfers 14 and 60 in replicate population 1. Yeast were incubated with the red dead-cell stain propidium iodide (PI) and the apoptosis stain DHR. Cells in the early stages of apoptosis stain green; cells dying from apoptosis stain with both PI and DHR, appearing yellow/orange; and necrotic cells stain red. (C) By 60 transfers, snowflake yeast had evolved to settle more rapidly than the 14 transfer isolate. Shown are stationary-phase cultures allowed to settle on the bench for 10 min. This is due to an increase in the size of the cluster at reproduction. Shown are the averages of seven single-cluster overnight life-history analyses, per genotype. Error bars are the SEM. (D) Apoptosis decreases propagule size. The 60 transfer strain, which has evolved a larger size at reproduction (C) and increased rates of apoptosis relative to the 14 transfer strain (A and B), produces proportionally smaller propagules. Experimental induction of apoptosis in the 14 transfer strain reduced propagule size. Shown are the averages of eight (14 and 60 transfers, apoptosis not induced) and seven (14 transfers, apoptosis induced) single-cluster overnight life-history analyses. Error bars are SEM.

Rapid and convergent evolution of the multicellular “snowflake” phenotype. All 10 replicate populations (replicate population number in lower right corner) evolved similar multicellular phenotypes after 60 rounds of selection for rapid settling (shown are replicate populations 1–5; see Fig. S2 for replicate populations 6–10). These genotypes display a similar growth form: the cluster is composed of related cells that do not disassociate after budding, resulting in branched multicellularity.

Snowflake-phenotype yeast have a novel multicellular life history that responds to selection. (A) Time-lapse microscopy of a small cluster shows that 300 min of growth and numerous cell divisions are required before the cluster first reproduces (arrow points to propagule separation). Small clusters are thus functionally juvenile, requiring further growth before becoming reproductively competent. (B) Analysis of cluster size at reproduction (dark blue bars) and offspring size (open bars, overlap shown in light blue) for the same genotype demonstrates that propagules nearly always start out functionally juvenile. (D) A single population of snowflake yeast was exposed to divergent selection for settling rate by allowing yeast to settle for either 5, 15, or 25 min at 1 × g before transfer. A shorter period before transfer imposes stronger selection for rapid settling. After 35 transfers, settling rate was assayed by examining the fraction of yeast biomass in the lower 30% of the culture after 7 min of settling at 1 × g. Populations transferred with strong selection for settling (5 min) evolved to settle more rapidly than populations exposed to weaker selection for rapid settling (15 and 25 min). Error bars are the SEM of three replicate populations. (C and E) The adaptations that resulted in the evolution of faster settling occurred as a result of a change in the cluster-level, not in unicellular life history. Populations selected for more rapid settling (5 min) evolved to delay reproduction until they reached a significantly larger size than the ancestral genotype, whereas relaxed selection for rapid settling resulted in the evolution of clusters that reproduced at a smaller size than the ancestor. Error bars are the SEM of a randomly selected genotype from the population.

Apoptosis is not a side effect of large cluster size. (A) We measured the relationship between the size of individual clusters and the percentage of cells that were apoptotic. Among clusters of a single genotype (isolated from either 14 or 60 transfers), there was no measurable effect of cluster size on apoptosis frequency (P = 0.36, ANCOVA with yeast strain as the cofactor) among snowflake yeast isolated after either 14 or 60 transfers from replicate population 1. (B) To determine if cluster size and apoptosis frequency are independently heritable, we selfed an isolate that forms large clusters with high rates of apoptosis and then assayed the resulting fast-settling progeny for apoptosis. Again, there was no relationship between cluster size (measured by settling rate) and apoptosis frequency (P = 0.55, linear regression). These results demonstrate that apoptosis is not simply a side effect of large cluster size, but rather that isolates evolving larger cluster size also evolved higher rates of apoptosis.