The story of the experiment

Snowflake yeast were made in Michael Travisano's lab at the University of Minnesota, where Will Ratcliff was a postdoc. Out of a conversation about experiments that could address the major evolutionary transitions, the two designed a simple experiment around a new selection regime: let yeast cells settle through the liquid culture, transfer the bottom fraction into the next day's fresh tubes, and see whether yeast could evolve multicellular clusters. The design was remarkably effective. Ten replicate populations of unicellular Saccharomyces cerevisiae Y55 were grown in liquid and settled daily, and all ten evolved clonal snowflake clusters within just 60 transfers, about 300 generations. Clusters form because mother and daughter cells fail to separate after budding, so every cluster is clonal. Under settling selection the multicellular genotypes had a 34% fitness advantage; without settling selection they appeared to pay a cost of about 10% (P = 0.06).

Two results from this period were important for the work that followed. The first was genetic. RNA-seq identified 1,035 differentially expressed genes, and seven of the ten most strongly downregulated were targets of the transcription factor ACE2. Knockout and complementation confirmed that loss of ACE2 is both necessary and sufficient for the snowflake phenotype. Because a single gene deletion produces the multicellular phenotype, later experiments could begin from a constructed ace2Δ ancestor rather than waiting for the mutation to arise.

The second was methodological: settling selection screens enormous numbers of clusters for size every day, and its strength can be tuned by shortening the settling window. That made snowflake yeast a practical system for evolving increased multicellular size (how and why we select for size).

Plate 01

Imaging

Differential interference contrast

Subject

Snowflake yeast cluster

A snowflake yeast cluster imaged in transmitted light, with no fluorescent label. Cells remain attached to the mothers that budded them, so the branched form is a consequence of how the group grows.

Ratcliff moved to Georgia Tech in 2014 and continued this work there. Over the next few years the lab studied how high rates of cell death promote the formation of multicellular aggregates, how aggregative and clonal multicellularity compete against one another, and the biophysics behind the first increases in cluster size. This work was done through short-term evolution experiments and modelling. There was no long-term evolution experiment, since size gains in the earlier work had been modest.

In these early experiments, the model system appeared to have an upper limit on size. Size increase flattened after a few hundred generations, and clusters remained microscopic. The interpretation available at the time was that snowflake yeast could evolve into multicellular clusters but could not evolve much beyond that, and that the system was therefore not well suited to studying open-ended multicellular evolution.

Ozan Bozdag joined the Ratcliff lab as a postdoc and proposed a new project exploring the relationship between multicellular size, oxygen concentration, and carbon metabolism, building on his doctoral work growing yeast under different metabolic regimes. In an organism that respires, oxygen reaches interior cells by diffusion, and diffusion limits how large a mass of respiring cells can become. If oxygen delivery was what constrained cluster size, then changing the growth conditions should remove the limit.

This can be tested directly, because metabolism in yeast can be manipulated. Respiration-deficient petite mutants cannot use oxygen at all, so increased cluster size carries no cost in oxygen delivery. Sparging air through the medium raises dissolved oxygen for populations that do respire.

Bozdag's postdoc project comprised twenty populations across four metabolic and oxygen regimes: anaerobic petites, strictly aerobic yeast at intermediate oxygen (about 24–25% of present atmospheric level), strictly aerobic yeast at high oxygen (about 72–75% PAL), and mixotrophic yeast at intermediate oxygen. All twenty were maintained under identical daily growth and settling selection for 145 transfers, roughly 812 generations.

Oxygen strongly affected size evolution, and the effect was not monotonic. Strictly aerobic populations at intermediate oxygen increased in mean size by only 8.9%, while anaerobic populations increased by 93% and high-oxygen populations by 97%. The size limit seen in the earlier experiments was set by the growth conditions, and it could be removed.

Bozdag, Libby, Pineau, Reinhard & Ratcliff 2021 · Nature Communications 12:2838

After the first 145 transfers Bozdag kept transferring the populations. That decision is what turned a postdoc project into a long-term evolution experiment.

There was no signal in the data at the time to justify continuing. For a long stretch after the first 145 transfers the anaerobic populations did not increase in size. A plateau in a selection experiment does not necessarily mean that adaptation has stopped. The experiment's name later honoured Richard Lenski's long-term evolution experiment with E. coli, which has been running since 1988 and has produced many of its most important results decades after it began.

Eventually the anaerobic populations began increasing in size again, and continued to do so. Around day 350 Bozdag saw clusters visible to the naked eye, and within about 50 more transfers all five anaerobic lines had them.

Fifteen of the original twenty populations are the ones still running: five anaerobic petite (PA1–5), five mixotrophic (PM1–5) and five obligately aerobic (PO1–5), all founded in 2018 from one isogenic ace2Δ Y55 clone (the anaerobic lines from a petite derivative of it), so the starting genotype is known exactly.

By 600 transfers, about 3,000 generations, all five anaerobic populations had become macroscopic. Mean cluster radius increased from 16 µm to 434 µm, roughly a 20,000-fold increase in volume, and biophysical toughness increased about 10,000-fold. These results were published in Nature in 2023, with Bozdag as first author and Ratcliff as senior author. The experiment was named the MuLTEE in the 2021 preprint of that paper.

Bozdag, Zamani-Dahaj, Day et al. 2023 · Nature 617:747–754

The experiment has since become the central project of the Ratcliff Lab and the basis of a decadal research program. Bozdag is principal investigator on the NSF LTREB award that funds it and Ratcliff is co-principal investigator.