In 2018 we founded twenty populations of ace2Δ snowflake yeast from one isogenic Saccharomyces cerevisiae Y55 clone (the anaerobic lines from a petite derivative of it). Fifteen of them, five in each of three metabolic environments, were kept running and form the MuLTEE. The daily growth-and-settling cycle is the same across all fifteen, with shorter settling once clusters become macroscopic; the treatments differ in metabolism.
PA, anaerobic. PM, mixotrophic. PO, obligately aerobic. Five replicates per treatment. Strains from any population and timepoint can be requested on the strain request page.
Treatment one · five replicates
Plate 01
The PA lines are petite mutants: mitochondrially dysfunctional, unable to grow on glycerol, and confirmed non-respiring by direct oxygen measurement with optodes. Because they never use oxygen, oxygen cannot become scarce inside a large cluster, so the diffusion cost that normally penalises large size is absent. This treatment was designed to test whether size evolution proceeds once that cost is removed. It did, in all five replicates, producing organisms roughly 20,000-fold larger by volume.
Biomass-weighted cluster radius increased about 27-fold in linear dimension, an estimated volume increase of roughly 2 × 104. Groups went from around 100 cells to around 450,000. All five anaerobic populations became macroscopic, visible without a microscope, and the clonal life cycle was retained throughout.
The ancestor fractures at about 240 Pa with toughness as low as 8.9 J m−3. Evolved macroscopic clusters exceed 0.6 MJ m−3. Individual-cell stiffness did not measurably change, so the gain is architectural rather than material.
Super-resolution panoramic integration imaging across the archived timepoints puts cellular aspect ratio at about 1.30 in the ancestor and 3.28 by 1,000 transfers, while cross-sectional module size grew from 30.7 to 87.1 µm. The two do not track each other perfectly: there are deviations from simple aspect-ratio scaling at t400 and t1000.
Segmented three-dimensional electron-microscopy reconstructions of evolved branches show branches held by two or more neighbours in configurations that rigid-body translation and rotation cannot undo without deformation or bond breakage.
All five anaerobic populations doubled their genome within the first 50 days, fixed tetraploidy by day 100, and held it for the next 950 days despite genomic instability. The anaerobic tetraploids then accumulated extensive aneuploidy that tracked the evolution of macroscopic size and helped sustain it.
Reduced HSC82 expression destabilises the Hsp90 client Cdc28 at the protein level without changing CDC28 transcript, which delays mitosis and prolongs polarized growth, and that is what makes cells longer. Restoring either gene shortened cells, shrank clusters and lowered multicellular fitness. The focal line was PA5 at 600 transfers.
The petite ancestor divides asynchronously because daughter cells take about 25% longer to complete their first division than their later ones. The anaerobic populations had lost that delay by day 200 and were still dividing synchronously at day 1,000. Synchrony is favoured during growth and during settling, so one cell-level timing change is visible to selection at both levels.
Evolved PA5 isolates from days 200, 400, 800 and 1,000 keep growing exponentially in static liquid at millimetre scale, and only sub-exponentially on agar. Above a threshold cluster size their own metabolism generates buoyancy-driven flows that enter from the sides and exit from the top, at speeds comparable to those extant organisms produce with cilia. The flows stop in dead or glucose-starved clusters and reverse when gravity is reversed.
Across the five replicate anaerobic populations the mutational targets were enriched in the same functional categories, including parallel changes in GIN4 and PHO81.
Treatment two · five replicates
In our 2021 oxygen experiment this regime was the control for the change in metabolism, and over the first 145 transfers it evolved to be 37% larger. The PM lines went through the same whole-genome duplication as the anaerobic lines, on the same schedule.
Diploid ancestors became tetraploid by day 50, tetraploidy fixed by day 100, and it persisted for the next 950 days. Whatever drives the genome duplication, it is not specific to the lineages that went on to become macroscopic.
Where the anaerobic tetraploids accumulated extensive aneuploidy alongside increasing size, the mixotrophic tetraploids did not, and they stayed microscopic. This suggests the aneuploidy is linked to the evolution of large size, since tetraploidy alone did not produce it.
The mixotrophic line tested did not show the reduced HSC82 expression that all five macroscopic anaerobic lineages converged on, which is the expected result if reduced Hsp90 is specifically associated with the evolution of large size.
Treatment three · five replicates
Plate 02
Glycerol cannot be fermented. Growth on it requires functional mitochondria and dissolved oxygen, so clusters compete for oxygen during the 24-hour growth phase, which does not happen in the other two treatments. Small clusters win the competition for oxygen, large clusters win the settling step, and neither can exclude the other. Group size alone, with no differentiated tissues, was enough to partition a niche.
The split arose from a single ancestor within 715 daily transfers. Genomic data suggest the two forms diverged early and have coexisted for most of that time, roughly 4,300 generations.
Under the standard growth-plus-settling regime, mixtures started across a wide range of initial frequencies return to the same equilibrium, the signature of negative frequency-dependent selection. The Small and Large genotypes were isolated from population PO4 after 715 daily transfers.
This identifies dissolved oxygen as the axis of the trade-off. Competition for oxygen is what keeps the small growth specialist in the population despite a daily selection step that rewards nothing but size.
Grown in monoculture without the opposite-sized competitor, both lineages evolve back toward intermediate size, and small-lineage monocultures re-evolve the two forms quickly. This is character displacement operating in both directions.
The aerobic ancestor divides synchronously, unlike the petite anaerobic ancestor, whose daughter cells take about 25% longer to complete their first division. The two treatments therefore started from developmentally different founders, which matters for any comparison of their branching topologies.
Strain codes
A strain name gives the treatment, the population and the timepoint. PA5 t600 is anaerobic population 5 at transfer 600.
The imaging archive
Plate 03
Plate 04
Plate 05
Plate 06
Plate 07
Plate 08
Plate 09
Plate 10