Publications
Papers that use MuLTEE populations or material derived from them, followed by other key snowflake yeast work the experiment relies on. For the full lab publication list, see the Ratcliff Lab site.
Papers over time
The interactive timeline needs JavaScript. The papers are listed below.
MuLTEE
MuLTEE papers
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Cell division timing shapes the morphology and size of nascent multicellular organisms
In the anaerobic MuLTEE ancestor, a daughter cell's first division takes about 25% longer than its later ones. The delay makes clusters smaller and more branched, and synchronous division improves both growth and survival of settling selection. The anaerobic populations lost the delay by day 200 and were still dividing in step at day 1,000.
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Genome duplication in a long-term multicellularity evolution experiment
In all five mixotrophic and all five anaerobic populations, the diploid yeast became tetraploid by day 50, and tetraploidy was fixed by day 100. Tetraploid cells are larger and longer, so they immediately form larger clusters. Selection for size kept the populations tetraploid for nearly 5,000 generations, and in the anaerobic lines it later opened the way to aneuploid routes to macroscopic size.
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Real-time, high-throughput super-resolution microscopy via panoramic integration
A new super-resolution microscopy method (SPI) that roughly doubles resolution while imaging large areas quickly. Applied to ancestral and evolved anaerobic MuLTEE yeast, it measured cell aspect ratio rising from about 1.30 to 3.28 over 1,000 transfers, and module size from 30.7 µm to 87.1 µm.
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Metabolically driven flows enable exponential growth in macroscopic multicellular yeast
Millimetre-scale anaerobic MuLTEE clusters keep growing exponentially in still liquid, but not on agar. Their metabolism drives buoyant flows that carry nutrients through the porous, entangled cluster. The flows stop in dead or glucose-starved clusters, reverse direction when gravity is reversed, and appear only above a threshold cluster size.
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Morphological Entanglement in Living Systems
3D electron-microscope reconstructions show that branches of evolved MuLTEE clusters are caged by their neighbours and cannot be pulled free. Simulations and experiments show that growing branches reach these entangled states, but shaking alone does not. Entanglement depends on growth over time, which is how these clusters become tough.
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Proteostatic tuning underpins the evolution of novel multicellular traits
All five anaerobic MuLTEE lineages that evolved macroscopic size convergently down-regulated the chaperone Hsp90 (HSC82), which destabilizes the cyclin-dependent kinase Cdc28, delays mitosis and prolongs polarized growth to elongate cells. Reinstating Hsp90 or Cdc28 expression shortened cells, shrank clusters and reduced multicellular fitness.
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Emergence and maintenance of stable coexistence during a long-term multicellular evolution experiment
Three of five obligately aerobic MuLTEE populations evolved stably coexisting small and large snowflake yeast lineages that persisted for nearly all of the 715 transfers studied (about 4,300 generations). The split is maintained by negative frequency-dependent selection converging on roughly 9% Large and 91% Small, with oxygen competition during growth favouring small clusters and settling selection favouring large ones.
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De novo evolution of macroscopic multicellularity
After 600 rounds of daily growth and settling selection, all five anaerobic MuLTEE populations evolved millimetre-scale bodies. Mean cluster radius rose from 16 µm to 434 µm, about 20,000 times larger by volume, and clusters became about 10,000 times tougher through cell elongation and branch entanglement. Yeast that relied on scarce oxygen stayed microscopic.
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Oxygen suppression of macroscopic multicellularity
Across roughly 812 generations of settling selection in 20 populations under four oxygen and metabolic regimes, anaerobic and high-oxygen snowflake yeast increased mean size by 93% and 97% respectively while strictly aerobic yeast at intermediate oxygen increased only 8.9%, with cellular aspect ratio explaining 92% of the variation in cluster size. These twenty populations were founded in 2018, and fifteen of them have been transferred daily ever since. This is the experiment that became the MuLTEE.
Snowflake yeast
Other key work relevant to the MuLTEE
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Oxygen-binding proteins aid oxygen diffusion to enhance fitness of a yeast model of multicellularity
Snowflake yeast engineered to make sperm whale myoglobin or peanut worm myohemerythrin let oxygen reach deeper into clusters (16 µm to 21 µm under low oxygen). The proteins gave a larger fitness benefit when extra oxygen was supplied, and none in small bud8Δ clusters.
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Cellular organization in lab-evolved and extant multicellular species obeys a maximum entropy law
In lab-evolved snowflake yeast and in the green alga Volvox carteri, the free space around each cell follows the same statistical distribution predicted for randomly packed granular materials. This makes the arrangement of cells predictable despite noisy growth, so offspring resemble their parents even without developmental regulation.
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Cellular packing, mechanical stress and the evolution of multicellularity
Snowflake yeast clusters fracture from crowding-induced internal stress rather than bond weakness, and over seven weeks (roughly 291 generations) of size selection clusters increased average radius 1.7-fold by evolving an 8% larger cell aspect ratio that dropped cluster volume fraction from 0.32 ± 0.04 to 0.22 ± 0.03, while chitin bond size did not change.
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Geometry, packing, and evolutionary paths to increased multicellular size
A geometric model of snowflake yeast shows that making cells longer is always a more efficient way to grow larger clusters than making bonds stronger, adding on average about 13 times more cells for the same change. Cluster size peaks near a 54° attachment angle.
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Origins of multicellular evolvability in snowflake yeast
Disruption of the transcription factor ACE2 is sufficient to produce the snowflake phenotype (non-synonymous ACE2 mutations in 5 of 10 independently evolved lineages; ACE2 knockout creates clusters, complementation restores unicellularity), and the resulting clonal branching growth form follows Pascal's triangle and yields broad-sense heritability of H2 = 0.84 for cluster size at reproduction.
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Geometry Shapes Evolution of Early Multicellularity
A mathematical model of snowflake cluster geometry shows that the branching tree topology imposes interior space constraints, so elevated cell death is paradoxically adaptive: in simulated populations the probability of cell death rises from 10−8 to about 10−2 within roughly 60 transfers, matching the experimental observation.
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Tempo and Mode of Multicellular Adaptation in Experimentally Evolved Saccharomyces cerevisiae
Over 227 days settling speed increased 44% through three temporally distinct phases: more cells per cluster (42.6 at day 7 to 114.5 at day 227), then a 2.21-fold increase in cell volume, then rounder, more hydrodynamic clusters (5.7% more round at day 227 than day 65).
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Experimental evolution of multicellularity
All 10 replicate populations evolved clonal multicellular snowflake clusters within 60 transfers of gravity-based settling selection, displaying a novel multicellular life history with a juvenile phase, propagules consistently less than half the parent cluster size, and rudimentary division of labor via evolved apoptosis.