In 2018, I started a project that is now known as the Multicellularity Long-Term Evolution Experiment (MuLTEE), which explores the very early steps of multicellular adaptation. Now at 1500+ days (7,500 generations), MuLTEE is the longest-running evolution experiment on nascent multicellularity and has generated various research papers and thesis chapters for scientists at the Ratcliff Lab, Georgia Tech, and universities outside of the US.
Below is a short summary of the genesis of the MuLTEE and a few topics we’ve explored so far.
Low-oxygen is a key limitation in snowflake yeast size evolution:
Early work on the evolution of multicellularity focused on group formation, including studies on ‘snowflake’ yeast where single-celled yeast evolved multicellular clusters. However, these studies were kept short as the ‘multicellular size’ -a key trait for the transition from simple cell clusters to integrated multicellular individuals- remained constrained despite strong daily selection for large groups.
In 2018, as I transitioned from my PhD work to postdoctoral research at Will Ratcliff’s Lab, I came up with an idea to address why the snowflake yeast used in previous work was limited to microscopic size. Inspired by Andy Knoll’s work (whom I was lucky to meet a couple of times!) and leveraging my diverse experience on yeast metabolism from Duncan Greig’s Lab, I designed this experiment to test the constraints on the evolution of multicellular size in snowflake yeast under different oxygen and metabolic conditions.
Did oxygen have any impact on the evolution of multicellular size? Yes! In fact, oxygen was the major factor in size evolution in snowflake yeast. Low oxygen concentrations strongly suppressed multicellular size evolution over 5,000 generations, the environment in which these yeast were previously evolved. In contrast, snowflake yeast evolved in supplemental (near modern-day level) oxygen showed the fastest increase in average multicellular size. Furthermore, unlike obligate multicellular eukaryotes from 600 million-year-old oceans that relied on oxygen, yeast can also grow without respiring oxygen. Under these fermentative conditions, simulating zero-oxygen environments, the cluster size of the yeast populations doubled in radius within just 150 days, outpacing the size increase seen in previous studies!
Evolution of macroscopic multicellularity in snowflake yeast:
Evolving larger size is crucial for multicellular organisms to undergo significant shifts in biophysical properties, cell biology, and genomics/proteomics. Having identified the constraint on size evolution and inspired by Richard Lenski and colleagues’ LTEE with bacteria, I decided snowflake yeast was now “ready” for a long-term evolution experiment. Fast-forward to the 600-day (3,000 generation) mark: five snowflake yeast populations evolved macroscopic multicellular clusters 20,000 times larger in volume, reaching millimeter scales visible to the naked eye! These macroscopic yeast grew clonally, starting from single cells, forming clusters containing an average of 300,000 cells—a dramatic shift, considering snowflake yeast in previous work had only a few hundred cells on average.
Bringing the results from the first two studies, our first paper that uncovered the constraint on snowflake yeast size evolution, and our second paper, that displays the evolution of macroscopic multicellularity, can be seen as the groundwork that made the MuLTEE and the subsequent research possible.
The now and the future of the MuLTEE:
In our subsequent work, we investigated the biophysics of branch entanglement, the coexistence of distinct phenotypic size classes of snowflake yeast, the molecular mechanisms behind cellular elongation and macroscopic multicellularity (such as the chaperone protein Hsp90), and emergent biophysical mechanisms like spontaneous fluid flows that may help alleviate constraints on nutrient transport.
Recently, we discovered that yeast in the MuLTEE have undergone whole-genome duplication and further increases in chromosome copy number (aneuploidies), aiding cell-level changes that drive macroscopic multicellular adaptations—all explored in our most recent Nature paper (also see the nice ‘behind the paper’ post from Kai about the story of this paper, which shows that studies at this scale are only possible through the dedicated work of many individuals, many of whom are almost always young scientists!). Unlike previous studies where tetraploid unicellular yeast are introduced through genetic engineering and revert to diploidy in benign environments, our experiment uniquely shows tetraploidy and aneuploidies evolving spontaneously and persisting long-term, driven by selection pressure shifting from single cells to multicellular individuals.
While the MuLTEE does not recapitulate the exact historical path of multicellular evolution, it showcases the first real-time evidence of the transition from single-celled individuals to multicellular individuals with novel cell biology, biophysics, and cluster size. These changes occur at the cost of single-cell fitness and lead to the entrenchment of the multicellular phenotype - all of which are now explored in the lab.
Furthermore, the MuLTEE is not only an experiment about how yeast could have evolved multicellular individuals but also a long-term evolution experiment of whole-genome duplication, aneuploidy, metabolic divergence (through fermentation vs respiration vs mixotrophy), and reductive evolution of mitochondria (akin to mitochondrion-related organelles or MROs).
In conclusion, this experiment is likely to generate more exciting research, which I am super excited to see by continuing it for the decade and beyond.
Here is a research highlight about key aspects of this work, written by Or Shalev and colleagues: Replaying the Evolution of Multicellularity
Other research:
Additionally, I am interested in understanding the genetic mechanisms of reproductive isolation and species formation in yeast. In a key study, we discovered that by repressing just two genes (SGS1 and MSH2) during meiosis, we could dramatically increase hybrid fertility between two diverged yeast species, Saccharomyces cerevisiae and S. paradoxus. This genetic manipulation raised hybrid gamete survival from less than 1% to 32%, comparable to non-hybrid crosses. This finding demonstrates that anti-recombination is the principal cause of hybrid sterility between these species.
By overcoming this barrier, we produced viable, euploid hybrid gametes with recombinant genomes from highly diverged parents. This discovery not only advances our understanding of speciation but also provides a valuable tool for yeast researchers. It enables the creation of hybrid strains with novel properties, facilitates trait mapping across species, and opens new possibilities for both basic research and biotechnology applications.
Here is a review paper exploring this topic, written by Jasmine Ono and me: Evolution and Molecular Bases of Reproductive Isolation
Press coverage about our work
• The Atlantic:
One of evolution’s biggest moments was re-created in a year
• National Geographic:
Evolving globs of yeast may unlock mysteries of multicellular life
• The New York Times:
An experiment repeated 600 times finds hints to evolution’s secrets
• Science Daily:
Did Earth’s early rise in oxygen help multicellular life evolve?
• Quanta Magazine:
Single cells Evolve large multicellular forms in just two years