G. Ozan Bozdag

Research

My research started with the genetics of stress tolerance in plants and yeast, moved to evolutionary genetics and speciation during my Ph.D., and since 2018 has mostly focused on the Multicellularity Long-Term Evolution Experiment (MuLTEE).

Early work

For my M.Sc. at Izmir Institute of Technology, I worked on the functional genetics of adaptation to boron and metal stress in plants and yeast. For my Ph.D., I joined Duncan Greig’s lab at the Max Planck Institute for Evolutionary Biology in Plön, where I studied the roles of environmental adaptation and microbial cooperation in the evolution of gene duplication (Molecular Ecology 2014) and mitochondrial-nuclear coevolution in yeast.

Speciation genetics

Also in Duncan Greig’s lab, I worked on why hybrids between two yeast species, Saccharomyces cerevisiae and S. paradoxus, are sterile. The two species differ by about 12% at the single-nucleotide level, and their hybrids produce almost no viable gametes. By repressing two DNA repair genes, SGS1 and MSH2, during meiosis, we raised hybrid gamete viability from 0.5% to 33%. This showed that anti-recombination is the principal cause of hybrid sterility between these species (Current Biology 2021).

The resulting hybrid gametes are viable, euploid, and carry recombinant genomes from two highly diverged parents. That makes them useful beyond speciation research: they can be used to map traits across species and to build hybrid strains with new properties. Jasmine Ono and I reviewed this field in Current Opinion in Genetics & Development (2022).

The MuLTEE

A size limit in snowflake yeast

Snowflake yeast grow as branched clusters because daughter cells stay attached to their mothers after division. Earlier work by Will Ratcliff and Michael Travisano showed that daily selection for fast settling can evolve these clusters from single-celled yeast in a few hundred generations. But cluster size then stopped increasing. Even after 400 days of selection, the clusters were still microscopic, so the system looked unsuitable for studying open-ended multicellular evolution.

When I joined Will Ratcliff’s lab as a postdoc, I proposed testing whether oxygen was the limit. I was inspired by Andy Knoll’s work on oxygen and early multicellular life, and by my Ph.D. work growing yeast under different metabolic regimes in Duncan Greig’s lab. In an organism that respires, oxygen reaches interior cells only by diffusion, which limits how large a mass of respiring cells can get. If that was the constraint, changing metabolism should remove it.

In 2018, I founded twenty populations from a single ancestor under four regimes: anaerobic (petite mutants that cannot use oxygen at all), aerobic at intermediate oxygen, aerobic at high oxygen, and mixotrophic. After 145 transfers (about 800 generations), populations at intermediate oxygen, the condition used in earlier work, had increased in size by only 9%. Anaerobic populations increased by 93% and high-oxygen populations by 97%. The size limit came from the culture conditions, not from the yeast (Nature Communications 2021).

Why I kept transferring

At 145 transfers the experiment had answered its question. I kept transferring anyway, mostly because of Richard Lenski’s long-term evolution experiment with E. coli, which has produced some of its most important results decades after it started. A plateau in a selection experiment does not necessarily mean adaptation has stopped. Keeping the populations going changed my career, and the research program of the Ratcliff Lab.

For months, the anaerobic populations didn’t get any bigger. Then they started growing again, and within a few hundred more transfers the clusters were visible without a microscope. By 600 transfers (about 3,000 generations), all five anaerobic populations were macroscopic. Mean cluster radius went from 16 µm to 434 µm, a roughly 20,000-fold increase in volume, and the clusters became about 10,000-fold tougher, comparable to wood (Nature 2023).

We named the experiment the MuLTEE while writing that paper, as a nod to Lenski’s LTEE. To keep it running past a postdoctoral timeline, I moved into a Research Scientist position.

What we’ve found since

The MuLTEE doesn’t recapitulate the exact historical path of multicellular evolution. What it does show, in real time, is a transition from single cells to multicellular individuals with new cell biology, biophysics, and size. It is also, by now, a long-term experiment on several other things: whole-genome duplication, aneuploidy, metabolic divergence across fermentation, respiration, and mixotrophy, and reductive evolution of mitochondria.

The experiment today

The MuLTEE is past 10,000 generations (2,000+ days). Fifteen populations are transferred every day, five each in three metabolic treatments: anaerobic, mixotrophic, and obligately aerobic. Every 25 days, all fifteen are frozen, and these more than 3,000 samples form a frozen fossil record that can be revived and competed against its own descendants.

The experiment is funded by an NSF Long-Term Research in Environmental Biology (LTREB) award on which I am PI, with Will Ratcliff as co-PI. So far it has produced papers in Nature, Nature Ecology & Evolution, Nature Communications, Science Advances, and Physical Review X, 16 Ph.D. thesis chapters, and collaborations with physicists and cell biologists in four countries. We have shared strains with 25 groups in nine countries, and any population from any archived timepoint can be requested through multee.org.

Or Shalev and colleagues wrote a research highlight on the work: Replaying the evolution of multicellularity.

Press

More coverage is listed on the Ratcliff Lab press page.