Constraints on the G1/S transition pathway may favor selection of multicellularity as a passenger phenotype

Reviewer #1 (Public review):

Summary:

Ducrocq et al. present research exploring the genetic link between simple multicellular group formation (ace2Δ/ace2Δ) and its interaction with cell-cycle progression mutants (e.g., cln3Δ/cln3Δ), demonstrating that this combination can provide fitness benefits during fluctuating resource conditions, resulting in a rapid increase in the fraction of multicellular cell-cycle mutants over unicellular yeast without selection for multicellular size. Because both the multicellular phenotype and the regulatory link enabling faster escape from the stationary phase are controlled by the Ace2 transcription factor, this work demonstrates that multicellularity can arise as a side-effect of a completely independent fitness advantage unrelated to the benefits of group formation itself. As a "passenger phenotype," multicellularity could thus emerge for other selective reasons, potentially facilitating a later transition to more entrenched multicellularity if novel conditions arise where group formation becomes directly beneficial.

Strengths:

This work is novel and exciting for research exploring the very first steps of the transition from unicellularity to simple multicellularity. This is particularly significant because the formation of multicellular groups is almost always assumed to come at a cell-level fitness cost due to reduced reproductive fitness compared to remaining unicellular. This cell-level fitness cost generally needs to be outweighed by the benefits of multicellular group formation (e.g., large size escaping predation) for the multicellular phenotype to be stable, which is true for a large number of cases studied in the literature, where the multicellular phenotype can only evolve over unicellular competitors under strong selection for multicellular groups. However, this study presents an interesting case of a genetic and environmental condition under which individual cells (forming simple multicellular clusters) can actually have higher reproductive fitness than unicellular yeast. This demonstrates that the assumed cost at the single-cell level does not always apply. In summary, this work represents a unique example contrary to common assumptions regarding the costs of multicellular phenotypes, showing that simple multicellular phenotypes can evolve and remain stable without requiring strong selection for multicellular size or other benefits of group formation.

The claims and interpretation of the results align well with the data presented. This is due to the careful and straightforward experimental design testing predictions with a clear, stepwise methodology, ruling out alternative explanations and providing support for the proposed link between the mutations (ace2, cln3, and others), their impact on faster exit from quiescence, and thus earlier entry into reproduction in fresh media, resulting in higher fitness in the snowflake yeast phenotype compared to unicellular yeast.

Weaknesses:

The authors show that the same multicellular phenotype with higher cell-level fitness due to faster exit from the stationary phase can also be observed with alleles found at other loci in non-laboratory yeast strains, implying that the results are likely not specific to a peculiar case genetically engineered in laboratory strains, but that similar phenotypes may be present in nature. However, this remains to be explored further by examining the natural ecology of commercially available or wild yeast isolates and their genomes. This is by no means a weakness of this study and, therefore, not necessarily something the current work can improve. It does mean, however, that the relevance of these findings for early multicellularity in yeast, and even more so for nascent multicellularity in distinct taxa, remains to be explored in the future. Until then, it is difficult to make strong claims about how applicable these results would be for non-laboratory yeast and other taxa. Regardless, this work does its part by representing a very exciting finding.

Reviewer #2 (Public review):

Summary:

Here, the authors attempt to demonstrate that a simple model of multicellularity - snowflake yeast - exhibits key ecologically relevant changes in the regulation of the cell cycle. By examining the effects of the ace2 mutation in environments where multicellularity is not directly selected for or against, and combining it with mutations in key cell cycle regulators, they hope to show that mutations driving simple multicellularity can be selectively favored due to their effects on the release from quiescence rather than their effects on multicellularity itself.

Strengths:

The experiments performed are extensive and thorough. The yeast genotypes examined are judiciously chosen, so as to map out a functional model of the relationship between alterations to cell cycle control and changes to multicellularity phenotypes. Multiple possible interactions are examined, with the causal link and model of the relationship between the multicellular passenger phenotype and the selectable quiescence-release phenotype being well-supported. There are extensive controls demonstrating the separation between the 'passenger' multicellular phenotype and the cell cycle regulation phenotypes examined, including haploid/diploid strains with different multicellular phenotypes but similar cell cycle regulation phenotypes, and phenocopy strains in which downstream enzymes are deleted rather than key central regulators.

Weaknesses:

My only concerns about these results relate to the focus on selection on cell cycle control being examined in a model of multicellularity with key core cell cycle mutations rather than in a wild-type background, as this is a somewhat artificial system.

I believe, however, that the authors convincingly make their case that this work on the multicellular phenotypes of yeast represents a potent proof-of-concept that simple multicellularity can be driven into existence or selected for as a passenger phenotype due to pleiotropic effects of mutations under selection from real-world ecological pressures. They are able to connect this phenotype back to known mutations of particular cell cycle regulators (RB) in other multicellular lineages and demonstrate that ecologically relevant changes to the cell cycle are connected to multicellular phenotypes. As a proof of concept of the connection between these phenotypes, rather than a study of a particular event in the past of a living lineage, it makes a strong case.

A longstanding question in the field of multicellularity is the selective pressures that can drive simple multicellularity into existence and then act on simple multicells to drive their increased size and complexity. This work brings to the table tangible evidence of the possibility that, instead of being selected for on its own, simple multicellularity can be a side-effect of selection on other key phenotypes.

This separates the question of the origins of multicellularity and the forces that drive its further evolution. This separation can reframe how the field is studied, especially in the context of the apparent dichotomy between dozens of origins of 'simple' multicellularity across the tree of life and a few origins of 'complex' multicellularity in the history of Earth. Especially in light of other evidence that multicellularity is connected to changes in cell cycle regulation, I believe that this is an important insight that will alter the way we think about the origins of this key evolutionary transition.

Author response:

We thank the reviewers for their insightful comments on our work.

We agree with reviewer #1 that further experiments would be needed to figure out how the observations done on lab strains can apply to yeast in various ecological conditions and particularly in the wild. We here provide a proof of principle that multicellularity selection can arise as a side-effect. It obviously does not prove that it took place during yeast evolution, but we would like to emphasize that resource fluctuations are very common in ecological conditions, making it highly likely that the environmental conditions necessary for the selection of the side effects described have arisen.

We agree with reviewer #2 that our work on yeast strains is “somewhat artificial” as often the case with model organisms under laboratory conditions. Importantly though, we showed that the effect found with the cln3 knock-out mutation can be phenocopied by overexpression of WHI5 (encoding the yeast equivalent of Rb). We propose that variations in the levels of cell cycle regulators during evolution may have played a role in multicellularity selection as a side effect. We agree that this is merely a hypothesis to explain the selection of multicellularity (just like predator escape) and that there is no direct evidence that this occurred in the history of the lineage. Nevertheless, our work provides a first evidence that such a selection of multicellularity as a side effect could be possible, and gives a framework to understand how multicellularity can persist in the wild, even when it is not the primary target of selection.

We are currently working on the text and figure revisions suggested by the reviewers.