Evolving Bodies: A Storify follow-up

By Carl Zimmer | January 18, 2012 12:51 pm

In yesterday’s New York Times, I wrote about a new paper in which scientists report the evolution of single-celled yeast into multicellular snowflake-like “bodies.” Most (but not all) of the experts I contacted for the story had high praise for the study. (It also won an award when it was presented as a talk over the summer at the Society for the Study of Evolution.) Once the story appeared, however, some scientists took to Twitter to express their skepticism. As much as I like Twitter, this is one of the situations where it fails. You can’t have a conversation about genetics, lab strains versus wild types, etc., in 140 character chunks. At least not very satisfying ones.

So here’s what I decided to do last night. I used Storify to collect the comments of Leonid Kruglyak of Princeton and Michael Eisen of Berkeley, and then passed them on to Will Ratcliff, the lead author of the new study. He then responded. Below you’ll find the Storify tweets, and then Ratcliff’s response. Please continue the conversation in the comment thread. (And be sure to download the paper–it’s open access.)

[View the story “Yeast evolving” on Storify]

Will Ratcliff responds:

Well, I don’t buy it that yeast are multicellular in nature. Certainly some yeast in nature form small clusters (like strain RM11), but as far as I know, these are the exception to the rule. Most strains isolated in nature are unicellular, or at most, flocculating (which I still count as unicellular but social). [CZ: “Flocculating” refers to the clumps that unrelated yeast cells form when they starve.]

In our case, we’re working with strain Y55, a yeast that is is not highly lab adapted (we know this because it still sporulates at nearly 100% efficiency. Sporulation efficiency is typically lost after long periods of lab adaptation.) We’ve known through knockout mutation libraries that breaking the ability to release daughter cells after mitosis gives you a snowflake-shaped cluster. We’re not claiming that we’re the first to observe this phenotype. What we claim is that we’re the first to systematically examine the transition to multicellularity. We see the evolution of clusters from single cells as a result of selection acting on de novo mutations, we see a shift to between-cluster selection, and we see subsequent adaptation occurring cluster-level traits (like division of labor).

Our yeast are not utilizing ‘latent’ multicellular genes and reverting back to their wild state. The initial evolution of snowflake yeast is the result of mutations that break the normal mitotic reproductive process, preventing daughter cells from being released as they normally would when division is complete. Again, we know from knockout libraries that this phenotype can be a consequence of many different mutations. This is a loss of function, not a gain of function. You could probably evolve a similar phenotype in nearly any microbe (other than bacteria, binary fission is a fundamentally different process). We find that it is actually much harder to go back to unicellularity once snowflake yeast have evolved, because there are many more ways to break something via mutation than fix it. The amazing thing we see is that we rapidly see adaptations to this adaptation. If we select for more rapid settling, snowflake yeast evolve to delay reproduction until the parent is larger, allowing it settle more quickly. We see the evolution of higher rates of apoptosis as a way to regulate the size and number of propagules produced. We show that the transition to multicellularity in yeast is surprisingly easy, and have no reason to suspect it would be any harder in other microbes with a reproductive process similar to yeast.

Comments (16)

  1. Jim Johnson

    For Mr. Ratcliff: This strikes me as an experiment in whose ongoing investigation, local high school classes might participate.

    Each participating class would attempt (perhaps with baker’s, vintner’s , or laboratory yeast) to replicate your results. Those classes which successfully replicate an evolution to a strain exhibiting multicellular characteristics could report their results (with photos) on a website set up for the purpose.

    Going further, dried samples of the resulting strains could be sent to some central point (such as your lab, or perhaps to a participating university) where characteristics might be examined of the snowflakes produced by each culture, and perhaps further experiments performed, including multicultural environments where the relative fitness of various snowflake cultures in different simulated environments could be examined.

    Beyond engaging the students in the science, this might produce results with direct bearing on your current project.

  2. ST

    The paper certainly raises interesting questions how multicellularity may have arisen and how we should define multicellularity in the first place.

    Other than that the paper is very much oversold.
    Given that S. cerevisiae arose from a multicellular ancestor, it appears the paper describes the reversion of a unicellular organism to multicellularity, rather than the evolution of multicellularity. The multicellular ancestor makes the last sentence of the paper (“The potential for the evolution of multicellularity may be less constrained than is frequently postulated.”) rather ludicrous. In his response above, Will Ratcliff points out that many loss of function mutations can give rise to this phenotype. Exactly. But unfortunately the paper suggests that a novel trait was evolved and not that multiple loss of function mutations were isolated.
    In that regard the title of Figure 1 states “Rapid and convergent evolution…”. The rapid is already debunked and convergent would require starting with different organisms (http://www.pbs.org/wgbh/evolution/library/01/4/l_014_01.html). Mere reproducibility does not equal convergent evolution.

    Secondly, the argument that these clusters are indeed a multicellular organisms depends on the authors’ point that they see “division of labor”. What do they mean by division of labor? That cells differentiate, have different functions and in the end are dependent upon each other? No. Rather some cells die while others don’t. Cells dying isn’t much of a specialization, hence the authors emphasis on cells dying as part of *Programmed* cell death (apoptosis). *Programmed* cell death means that cells do differentiate and make a cell fate decision to die. But unfortunately the authors provide no evidence that these dying cells undergo *Programmed* cell death (staining for reactive oxygen species, a marker of cell “sickness”, and cell permeability, a late marker of cell death, is no evidence of a cell fate decision). Within any population of yeast, a fraction of cells will undergo precisely this form of cell death and eventually every single cell will die this way.

    Other points: “No propagules were produced by clusters less than a minimal size, demonstrating that the snowflake phenotype exhibits juvenile/adult life stage differentiation (Fig. 3B).” – Yeast cells die as they age. The snowflake cells grow outwards, meaning old cells will be towards the center and result in the release of “branches” when they do die. Saying this demonstrates a juvenile/adult life stage differentiation is a huge leap.
    Lots of talk about “cluster-level traits”, but no discussion of how they may simply arise from variations in single cell fitness. All these “cluster-level traits” appear to boil down to the rate at which single cells die.
    Flocculation also occurs between genetically identical cells that express the same cell surface proteins.
    Through their use of anthropomorphisms (“division of labor”, “suicide”, “junvenile/adult stage”) the authors suggest that their yeast isolates evolved novel and highly complex traits. But at no point do they acknowledge that there are simpler explanations.

  3. John Kubie

    Off Topic: Last summer I found Google+ very good for scientific discussions. Now that the user-base has expanded, not sure it still is. But it seemed to work much better than twitter or facebook. Very readable, and lengths are good. Discussions are generally high-level and polite. And you (generally) know who you’re talking to.

  4. @ Jim. This is a really cool idea, I’d like to see it expanded to different species of unicellular organisms too!

    @ST. Saccharomyces cerevisiae lost multicellularity hundreds of millions of years ago. I explained in the prior post why we are confident that the traits that we see evolving are not a reversion to ancestral multicellularity.

    I’ll address your points above sequentially. My responses in bold.

    1- ST- In his response above, Will Ratcliff points out that many loss of function mutations can give rise to this phenotype. Exactly. But unfortunately the paper suggests that a novel trait was evolved and not that multiple loss of function mutations were isolated.

    Only the first step, the evolution of clusters through post-division adhesion appears to be the result of a loss of function (the loss of the ability to separate off daughter cells after mitosis). After this, we see several adaptations that improve the fitness of clusters. We see an increase in cluster size, due to an increase in both the number of cells in the cluster as well as the size of those cells. We see the evolution of higher rates of apoptosis (I’ll explain below why this is not synonymous with ‘aged’ cells). These traits are novel, and are interesting to us because they improve the fitness of whole clusters, not their component cells. This means that adaptation is occurring in cluster-level traits, and that the yeast are evolving in a multicellular manner.

    2-ST-“Rapid and convergent evolution…”. The rapid is already debunked and convergent would require starting with different organisms.

    The rapid part is debunked? What? We started with a single genotype that did not form clusters. Mutations that result in clustering occur, and then increase in frequency due to selection. This is textbook evolution, and it occurs rapidly. As for convergence, 10 independent populations that started with the same ancestor evolved the snowflake phenotype, not, say huge cell size or flocculation. This is textbook convergent evolution.

    3-ST-Secondly, the argument that these clusters are indeed a multicellular organisms depends on the authors’ point that they see “division of labor”. What do they mean by division of labor? That cells differentiate, have different functions and in the end are dependent upon each other? No. Rather some cells die while others don’t. Cells dying isn’t much of a specialization, hence the authors emphasis on cells dying as part of *Programmed* cell death (apoptosis). *Programmed* cell death means that cells do differentiate and make a cell fate decision to die.

    I disagree with your assessment about what defines multicellularity, but that’s a matter for another time. A key step in programmed cell death in yeast is the production of reactive oxygen species (Frank Madeo has many papers discussing the finer points of yeast apoptosis, we’re using his procedure to measure ROS). Here’s what we see- the unicellular ancestor and snowflake yeast have very low levels of ROS induction, and very low levels of cell death during the first 24 hours of growth. During our experiment, they evolve a nearly 10-fold increase in the number of cells that are producing high levels of ROS during the exponential phase of growth. Because it was measured at the same time in the culture cycle, we know it’s not due to, say, differences in the number of old cells in the cluster. Further, we showed that this trait segregates independently of cluster size during classical mating experiments, so it’s heritable (almost certainly genetic). Even cells that are just one division old (about 1.5 hours) can begin the process of apoptosis, so it’s not restricted to ‘old aged’ cells. We show that by dying, these cells serve a function that benefits the cluster, allowing a cluster to produce a greater number of small, fast growing propagules. This function is different from the other genetically-identical cells in the same cluster that reproduce: it is thus division of labor. From an evolutionary perspective, the fact that the cells that perform this function give up their lives is significant, demonstrating that selection is acting at the level of the whole cluster, not it’s component cells.

    4-ST-“No propagules were produced by clusters less than a minimal size, demonstrating that the snowflake phenotype exhibits juvenile/adult life stage differentiation (Fig. 3B).” – Yeast cells die as they age. The snowflake cells grow outwards, meaning old cells will be towards the center and result in the release of “branches” when they do die. Saying this demonstrates a juvenile/adult life stage differentiation is a huge leap.

    We’re not claiming that this trait evolved specifically; rather we think that the juvenile/adult distinction is an emergent property of the way that clusters form. The fact of the matter is that propagules don’t make their own propagules until they grow up to their parent’s size. This is a exerts a real, tangible constraint on their evolution. For example, we see adaptation occurring in this emergent multicellular life history. In our divergent selection experiment, we found that snowflake yeast adapt to stronger selection for settling speed (less time to get to the bottom) by delaying reproduction until the cluster is larger, which increases settling speed.

    5- ST-Flocculation also occurs between genetically identical cells that express the same cell surface proteins.

    It can, yes, but nonrelatives that are express the same surface proteins can also stick together. See the excellent paper by Scott Smukalla et al., “FLO1 Is a Variable Green Beard Gene that Drives Biofilm-like Cooperation in Budding Yeast.” With post-division adhesion, this possibility for within-group chimerism is removed.

    If you are not satisfied with these explanations, you can simply wait for more data. We’re working out the molecular genetics of the transition described in the paper, including apoptosis (preliminary results suggest that it is indeed an active process, not simply the result of ‘old age’). We’re redoing our experiment with other microbes that never had a multicellular ancestor, such as Chlamydomonas. Finally, and perhaps most importantly, we’re continuing to evolve our yeast in a long-term experiment- my expectation is that this is just the beginning of their multicellular evolution.

  5. I’d like to add just a bit to Will’s excellent responses. Major morphological changes in microbial form and function have previously been observed. Perhaps the best example is that of the adaptive radiation of the bacterium Pseudomonas fluorescens in as little as three days! The first publication of this was in 1998 (Rainey and Travisano http://www.nature.com/nature/journal/v394/n6688/abs/394069a0.html). The phenotypes in that study are native to the laboratory conditions in which they were evolved, just as in our current research. A take home message from our current work, and previous studies, is that dramatic evolutionary outcomes can rapidly evolve under the appropriate environmental conditions.

  6. ST

    Will,
    Thank you for responding to my post and explaining several points in finer detail than you could have in the paper. Your paper is tremendously interesting and, as I said above raises interesting questions how multicellularity may have arisen. I personally would have appreciated your paper a lot more if the language was more tempered, especially in regards to apoptosis.

    1-If the multicellular ancestor of yeast became unicellular through a gain of function mutation, then loss of this function could easily result in multicellularity again. The problem is that your paper states: “The potential for the evolution of multicellularity may be less constrained than is frequently postulated.” No matter how you put it, starting with an organism that has multicellular ancestors is different from evolving multicellularity entirely de novo.

    2- See above why rapid is ill fitting. See this link (http://www.pbs.org/wgbh/evolution/library/01/4/l_014_01.html) regarding convergent evolution. (“This is a dramatic example of convergent evolution, when organisms that aren’t closely related evolve similar traits as they both adapt to similar environments.”) You started with the same strain of one species. Did you test different strains? Is Y55 potentially predisposed to this phenotype?

    3- I did not intend to define multicellularity, but rather what one might understand as “division of labor”. ROS maybe a step in apoptosis, although apoptosis in yeast certainly isn’t a clear cut case, but regardless showing the presence of ROS is not equivalent to demonstrating apoptosis.
    Thanks for sharing the comparison to the similarly cultured single cell yeast. I know you are limited in what you can include in a paper, but showing this is actually a heritable trait seems very important.
    To me a semi-penetrant lethal mutation that stochastically leads to cell death is the simplest explanation. Is that division of labor? Maybe, and with subsequent modifications it certainly could become it.

    4- Above you mentioned that “Even cells that are just one division old (about 1.5 hours) can begin the process of apoptosis, so it’s not restricted to ‘old aged’ cells.” and parent size does vary a lot. More importantly, you are selecting for something that settles in the culture. Your scheme likely selected against clusters smaller than a minimal size as they would have been to soluble to be captured by your transfer.
    Along those lines: Is the size of clusters limited by branches breaking off? I.e. does the size of a propagule plateau as the rate of cell death and branch dispersal reaches equilibrium with cluster growth. Also, does the transfer of the settled cells by pipette limit their size? Presumably clusters could get too large to be transferred in this manner.

    5- I included this as Carl stated above “”Flocculating” refers to the clumps that unrelated yeast cells form when they starve.”

    Will, I greatly appreciate your explanations and am looking forward to Michael’s and your future publications. As mentioned above, it would be very interesting to see if other S. cerevisiae isolates and close relatives of this species can make the transition as easily.
    Best of luck with your experiments.

  7. PA

    These are all very interesting points, however ST’s comment on the level of selection has not yet been addressed, and in my view this is the most critical and fascinating issue in the area of major evolutionary transitions. To claim that the level of selection has shifted from the single cell to the level of the group (the snowflake), it is necessary to show that the “higher level adaptions” observed here not only improve the fitness of the group, but that this is not simply a by-product of selection acting on the individual cells.
    Here, only cell fitness was measured – and shown to increase. This means that the “cluster-level traits” actually benefit the lower level. Will states in the paper that the cell and group fitness are aligned: “Snowflake yeast that produce smaller propagules can make more of them, increasing a cluster’s fecundity, and smaller propagules will be relatively faster growing than larger propagules” i.e. higher propagule production results in higher cell fitness.
    By forming snowflakes which produce altruistic cell types, cells can grow faster than their non-cooperative ancestors. This is an interesting example of the evolution of cooperation, not an evolutionary transition.

  8. Two points: skepticism is great in science, but it’s not necessary to be skeptical of absolutely everything all the time. It can really get in the way of learning about the research.

    The other point is that this is not about yeast: it’s about the evolution of multicellularity. The fact (or non-fact) of ancestral yeast multicellularity is irrelevant. This is an illustration of how multicellularity can evolve, and the conditions under which it evolves (I mean the entire body of research, not just this particular experiment). Every “major transition” in evolution began within a single species.

  9. Hi ST,

    Thanks for your follow-up, I appreciate the response and positive tone. A few brief responses to your additional questions:

    1) We already know from classical yeast genetics that there are multiple ways to get a cluster. Since this work was carried out in different strains than the one we used in our experiment, I’d be confident in guessing that snowflake yeast could be evolved in any Saccharomyces cerevisiae strain. Simply getting clusters through post-division adhesion strikes me as quite easy and general (and has been seen in other microbes), which is why Carl quoted me as saying that it’s not “some freaky yeast thing”.

    2) The evolution of clusters and subsequent cluster-level adaptation occurs very quickly, which is why we call it rapid. Even if you think this is affected by ancient multicellular ancestors (which I do not), it is still rapid. The link you provide is looking at convergent evolution among different animals, but that is not the definition of convergent evolution, it is a special case. The definition of convergent evolution (according to Doug Futuyma) is “Evolution of similar features independently in different evolutionary lineages, usually from different antecedent features or by different developmental pathways”.

    3) All we’re saying by “division of labor” is that dead cells play a beneficial, functional role that is different from live cells (like the outer layer of skin, say). We see that the rate of apoptosis increases through evolutionary time, showing that selection acts on this benefit.

    4) Yes, cluster size is limited by branches breaking off. You can see this in some of the videos (like S2).

    Finally, I’d like to note that language is an imprecise form of communication. You may not like the term ‘apoptosis’, but that’s what yeast people call the process by which cells produce tons of ROS then quickly die. If they called it ROS-Mediated Catastrophic Yeast Implosion or something like that I’d have used that term.

    Cheers,

    Will

  10. PA-

    We spend most of the paper looking at how the level of selection shifts from the uni- to the multicellular level. I’ll briefly summarize our arguments. Once clusters evolve, we see a shift to among cluster selection: they either get to the bottom of the tube and survive as a whole, or they fail to do so and die as a whole. If clusters vary in traits that affect their fitness, and if this variation is heritable, then cluster level traits can evolve in response to between-cluster selection. We have two different experimental results that confirm this:

    1) Divergent selection experiment. We see that in response to an increased strength of selection for rapid settling, snowflake yeast evolve faster settling by growing to a larger size. This is accomplished by delaying the onset of ‘reproductive maturity’, the minimum size of propagule production. This is a change in cluster-level traits that only makes sense in the context of a cluster of cells (single cells from larger, faster settling clusters probably don’t settle any faster than single cells from smaller, slower settling clusters).

    2) The evolution of increased apoptosis. Apoptotic cells die, reducing fitness at the single-cell level, but increase the fitness of the cluster. Here the two levels of selection are directly opposing each other, and the cluster-level benefit beats out the single-cell level cost. There is no better test over which level selection is acting.

    Of course, you can model this all thorough Hamiltonian kin selection (single cells that commit suicide help their clonemates in the same cluster increasing their inclusive fitness). This is not a problem for major transitions, though, as you can also do the same thing for frogs or redwood trees.

  11. Leonid

    “Certainly some yeast in nature form small clusters (like strain RM11), but as far as I know, these are the exception to the rule.” This is incorrect. The cluster-like “clumpy” growth with incomplete mother-daughter separation is the natural, wild-type state characteristic of most of non lab strains of S. cerevisiae.

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The Loom

A blog about life, past and future. Written by DISCOVER contributing editor and columnist Carl Zimmer.

About Carl Zimmer

Carl Zimmer writes about science regularly for The New York Times and magazines such as DISCOVER, which also hosts his blog, The LoomHe is the author of 12 books, the most recent of which is Science Ink: Tattoos of the Science Obsessed.

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