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    Reply to the reviewers

    Point-by-point description of the revisions

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Hello, we wrote our review before seeing that you have special formatting requirements. We're just going to post our review in it's entirety rather than rewrite it based on these suggestions. It encompasses the above content, it's just not formatted in the suggested order. We hope that's OK!

    **Full review:**

    This manuscript makes a strong case for the evolvability of multicellular size via selection for settling rate in the icthyosporea. The use of an experimental evolution framework to assess the evolvability of multicellular phenotypes, using sedimentation rate as a selective pressure, extends the previous work of others into a new domain within the holozoan and the closest living relatives of animals. The natural, ecological significance of selection for sedimentation rate is a novel idea, and the connection between sedimentation rate and multicellular evolution in natural as opposed to contrived experimental circumstances is an interesting idea. The results are striking and well supported, with laboratory evolution rapidly adjusting both the cellular composition and the multicellular phenotypes of the organisms involved in ways that are well explained. This is an important result that brings the laboratory study of the evolution of multicellularity forward, into a different branch of the tree of life and showing its broad applicability.

    Sequencing of evolved lines adds significantly to the completeness of the story. While the causal role of these mutations in the production of the observed multicellular phenotypes are not demonstrated via manipulation or breeding, this is quite understandable in the light of the unusual model organism and the observed homologies and role of the genes involved. While this is largely clear from a reading, we believe the manuscript would benefit from a brief analysis of the numerical enrichment of genes with homologs involved in cytokinesis, cell membrane composition, and cell cycle control relative to the null hypothesis of genes picked randomly from the genome. If this is beyond the scope of this research in an unusual model organism with many poorly annotated genes, then a slightly expanded verbal discussion of the potential roles of the apparent functions of these genes in the evolution of multicellular clumping would be an appropriate substitute.

    We wholeheartedly recommend the publication of this manuscript with a number of minor revisions, which while not affecting the main conclusions or points of the manuscript will clarify important points, adjust small errors, and point the reader at relevant literature and concepts.

    __ANSWER____: We would like to heartily thank the reviewers for their appreciation of our work. __

    **Major points:**

    none.

    **Minor points:**

    Line 79 - is sedimentation rate really invariably associated with multicellularization? Active swimming would seem to prevent this.

    ANSWER____: We meant to refer to the fact that all published examples of the emergence of multicellularity from unicellular ancestors have been accompanied by an increased sedimentation rate. Active swimming alone would just increase the diffusion rate of cells and not counteract the effects of increased size and density; such an active mechanism would also require directionality away from the tendency to sediment. A more passive mechanism, whereby a genetic variant, or cell cycle transition, which simultaneously causes a relative decrease in density while increasing cell size, leaving the net sedimentation rate the same as the ancestor, while conceivable, has not been observed in the literature. We changed the text from “invariably” to “frequently” at line 80 to emphasize how this is an empirical observation.

    Line 164 - the precise phenotype in the evolution experiment being referred to is unclear without further context, with the ordering of paragraphs possibly needing a little work.

    ANSWER____: We tightened the paragraphs and merged both, the sentence containing “this phenotype” was removed.

    Line 178 - is sorting them into three classes informative? Are there different mutations associated with these, or is it just visual clumping on the numberline? Perhaps not a useful classification, but the existence of great variation is an important point to get across. A more useful classification might be those that increase sedimentation with large density changes versus exclusively by clumping.

    ANSWER____: We agree with this argument and ultimately decided to remove the visual classification. We revised the text and figures accordingly.

    Line 254 - excess cellular density is referred to interchangeably with density, when these are very different figures. This continues in line 269, and in the figure legends of Figure 4.

    ANSWER____: We fixed this.

    Line 341 - the rule of RCC1 homolog in other organisms could be expanded on in slightly more detail. Similarly, other mutations in this same section known to affect cytokinesis could have potential mechanisms for affecting clumping commented upon, especially given the cell membrane results in the figures.

    ANSWER____: We share the reviewer’s enthusiasm about some of these mutations. We, however, try to be very conservative about what each gene or protein could be doing. Indeed, the absence of genetic tools does not allow us to directly test the effect of each mutation. We added a couple of extra sentences about RCC1 as well as about cytokinetic proteins and their potential role in clumping phenotypes.

    Line 387 - awkward formatting or sentence structure, with dashes and commas.

    ANSWER____: We fixed the sentence structure.

    Line 395 - this cellular process, or this evolutionary process of selection for faster settling?

    ANSWER____: We revised this appropriately.

    Line 408 - per unit volume

    ANSWER____: Fixed.

    Line 425 - the idea of clumpiness as ancestral is quickly put forward and dismissed within a single sentence. This could be explored in slightly more detail as an option, before concluding that what is clear is that the phenotype is easy to change.

    ANSWER:____ We agree that it would be interesting to pursue the ecological role and distribution of clumping and cell cycle phenotypes for other species in the Ichthyosporea genus. We could propose alternative scenarios of which trait came or went first and test this hypothesis by calculating the correlation of the presence or absence of the trait with the branch lengths and branching patterns of phylogenetic trees we have built using genome sequences. However, for our dataset, this would nonetheless remain a fragile correlation consisting of five data points. We do not feel such speculation is helpful for the text.

    __However, because two reviewers have mentioned or suggested in this direction, we expanded the discussion and annotated the tips of the species tree in figure 5 with the traits of interest. The result shows that S. gastrica, S. tapetis and S. nootkatensis species exhibit clumpiness as a trait. However, the data is not enough to resolve whether the traits are “derived” or “ancestral”. __

    Line 437 - sedimentation as a highly variable trait, or a highly evolvable trait?

    ANSWER____: Evolvable trait. We fixed it in the text.

    Figure 1G, 1H: We are fairly certain that the logarithmic scale of DNA content and coenocyte volume are mislabeled. The scale that is labeled log2 in 1G in the legend goes up by factors of 2 rather than single digits. The axis is obviously logarithmic, and the log2 in the legend is superfluous and misleading. Similarly, in 1H a scale labeled as log10 goes from 1 to 30, which on a logarithmic scale would be a sphere approximately 100 kilometers wide. The numbers can remain, but the legend should remove the log10.

    ANSWER____: Fixed. It is indeed a log scale. We made sure to remove the confusing log2 and log10 from figure and legend.

    **General:**

    Were there any head to head competitions performed? Not suggesting you need to, but it's a nice way to directly examine fitness consequences of multicellularity, and is commonly done in the field. If you have done this it wasn't clear to us.

    __ANSWER____: We now included a fitness experiment previously performed using the clumpy S01 and S03 in a head-to-head competition with the Ancestor (AN). The results are shown in Figure 2E and Figure 2 – figure supplement 1D. __ The results reflect how the fast-sedimenting clumpy phenotype is highly advantageous in our experimental evolution selection procedure, however deleterious in the absence of selection.

    Reviewer #1 (Significance (Required)):

    see the above comments about writing the review before realizing there were specific formatting suggestions. I hope you understand us not wanting to re-write the review having already written it once.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    The present work adds to the growing literature on sedimentation rate as a major player in the evolution of multicellularity. Via rigorous experimentation, the authors convincingly show that they can select for increase sedimentation rate and identify two mechanisms underlying this increase: incomplete cellular separation leading to multicellular groups and increases in cellular density. They also show surprising natural variation in sedimentation and argue that, along with similar evidence from other organisms, their findings cement the likely major role of sedimentation and go farther by revealing the tight genetic control that it is under.

    Reviewer #3 (Significance (Required)):

    This is a very significant study because it illuminates processes and underlying mechanisms that could have played a major role in the transition to multicellularity. Their result will likely greatly influence the conceptual and theoretical thinking and will foster additional empirical directions. My only quibble with the manuscript is that I wished for a bit more ecological context and grounding of the main findings: in that respect, both the abstract and the last paragraph of the discussion leave me wanting and occasionally puzzled. If maintaining buoyancy is such a strong selective pressure and the variation in sedimentation rate is such a challenge to it, then I think explaining a bit more exactly why sedimentation would evolve, why so much variation would exist etc etc would be really helpful to the more naive reader. Just a bit further elaboration on selective pressures (even presumed ones and even if speculative) would be helpful to put the picture together.

    ANSWER____: We would like to thank Reviewer #3 for his/her comments. We do believe that extensive ecological context is highly relevant. Throughout the manuscript, we strived to be conservative in the way we describe both our model system and its experimental and natural settings, perhaps to a fault, but we now do offer an evolutionary model that tries to shed light into the phenotypic evolution of the various species through different routes (Fig. 5H). To elaborate more on the rationale behind this strategy, we offer the following two aspects:

    1. we are investigating a sizeable, but still a very limited number of six *Sphaeroforma * Therefore, we feel that explaining what trait may be considered ancestral is speculative based on the known species tree (we revised our Discussion in this regard and update figure 5A).
    2. __our knowledge about the ecological niches of Sphaeroforma species is limited. We avoid extensive speculation, and while inference of the potential ecological context is part of the scope of this study, we relied on an experimental approach to tackle our questions, rather than ecological observation or computational modeling. __
    • throughout the text we aimed to avoid taking a strong stance on the “adaptiveness” of the traits which we are measuring. This is because, depending on the model specification and parameters, ecological models could be made for or against whether the cellular traits of size and density, and their effects on the higher-level trait of sedimentation rate, might be adaptive “in the wild”.

    We hope that future studies will be able to tackle any open questions on the understanding of the ecology of ichthyosporeans, hopefully benefitting from our inferred evolutionary insights in this study.

    **A more minor point:**

    I remember seeing a talk by Will Ratcliff a while back in which he showed that in S cerevisiae they also see the two mechanisms of increased sedimentation: increased cellular size and clumping. Yet, I didn't see a reference to that work in the context of the cell density mechanism discussion and wondered why.

    __ANSWER____: We do believe to have cited the relevant papers from the Ratcliff lab. To be clear, we observed two separate physical mechanisms for fast-sedimentation: __


    1. by cell-clumping (increasing size),
    2. __by increasing the number of nuclei per unit volume (increasing density). __

    To our knowledge the 1st mechanisms was indeed observed in snowflake yeasts (for which we referenced all relevant studies), whereas the 2nd, which we believe might be specific to multinucleated cells, while a conceivable variable affected by mutations in the organisms from these studies, has not been measured to our knowledge. We added a new model figure (Figure5H) to hopefully better get this message across.


    Reviewer #4 (Evidence, reproducibility and clarity (Required)):

    In this study Dudin et al. explored the variability of sedimentation rates in members of the Sphaeroforma genus and found that sedimentation rates are very variable between different isolates as well as during the life cycle of each isolates. Following this observation Dudin et al. evolved S. arctica under a regime favoring fast settling objects. After a few hundred generations they observed that most lineages increased their sedimentation rate. Characterization of some of these evolved population suggests two distinct mechanisms allowing fast sedimentation: cluster formation by non-separation of cells post-cellularization and increase in object density. By sequencing the evolved lines Dudin et al. were able to identify that several mutations has been under the effect of positive selection and that some of the mutations relate to mechanisms involved in cell separation and cellularization.

    ANSWER____: We dearly thank Reviewer #4 for his/her time and efforts.

    **Major comments: **

    • Line 143, I don't understand how figure 1G shows that "nuclear division cycles were periodic...".

    ANSWER____: From previous published results (Ondracka et al 2018 & Dudin et al 2021), we know that nuclear divisions in S. arctica are strictly synchronized and occur within defined time-intervals. As can be seen in Figure 1G, DNA content doubles with a constant interval of about 9 hrs. Likewise, this phenomenon is clearly depicted in Figure 4F and Figure S4H. These results combined with results shown in Figure 1F, demonstrate that division cycles are still periodic in our experimental setting and are not occurring asynchronously as no odd number of nuclei per cell was observed.

    • When characterizing the evolved lines, the authors display (and measure?) separately the size and the sedimentation rate, but don't directly compare them. If the statement that density plays a role in the sedimentation rate of S4 and S9 but not S1, then correlation between size and sedimentation should be similar between AN and S1 and changed in S4 and S9. It would be nice to see these relationships and the correlations.

    __ANSWER____: We do indeed measure the size and the sedimentation rate of each fast-settling mutant separately. This is shown in figure 1C, where sedimentation rate is plotted against cell size for our dataset and the older Smayda (1973) data. Further, both measurements, directly, feed in the estimation of cellular density in Figures 4C and S4D (explained extensively in the methods). Cellular density estimations show the correlations and relationships between S1 and AN as well as between S4 and S9. __

    • Line 288: "surviving 780 generations of passaging for all 10 isolates" what data is this referring to?

    ANSWER____: This refers to growing cultures in the lab of fast-settling mutants with tens of passages done without any selection. These growing cultures maintained their clumping phenotypes even without a constant selection, suggesting they are due to a genetic modification. We are unsure about how to answer reviewer #4 as this is the data we are mentioning. We however changed “surviving” to “persisting for”, and hope it better clarified the sentence.

    • The weakest aspect of the paper is that there is neither a statistical argument (with a single anecdotal exception), from seeing the same genes or pathways mutated in parallel experiments, or experimental reconstruction that argues that any of the observed mutations were selected as opposed to being neutral mutations that hitch-hiked with adaptive mutations. One strongly suspect that some of the observed mutations were selected, but from the available data, it is impossible to know which were selected and which were hitch-hiking.

    ANSWER____: We agree that our draft did not elaborate in-depth if mutations were drivers versus passengers, a fact also mentioned by another reviewer. To be fair however, there are several important considerations to make.

    __First, and most importantly, we do offer an unprecedented look into the genetic underpinnings of this novel model organism, and demonstrate highly parallel phenotypic evolution in response to selection. The molecular genetic signal reflects this finding given a skewed dN/dS-ratio > 1. While the precise molecular changes are not as easy to interpret, molecular parallelism at the level of genes is not a prerequisite for directional selection in repeat lineages, especially given the complex genomic architecture of S. arctica. __

    Second, while we didn’t emphasize this a lot, the results from our bioinformatic analyses are pretty unique. We are dealing with a non-standard model organism here, with highly intriguing placement in the tree of life, but with big genome size, at >140 Mbp. This is 1-2 orders of magnitude larger than that of other single-celled model systems used in evolution experiments, including E. coli or S. cerevisiae. Unlike the latter two, this organism’s genome contains extensive levels of intergenic and intronic sequence, as well as a high amount of (simple sequence) duplication. Hence, the analyses of the resequencing data were a major effort, and it took an extensive amount of time to identify the mutations.

    Third, there are no genetic tools that would allow us to either perform molecular genetics or crossing with S. arctica as of now. This will change in the future, and in this event, our comprehensive list of target genes will be hopefully valuable to the field and beyond.

    • Even if the authors knew which mutations were selected, it is not possible to say if the mutations that have been selected are directly advantageous in the settling regime, they could be due to adaptation to lab conditions and higher temperatures, etc. Having a control evolution experiment with no settling selection would be required to reach the conclusion that the mutants were selected for faster sedimentation.

    __ANSWER____: We agree that a “no-selection”-control experiment would have been helpful for the molecular interpretation. But the clumping phenotype has never been observed to occur in many generations of passaging in any of the labs culturing these organisms and at different temperatures (we made sure to specify this in the text) As such, we argue that any adaptation to laboratory conditions must have happened before we conducted our selection experiment. Given that the molecular signals were unique (with one exception), we have reason to believe that the highly controlled nature of the experiment with a constant environment throughout, did at least not bias the molecular signals toward extensive genetic parallelism. __


    **Minor comments:**

    • Line 164, the authors write "this phenotype", it is unclear what phenotype is referred to as.

    ANSWER____: Fixed

    • Line 187: the authors use the word "radius" in the text, while using "perimeter" in the figure.

    ANSWER____: Fixed

    • Line 224: Is the use of the expression "incomplete detachment between daughter and mother cell" appropriate given that all cells emerge from a multinucleated cell?

    ANSWER____: Fixed – “incomplete detachment between cells.”

    • Line 151, typo, the "with" should be removed.

    ANSWER____: We believe the reviewer wanted to point out the “with” in line 251, which we fixed.

    • The intro about changes in ecology is nice but does not make sense given the rest of the paper, I would add it to the discussion.

    ANSWER____: We beg to differ with Reviewer#4 here, as the water column distribution for plankton in marine environment is one of the key aspects of our paper and is a critical parameter in models of water body ecology.

    • Line 399 "increase their cell size by increasing cell-cell adhesion post-cellularization" the first use of "cell" is misleading because the objects are now a collection of cells rather than a single cell.

    ANSWER____: Fixed

    Reviewer #4 (Significance (Required)):

    Most of the findings made in this study have been obtained in previous studies done with more genetically tractable organisms, however this is the first time that such experimental evolution was made on a unicellular non-model system organism closely related to animals. The significance of the work is reduced by the failure to produce evidence to answer two critical questions about the observed mutations: 1) were they selected during the experiment or did they hitch-hike with other selected mutations, and 2) if they were selected, were they selected because they led to faster sedimentation or some other aspect of the conditions in which they were passaged. It would take serious effort to perform additional experiments to address these questions and thus the authors are likely to be better off explaining that their work is unable to answer the questions and thus they are speculating about both the causality of the mutants and the nature of the advantage they conferred.


    ANSWER____: We beg to differ with the reviewer’s argument.

    __We believe that our study demonstrates heritable phenotypic changes for an evolvable, ecologically relevant trait, and their tight cellular regulation. We identify and carefully quantify how two cellular growth phenotypes – the nuclear division rate and cell size control –– can vary heritably and independently of one another, and together directly shape variation in a critical ecological parameter of a marine organism. Therefore, in addition to the fact that the work was performed in an emerging model marine organism, this work provides fundamental “novel” insight into cellular trait evolution more generally. __

    __Our results do not depend upon knowing the exact genetic mutations or molecular mechanisms which have caused these phenotypic changes. Nor, as the reviewer implies, do we claim to have identified particular mutations that were selected, or their effects on particular cellular phenotypes. We do, however, provide a large amount of evidence that the changes are likely genetic. With our sequencing effort, we find a strong, statistically significant, molecular signal of adaptation in the lineages (dN/dS > 1), and we publish a curated list of affected genes which are potentially causative for the phenotypes we observe. __

    Because we did not observe frequently recurrent mutations, as most directed (and cancer, antimicrobial resistance, etc.) evolution studies find, our results suggest that there is a large mutational target size affecting the phenotype of interest, reflecting its potentially broad genetic and molecular control mechanisms. We view these results as a great strength of the study, and consider this result in and of itself “novel”. Furthermore, we have now added and ____used a statistical genetic approach to quantify the heritability of traits, or what proportion of the variance in phenotype is due to an individual’s inherited state____ (Figure 1 – figure supplement 1A). The results show that Heritability exceeds 95% across phenotypes, and across the entire dataset, H exceeded 99% of the total phenotypic variance (ANOVA F = 1118 on 252 and 735 DF, p = 0). This means that for a typical individual genotype in a given environment, we could predict its average phenotypic measurement with >97% accuracy.

    The fact that we do not conclusively identify which particular mutations are causative does not obviate the overwhelming evidence that heritable changes occurred in our samples, leading to repeated phenotypic convergence affecting the trait of sedimentation rate. We believe these phenotypic changes, and our quantification of their magnitude, to be a “novel” and “significant” contribution to the literature on cellular trait evolution, ecology, and multicellularity.





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    Referee #3

    Evidence, reproducibility and clarity

    In this study Dudin et al. explored the variability of sedimentation rates in members of the Sphaeroforma genus and found that sedimentation rates are very variable between different isolates as well as during the life cycle of each isolates. Following this observation Dudin et al. evolved S. arctica under a regime favoring fast settling objects. After a few hundred generations they observed that most lineages increased their sedimentation rate. Characterization of some of these evolved population suggests two distinct mechanisms allowing fast sedimentation: cluster formation by non-separation of cells post-cellularization and increase in object density. By sequencing the evolved lines Dudin et al. were able to identify that several mutations has been under the effect of positive selection and that some of the mutations relate to mechanisms involved in cell separation and cellularization.

    Major comments:

    • Line 143, I don't understand how figure 1G shows that "nuclear division cycles were periodic...".
    • When characterizing the evolved lines, the authors display (and measure?) separately the size and the sedimentation rate, but don't directly compare them. If the statement that density plays a role in the sedimentation rate of S4 and S9 but not S1, then correlation between size and sedimentation should be similar between AN and S1 and changed in S4 and S9. It would be nice to see these relationship and the correlations.
    • Line 288: "surviving 780 generations of passaging for all 10 isolates" what data is this referring to?
    • The weakest aspect of the paper is that there is neither a statistical argument (with a single anecdotal exception), from seeing the same genes or pathways mutated in parallel experiments, or experimental reconstruction that argues that any of the observed mutations were selected as opposed to being neutral mutations that hitch-hiked with adaptive mutations. One strongly suspect that some of the observed mutations were selected, but from the available data, it is impossible to know which were selected and which were hitch-hiking.
    • Even if the authors knew which mutations were selected, it is not possible to say if the mutations that have been selected are directly advantageous in the settling regime, they could be due to adaptation to lab conditions and higher temperatures, etc. Having a control evolution experiment with no settling selection would be required to reach the conclusion that the mutants were selected for faster sedimentation.

    Minor comments:

    • Line 164, the authors write "this phenotype", it is unclear what phenotype is referred to as.
    • Line 187: the authors use the word "radius" in the text, while using "perimeter" in the figure.
    • Line 224: Is the use of the expression "incomplete detachment between daughter and mother cell" appropriate given that all cells emerge from a multinucleated cell?
    • Line 151, typo, the "with" should be removed.
    • The intro about changes in ecology is nice but does not make sense given the rest of the paper, I would add it to the discussion.
    • Line 399 "increase their cell size by increasing cell-cell adhesion post-cellularization" the first use of "cell" is misleading because the objects are now a collection of cells rather than a single cell.

    Significance

    Most of the findings made in this study have been obtained in previous studies done with more genetically tractable organisms, however this is the first time that such experimental evolution was made on a unicellular non-model system organism closely related to animals. The significance of the work is reduced by the failure to produce evidence to answer two critical questions about the observed mutations: 1) were they selected during the experiment or did they hitch-hike with other selected mutations, and 2) if they were selected, were they selected because they led to faster sedimentation or some other aspect of the conditions in which they were passaged. It would take serious effort to perform additional experiments to address these questions and thus the authors are likely to be better off explaining that their work is unable to answer the questions and thus they are speculating about both the causality of the mutants and the nature of the advantage they conferred.

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    Referee #2

    Evidence, reproducibility and clarity

    The present work adds to the growing literature on sedimentation rate as a major player in the evolution of multicellularity. Via rigorous experimentation, the authors convincingly show that they can select for increase sedimentation rate and identify two mechanisms underlying this increase: incomplete cellular separation leading to multicellular groups and increases in cellular density. They also show surprising natural variation in sedimentation and argue that, along with similar evidence from other organisms, their findings cement the likely major role of sedimentation and go farther by revealing the tight genetic control that it is under.

    Significance

    This is a very significant study because it illuminates processes and underlying mechanisms that could have played a major role in the transition to multicellularity. Their result will likely greatly influence the conceptual and theoretical thinking and will foster additional empirical directions. My only quibble with the manuscript is that I wished for a bit more ecological context and grounding of the main findings: in that respect, both the abstract and the last paragraph of the discussion leave me wanting and occasionally puzzled. If maintaining buoyancy is such a strong selective pressure and the variation in sedimentation rate is such a challenge to it, then I think explaining a bit more exactly why sedimentation would evolve, why so much variation would exist etc etc would be really helpful to the more naive reader. Just a bit further elaboration on selective pressures (even presumed ones and even if speculative) would be helpful to put the picture together.

    A more minor point:

    I remember seeing a talk by Will Ratcliff a while back in which he showed that in S cerevisiae they also see the two mechanisms of increased sedimentation: increased cellular size and clumping. Yet, I didn't see a reference to that work in the context of the cell density mechanism discussion and wondered why.

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    Referee #1

    Evidence, reproducibility and clarity

    Hello, we wrote our review before seeing that you have special formatting requirements. We're just going to post our review in it's entirety rather than rewrite it based on these suggestions. It encompasses the above content, it's just not formatted in the suggested order. We hope that's OK!

    Full review:

    This manuscript makes a strong case for the evolvability of multicellular size via selection for settling rate in the icthyosporea. The use of an experimental evolution framework to assess the evolvability of multicellular phenotypes, using sedimentation rate as a selective pressure, extends the previous work of others into a new domain within the holozoan and the closest living relatives of animals. The natural, ecological significance of selection for sedimentation rate is a novel idea, and the connection between sedimentation rate and multicellular evolution in natural as opposed to contrived experimental circumstances is an interesting idea. The results are striking and well supported, with laboratory evolution rapidly adjusting both the cellular composition and the multicellular phenotypes of the organisms involved in ways that are well explained. This is an important result that brings the laboratory study of the evolution of multicellularity forward, into a different branch of the tree of life and showing its broad applicability.

    Sequencing of evolved lines adds significantly to the completeness of the story. While the causal role of these mutations in the production of the observed multicellular phenotypes are not demonstrated via manipulation or breeding, this is quite understandable in the light of the unusual model organism and the observed homologies and role of the genes involved. While this is largely clear from a reading, we believe the manuscript would benefit from a brief analysis of the numerical enrichment of genes with homologs involved in cytokinesis, cell membrane composition, and cell cycle control relative to the null hypothesis of genes picked randomly from the genome. If this is beyond the scope of this research in an unusual model organism with many poorly annotated genes, then a slightly expanded verbal discussion of the potential roles of the apparent functions of these genes in the evolution of multicellular clumping would be an appropriate substitute.

    We wholeheartedly recommend the publication of this manuscript with a number of minor revisions, which while not affecting the main conclusions or points of the manuscript will clarify important points, adjust small errors, and point the reader at relevant literature and concepts.

    Major points:

    none.

    Minor points:

    Line 79 - is sedimentation rate really invariably associated with multicellularization? Active swimming would seem to prevent this.

    Line 164 - the precise phenotype in the evolution experiment being referred to is unclear without further context, with the ordering of paragraphs possibly needing a little work.

    Line 178 - is sorting them into three classes informative? Are there different mutations associated with these, or is it just visual clumping on the numberline? Perhaps not a useful classification, but the existence of great variation is an important point to get across. A more useful classification might be those that increase sedimentation with large density changes versus exclusively by clumping.

    Line 254 - excess cellular density is referred to interchangeably with density, when these are very different figures. This continues in line 269, and in the figure legends of Figure 4.

    Line 341 - the rule of RCC1 homolog in other organisms could be expanded on in slightly more detail. Similarly, other mutations in this same section known to affect cytokinesis could have potential mechanisms for affecting clumping commented upon, especially given the cell membrane results in the figures.

    Line 387 - awkward formatting or sentence structure, with dashes and commas.

    Line 395 - this cellular process, or this evolutionary process of selection for faster settling?

    Line 408 - per unit volume

    Line 425 - the idea of clumpiness as ancestral is quickly put forward and dismissed within a single sentence. This could be explored in slightly more detail as an option, before concluding that what is clear is that the phenotype is easy to change.

    Line 437 - sedimentation as a highly variable trait, or a highly evolvable trait?

    Figure 1G, 1H: We are fairly certain that the logarithmic scale of DNA content and coenocyte volume are mislabeled. The scale that is labeled log2 in 1G in the legend goes up by factors of 2 rather than single digits. The axis is obviously logarithmic, and the log2 in the legend is superfluous and misleading. Similarly, in 1H a scale labeled as log10 goes from 1 to 30, which on a logarithmic scale would be a sphere approximately 100 kilometers wide. The numbers can remain, but the legend should remove the log10.

    General:

    Were there any head to head competitions performed? Not suggesting you need to, but it's a nice way to directly examine fitness consequences of multicellularity, and is commonly done in the field. If you have done this it wasn't clear to us.

    Significance

    see the above comments about writing the review before realizing there were specific formatting suggestions. I hope you understand us not wanting to re-write the review having already written it once.

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