Somatic drift and rapid loss of heterozygosity suggest small effective population size of stem cells and high somatic mutation rate in asexual planaria

  1. Hosseinali Asgharian
  2. Joseph Dunham
  3. Paul Marjoram
  4. Sergey V. Nuzhdin

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Planarian flatworms have emerged as highly promising models of body regeneration due to the many stem cells scattered through their bodies. Currently, there is no consensus as to the number of stem cells active in each cycle of regeneration or the equality of their relative contributions. We approached this problem with a population genetic model of somatic genetic drift. We modeled the fissiparous life cycle of asexual planarians as an asexual population of cells that goes through repeated events of splitting into two subpopulations followed by population growth to restore the original size. We sampled a pedigree of obligate asexual clones of Girardia cf. tigrina at multiple time points encompassing 14 generations. Effective population size of stem cells was inferred from the magnitude of temporal fluctuations in the frequency of somatic variants and under most of the examined scenarios was estimated to be in the range of a few hundreds. Average genomic nucleotide diversity was 0.00398. Assuming neutral evolution and mutation-drift equilibrium, the somatic mutation rate was estimated in the 10 −5 − 10 −7 range. Alternatively, we estimated N e and somatic μ from temporal changes in nucleotide diversity π without the assumption of equilibrium. This second method suggested even smaller N e and larger μ . A key unknown parameter in our model on which estimates of N e and μ depend is g , the ratio of cellular to organismal generations determined by tissue turnover rate. Small effective number of propagating stem cells might contribute to reducing reproductive conflicts in clonal organisms.

Article activity feed

  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

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

    Many flatworm species reproduce asexually, by fission, and the process relies on the activity of stem cells (neoblast), which drive regeneration. The question that this work tries to address is what is the dynamics of stem cells in this process, including how many stem cells contribute to regeneration, what are the mutation rates and selection mechanisms, if any. Towards this, the authors tracked one specimen of planarian Girardia tigrina for more than ten rounds of fission, and re-sequenced its genome at multiple time points and applied methods of population genetics to analyze and model the data. The main conclusion of the work is that there is high somatic mutation rate, rapid loss of heterozygosity, and a small size of the stem cell population that contributes to regeneration after fission.

    Reviewer #1 (Significance (Required)):

    The work has value, since it provides a framework to address the evolutionary aspects of stem cell dynamics in flatworms. However, as the authors point multiple times, there are many unknown biological parameters, such as, for example, the ratio of cell to organism regeneration (g), and simplifications, which can significantly influence the results. For this reason, the authors provide a range of estimates for somatic mutation rates and the effective stem cell population size, rather than some final conclusions. As the authors point out, further work will be needed to refine the model but generating new data for that is beyond the scope of this manuscript. As such, I find this manuscript is an important initial contribution to the field of stem cell population dynamics in flatworms, and its methods, results and conclusions convincing. I don't have further suggestions for improving this manuscript.

    Thank you very much for your positive assessment of our work.

    **Referees cross-commenting**

    I agree with the suggestion of Reviewer #2 that repeating the analysis on additional contings, instead of focusing only on one longest contig in the assembly, will be useful.

    We will process and analyze a few additional contigs to evaluate genomic variation in transmission of somatic variants in this system.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    **Summary:**

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    The authors aimed to use population genetics to determine the number of stem cells active in each cycle of regeneration or the equality of their relative contributions in planarians. They approached this by establishing a population with serial fission from one wild isolate of Girardia cf. tigrina collected in Italy. They used next generation sequencing to sample variants of regenerated worms at different generations of fissioning. They estimated the effective population size of stem cells to be a few hundreds, besides calculation of nucleotide diversity and somatic mutation rate. They propose small effective number of propagating stem cells might contribute to reducing reproductive conflicts in clonal organisms.

    **Major comments:**

    • Are the key conclusions convincing?

    The mutation rate is reasonable. The effective stem cell population size and the genetic diversity may vary between different species. A small effective stem cell population size is not counter intuitive.

    Generally, the work is interesting and deserves to be published.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The current analysis is based on many assumptions, one single set of experiments and a genome that is not well assembled. The authors have been careful with their language and documented the limitations in discussion.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    I will feel more comfortable if the authors can repeat the analysis with two more random long contigs to have a better idea if the localization of markers impacts the conclusion. The concern is if different parts of the genome behave differently and if the Girardia genome is highly repetitive. As the pipeline of analysis is established, I expect this can be completed in a month with no experimental cost.

    We will process and analyze a few additional contigs to evaluate genomic variation in transmission of somatic variants in this system.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Yes

    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes

    • Are the experiments adequately replicated and statistical analysis adequate?

    Yes

    **Minor comments:**

    • Specific experimental issues that are easily addressable.

    Yes.

    • Are prior studies referenced appropriately?

    Yes.

    • Are the text and figures clear and accurate?

    Yes.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Yes. Please also see the significance section.

    Specifically, my concerns are about writing and the context of current study.

    "Small effective number of propagating stem cells might contribute to reducing reproductive conflicts in clonal organisms." is confusing. Not a good abstract ending sentence for the work presented. Reproductive conflicts need clarification. How the work relates to that concept needs support. I recommend keeping the interpretations simple and focused on the data.

    Please see response under “Significance”.

    Reviewer #2 (Significance (Required)):

    Both questions the authors attempt to address, the genetic diversity of clonal animals and the number of stem cells contributing to regeneration, are interesting and important. The combination of these two is a bit odd in the manuscript. In other words, the population genetics approach did not address the cell biology question:how many or what proportion of stem cells are active in each cycle of regeneration. I would recommend the authors to focus the writing on one question only: the genetic diversity and evolution of a clonal species, which is driven by stem cell genome evolution and the process of regeneration. The cell biology question, phrased by the author in the abstract and introduction, need to be resolved by cell biologists. I understand the appeal to put the current study in the context of regeneration research. A balance should be achieved. Currently, the second sentence of the abstract and the first paragraph of introduction are odd and misleading. The first paragraph of the introduction can be a second paragraph to introduce the planarian system for the study.

    We will restructure the manuscript to clearly separate the findings that arose directly from experimental (sequencing) data i.e. magnitude and inheritance pattern of somatic variation, and the findings that were inferred from our approximate population genetic model and depend on the unknown parameter g i.e. the effective number of stem cells and the somatic mutation rate. We will emphasize the distinction. The statements that are tangentially relevant and are not directly supported by our analyses will be modified or removed.

    In the context of genetic diversity of clonal species, many studies shall be referenced. It is interesting as well to draw comparisons with other species. Asexual planarians are unique and interesting in that space.

    Thus said, the attempt to examine stem cell population genetics is especially interesting and important as the fissiparous planarians do not undergo bottleneck selection by zygotes. In the context of recent progress studying planarian genetic diversity (Nishimura, O. et al. 2015, Guo, L. et al. 2016), Asgharian H. et al.'s work is timely and an important contribution to planarian researchers and evolutionary biologists. The question has general interest to cancer biologists as well. The manuscript does not have the data and is not written in a way to reach such broader audiences yet. A community is growing to address these questions.

    We agree with the reviewer’s point about the pioneering works of Nishimura et al. 2015 and Guo et al. 2016. Both papers were indeed cited in our manuscript. We will cite more studies pertaining to the question of somatic genetic diversity in planarians.

    The study of planarian genetic diversity has just started with two publications (Nishimura, O. et al. 2015, Guo, L. et al. 2016). It is reasonable to have lots of limitations and assumptions in the manuscript. The work is an interesting piece to be published, assuming the major points listed in the review is addressed. The reported findings will be part of the early literature and inspiration for planarian researchers and evolutionary biologists. I expect many more future manuscripts will be published, either to reexamine the reported findings or to push our understanding of the question deeper.

    Thank you very much for this assessment. We fully agree.

    My expertise is with planarian biology, genome, genetics, and diversity. I do not have sufficient expertise to evaluate the equations used in the study.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    The authors aimed to use population genetics to determine the number of stem cells active in each cycle of regeneration or the equality of their relative contributions in planarians. They approached this by establishing a population with serial fission from one wild isolate of Girardia cf. tigrina collected in Italy. They used next generation sequencing to sample variants of regenerated worms at different generations of fissioning. They estimated the effective population size of stem cells to be a few hundreds, besides calculation of nucleotide diversity and somatic mutation rate. They propose small effective number of propagating stem cells might contribute to reducing reproductive conflicts in clonal organisms.

    Major comments:

    • Are the key conclusions convincing?

    The mutation rate is reasonable. The effective stem cell population size and the genetic diversity may vary between different species. A small effective stem cell population size is not counter intuitive.

    Generally, the work is interesting and deserves to be published.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The current analysis is based on many assumptions, one single set of experiments and a genome that is not well assembled. The authors have been careful with their language and documented the limitations in discussion.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    I will feel more comfortable if the authors can repeat the analysis with two more random long contigs to have a better idea if the localization of markers impacts the conclusion. The concern is if different parts of the genome behave differently and if the Girardia genome is highly repetitive. As the pipeline of analysis is established, I expect this can be completed in a month with no experimental cost.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Yes

    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes

    • Are the experiments adequately replicated and statistical analysis adequate?

    Yes

    Minor comments:

    • Specific experimental issues that are easily addressable.

    Yes.

    • Are prior studies referenced appropriately?

    Yes.

    • Are the text and figures clear and accurate?

    Yes.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Yes. Please also see the significance section. Specifically, my concerns are about writing and the context of current study.

    "Small effective number of propagating stem cells might contribute to reducing reproductive conflicts in clonal organisms." is confusing. Not a good abstract ending sentence for the work presented. Reproductive conflicts need clarification. How the work relates to that concept needs support. I recommend keeping the interpretations simple and focused on the data.

    Significance

    Both questions the authors attempt to address, the genetic diversity of clonal animals and the number of stem cells contributing to regeneration, are interesting and important. The combination of these two is a bit odd in the manuscript. In other words, the population genetics approach did not address the cell biology question:how many or what proportion of stem cells are active in each cycle of regeneration. I would recommend the authors to focus the writing on one question only: the genetic diversity and evolution of a clonal species, which is driven by stem cell genome evolution and the process of regeneration. The cell biology question, phrased by the author in the abstract and introduction, need to be resolved by cell biologists. I understand the appeal to put the current study in the context of regeneration research. A balance should be achieved. Currently, the second sentence of the abstract and the first paragraph of introduction are odd and misleading. The first paragraph of the introduction can be a second paragraph to introduce the planarian system for the study.

    In the context of genetic diversity of clonal species, many studies shall be referenced. It is interesting as well to draw comparisons with other species. Asexual planarians are unique and interesting in that space.

    Thus said, the attempt to examine stem cell population genetics is especially interesting and important as the fissiparous planarians do not undergo bottleneck selection by zygotes. In the context of recent progress studying planarian genetic diversity (Nishimura, O. et al. 2015, Guo, L. et al. 2016), Asgharian H. et al.'s work is timely and an important contribution to planarian researchers and evolutionary biologists. The question has general interest to cancer biologists as well. The manuscript does not have the data and is not written in a way to reach such broader audiences yet. A community is growing to address these questions.

    The study of planarian genetic diversity has just started with two publications (Nishimura, O. et al. 2015, Guo, L. et al. 2016). It is reasonable to have lots of limitations and assumptions in the manuscript. The work is an interesting piece to be published, assuming the major points listed in the review is addressed. The reported findings will be part of the early literature and inspiration for planarian researchers and evolutionary biologists. I expect many more future manuscripts will be published, either to reexamine the reported findings or to push our understanding of the question deeper.

    My expertise is with planarian biology, genome, genetics, and diversity. I do not have sufficient expertise to evaluate the equations used in the study.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Many flatworm species reproduce asexually, by fission, and the process relies on the activity of stem cells (neoblast), which drive regeneration. The question that this work tries to address is what is the dynamics of stem cells in this process, including how many stem cells contribute to regeneration, what are the mutation rates and selection mechanisms, if any. Towards this, the authors tracked one specimen of planarian Girardia tigrina for more than ten rounds of fission, and re-sequenced its genome at multiple time points and applied methods of population genetics to analyze and model the data. The main conclusion of the work is that there is high somatic mutation rate, rapid loss of heterozygosity, and a small size of the stem cell population that contributes to regeneration after fission.

    Significance

    The work has value, since it provides a framework to address the evolutionary aspects of stem cell dynamics in flatworms. However, as the authors point multiple times, there are many unknown biological parameters, such as, for example, the ratio of cell to organism regeneration (g), and simplifications, which can significantly influence the results. For this reason, the authors provide a range of estimates for somatic mutation rates and the effective stem cell population size, rather than some final conclusions. As the authors point out, further work will be needed to refine the model but generating new data for that is beyond the scope of this manuscript. As such, I find this manuscript is an important initial contribution to the field of stem cell population dynamics in flatworms, and its methods, results and conclusions convincing. I don't have further suggestions for improving this manuscript.

    Referees cross-commenting

    I agree with the suggestion of Reviewer #2 that repeating the analysis on additional contings, instead of focusing only on one longest contig in the assembly, will be useful.

  4. Version published to 10.1101/665166v2 on bioRxiv
  5. Version published to 10.1101/665166v1 on bioRxiv