Hidden paths to endless forms most wonderful: parasite-blind diversification of host quality

Evolutionary diversification can occur in allopatry or sympatry, can be driven by selection or unselected, and can be phenotypically manifested immediately or remain latent until manifested in a newly encountered environment. Diversification of host–parasite interactions is frequently studied in the context of intrinsically selective coevolution, but the potential for host–parasite interaction phenotypes to diversify latently during parasite-blind host evolution is rarely considered. Here, we use a social bacterium experimentally adapted to several environments in the absence of phage to analyse allopatric diversification of host quality—the degree to which a host population supports a viral epidemic. Phage-blind evolution reduced host quality overall, with some bacteria becoming completely resistant to growth suppression by phage. Selective-environment differences generated only mild divergence in host quality. However, selective environments nonetheless played a major role in shaping evolution by determining the degree of stochastic diversification among replicate populations within treatments. Ancestral motility genotype was also found to strongly shape patterns of latent host-quality evolution and diversification. These outcomes show that (i) adaptive landscapes can differ in how they constrain stochastic diversification of a latent phenotype and (ii) major effects of selection on biological diversification can be missed by focusing on trait means. Collectively, our findings suggest that latent-phenotype evolution should inform host–parasite evolution theory and that diversification should be conceived broadly to include latent phenotypes.

Supplementary material -Statistical analysis: -I think it would be better for the statistical analysis to be included in the main manuscript.
-Where the authors fit random effects in their models -they should clearly state whether these were random effects on the intercept or random effects on the gradient. - In the second paragraph of statistical analysis the authors don't explicitly state what the response variable is. - In the second paragraph of the statistical analysis, the authors fit two fixed effects, but did they fit an interaction between these effects? And if not, why did the authors choose to not fit an interaction?
Minor comments: -Line 22: there is a 'II)' but no 'I)' -Line 94: 'has been little investigated' -I understand what the authors mean here but the phrasing is a little odd. I think that this is a paper of interest and is generally well written. I enjoyed reading it and I like how the latent phenotype and LPE were discussed and defined.
However, I have struggled to understand the experimental set-up and rationale for using the DZ strain for quantifying phages after the 24 hour "host quality experiment"….that is if I have interpreted the experimental set up correctly. Please see my comments on this below. Until I could get some clarity on the experimental setup and the rationale behind it is hard to properly evaluate the results and discussion.
In line 90-91… you define host quality as the degree to which a host genotype or population facilities parasite growth. Am I correct in thinking that for phage quantification following your 24 hour phage propagation/host quality experiment that you used strain DZ1 as the only host to quantify the phages?
If this is the case then I think that there may be a fundamental problem with this approach due to efficiency of plaquing (EOP) which can vary widely between bacterial hosts. With EOP you can get huge variance in phage titres (by many orders of magnitude) for a single phage stock when tested on highly related hosts.
For this reason you need to quantify phage on the host that you are interested in assessing for host quality not on a "common garden" host e.g. DZ2. By using DZ2 for quantification of the phage populations size on a given host (as a proxy for host quality) you do not have a measure of host quality on that host, you only have a measure of how it titres on DZ2 -i.e. you could have some phages that grow really well on their own host but not on DZ2 and vice versa. I hope this point makes sense as it is key to the experiment but please correct me if I have misinterpreted the design.
In addition to this, another key issue for me is the use of the 24 hour phage propagation experiment as a measure of host quality. 24 hours is a long time from a bacterial and phage perspective and you could get selection for resistance amongst other things during this time frame. I am not sure that it is a good and accurate measure of host quality as you define it. Perhaps a one more useful measure(s) of host quality would be to take the hosts you are interested in and doing a one-step growth curve with them… I think this would provide a much better measure of host quality as it you will get information on phage adsorption, length of infection cycle and burst size which will allow you measure the number of progeny produced per host and over what time-scale. I think this would really bolster your study as it would remove any ambiguity or alternative explanation as to why you see lower populations on different evolved hosts.
Another issue was with the initial experimental set-up that I am not sure I understood properlywere six different ancestral variants used to initiate the experiment? Line 221 Also, Line 104 onwards -you discuss the potential receptors sites of phages but do you have any detailed information on how the phage used in this study infects M. xanthus and how resistance evolves -this might help inform the data that you have observed for certain selective environments? For example you investigate the M. xanthus motility systems -is this because phage bind to flagella or pilli or what was the motivation for using these variants in the analysis.

06-Feb-2021
Dear Ms Freund: I am writing to inform you that your manuscript RSPB-2020-2773 entitled "Hidden paths to endless forms most wonderful: Parasite-blind diversification of host quality" has, in its current form, been rejected for publication in Proceedings B. Both reviewers and the AE have provided comments on your manuscript (which you can find below), and while they, and I, like your premise, there are a number of issues that need to be clarified before we can further consider your manuscript. With this in mind we would be happy to consider a resubmission, provided the comments of the referees are fully addressed. However please note that this is not a provisional acceptance and we will send the revision back to the original reviewers, should the be available.
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Sincerely, Dr Sarah Brosnan Editor, Proceedings B mailto: proceedingsb@royalsociety.org Associate Editor Board Member: 1 Comments to Author: This paper presents interesting data on how latent phenotypes that evolve (either neutrally or non-neutrally) in non-focal environments are then expressed and thus shape interspecific outcomes in novel environments where the interactor is present, delving into an interesting question in evolution and ecology. Both reviewers found the topic addressed and the findings interesting. Reviewer 1 had several important questions about the statistical analyses and ways to clarify what was done and why, while Reviewer 2 had several fundamental questions on how the work was performed and the implications of these choices, all of which should be addressed. I echo the comments of Reviewer 1 that the paper will need some reworking for clarification and to have the impact required for publication in Proc B.

Reviewer(s)' Comments to Author:
Referee: 1 Comments to the Author(s) The authors use experimental evolution of a social bacterium (Myxococcus xanthus) to analyse the allopatric diversification of latent host quality. They found that evolving the bacterium in the absence of phage reduced the quality of the host, from the perspective of the phage. In addition, the authors highlight that latent-phenotype evolution, a term they coin, should be used to inform host-parasite evolutionary theory.
-Need more information on statistical analysis in the main body of the paper (line 267-268). Supplementary material -Statistical analysis: -I think it would be better for the statistical analysis to be included in the main manuscript.
-Where the authors fit random effects in their models -they should clearly state whether these were random effects on the intercept or random effects on the gradient. -In the second paragraph of statistical analysis the authors don't explicitly state what the response variable is. -In the second paragraph of the statistical analysis, the authors fit two fixed effects, but did they fit an interaction between these effects? And if not, why did the authors choose to not fit an interaction?
Minor comments: -Line 22: there is a 'II)' but no 'I)' -Line 94: 'has been little investigated' -I understand what the authors mean here but the phrasing is a little odd.
-Line 273-285 -Perhaps this paragraph would be better suited for the discussion for discussion as there are no results here. Results don't begin until line 288.
-Line 295-309 and Line 430-444 -Again these sections are more discussion based than results focussed.
-Line 471 -write out "long term evolution experiment" before the acronym.

Referee: 2
Comments to the Author(s) Dear Authors, I think that this is a paper of interest and is generally well written. I enjoyed reading it and I like how the latent phenotype and LPE were discussed and defined.
However, I have struggled to understand the experimental set-up and rationale for using the DZ strain for quantifying phages after the 24 hour "host quality experiment"….that is if I have interpreted the experimental set up correctly. Please see my comments on this below. Until I could get some clarity on the experimental setup and the rationale behind it is hard to properly evaluate the results and discussion.
In line 90-91… you define host quality as the degree to which a host genotype or population facilities parasite growth. Am I correct in thinking that for phage quantification following your 24 hour phage propagation/host quality experiment that you used strain DZ1 as the only host to quantify the phages?
If this is the case then I think that there may be a fundamental problem with this approach due to efficiency of plaquing (EOP) which can vary widely between bacterial hosts. With EOP you can get huge variance in phage titres (by many orders of magnitude) for a single phage stock when tested on highly related hosts. For this reason you need to quantify phage on the host that you are interested in assessing for host quality not on a "common garden" host e.g. DZ2. By using DZ2 for quantification of the phage populations size on a given host (as a proxy for host quality) you do not have a measure of host quality on that host, you only have a measure of how it titres on DZ2 -i.e. you could have some phages that grow really well on their own host but not on DZ2 and vice versa. I hope this point makes sense as it is key to the experiment but please correct me if I have misinterpreted the design.
In addition to this, another key issue for me is the use of the 24 hour phage propagation experiment as a measure of host quality. 24 hours is a long time from a bacterial and phage perspective and you could get selection for resistance amongst other things during this time frame. I am not sure that it is a good and accurate measure of host quality as you define it.
Perhaps a one more useful measure(s) of host quality would be to take the hosts you are interested in and doing a one-step growth curve with them… I think this would provide a much better measure of host quality as it you will get information on phage adsorption, length of infection cycle and burst size which will allow you measure the number of progeny produced per host and over what time-scale. I think this would really bolster your study as it would remove any ambiguity or alternative explanation as to why you see lower populations on different evolved hosts.
Another issue was with the initial experimental set-up that I am not sure I understood properlywere six different ancestral variants used to initiate the experiment? Line 221 Also, Line 104 onwards -you discuss the potential receptors sites of phages but do you have any detailed information on how the phage used in this study infects M. xanthus and how resistance evolves -this might help inform the data that you have observed for certain selective environments? For example you investigate the M. xanthus motility systems -is this because phage bind to flagella or pilli or what was the motivation for using these variants in the analysis.
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Comments to the Author I am satisfied that the authors addressed my comments and have revised the manuscript appropriately.

Review form: Reviewer 2
Recommendation Accept as is

Scientific importance: Is the manuscript an original and important contribution to its field? Excellent
General interest: Is the paper of sufficient general interest? Good Quality of the paper: Is the overall quality of the paper suitable? Excellent Is the length of the paper justified? Yes

Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Comments to the Author Thank you for your detailed clarification, it has greatly helped with the interpretation of the results and conclusions reached. within the confines of your definition of host quality I think the experiments are perfectly fine but perhaps in future studies some one step growth curves could provide more detailed insight.

17-Mar-2021
Dear Dr Freund I am pleased to inform you that your Review manuscript RSPB-2021-0456 entitled "Hidden paths to endless forms most wonderful: Parasite-blind diversification of host quality" has been accepted for publication in Proceedings B.
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If you have already submitted your data to dryad you can make any necessary revisions to your dataset by following the above link. Comments to the Author(s). Thank you for your detailed clarification, it has greatly helped with the interpretation of the results and conclusions reached. within the confines of your definition of host quality I think the experiments are perfectly fine but perhaps in future studies some one step growth curves could provide more detailed insight.

24-Mar-2021
Dear Ms Freund I am pleased to inform you that your manuscript entitled "Hidden paths to endless forms most wonderful: Parasite-blind diversification of host quality" has been accepted for publication in Proceedings B.
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RSPB-2020-2733
Hidden paths to endless forms most wonderful: Parasite-blind diversification of host quality Lisa Freund, Marie Vasse and Gregory J. Velicer,

Response to Referees
Associate Editor Board Member: 1 Comments to Author: This paper presents interesting data on how latent phenotypes that evolve (either neutrally or nonneutrally) in non-focal environments are then expressed and thus shape interspecific outcomes in novel environments where the interactor is present, delving into an interesting question in evolution and ecology. Both reviewers found the topic addressed and the findings interesting. Reviewer 1 had several important questions about the statistical analyses and ways to clarify what was done and why, while Reviewer 2 had several fundamental questions on how the work was performed and the implications of these choices, all of which should be addressed. I echo the comments of Reviewer 1 that the paper will need some reworking for clarification and to have the impact required for publication in Proc B.
We thank the editor and both referees for their thoughtful and positive comments. We respond to the referee comments below and have made corresponding manuscript changes in most cases. In particular, we have sought to make our statistical analyses clearer in response to Referee 1 and, in response to Referee 2, have sought to better clarify our experimental procedures and the importance of using a single highly susceptible strain of M. xanthus (DZ1) to quantify populations sizes of Mx1 phage after 24 h of growth in liquid cultures of various MyxoEE-3 populations, which is our measure of host quality in the context of this study.

Referee: 1
Comments to the Author(s) The authors use experimental evolution of a social bacterium (Myxococcus xanthus) to analyse the allopatric diversification of latent host quality. They found that evolving the bacterium in the absence of phage reduced the quality of the host, from the perspective of the phage. In addition, the authors highlight that latent-phenotype evolution, a term they coin, should be used to inform host-parasite evolutionary theory.
-Need more information on statistical analysis in the main body of the paper (line 267-268). We would have liked to move the statistical material to the main text, but we consulted the editor on this point and were told that the restriction on the page number is very strict, and therefore we regretfully need to keep the statistics in the supplement.
- Figures 1-4: The figures are extremely small. We noticed the same after submission and regret this. The figures were regular size when we uploaded them and were somehow made smaller by the Proc B file conversion process. We will discuss this issue with the production editor to make sure the figures are adequately sized for publication if the paper is accepted.

Supplementary material -Statistical analysis: -I think it would be better for the statistical analysis to be included in the main manuscript.
See reply above.
-Where the authors fit random effects in their models -they should clearly state whether these were random effects on the intercept or random effects on the gradient. We fitted on the intercept and now indicate this in the manuscript.

-In the second paragraph of statistical analysis the authors don't explicitly state what the response variable is.
We now indicate that the response variable is the final number of phage particles after 24 h of growth in liquid cultures of various M. xanthus populations (log-transformed data).
-In the second paragraph of the statistical analysis, the authors fit two fixed effects, but did they fit an interaction between these effects? And if not, why did the authors choose to not fit an interaction? We did fit the interaction between the two fixed effects and now clearly indicate this in the text.

Minor comments: -Line 22: there is a 'II)' but no 'I)'
This has been corrected.
-Line 94: 'has been little investigated' -I understand what the authors mean here but the phrasing is a little odd. We prefer this phrasing over alternatives.
-Line 273-285 -Perhaps this paragraph would be better suited for the discussion for discussion as there are no results here. Results don't begin until line 288.
-Line 295-309 and Line 430-444 -Again these sections are more discussion based than results focussed. We think keeping this material in the Results section is helpful for assisting readers with interpreting the results as we describe them.
-Line 471 -write out "long term evolution experiment" before the acronym. We have written this out.
- Fig 1B and 2A -include "HA" and "SA" in figure legend -I assume these refer to hard and soft agar, but it would be good to include this in the figure legend for clarity. We now explain these abbreviations in the figure legend.

-Supplementary info: Final line of the statistical analysis -the references are not provided for all of the r packages (ggpubr and ggsignif).
We added the missing references.

Referee: 2
Comments to the Author(s) Dear Authors, I think that this is a paper of interest and is generally well written. I enjoyed reading it and I like how the latent phenotype and LPE were discussed and defined.
We thank the reviewer for their interest and positive comments.
However, I have struggled to understand the experimental set-up and rationale for using the DZ strain for quantifying phages after the 24 hour "host quality experiment"….that is if I have interpreted the experimental set up correctly. Please see my comments on this below. Until I could get some clarity on the experimental setup and the rationale behind it is hard to properly evaluate the results and discussion.
We apologize that we failed to explain the rationale for the choice of M. xanthus strain DZ1 to quantify the number of viable Mx1 phage particles after Mx1 growth on MyxoEE-3 populations, as well as for any other aspects of our experiment descriptions that may not have been clear. We have revised the manuscript to include thise rationale for using DZ1 as an indicator strain for phage quantification and to enhance the clarity of relevant experiment descriptions. DZ1 was chosen for the plaque assay because it was previously known to be extremely susceptible to lysis by Mx1, which readily forms plaques within lawns of DZ1. We use well-established plaque assays for our quantification of Mx1 phage (the Double Agar Overlay method, Kropinski et al. 2009), as elaborated further below and in our Methods section. In line 90-91… you define host quality as the degree to which a host genotype or population facilities parasite growth. Am I correct in thinking that for phage quantification following your 24 hour phage propagation/host quality experiment that you used strain DZ1 as the only host to quantify the phages? If this is the case then I think that there may be a fundamental problem with this approach due to efficiency of plaquing (EOP) which can vary widely between bacterial hosts. With EOP you can get huge variance in phage titres (by many orders of magnitude) for a single phage stock when tested on highly related hosts.
Yes, only DZ1 was used for the phage-quantification step at the end of the assay and we now state this more explicitly in the Methods. We define host quality as the degree to which a host population supports phage growth in liquid. Importantly, we are not quantifying titres resulting from growth on DZ1. in liquid and DZ1 lawns serve merely to allow us to estimate the number of viable phage particles after growth in liquid on distinct MyxoEE-3 populations. With our approach, the variance in phage numbers comes solely from differences in Mx1 growth on distinct MyxoEE-3 populations in liquid. It is precisely because we use only one phage genotype and only one M. xanthus plaque-permissive strain -DZ1 -that we can quantitatively compare how well Mx1 grows on the different MyxoEE-3 populations and ancestors during the liquid phase.
For clarity, we here briefly summarize the assay again. (Apologies for repeating points already understood by the referee, but we err on the side of thoroughness to insure clarity.) First, we distinguish between two major phases of our assays to quantify the quality of MyxoEE-3 populations as Mx1 hosts, namely i) the growth phase in liquid, which is the context of host quality in this study and ii) the post-growth quantification phase, in which we determine the number of viable phage particles resulting from growth in the liquid phase.
Growth phase -growth of a clonal Mx1 stock on diverse MyxoEE-3 bacterial populations A single phage stock was used as the common source for all of our Mx1 growth assays. Importantly, Mx1 was not part of the MyxoEE-3 evolution experiment, in which only M. xanthus evolved.
To initiate the host-quality growth assays, aliquots of our Mx1 source stock of identical volume and Mx1 density (~2 x 10 6 particles/ml) were added to cultures of different MyxoEE-3 populations (or their ancestors) of standardised bacterial density (~2 x 10 8 cells/ml) at T0. These bacteria-phage communities in liquid culture were then incubated for 24 h under the specified conditions to assess host quality.
Quantification phase -dilution-plating of post-growth Mx1 populations and counting the resulting plaques Importantly, the Mx1 quantification phase was not part of the context in which host-quality was assessed. We were only interested in host quality during the liquid phase. The Mx1 quantification phase is simply an approach to quantify the size of Mx1 populations after the 24 h liquid growth phase. First, the phage were separated from the MyxoEE-3 bacteria by the specified protocol. The separated phage then underwent serial dilution followed by mixture with aliquots of strain DZ1 and these mixtures were then embedded within soft-agar overlaid on hard-agar nutrient medium. Within the soft-agar overlay, DZ1 grows into a thick lawn in the absence of Mx1. When Mx1 is present, an individual particle will grow by lysing DZ1 cells until a round, clear plaque is formed. Each plaque represents a single phage particle that was present at the end of the growth phase. By counting the plaques from a plate with an appropriate number and multiplying by the relevant dilution factor, the total number of phage present after the 24 h of growth on an MyxoEE-3 population in liquid could be calculated. As DZ1 is a highly susceptible indicator strain, the number of phage able to form plaques on a DZ1 lawn is close to the total number of viable phage. Most importantly, using DZ1 as the indicator strain allows for the comparison between the different phage samples amplified in the MyxoEE-3 populations.
To summarize, separate samples of single phage stock are inoculated into different bacterial cultures, allowed to grow as they are able on various MyxoEE-3 cultures for 24 h in liquid and then their respective populations sizes are assessed by a plaque-number assay using DZ1, an M. xanthus genotype highly susceptible to lysis by Mx1.
For this reason you need to quantify phage on the host that you are interested in assessing for host quality not on a "common garden" host e.g. DZ2. By using DZ2 for quantification of the phage populations size on a given host (as a proxy for host quality) you do not have a measure of host quality on that host, you only have a measure of how it titres on DZ2 -i.e. you could have some phages that grow really well on their own host but not on DZ2 and vice versa. I hope this point makes sense as it is key to the experiment but please correct me if I have misinterpreted the design. Actually, in the context of our experiment the opposite is correct. The different MyxoEE-3 populations cannot be used for both the phage 24-h growth assays in liquid (i.e. the host quality assays) and postgrowth plaque-count assays for precisely the reason raised by the reviewer, because Mx1 might have different EOPs across different MyxoEE-3 populations and thus we would not have an identical method for quantifying viable phage particles after the 24 h liquid assays. Because we want to assess host quality in liquid culture, it is in fact very important that we use only one highly susceptible M. xanthus genotype for the plaque counts to avoid confounding variation in EOP. We modified the Methods to clarify this point.
In addition to this, another key issue for me is the use of the 24 hour phage propagation experiment as a measure of host quality. 24 hours is a long time from a bacterial and phage perspective and you could get selection for resistance amongst other things during this time frame. I am not sure that it is a good and accurate measure of host quality as you define it.
In the context of this study, we define host quality as simply the degree to which a host population facilitates parasite growth during 24 h in liquid culture. 24 hours is in fact a very short time with regard to the potential for resistance to Mx1 to appear and rise to appreciable frequency. Our M. xanthus cultures started the growth assays already at ~2 x 10 8 cells/ml when we added the phage for the growth assays. In the medium used, M. xanthus can only grow to a maximum density of ~2 x 10 9 cells/ml and does so within ~12 hours in the absence of phage.
M. xanthus grows slowly compared to most model-system bacteria with a doubling time of ~4 hrs under our experimental conditions (Velicer et al. 1998, PNAS). This means that even if an Mx1-resistant mutant of M. xanthus were already present among the ~1.6 x 10 9 cells (8 ml of culture at ~2 x 10 8 cells/ml) to which Mx1 was added to initiate each Mx1-growth assays, that mutant could only grow to a maximum of ~100 cells within the 24 hours of the assay, a negligible number relative to the initial bacterial population size. Moreover, even a number of ~100 cells could be reached by the mutant in 24 h only if the total population was prevented by phage from reaching stationary phase and thereby ceasing all population growth, including that of the mutant. (Stationary phase would normally be reached in ~12 h from the starting density of ~2 x 10 8 cells/ml in the absence of phage.) Thus, we argue that it is not quantitatively plausible that Mx1-resistant mutants that first appear in the cultures used for our growth assay could have significantly impacted our results.
[However, we would note that even if this were a realistic scenario, differences in the rate at which different MyxoEE-3 populations mutate to Mx1-resistance would be fascinating and would fall within the scope of our analysis. We are interested in the relative degree to which different MyxoEE-3 populations support Mx1 growth, irrespective of the precise mechanisms that generate such differences. That being said, given what we know about M. xanthus growth, we reiterate that we think it is implausible that differences between MyxoEE-3 populations in rates of mutation to Mx1-resistance during our growth assays might explain the differences we observe in Mx1 population sizes after growth on different MyxoEE-3  Perhaps a one more useful measure(s) of host quality would be to take the hosts you are interested in and doing a one-step growth curve with them… I think this would provide a much better measure of host quality as it you will get information on phage adsorption, length of infection cycle and burst size which will allow you measure the number of progeny produced per host and over what time-scale. I think this would really bolster your study as it would remove any ambiguity or alternative explanation as to why you see lower populations on different evolved hosts. We agree with the reviewer that descriptions of more detailed components of Mx1 growth on various MyxoEE-3 populations in liquid would be interesting. However, these sub-components of phage growth are not necessary for our clearly defined measure of host quality and thus are not necessary for any of the inferences we make in this study. It could be interesting to see more detailed descriptions of Mx1 growth components pursued in a future study. We specify the focus of this study early on, namely, comparing how well a single phage genotype grows on ancestral vs evolved M. xanthus host genotypes after the evolved hosts underwent evolution during MyxoEE-3 without interacting with the focal phage genotype.
Another issue was with the initial experimental set-up that I am not sure I understood properly -were six different ancestral variants used to initiate the experiment? Line 221 Yes, as described in our manuscript (see Table S1), six ancestral M. xanthus genotypes were used to establish the MyxoEE-3 evolution experiment. Among these six, there were three distinct motility genotypes that were each represented by sister clones of two distinct marker states -rifampicin-sensitive and rifampicin-resistant. GJV1 and GJV2 were each ancestral to 36 of the MyxoEE-3 populations examined here, which represent the bulk of our study (Figs. 1-3, S1-S3). Additionally, GJV3-GJV6 were each the ancestors of 8 of the MyxoEE-3 populations, which were only examined in assays reported in Fig. 4.
Also, Line 104 onwards -you discuss the potential receptors sites of phages but do you have any detailed information on how the phage used in this study infects M. xanthus and how resistance evolves -this might help inform the data that you have observed for certain selective environments? For example you investigate the M. xanthus motility systems -is this because phage bind to flagella or pilli or what was the motivation for using these variants in the analysis. The motivation for examining MyxoEE-3 populations descended from the motility mutants was an interest in the possibility of i) immediate effects of removing one motility system or the other on host quality, especially with respect to removal of Type-4-pili-based motility and ii) historical genotype effects on the evolution of latent of host quality, particularly with respect to variable motility genotypes. Prior to this study it was unknown whether Mx1 phage interact with Type 4 pili. However, this was a plausible possibility given known interactions between phage and Type 4 pili in other species. In our case, we find that production of pili does not promote phage infection but rather hinders it, presumably via production of exopolysaccharides, which is known to be positively regulated by pilin production.