Disentangling the mechanisms underpinning disturbance-mediated invasion

Disturbances can play a major role in biological invasions: by destroying biomass, they alter habitat and resource abundances. Previous field studies suggest that disturbance-mediated invader success is a consequence of resource influxes, but the importance of other potential covarying causes, notably the opening up of habitats, have yet to be directly tested. Using experimental populations of the bacterium Pseudomonas fluorescens, we determined the relative importance of disturbance-mediated habitat opening and resource influxes, plus any interaction between them, for invader success of two ecologically distinct morphotypes. Resource addition increased invasibility, while habitat opening had little impact and did not interact with resource addition. Both invaders behaved similarly, despite occupying different ecological niches in the microcosms. Treatment also affected the composition of the resident population, which further affected invader success. Our results provide experimental support for the observation that resource input is a key mechanism through which disturbance increases invasibility.


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 1. The invasion success is here defined as the frequency of invader in the final population. A more informative measure would be the change in frequency in the population from addition to end. This would of course be problematic as it is difficult to measure the starting population size, but it would be easy to get an idea of the typical starting population size and its variation simply by plating at least 10 tubes at the same time as the invaders were added. Also of course the number of invaders should be quantified. This would give an opportunity to say something more about what happens during the invasion (for example if cell number decrease or increase). Trivially a lower resident population at the start will give a higher invasion success at the end as the starting percentage will be higher. This would need to be clarified in the manuscript.
2. I have a lot of concerns about the experimental data that would need to be explained in some way. To me it seems very odd that there can be a 200-fold difference in cfu for the same treatment (WS invader treatment 3). There is no discussion of this. In addition there is a lot of variation in the averages between very similar treatments that is not discussed. For example there is a 200fold difference in cfu for treatment 3 depending on if the invader is a SM or WS.
3. What is the basis for the fitness increase in the lacZ strain (as shown in the competition experiment) and how large is the increase in fitness? Was part of the experiment to isolate mutants with increased fitness to use in the invasion and therefore it was allowed to diversity for 6 days before the invasion? It seems trivial that the higher fitness SM lacZ would increase more in frequency with resources added simply due to a greater number of generations of growth. As a main conclusion of the paper is to say that resource addition increase invasibility this need to be carefully dissected.
4. Even though SM lacZ is fitter and as expected can increase in density (i.e. invade) there is no reason to believe that the WS lacZ also has increased fitness relative to non-marked WS. If it does not (and it might very well have lower fitness) the expectation would in the simplest case be that it will not invade. Possibly a lower fitness genotype could invade due to a physiological advantage (i.e. not spending as long in stationary phase), but if this was the case there can also be major differences between the SM and WS invaders in this regard. It is simply not clear why there is an expectation that both the WS and SM lacZ will invade (or in fact that they even increase in frequency after being added).
5. Dryad data would ideally contain colony counts (i.e. raw data), as it seems that there were very few for some (for example a zero for number of WS for SM invader treatment 2). It would also help to clarify that this is per 25 ul (or simply standardize to per ml or give the total population size). According to the methods 10-5 and 10-6 dilution were plated while some data say 25000, which of course would need colony counts of <1.
6. Why is no data on the amount (now it is only volume) of cells added for WS lacZ and SM lacZ included (are numbers of WS and SM similar)? This in combination with an approximate value of cfu at the time of treatments could tell us something about what happens. For example are total cell densities decreasing or increasing. Is the difference mainly in death rates or growth rates? 7. I fail to see that addition of 2 ml of liquid would not result in some disruption of the WS pellicle at the air-liquid interface. Are the pellicles not in any way attached to the walls of the tube?
8. My expectation would be that there is a strong time dependence on how successful WS types will be after habitat disruption. If the supply of nutrients are not enough to promote growth for long enough to start to form a pellicle, there will be no advantage of being WS.

Recommendation
Major revision is needed (please make suggestions in comments)

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

Is the length of the paper justified? Yes
Should the paper be seen by a specialist statistical reviewer? No

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.

Comments to the Author
The authors use a model experimental system to explore the effects of disturbance on invasion success. This is an interesting experiment and I enjoyed reading the paper. I have a few comments, mainly focused many on how best to interpret the data and some unexpected results.
I'm concerned that the measure of "invasion success" (invader cfu / total cfu ) used in this paper is not a good representation of "success". With this measure, an invader with the same population growth rate will appear less successful if it has invaded a community made up of more individuals (or total cfu) than if it has invaded a community with fewer individuals. This doesn't make sense to me. If every invader was inoculated into a same-sized resident community (total cfu), then this wouldn't matter, but that's not the case here. "All microcosms were then immediately invaded with 60 ul (1% of resident population volume) of either the SM or WS invader." (lines 108-110) -This means that the starting ratio of invader cfu to total cfu may have varied quite a bit. Instead of a ratio of (final invader cfu)/(final total cfu), why not just use a measure of invader population increase instead? E.g. (final invader cfu)/(initial invader cfu) . This new measure of success (invader population increase) may very well be driven by resident community size, but I think it would be better to explore this potential relationship with linear models, instead of embedding it in the measure itself.
The resident communities evolved for 7 days in the static microcosms before disturbance/ invasion. The invaders, on the other hand were isolated from a 6-day evolved community. What was the rational for this difference? If the invader had been adapted for longer, would we expect to see a significant effect of "habitat opening" on invasion success?
The authors report that their ancestral lacZ+ strain has a higher fitness than the ancestral lacZstrain. This is a surprising (and possibly concerning) result given that lacZ has always been described as a neutral marker in previous Pseudomonas fluorescens studies. Can the authors please comment on this surprising result? Is the strain used here different from that in previous studies?
Lines 171-176: If there are significant effects that depend on invader morphotype and its interactions, why not display these differences in a plot, instead of pooling everything together in Fig 3 so that we see no effects of treatment?
Lines 178-187: This paragraph is confusing because I'm not always sure which SM and WS (invader, resident, or total) are being referred to. E.g. "… we included final SM and WS densities as terms in the statistical model for invasion success." Do you mean both resident and invader? Please clarify. Related to the statistical models discussed in this paragraph -did the authors test the effects of total cfu on invasion success? I understand that the focus here is on looking for effects of resident SM on invader SM, and resident WS on invader WS, but perhaps it's just a total population size effect? Particularly since resource influx seems to be the important treatment here and those resources are used by the entire community. Lines 218-220: "This lack of difference between invasion success when the residents were occupying the niche as opposed to being present in the wider environment suggests an absence of priority for this particular niche (22)." This is pretty surprising to me (as the authors acknowledge it may be), especially given previous studies done in this system (albeit testing priority effects in different ways from this study) that do suggest there are priority effects (e.g. Zee & Fukami 2018). Can the authors comment on this finding in the context of previous work in this system?

29-Jul-2019
Dear Mr Lear: I am writing to inform you that your manuscript RSPB-2019-1549 entitled "Disentangling the mechanisms underpinning disturbance-mediated invasion" has, in its current form, been rejected for publication in Proceedings B.
This action has been taken on the advice of referees, who have recommended that substantial revisions are necessary. 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.
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Please find below the comments made by the referees, not including confidential reports to the Editor, which I hope you will find useful. If you do choose to resubmit your manuscript, please upload the following: 1) A 'response to referees' document including details of how you have responded to the comments, and the adjustments you have made.
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Sincerely, Professor Gary Carvalho mailto: proceedingsb@royalsociety.org Associate Editor Comments to Author: Your ms has been reviewed by 2 expert reviewers. Both find the research questions worthwhile and the findings potentially interesting. However, some important technical issues with the experiments are raised that will require some additional work. The main problem identified by both reviewers is with your measure of invasion success (end-point proportion of the invader) which instead needs to be recalculated as a growth rate (e.g. Malthusian parameter). There are a number of other issues raised by the reviewers that require additional clarification/explanation.

Reviewer(s)' Comments to Author:
Referee: 1 Comments to the Author(s) 1. The invasion success is here defined as the frequency of invader in the final population. A more informative measure would be the change in frequency in the population from addition to end. This would of course be problematic as it is difficult to measure the starting population size, but it would be easy to get an idea of the typical starting population size and its variation simply by plating at least 10 tubes at the same time as the invaders were added. Also of course the number of invaders should be quantified. This would give an opportunity to say something more about what happens during the invasion (for example if cell number decrease or increase). Trivially a lower resident population at the start will give a higher invasion success at the end as the starting percentage will be higher. This would need to be clarified in the manuscript.
2. I have a lot of concerns about the experimental data that would need to be explained in some way. To me it seems very odd that there can be a 200-fold difference in cfu for the same treatment (WS invader treatment 3). There is no discussion of this. In addition there is a lot of variation in the averages between very similar treatments that is not discussed. For example there is a 200fold difference in cfu for treatment 3 depending on if the invader is a SM or WS.
3. What is the basis for the fitness increase in the lacZ strain (as shown in the competition experiment) and how large is the increase in fitness? Was part of the experiment to isolate mutants with increased fitness to use in the invasion and therefore it was allowed to diversity for 6 days before the invasion? It seems trivial that the higher fitness SM lacZ would increase more in frequency with resources added simply due to a greater number of generations of growth. As a main conclusion of the paper is to say that resource addition increase invasibility this need to be carefully dissected.
4. Even though SM lacZ is fitter and as expected can increase in density (i.e. invade) there is no reason to believe that the WS lacZ also has increased fitness relative to non-marked WS. If it does not (and it might very well have lower fitness) the expectation would in the simplest case be that it will not invade. Possibly a lower fitness genotype could invade due to a physiological advantage (i.e. not spending as long in stationary phase), but if this was the case there can also be major differences between the SM and WS invaders in this regard. It is simply not clear why there is an expectation that both the WS and SM lacZ will invade (or in fact that they even increase in frequency after being added).
5. Dryad data would ideally contain colony counts (i.e. raw data), as it seems that there were very few for some (for example a zero for number of WS for SM invader treatment 2). It would also help to clarify that this is per 25 ul (or simply standardize to per ml or give the total population size). According to the methods 10-5 and 10-6 dilution were plated while some data say 25000, which of course would need colony counts of <1.
6. Why is no data on the amount (now it is only volume) of cells added for WS lacZ and SM lacZ included (are numbers of WS and SM similar)? This in combination with an approximate value of cfu at the time of treatments could tell us something about what happens. For example are total cell densities decreasing or increasing. Is the difference mainly in death rates or growth rates? 7. I fail to see that addition of 2 ml of liquid would not result in some disruption of the WS pellicle at the air-liquid interface. Are the pellicles not in any way attached to the walls of the tube?
8. My expectation would be that there is a strong time dependence on how successful WS types will be after habitat disruption. If the supply of nutrients are not enough to promote growth for long enough to start to form a pellicle, there will be no advantage of being WS.
Referee: 2 Comments to the Author(s) The authors use a model experimental system to explore the effects of disturbance on invasion success. This is an interesting experiment and I enjoyed reading the paper. I have a few comments, mainly focused many on how best to interpret the data and some unexpected results.
I'm concerned that the measure of "invasion success" (invader cfu / total cfu ) used in this paper is not a good representation of "success". With this measure, an invader with the same population growth rate will appear less successful if it has invaded a community made up of more individuals (or total cfu) than if it has invaded a community with fewer individuals. This doesn't make sense to me. If every invader was inoculated into a same-sized resident community (total cfu), then this wouldn't matter, but that's not the case here. "All microcosms were then immediately invaded with 60 ul (1% of resident population volume) of either the SM or WS invader." (lines 108-110) -This means that the starting ratio of invader cfu to total cfu may have varied quite a bit. Instead of a ratio of (final invader cfu)/(final total cfu), why not just use a measure of invader population increase instead? E.g. (final invader cfu)/(initial invader cfu) . This new measure of success (invader population increase) may very well be driven by resident community size, but I think it would be better to explore this potential relationship with linear models, instead of embedding it in the measure itself.
The resident communities evolved for 7 days in the static microcosms before disturbance/ invasion. The invaders, on the other hand were isolated from a 6-day evolved community. What was the rational for this difference? If the invader had been adapted for longer, would we expect to see a significant effect of "habitat opening" on invasion success?
The authors report that their ancestral lacZ+ strain has a higher fitness than the ancestral lacZstrain. This is a surprising (and possibly concerning) result given that lacZ has always been described as a neutral marker in previous Pseudomonas fluorescens studies. Can the authors please comment on this surprising result? Is the strain used here different from that in previous studies?
Lines 171-176: If there are significant effects that depend on invader morphotype and its interactions, why not display these differences in a plot, instead of pooling everything together in Fig 3 so that we see no effects of treatment?
Lines 178-187: This paragraph is confusing because I'm not always sure which SM and WS (invader, resident, or total) are being referred to. E.g. "… we included final SM and WS densities as terms in the statistical model for invasion success." Do you mean both resident and invader? Please clarify. Related to the statistical models discussed in this paragraph -did the authors test the effects of total cfu on invasion success? I understand that the focus here is on looking for effects of resident SM on invader SM, and resident WS on invader WS, but perhaps it's just a total population size effect? Particularly since resource influx seems to be the important treatment here and those resources are used by the entire community. Lines 218-220: "This lack of difference between invasion success when the residents were occupying the niche as opposed to being present in the wider environment suggests an absence of priority for this particular niche (22)." This is pretty surprising to me (as the authors acknowledge it may be), especially given previous studies done in this system (albeit testing priority effects in different ways from this study) that do suggest there are priority effects (e.g. Zee & Fukami 2018

Quality of the paper: Is the overall quality of the paper suitable? Good
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 The authors have addressed my main concerns and questions, and improved the manuscript.

Review form: Reviewer 2
Recommendation Accept with minor revision (please list in comments)

Quality of the paper: Is the overall quality of the paper suitable? Good
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. I have a couple of minor comments: 1) On the previous submission, I asked why the invaders were isolated after 6 days. I don't think the authors have answered my question. What I was trying to get at was: why were the invaders isolated after 6 days of evolution instead of 7? The invaders were then placed into microcosms that have been evolving for 7 days, so why weren't the invaders allowed to pre-evolve for 7 days as well? I just seems like a strange mis-match in the experiment. Perhaps it's the case that a single day difference in amount of pre-evolution is not enough to matter, but I'd expect that a better adapted invader would have more of an advantage in the "habitat opening" treatment.
2) I do think that an un-pooled version of Fig 3 would  Your manuscript has now been peer reviewed and the reviews have been assessed by an Associate Editor. The reviewers' comments (not including confidential comments to the Editor) and the comments from the Associate Editor are included at the end of this email for your reference. As you will see, the reviewers and the Editors have raised some concerns with your manuscript and we would like to invite you to revise your manuscript to address them.
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Overall, I am satisfied with how the authors have addressed my concerns from the previous submission. In particular, I appreciate the change made to their measure of invasion success as well as the additional point the authors now include in their discussion of presence/ absence of priority effects in this study. I have a couple of minor comments: 1) On the previous submission, I asked why the invaders were isolated after 6 days. I don't think the authors have answered my question. What I was trying to get at was: why were the invaders isolated after 6 days of evolution instead of 7? The invaders were then placed into microcosms that have been evolving for 7 days, so why weren't the invaders allowed to pre-evolve for 7 days as well? I just seems like a strange mis-match in the experiment. Perhaps it's the case that a single day difference in amount of pre-evolution is not enough to matter, but I'd expect that a better adapted invader would have more of an advantage in the "habitat opening" treatment.

07-Jan-2020
Dear Mr Lear I am pleased to inform you that your manuscript entitled "Disentangling the mechanisms underpinning disturbance-mediated invasion" has been accepted for publication in Proceedings B.
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Dear Professor Carvalho, We are very grateful to the insightful comments from the Associate Editor and reviewers, which has improved our manuscript. Please find our point by point responses below. Where both referees made similar points, their comments have been added together.

Measure of invasion success
Referee 1. The invasion success is here defined as the frequency of invader in the final population. A more informative measure would be the change in frequency in the population from addition to end. This would of course be problematic as it is difficult to measure the starting population size, but it would be easy to get an idea of the typical starting population size and its variation simply by plating at least 10 tubes at the same time as the invaders were added. Also of course the number of invaders should be quantified. This would give an opportunity to say something more about what happens during the invasion (for example if cell number decrease or increase). Trivially a lower resident population at the start will give a higher invasion success at the end as the starting percentage will be higher. This would need to be clarified in the manuscript.
Referee 2. I'm concerned that the measure of "invasion success" (invader cfu / total cfu ) used in this paper is not a good representation of "success". With this measure, an invader with the same population growth rate will appear less successful if it has invaded a community made up of more individuals (or total cfu) than if it has invaded a community with fewer individuals. This doesn't make sense to me. If every invader was inoculated into a same-sized resident community (total cfu), then this wouldn't matter, but that's not the case here. "All microcosms were then immediately invaded with 60 ul (1% of resident population volume) of either the SM or WS invader." (lines 108-110) -This means that the starting ratio of invader cfu to total cfu may have varied quite a bit. Instead of a ratio of (final invader cfu)/(final total cfu), why not just use a measure of invader population increase instead? E.g. (final invader cfu)/(initial invader cfu) . This new measure of success (invader population increase) may very well be driven by resident community size, but I think it would be better to explore this potential relationship with linear models, instead of embedding it in the measure itself.
Response: Thank you for raising this pointwe agree our measure of invader success could have been confounded by differences in starting ratios of the residents and invaders. We therefore have changed our measure to vthe proportional change in invader density (Ross-Gillespie, Gardner et al. 2007) between the time of inoculation and the end of the experiment. We already had directly measured starting invader density from plate counts, which was the same for all replicates for each of the invaders, as the same source populations were used. Unfortunately, as recognised by Referee 1, it was impossible to directly measure starting resident density. This would require disrupting the biofilms of the diversified microcosms at the time of invasion; and biofilm disruption was one of our experimental treatments. In order to get an idea of day-7 resident density to fulfil this measure, we followed Referee 1's suggestion of re-evolving the residents (12 replicates) for 7 days under the same conditions as the original experiment, and using this mean density for all starting resident densities. Given that all resident communities for all treatments were propagated under the same conditions prior to invasion, using this mean value will not create any systematic bias across treatments,