Mechanisms Generating Dichotomies in the Life Strategies of Heterotrophic Marine Prokaryotes

Round 1

Reviewer 1 Report

A prominent professor and his team author the manuscript. I do not doubt the knowledge/information provided in the manuscript. And I'm confident that this will be an good Review after correction. Nevertheless, I find it hard to follow. The majority of the comments are related to the clarity and the flow of messages. After reading the manuscript, readers with minimal background on the jargon will have flooded with hundreds of question marks. The following are just some examples. Please ask someone to proofread the manuscript after considering the following comments.

1. Title and L9-11. By reading the title and the 1st line in the Abstract, I cannot understand the scope of Review. Please do it 'slowly' before entering the main core business (idea). There are many jargons (i.e. background theories) that not all readers may understand. Consider rewriting/rearranging and delivering the messages in step by step manner.
2. Rewrite the whole Introduction. After reading Introduction for the 1st time, I'm not able to understand the main scope, the purpose of the Review, and many sentences are ambiguous. And the Introduction did not help me to understand the subsequent topics. Examples are:

• L34-35: I need to read three times before guessing the meaning. Please use a proper term such as metataxonomy instead of the metagenome. Metataxonomy may or may not necessarily relate to the 'richness of species'. 
• L35-36. The connection to the earlier sentence is odd. And the following line is talking something else (WGS). 
• L44: What do you mean by clonal here?
• Please define L-strategist, etc., briefly in the Introduction and other terminology as you move on subsequently.
• Introduction only takes SAR11 as an example, then in L80 onwards, authors wrote about Vibrio. It isn't obvious to follow what is the focus and what is the connection between SAR11 and Vibrio

3. L102-105, L121-123, etc. Avoid a 'lonely' paragraph that hardly fits the story's flow.

4. Major comment for the whole manuscript. Another reason it is very challenging to follow the Review is messages are choppy or jumpy. The connection within and across paragraph(s) is weak. I’m not able to enjoy reading the review before this point is well addressed.

5. Please provide higher resolutions for Figure 1.

6. L362 onward. How about Vibrio? Why do authors select Vibrio and SAR11 only? From a taxonomy perspective, one is Gamma- and the other is AlphaProteobacteria. I wasn't sure and confused because it is not stated clearly from the start! Is 'idealized model in Fig. 1' applicable to SAR11 only, Vibrio, or limited to Proteobacteria or ALL heterotrophic aquatic prokaryotes? If the scope is 'narrow' (or unable to be proven for all heterotopic aquatic prokaryotes), please consider a better title. I hope to read again to learn from the Review.

Author Response

Reply to Reviewer 1.

We thank the reviewer for his/her appreciation of the significance of the contribution. We also acknowledge the point that the flow of arguments is difficult to follow.

Our revision in response to this mainly affects the order of presentation and addition of explanatory text to ease the transition between paragraphs and sections.

1: Introduction is re-written to contain more arguments for the sections to come. Acknowledging that readers of Diversity may have slightly different backgrounds (taxonomy, molecular biology, evolution, ecology….) we have also tried to avoid specialized terminology whenever possible. We have added text to emphasize that SAR11 and Vibrio are chosen as (important) examples of the S- and L-strategies, respectively. We have also highlighted in the introduction how the S-strategists are dominated by alpha-proteobacteria, while the L-strategist group is more diverse, including both gamma-proteobacteria and Bacteriodetes.

2: With its length constraints, we found the Abstract more difficult to revise according to the philosophy above,  but have added an introductory sentence to “soften” the introduction of the subject. Other slight modifications have been done to improve language.

3: The order of sections 2 (observational studies of Vibrio) and 3 (describing the connection between organic-C, diffusion theory and S/L-strategies) is reversed. The theoretical foundation is now introduced first, followed by the three sections on observational studies, mesocosm studies and aquaculture applications.  Also, for the observational studies, evidence for a role of organic-C is now reviewed first, the role of temperature follows as additional information.

Reviewer 1

A prominent professor and his team author the manuscript. I do not doubt the knowledge/information provided in the manuscript. And I'm confident that this will be an good Review after correction. Nevertheless, I find it hard to follow. The majority of the comments are related to the clarity and the flow of messages. After reading the manuscript, readers with minimal background on the jargon will have flooded with hundreds of question marks. The following are just some examples. Please ask someone to proofread the manuscript after considering the following comments.

1. Title and L9-11. By reading the title and the 1st line in the Abstract, I cannot understand the scope of Review. Please do it 'slowly' before entering the main core business (idea). There are many jargons (i.e. background theories) that not all readers may understand. Consider rewriting/rearranging and delivering the messages in step by step manner.

 * *See comments above

1. Rewrite the whole Introduction. After reading Introduction for the 1st time, I'm not able to understand the main scope, the purpose of the Review, and many sentences are ambiguous. And the Introduction did not help me to understand the subsequent topics. Examples are:

• L34-35: I need to read three times before guessing the meaning. Please use a proper term such as metataxonomy instead of the metagenome. Metataxonomy may or may not necessarily relate to the 'richness of species'. 

** The “metagenomics – metataxonomy” issue is avoided by re-writing

• L35-36. The connection to the earlier sentence is odd. And the following line is talking something else (WGS). 

** re-written

• L44: What do you mean by clonal here?

** term avoided

• Please define L-strategist, etc., briefly in the Introduction and other terminology as you move on subsequently.

** Both the importance of size, and the L-strategists’ use of organic-C to increase size are now described in the introduction.

• Introduction only takes SAR11 as an example, then in L80 onwards, authors wrote about Vibrio. It isn't obvious to follow what is the focus and what is the connection between SAR11 and Vibrio

**It should now be clear that Vibrio is discussed as an example of the L-strategists.

3. L102-105, L121-123, etc. Avoid a 'lonely' paragraph that hardly fits the story's flow.

***reformulated

4. Major comment for the whole manuscript. Another reason it is very challenging to follow the Review is messages are choppy or jumpy. The connection within and across paragraph(s) is weak. I’m not able to enjoy reading the review before this point is well addressed.

**hopefully improved by the general revision described above

5. Please provide higher resolutions for Figure 1.

6. L362 onward. How about Vibrio? Why do authors select Vibrio and SAR11 only? From a taxonomy perspective, one is gamma- and the other is alpha-Proteobacteria. I wasn't sure and confused because it is not stated clearly from the start! Is 'idealized model in Fig. 1' applicable to SAR11 only, Vibrio, or limited to Proteobacteria or ALL heterotrophic aquatic prokaryotes? If the scope is 'narrow' (or unable to be proven for all heterotopic aquatic prokaryotes), please consider a better title. I hope to read again to learn from the Review.

** The diversity among L-strategists is discussed already in the introduction and examples of gamma-proteobacteria and Bacteriodetes are discussed later (e.g. section om mesocosms). The legend to Figure 1 refers only to generic aspects of competition and predation with no reference to SAR11 or Vibrio. Hopefully, the generic nature of the two strategies will now be evident to the reader

Author Response File: Author Response.pdf

Reviewer 2 Report

It would be good if the authors could clarify or correct the below:

Line 83 V. cholerae

Line 109, V. harveyi

Line 176, please elaborate on the "Winnie=the-Pooh" strategists

In box 1, please replace the a with alpha symbol.

The last statement in box 1, assumes the similar requirements for both S- and L- strategists. However the difference in the genome size could also have significantly different requirements (due to the functionalities encoded in the genome).

Line 241, Vibrio

Line 247, reviewed

Line 288, and

Line 318, Bacteroidota phylum

Figure 2, what about r- and K strategists within the same Vibrio genus used to explain the predominance of certain Vibrio species (cf. Wong et al. 2019 Environmental control of Vibrio spp. abundance and community structure in tropical waters. FEMS Microbiology Ecology 95: fiz176)

Author Response

Reviewer 2

We thank the reviewer for the generally positive evaluation and the detailed corrections.

All recommendations have been followed.

It would be good if the authors could clarify or correct the below:

Line 83 V. cholerae

** done

Line 109, V. harveyi

** done

Line 176, please elaborate on the "Winnie=the-Pooh" strategists

** An explanation for the terminology is now added in paranthesis.

In box 1, please replace the a with alpha symbol.

** problem seemingly arise during file conversions. Hope the correction now is retained

The last statement in box 1, assumes the similar requirements for both S- and L- strategists. However the difference in the genome size could also have significantly different requirements (due to the functionalities encoded in the genome).

** Also here, the Greek letter rho seem either to have removed or converted to an r, perhaps producing some of the confusion? The basic hypothesis is the assumption of either a constant cell quota Q of the limiting element, independent of size (L-strategists) or a constant volume-specific content rho (S-strategists), making cell quota size-dependent. A sentence is added in Box 1, pointing out that this is an assumption open to further research.

Line 241, Vibrio

**Corrected

Line 247, reviewed

**Corrected

Line 288, and

**Corrected

Line 318, Bacteroidota phylum

**Bacteriodetes replaced with Bacteriodotaa

Figure 2, what about r- and K strategists within the same Vibrio genus used to explain the predominance of certain Vibrio species (cf. Wong et al. 2019 Environmental control of Vibrio spp. abundance and community structure in tropical waters. FEMS Microbiology Ecology 95: fiz176)

** Thank you for this relevant reference. Incorporated in a comment of possible r/K differences within the L-strategists (l. 411-412)

Author Response File: Author Response.pdf

Reviewer 3 Report

The Authors provided a review of life strategies of some marine bacteria, e.g. Vibrio spp. and members of SAR11. In general, the review is comprehensive. It is in scope of the journal, but some issues must be fixed.

Figure 1: axes should be labeled.

Following issues have to be discussed: who is SAR11, who lives in marine water - at least at the level of phyla, what is the place and the role of SAR11 and Vibrio spp.

How did the Authors explain comparison of bacterial groups at different levels of taxa?

-l. 53-70. To justify this, you must provide the data on average sizes of the genomes of SAR11 and Vibrio.

-l. 35. "as defined by their 16 rRNA gene" should be "as defined by their 16 rRNA gene amplicons".

-l. 102-106. references are missed.

-l. 36. "the evenness component of diversity" - what is it? please, describe.

-l. 38. surface of what?

-l. 39. Strategies of what?

-l. 53-61. Do you discuss here the S strategy? Mention it as in the next paragraph.

-l. 83, 97, 115. cholera should be cholerae.

Author Response

Reviewer 3

The Authors provided a review of life strategies of some marine bacteria, e.g. Vibrio spp. and members of SAR11. In general, the review is comprehensive. It is in scope of the journal, but some issues must be fixed.

Figure 1: axes should be labeled.

** Done

Following issues have to be discussed: who is SAR11, who lives in marine water - at least at the level of phyla, what is the place and the role of SAR11 and Vibrio spp.

How did the Authors explain comparison of bacterial groups at different levels of taxa?

** I am not quite sure how to respond to this comment since I see “the place and the role of SAR11 and Vibrio spp. as a (the) central theme of the submitted version. What may be confusing is perhaps (?) the approach where focus on the basic selection mechanisms of competition and (predator)defence and claim that SAR11 and Vibrio solve this by very different strategies; yet these strategies can be understood from the same physical basis (diffusion limited uptake). The larger issue of who lives in seawater is discussed in the introduction from this perspective (e.g. the genetic evidence of two strategies). We acknowledge that there is no attempt to give any phylogenetic review of who lives in seawater. There are two reasons for this: 1) Diversity only allows condensed reviews and 2)it would blur the focus on basic selection mechanisms which we want to be the essence of our focussed review. The revision attempts to clarify this purpose by some rearrangement of the order of arguments and by more text explaining the connections between sections.

The issue of comparison between different levels of taxa is briefly discussed at the end where diversification at strain level is proposed to be more dominant in S-strategists as compared to diversification at species level for the L-strategists; suggesting that the claim for higher diversity among L-strategists may be rooted in our tendency to associate diversity with the species level-

-l. 53-70. To justify this, you must provide the data on average sizes of the genomes of SAR11 and Vibrio.

** The average genome size of SAR11 is provided. The ideal comparison would be an average for the L-strategists. No such numbrr is, however, available, and a comparison to Vibrio is included as an illustration. (line 55)

-l. 35. "as defined by their 16 rRNA gene" should be "as defined by their 16 rRNA gene amplicons".

**Corrected

-l. 102-106. references are missed.

**This is the very general argument that “temperature affects everything”. Separating direct temperature effects from indirect in natural systems is then notoriously difficult. This is so close to self-evident that I am not aware of a good reference to anybody that have treated it explicitly. It is therefore left as a statement of our own responsibility.

-l. 36. "the evenness component of diversity" - what is it? please, describe.

** Re-written to avoid a non-essential term

-l. 38. surface of what?

**Corrected to “ocean surface”

-l. 39. Strategies of what?

** Corrected to “life strategies”. This is not very illuminating at this point, but the main theme of the rest of the manuscript is to explain these two life strategies.

-l. 53-61. Do you discuss here the S strategy? Mention it as in the next paragraph.

** This part is re-written in the revision. The paragraph l.72-85 hopefully clarifies how the existing streamlining hypothesis fits the trade-off considerations and the S-strategy discussed here.

-l. 83, 97, 115. cholera should be cholerae.

**corrected

Author Response File: Author Response.pdf

Reviewer 4 Report

This is a review and theoretical summary of two dimensions of prokaryote adaptive strategies: The well-established r/K strategy, and a more novel perspective on size Large v. Small or (S/L) strategy, including theoretical relationships between the two drawing on published evidence for two representative prokaryotes  small, genomic streamlined oceanic SAR 11 and Vibrio spp.

Using these two taxa, the authors have garnered evidence broadly from many different aquatic studies spanning oceanic gyres to infectious diseases of marine organisms. Overall, this offers a novel perspective, although largely through the lens of these specifically selected two prokaryote taxa, and is a useful contribution.

In addition to a few minor editorial type of suggestions, my only other recommendation would be for the authors to consider adding somewhat more information at the end, to indicate what further research studies might be done to more fully explore and validate the theoretical assumptions concluded in this study. For example, are there additional microcosm or mesocosm studies that can be done using a broader range of prokaryotes of different sizes and different combinations of taxa, thus looking more fully at community level responses beyond the two taxa highlighted here.

That is, in general, what advice for further research can the authors recommend to more fully explore and amplify the theoretical ideas and model presented in this paper. Regarding the Conclusions,  I also make a final suggestion below for an additional citation that may help to bolster the conclusion section. There has been a long and varied literature on whether prokaryote size is related to ecological function, particularly strategies such as r/K selection, and the recommended article seems to be more consistent with the position taken by the authors of this manuscript.

Line     Comment

20        “------ using non-limiting organic-C to increase size.  The “streamlining” theory that has been suggested for---”

35        “---(as defined by their 16S  rRNA gene)—”

81        recommend using full generic name for first use (if this is required by this journal) : “---with Vibrio splendidus  and Vibrio anguillarum as examples of the causative agents. Vibrio also include human pathogens such as Vibrio cholera [10] and Vibrio vulnificus. Typically in journals, the first use of a taxonomic species name should be spelled out in full, thereafter abbreviated.

“---with human pathogenic Vibrio spp. in the Baltic region,---” spp. should not be  italicized.

105      misspelled word         autochthonous supply  NOT    authochtonous supply

157      ---“---If this was the only reason, however, such organisms should---” Au: need to check for this minor irregularity in use of  ‘however’ some places it is correctly punctuated.

213      “---experimental conditions can select for-----"

215      “ Like the Vibrionaceae, genera in the  Psychromonadaceae also belong to the  g-Proteobacteria [49].”   Psychromonadaceae is a family, not a genus.

288      “---of top-down and bottom-up control strongly reduced bacterial glucose consumption;--”

302      “--as Polaribacter spp., which have the complex enzyme machinery---”

341      The overall conclusion included by the authors that the suggested L-strategists do not fit a dichotomy (i.e., r & K) since they combine K-defining traits (high defensive and competitive abilities at low concentration of limiting nutrient) with r-defining traits (i.e. C-storage and rapid growth) is interesting, and intersects with a long history of discussion and commentary on whether the size of bacteria across broad ranges correlates with the degree of r- vs. K-strategy of the size groups. These authors’ conclusion of some independence, with L varying across both is bolstered by recent evidence of a lack of correlation generally between variation broadly in  size and the r- to K- strategy continuum (Westoby M., Nielsen D.A., Gillings M. R., Litchman E., Madin J. S., Paulsen I. T., Tetu S. G. (2021) Cell size, genome size, and maximum growth rate are near-independent dimensions of ecological variation across bacteria and archaea. Ecology and Evolution 11:3956-3976.).

Author Response

Reviewer 4

This is a review and theoretical summary of two dimensions of prokaryote adaptive strategies: The well-established r/K strategy, and a more novel perspective on size Large v. Small or (S/L) strategy, including theoretical relationships between the two drawing on published evidence for two representative prokaryotes  small, genomic streamlined oceanic SAR 11 and Vibrio spp.

Using these two taxa, the authors have garnered evidence broadly from many different aquatic studies spanning oceanic gyres to infectious diseases of marine organisms. Overall, this offers a novel perspective, although largely through the lens of these specifically selected two prokaryote taxa, and is a useful contribution.

In addition to a few minor editorial type of suggestions, my only other recommendation would be for the authors to consider adding somewhat more information at the end, to indicate what further research studies might be done to more fully explore and validate the theoretical assumptions concluded in this study. For example, are there additional microcosm or mesocosm studies that can be done using a broader range of prokaryotes of different sizes and different combinations of taxa, thus looking more fully at community level responses beyond the two taxa highlighted here.

That is, in general, what advice for further research can the authors recommend to more fully explore and amplify the theoretical ideas and model presented in this paper. Regarding the Conclusions,  I also make a final suggestion below for an additional citation that may help to bolster the conclusion section. There has been a long and varied literature on whether prokaryote size is related to ecological function, particularly strategies such as r/K selection, and the recommended article seems to be more consistent with the position taken by the authors of this manuscript.

**We thank the reviewer for the generally positive evaluation and constructive suggestions.

Line     Comment

20        “------ using non-limiting organic-C to increase size.  The “streamlining” theory that has been suggested for---”

**Punctuation mark inserted

35        “---(as defined by their 16S  rRNA gene)—”

**Reformulated according to suggestion from other reviewer

81        recommend using full generic name for first use (if this is required by this journal) : “---with Vibrio splendidus  and Vibrio anguillarum as examples of the causative agents. Vibrio also include human pathogens such as Vibrio cholera [10] and Vibrio vulnificus. Typically in journals, the first use of a taxonomic species name should be spelled out in full, thereafter abbreviated.

** Revised accordingly

“---with human pathogenic Vibrio spp. in the Baltic region,---” spp. should not be  italicized.

** Corrected

105      misspelled word         autochthonous supply  NOT    authochtonous supply

**Corrected

157      ---“---If this was the only reason, however, such organisms should---” Au: need to check for this minor irregularity in use of  ‘however’ some places it is correctly punctuated.

** Re-formulated to reduce the rather frequent use of “however”.

213      “---experimental conditions can select for-----"

**Corrected

215      “ Like the Vibrionaceae, genera in the  Psychromonadaceae also belong to the  g-Proteobacteria [49].”   Psychromonadaceae is a family, not a genus.

** Thanks, “genus” removed

288      “---of top-down and bottom-up control strongly reduced bacterial glucose consumption;--”

** an replaced with and

302      “--as Polaribacter spp., which have the complex enzyme machinery---”

**Comma inserted

341      The overall conclusion included by the authors that the suggested L-strategists do not fit a dichotomy (i.e., r & K) since they combine K-defining traits (high defensive and competitive abilities at low concentration of limiting nutrient) with r-defining traits (i.e. C-storage and rapid growth) is interesting, and intersects with a long history of discussion and commentary on whether the size of bacteria across broad ranges correlates with the degree of r- vs. K-strategy of the size groups. These authors’ conclusion of some independence, with L varying across both is bolstered by recent evidence of a lack of correlation generally between variation broadly in  size and the r- to K- strategy continuum (Westoby M., Nielsen D.A., Gillings M. R., Litchman E., Madin J. S., Paulsen I. T., Tetu S. G. (2021) Cell size, genome size, and maximum growth rate are near-independent dimensions of ecological variation across bacteria and archaea. Ecology and Evolution 11:3956-3976.).

**Thank you for this interesting and relevant reference. It is included as a discussion point in the revision (l.423.434).

The proposal to include a discussion of future research, we find a bit more difficult. There is a comment added at the end of Box 1, pointing out that variation in cell quota  Q versus variations in volume specific content sigma is the essential physiological difference between the S- and the L-strategy. This assumption is mentioned as an obvious point for further research. We believe we have shown in this review how the range of implications is very broad (and therefore opens a wide range of research questions). We have chosen to add a final concluding paragraph emphasizing this point.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Congratulations. I am sure that authors have tries the best to make this review meaningful. I enjoy reading this version. 

Reviewer 3 Report

I suggest acceptance.