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Observations on the Antagonistic Relationships between Fungi, Archaea, and Bacteria in Livingston Island (Maritime Antarctica) with the Use of Amplicon-Based Metagenomics

Diversity 2024, 16(1), 56; https://doi.org/10.3390/d16010056

by Svetoslav G. Dimov^1,*,†

, Vesselin V. Doytchinov^1,†

, Neyko Neykov², Tsvetana Muleshkova¹

, Lyubomir Kenderov¹, Ralitsa Ilieva¹, Dimitrina Georgieva Miteva¹

, Meglena Kitanova¹

, Slavil Peykov¹ and Mihail Iliev¹

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Reviewer 3:

Virginia De Garcia

Diversity 2024, 16(1), 56; https://doi.org/10.3390/d16010056

Submission received: 12 October 2023 / Revised: 13 January 2024 / Accepted: 14 January 2024 / Published: 16 January 2024

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The title is appropriate to the content of the ms, but in my opinion too dramatic. In my opinion more adequate is: “Observation on the antagonisms between Fungi, Archaea, and Bacteria in Livingston Island (Maritime Antarctica) with use amplicon-based metagenomic study”.

The abstract is properly written and focus on the results obtained.

In my opinion the introduction requires significant changes. This very short chapter contains very general information in a few sentences. In my opinion, the introduction is too general and does not sufficiently introduce the reader to the issues described. There is no description of the importance of such an important and common phenomenon as relationships between the components of the microbiome in natural environments, especially antagonistic relationships. A brief list of several publications does not exhaust the requirements for introducing the field of research and justifying the topic. Perhaps, as the authors point out, there are not many reports on the relationships in microbiocenoses of polar environments regarding metagenomic analyses, but these phenomena have been quite well described in relation to strains isolated from these environments. This allows us to put forward valuable hypotheses regarding the overall functioning of these relationships and, consequently, verify them using the methods described in the manuscript. In this context, this chapter lacks a clearly formulated research hypothesis that is adequate to the research conducted.

Materials and Methods

In my opinion, the material for DNA isolation from solid surfaces should be taken in three independent samples and then the DNA pooled. This would allow obtaining more reliable results of the structure of microbiocenoses. One randomly collected sample limits the possibility of drawing conclusions about the entire environment.

Results

The results section is poorly described. It consists only of tables and figures that are not described in detail.

Table 1 does not include a description of the W06 water sample, which is included in table 2 of the results.

Figure 2 To compare the values of indicators, the scale values for the three described taxa should be unified.

Table 5 - it is good if the Pearson correlation values are marked with one star and the Sperman correlation values with two stars next to the first value, instead of "upper" and "below"

Discussion

P4 L33-42 - This text should be moved to the Introduction chapter.

P4 L43-49 - This text should be moved to the Material and Methods chapter, and the goal of the research should be moved to the end of Introduction chapter

The discussion should be re-written. This chapter contains numerous references that should be included in the results section, e.g. P4 L50-59.

References should include publications on antagonistic interactions among the components of microbiocenoses in polar environments, even if they do not concern metagenomic studies.

Author Response

Dear Reviewer,

Please, allow me first to thank you for your efforts which significantly increased the quality of our manuscript. We concurred with most of your remarks, so we hope you will accept our work in its current form.

Please, find bellow our responses and addresses:

“The title is appropriate to the content of the ms, but in my opinion too dramatic. In my opinion more adequate is: “Observation on the antagonisms between Fungi, Archaea, and Bacteria in

Livingston Island (Maritime Antarctica) with use amplicon-based metagenomic study”.”

We are thankful for the suggestion and changed the title.

“The abstract is properly written and focus on the results obtained.”

We appreciate the esteem.

“In my opinion the introduction requires significant changes. This very short chapter contains very general information in a few sentences. In my opinion, the introduction is too general and does not sufficiently introduce the reader to the issues described. There is no description of the importance of such an important and common phenomenon as relationships between the components of the microbiome in natural environments, especially antagonistic relationships. A brief list of several publications does not exhaust the requirements for introducing the field of research and justifying the topic. Perhaps, as the authors point out, there are not many reports on the relationships in microbiocenoses of polar environments regarding metagenomic analyses, but these phenomena have been quite well described in relation to strains isolated from these environments. This allows us to put forward valuable hypotheses regarding the overall functioning of these relationships and, consequently, verify them using the methods described in the manuscript. In this context, this chapter lacks a clearly formulated research hypothesis that is adequate to the research conducted.”

We only partially agree with this remark. The reasons are: 1) Investigations on the antagonisms between isolates does not have the potential to be relevant if transposed to the different life domains of eubacteria, archaea and fungi, especially in the light that other ecological relationships between representatives of these domains for sure cannot be excluded; 2) as it is indicated within the Introduction section, the main goal of the research project was not to investigate the phyla’ antagonisms but the microbiotas composition, however we were surprised by the observations on which this manuscript is based, and decided to try extract some informative correlations; and 3) we were surprised by the lack of such comparative studies even for other much accessible for the research community environments. Still we agree with the remark of the formulated hypothesis, so, we made appropriate changes.

“Materials and Methods

We completely concur that it would be much better to have three or more repetitions. However, the short periods of the expeditions (about 20-25 days), combined with the need of logistical support (boats and snowmobiles together with the logistics personal) which is also needed for the other research projects, as well as the periods of bad weather, made impossible to have repetitions of many sampling sites. So, instead of independent samplings of a few sites we decided to cover more sampling sites.

“Results

The results section is poorly described. It consists only of tables and figures that are not described in detail.”

The policy of the “Diversity” journal is to have separate “Results” and “Discussion” sections. So, to respect this policy, as well as to avoid redundancies, ww kept this section only for listing the experimental results. As our results are mostly numerical, we believe that presenting them under the form of tables is appropriate.

“Table 1 does not include a description of the W06 water sample, which is included in table 2 of the results.”

We do not agree with this remark, as the geographic coordinates are listed within the table. However, to make it more informative, we specified the type of water.

Figure 2 To compare the values of indicators, the scale values for the three described taxa should be unified.

We are sorry, but this cannot be done because the main idea is to directly observe if the value is greater or lesser in comparison to the arbitrary chosen median value of the index. If we do this the figure will be less informative.

“Table 5 - it is good if the Pearson correlation values are marked with one star and the Sperman correlation values with two stars next to the first value, instead of "upper" and "below"”

We are thankful for the suggestion, and therefore made the changes.

“Discussion

P4 L33-42 - This text should be moved to the Introduction chapter.”

We made the proposed change.

“P4 L43-49 - This text should be moved to the Material and Methods chapter, and the goal of the research should be moved to the end of Introduction chapter”

We made the proposed changes.

“The discussion should be re-written. This chapter contains numerous references that should be included in the results section, e.g. P4 L50-59.”

The references were included.

“References should include publications on antagonistic interactions among the components of microbiocenoses in polar environments, even if they do not concern metagenomic studies.”

Unfortunately, with a single exception which is cited (Li et al., 2020) we didn’t find any data within the largest scientific publications databases (such as PubMed, Google Scholar, and some others) of similar studies in any environments performed by any means, not only by the tools of the metagenomics. We will be very grateful if the reviewer could share information of such publications.

On behalf of all the co-authors, allow me to express once again our gratitude for your work and time spent for the evaluation of the manuscript.

Svetoslav G. Dimov

November 12, 2023

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Authors of that manuscript attempted to investigate patterns in abundance and diversity of three different taxa, including Archaea, Bacteria and Fungi, in the Antarctic environments. Considering very low number of samples investigated, 14 from terrestrial habitats and 4 from marine environment, this work can be considered as only preliminary insight into the investigated problem. Still, their work is valuable, as our knowledge on microbial communities existing in the extreme environmental condition in Antarctica, but also elsewhere, is still very limited. Personally, being involved in the Antarctic research I became truly interested in this work after reading its abstract. However, to my great disappointment I see that the manuscript still needs a lot of effort before its publication, and surely should not be published in its present form.

Firstly, authors claim that they investigated antagonisms among Archaea, Bacteria and Fungi. This is not true. As I have purposely indicated at the very beginning, authors have investigated patterns in abundance and diversity. Surely, these patterns can be interpreted as antagonism, but there are also other possible and I would say even more likely explanations of mechanisms that can drive the observed patterns. For instance, environmental requirements and habitat suitability or stochastic events and random distribution. In case of the Antarctic environments, adaptations to the extreme environmental condition and legacy of historical events are particularly often emphasized in numerous literature. Unfortunately, authors has only briefly mentioned possible role of environmental requirements, still omitting any discussion of that topic.

Secondly, methodology has to be described in more details with usage of appropriate terminology. Authors have mentioned, that they collected “solid samples … of biomass or sediments” or “microbial surface contamination”. To be honest, I do not understand what kind of samples were collected for that work. Maybe this is the result of some mistakes related to direct translation from the authors language into English. This have to be corrected and authors have to provide clear and understandable terminology.

Finally, the section Discussion is limited to quite detailed discussion of methodology and then recapitulation of data; but completely lacks Discussion of results from other research. There is a vast literature on mechanism underlying drivers of patterns in microbial (and other biota) abundance and diversity, both from Antarctica and elsewhere. Moreover, Discussion virtually lacks any citations, still it contains a number of statements that are not a new discovery and/or were discussed in other literature, so they need appropriate literature citations. Without that they can be considered as plagiarism, hopefully not intended by the authors, still not acceptable without proper literature citations. This is the main reason why, with my big disappointment, I cannot suggest anything more but major revision, thorough major revision.

More detailed comments.

As the first part of the manuscript (pages 1-4) lack line numbering, for that part I am referring to particular parts of the manuscript.

Page 1.

1. Introdyction

Page 1, Paragraph 1: “they are also at the basis and at the end of every food chain

This is not true, at these biota are not authotrophs they are not at the basis of the food chain, and surely they are also not at its end. Considering only terrestrial biota, that is omitting birds, in case of the Antarctic ecosystems Acari and Collembola are generally considered to be at the end of the food chain. However, nematodes, tardigrades and rotifers are in some habitats also considered to be at the end of the food chain.

Page 2.

The end of second paragraph of Introduction:

The last two lines of the second paragraph (the first two line of the text on the page 2) need editing of its format

2. Materials and Methods

2.1.Sampling sites and sampling methods.

“For the solid samples, 150-250 mg of biomass or sediment were collected”

To be honest I have no idea what substratum was collected as these “solid samples”? Was that soil, cryptogamic crust, some algal/cyanobacterial mats or something else? This has to be clearly specified. The terminology used, that is “solid samples”, “biomass” or “sediment” is completely unclear.

Table 1.

What does “Microbial surface contamination” mean? What is that? In case of the samples S13 and S22 it has to be specified what king of biomass was collected. As I indicated above clear terminology has to be used.

Page 3.

2.2. DNA extraction

Again, terminology regarding “biomass” has to be changed or clearly defined.

2.4. Data processing and operational taxonomic units (OTUs) analyses

At the end of that section four links to external sources are given. But none of these links work. By the way citation of appropriate literature in that case would be sufficient, and I am not sure why authors provided links to some external web sources.

Page 4.

2.4. Antagonism analyses and relative measuring

“The mean values of the number of the total tags, the number of the OTUs, the Shannon, the Simpson, The Chao1, and the ACE indices were calculated”

Grammar correction for the above quotation needed. The following reads better:

“The mean values of the number of total tags, number of OTUs, and Shannon, Simpson, Chao1 and ACE indices were calculated”

“binome antagonisms in each sample were relatively quantified”

The authors did not quantified antagonisms, but they quantified relative values of diversity measures. Then, the relative value was interpreted as an indication of antagonisms. This is very significant difference. Thus, the quoted above statement has to be modified. That is, authors should clearly describe their calculations and only after that explain their interpretation of the results obtained.

3. Results

3.1. Sequencing statistics and alpha diversity indices

“and some of the alpha diversity indices are presented in Table 2”

As I see not “some” but all calculated indexes are given in Table 2. So, this sentence has to be modified. For instance: “and calculated alpha diversity indices are presented in Table 2”

Page 6.

Line 6:

3.2. Antagonism study

Authors did not investigated antagonisms directly, thus the following would probably be more appropriate for the title of the section 3.2.

3.2. Antagonism assessment

Lines 7-8: “The numerical values for each index (the number of the total tags, the number of the OTUs, the Shannon, the Simpson, the Chao1, and the ACE indices)”

Grammar correction needed. The following reads better:

“The numerical values for each index (i.e.: number of total tags, number of OTUs, and Shannon, Simpson, Chao1 and ACE indices)”

Lines 10-11: “for each index according to its proper value in comparison to the mean value for this index”

Correction of grammar and style is needed. Values obtained in research are always “proper”, there is nothing like “improper” about values, but surely about such terminology.

Table 3.

The Table Caption should clearly explain what matrix is presented in the Table; it may of course also refer to data source (i.e. Figure 2 in that case) but this cannot be the main information given.

Table 4.

Provide in the Table Caption information on “n”, that is the total number of samples (i.e.: “n = 18”).

Table 5.

Significant correlation has to be distinguished from insignificant, e.g. using * by the significant correlation values. Explanation of such a symbol has to be also added into the Table caption.

“The upper shows the Pearson correlation, while the number below presents the Spearman correlation.” The following reads better: “The upper value represents Pearson correlation coefficient, while the value given bellow represents Spearman correlation coefficient.”

Lines 53-54: “the number of the Archaea (as total tags as well as the number of OTUs) was much lower than those of Fungi and Bacteria.”

This is not true, at least not in the case of OTUs. While the total number of tags in Archaea was indeed considerable lower than the number of tags in Fungi and Bacteria, and the number of OTUs in Archaea was also considerably lower than that of Bacteria, but the value for Archaean OTUs was relatively similar to that in Fungi (or in most cases and on average even greater).

Lines 64-65: “one of the domains is more than the observed average, the other is less than the observed average”

Grammar correction needed.

Line 77: “in the water samples”

Does that refer to “the sea water samples”? If so, add the word “sea” to make that clear.

Lines 83-85:

“The observed discrepancies in all the three binomes concerning the Shanon and the Simposon indices [21,22,29] varied between 17% and 50%.”

This sentence presents the authors own results, so I do not really understand what the meaning of the citations given here is. They seem to be redundant in that sentence.

Lines 112-114: “surface contamination samples” & “a type of surface contamination”

What does that mean? What is a surface sample? With what these samples were contaminated? This is clearly some wired terminology.

Line 119: “could be attributed to the Antarctic climate as a whole”

What is this conclusion based on?

Line 123: “is sample S22” --> “is the sample S22”

Line 123: “was obtained from biomass”

Again, what biomass?

Lines 125-129:

“This is the only sample where the numbers of the total tags and the OTUs in Archaea exceed those of Fungi, and approach those of Bacteria. More interestingly, the number of the archaeal total tags is some 16% greater than the fungal one. However, the archaeal OTUs are more than 6 times more numerous than the fungal ones.”

Comparing the sample S22 with other samples it seems to me that value for Archaea are comparable to values in other samples. On the other hand this sample is characterized by exceptionally low value of OTUs for Fungi, also Fungal total tags in that sample are the lowest compared to other samples. So, there is nothing exceptional about Archaea in that samples but described relationships are the results of exceptionally value for Fungi. So, in their discussion the authors should focus on Fungi rather than Archaea.

Lines 131-136:

Very long and stylistically complicated sentence. Grammar and style correction needed.

Lines 144-146:

“If this hypothesis is true, it would explain why in the “old” ecological niches in our study in general Fungi dominate over Archaea.”

Honestly, I do not understand why being late colonizer would explain ability of having greater abundance in latter concessional stages. Moreover, some literature sources citation is clearly needed here.

Lines 158-159: “could impact the observed antagonisms”

This work does not observed antagonism but patterns that can be interpreted as antagonisms, though other interpretations are also possible, such as stochastic events or ecological preferences. Therefore, this statement should be replaced with “could impact the observed patters”. Then, this should be followed by discussion on the possible mechanisms, such as antagonisms, but also stochastic events with random distribution and ecological preferences should also be discussed, and supported by citations of appropriate literature.

Line 179: “causes of the observed antagonism”

As I have already indicated above, the authors did not observed antagonism; they only observed patterns in abundance and diversity. Surely, these patterns can be interpreted in context of antagonism, but this is not the only likely mechanism and other mechanisms should also be considered and discussed.

Lines 187-188: “Unfortunately, because of the general lack of similar studies”

While there indeed might similar studies might be lacking, particularly in the Antarctic. However, but there is vast amount of literature concerning mechanisms underlying differences in abundance and diversity patterns, also with focus on the Antarctic biota. Antagonism is only one of a few different mechanisms that can underline the observed patterns. I am truly surprised, that authors did not consider that. Moreover, their work completely lack citation of appropriate literature, even when concerning antagonisms among biota.

------------------------------------------------------------------

Comments on the Quality of English Language

In general moderate editing of English language would be sufficient. However, detailed verification of scientific terminology is needed, particularly concerning methodology.

---------------------------------------------

Author Response

Dear Reviewer,

Please, find bellow our responses and addresses:

“Authors of that manuscript attempted to investigate patterns in abundance and diversity of three different taxa, including Archaea, Bacteria and Fungi, in the Antarctic environments. Considering very low number of samples investigated, 14 from terrestrial habitats and 4 from marine environment, this work can be considered as only preliminary insight into the investigated problem. Still, their work is valuable, as our knowledge on microbial communities existing in the extreme environmental condition in Antarctica, but also elsewhere, is still very limited. Personally, being involved in the Antarctic research I became truly interested in this work after reading its abstract. However, to my great disappointment I see that the manuscript still needs a lot of effort before its publication, and surely should not be published in its present form.”

We deeply appreciate the reviewer’s esteem of our pioneering work.

“Firstly, authors claim that they investigated antagonisms among Archaea, Bacteria and Fungi. This is not true. As I have purposely indicated at the very beginning, authors have investigated patterns in abundance and diversity. Surely, these patterns can be interpreted as antagonism, but there are also other possible and I would say even more likely explanations of mechanisms that can drive the observed patterns. For instance, environmental requirements and habitat suitability or stochastic events and random distribution. In case of the Antarctic environments, adaptations to the extreme environmental condition and legacy of historical events are particularly often emphasized in numerous literature. Unfortunately, authors has only briefly mentioned possible role of environmental requirements, still omitting any discussion of that topic.”

We completely agree that our study is a preliminary one, and hope in the future to investigate in depth the relationships between the different types of microorganisms. This could be achieved for example by the means of the shotgun metagenomics approach which, unfortunately, requires for easier analysis isolation of high molecular weight DNA which was not possible because of the limited available laboratory infrastructure in our Polar base.

“Secondly, methodology has to be described in more details with usage of appropriate terminology. Authors have mentioned, that they collected “solid samples … of biomass or sediments” or “microbial surface contamination”. To be honest, I do not understand what kind of samples were collected for that work. Maybe this is the result of some mistakes related to direct translation from the authors language into English. This have to be corrected and authors have to provide clear and understandable terminology.”

We changed “microbial surface contamination” to “microbial surface biofilm” and “biomass” to “sludge biomass”.

“Finally, the section Discussion is limited to quite detailed discussion of methodology and then recapitulation of data; but completely lacks Discussion of results from other research. There is a vast literature on mechanism underlying drivers of patterns in microbial (and other biota) abundance and diversity, both from Antarctica and elsewhere. Moreover, Discussion virtually lacks any citations, still it contains a number of statements that are not a new discovery and/or were discussed in other literature, so they need appropriate literature citations. Without that they can be considered as plagiarism, hopefully not intended by the authors, still not acceptable without proper literature citations. This is the main reason why, with my big disappointment, I cannot suggest anything more but major revision, thorough major revision.”

Unfortunately, with an exception of the study of Li et al.2020 which is cited, we didn’t find any publications on the relationships between the three microbial domains as a whole within the largest scientific publications databases (such as PubMed, Google Scholar, and some others) of similar studies in any environments performed by any means, not only by the tools of the metagenomics. We will be very grateful if the reviewer could share information of such publications. Indeed, there are lot of publications on the relationships of the lower taxonomic ranks, though, we believe it will be incorrect to transpose them to the whole domains.

More detailed comments.

As the first part of the manuscript (pages 1-4) lack line numbering, for that part I am referring to particular parts of the manuscript.

“Page 1.

Introdyction

Page 1, Paragraph 1: “they are also at the basis and at the end of every food chain

We are thankful for this remark and we removed the statement.

Page 2.

The end of second paragraph of Introduction:

“The last two lines of the second paragraph (the first two line of the text on the page 2) need editing of its format”

This was corrected.

“2. Materials and Methods

2.1.Sampling sites and sampling methods.

“For the solid samples, 150-250 mg of biomass or sediment were collected”

This was corrected as mentioned above.

“Table 1.

This was corrected as mentioned above.

“Page 3.

2.2. DNA extraction

Again, terminology regarding “biomass” has to be changed or clearly defined.”

This was corrected as mentioned above.

“2.4. Data processing and operational taxonomic units (OTUs) analyses

During the current revision we found unfortunately that the hosting of the tools changed. We also agree that the presence of the links is redundant, so we removed them, leaving only the citations.

“Page 4.

2.4. Antagonism analyses and relative measuring

“The mean values of the number of the total tags, the number of the OTUs, the Shannon, the Simpson, The Chao1, and the ACE indices were calculated”

Grammar correction for the above quotation needed. The following reads better:

“The mean values of the number of total tags, number of OTUs, and Shannon, Simpson, Chao1 and ACE indices were calculated””

We are deeply thankful for this correction!

““binome antagonisms in each sample were relatively quantified”

We are thankful for suggesting this clarification! We agree and clarified this.

“3. Results

3.1. Sequencing statistics and alpha diversity indices

“and some of the alpha diversity indices are presented in Table 2”

As I see not “some” but all calculated indexes are given in Table 2. So, this sentence has to be modified. For instance: “and calculated alpha diversity indices are presented in Table 2””

We are grateful for this suggestion!

“Page 6.

Line 6:

3.2. Antagonism study

Authors did not investigated antagonisms directly, thus the following would probably be more appropriate for the title of the section 3.2.

3.2. Antagonism assessment””

Again, we are grateful for the suggestion!

“Lines 7-8: “The numerical values for each index (the number of the total tags, the number of the OTUs, the Shannon, the Simpson, the Chao1, and the ACE indices)”

Grammar correction needed. The following reads better:

“The numerical values for each index (i.e.: number of total tags, number of OTUs, and Shannon, Simpson, Chao1 and ACE indices)””

Again, we are grateful for the suggestion!

“Lines 10-11: “for each index according to its proper value in comparison to the mean value for this index”

Correction of grammar and style is needed. Values obtained in research are always “proper”, there is nothing like “improper” about values, but surely about such terminology.”

The word “proper” was deleted.

“Table 3.

The Table Caption should clearly explain what matrix is presented in the Table; it may of course also refer to data source (i.e. Figure 2 in that case) but this cannot be the main information given.””

We changed the table’s caption as suggested.

““Table 4.

Provide in the Table Caption information on “n”, that is the total number of samples (i.e.: “n = 18”).””

We clarified this and made the change.

““Table 5.

Significant correlation has to be distinguished from insignificant, e.g. using * by the significant correlation values. Explanation of such a symbol has to be also added into the Table caption.

We changed the table accordingly to the suggestions.

“Lines 53-54: “the number of the Archaea (as total tags as well as the number of OTUs) was much lower than those of Fungi and Bacteria.”

We removed the OTUs.

“Lines 64-65: “one of the domains is more than the observed average, the other is less than the observed average”

Grammar correction needed.”

We rephrased the sentence.

“Line 77: “in the water samples”

Does that refer to “the sea water samples”? If so, add the word “sea” to make that clear.”

All water samples but we clarified this within the text.

““Lines 83-85:

“The observed discrepancies in all the three binomes concerning the Shanon and the Simposon indices [21,22,29] varied between 17% and 50%.”

This sentence presents the authors own results, so I do not really understand what the meaning of the citations given here is. They seem to be redundant in that sentence.”

The citations were removed.

“Lines 112-114: “surface contamination samples” & “a type of surface contamination”

What does that mean? What is a surface sample? With what these samples were contaminated? This is clearly some wired terminology.”

It was corrected already as previously suggested.

“Line 119: “could be attributed to the Antarctic climate as a whole”

What is this conclusion based on?”

We removed “as a whole” and changed the sentence=

Line 123: “is sample S22” --> “is the sample S22”

We are grateful for pointing us this error!

“Line 123: “was obtained from biomass”

Again, what biomass?”

It was corrected already as previously suggested.

“Lines 125-129:

We are sorry but we do not agree with this argument. As it is supported by the citations within this paragraph, there are scientific evidences that Bacteria and Archaea are the first colonizers of new and pristine ecological niches. So, our observation support the cited findings, therefore, the focus should not be put on fungi.

Lines 131-136:

Very long and stylistically complicated sentence. Grammar and style correction needed.

We made some changes.

“Lines 144-146:

“If this hypothesis is true, it would explain why in the “old” ecological niches in our study in general Fungi dominate over Archaea.”

We enlarged this paragraph supporting our statements by introducing new citations.

“Lines 158-159: “could impact the observed antagonisms”

We replaced “antagonisms” with “patterns interpreted as antagonistic”. Unfortunately, without whole shotgun metagenomic sequencing all hypotheses would be more or less speculative, and this is the reason we restrained ourselves to speculate.

Line 179: “causes of the observed antagonism”

We replaced “antagonisms” with “patterns interpreted as antagonistic”.

“Lines 187-188: “Unfortunately, because of the general lack of similar studies”

Unfortunately, with a single exception which is cited (Li et al., 2020) we didn’t find any data within the largest scientific publications databases (such as PubMed, Google Scholar, and some others) of similar studies in any environments performed by any means, not only by the tools of the metagenomics. We will be very grateful if the reviewer could share information of such publications.

“Comments on the Quality of English Language

In general moderate editing of English language would be sufficient. However, detailed verification of scientific terminology is needed, particularly concerning methodology.”

The manuscript passed through another round of grammar and spell check. The terminology was changed accordingly to the suggestion.

On behalf of all the co-authors, allow me to express once again our gratitude for your work and time spent for the evaluation of the manuscript.

Svetoslav G. Dimov

November 12, 2023

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors, congratulation on you research work, this study of antagonisms between Fungi, Archaea, and 2 Bacteria in Livingston Island (Maritime Antarctica) with use 3 amplicon-based metagenomic study, its very interesting and necessary to try to undestand what happend with the microbial comunities in extreme environments.

I attach the pdf file with some comments.

I do have a few comments, I think though the manuscript there is a several concepts that repete several time. This should be corrected, in introduction, results and discussion.

I have a question regarding the analysis of the data, because you analyze all samples toguether, I beleaved each substrate must be analyzed separately. You have coment that the low numers of replica can be a problem, but i think it could be interest see the results of the different substrats and if they have differences (ex: soil - water). Regarding fungi exists reports that showed significant differences among comunnities when compare soil and ice/water.

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Thank you for your effort which raised the quality of the manuscript.

Please, find below our responses to your comments:

The paragraph within lines 49-58 can be shortened.

The paragraph was shortened.

Line 88.

We are grateful for this clarification, and the phrase was changed.

Lines 90-92.

The sentence was re-phrased according to the reviewer’s suggestion.

Figure 2.

The figure was replaced with one with higher resolution and vertical arrangement to make the parts bigger.

Lines 312-313.

We are very grateful for this suggestion and have made the change.

6. Lines 331-344.

I am also uploading the pdf file with your comments and our responses.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

As I have clearly indicated previously, the manuscript contains several issues that have to be considered and revise thoroughly. Therefore, I was kind of surprised when I was asked to revise supposedly revised manuscript just after a few days. As I see, the authors have mostly limited their revision to editorial corrections and shifting position of some paragraphs. Unfortunately, they did not implement virtually any of the most important suggestions regarding both my and the other reviewer comments. It is really frustrating when authors ignore work and efforts of reviewers to help improve a manuscript.

I have to clearly indicate again, that one of the biggest problems with this work is that the authors’ presented the results of their investigation as if they documented outcome of antagonistic interactions among different biota; that is among Archaea, Bacteria and Fungi. This is not only misleading but such indication is false, and as such not acceptable. As I have indicated clearly this work documents nothing more but only patterns in abundance and distribution of the investigated taxa. Surely, these patterns can be interpreted as a result of antagonisms. However, as I have clearly indicated other mechanisms are also possible, for instance environmental requirements and habitat suitability or even stochastic events and random distribution. Therefore, the potential role of these mechanisms cannot be ignored but have to be considered and discussed at least briefly. Without that the manuscript as misleading, and, regrettably, I cannot recommend its publication.

Secondly, the section Discussion is limited to quite detailed comments on methodology and then recapitulation of data; but completely lacks true Discussion of results in the context of other research. There are also numerous statements, which clearly need appropriate literature citation, otherwise they might be considered as plagiarism. Contrary, to authors rebuttals, both to my and the other reviewer suggestion, there is abundant literature that do have relevance for that investigation and interpretation of results, and, therefore, cannot be ignored but discussed at least briefly and quoted accordingly. Both in the discussion and as suggest by the other reviewer also in introduction. At present it seems as if authors did not bother to check any literature beyond their own very narrow focus. This substantially lowers quality and appeal of their work.

Finally, the work need very detail linguistic correction. Hope this time authors will consider my comments and suggestions, and my time devoted to help them improve the manuscript, more seriously and conduct true thorough revision of their manuscript.

Some more detailed comments.

Introduction

Line 35: “energy and substances circulation”

While matter does circulate in ecosystems, energy does not circulate but flows across the ecosystems. So, this statement, being false, has to be rephrased.

Lines 33-35:

This statement on the importance of microbiota in the Antarctic ecosystems needs literature citation. The literature quoted after the next sentence, is not adequate in that case.

Lines 44-45:

“Still, with very few exceptions, the mutual ecological relationships between these three domains of microorganisms as a whole are poorly investigated.”

Literature citation needed, either presenting these “exception” or for the whole statement.

Lines 47-48:

“most of the studies are focused on research within a single domain in a given ecological niche.”

Literature citation would be desirable, either for the whole statement or giving examples of studies focusing only on “a single domain” or “a given ecological niche”.

Lines 49-51:

“Unfortunately, Antarctic microbial communities are still poorly studied, mostly because of the challenging environments and the remoteness from the needed adequate laboratory infrastructure.”

This is not a novel statement, but these problems were already discussed in numerous literature. Thus, to avoid plagiarism, citation of an appropriate literature is needed.

Lines 55-58:

“Yet, the focus of these studies was put on the characterization of the fungal, archaeal, and bacterial compositions within the soils and the aquatic environments but not on the existing relationships between these three domains.”

This statement is not quite true or at least misleading. While these works indeed did not investigate relationship between Archaea, Bacteria and Fungi, they provided significantly more than just characterization of the investigated taxa composition. For instance, Picazo et al. (2019) and Kim et al. (2020) demonstrated importance of environmental factors, particularly nutrients and salinity, as major factors driving patterns in structure and distribution of their assemblages. This is not a novel discovery but a well known phenomenon, and many works has already pointed to the importance of environmental factors as the major driver of local patterns in structure and distribution of the Antarctic biota. Surely, biological interactions, while poorly investigated and understood are also significant. This was documented and discussed in numerous investigations and in several reviews virtually for all Antarctic biota. Moreover, contrary to the authors’ rebuttals, mechanisms that underline distribution patterns for other biota and/or conducted with application of different methodology do have relevance for their own investigation and interpretation of results, and, therefore, cannot be ignored but discussed at least briefly and quoted accordingly.

Line 67 & 72: “patterns of antagonism”

Again, authors did not investigated antagonism or patterns of antagonisms. Thus, this terminology is misleading and have to change.

Lines 69-71:

“We stated the hypothesis that in these inhospitable environments the different life domains as a whole should probably be concurrent for the occupations of the ecological niches.”

It seems to me that formulation of the investigated hypothesis would read better when placed at the very end of the last paragraph. Moreover, it might be good if this would be followed by a clear statement that that applied methodology provides only indirect evidence.

Lines 76-77:

“submerged microbial surface biofilm of rocks and algae”

“biofilm (or microbial mats) on submerged roks” would read better, though I am not sure what authors had in mind in case of algae, if this was “algal mat” or some biofilm collected from the top of an algal mat. This should all be clarified using established terminology and clearly defined in the Material and Methods section.

Line 77:

“biomass sludges”

Sluge is generally defined as:

(1) thick, soft, wet mud or a similar viscous mixture of liquid and solid components, especially the product of an industrial or refining process;

(2) dirty oil, especially in the sump of an internal combustion engine;

(3) sea ice newly formed in small pieces.

With the exception of the sea ice sludge, this terminology is generally associated with industrial contamination, and I truly doubt if it usage is appropriate in that case.

“Microbial mats”, which is widely used terminology in the Antarctic literature, would probably be more appropriate in that case.

Material and Methods

Lines 83-84: “four water and fourteen terrestrial samples”

This statement is not precise and a bit misleading. For instance, samples of microbial mat/biofilm or sediment collected from the bottom of freshwater pond or from submerged rocks are not really terrestrial samples. Considering the scale of the investigated biota, this is not a terrestrial environment and biota in these samples surely does not represent terrestrial biota. Similarly, clear information that both freshwater and seawater samples were collected. I did noticed, that each simple is relatively clearly described in Table 1. But clear information on sampling sites and sampling scheme should also be presented in the text. Moreover, for clarity I would strongly suggest providing a Figure that will show a map of the study area and location of the sampling sites.

Line 87:

“biomass sludges”

Terminology correction needed.

Table 1.

Firstly, I would suggest some effort to format Table for a better appearance, e.g. remove redundant spacing before the last row. On the other hand inserting a space that would separate water samples from the rest of samples given above might a good idea.

Secondly, descriptions of the samples should be on one hand more precise regarding type environment but simplified in case of sample descriptions. For instance, instead of “Microbial surface biofilm from a rock submerged in the lagoon” the following reds better “lagoon, microbial biofilm from submerged rocks”. However, in that particular case it is necessary to provide more precise information on the lagoon. Was that a marine lagoon of a lagoon in freshwater lake? In fact it would be good to split the second column into two separate columns; one with the information on the type of environment (Environment) and the other describing the sample (Sample Type).

Results

Lines 167-168:

“according to its value in comparison to the mean value”

Rephrasing, to correct style and grammar of that statement is needed.

Table 4.

As authors inserted the information on the sample size in the figure caption it was unnecessary to insert that within the table. I would also suggest simplification of the information in the rows as repeating information that data present “discrepancies” is redundant, that is:

“Number of cases of discrepancies in the total number of samples n=18” –> “Number of cases”

“Percentages of cases of discrepancies” –> “Percentages of cases”

Table 5.

The highest positive and negative correlations coefficients are marked with “*” and “**” for the Pearson and the Spearman correlations respectively

I have suggest to mark with a star (*) significant correlation as it is a general practice in scientific literature. On the other hand the other reviewer suggest to distinguish the two correlation coefficient by adding one or two stars, which implies removing unnecessary in such case footnote. Authors decided to do something in between, which provided a very misleading outcome. Firstly, it does not really make any sense to mark “the highest correlations” as it is written in the figure captions, buy the way not all the highest values were marked (e.g. the highest value of Pearson correlation for Fungi – Bacteria OUTs and Chao1 were not marked). Secondly, different number of stars is usually used in the scientific literature to mark different levels of significance, e.g.: * for p<0.5, ** for p<0.1 and *** for p<0.001. As I have suggested previously “statistically significant values should be clearly distinguished. Unless there are clear differences in the significance level (as I have indicated above) providing different number of stars is misleading and should be avoided, despite the other reviewer suggestion. Adding an additional column (and placing it as a second column) with the names of appropriate correlation indexes for each of the relationship would not be complicated and should resolve the concerns of the other reviewer.

Discussion

Firstly, I would strongly suggest beginning Discussion with a general remark on the research goals and working hypothesis. This should be also followed by a at least short statement that while authors hypothesize that the documented patterns in abundance and diversity reflect antagonistic interactions, other mechanisms such as environmental requirements and habitat suitability or even stochastic events and random distribution, as it was documented in other studies of microbial (e.g.: Picazo et al. 2019; Kim et al. 2020) and other Antarctic biota. Of course, citation of appropriate literature would be needed. As I have already indicated, contrary to the authors’ rebuttals, there is a vast literature on mechanisms that underline distribution patterns for the Antarctic biota, even if they were conducted with application of other methodology or focused on other biota they are still highly significant for results presented in that work and have to be quoted accordingly. Moreover, this is authors work to search for an appropriate and relevant literature and not the role of a reveres to that for them. The literature is vast; the authors just have to look a bit wider beyond the narrow methodology they applied.

Lines 197-200:

“the number of the Archaea as total tags [18-20] was much lower than those of Fungi and Bacteria … the archaeal alpha diversity indices (Shannon, Simpson Chao1, and ACE) [21-25] were from the same order as those of the two other studied domains.”

These sentences provides nothing more than quotation of the authors own data. Therefore, I do not understand what the cited literature [i.e.: 18-20 and then 21-25] refers to. Placing citation within these sentences does not seem to be appropriate. The cited references should be removed from here and placed after statements that truly refer to cited literature.

Lines 204-206:

“One of the most probable explanations of this disparity is the assumption that some antagonism between Archaea and the other two domains should exist, in turn probably caused by the harsh, and in some cases oligotrophic, Antarctic conditions”.

Some of the above statements require literature citation.

Lines 206-209:

“So, we decided to measure relatively this antagonistic relationship most simply - by comparing in binomes: in how many samples when one of the domains was more represented than the observed on average, the other was less represented than the observed on average.”

Grammar and/or style correction needed.

Line 214: “the most primary parameter”

This sound kind of wired, linguistic correction is needed.

Line 216: “antagonism was observed”

No, this is not true. Authors did not observe any antagonism; they observed differences (discrepancies) in abundance and/or diversity data compared to expected data, these discrepancies can be interpreted as a result of antagonisms. This is a big difference in comparison to the authors’ statement which is to simplified and might be misleading.

Lines 235-236:

“This can be explained by the fact that this index is based on the singletons and the doubletons, thus estimating the “missed” species”

Literature citation needed.

Lines 236-237:

“Bacteria being largely the most species-abundant superkingdom”

Literature citation needed.

Lines 237-238:

“On the other hand, discrepancies in 56% and 61% of the cases were observed respectively for the binomes Fungi-Archaea and Archaea-Bacteria.”

This statement refers for ACE index. Previous two sentences refer to Chao1, so without indication that these data refer to ACE readers would understand this also refers to Chao1, which is misleading.

Line 257:

“biomass sludges”

Terminology correction needed.

Lines 273-274:

“However, the archaeal OTUs are more than 6 times more numerous than the fungal ones.”

I would strongly suggest adding an explanation, that to a large extend such big difference is due to exceptionally low OUT for fungi. Without that such statement might be misleading.

Lines 284-286:

“It has been observed that microorganisms are the first colonizers after the receding glaciers in the mountains [29]”

I would suggest checking literature on microbial succession or on variation in microbial communities on glacier forelands in Antarctica and Arctic, and citing them accordingly.

Lines 291-292:

“Our observation concurs with the general logic that pristine environments are firstly colonized by autotrophs, the heterotrophs coming somewhat later.”

Literature citation needed.

Line 293:

“such studies in Antarctica are rather scarce”

Still, should not be omitted be cited acordingly.

Lines 311-213:

“active, due to the synthesis of different types of antimicrobials, and passive, resulting from the concurrence for nutrients and energy between the different types of microorganisms.”

Literature citation needed, preferably with quotation of some examples.

Line 319:

“biomass sludges”

Terminology correction needed.

Lines 320-223:

“the lack of a well established methodology for quantification of the antagonisms between the three do mains. Unfortunately, this drawback could not be adequately addressed because of the very scarce publications of similar studies.”

Literature citation needed, some of these “scarce publications” should be quoted.

Conclusions

I would strongly suggest indicating in the very first sentence what is the main outcome/result, and only after that providing interpretations. Otherwise this might be very misleading. For instance:

Results of our study demonstrated differences in abundance and distribution patterns among Archaea, Bacteria and Fungi. While this might be related to differences in environmental requirements, that were not investigated in our study, we believe that antagonistic interaction play significant role.

Literature

Format of the cited literature is not consistent. In particular, considering DOI number, while majority of cited works is listed with some do not provide DOI. I believe, this should be unified.

-------------------------------------

Comments on the Quality of English Language

As I have indicated above, the work need very detail linguistic correction, including terminology, grammar and style.

Author Response

Dear reviewer,

Please accept our gratitude for your work. We address your remarks as follows:

“As I have clearly indicated previously, the manuscript contains several issues that have to be considered and revise thoroughly. Therefore, I was kind of surprised when I was asked to revise supposedly revised manuscript just after a few days. As I see, the authors have mostly limited their revision to editorial corrections and shifting position of some paragraphs. Unfortunately, they did not implement virtually any of the most important suggestions regarding both my and the other reviewer comments. It is really frustrating when authors ignore work and efforts of reviewers to help improve a manuscript.”

We are sorry you remained with an erroneously frustration feeling we do not respect your work. Please, let me reassure you that this is not the case! We do sincerely believe that after agreeing with about 88% of your remarks the quality of the manuscript increased – something we noted in our previous responses to you. You had in total 33 remarks and suggestions to the manuscript, and we agreed with you in 29 of the cases making the changes and the corrections.

One of your remarks we couldn’t address according to your desire was that we didn’t discuss the mechanisms leading to the observed patterns. Making whole-metagenome shotgun sequencing which could respond to your remarks by meta-metabolomic analyses was not within the scope of the project in one hand, while in the other hand, we believe you will agree that we just can’t return in Antarctica for additional experiments even we found the necessary founding for additional experiments within an already finished project.

The second of your remarks we couldn’t address accordingly to your suggestions was that the “Discussion” section lacks enough citations. We suppose you will agree that this section of the manuscript should not be limited only to comparisons to similar works – that is why it is called that. Of course, the proper scientific approach requires that the obtained results should be compared to other similar works. That is why we included only one proper citation of somewhat similar research which was THE ONLY ONE WE COULD FIND. Further in your review you suggest comparing our results to those of Picazo et al., 2019 and Kim et al., 2020, however, these two works deal with the eubacterial composition and reflects of the environmental factors on it, which is out of the main idea and scope of our manuscript.

The third remark concerned sample 22. We are sorry but we still do not agree for this with you, being supported by the scientific publications we cited.

The fourth remark in your previous review was again about the lack of discussion on the possible mechanisms of antagonism. We already addressed this issue several lines above.

“I have to clearly indicate again, that one of the biggest problems with this work is that the authors’ presented the results of their investigation as if they documented outcome of antagonistic interactions among different biota; that is among Archaea, Bacteria and Fungi. This is not only misleading but such indication is false, and as such not acceptable. As I have indicated clearly this work documents nothing more but only patterns in abundance and distribution of the investigated taxa. Surely, these patterns can be interpreted as a result of antagonisms. However, as I have clearly indicated other mechanisms are also possible, for instance environmental requirements and habitat suitability or even stochastic events and random distribution. Therefore, the potential role of these mechanisms cannot be ignored but have to be considered and discussed at least briefly. Without that the manuscript as misleading, and, regrettably, I cannot recommend its publication.”

Qualifying our text as misleading and false without any support for these statements, to our opinion, goes far beyond good academic language, so we will pass it. We will only respond to you that in the previous revision we changed “antagonisms” to “patterns of antagonisms”, even though we didn’t agree completely with this because our experimental data suggested otherwise. Additionally, there is not a single sentence within our manuscript dealing with an “outcome of antagonistic relationships” – we only took a snapshot picture and discuss it. Again, as we already responded to you, studying the antagonistic mechanisms was out of the scope of the already finished research project, and it is possible if an entirely new project is made, based on a different concept and methodology.

“Secondly, the section Discussion is limited to quite detailed comments on methodology and then recapitulation of data; but completely lacks true Discussion of results in the context of other research. There are also numerous statements, which clearly need appropriate literature citation, otherwise they might be considered as plagiarism. Contrary, to authors rebuttals, both to my and the other reviewer suggestion, there is abundant literature that do have relevance for that investigation and interpretation of results, and, therefore, cannot be ignored but discussed at least briefly and quoted accordingly. Both in the discussion and as suggest by the other reviewer also in introduction. At present it seems as if authors did not bother to check any literature beyond their own very narrow focus. This substantially lowers quality and appeal of their work.”

We believe we have already discussed these issues previously.

“Finally, the work need very detail linguistic correction. Hope this time authors will consider my comments and suggestions, and my time devoted to help them improve the manuscript, more seriously and conduct true thorough revision of their manuscript.”

The manuscript passed another round of language polishing.

Some more detailed comments.

Introduction

Line 35: “energy and substances circulation”

“While matter does circulate in ecosystems, energy does not circulate but flows across the ecosystems. So, this statement, being false, has to be rephrased.”

Corrected

Lines 33-35:

This statement on the importance of microbiota in the Antarctic ecosystems needs literature citation. The literature quoted after the next sentence, is not adequate in that case.

Corrected

Lines 44-45:

“Still, with very few exceptions, the mutual ecological relationships between these three domains of microorganisms as a whole are poorly investigated.”

Literature citation needed, either presenting these “exception” or for the whole statement.

Corrected

Lines 47-48:

“most of the studies are focused on research within a single domain in a given ecological niche.”

Literature citation would be desirable, either for the whole statement or giving examples of studies focusing only on “a single domain” or “a given ecological niche”.

Corrected

Lines 49-51:

“Unfortunately, Antarctic microbial communities are still poorly studied, mostly because of the challenging environments and the remoteness from the needed adequate laboratory infrastructure.”

This is not a novel statement, but these problems were already discussed in numerous literature. Thus, to avoid plagiarism, citation of an appropriate literature is needed.

These are general considerations and well known facts, not scientific findings or theories, so to our opinion no citations are needed.

Lines 55-58:

As we already said, the objective of our paper was not to study the impact of the environmental factors on the microbiota. The works of Kim and Picazo are excellent, however we cited them in another context. The goal of our study was to report our finding on the observed antagonistic patterns between archaea, fungi, and bacteria.

Line 67 & 72: “patterns of antagonism”

Again, authors did not investigated antagonism or patterns of antagonisms. Thus, this terminology is misleading and have to change.

We do not agree that the terminology is misleading.

Lines 69-71:

“We stated the hypothesis that in these inhospitable environments the different life domains as a whole should probably be concurrent for the occupations of the ecological niches.”

We believe that if we move the sentence, the text will become logically inconsistent because just this hypothesis motivates our further investigations.

Lines 76-77:

“submerged microbial surface biofilm of rocks and algae”

We are thankful for suggesting to use “microbial mats” and made the changes.

Line 77:

“biomass sludges”

Sluge is generally defined as:

(1) thick, soft, wet mud or a similar viscous mixture of liquid and solid components, especially the product of an industrial or refining process;

(2) dirty oil, especially in the sump of an internal combustion engine;

(3) sea ice newly formed in small pieces.

With the exception of the sea ice sludge, this terminology is generally associated with industrial contamination, and I truly doubt if it usage is appropriate in that case.

“Microbial mats”, which is widely used terminology in the Antarctic literature, would probably be more appropriate in that case.

The notion “sludge” is widely accepted for describing microbial biomass in aquatic environments, for example in sewage systems (https://doi.org/10.3389/fmicb.2018.01462 , https://doi.org/10.1016/j.scitotenv.2022.157384 , https://doi.org/10.1016/j.watres.2022.119055 , https://doi.org/10.1016/j.watres.2022.118646 , https://doi.org/10.1016/j.biortech.2022.127201 ).

Material and Methods

Lines 83-84: “four water and fourteen terrestrial samples”

We agree that “solid” which was further used is more adequate and made the change. A figure was included.

Line 87:

“biomass sludges”

Terminology correction needed.

We disagree as we motivated us previously.

Table 1.

We changed the table according to your suggestions.

Results

Lines 167-168:

“according to its value in comparison to the mean value”

Rephrasing, to correct style and grammar of that statement is needed.

Done.

Table 4.

“Number of cases of discrepancies in the total number of samples n=18” –> “Number of cases”

“Percentages of cases of discrepancies” –> “Percentages of cases”

We made the corrections

Table 5.

The highest positive and negative correlations coefficients are marked with “*” and “**” for the Pearson and the Spearman correlations respectively

We apologize for the previous misunderstanding in relation to the table, and the mix-up that happened with the requests of both reviewers. A single star has been added to the statistically significant correlations (using a significance level of α=0.05). As suggested, we have also added a second column to help clarify which values belong to which coefficient.

Discussion

We believe that we made the required changes.

Lines 197-200:

The citations were included to ease the interpretation of the indices, however, according to the suggestion, they were removed.

Lines 204-206:

Some of the above statements require literature citation.

We are sorry but we believe it is impossible to find citations for our own assumptions. Especially in the light that there is only one study on whole-domains antagonisms dealing with arable soils and not Antarctica.

Lines 206-209:

Grammar and/or style correction needed.

We made a correction.

Line 214: “the most primary parameter”

This sound kind of wired, linguistic correction is needed.

Changed to “informative”.

Line 216: “antagonism was observed”

It was changed to “patterns of antagonism”, however, we do not agree with you because of the arguments we stated above.

Lines 235-236:

“This can be explained by the fact that this index is based on the singletons and the doubletons, thus estimating the “missed” species”

Literature citation needed.

The citation was included.

Lines 236-237:

“Bacteria being largely the most species-abundant superkingdom”

Literature citation needed.

No, it is neither needed or possible because this sentence deals with our own observations.

Lines 237-238:

“On the other hand, discrepancies in 56% and 61% of the cases were observed respectively for the binomes Fungi-Archaea and Archaea-Bacteria.”

This statement refers for ACE index. Previous two sentences refer to Chao1, so without indication that these data refer to ACE readers would understand this also refers to Chao1, which is misleading.

We included clarification.

Line 257:

“biomass sludges”

Terminology correction needed.

We motivated ourselves above why we do not agree with this remark.

Lines 273-274:

“However, the archaeal OTUs are more than 6 times more numerous than the fungal ones.”

I would strongly suggest adding an explanation, that to a large extend such big difference is due to exceptionally low OUT for fungi. Without that such statement might be misleading.

If this is not a pattern of antagonism, what would be? We do not agree that the statement is misleading.

Lines 284-286:

“It has been observed that microorganisms are the first colonizers after the receding glaciers in the mountains [29]”

I would suggest checking literature on microbial succession or on variation in microbial communities on glacier forelands in Antarctica and Arctic, and citing them accordingly.

We cited some studies on primary microbial successions.

Lines 291-292:

“Our observation concurs with the general logic that pristine environments are firstly colonized by autotrophs, the heterotrophs coming somewhat later.”

Literature citation needed.

Done.

Line 293:

“such studies in Antarctica are rather scarce”

Still, should not be omitted be cited acordingly.

Lines 311-213:

Done.

“active, due to the synthesis of different types of antimicrobials, and passive, resulting from the concurrence for nutrients and energy between the different types of microorganisms.”

Literature citation needed, preferably with quotation of some examples.

It is our own hypothesis. Discussing mechanisms of active or passive antagonisms among microorganisms is out of the scope of the manuscript, especially in the case where we don’t have any information due to the lack of meta-metabolomic studies.

Line 319:

“biomass sludges”

Terminology correction needed.

We already addressed this.

Lines 320-223:

Literature citation needed, some of these “scarce publications” should be quoted.

How could one cite something which does not exist? All works which could be related to ours do not attempt to quantify antagonistic patterns.

Conclusions

I would strongly suggest indicating in the very first sentence what is the main outcome/result, and only after that providing interpretations. Otherwise this might be very misleading. For instance:

We are sincerely grateful for this suggestion, so, we included the sentence.

Literature

Format of the cited literature is not consistent. In particular, considering DOI number, while majority of cited works is listed with some do not provide DOI. I believe, this should be unified.

We are using a references manager, so, we don’t know why the DOI number is missing for some of the references. Our previous experience shows that this should not be a problem, the editorial manager correcting this before publications.

On behalf of the coauthors, I would like to thank you for this second round of review.

Best regards,

Assoc. Prof. Svetoslav Dimov, Ph.D.

Chairman of the Department of Genetics

Faculty of Biology, Sofia University "St. Kliment Ohridski"

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Authors of that work documented patterns of distribution, abundance and diversity of Archaea, Bacteria and Fungi in Antarctic environments. While the recorded patterns can be interpreted in terms of antagonistic interactions, or even may suggest such interaction, the authors in their statements have to make clear that their work does not provide evidence for occurrence of antagonistic interactions (or even any biotic interactions) but their work only provides grounds for hypothesis that the recorded patterns of distribution, abundance and diversity seems to driven by antagonistic interactions. As I have indicated previously, one of the major problems of this work was (and still is) that authors provided numerous statements as if they investigated and documented antagonistic interactions (or patterns of antagonistic interactions). Such statements are misleading and have to be corrected.

I do admit that authors have improved in that respect some parts of the manuscript significantly. In particular, as suggested, they added into Discussion and Conclusions some statements on mechanisms other than antagonisms that could affect abundance and diversity patterns that they recorded during their investigation. The problem is that the manuscript still contains a number of statements and terminology that can be confusing and misleading and should be corrected accordingly. For instance, the Abstract should also be corrected accordingly. I did not point to that previously, but this is kind of obvious that the Abstract has to comply with the rest of the work. It has to be remembered that Abstract present separate part of the work which is usually read first, unfortunately sometimes even without detailed examination of the other parts of the work; therefore, quality of its contents is particularly important. For some more detailed comments related to that issue see bellow.

Moreover, I would strongly suggest changing the title, so it would reflect real findings of that investigation. For instance:

Diversity and abundance patterns suggest antagonistic interactions among Fungi, Archaea, and Bacteria in Antarctic environments

Diversity and abundance patterns suggest importance of antagonistic interactions in driving composition of the Antarctic microbial assemblages

I would like to indicate here, that there is a long lasting debate in scientific literature concerning mechanisms driving composition and distribution of biotic communities existing in the Antarctic environments. For long it was assumed that, due to harsh environmental conditions, these communities are driven primarily by environmental factors and biotic interactions are not important factor. Only relatively recently a few studies provided evidence that this long lasting view is too simplified and biotic interactions do play significant role. This is why I was truly excited when I have noticed this manuscript. Unfortunately, here I relate to the second major issue with that manuscript, it seems that authors are completely unfamiliar with adequate literature and the manuscript lacks adequate discussion. As I have indicated previously, and I have to emphasize again, literature concerning other biota and other methods are often essential. Well, if authors are not willing to go beyond a narrow focused case study this might be acceptable. Still, lack of citation of virtually any literature (which was also pointed by the other reviewer) is a major issue of that manuscript that cannot be simply ignored. I would strongly advise that authors check literature on drivers of species distribution in Antarctica, and particularly on biotic interactions. Searching literature databases for “biological interactions”, “biotic factors” and “Antarctic” would surely list some essential literature.

Moreover, poor quality of English style and grammar is still an issue. Well, the text is understandable but far from being written in correct English. However, as I have indicated previously, this is not a role of a reviewer to provide detailed suggestions or correction on that. Anyway, I believe that assistance from the journal can help with that issue.

Finally, I would like to indicate, that I do find presented results truly interested, particularly in the context of our current knowledge on factors driving patterns of species distribution in the Antarctic environments. I would therefore truly like to congratulate the authors on good idea and well done research. However, obtaining interested data is only half of the way. Preparing a good scientific article is also challenging and often even more difficult. I am truly sorry to say, but authors still have some essential work to do regarding that. Because of the two major issues that I have pointed above, I cannot recommend that manuscript for publication. I still believe that the data and the hypothesis on antagonistic interactions formulated by authors is important and very interesting, particularly for polar scientists working in Antarctica. I therefore, truly look forward to see that work published, but as I have indicated above I cannot recommend its publication in its current form.

Some more detailed comments:

Abstract

Lines 13-14: “patterns of antagonism between these three super kingdoms were observed”

This statement is not true and might be misleading. Authors did not observed “antagonisms” or any “pattern of antagonism” but they recorded “patterns of distribution, abundance and diversity”. So this has to be change accordingly.

Lines 19-20: “The antagonism between Archaea and Fungi was strongest”

Again, the authors did not measure antagonism or its strength.

Line 21: “antagonism was slightly detectable”

See above.

I believe the whole Abstract have to be carefully rewritten, with information on obtained results given first (i.e., patterns of distribution, abundance and diversity), only after that conclusions and interpretation of results can be given.

Line 61: “was focused” –> “focused” would probably sound better.

Line 63: “inversely proportional dependence”

The word “dependence” implies that one variable depends (is determined) by another. There is no evidence for that in that case. These variables just correlate, which is not the same as being dependent.

Lines 64-65: “… and to a lesser extent between bacteria and archaea, and fungi and bacteria”.

I would suggest adding at the end of that sentence the following or similar statement: “suggesting occurrence of antagonistic interactions”

Line 67: “we decided to investigate these domains’ patterns of antagonism”

The authors have rebutted <… we changed “antagonisms” to “patterns of antagonisms”, even though we didn’t agree completely with this because our experimental data suggested otherwise.>

Firstly, I do not understand why authors have changed “antagonism” into “patterns of antagonisms”. As I have indicated, when referring to their own data they should refer to “patterns of distribution” or “patterns of abundance and diversity” and only when providing interpretation can refer to “antagonism”. By the way, “patterns of antagonisms” or “antagonistic patterns” (see Line 72) sounds wired.

Line 82-83: “solid samples”

Sounds strange, I would suggest replace that with “soil and microbial mat samples”, here and whenever “solid samples” is used.

Line 67: “sludge biomass”

As I have indicated previously “With the exception of the sea ice sludge, this terminology is generally associated with industrial contamination, and I truly doubt if it usage is appropriate in that case.”

The authors have rebutted < The notion “sludge” is widely accepted for describing microbial biomass in aquatic environments, for example in sewage systems > which only confirms what I have indicated that this is terminology related to the “industrial contamination”. From authors rebuttal I see that they relate to microbial biomass. As I have indicated previously in the Antarctic literature “microbial mats” is widely used terminology. As authors have adopted that for other samples I do not really understand why they cannot adopt that also in that case. By the way, “microbial mats” often contain incorporated soil/sediment particles.

Figure 1.

The location of S18 sample is missing. I am aware that this sample was collected at some larger distance from the other samples. Still it should not be omitted from the map and at least an arrow pointing to that sampling site with information on the distance should be given.

By the way, I would advise to check in elementary school books what makes a map.

I am sorry for sarcasm, but I would not accept such “map” from my students for a seminar presentation. Having kids I am also vary familiar what knowledge and skills are required at elementary school level and I believe that scientific literature should not fall below that even in such small and seemingly insignificant details as quality of a map showing the study area. Anyway, the “map” presented by authors lack essential elements (such as a scale bar, orientation) to be even called a map, and because of that is not really informative. Moreover, it would be nice if a reader could place the study site in some broader geographical settings. Therefore, adding an insert (as it is usually done in scientific literature) showing position of the area within the island or even with the Antarctic would be desirable. Maps of Livingston Island were already presented in a number of different publications, so this should not be really difficult to adopt one from some previous publication.

Line 171: “lesser then” –> “lower/smaller than”

Line 183: “lesser value” –> “lower/smaller value”

Line 196: “we hypothesized that they could witness patterns of antagonistic relationships” –>

One of the following reads better:

“we hypothesized that they reflect antagonistic relationships” or

“we hypothesized that they are due to antagonistic relationships”

By the way, this sentence, added following my previous suggestions, clearly reflects the true results of that work, which documented nothing more but patterns in abundance and diversity based on which authors hypothesized on possible importance of “antagonistic relationships”. That is the “antagonistic relationships” is only a hypothesis resulted from the recorded patterns of abundance and diversity.

Line 227: “These patterns of antagonism” –> “Such discrepancy”

Lines 248-249: “Bacteria being largely the most species-abundant superkingdom according to our own observations.”

Does this statement refer to the authors own data presented in this work? If so this should be made clear. However, if this refers to generally known fact literature sources has to be given, because the authors are not the first to report that. Without literature citation this can be considered as plagiarism.

Line 270: “are from a type of mat” –> “represent microbial mats”

Lines 284-285: “are more than 6 times more numerous

Lines 284-285: “the archaeal OTUs are more than 6 times more numerous than the fungal ones”

The following reads better:

“the archaeal OTUs are over 6 times more numerous than the fungal ones, though this is mostly due to exceptionally low number of fungal OTUs.”

The other thing is that such high proportion of archaea compare to fungi is due only to the exceptionally low number of fungal OTUs. This information is important for interpretation of data and should be added as I have indicated previously. Hiding that information might be misleading, and, therefore, I do not really understand why despite my previous suggestion the authors are refusing to indicate that potentially very important fact.

Lines 329-331: “… active, due to the synthesis of different types of antimicrobials, and passive, resulting from the concurrence for nutrients and energy between the different types of microorganisms.”

This statement does not belong to original finding of that work, but these mechanisms were described in previous literature. Therefore, citation of an appropriate literature is obligatory, otherwise such statement is nothing but plagiarism.

Lines 334-335: “… the polar bases are seasonal meaning that they are accessible only during the Antarctic summer”

This statement is not true and has to be modified. Firstly, many baser are year round. Secondly, if a base is seasonal it does not mean it is “accessible only during the Antarctic summer”, it only means it is not used permanently but in general only seasonally.

Finally, I would like to comment on one of the authors’ rebuttal.

The authors have rebutted writing: “We do sincerely believe that after agreeing with about 88% of your remarks the quality of the manuscript increased ... You had in total 33 remarks and suggestions to the manuscript, and we agreed with you in 29 …”

There is a saying that there is „truth, lie and statistics”. This reflect situations when statistics is used not demonstrate the truth but for manipulation. Majority of my previous comments concerned editorial matters and/or linguistic corrections, and were therefore, completely unimportant for the work merit. Using their number to support statement as if virtually all comments were implemented, when in fact the major concerns were not addressed, is nothing more but the use of “statistic” in a way reflected in the saying that I quoted.

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Comments on the Quality of English Language

As I have indicated, poor quality of English style and grammar is still an issue. Well, the text is understandable but far from being written in correct English. However, as I have indicated previously, this is not a role of a reviewer to provide detailed suggestions or correction on that. Anyway, I believe that assistance from the journal can help with that issue.

Author Response

please see the latest version of our manuscript

Author Response File: Author Response.pdf

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Observations on the Antagonistic Relationships between Fungi, Archaea, and Bacteria in Livingston Island (Maritime Antarctica) with the Use of Amplicon-Based Metagenomics

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