Microbial Communities in Permafrost, Moraine and Deschampsia antarctica Rhizosphere Soils near Ecology Glacier (King George Island, Maritime Antarctic)
, Alexis Gaete 1, Dariel López 2, Andrés E. Marcoleta 3, Francisco P. Chávez 4, León A. Bravo 2,5, Jacquelinne J. Acuña 5,6,7, Verónica Cambiazo 1,6,* and Milko A. Jorquera 5,7,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsResearch is undoubtedly relevant. Changes in polar ecosystems under conditions of global warming undoubtedly need to be recorded using advanced methods.
However, there are a number of questions and comments about the study
The information from the abstract and introduction orients the reader to the fact that all components of the microbial community are analyzed in the work. Meanwhile, the study itself is devoted to the analysis of three groups: Bacteria, Archaea, Fungi. It seems to me that the authors need to specify in the Abstract and Introduction which components of the microbial community are emphasized. It is also desirable to make a small explanation why Fungi were analyzed out of the whole variety of eukaryotic microorganisms, since the primers used make it possible to evaluate the general structure of a number of other eukaryotic organisms (for example, these primers are quite suitable for many eukaryotic algae, and a number of infusoria).
The section 2.4. 'Amplicon sequencing and data analysis' ('Materials and methods') , I recommend specifying not only the primers, but also the amplification conditions. It is also necessary to visit whether the protocols were standard, or the authors optimized them
Figure 3. The captions to the figures are very small and hard to read. Could the authors distinguish the taxa of the Uniculture d group from the Other Group so that readers can navigate the degree of knowledge of these ecosystems? Would you also like to see a small discussion of the problem of uncultivated diversity for polar ecosystems?
Have similar studies been conducted for other polar ecosystems? For example, The Ecosystems Of The Arctic. If such studies have been conducted, then we would like to see a comparison of the results obtained.
Author Response
Reviewer #1
Research is undoubtedly relevant. Changes in polar ecosystems under conditions of global warming undoubtedly need to be recorded using advanced methods.
However, there are a number of questions and comments about the study
Response: We thank to Reviewer #1 for the revision and this positive comment given. All remarks and suggestions listed by the Reviewer #1 were considered, and the manuscript was accordingly improved. Our responses are listed point-by-point below.
The information from the abstract and introduction orients the reader to the fact that all components of the microbial community are analyzed in the work. Meanwhile, the study itself is devoted to the analysis of three groups: Bacteria, Archaea, Fungi. It seems to me that the authors need to specify in the Abstract and Introduction which components of the microbial community are emphasized.
Response: Based on the recommendation, the Abstract and Introduction was accordingly modified, specificizing the microbial groups analyzed in the literature and in our study (Page 1, lines 27-28, Page 2, line 51, Page 2, line 65, and Page 2, line 81-82).
It is also desirable to make a small explanation why Fungi were analyzed out of the whole variety of eukaryotic microorganisms, since the primers used make it possible to evaluate the general structure of a number of other eukaryotic organisms (for example, these primers are quite suitable for many eukaryotic algae, and a number of infusoria).
Response: We understand the Reviewer’s concern. In this study, we used 16S/18S/ITS Amplicon Metagenomic Sequencing service by Novogen Co. (https://www.novogene.com/us-en/services/research-services/metagenome-sequencing/16s-18s-its-amplicon-metagenomic-sequencing/) with the primer set (ITS3-2024F and ITS4-2409R). According to the information on the Novogen Co. website the primer set is specific to cover Fungi. Primer sets to cover other specific Eukaryote groups (e.g., metazoa, protists, endophytic fungi and arbuscular mycorrhizal fungi) are also available in the Novogen Co. website but not used in our study.
The section 2.4. 'Amplicon sequencing and data analysis' ('Materials and methods'), I recommend specifying not only the primers, but also the amplification conditions. It is also necessary to visit whether the protocols were standard, or the authors optimized them
Response: Amplicon sequencing was performed based on standardized protocol by Novogen Co. We only provided appropriated DNA extracts to the Amplicon Sequencing Service. However, more detailed information was added in the text (Page 4, lines 148-153).
Figure 3. The captions to the figures are very small and hard to read. Could the authors distinguish the taxa of the Uncultivated group from the Other Group so that readers can navigate the degree of knowledge of these ecosystems?
Response: The size of most of Figures was increased to a better reading and visualization of their information. We hope that these changes satisfy the Reviewer’s comment. In relation to the “unculturable” bacterial groups, the taxa with minor relative abundances were grouped and categorized as “Other”, independently of their culturability; therefore, differentiation and analysis between members in culturable and unculturable bacterial taxa were not in our study. However, we understand the relevance of culturability in Antarctic microbiology (and other extreme environments), and we hope include this parameter in further studies.
Would you also like to see a small discussion of the problem of uncultivated diversity for polar ecosystems?
Response: A brief discussion about cultivability and in-situ cultivation was added in text as requested (Page 14, lines 412-418).
Have similar studies been conducted for other polar ecosystems? For example, The Ecosystems Of The Arctic. If such studies have been conducted, then we would like to see a comparison of the results obtained.
Response: The discussion was completely revised and improved, where this commentary was also considered (Page 13, lines 353-359; Page 14, lines 428-434). We hope these changes solve the Reviewer’s comment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsJournal: Diversity
The paper submitted for review entitled "Microbial community in permafrost, moraine and Deschampsia antartica rhizosphere soils near Ecology Glacier (King George Island, Maritime Antarctic)" written by Daniel E. Palma and co-authors fits well into the contemporary problems of polar ecomicrobiology. The paper concerns the structure of the microbiome of polar soils and expands knowledge on the properties of the soil microbiome in an area subjected to rapid deglaciation, contributing to a better understanding of the dynamics of soil microbiocenoses occurring under the influence of climate change.
The abstract is synthetic, written exhaustively and contains all the information relevant to the content of the paper from the reader's point of view. The introduction is written clearly and refers well to the scope of the research. The aim of the research is clearly described, although the research hypothesis is not formulated.
The Materials and Methods chapter does not clearly and unambiguously formulate the sampling policy in the ASPA area. I also did not find such information in the provided link on the INACH website. This should be explained in more detail.
The sampling method is correct, although it is not clear how many soil samples were taken for testing. The text and Figure 1 suggest that samples P and M were taken pointwise, each in one place? Please explain.
I have no objections to subsections 2.3 and 2.4, the procedures are described clearly and legibly enough.
The results obtained are described clearly and presented in "self-reading" graphs, but I have a few reservations about their interpretation:
Subsection 2.2 states: "Total carbon (TC) and total nitrogen (TN) were determined on an automated elemental analyzer EA 3000..." however, the results of carbon determinations are not included in Table 2.
It seems that the poor results of DNA isolation from soil material at site P are a result of the sampling method. Pooled samples from a square area of 1 meter side provide much better and more reliable results.
Figure 2 shows the numbering of samples P1, P4, P5 and P6 and M1, M2, M4, M5 and M6 – this numbering is not referenced in the Materials and Methods section.
How can the increase in the share of Actinobacteria in the soil microbiome from sample P to R be explained?
In my opinion, it is not necessary to include the sentence: “…soil samples collected …… during the 58th Scientific Antarctic 250 Expedition (2021–2022) organized by the Chilean Antarctic Institute (INACH)” under each figure, because this was included in the Materials and Methods section.
The high frequency of Chitinophagaceae in the analyzed soil samples in this particular area is definitely related to the penguins’ breeding grounds and the deposition of chitin into the soil from their main food source, which is krill.
The discussion is written exhaustively and substantively, referring to the results of other authors' work, although I missed the reference to the differences in the physicochemical parameters of the analyzed soils, especially in relation to phosphorus. How can we explain the similar content of phosphorus in the subglacial soil and on the moraine (samples P and M)?
The cited references fit well into the research problem presented in the publication, are adequate and justified.
Author Response
Reviewer #2
The paper submitted for review entitled "Microbial community in permafrost, moraine and Deschampsia antartica rhizosphere soils near Ecology Glacier (King George Island, Maritime Antarctic)" written by Daniel E. Palma and co-authors fits well into the contemporary problems of polar ecomicrobiology. The paper concerns the structure of the microbiome of polar soils and expands knowledge on the properties of the soil microbiome in an area subjected to rapid deglaciation, contributing to a better understanding of the dynamics of soil microbiocenoses occurring under the influence of climate change.
The abstract is synthetic, written exhaustively and contains all the information relevant to the content of the paper from the reader's point of view. The introduction is written clearly and refers well to the scope of the research. The aim of the research is clearly described, although the research hypothesis is not formulated.
Response: We thank to Reviewer #2 for the revision and this positive comment given. All remarks and suggestions listed by the Reviewer #2 were considered, and the manuscript was accordingly improved. Our responses are listed point-by-point below. As requested, the hypothesis of this study was also added in the Introduction (Page 2, lines 78-81).
The Materials and Methods chapter does not clearly and unambiguously formulate the sampling policy in the ASPA area. I also did not find such information in the provided link on the INACH website. This should be explained in more detail.
Response: For sampling, a certificate of permission is given to the researcher by the Chile’s Antarctic Institute (INACH). This certificate must be kept and presented to the Head of scientific Antarctic Station by the researchers during sampling on Antarctic Specially Protected Areas (ASPA). The certificates are privately managed, and they are not posted and opened in INACH website. However, additional information about ASPA permission and sampling protocol in website of INACH and Secretariat of the Antarctic Treaty was added in the text (Page 2, lines 90-94; Page 14, line 470).
The sampling method is correct, although it is not clear how many soil samples were taken for testing. The text and Figure 1 suggest that samples P and M were taken pointwise, each in one place? Please explain.
Response: Thank you for this comment. The sampling explanation was revised and improved in the text (Page 3, lines 96-97).
I have no objections to subsections 2.3 and 2.4, the procedures are described clearly and legibly enough.
Response: We thank to Reviewer #2 for the revision and this positive comment given.
The results obtained are described clearly and presented in "self-reading" graphs, but I have a few reservations about their interpretation:
Subsection 2.2 states: "Total carbon (TC) and total nitrogen (TN) were determined on an automated elemental analyzer EA 3000..." however, the results of carbon determinations are not included in Table 2.
Response: Sorry for our mistake. The Table 2 and their title was corrected (Page 6, lines 196-197).
It seems that the poor results of DNA isolation from soil material at site P are a result of the sampling method. Pooled samples from a square area of 1 meter side provide much better and more reliable results.
Response: We appreciate this information, which will be considered in further studies using samples from Antarctic poor-DNA soil samples.
Figure 2 shows the numbering of samples P1, P4, P5 and P6 and M1, M2, M4, M5 and M6 – this numbering is not referenced in the Materials and Methods section.
Response: The information was added in the text of Methods section as requested (Page 3, line 96; Page 3, lines 100-101)
How can the increase in the share of Actinobacteria in the soil microbiome from sample P to R be explained?
Response: This statement was added and discussed (Page 12, lines 328-331).
In my opinion, it is not necessary to include the sentence: “…soil samples collected …… during the 58th Scientific Antarctic 250 Expedition (2021–2022) organized by the Chilean Antarctic Institute (INACH)” under each figure, because this was included in the Materials and Methods section.
Response: As recommended the sentence was deleted in all figure’s captions.
The high frequency of Chitinophagaceae in the analyzed soil samples in this particular area is definitely related to the penguins’ breeding grounds and the deposition of chitin into the soil from their main food source, which is krill.
Response: We appreciate this comment, the paragraph was improved (Pages 12-13, lines 339-346).
The discussion is written exhaustively and substantively, referring to the results of other authors' work, although I missed the reference to the differences in the physicochemical parameters of the analyzed soils, especially in relation to phosphorus. How can we explain the similar content of phosphorus in the subglacial soil and on the moraine (samples P and M)?
Response: The discussion was completely revised and improved, where this commentary was also considered (Page 12, lines 300-307).
The cited references fit well into the research problem presented in the publication, are adequate and justified.
Response: We thank to Reviewer #2 for the revision and this positive comment given.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsPalma et al. examined the bacterial, archaeal, and fungal diversity and community structure in three different succession stages in the glacier forefields near Ecology Glacier, Antarctic. The work is descriptive, and improvements are needed for the acceptance for publication. The main issue was the discussion, which merely compared their results with other studies, without discussing the mechanisms and implications of the findings. Thus, further in-depth discussion would be required.
Specific comments:
Introduction
Line 47, why coevolve?
Line 54, what is plant‒soil‒microbe continuum?
Line 55, rephrase sentence.
Line 66 what is nutrient speciation?
Methods
Line 97, specifies the storage temperature
Line 114, spell FAAS in full
Figure 1. The information in the figure is unreadable due to font size
Table 1 archaea primer, the amplicon length is 400-500. The sequencing platform the author used (NovaSeq 6000), typically supports a 250 bp x 2 reaction, thus the maximum amplicon length it can handle is approximately 480 bp. Thus, please check the amplicon length distribution and whether sequences were discarded due to failed merging. This may explain the low reads obtained for the archaea.
Results
Line 175 and other places, please specify the exact p-value, instead of using < 0.05
Table 2, 7.6 ± 0.3* a**, is the first ‘*’ redundant?
Line 195, is it among the samples or among succession stages?
Line 236, change to Fig. 4B
Line 256, “with narrower ranges for the M samples”, please rephrase
Line 290-292, please add references
Line 304, please check whether the study in reference 51 was based on OTU or ASV.
Line 378-380, please add more discussion on why temporal changes happened.
Comments on the Quality of English LanguageThe manuscript is generally well-written, but improvement on writing clarity are required.
Author Response
Reviewer #3
Palma et al. examined the bacterial, archaeal, and fungal diversity and community structure in three different succession stages in the glacier forefields near Ecology Glacier, Antarctic. The work is descriptive, and improvements are needed for the acceptance for publication. The main issue was the discussion, which merely compared their results with other studies, without discussing the mechanisms and implications of the findings. Thus, further in-depth discussion would be required.
Response: The discussion section was completely revised and accordingly improved based on this and other reviewer’s comments. We hope that changes done solve the reviewer’s comment.
Specific comments:
Introduction
Line 47, why coevolve?
Response: The sentence was changed and “coevolve” was deleted from the sentence (Page 2, line 48).
Line 54, what is plant‒soil‒microbe continuum?
Response: The sentence was revised and changed (Page 2, line 54-55).
Line 55, rephrase sentence.
Response: The sentence was revised and rephrased (Page 2, lines 56-57)
Line 66 what is nutrient speciation?
Response: The sentence was revised and changed (Page 2, lines 65-67)
Methods
Line 97, specifies the storage temperature
Response: The sentence was revised and accordingly improved (Page 3, line 104-105).
Line 114, spell FAAS in full
Response: FAAS was spelled out in the text as requested (Page 3, lines 122).
Figure 1. The information in the figure is unreadable due to font size
Response: The size of all Figures was increased to a better reading and visualization of their information. We hope that these changes satisfy the Reviewer’s comment.
Table 1 archaea primer, the amplicon length is 400-500. The sequencing platform the author used (NovaSeq 6000), typically supports a 250 bp x 2 reaction, thus the maximum amplicon length it can handle is approximately 480 bp. Thus, please check the amplicon length distribution and whether sequences were discarded due to failed merging. This may explain the low reads obtained for the archaea.
Response: We understand the revoewer’s concern. The amplicon length is according to the information supplied by Novogen Co. website (https://www.novogene.com/us-en/services/research-services/metagenome-sequencing/16s-18s-its-amplicon-metagenomic-sequencing/) base on its standardized protocol and the alignment position of primers on archaeal 16S rRNA gene (Arch519F and Arch915R)
Results
Line 175 and other places, please specify the exact p-value, instead of using < 0.05
Response: p-value was added in the text (Page 5, line 174).
Table 2, 7.6 ± 0.3* a**, is the first ‘*’ redundant?
Response: The Table 2 was accordingly corrected.
Line 195, is it among the samples or among succession stages?
Response: The sentence was revised and changed (Page 6, line 204).
Line 236, change to Fig. 4B
Response: Revised and homogenous in whole manuscript.
Line 256, “with narrower ranges for the M samples”, please rephrase
Response: The sentence was revised and rephrased (Page 10, lines 257-261).
Line 290-292, please add references
Response: Reference was added (Page 12, line 292)
Line 304, please check whether the study in reference 51 was based on OTU or ASV.
Response: The Reference was revised and requested information was added in text (Page 12, lines 310).
Line 378-380, please add more discussion on why temporal changes happened.
Response: The discussion was completely revised and improved, where this commentary was also considered (Page 14, 403-407). We hope these changes solve the Reviewer’s comment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors made the major part of necessary changes and responded to the comments.
Minor remarks:
1. The letters a and b are present in table 2, but the note does not indicate what they mean. Please add.
2. The symbols in Figure 3 (A) cannot be read in the pdf.
3. The symbols in Figure 5 (A) cannot be read in the pdf.
Author Response
Manuscript ID: diversity-3384311
Title: Microbial community in permafrost, moraine and Deschampsia antarctica rhizosphere soils near Ecology Glacier (King George Island, Maritime Antarctic)
Response: We would like to thank the Editor and three anonymous reviewers for their helpful and constructive criticism that have significantly contributed to improve the quality of our manuscript. We considered all remarks and suggestions listed by the reviewers, and the manuscript was newly revised and corrected according to the recommendations. Our responses appear point by point below.
Reviewer #1
Research is undoubtedly relevant. Changes in polar ecosystems under conditions of global warming undoubtedly The authors made the major part of necessary changes and responded to the comments.
Response: We thank to Reviewer #1 for the revision and this positive comment given. All remarks and suggestions listed by the Reviewer #1 were considered, and the manuscript was accordingly improved. Our responses are listed point-by-point below.
Minor remarks:
- The letters a and b are present in table 2, but the note does not indicate what they mean. Please add.
Response: Sorry for our mistake. The Table 2 was emended.
- The symbols in Figure 3 (A) cannot be read in the pdf.
Response: The Figures were modified and replaced. I hope these changes satisfy the reviewer’s comment.
- The symbols in Figure 5 (A) cannot be read in the pdf.
Response: The Figures were modified and replaced. I hope these changes satisfy the reviewer’s comment.
Reviewer #3
All my concerns have been properly addressed
Response: We thank to Reviewer #3 for the revision and this positive comment given.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsAll my concerns have been properly addressed.
Author Response
Manuscript ID: diversity-3384311
Title: Microbial community in permafrost, moraine and Deschampsia antarctica rhizosphere soils near Ecology Glacier (King George Island, Maritime Antarctic)
Response: We would like to thank the Editor and three anonymous reviewers for their helpful and constructive criticism that have significantly contributed to improve the quality of our manuscript. We considered all remarks and suggestions listed by the reviewers, and the manuscript was newly revised and corrected according to the recommendations. Our responses appear point by point below.
Reviewer #1
Research is undoubtedly relevant. Changes in polar ecosystems under conditions of global warming undoubtedly The authors made the major part of necessary changes and responded to the comments.
Response: We thank to Reviewer #1 for the revision and this positive comment given. All remarks and suggestions listed by the Reviewer #1 were considered, and the manuscript was accordingly improved. Our responses are listed point-by-point below.
Minor remarks:
- The letters a and b are present in table 2, but the note does not indicate what they mean. Please add.
Response: Sorry for our mistake. The Table 2 was emended.
- The symbols in Figure 3 (A) cannot be read in the pdf.
Response: The Figures were modified and replaced. I hope these changes satisfy the reviewer’s comment.
- The symbols in Figure 5 (A) cannot be read in the pdf.
Response: The Figures were modified and replaced. I hope these changes satisfy the reviewer’s comment.
Reviewer #3
All my concerns have been properly addressed
Response: We thank to Reviewer #3 for the revision and this positive comment given.
Author Response File: Author Response.pdf
