Structural Shifts in the Soil Prokaryotic Communities Marking the Podzol-Forming Process on Sand Dumps

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Soil Systems 2647478

 

Structural shifts in the soil prokaryotic communities marking the podzol-forming process on sand dumps.

 

Evdokimova et al. present and analysis of the microbiota of podzols from a former sand-mining location that has undergone spontaneous revegetation with pine forest in the past 70 to 100 years. Unlike many studies, this study analysed the recovered DNA sequences in the context of soil horizons, rather than simply depth or other measurements that do not correspond to patterns and processes of soil genesis.

The study site is fascinating and I agree with the author’s conclusions that suggest future studies should include greater detail in the chronosequence, with additional sites/ages as well as investigation of factors such as tree density and plant species diversity.

I found no serious problems with this manuscript and I believe it is quite suitable for publication.

Author Response

Reviewer 1

 

Evdokimova et al. present and analysis of the microbiota of podzols from a former sand-mining location that has undergone spontaneous revegetation with pine forest in the past 70 to 100 years. Unlike many studies, this study analysed the recovered DNA sequences in the context of soil horizons, rather than simply depth or other measurements that do not correspond to patterns and processes of soil genesis.

The study site is fascinating and I agree with the author’s conclusions that suggest future studies should include greater detail in the chronosequence, with additional sites/ages as well as investigation of factors such as tree density and plant species diversity.

I found no serious problems with this manuscript and I believe it is quite suitable for publication.

Dear reviewer! Thank you for your comments and evaluation of our manuscript.

Evgeny Abakumov, corresponding author

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The article is interesting, relevant, with an analysis of extensive literature. I have the following questions and comments:

1. There is very little information about vegetation (it is advisable to cover at least the dominant plants of ground cover).

2. At least general descriptive information about soil fauna is desirable. Large soil invertebrates, on which soil formation largely depends, may have been observed. Perhaps this would help explain the rate of soil recovery.

3. The abstract states that during the work it was possible to identify microbial indicators and driving forces of primary pedogenesis. But there is no information about this in the conclusion. I would like to see this in Conclusions.

4. The work actually deals with primary succession - soil formation in a sand quarry. Indeed, for such conditions, almost complete restoration of the soil in less than 100 years is very fast. This phenomenon needs a more detailed explanation. Perhaps the reason is unaccounted for biotic factors.

Author Response

Reviewer 2

 

The article is interesting, relevant, with an analysis of extensive literature. I have the following questions and comments:

1. There is very little information about vegetation (it is advisable to cover at least the dominant plants of ground cover).

Thank you, information about vegetation has been added.

2. At least general descriptive information about soil fauna is desirable. Large soil invertebrates, on which soil formation largely depends, may have been observed. Perhaps this would help explain the rate of soil recovery.

Thank you for your clarification. Representatives of soil fauna - macro-, meso- and microfauna, mainly various invertebrates - are undoubtedly important for understanding the whole reperesentation of soil ecosystem restoration as a whole, as they take an active part in transformation of soil structure and thus perform a habitat-forming function for soil microorganisms in particular. Thus, it is known that predatory consumers (nematodes, acarids) appear when there is enough prey in the food web, i.e. at later stages of succession. Similarly, collembolas and root-feeding nematodes appear later when there is sufficient root biomass. However, our study is focused specifically on the search for indicators of soil regeneration within the framework of prokaryotic community analysis, and the main objective is to find groups of prokaryotes marking the stages of soil regeneration. Perhaps, in the future, the data on the study of the chronology of restoration of spoil pits should be supplemented by the study of soil biota at all levels, including representatives of soil fauna as well, with the construction and analysis of morphology and structure of food-webs formed in the process of soil formation of disturbed lands, but this is already the task of the next generation of research. 

3. The abstract states that during the work it was possible to identify microbial indicators and driving forces of primary pedogenesis. But there is no information about this in the conclusion. I would like to see this in Conclusions.

Conclusion has been amended by additional information

4. The work actually deals with primary succession - soil formation in a sand quarry. Indeed, for such conditions, almost complete restoration of the soil in less than 100 years is very fast. This phenomenon needs a more detailed explanation. Perhaps the reason is unaccounted for biotic factors.

Following information has been added about the rate of soil formation:

Podzol formation is one of the fastest processes of soil formation, especially on mineralogically poor sands of fluviglacial origin. Differentiation of soil profile by Al-Fe-humus type occurs rapidly and preclimax soil can be formed in the first hundred years after the beginning of soil formation. [3, 4].

 

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

an interesting manuscript. I've provided edits and suggestions in the PDF file.

The focus is on recovery from anthropogenic disturbances, not natural disturbances. Is there a different recovery path/trajectory because of this?

have the authors considered just stating the five soil-forming factors at the beginning of the manuscript?

Does plant litter include increased root biomass? With an increase in plant density and biomass, is there an increase in rhizosphere habitat complexity and sloughing of root cells during growth which will provide more and different food resources for the increasing diversity of the microbial community?

Comments for author File: Comments.pdf

Comments on the Quality of English Language

some editing is required

Author Response

Reviewer 3

 

an interesting manuscript. I've provided edits and suggestions in the PDF file.

The focus is on recovery from anthropogenic disturbances, not natural disturbances. Is there a different recovery path/trajectory because of this?

Anthropogenic disturbance in this case leads to complete disturbance of the land cover and soil formation starts from the so-called "zero stage" [1]. In the case of natural disturbances, for example, windthrow successions, the soil layer is not completely destroyed and this model of chronoseries is much less inofrmative than the first one.

This paragraph has been added to introduction.

 

Figure 1. need a little more detail in the caption.

The figure caption was detalized.

have the authors considered just stating the five soil-forming factors at the beginning of the manuscript?

Thank you for this comment; I have added information about 4 factors of soil formation within the time

We have also followed all the comment in pdf file and improved the manuscript

 

Does plant litter include increased root biomass? With an increase in plant density and biomass, is there an increase in rhizosphere habitat complexity and sloughing of root cells during growth which will provide more and different food resources for the increasing diversity of the microbial community?

 

Thank you. Changes in edaphic factors determine the composition of microbiota during pedogenesis and primarily contribute to the availability of microorganisms for recruitment by plant roots in the rhizosphere. In turn, recruitment of rhizosphere microbiota by developing plant community is determined by changing environmental conditions at different stages of soil development during the soil formation chronosequences. As soils develop, accumulation of organic matter, including readily available organic matter of the litter and root mass the microbial diversity becomes higher in the rhizosphere than in the bulk soils at each developmental stage. Our studies demonstrate the stratification of the prokaryotic community along soil horizons, including the groups of microorganisms which occurrence is apparently associated with rhizosphere loci. However, since a separate consideration of the rhizosphere and the bulk soil was beyond the scope of our study, we did not study in detail the features of the formation of rhizosphere communities of prokaryotes during the ecogenesis of soils in the investigated sandy quarry - we thank you for suggesting the further direction of our research in this area.

Thank you for your comments,

E. Abakumov, corresponding author

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I carefully read the manuscript of Evdokimova and co-authors “Structural shifts in the soil microbiome marking the podzol-forming process on sand dumps” submitted to Soil System. The paper describes the pedogenesis process from a sand quarry and the changes in the microbial community in the course of soil profile evolution. Molecular techniques such as quantitative real time PCR and NGS were applied to analyses the soil microbiota. In general, the paper was well written providing a general view of microbiota diversity and which microbial groups can potentially be involved in soil profile formation.  

I noticed several issues that need to be corrected to improve the editing and readability of the paper: 1) the statistical analysis is insufficient and an analysis of the variance (ANOVA) must be conducted on all the data characterizing the soil, both chemical and microbiological; 2) the experimental design is not fully clear: why were different depths used in the M1 stage if it is a single profile? 3) there is much confusion in the numbering of the figures and in the combination of the caption; 4) some references are put in full while others as number; 5) the figure of the PCA can be very informative but needs to be further explained: what are the gray dots? why do some pedogenesis stages have three points for depth while others have five? 6) there is also some taxonomic confusion between family and order. Moreover, Italic is generally used for taxa at the level of family and below; 7) clarify what you mean regard pre-climax and climax by thinking of an audience not familiar with this topic; 8) the discussion needs to be improved. Sometime, there is not a clear link between the data obtained and those obtained from other authors.

Other specific comments:

Keywords: move in alphabetic order

Line 60: add reference to Dobzhnsky’s word

Line 124: here and throughout the text, use abbreviation for carbon (C) and nitrogen (N)

Line 169: Pinus Sylvestri in italic

Line 184: it is not clear, use M1, M2……

Line 186: explain which horizon you sampled within each site

Line 193: add reference

Lines 195-199: add reference

Line 201: are soil sample for DNA extraction collected in the same way as samples for chemical analysis?

Line 206: How did you evaluate the purity of DNA by electrophoresis? and the quantity? You determined the DNA concentration by SPECTROStar Nano.

Line 208: Compared to what? Soil dry weight/fresh weight? Do you think it makes sense to report both yield and final concentration?

Line 210: add the primers used with relative references

Line 246: why did you use t-test and you didn’t use ANOVA? I think that ANOVA is more correct to compare more than two samples. Moreover, where are the results of significance?

Line 248: and archaeal and fungal dissimilarity?

Results: you must better clarify what changes according to the development of the soil and what according to the depth

Line 284: where is the method used for exchange capacity explained? And where are the results obtained?

Line 285: from M1 to M5? Try to be clearer

Line 294: generally, the percentage of two particle size were reported, clay and silt or clay and sand, thus the third particle will be easily determined

Table 1: explain why for M1 stage you analysed soil at four different depth. Were the samples collected at: 0-5 cm; 5-10 cm; 10-20 cm, and 20-30 depth? How did you choose these depth? Use a depth range for each profile. There isn’t statistical analysis.

Line 301: the alpha diversity was linked to Illumina results, thus move after the real time results

Table 2: distance better each stage of soil development, otherwise it is no clear. There isn’t statistical analysis.

Figure 3: are you sure this is the correct figure?

Line 333: where is it written that you did

PCA Figure: I’d put the data labels from the most superficial to the deepest horizon, in the same order as the caption

Line 353: where is the list? Add it in supplementary materials

Line 369-371: are these OTU referred to a specific phylum? if yes, you must specify.

Line 409: where is the list? Add it in supplementary materials

Ligure CCA: the vision of this figure is not immediate; the horizons could be placed as symbols with different colors based on the two different stages of development

Line 503: where does it come from? The purpose of the Discussion to give evidence that supports the obtained data.

 

 

 

Author Response

Rev 1

Dear editot!

thank you for your comment and suggestions, all changes are trached in yellow color in updated version of the manuscript.

• the statistical analysis is insufficient and an analysis of the variance (ANOVA) must be conducted on all the data characterizing the soil, both chemical and microbiological; 

Done

2) the experimental design is not fully clear: why were different depths used in the M1 stage if it is a single profile? 

Yes, this is one profile where horizons are not developed, because this is «zero» stage of soil formation, but, in with aim to compare microbiome of developed horiozons on the same depths on the next stages of soil formation we have compared they with various depth on very initial stage of soil development — on the M1 stage.

 

3) there is much confusion in the numbering of the figures and in the combination of the caption; 

Yes. There was a mistake - one figure has lost its caption. We also added some figures to the supplement.

4) some references are put in full while others as number; 

Done

5) the figure of the PCA can be very informative but needs to be further explained: what are the gray dots? 

We fixed it.

5) why do some pedogenesis stages have three points for depth while others have five?

The soil development within time is a dynamic process, thus, some stages have only one horizon, another stage have 2, the next 3 etc. The mature soil usually includes up to 5-6 horizons. This process is called soil evolution under the ecogenetic sucsession.

6) there is also some taxonomic confusion between family and order. Moreover, Italic is generally used for taxa at the level of family and below; 

We changed it in the text

7) clarify what you mean regard pre-climax and climax by thinking of an audience not familiar with this topic; 

We made a short description of the climax term in the text and supplied it with the citation. 

8) the discussion needs to be improved. Sometimes, there is not a clear link between the data obtained and those obtained from other authors.

Done

Other specific comments:

Keywords: move in alphabetic order

Done

Line 60: add reference to Dobzhnsky’s word 

We put the corresponding citation. 

Line 124: here and throughout the text, use abbreviation for carbon (C) and nitrogen (N)

Done

Line 169: Pinus Sylvestri in italic

Done

Line 184: it is not clear, use M1, M2……

Done

Line 186: explain which horizon you sampled within each site

We added the description of the horizons to 169-170 and the selection scheme: M1––the initial (zero) stage, lacking any evidence of vegetation (samples of C1-C2-C3-C4); M2––sandy 3-year-old primitive sod gray humus soil (samples of Oi-Ay-BF-C) ; M3 (samples of Oi-E-BF-BC-C) and M4  (Oi-Oe-E-BHF-BF-C) ––30- and 70-year-old Embryopodzols, respectively; and, finally, M5––mature (benchmark) (Oi-E-BF-C)

Line 193: add reference

Done

Lines 195-199: add reference

Done

Line 201: are soil sample for DNA extraction collected in the same way as samples for chemical analysis?

The corresponding description was written in 170-180 materials and methods

Line 206: How did you evaluate the purity of DNA by electrophoresis? and the quantity? You determined the DNA concentration by SPECTROStar Nano.

The quality of DNA extraction was assessed on the electrophoregram by the presence of a clear bright band relative to the marker (lambda-hindIII). Added in the text. 

Line 208: Compared to what? Soil dry weight/fresh weight? Do you think it makes sense to report both yield and final concentration?

Yes, the final concentration is enough. 

Line 210: add the primers used with relative references 

The text of the Manuscript was added by the following: 

The pair of primers Eub338 / Eub518 (Lane, D.J., 1991. 16S/23S rRNA sequencing. In: Stackebrandt, E., Goodfellow, M. (Eds.), Nucleic acid Techniques in Bacterial Systematics. Wiley, New York, pp. 115–175.) and ITS1f/5.8s (Fierer N, Jackson JA, Vilgalys R, Jacksson RB. Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays. Appl Environ Microbiol. 2005;71: 4117. pmid:16000830).

Line 246: why did you use t-test and you didn’t use ANOVA? I think that ANOVA is more correct to compare more than two samples. Moreover, where are the results of significance?

Thank you. We used ANOVA to calculate the significance of the difference in alpha-diversity indices of the samples. The significantly different samples are marked by letters “a-e” in Table 2. 

Line 248: and archaeal and fungal dissimilarity?

Beta diversity as well as the amount of dissimilarity (distance) between samples was calculated only for prokaryotic communities (on the base of 16 S rRNA sequence data). The “bacterial” has changed to the “prokaryotic” in the text of the Manuscript.

Results: you must better clarify what changes according to the development of the soil and what according to the depth

This is probably the cornerstone of the present work. It is hard to separate soil profile (here we can’t put the formal depth) from the time point (site), because they are deeply connected. The soil profile demonstrates the final stage of the certain soil-forming processes, threatening mineral matrix and forming the characteristic soil structure in time. 

Line 284: where is the method used for exchange capacity explained? And where are the results obtained?

Cation exchange capacity was not determined, this was mistake, mentioning is deleted.

Line 285: from M1 to M5? Try to be clearer

Yes, you’re right. Fixed.

 

Line 294: generally, the percentage of two particle size were reported, clay and silt or clay and sand, thus the third particle will be easily determined

we have wrote in the text:

«The soils were classified as sandy textured due to the very low clay content»

it does not mean that we determined 2 or 3 fractions of grain size, we only mention the soil texture class.

Table 1: explain why for M1 stage you analysed soil at four different depth. Were the samples collected at: 0-5 cm; 5-10 cm; 10-20 cm, and 20-30 depth? How did you choose these depth? Use a depth range for each profile. There isn’t statistical analysis.

These are general routine characteristics of differently-aged soils which are needed only for diagnosing different stages of the soil development process. M1 stage is lacking any morphologically separate horizons, so there we operate with depths, other stages can be divided into morphologically defined horizons and reasonably must be described in these terms. This is the same question you have asked below in the beginning of the results section - we concentrate not even on depths, but on the soil morphology. 

Line 301: the alpha diversity was linked to Illumina results, thus move after the real time results

Done

Table 2: distance better each stage of soil development, otherwise it is no clear. There isn’t statistical analysis.

We performed ANOVA and added the result to the table2.

Figure 3: are you sure this is the correct figure?

We changed the figures’ order and rewrote the caption. We are really sorry for these mistakes. 

Line 333: where is it written that you did

The text was added by the following: “PERMANOVA was applied to calculate the significant differences between samples in beta-diversity analysis.”

PCA Figure: I’d put the data labels from the most superficial to the deepest horizon, in the same order as the caption

Again, they are ranged according to soil developmental process, first there are mineral (C, BC horizons), on the latest stages the organic matter starts to accumulate within the soil profile and we can distinguish first organo-mineral horizons (like AY, BF) and then - the true organogenic layers (O) and podzolization markers (E). 

Line 353: where is the list? Add it in supplementary materials

The list is shown on the figure - the column with the abbreviations of the bacterial and archaeal names on the right side. 

Line 369-371: are these OTU referred to a specific phylum? if yes, you must specify.

The specific groups can be also found on the corresponding figure directly on the circular diagram. Of course it is better to write them in the text.

Line 409: where is the list? Add it in supplementary materials

We added the list of the taxa to the supplementary materials. 

Ligure CCA: the vision of this figure is not immediate; the horizons could be placed as symbols with different colors based on the two different stages of development

Done

Line 503: where does it come from? The purpose of the Discussion to give evidence that supports the obtained data.

These are our hypotheses. We added this remark to the text. We also added some missed references to the discussion section. 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript Structural shifts in the soil microbiome marking the podzol-  forming process on sand dumps by Evdokimova et al., written well and reported the interesting findings. However, introduction is lengthy and it has to be reduced.

In sampling, if we collect samples today that will represent present condition of the site, if this is the case, How samples belong to different chronology has been obtained

If KEGG analysis is given it will be good to understand the functions of microbiota in Podozol development

Author Response

Dear reviwer!

 

thank you for your comment and suggestions, all changes are trached in yellow color in updated version of the manuscript.

 

sincerely yours,

Evgeny Abakumov

 

Rev_2

1)However, introduction is lengthy and it has to be reduced.

We rewrote the introduction and added some extra references

2) In sampling, if we collect samples today that will represent present condition of the site, if this is the case, How samples belong to different chronology has been obtained 

Quarry development has i, in which some dumps are younger and others older, if it is known that different ages of dumps correspond to different age stages of soil formation.

This type of soil dating chronology method is widely used in current soil science

Mokma, D.L.; Yli-halla, M.; Lindqvist, K. Podzol Formation in Sandy Soils of Finland. Geoderma 2004, 120, 259– 663 272, doi:10.1016/j.geoderma.2003.09.008.

 

3) If KEGG analysis is given it will be good to understand the functions of microbiota in Podozol development

Unfortunately in this work we didn’t perform the KEGG analysis. Recently we performed the whole-metagenome sequencing of DNA, we hope this analysis will be published soon.  

Reviewer 3 Report

Comments and Suggestions for Authors

Manuscripter Recommendation Term: Receive

Overall Manuscripter Manuscript Rating: 60

Thank you for inviting me to evaluate the manuscript titled “Structural shifts in the soil microbiome marking the podzol-forming process on sand dumps”. This manuscript summarizes the model of podzol soil genesis showed the continuous evolution of the microbiome structure, which corresponded with the process of soil organic-matter redistribution and formation of the vertical soil profile.

However, there are several points where this manuscript could be improved:

1.   The manuscript mentioned that "5 time points in whole layers of collecting samples (from 0 to 70 years)". Please add the description of time point of M5 in order to distinguish the time point of M4. Or modify the sampling time range in the abstract.

2.   It is suggested to add the relevant pictures or description of "soil horizon" in the sampling site, and make a simple explanation in the Materials and Methods (such as horizon in Table 1).

3.   Page 4, Line 166, 172-173 and Table 1: The interpretation of the M2 should be complete and uniform when it first appears.

4.   Page 7, Line 286-289: Please evident that the trend mentioned here is directly related to "organic-matter accumulation and redistribution".

5.   Page 19, Line 460-462: The model includes total C_org, depth, clay content and pH and other factors. It is suggested that a comprehensive analysis about the influencing factors of each region in Figure 8 should be added to the discussion.

6.   Page 22, Line 624-626: It is mentioned that the chronosequence was completed with at least one intermediate stage of soil formation, which ranged from 40 to 45 years old. Please supplement relevant references.

7.   The font and format of the chart are not unified. Please adjust the size of the picture appropriately, such as the title in Figure 8 is not bold.

8.   There are some grammatical mistakes in the manuscript. (e.g., "The rate of succession decreases in the final stages of soil development" in line 118, page 3,"The pH values were determined using a routine method in water and calcium chloride suspensions" in line 192, page 5, "Beginning" in line 542, page 20).

9.   Please check the text for spelling mistakes.

10. Please check again the format and the order of the reference. Please be selective but representative with referencing.

Despite this, the results are promising and have guidance to the relationship between the structure of the soil microbiome and the peculiarities of the evolution of the podzolic soil profile. Accordingly, I would like to recommend the manuscript be accepted after a major revision.

Comments on the Quality of English Language

The quality of the English language is fine, with only minor changes.

Author Response

Dear reviwer!

 

thank you for your comment and suggestions, all changes are trached in yellow color in updated version of the manuscript.

 

sincerely yours,

Evgeny Abakumov

 

Rev_3

1. The manuscript mentioned that "5 time points in whole layers of collecting samples (from 0 to 70 years)". Please add the description of time point of M5 in order to distinguish the time point of M4. Or modify the sampling time range in the abstract.

 

 

Samples were collected at 5 time points (0,  3, 30.  70 years and mature soil) from the entire set of soil horizons revealing the history of pedogenesis. This sentence was added to the abstract.

 

2. It is suggested to add the relevant pictures or description of "soil horizon" in the sampling site, and make a simple explanation in the Materials and Methods (such as horizon in Table 1).

 

We have supplied the text with better explanation of the horizon properties.  

C — intial parent material — not altered by pedogeneis

Oi- initial litter, not altered by organic matter transformation process

Oe- littes, sligthly transformed by ensimatic processes

AY- humus accumulative superficial soil horizon

BF- illuvial iron-oxide horizon

(e)— entic podzol horizon

BHF — illuvial iron-oxide-humic complexes horizon.

Also we modified the sampling description in the materials and methods section. 

3. Page 4, Line 166, 172-173 and Table 1: The interpretation of the M2 should be complete and uniform when it first appears.

 

M2––sandy 3-year-old primary sod gray humus soil with developed humus layer and first features of initial parent material alteration. Added to the text. 

4. Page 7, Line 286-289: Please evident that the trend mentioned here is directly related to "organic-matter accumulation and redistribution".

 

This tendency may be closely associated 286 with organic-matter accumulation and redistribution, and particularly in the observed 287 peak in organic-matter content in the 30-year-old site, leading to a substantial decrease in 288 the corresponding pH values.

5. Page 19, Line 460-462: The model includes total C_org, depth, clay content and pH and other factors. It is suggested that a comprehensive analysis about the influencing factors of each region in Figure 8 should be added to the discussion.

The text of the Manuscript was added  by the following: “Detailed analysis of the transition from the M3 stage of embryopodzol to the mature podzol profile at the M4 stage demonstrated the continuation of the process of acidification of the upper part of the soil profile (Figure c CCA), associated with the accumulation of organic matter in the O horizon of the M4 stage. Also, in relation to mineral horizons, their preferential enrichment with clay particles was observed, compared to the upper part of the profile, which was also more characteristic of the more mature preclimax soil (M4).”

6. Page 22, Line 624-626: It is mentioned that the chronosequence was completed with at least one intermediate stage of soil formation, which ranged from 40 to 45 years old. Please supplement relevant references.

The sentence was changed to “Thus, in further research it will be reasonable to complete the chronosequence with at least one intermediate stage of soil formation, ranging from 40 to 45 years old. 

7. The font and format of the chart are not unified. Please adjust the size of the picture appropriately, such as the title in Figure 8 is not bold.

The picture was modified.

8. There are some grammatical mistakes in the manuscript. (e.g., "The rate of succession decreases in the final stages of soil development" in line 118, page 3,"The pH values were determined using a routine method in water and calcium chloride suspensions" in line 192, page 5, "Beginning" in line 542, page 20).

Sorry for these mistakes, we will check the text again.

9. Please check the text for spelling mistakes.

Done

10. Please check again the format and the order of the reference. Please be selective but representative with referencing.

Done

 

 

 

Reviewer 4 Report

Comments and Suggestions for Authors The article "Structural shifts in the soil microbiome marking the podzol- forming process on sand dumps" deals with the description of microbiome evolution throughout different stages of soil formation. I find the idea of this work nice for the readership of Soil Systems. The methods are adequate to conduct this study and the conclusions are supported by the results. However, there are several issues, mainly related with the presentation of data that should be addressed to facilitate an easier understanding of the story of this work:     Methods and/or introduction. Please, define the terms used for soil horizons. The codes used (A, B, Oi, BHF, etc.) are not explained together within the manuscript and is too difficult to read as it is. The result section usually is referred to such kind of letter codes and the reader of the manuscript may be not so used to these terms. Hence, a single section or table or figure that explain this would facilitate to understand this study. L. 117. Please, define "specific" in this sentence L. 138-142. Through 16S rRNA metabarcoding you can only study the prokaryotic communities, not the entire soil "microbiome". Please, revise here and along the manuscript the incorrect use of the terms "microbiome", "microorganisms" , and others that include non-prokaryotic microbes when referred to the output of the 16S rRNA metabarcoding Figure 2 and sampling methods. It seems that there are some roots from trees that reach the sampling sites. How was the effect of the plant's rhizospheres taken into account? Were all the sampling sites ~equally distant from trees? If so, please, specify this within the methods. If not, please, provide discussion on how these rhizospheres may bias the microbial composition. L 211. The reference (Ivanova et al) should be cited with the number style (i.e.: [67]). In any case, please, provide more details, even brief, within this manuscript on qPCR methods, such as the primers used. Here it is not referred to any methods to estimate the archaeal abundance, but the results include this. How was that achieved? L 241. Please, specify the Greengenes version   L. 281. The sequences are not available. Please, release the sequences. Table 1. Please,  explain the abbreviations as part of the table heading (i.e.: "C", "TOC", "AY") L 304-305. Please, provide statistics (i.e.: Kruskal-Wallis test) for these comparisons Table 2. I think this data would be more informative if presented as boxplots, at least for one of the metrics. Figure 3. "Metabolic" rather than "Methabolic", right?   Figures issue: It seems that you missed a figure and mixed the legends among figures. Caption for Figure 3 should be placed within Figure 4. Caption for Figure 4 should be linked to Figure 5. Caption for Figure 5 should be linked with some missing figure? Also, Figure 3 lacks its own caption   Figure of the number of microbes: The error bars are not shown.

L 352: "...but some were specific bacteria...". This is not informative, please, specify the taxa or remove this sentence.

Results: The alpha rarefaction plot is not shown, but according to the methods it was performed. Please, provide this figure as a supplementary file.   L375. Enriched compared to what? Figure 7. The units for the heatmap are missing. Does it represent relative abundance (%)? What represents the size of each bar? This figure is not easily understood       Figure 8. The Figure shows ASVs, but the study is based on OTUs. How can it be explained? Also, it would be better to use idfferent colors to depict ASVs/OTUs than variables, such as Clay, Depth... Finally: why are horizons placed at the level of ASVs? Maybe some points or colors were lost at the submission step.  

Discussion: Please, add references to each sentence that is related with knowledge not obtained from this work. E.g.: the catabolic role of fungi, the ability of some bacteria to hidrolyze simply components of organic matter, etc.

 

Author Response

Dear reviwer!

thank you for your comment and suggestions, all changes are trached in yellow color in updated version of the manuscript.

sincerely yours,
Evgeny Abakumov

 

Rev_4

1)Methods and/or introduction. Please, define the terms used for soil horizons. The codes used (A, B, Oi, BHF, etc.) are not explained together within the manuscript and is too difficult to read as it is. The result section usually is referred to such kind of letter codes and the reader of the manuscript may be not so used to these terms. Hence, a single section or table or figure that explain this would facilitate to understand this study.

The requested description was put in the text. 

 

C — intial parent material — not altered by pedogeneis

Oi- initial litter, not altered by organic matter transformation process

Oe- littes, sligthly transformed by ensimatic processes

AY- humus accumulative superficial soil horizon

BF- illuvial iron-oxide horizon

(e)— entic podzol horizon

BHF — illuvial iron-oxide-humic complexes horizon

 

2)  L. 117. Please, define "specific" in this sentence 

 

It was changed to “dominate in mature soils”

 

3) L. 138-142. Through 16S rRNA metabarcoding you can only study the prokaryotic communities, not the entire soil "microbiome". Please, revise here and along the manuscript the incorrect use of the terms "microbiome", "microorganisms" , and others that include non-prokaryotic microbes when referred to the output of the 16S rRNA metabarcoding 

 

Done

 

4) Figure 2 and sampling methods. It seems that there are some roots from trees that reach the sampling sites. How was the effect of the plant's rhizospheres taken into account? Were all the sampling sites ~equally distant from trees? If so, please, specify this within the methods. If not, please, provide discussion on how these rhizospheres may bias the microbial composition.

 

Yes, the rhizospheric effect is quite important. It was mentioned in the discussion section. 

 

5) L 211. The reference (Ivanova et al) should be cited with the number style (i.e.: [67]). In any case, please, provide more details, even brief, within this manuscript on qPCR methods, such as the primers used. Here it is not referred to any methods to estimate the archaeal abundance, but the results include this. How was that achieved? 

 

We added required references and modified this part.

The text of the Manuscript was added by the following: “The pairs of primers Eub338 / Eub518 [60],  915f/1059r (61) and ITS1f/5.8s (62). was used for bacteria, archaea and fungi correspondingly.

 

6) L 241. Please, specify the Greengenes version   

 

The mistake appeared here. We specified the classification procedures. 

 

7) There appeared a problem with the access, we wrote to the supply team, but they didn’t answer. Yesterday we sent an additional request for sequence accommodation. He hope that this problem will be solved during paper reconsideration.

 

8) Table 1. Please,  explain the abbreviations as part of the table heading (i.e.: "C", "TOC", "AY") 

 

Done

 

9) L 304-305. Please, provide statistics (i.e.: Kruskal-Wallis test) for these comparisons 

 

The ANOVA statistic method was applied to these data

 

10) Table 2. I think this data would be more informative if presented as boxplots, at least for one of the metrics. 

 

The box-plots will be attached to this work as a supplement figure. 

 

11) Figure 3. "Metabolic" rather than "Methabolic", right?  

 

Yes, it was a mistake.

 

12) Figures issue: It seems that you missed a figure and mixed the legends among figures. Caption for Figure 3 should be placed within Figure 4. Caption for Figure 4 should be linked to Figure 5. Caption for Figure 5 should be linked with some missing figure? Also, Figure 3 lacks its own caption  

 

Sorry for this mistake, everything was turned to the right order

 

13) Figure of the number of microbes: The error bars are not shown.

 

Done

14) L 352: "...but some were specific bacteria...". This is not informative, please, specify the tax``````````````a or remove this sentence.

This sentence was modified

Results: 

15) The alpha rarefaction plot is not shown, but according to the methods it was performed. Please, provide this figure as a supplementary file.  

 

We provide rarefaction curves in supplementary materials.

 

16) L375. Enriched compared to what? 

 

“Enriched” were displaced with “dominated”

 

17) Figure 7. The units for the heatmap are missing. Does it represent relative abundance (%)? What represents the size of each bar? This figure is not easily understood    

 

The description was added to the caption.   

 

18) Figure 8. The Figure shows ASVs, but the study is based on OTUs. How can it be explained? Also, it would be better to use idfferent colors to depict ASVs/OTUs than variables, such as Clay, Depth... Finally: why are horizons placed at the level of ASVs? Maybe some points or colors were lost at the submission step.  

 

We used Silva release for identification of our sequences, and ASV is a typical designation for that. OTU is a more general word, which includes ASV. We think it is better to uniform everything, so we displaced ASV with OTU. 

 

The text of the Manuscript was added by the following: OTU picking was performed using a close-reference picking algorithm based on 97% sequence similarity with the SILVA database, version 132, 2018 (https://www.arb-silva.de/download/archive/qiime, accessed on 28 November 2019). 

 

Discussion: 

19) Please, add references to each sentence that is related with knowledge not obtained from this work. E.g.: the catabolic role of fungi, the ability of some bacteria to hidrolyze simply components of organic matter, etc.

The discussion section was modified accordingly suggestions

 

 

 

Reviewer 5 Report

Comments and Suggestions for Authors

Overall, this manuscript is well prepared. Besides a few minor issues, there are two main problems:

1. Since the authors have already performed 16S high-throughput sequencing, why didn't they also do ITS sequencing? If you don't consider the changes in fungal communities, your succession study will be incomplete. Moreover, you mentioned that fungi play a significant role in the later stages of succession. Therefore, I strongly suggest that you supplement your data with ITS high-throughput sequencing.
2. In the Materials and Methods section, you stated that you used OTUs (Operational Taxonomic Units) for classification, but in the Results section and figure legends, you mentioned the use of ASVs (Amplicon Sequence Variants). So, which one did you actually use?

You must address these two major issues before the manuscript can be published.

Comments on the Quality of English Language

Well prepared

Author Response

Dear reviwer!

thank you for your comment and suggestions, all changes are trached in yellow color in updated version of the manuscript.

sincerely yours,
Evgeny Abakumov

 

Rev_5

1. Since the authors have already performed 16S high-throughput sequencing, why didn't they also do ITS sequencing? If you don't consider the changes in fungal communities, your succession study will be incomplete. Moreover, you mentioned that fungi play a significant role in the later stages of succession. Therefore, I strongly suggest that you supplement your data with ITS high-throughput sequencing.

 

This is a valuable comment. Fungi community play a crucial role in soil functioning. But, unfortunately for today we didn’t have an opportunity to perform this analysis. We hope to do it in further research. 

To fix this unfortunate omission we changed the title of the article replacing “microbiome” with  “procaryotic community”. 

2. In the Materials and Methods section, you stated that you used OTUs (Operational Taxonomic Units) for classification, but in the Results section and figure legends, you mentioned the use of ASVs (Amplicon Sequence Variants). So, which one did you actually use?

We used Silva release for identification of our sequences, and ASV is a typical designation for that. OTU is a more general word, which includes ASV. We think it is better to uniform everything, so we displaced ASV with OTU. 

 

Reviewer 6 Report

Comments and Suggestions for Authors

General comments:

Abstract, Introduction, Reference list: authors have omitted a number of the current publications directly related to the study (e.g. Geisen, 2021; Xiong, Lu, 2022; FAO, ITPS, GSBI, SCBD and EC. 2020; Baruch et al 2020; etc.).

Authors mixed the conception of “biodiversity” and “diversity” (e.g. Line 52, 67, 125,

Soil biodiversity comprises a huge part of global diversity, no precise data are available. Recent estimates of total diversity suggest that both bacteria and fungi may each comprise as many as 1.5 million species. According to FAO, soil biodiversity is “the variety of life belowground, from genes and species to the communities they form, as well as the ecological complexes to which they contribute and to which they belong, from soil micro-habitats to landscapes”.

Suggestion: moderate changes

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Extensive editing of English language required

Author Response

Dear reviwer!

thank you for your comment and suggestions, all changes are trached in yellow color in updated version of the manuscript.

sincerely yours,
Evgeny Abakumov

 

Rev_6

1. Abstract, Introduction, Reference list: authors have omitted a number of the current publications directly related to the study (e.g. Geisen, 2021; Xiong, Lu, 2022; FAO, ITPS, GSBI, SCBD and EC. 2020; Baruch et al 2020; etc.).

 

Soil microbial diversity is largely discussed in plenty of modern publications. We tried to concentrate only on the articles, which are closely related to the microbial (prokaryotic) succession occurring in pedogenesis. Thank you for the reference list, we will rewrite the introduction section. 

2. Authors mixed the conception of “biodiversity” and “diversity” (e.g. Line 52, 67, 125,

Soil biodiversity comprises a huge part of global diversity, no precise data are available. Recent estimates of total diversity suggest that both bacteria and fungi may each comprise as many as 1.5 million species. According to FAO, soil biodiversity is “the variety of life belowground, from genes and species to the communities they form, as well as the ecological complexes to which they contribute and to which they belong, from soil micro-habitats to landscapes”.

 

Yes, you are right. It is better to use “diversity” here. We fixed it. 

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I still don't see the results of the ANOVA statistical analysis conducted on the chemical data (Table 1 general soil properties) and bacterial, archaeal, and fungal quantification (Figure 4). In Materials and Methods it is not mentioned that ANOVA analysis was done on chemical and biochemical data while in the authors’ report, they claimed yes. However, if it is true that three replicates were collected for each condition, statistical analysis is necessary. Thus, I cannot accept the publication of this manuscript until the analysis is completed and shown. Moreover, the meaning of figure 3 and its caption is not clear and supplementary materials need a few lines of description.

Author Response

I still don’t see the results of the ANOVA statistical analysis conducted on the chemical data (Table 1

general soil properties) and bacterial, archaeal, and fungal quantification (Figure 4). In Materials and

Methods it is not mentioned that ANOVA analysis was done on chemical and biochemical data while in

the authors’ report, they claimed yes. However, if it is true that three replicates were collected for each

condition, statistical analysis is necessary. Thus, I cannot accept the publication of this manuscript until

the analysis is completed and shown. Moreover, the meaning of figure 3 and its caption is not clear and

supplementary materials need a few lines of description.

 

We apologize for the mistake. Supplementary material weren’t attached carefully. We fixed it now and added the ANOVA analysis for the real time results (see Table S1 and S2).

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have already answered questions I asked previously. It can be considered to be published.

 

Author Response

Dear reviwer!

thank you for you comments!

Reviewer 4 Report

Comments and Suggestions for Authors

The authors fulfill all my suggestions

Author Response

Thank you for your reply!

Reviewer 5 Report

Comments and Suggestions for Authors

Your response did not resolve my doubts. Instead, it has increased my doubts.

1.     In your reply, you mentioned that you did not perform ITS sequencing and therefore modified the title. However, you included the ITS sequencing method in the Methods section and used words such as microbial diversity and microbial community extensively in the main text. If you added the ITS sequencing content later due to the request of other reviewers, why didn't you discuss the changes in fungal communities in the Results and Discussion sections? My suggestion is either to remove the content about ITS sequencing from the Methods section and change all “microbial” to “prokaryotic”, or revert to the original title and include discussions about fungal communities in the Results and Discussion.

2.     OTU and ASV are two different methods for classifying microbial taxa. Please refer to the following references to confirm whether it is OTU or ASV.

Callahan B J, McMurdie P J, Rosen M J, et al. DADA2: high-resolution sample inference from Illumina amplicon data. Nature methods, 2016, 13(7): 581-583.

UNOISE2: Improved error-correction for Illumina 16S and ITS amplicon read. bioRxiv, 2016

Comments on the Quality of English Language

Overall, there are no major issues with English and it is completely understandable.

Author Response

 

Comments and Suggestions for Authors

 

Your response did not resolve my doubts. Instead, it has increased my doubts.

1. In your reply, you mentioned that you did not perform ITS sequencing and therefore modified the title.

However, you included the ITS sequencing method in the Methods section and used words such as

microbial diversity and microbial community extensively in the main text. If you added the ITS sequencing

content later due to the request of other reviewers, why didn’t you discuss the changes in fungal

communities in the Results and Discussion sections? My suggestion is either to remove the content about

ITS sequencing from the Methods section and change all “microbial” to “prokaryotic”, or revert to the

original title and include discussions about fungal communities in the Results and Discussion.

 

Unfortunately we are unable to perform ITS sequencing, so we excluded all the information on the fungal communities and changed the corresponding terminology in the manuscript.

 

2. OTU and ASV are two different methods for classifying microbial taxa. Please refer to the following

references to confirm whether it is OTU or ASV.

Callahan B J, McMurdie P J, Rosen M J, et al. DADA2: high-resolution sample inference from Illumina

amplicon data. Nature methods, 2016, 13(7): 581-583.

UNOISE2: Improved error-correction for Illumina 16S and ITS amplicon read. bioRxiv, 2016

Thank you for the references, we performed OTU analysis, ASV abbreviations were removed from the text.  

 

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Little has changed compared to the old version, the ANOVA analysis is still not reported in table 1 (General soil properties), both standard deviation and significance test are missing.

 

Figure 3 is still not clear; how does such a figure represent the statistical analysis of soil physicochemical data? In Material and Methods (paragraph 2.2. Soil analysis) was reported that ANOVA was added, but where are the significant differences reported between the various samples?

Reviewer 5 Report

Comments and Suggestions for Authors

This manuscript can be accepted.