Soil Nutrient Availability Regulates Microbial Community Composition and Enzymatic Activities at Different Soil Depths along an Elevation Gradient in the Nanling Nature Reserve, China

Round 1

Reviewer 1 Report (New Reviewer)

The manuscript “Soil nutrient availability regulates microbial community composition and enzymatic activities along an altitude gradient in the Nanling Nature Reserve, China” is aimed on better understanding of soil microbial community composition and enzyme activities depending on different altitudes in the light of ecosystem functioning under climate changes.

the topic of the manuscript is relevant for better understanding the dynamic of soil microorganisms in the light of climate changes.

 The main lack of the research is short period of observation. As well consideration of the types of vegetation biomass would help for better discussions and understanding of the processes of organic substrate decomposition and turnover. However, the authors acknowledge that the conclusions of the study are based on measurements of only one season, which may affect the objective assessment of the situation and processes. Therefore, they are intended to continue their study.

 

All other aspects, such as methodology and data processing are well done and presented.

 The main conclusion should be adjusted as for a single observation:

 

Enzyme vector analysis documented the response of the microbial community to the presence of nutrients in the soil, and enzymatic activity varied with altitude.

Author Response

The manuscript “Soil nutrient availability regulates microbial community composition and enzymatic activities along an altitude gradient in the Nanling Nature Reserve, China” is aimed on better understanding of soil microbial community composition and enzyme activities depending on different altitudes in the light of ecosystem functioning under climate changes.

the topic of the manuscript is relevant for better understanding the dynamic of soil microorganisms in the light of climate changes.

 The main lack of the research is short period of observation. As well consideration of the types of vegetation biomass would help for better discussions and understanding of the processes of organic substrate decomposition and turnover. However, the authors acknowledge that the conclusions of the study are based on measurements of only one season, which may affect the objective assessment of the situation and processes. Therefore, they are intended to continue their study.

All other aspects, such as methodology and data processing are well done and presented.

 The main conclusion should be adjusted as for a single observation:

Enzyme vector analysis documented the response of the microbial community to the presence of nutrients in the soil, and enzymatic activity varied with altitude.

 A: Thank you again for your constructive comments for improving the manuscript!

We have checked the manuscript carefully and revised it according to the comments.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

The paper is devoted to ecologically interesting and important topic – the influence of altitude and soil depth on some aspects of nutrient availability, enzymatic activity, and the composition of microbial communities in a Chinese subtropical forest. The authors performed a lot of analyses and measurements, and collected the rich primer data. However, in order to be published, the paper must be substantially revised and improved.

The title does not fully correspond to the paper topic because it even does not mention the impact of soil depth on the studied characteristics 

Introduction

The authors use here and throughout the text the expression "soil carbon (C) and nutrients" (line 38). Such expression is also used in some papers cited in the submitted manuscript. However, I agree with the definition, that C, as well as N and P is a macronutrient (https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book%3A_General_Biology_(Boundless)/31%3A_Soil_and_Plant_Nutrition/31.01%3A_Nutritional_Requirements_of_Plants/31.1C%3A_Essential_Nutrients_for_Plants#:~:text=The%20essential%20elements%20can%20be,calcium%2C%20magnesium%2C%20and%20sulfur. )

Line 49: "… plant-derived C sources that are generally labile". It is unclear - whether plant-derived C sources like cellulose and lignin are labile and not recalcitrant.

In the last paragraph the authors gave a set of hypotheses they tested, but they did not describe the objectives of the study – which parameters they determined in order to test the hypotheses.

Material and Methods, the subsection 2.1. – the authors should specify why namely these altitudes were chosen (differences-similarities among them)

Results, the subsection 3.1 – the authors did not measure any soil physical parameters (color, texture, structure, porosity, density, consistence, aggregate stability, and temperature), only chemical parameters.

Lines 166-177 – hard to follow the text, which 8 times repeats "was significantly higher (lower)" instead of writing, for example, "Soil C:N ratio significantly increased with altitude".

Table 1. Why is SE given in parentheses and not as ±?

Table 2. - the title does not correspond to the table content. The table shows the concentrations of phospholipid fatty acids as biomarkers of Gram-positive (GP), Gram-negative (GN) bacteria, and their ratio in soil profiles along the altitudinal gradient.

Concerning statistical tests – strangely, the authors did not perform an important two-way unbalanced ANOVA with interactions in order to test the effect of altitude and soil depth, separately and in interaction, on the studied characteristics.

Discussion is superficial, chaotically written, and almost does not touch the topic announced in the title – the effect of altitude on the studied soil characteristics. Conclusions have not concluded the results obtained and focused almost only on the further studies needed. Overall, the text contains a lot of unclear, uncertain, and awkward statements and expressions and must be thoroughly checked by a language editor.

All other numerous comments and suggestions are inserted into the attached PDF version of the manuscript.

Comments for author File: Comments.pdf

Author Response

The paper is devoted to ecologically interesting and important topic – the influence of altitude and soil depth on some aspects of nutrient availability, enzymatic activity, and the composition of microbial communities in a Chinese subtropical forest. The authors performed a lot of analyses and measurements, and collected the rich primer data. However, in order to be published, the paper must be substantially revised and improved.

The title does not fully correspond to the paper topic because it even does not mention the impact of soil depth on the studied characteristics 

A: Thanks for your comments. We have added soil depth information in the title.

Introduction

The authors use here and throughout the text the expression "soil carbon (C) and nutrients" (line 38). Such expression is also used in some papers cited in the submitted manuscript. However, I agree with the definition, that C, as well as N and P is a macronutrient (https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book%3A_General_Biology_(Boundless)/31%3A_Soil_and_Plant_Nutrition/31.01%3A_Nutritional_Requirements_of_Plants/31.1C%3A_Essential_Nutrients_for_Plants#:~:text=The%20essential%20elements%20can%20be,calcium%2C%20magnesium%2C%20and%20sulfur. )

Line 49: "… plant-derived C sources that are generally labile". It is unclear - whether plant-derived C sources like cellulose and lignin are labile and not recalcitrant.

A: Thanks for your comments. We have revised this sentence for more accurate.

“…plant-derived C sources contained some labile carbon”

In the last paragraph the authors gave a set of hypotheses they tested, but they did not describe the objectives of the study – which parameters they determined in order to test the hypotheses.

A: Thanks for your comments. We have described the objectives of the study in the last paragraph.

“To understanding how soil microbial community composition and enzyme activities vary according to altitude, we analyzed the microbial community, soil-nutrient availa-bility and enzymatic activity along an altitude gradient in the Nanling Nature Reserve, China.”

Material and Methods, the subsection 2.1. – the authors should specify why namely these altitudes were chosen (differences-similarities among them)

A: Thanks for the nice suggestions. To explore the effects of altitude changes on soil nutrients and microorganisms (below-ground ecosystems), we selected these altitudes (ranges from 1000m to 1400m) according to their similar vegetation habitat (broad-mixed forests and less disturbed).

Results, the subsection 3.1 – the authors did not measure any soil physical parameters (color, texture, structure, porosity, density, consistence, aggregate stability, and temperature), only chemical parameters.

A: Thanks for your comments. Now we included additional information about the soils, such as “soils in those forests are typical yellow containing granite, metamorphic rock, and sandstone (Gan et al. 2016).”

Lines 166-177 – hard to follow the text, which 8 times repeats "was significantly higher (lower)" instead of writing, for example, "Soil C:N ratio significantly increased with altitude".

A: Thanks for your comments. We have revised the repetitive writing in the article.

Table 1. Why is SE given in parentheses and not as ±?

A: Thanks for your comments. To keep the data in one table, we chose to display SE in parentheses.

Table 2. - the title does not correspond to the table content. The table shows the concentrations of phospholipid fatty acids as biomarkers of Gram-positive (GP), Gram-negative (GN) bacteria, and their ratio in soil profiles along the altitudinal gradient.

A: Thanks for your comments. We have revised it according to your suggestion.

Concerning statistical tests – strangely, the authors did not perform an important two-way unbalanced ANOVA with interactions in order to test the effect of altitude and soil depth, separately and in interaction, on the studied characteristics.

A: Thanks for your comments. We did the two-way ANOVA analysis to explore altitude, soil depth, the interactions of altitude and soil depth effects on soil characteristics, respectively. Please see the detailed information in supplement in this manuscript.

Discussion is superficial, chaotically written, and almost does not touch the topic announced in the title – the effect of altitude on the studied soil characteristics. Conclusions have not concluded the results obtained and focused almost only on the further studies needed. Overall, the text contains a lot of unclear, uncertain, and awkward statements and expressions and must be thoroughly checked by a language editor.

A: Thanks for your comments. We thought there are various factors drive soil characteristics along the altitude changes (e.g. vegetation, temperature, slop...), and it is hard to clarify or statement how the altitude change effects on each soil character according to our data (also this is not we want). Our research mainly focused on how the relationships between soil nutrients and microbial communities responding to different altitude gradients.

All other numerous comments and suggestions are inserted into the attached PDF version of the manuscript.

A: Thanks for your comments. We have revised it according to the attached PDF version of the manuscript.

Author Response File: Author Response.pdf

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

This study observed the relationship between soil microbial community composition, enzymatic activity, and altitude in a subtropical forest. However, there are short points as below:

The scope of this study is limited to only three specific locations and does not encompass a full gradient of altitude. Additionally, the study does not account for seasonal changes, which could significantly impact the results. To compensate the lack of seasonal variation, I want to recommend for the researchers to conduct measurements over multiple years. I recommend submitting this ms to a local journal.

Reviewer 2 Report

I have taken the time to review the manuscript forests-2391043 titled “Soil nutrient availability regulates microbial community com-2 position and enzymatic activities along an elevational gradient 3 in the Nanling Nature Reserve, China”. This article studied how changes in soil nutrient content along an altitude gradient affect soil microbial community composition, specifically gram-positive and gram-negative bacteria, and enzyme activity.

The subject is of interest and the manuscript has potential, but it fits the scope of the journal only in that the soil samples originated from a forest habitat; beyond this, there is no further reference to forests. Moreover, there are considerable problems in all sections of this manuscript.

The introduction is the most developed section and presents the subject well. However, there is no reference to the habitat(s) and how this influences microbial community composition and activity. The material and methods section is sorely insufficient. It is missing individual descriptions for each of the sites, the plant community where the samples were taken, differences in climate, temperature, precipitation, etc. The descriptions for most of the analyses is also insufficient. The reader should be able to replicate the analyses conducted with the information provided in the article; that is not the case here. The statistical section is also does not have enough information to understand what they did. The results section is riddled with issues, reflecting poor proofreading.

Similarly, there are incorrect statements in the discussion, which I found to be generally poorly written. The beginning reads like a continuation of the results section and has minimal development, extrapolation, or comparing to the literature. The structure of the discussion also does not lend itself to clearly addressing the hypotheses. Whether the hypotheses were supported or not should also be stated at the beginning of the paragraphs and then elaborated more in the subsequent sentences/paragraphs. This was not the case. It is also good practice to restate the hypotheses in the discussion.

Moreover, there was also no discussion on the potential influences of the habitat, e.g. under which plants, the soil was taken or the differences of the environmental conditions, e.g. temperature and precipitation, between the sampling points. There should also be included a section on the limitations of their experiment. Differences in soil C and nutrient availability are not the only factors that change with altitude, and the myriad of factors that change were not controlled neither mentioned in this manuscript. This is a major caveat and limitation of their study.

For these reasons, I cannot recommend this manuscript for publication unless considerable changes are made.

 

Title

Throughout the manuscript ‘altitude’ is used, but in the title ‘elevation’ is used.

Introduction

Line 22: It would be good to indicate the three altitudes earlier in the abstract, because here I have no idea whether 1400 m is the highest, lowest, or middle altitude studied.

Line 37-39: there is a comma missing that delineates the end of the examples. I imagine it should go after ‘nutrient availability’?

Line 51-52: there are also a myriad of other drivers driving GP:GN ratios beyond soil C availability.

Line 51: In the manuscript, sometimes a space is included after the colon and sometimes not, this needs to be standardized throughout the document. Normally, there is no space, e.g. GP:GN.

H2: GP and GN don’t have the same physiology and thus do not have the same tolerance to different stresses, GP bacteria being considered the more resistant of the two. Differences in altitude are associated with large differences in environmental stresses such as temperature, water availability, and season length. Would the higher stress levels of higher elevations not favor GP bacteria?

I find the introduction is missing information on the habitats this soil is coming from. The plant communities have a huge affect on the microbial community composition.

 

Material and Methods

Line 95: are all the sites sampled forested? Were there the same plant species present at each site? Were the samples taken under the same plant species? This plays a huge role in the community composition.

Line 94: what was the average temperature per site?

Line 96: So, all elevations were sampled at the same time? The higher altitudes are not going to be at the same physiological stage as the lower altitudes. How did you take this into account?

Line 96: I don’t follow how your sites were distributed. The reserve ranges from 1000-1400 m and you say that you sampled every 200 m; that would give you three sites: 1000 m, 1200 m, and 1400 m. More description is needed here.

Line 97: what is the slope aspect at each sampling location? This plays a large role in nutrient retention, particularly in areas of heavy precipitation such as monsoon climates.

Line 100-101: I don’t understand what you mean by ‘soil samples were homogenized in each layer’. Do you mean the 5 soil cores at each site were homogenized by depth?

Line 105: Do you have the stone content, bulk density, and organic material content, or general estimations, per soil layer and per site? Was the soil more stoney at higher elevations? What is the soil texture and soil type? These are very important variables influencing microbial community, are highly heterogeneous over small spatial scales, and could have influenced your results.

Line 108: what solution was used for the pH measurement and what was the concentration of the solution?

Line 108: The TOC, TN, and TP was measured on how much soil? was this done on dried soil? Please provide more details on these methods.

Line 113: How much soil was used for the PLFA? How were the PLFA extracts obtained? Please provide more information on this analysis and a reference for the method/protocol followed.

Line 128: oxford/Harvard comma missing before the ‘and’.

Line 127-131: This sentence is long and difficult to follow, particularly with all the with all the ‘respectively’ sections. Please restructure.

Line 144-3: there should be a space between “C-“ and “versus” on these two lines.

Line 147: Please add package references for all packages used.

Line 148: All data were log-transformed or data were log-transformed when necessary? If all data was log-transformed, it is not necessary to state “when necessary”.

Line 149: It is not clear to me how exactly you applied the ANOVA tests. Was each plot, depth, etc. run individually? How did you account for non-independence? It is also not clear to me after reading the material and methods how many observations you are working with. You have 9 plots x 2 depths (the 5 cores were homogenized per plot), does that mean you only have 18 observations per analysis? Did you have laboratory replicates? There is a lot of information missing.

Line 154 soil microbial community composition soil microbial community composition

 

Results

Line 162-163: It is not completely clear what you mean by ‘at each soil depth’. The variables are similar along the altitudes, but are they different between the soil depths?

Line 163-164: Did the preceding sentence state that the variables were similar along the altitudes? But here you are saying they are different. The first sentence likely needs to be rephrased to be clearer. Also, there is no need to have ‘m’ in parentheses.

Line 164: Arriving at this point, it was never stated how many elevation levels you had. It would be good to state in the material and methods that you measured every 200 m for a total of 3 altitude points. It would then make this sentence clear that you are referring to all three points and not omitting any.

Line 174: Please move the legend above the table. The ‘notes’ should also just be a part of the table legend. There is not ‘±SE’ included in the table, is this suppose to be the values in parentheses? This is the first time I see any reference to the observation number (n=15), this should be indicated in the material and methods and where is this 15 coming from? You only mentioned 9 plots and had no references to replicates.

Line 174: Please indicate that the capital letters indicate significant differences between the averages; I was confused as to why there were not capital letters for each altitude. Also, why did you not compare the different depths at each altitude?  The basis of your hypotheses is comparing the different altitudes; exploring the difference in averages does not help you address your hypotheses.

Line 181: ‘across all soil depths’ is odd here, you only have two depths and you already indicated them in the sentence. Perhaps you meant ‘across all altitudes’, but you only looked at the average so you do not have this information.

Line 182: please remove the extra space after ‘Figure 1) .’

Line 186: If you say ‘at all altitudes’, it gives the reader the impression that you compared the different soil depths at all altitudes, which you did not. You should indicate more clearly that the when you compare the depths you are comparing the averaged values of all altitudes.

Line 188: You are also missing a period here, and the notes should be a part of the table legend above the table. There is also no need for a space between the parentheses and ‘nmol’. Also same issue with Table 1, there is no ‘±SE’.

Line 212: Reference to the two different panels (a) and (b) need to be included in the legend.

Line 215: ‘gradients’ is not correct here since it is a single altitude point. Best to have just ‘among altitudes’ or ‘altitude points’.

Line 219: it should be indicated that this is the percent explained by the two axes.

Line 221: It is not possible to state what bacterial group was correlated with what variables, you can only speak in terms of the ratio. For example (Fig 3a), there is a higher GP:GN ratio correlated with higher soil P and soil N, however you do not know what caused this change in ration. This increased ratio could be due to a decrease in GN with no change in GP, so GP is not positively correlated to soil PN but instead GN is negatively affected by soil PN.

Line 225: This should say ‘GP bacteria were positively correlation to the soil C:P and N:P ratios’. However, again, you cannot extrapolate to the bacterial group; you can only speak in terms of the influence on the ratio.

Line 221: There is no Figure 2c, this should be Fig. 2, but then you are referencing Fig. 3 before Fig. 2. Figures need to be reorganized and references in the text corrected.

Line231: what do you mean by ‘enzyme activity patterns were largely consistent across altitude at each soil depth’? Do you mean the patterns were consistent between the soil depths? This is not the case for BG activity.

Line 232: This is incorrect, BG increased with altitude in the surface soil, but was highest at 1200 m in subsurface soil.

Line 234: this should be 1000 m not 1200 m. The difference between 1200 and 1400 is not significant for AG. For this entire paragraph, the data does not show the differences to be as cut and dry as stated; there is a lot of overlap of the groups, i.e. lots of ‘ab’s. This nuance should be clearly stated in the text.

Line 235-237: There is no significant variation in vector angle so how can it be negatively related to vector length? Moreover, there was no statistical comparison of these two variables and Fig. 3 does not even show vector angle, only vector length.

Line 237: You cannot say ‘Regardless of altitude’ since you did not explore the differences between soil depths at different altitudes, you only explored the average of all altitudes.

Line 260, 262, and 365: Are the uppercase ‘B’ and ‘A’ here errors?

Line 241: missing period.

Line 270: again the note should be part of the table legend.

Line 290: ‘BG activity positively correlated with GP and GN bacteria and negatively correlated with the GP: GN ratio’. These results seem odd, though possible if the positive correlation between BG and GN is stronger than between BG and GP.

Line 292-294: vector length is positively correlated with GP:GN at the surface soil depth not at the subsurface depth.

Line 295: the comma before the ‘and’ should be removed.

Line 299: Period missing

Table 4: Why are the asterisk for AP – GN a light gray? Again, the note should be part of the legend.

 

Discussion

Line 303-304: Please provide the citations for the statement ‘consistent with previous studies’. Table and figure references do not belong in the discussion and should be removed. You also write ‘low temperatures simultaneously constraining…’, simultaneously as what?

Line 303-308: This could be a single sentence.

Line 308: it is typically better practice to state ‘contrary to H1’ instead of ‘unexpectedly’.

Line 309: You did not conduct any measurements to verify that C was actually limited in the system, therefore you should state ‘increasing potential C limitation’.

Line 310: again ‘contrary to our second hypothesis’ oppose to ‘unexpectedly.

Line 317-318: the second clause of this sentences is incomplete and grammatically incorrect.

Line 319: ‘This is reflected with the GP:GN ratio being the lowest…’ would be better than ‘thus’.

Line 323: This belongs in the results section. Also, please make the label ‘GP:GN ratio’ larger, it is barely visible.

Line 325: Indicate what analysis this is referring to. Is this the GP:GN ratio from the PLFA analysis?

Line 334: remove the table reference.

Line 336: Again, you cannot extrapolate that there is more P available from the N:P ratio. The lower ratio could be due to a decline in N and no difference in P. Refer to the P concentration value in Table 1 instead of trying to extrapolate from the ratio.

Line 338-339: H2 predicted ‘GP: GN ratio would decrease with altitude and increase with soil depths’. As you stated a few lines above, both GP and GN decreased and moreover Table 2 and Figure 1 both indicate that the GP:GN ratio was higher in the subsurface soil. Therefore your H2 was not supported.

Line 341: Please restate the H3 otherwise it is very confusing that it is just shoved in here with no elaboration. This is also where you should state whether the hypothesis was supported or not.

Line 342: ‘as’ should not be used as a replacement to ‘because’. Moreover, you can only extrapolate that this was seen due to enzyme stoichiometry. Therefore ‘likely because’ is more suited for this context.

Line 343-344: ‘confirmed’ is too strong, ‘support’ is better suited. Also, what do you mean by ‘dominated’, did you mean ‘determined’? ‘available soil C’

Line 345: it depends partially on microbial biomass, not completely.

Line 348: ‘Microbes produce 348 more enzymes targeting the increased abundance of substrates’ in what context or as compared to what group?

Line 353: There should not be references to figures in the discussion let alone figures. This goes in the results section.

Line 348-349: With the results you have, you cannot prove that specific enzyme activity decreased with soil depth due to decreased C and nutrient concentrations, correlation does not equal causation. You can merely state that it is likely due to or likely caused by.

Line 369-372: this statement is too strong, again correlation does not equal causation. The decline in soil C and nutrient concentrations likely caused or likely reduced the seen patters.

Line 378-382: this sentence is too long, please cut in two.

Line 388: The results support, they do not confirm.