Review Reports - How Elemental Stoichiometric Ratios in Microorganisms Respond to Thinning Management in <i>Larix principis-rupprechtti</i> Mayr. Plantations of the Warm Temperate Zone in China

Round 1

Reviewer 1 Report

Forests-1190098

How Elemental Stoichiometric Ratios in Microorganisms Respond to Thinning Managements in Larch Plantations of Warm Temperate Zone in China

Cai et al. present an analysis of the stoichiometric ratios of important elements among the microorganisms living soils under managed forests, and show that thinning of trees leads to changes in microbial relative abundances as well as above-ground changes. The use of English in the paper is generally good but does need some improvement, which I think could be achieved with thorough proof-reading, paying particular attention to scientific conventions such as the use of italics in organism names.

Detailed comments below.

Why is Larch in italics? Larch refers to trees in the genus Larix. If the authors are referring to a mixture of species within the genus, please change Larch to Larix spp. (in italics) or Larix sp. (in italics) if only one species in the genus is considered, but the exact species identity is unknown. The genus and species name is used correctly in the Introduction, but sometimes incorrectly in the Materials and Methods, and variably throughout the paper.

I would like to see the word “proved” removed from this manuscript. The results of this study are interesting and useful, and provide some good evidence regarding nutrient cycling in managed forests, but “proved” is too strong a word for a study like this. Words such as “demonstrated”, “suggested”, “indicated”, “showed” are more appropriate.

Abstract

The word nitrogen appears in a different font in the sentence “Combining Pearson correlation …. alteration of the microbial biomass carbon : nitrogen was primarily”…

Materials and Methods

I leave the decision to the type-setters at Forests, but the way the equations are presented is different from how I usually see equations in similar journals.

Results

This is the first mention of harvest year. Are you referring to the soil sampling in 2017 and 2018, or to an as-yet-undescribed removal of trees?

How does the variable Bacteria biomass differ from the variables Gram-positive bacteria biomass and Gram-negative bacteria biomass? Is Bacteria simply the sum of G+ and G-?

Discussion

I thought the discussion section was well-written, generally, and the ideas were developed nicely.

Author Response

Response to Reviewer Comments

(Manuscript No. Forests-1190098)

Dear Reviewer 1,

Thank you very much for the time you have spent handling our manuscript entitled “How Elemental Stoichiometric Ratios in Microorganisms Respond to Thinning Managements in Larch Plantations of Warm Temperate Zone in China”. The comments were very positive and constructive, and we appreciate the opportunity to address the large number of excellent suggestions and calls for clarification in a revised version of the manuscript.

We feel that the manuscript has now been substantially improved, and hope that this version will be suitable for publication in Forests.

General comments:

Point 1: Why is Larch in italics? Larch refers to trees in the genus Larix. If the authors are referring to a mixture of species within the genus, please change Larch to Larix spp. (in italics) or Larix sp. (in italics) if only one species in the genus is considered, but the exact species identity is unknown. The genus and species name is used correctly in the Introduction, but sometimes incorrectly in the Materials and Methods, and variably throughout the paper.

Response: Thanks very much for the useful suggestion. I'm sorry, sometimes the incorrectly names of genera and species are used in the Materials and Methods or elsewhere. The larch referred to in this paper is Larix principis-rupprechtii Mayr. So we have re-check the species names throughout the paper and rewrote them.

Point 2: I would like to see the word “proved” removed from this manuscript. The results of this study are interesting and useful, and provide some good evidence regarding nutrient cycling in managed forests, but “proved” is too strong a word for a study like this. Words such as “demonstrated”, “suggested”, “indicated”, “showed” are more appropriate.

Response: Thank you for your comments, and we totally agree that the word “proved” is too strong for our study. We used“demonstrated”, “suggested”, “indicated” or “showed” instead of “proved”. Please see line 768 and line 1130. .

Point 3: The word nitrogen appears in a different font in the sentence “Combining Pearson correlation …. alteration of the microbial biomass carbon : nitrogen was primarily”…

Response: Thank you for your comments. We have re-check the font in the sentence and rewrote the font of word nitrogen. Please see line 25.

Point 4: I leave the decision to the type-setters at Forests, but the way the equations are presented is different from how I usually see equations in similar journals.

Response: Thank you for your comments. We have re-check the equations and rewrote them. Please see line 395-399.

Point 5: This is the first mention of harvest year. Are you referring to the soil sampling in 2017 and 2018, or to an as-yet-undescribed removal of trees?

Response: Thanks you. In our study, the “harvest year” means sampling time. However, in paragraph 3.1, we would like to show the effect of thinning managements on soil physical and chemical properties, so we delete this sentence and rewrote paragraph 3.1. Please see line 531-532.

Point 6: How does the variable Bacteria biomass differ from the variables Gram-positive bacteria biomass and Gram-negative bacteria biomass? Is Bacteria simply the sum of G+ and G-?

Response: Thanks. We conducted PLFA analysis to evaluate the soil microbial community groups. The application of the PLFA method has limitations because the classification of PLFAs into microbial groups is imprecise. Nevertheless, it is still a simple and fast procedure and is proven to be sensitive to microbial community shifts. So we grouped PLFAs according to specific microbial community markers: gram-positive bacteria were the sum of i13:0, i14:0, a14:0, a15:0, i15:0, i16:0, a17:0, i17:0, i18:0; gram-negative bacteria were the sum of 10:0 2OH, 15:1 w4c, 15:1 w6c, 16:0 2OH, 16:1 w6c, 16:1 w7c, 16:1 w9c, cy17:0, 17:1 w8c, 18:1 w5c, 18:1 w7c, cy19:0; bacteria were the sum of gram-positive, gram-negative bacteria, 14:0, 15:0, 17:0, and 18:0. Gram-positive bacteria, gram-negative bacteria, bacteria, fungi, actinomycetes and their fatty acid biomarkers were presented in Table S2.

In fact, such application of the PLFA methods have recently been investigated at different forest ecosystems. For example, Qiu et al. (2019) found that the concentration of phospholipid fatty acids (PLFA) for gram-positive bacteria, gram-negative bacteria, bacteria varied among different stand density plantation. Yang et al. (2017) indicated that small gaps and closed canopies had a strong impact on the concentration of phospholipid fatty acids (PLFA) for gram-positive bacteria, gram-negative bacteria, bacteria, fungi, and total PLFAs. Therefore, we grouped PLFAs according to specific microbial community markers in this manuscript.

Qiu, X.C.; Peng, D.L.; Wang, H.B.; Wang, Z.Y.; Cheng, S. Minimum data set for evaluation of stand density effects on soil quality in Larix principis-rupprechtii plantations in North China. Ecol. Indic. 2019. 103, 236-247. doi: https://doi.org/10.1016/j.ecolind.2019.04.010.

Yang,Y.; Geng, Y.; Zhou, H.; Zhao,G.; Wang,L. Effects of gaps inthe forest canopy on soil microbial communities and enzyme activity in a Chinese pine forest. Pedobiologia 2017. 61, 51-60. doi: 10.1016/j.pedobi.2017.03.001.

Point 7: I thought the discussion section was well-written, generally, and the ideas were developed nicely.

Response: Thank you for your comments on my discussion.

Point 8: The use of English in the paper is generally good but does need some improvement.

Response: Thanks very much for the useful suggestion. Now we have re-checked the use of English in the manuscript, and rewrote them. The detailed modification has been clearly emphasized in the manuscript with the function of "tracking changes".

Author Response File: Author Response.doc

Reviewer 2 Report

Dear Authors,

the manuscript entitled “How Elemental Stoichiometric Ratios in Microorganisms Respond to Thinning Managements in Larch Plantations of Warm Temperate Zone in China” applied ecological stoichiometry to a larch plantation established in 1981, where tree density was lowered in 2007 and again in 2012, when 4 different thinning treatments were performed. The concept behind the work, to understand the how the different tree densities impact the microbiology and the elements cycling, is quite novel, fits the scope of the journal and may result of interest for the readers both in the microbiology field and from the biogeochemistry area.

The manuscript needs some major revisions: 1) reduce the introduction by 30% resuming important points and avoiding repetition (for example the concept of soil hydrothermal conditions affected by tree density, impacting on soil microorganisms is repeated at least three times!); 2) rethink the figures presented putting data of secondary importance in supplementary material (soil temperature) and presenting relevant parameters: I couldn’t access to supplementary material where the authors relegate very important results (total carbon (STC), nitrogen (STN), phosphorus (STP) and available nutrients (NO3--N, NH4+-N and SAP) ); 3) reduce the discussion, again, avoiding repeating concepts and taking more care of the correctness of the text: the discussion presents references to tables that aren’t in the text (Table 5, 6 and 7) or point to different results (Figure 1 is cited referring to P content while it represents soil temperature).

Some additional punctual comments:

- Unfortunately the text I’ve been able to download did not have line numbering-

First Line in paragraph 2.1 - Please define the tree dominant specie or species in your experimental field with full scientific name i.e Larix principis-rupprechtii.

First Line in paragraph 2.2 - Please provide the sampling depth. Also, the depth of soil temperature recording. Please provide the method you used to record soil water content (did you use a sensor?). In the same paragraph the sentence “However, test samples were need extracted with 0.5 M NaHCO3 ““ seems incorrect

Results, Paragraph 3.1: I suggest putting the Table S3 in the text and leave Figure 1 to supplementary material. The sentence “Soil moisture and pH value ranged from 31.6% to 53.3% and from 6.07 9.5 °C to 6.3 15.5 °C, respectively (Table S3).“ seems incorrect to me. How was air temperature in the field in correspondence with the soil temperature you recorded?

Figure 1 - The lettering seems wrong to me: How can MDP 2018 be statistically not different to LDP 2017? How can HDP2017 be not different to HDP2018, MDP2018, LDP 2018??

“The carbon content of litter was higher in MDP and LDP than that of high density plantation and control site. ” Given the averages you present this is not correct: LDP doesn’t present higher biomass than HDP (58.01 and 60.44 respectively).

Table 2: statistics here is not clear: did you compare all values in the two years or you compared within each year? For microbial biomass N how could 171.94± 1.49 be not different to 123.93±9.84 (same letter)?? Please double check all these data.

Figure 3: Again weird lettering for total PLFAs and Bacteria. In bacteria how can 2018 HDP be labelled ab when 2017 MDP value is labelled b? Please check again.

Figure 5 caption. Please change “Relationship” with “Pearson Correlation”

Table 4 caption. Please change “Linkage” with “Pearson Correlation”

Paragraph 4.2: please double check the table numbers you refer to.

Author Response

Response to Reviewer Comments

(Manuscript No. Forests-1190098)

Dear Reviewer 2,

We feel that the manuscript has now been substantially improved, and hope that this version will be suitable for publication in Forests.

Major comments:

Point 1: reduce the introduction by 30% resuming important points and avoiding repetition (for example the concept of soil hydrothermal conditions affected by tree density, impacting on soil microorganisms is repeated at least three times!);

Response: Thank you for your comments. We agree that part of the introduction may be repetition. Now we have checked the introduction and rewrote them. For example, “Moreover, thinning treatment could alter the composition of soil microorganisms by adjusting soil hydrothermal condition and light intensity [17, 18]. These results indicate that the concentration of soil elements and soil microbial communities may have a great response to changes of forest management and changes of plant and litter characteristics.” has been deleted, and added the sentence “As a result, soil microbial communities may responsed strongly the changes in plant and litter characteristics after forest thinning management.” Please see Line: 80-82. “Ren et al. (2016) [8] found that soil microbial biomass ratio can be very sensitive to variation in soil pH, moisture, and nutrient concentrations following afforestation.” has been deleted. Other detailed revisions have been clearly emphasized in "introduction" with the "Tracking Change" function.

Point 2: rethink the figures presented putting data of secondary importance in supplementary material (soil temperature) and presenting relevant parameters: I couldn’t access to supplementary material where the authors relegate very important results (total carbon (STC), nitrogen (STN), phosphorus (STP) and available nutrients (NO₃^--N, NH₄⁺-N and SAP) );

Response: Thank you for your comments, and we have put Table S3 in the manuscript, please refer to Table 1 in line 536-538. Meanwhile, Figure 1 has been moved to supplementary material.

Point 3: reduce the discussion, again, avoiding repeating concepts and taking more care of the correctness of the text: the discussion presents references to tables that aren’t in the text (Table 5, 6 and 7) or point to different results (Figure 1 is cited referring to P content while it represents soil temperature).

Response: Thank you for your comments. We agree that part of the discussion may be repetition. Now we have checked the discussion and rewrote them. For example, “Li et al. (2019) [60] results showed that fungi abundance, bacteria abundance, and nutrient element concentration were the important factors related to the microbial biomass stoichiometric ratio.” has been deleted. Other detailed revisions have been clearly emphasized in "discussion" with the "Tracking Change" function. Moreover, we have double check the table numbers in discussion, please see line: 769, line 771, line 1101-1102, and line 1106.

Point 4: Unfortunately the text I’ve been able to download did not have line numbering.

Response: Thanks you. Considering your comments, we have added the line numbers in the whole manuscript.

Point 5: First Line in paragraph 2.1 - Please define the tree dominant specie or species in your experimental field with full scientific name i.e Larix principis-rupprechtii.

Response: Thanks very much for the useful suggestions. We have add “Larix principis-rupprechtii Mayr.” at first line in paragraph 2.1 as suggestion, please see Line: 371. Moreover, we have re-check the species names throughout the paper and rewrote them.

Point 6: First Line in paragraph 2.2 - Please provide the sampling depth. Also, the depth of soil temperature recording. Please provide the method you used to record soil water content (did you use a sensor?). In the same paragraph the sentence “However, test samples were need extracted with 0.5 M NaHCO₃ ““ seems incorrect

Response: Thanks very much for the useful suggestions. In this study, soil samples were collected at the depth of 0-10 cm. We have provide the sampling depth at first line in paragraph 2.2. Please see line 401. Soil water content (SWC) was measured by oven-drying to constant mass at 105°C. We have add the detail methods in paragraph 2.2, please see line 440-441.

In addition, microbial biomass P was analyzed through fumigation–extraction method as well. Half of the fresh samples were needed to fumigate with chloroform in a vacuum (at least 24 h). However, the extraction process was used 0.5 M K₂SO₄ instead of 0.5 M K₂SO₄, and then used the colorimetric method to measure the concentration of biomass phosphorus. Now we have rewrote the sentence “However, test samples were need extracted with 0.5M NaHCO₃”, please see Line: 449-453.

Point 7: Results, Paragraph 3.1: I suggest putting the Table S3 in the text and leave Figure 1 to supplementary material. The sentence “Soil moisture and pH value ranged from 31.6% to 53.3% and from 6.07 9.5 °C to 6.3 15.5 °C, respectively (Table S3).“ seems incorrect to me. How was air temperature in the field in correspondence with the soil temperature you recorded?

Response: Thank you for your comments. We have put Table S3 in the manuscript, please refer to Table 1 in line 536-538. Meanwhile, Figure 1 has been moved to supplementary material. In addition, we have re-examined the sentence “Soil moisture and pH value ranged from 31.6% to 53.3% and from 6.07 9.5 °C to 6.3 15.5 °C, respectively (Table S3)”, and deleted “9.5 °C and 15.5 °C”. Please see line 531-532.

Point 8: Figure 1 - The lettering seems wrong to me: How can MDP 2018 be statistically not different to LDP 2017? How can HDP2017 be not different to HDP2018, MDP2018, LDP 2018??

Response: Thank you for your comments. In Figure 1, different lowercase letters mean the variation of soil temperature between treatments (P<0.05). We have re-wrote the note in Figure 1, please see Figure S1 in supplementary material.

Point 9: “The carbon content of litter was higher in MDP and LDP than that of high density plantation and control site. ” Given the averages you present this is not correct: LDP doesn’t present higher biomass than HDP (58.01 and 60.44 respectively).

Response: Thanks very much for the useful suggestions. In Figure 2, we showed the carbon, nitrogen, and phosphorus content of litter layer in L. principis-rupprechtii plantations with different density. In fact, Figure 2 does not shown the carbon storage in litter layer, but the carbon content of litter layer might not be proportional to the increase of litter layer biomass or thinning intensity. For example, Ge et al. found that maximum litter layer biomass was 14.538 t hm-2 in Eucalyptus grandis x E.urophylla plantation, but the highest carbon content of litter layer was found in Acacia cincinnata F.Muell. plantation.

Meanwhile, previous studies showed that ecological factors, such as aboveground plant composition, soil properties, microbial communities, and climate factors, primarily drive the nutrient contents (including carbon, nitrogen, and phosphorus) in litter layers (Rui et al. 2019; Morrison et al. 2019; Li et al. 2018). In this study, we found that the carbon content of litter was higher in MDP and LDP than that of high density plantation and control site (Figure 1). I feel sorry that figure 2 may be misleading the effect of thinning treatment on litter properties, so we have checked the data in figure 2 and re-made it. However, considering that Figure 1 should be moved to supplementary material. Now, we have replaced Figure 2 with Figure 1, please see Line: 613-615.

Ge L.L.; He Z.M. et al. Biomass and litter carbon and nitrogen return of different plantations in the sandy coastal plain area. Journal of Northwest Forestry University. 2019. 34(1): 39-46. (In Chinese)

Rui Y.; Nico E., et al. Additive effects of experimental climate change and land use on faunal contribution to litter decomposition. Soil Biology and Biochemistry. 2019. 131. doi: 10.1016/j.soilbio.2019.01.009.

Morrison E.W.; Pringle A.; Diepen, V.; et al. Warming alters fungal communities and litter chemistry with implications for soil carbon stocks. Soil Biology and Biochemistry. 2019. 132(4). doi: 10.1016/j.soilbio.2019.02.005

Li Y.B.; Bezemer T.M.; Yang J.J. et al. Change in litter quality induce by N deposition alter soil microbial communities. Soil Biology and Biochemistry. 2018. doi: 10.1016/j.soilbio.2018.11.025.

Point 10: Table 2: statistics here is not clear: did you compare all values in the two years or you compared within each year? For microbial biomass N how could 171.94±1.49 be not different to 123.93±9.84 (same letter)?? Please double check all these data.

Response: Thank you for your comments. Different lowercase letters indicate significant differences between treatments. So we have carefully checked all these data in Table 2 and re-made Table 2. As we have put Table S3 in the manuscript (Table 1), we have replaced Table 3 with Table 2, please see Table 3 in Line: 631-634.

Point 11: Figure 3: Again weird lettering for total PLFAs and Bacteria. In bacteria how can 2018 HDP be labelled ab when 2017 MDP value is labelled b? Please check again.

Response: Thank you for your comments. In Figure 3, different lowercase letters mean the variation of soil temperature between treatments (P < 0.05). We have re-wrote the note in Figure 3. But considering that Figure 1 should be moved to supplementary material. Now, we have replaced Figure 3 with Figure 2, please see Line: 662-664.

Point 12: Figure 5 caption. Please change “Relationship” with “Pearson Correlation”

Response: Thanks very much for the useful suggestions. In Figure 5, we used regression analyses to evaluate relationships between soil microbe element ratios and microbe community biomass. So we may use “Relationship” more suitable in the caption of Figure 5. Hope you can understand.

Point 13: Table 4 caption. Please change “Linkage” with “Pearson Correlation”

Response: Thanks very much for the useful suggestions. We quite agree that we may use “Pearson Correlation” more suitable in the caption of Table 4. We have re-wrote the caption of Table 4, please see Line: 740-742.

Point 14: Paragraph 4.2: please double check the table numbers you refer to.

Response: Thanks very much for the useful suggestions. We have double check the table numbers in Paragraph 4.2 and elsewhere, please see line: 769, line 771, line 1101-1102, line 1106.

Point 15: English language and style are fine/minor spell check required.

Author Response File: Author Response.doc

Round 2

Reviewer 2 Report

The manuscript has been improved. Now it's ready for publication

Author Response

Response to Reviewer Comments

(Manuscript No. Forests-1190098)

Dear Reviewer 2,

We feel that the manuscript has now been substantially improved, and hope that this version will be suitable for publication in Forests.

Point 1: The manuscript has been improved. Now it's ready for publication.

Response: Thank you for your comments. I'm very grateful to reviewer 2 for being satisfied with my revision.

Author Response File: Author Response.pdf