Review Reports - Effects of Simulated Warming on Bacterial Community Structure in Litters of Chinese Fir Based on Displacement Test

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Effects of Simulated Warming on Bacterial Community Structure in Litters of Chinese Fir Based on Displacement Test" is interesting.

In this study, a displacement test was used to simulate the effects of warming (+2.5, +4.6, and +6.5 ℃) on the composition and diversity of bacterial communities in litters of Chinese fir based on the natural temperature differences between the elevations of Mt. Wuyi. It is in line with the scope of the special issue of the journal Forests.

An abstract is a condensed summary of the manuscript. It does not raise any doubts.

The introduction is well written. It is a synthetic description of the literature on the researched topics and a good justification for the research conducted.

The Materials and Methods section needs some additions. Under Table 1, the abbreviation DBH should be explained. In Table 2, homogeneous groups should be indicated next to the standard deviations. In subsection 2.4, it is advisable to provide more details, as some statements are not understandable. In this subsection, the authors write that the incubation to determine enzyme activity was performed for 1 h and the results presented in Figure 6 are expressed in d. It would be good to provide units of activity, whether they refer to the amount of substrate used or the amount of product produced.

It would be good if the authors explained how they obtained the results shown in Figure 5.

Results is well and clearly edited. My only suggestion is to improve the quality of Figure 4.

The discussion is well developed. The results are well explained against the background of the literature on the subject.

The conclusions result from the scope of the research conducted and are a good summary of the issues examined.

References need to be corrected, because out of 45 cited publications, as many as 15 are from ten years ago.

Author Response

Response: Dear reviewer, thank you for your suggestions to make the article better. We are very grateful to the critical comments and thoughtful suggestions from reviewer and editor. Based on these comments and suggestions, we have carefully modified manuscript according to our best knowledge. All comments are addressed below in the revised manuscript. The line numbers referred to below are in the revised manuscript

Point 1: An abstract is a condensed summary of the manuscript. It does not raise any doubts.

The introduction is well written. It is a synthetic description of the literature on the researched topics and a good justification for the research conducted.

Response 1: Dear reviewer, thank you for your approval of this article.

Point 2: The Materials and Methods section needs some additions. Under Table 1, the abbreviation DBH should be explained. In Table 2, homogeneous groups should be indicated next to the standard deviations. In subsection 2.4, it is advisable to provide more details, as some statements are not understandable. In this subsection, the authors write that the incubation to determine enzyme activity was performed for 1 h and the results presented in Figure 6 are expressed in d. It would be good to provide units of activity, whether they refer to the amount of substrate used or the amount of product produced.

Response 2: Dear reviewer, thanks for your comments. Under Table 1, “DBH: diameter at breast height” has been added. (P3, L137); In Table 2, we have indicated homogeneous groups to the standard deviations. And more details information was added in subsection 2.4.

Urease activity was determined using the indophenol blue method [26], the litter homogenization was incubated with 0.5 mL of 10% urea solution and 1 mL of citric acid buffer at 37 ℃ for 24 h. (P4, L171-172);

The results of enzyme activity were calculated (1 h = 1/24 d). Since the activity of sucrase and peroxidase were cultured for 24 h, we uniformly used d to denote them.

Point 3: It would be good if the authors explained how they obtained the results shown in Figure 5.

Response 3: Dear reviewer, the Functional Annotation of Prokaryotic Taxa (FAPROTAX) database was used to annotate bacterial community functions and the functional abundance was subjected to a “Hellinger” transformation. (P5, L182-184)

Point 4: Results is well and clearly edited. My only suggestion is to improve the quality of Figure 4. The discussion is well developed. The results are well explained against the background of the literature on the subject.。

Response 4: Dear reviewer, thank you for your suggestions to make the article better. Figure 4 is not clear may probably caused by the large amount of content and the small size, which may be seen more clearly if you zoom in.

Point 5: The conclusions result from the scope of the research conducted and are a good summary of the issues examined.

Response 5: Dear reviewer, thank you for your approval of this article.

Point 6: References need to be corrected, because out of 45 cited publications, as many as 15 are from ten years ago.

Response 6: Dear reviewer, thank you for your suggestions to make the article better. I have added the recently published literature to the manuscript, and the ancient literature was replaced. More details of the revisions are provided in the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors presented a study of the effect of soil temperatures on bacterial communities of Chinese fir litter. The work is of great interest from a methodological point of view. The effect of warming here was modeled using high-altitude thermal gradients under natural conditions of accumulation and decomposition of forest litter. Nevertheless, there are some comments, the answers to which would help readers better understand the research done and the results presented.

lines 40-55. Are there other similar studies of bacterial communities using elevation gradients? Please add to this section or indicate that such a method has not been used directly for bacterial communities.

Section 2.1. Insufficient information about the location of the experiment. It is also necessary to specify the orientation of the slope, the altitudinal nature of soil changes, litter and water regime.

Section 3.2. There is no information about sampling and analysis of background bacterial communities at the experimental sites. There is also no information about the bacterial communities of the litter before placing it on the study sites in the Results.

Section 4. The influence or lack of influence of other important factors, such as moisture, solar radiation levels and undergrowth density, should be indicated. It is also necessary to discuss the relationship between agrochemical indicators of soil, undergrowth and the representation of the bacterial community. For example, the question is very interesting, why does the number of bacteria decomposing cellulose also increase with increasing temperature, and at the same time the nitrogen content also increases?

Author Response

Response: Dear reviewer, we are very grateful to the critical comments and thoughtful suggestions from reviewer and editor. Based on these comments and suggestions, we have carefully modified manuscript according to our best knowledge. All comments are addressed below in the revised manuscript. The line numbers referred to below are in the revised manuscript.

Point 1: lines 40-55. Are there other similar studies of bacterial communities using elevation gradients? Please add to this section or indicate that such a method has not been used directly for bacterial communities.

Response 1: Dear reviewer, we have added relevant similar studies in introduction.

Klimek et al. [11] found that temperature range was the main factor affecting litter respiration rate and microbial biomass in the field experiment of litter translocation along the altitudinal gradient, and microbial biomass increased with the extension of decomposition time. Bohara et al. [12] found that temperature was the best predictor of litter mass loss in the study of in-situ litter decomposition along the elevation gradient. (P2, L52-57)

Point 2: Section 2.1. Insufficient information about the location of the experiment. It is also necessary to specify the orientation of the slope, the altitudinal nature of soil changes, litter and water regime.

Response 2: Dear reviewer, we have supplemented the information about the test sites in Section 2.1.

The soil types from bottom to top are red soil, yellow–red soil, yellow soil, and meadow soil. The vegetation types are subtropical evergreen broad-leaved forest, coniferous mixed forest, coniferous forest, sub-alpine dwarf forest, and alpine meadow, respectively. (P3, L103-106)

Because the temperature between altitudes is highly dependent on slope and insolation, all plots were established on southeastern slopes. (P3, L107-109)

And the average annual precipitation information of each plot was added in Table 1.

Point 3: Section 3.2. There is no information about sampling and analysis of background bacterial communities at the experimental sites. There is also no information about the bacterial communities of the litter before placing it on the study sites in the Results.

Response 3: Dear reviewer, thanks for pointing out this. We did not sample and analyze the background bacterial community in the soil of the experimental sites, which is a pity of this study. There are differences in the abundance and diversity of bacterial communities in soil at different altitudes. But these differences are formed over a long period of time under different temperature conditions. In this study, we believe that when the temperature changes, the soil environment for litter decomposition will inevitably change. However, temperature is the main factor in explaining litter decomposition along an altitudinal gradient.

In this study, experimental materials were collected from a 15-year-old Chinese fir plantation at the Xinkou National Forest Farm. We took the litters back to the laboratory, separated the twigs and leaves, and mixed evenly to ensure consistency in the initial quality and bacterial communities of litter. However, we did not measure the initial bacterial community information of litter. Instead, the specific objectives of this study were to observe the differences in bacterial community structure in litters under different temperature conditions after 360 days of decomposition.

Point 4: Section 4. The influence or lack of influence of other important factors, such as moisture, solar radiation levels and undergrowth density, should be indicated. It is also necessary to discuss the relationship between agrochemical indicators of soil, undergrowth and the representation of the bacterial community. For example, the question is very interesting, why does the number of bacteria decomposing cellulose also increase with increasing temperature, and at the same time the nitrogen content also increases?

Response 4: Dear reviewer, thank you for your suggestions to make the article better. The possible influence of other important factors (soil quality, precipitation and UV-B radiation, forest gaps) was added in section 4.

Soil quality is an important factor affecting the abundance of bacterial communities, and the availability of soil nutrients significantly affects the metabolic turnover of microorganisms [33]. (P10, L316-318);

However, besides temperature among the climatic factors, humidity and radiation may also be relative factors that make bacterial community structure vary with altitudes. Different precipitation and UV-B radiation intensities significantly affect the release rate of easily decomposed components in the litter, and alter the composition of bacterial communities. Ren et al. [42] reported that increased precipitation significantly enhanced soil microbial biomass by 16.18%, which in turn accelerated the decomposition rate of forest litter. Different forest gaps along the altitude gradients significantly affect the microbial biomass in litters. Zhang et al. [43] found that a forest gap of 100–900 square metres was conducive to the increment of microbial biomass carbon and nitrogen during the decomposition of forest litter. (P11, L359-368)

Interestingly, with the rising of temperature, the lignin content, cellulose content and the relative abundance of Bacteroidetes showed a decreasing trend, while nitrogen content showed an increasing trend. This indicates that Bacteroidetes may not be the main bacterial community involved in the degradation of complicated organics in Chinese fir litter. Nitrogen content increased may result from an increase in the species richness and diversity of bacterial communities. Nitrogen is essential for the growth and turnover of microbial communities as it participates in the construction of proteins [36]. (P10, L330-336)

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The study presented here can be considered relatievely interesting, especially with regard to the possible impact that thermal changes associated with the climate crisis may have on the role that bacterial communities play in biodegradation cycles. However, the authors do not go into excessive depth and, in this sense, the article could be more ambitious, both in terms of the taxonomic level at which the analysis is carried out and in the discussion of the results obtained, which is excessively generalist. The authors include too much descriptive content from other works, in many cases without directly relating them to the results obtained. It would be much more appropriate to limit the descriptive content, including the results themselves, and to propose more potential explanations for these results, supported by other research.

Comments:

- The keywords are already, for the most part, included in the title.

- More information should be provided on the massive sequencing process: platform used, database used for taxonomic affiliation, bioinformatic processing of raw data, etc.

- References for enzymatic protocols should be included.

- L. 175 and 181: Planctomycetes and Candidatus_Solibacter do not increase significantly with temperature. Only the maximum level shows differences with the remaining sampling points.

- Some statements should be supported by references:

"Compared to fungi, bacteria play a more important role in the degradation of keratin and aromatic compounds"

"What is noteworthiness is that warming had no significant effects on the Proteobacteria, Actinobacteria, and Acidobacteria abundance, which may be due to their wide ecological ranges and strong adaptability to external temperature change"

- Section 4.3, does not contribute anything.

- In situ should appear in italics.

- Authors abuse the use of commas to the detriment of periods, which in many cases is inadequate.

Comments on the Quality of English Language

- Wuyi Mountain, as the first peak in East China and has obvious vertical distribution characteristics of climate zone [11], making it a good experimental site to carry out this study.

- Bacteria, as heterotrophic organisms, warming changes the content of organic compounds preferentially absorbed by the bacteria, causing changes in the abundance of bacterial communities.

- Sentences describing characteristics of phyla, since they include numerous species, should be formulated with plural verb forms (L. 299 and L. 301).

Author Response

Dear reviewer, we are very grateful to the critical comments and thoughtful suggestions from reviewer and editor. Based on these comments and suggestions, we have carefully modified manuscript according to our best knowledge. All comments are addressed below in the revised manuscript. The line numbers referred to below are in the revised manuscript.

Point 1: The keywords are already, for the most part, included in the title.

Response 1: Dear reviewer, thanks for pointing out this. We have replaced some of the keywords.

Keywords: warming; Chinese fir litters; α-diversity; litter decomposition enzyme activity; chemical property (P1, L28-29)

Point 2: More information should be provided on the massive sequencing process: platform used, database used for taxonomic affiliation, bioinformatic processing of raw data, etc.

Response 2: Dear reviewer, more information about the sequencing process has been added in Section 2.3.

DNA quality and concentration were assessed using 1% agarose gel electrophoresis and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). (P4, L150-151);

The amplified products were purified by AMPure XP Beads (Beckman Agency, Muskogee, OK, USA), and the ABI StepOnePlus Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) was used to quantify the products. The products were sequenced using an Illumina NovaSeq 6000. (P4, L156-159)

Point 3: References for enzymatic protocols should be included.

Response 3: Dear reviewer, thanks for your comment. We have added the references for enzymatic protocols in Section 2.4.

Sucrase activity assays were based on the ability to catalyze the degradation of sucrose to produce reducing sugars, which can produce brown-red amino compounds [25]. Urease activity was determined using the indophenol blue method [26] , the litter homogenization was incubated with 0.5 mL of 10% urea solution and 1 mL of citric acid buffer at 37 ℃ for 24 h. For peroxidase activity, 0.6 mL of 0.3% hydrogen peroxide was added. All reaction mixtures were incubated at 37 ℃ for 1 h and centrifuged for 2 min at 5000 rpm. It catalyzes the oxidation of organic matter to quinone [27]. (P4, L168-175)

Point 4: L. 175 and 181: Planctomycetes and Candidatus_Solibacter do not increase significantly with temperature. Only the maximum level shows differences with the remaining sampling points.

Response 4: Dear reviewer, we have revised the relevant statements.

With the rising of temperature, the relative abundance of Bacteroidetes decreased significantly. The differences of the relative abundance of Planctomycetes between W_6.5 and other conditions were significant. (P5, L194-197);

With an increase in temperature, the relative abundance of Mucilaginibacter and Granulicella significantly decreased, while that of Candidatus_Solibacter were highest at W_6.5. The order of the relative abundance of Acidothermus under different temperature conditions was W_6.5 > W_2.5> W_4.6 > W₀, and the differences between W_6.5, W_2.5, and W₀ were significant. (P5, L200-205);

Warming significantly decreased the relative abundance of Bacteroidetes and that of Planctomycetes significantly increased under 6.5 ℃ warming condition. (P10, L319-321)

Point 5: Some statements should be supported by references:

"Compared to fungi, bacteria play a more important role in the degradation of keratin and aromatic compounds"“

Response 5: Dear reviewer, thanks for your comment. We have cited references to support the relevant statements.

Compared to fungi, bacteria play a more important role in the degradation of keratin and aromatic compounds [29]. (P10, L298-299); 29. Witoon, P.; Tesfaye, W.; Guillaume, L.; Michael, S.; Marek, J.P.; Danuta, K.; Martin, H.; Dirk, K.; François, B. Life in leaf litter: novel insights into community dynamics of bacteria and fungi during litter decomposition. Mol. Ecol. 2016, 25, 4059-4074. (P14, L519-520);

What is noteworthiness is that warming had no significant effects on the Proteobacteria, Actinobacteria, and Acidobacteria abundance, which may be due to their wide ecological ranges and strong adaptability to external temperature change [32]. (P10, L312-315); 32. Jiang, X.; Ma, D.; Zang, S.; Zhang, D.; Sun, H. Characteristics of soil bacterial and fungal community of typical forest in the Greater Khingan Mountains based on high-throughput sequencing. Microbiol. Chin 2021, 48, 1093-1105. (P14, L525-526);

Point 6: Section 4.3, does not contribute anything.

Response 6: Dear reviewer, the main purpose of section 4.3 is to express the innovation of this study in using the natural differences in temperature between the elevation of Wuyi Mountain to carry out the field experiment of litter translocation. And we believe that the natural environment gradient test is better than indoor single-factor controlled simulation experiments.

Point 7: In situ should appear in italics.

Response 7: Dear reviewer, thanks for your comment. We have corrected it in the manuscript.

such as indoor microenvironment simulation (artificial climate chamber [5] and greenhouse [6]) and in situ heating methods (resistance heating cables [7] and open-top chamber [8]). (P1, L43-45);

which can be mainly divided into indoor microenvironment, in situ heating, and natural environment gradient tests [52]. (P12, L411-413);

The in situ heating test is a research method for in situ controlled warming on a small scale. (P12, L417-418);

Point 8: Authors abuse the use of commas to the detriment of periods, which in many cases is inadequate.

Response 8: Dear reviewer, we're sorry about that. We have found relevant organizations to edit the article.

Point 9: Wuyi Mountain, as the first peak in East China and has obvious vertical distribution characteristics of climate zone [11], making it a good experimental site to carry out this study.

Response 9: Dear reviewer, thanks for your comment.

Wuyi Mountain is known as the first peak in East China and has obvious vertical distribution characteristics of climate zone [13], making it a good experimental site for this study. (P2, L59-61)

Point 10: Bacteria, as heterotrophic organisms, warming changes the content of organic compounds preferentially absorbed by the bacteria, causing changes in the abundance of bacterial communities.

Response 10: Dear reviewer, thanks for your comment.

Bacteria as a heterotrophic organism, warming alters the content of organic compounds preferentially absorbed by the bacteria, causing changes in the abundance of bacterial communities. (P10, L324-326)

Point 11: Sentences describing characteristics of phyla, since they include numerous species, should be formulated with plural verb forms (L. 299 and L. 301).

Response 11: Dear reviewer, thanks for your comment.

A recent study has shown that Proteobacteria are crucial in the oxidation and energy metabolism of inorganic and organic compounds, whereas Actinobacteria are mainly involved in the degradation of complex carbohydrate, both of which are important saprophytic bacteria in the litter decomposition process [31]. (P10, L309-312);

Previous studies have found that Planctomycetes are primarily responsible for the degradation of polycyclic aromatic compounds and promote the mineralization of organic carbon [34], whereas Bacteroidetes are mainly involved in nitrification and the degradation of complex biomacromolecules [35] (P10, L326-329);

The Mucilaginibacter abundance were closely related to lignin and cellulose contents, and Acidothermus were related to N/P and K content (Figure 7B). (P10, L338-340)

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have made certain changes in consideration of the suggestions received. Some of them are correct, while others are less so.

- The use of plural verb forms referring to filos has been modified, but also in reference to genders, even when these are modified by singular nouns. Please correct that (example: the abundance of Mucilaginibacter was related...).

- Scientific names are only italicized for the taxonomic level of genus and below. This is not the case for phyla.

- Although some additional information has been provided regarding the massive sequencing process, it is still insufficient. It is not indicated whether OTUs or ASVs have been used, with which platform the taxonomic assignments have been made, etc.

- Regarding section 4.3., I understand the authors' argumentation. However, since no comparative study with other methodologies is made, it would be more appropriate to comment on it in the Introduction.

Comments on the Quality of English Language

See comments for authors

Author Response

The authors have made certain changes in consideration of the suggestions received. Some of them are correct, while others are less so.

Point 1: The use of plural verb forms referring to filos has been modified, but also in reference to genders, even when these are modified by singular nouns. Please correct that (example: the abundance of Mucilaginibacter was related...).

Response 1: Dear reviewer, thanks for pointing out this. We are sorry about that, the relevant content had been corrected.

With an increase in temperature, the relative abundance of Mucilaginibacter and Granulicella significantly decreased, while that of Candidatus_Solibacter was highest at W_6.5.(P5, L206-208);

The abundance of Mucilaginibacter was closely related to lignin and cellulose contents, and the abundance of Acidothermus was related to N/P and K content (Figure 7B). (P11, L345-346)

Point 2: Scientific names are only italicized for the taxonomic level of genus and below. This is not the case for phyla.

Response 2: Dear reviewer, thanks for pointing out this. we have revised the relevant statements.

Warming significantly increased the relative abundance of Acidothermus and decreased that of Bacteroidetes and Mucilaginibacter. The relative abundance of Planctomycetes exhibited a positive association with nitrogen content and a negative association with cellulose content, whereas phosphorus content was the main driving factor of Acidothermus abundance. (P1, L19-24);

At the phylum level, the relative abundance of Planctomycetes, Proteobacteria, Actinobacteria, and Acidobacteria were more than 10%. With the rising of temperature, the relative abundance of Bacteroidetes decreased significantly. The differences of the relative abundance of Planctomycetes between W_6.5 and other conditions were significant. Warming had no significant effects on the relative abundance of Actinobacteria, Proteobacteria, and Acidobacteria. (P5, L199-205);

Some phyla had a strong association with chemical properties of the litter, such as Planctomycetes, Bacteroidetes, and Verrucomicrobia. (P8-9, L268-269)

Song [30] simulated the effects of soil warming on bacterial communities in Chinese fir leaf litter by embedding resistance heating cables and found that Acidobacteria, Proteobacteria, and Actinobacteria were the most abundant phyla, soil warming significantly affect the relative abundance of Actinobacteria and Proteobacteria. After 360 days of decomposition, we found that Planctomycetes, Proteobacteria, Acidobacteria, and Actinobacteria were the dominant phyla in Chinese fir litters under different temperature conditions… (P10, L308-314)

Point 3: Although some additional information has been provided regarding the massive sequencing process, it is still insufficient. It is not indicated whether OTUs or ASVs have been used, with which platform the taxonomic assignments have been made, etc.

Response 3: Dear reviewer, more information about the sequencing process has been added in Section 2.3.

FASTP (version 0.18.0) was used to further filter raw reads, and QIIME (version 1.9.1) was used to spliced the bidirectional sequences to obtain high-quality effective tags. The effective tags were clustered into operational taxonomic units (OTUs) at 97% identity using UPARSE software (version 9.2.64). The tag sequence with the highest abundance was selected as a representative within each cluster. Bacterial OTUs were taxonomically assigned with the Silva database (http://www.arb-silva.de). (P4, L160-165)

Point 4: Regarding section 4.3., I understand the authors' argumentation. However, since no comparative study with other methodologies is made, it would be more appropriate to comment on it in the Introduction.

Response 4: Dear reviewer, thanks for your suggestion. We agree with you. In this study, we did not use other methodologies to carry out comparative study, so it is more appropriate to put this part in the Introduction.

Over the past decade, the responses of litter decomposition to warming in forest ecosystems have been widely studied. However, most of these studies are based on human-controlled experiments, such as indoor microenvironment simulation (artificial climate chamber [5] and greenhouse [6]) and in situ heating methods (resistance heating cables [7] and open-top chamber [8]), which have defects such as large differences between test and natural conditions in the field, as well as being underrepresented. Elevation gradient research was recognized as a powerful tool for exploring the processes of warming impact on ecosystems [9]. The process of litter decomposition in a field environment can be more accurately reconstructed using the natural temperature difference between altitudinal gradients to perform a displacement test of litter decomposition. (P1-2, L41-51)

Author Response File: Author Response.pdf