Slope Position Rather Than Thinning Intensity Affects Arbuscular Mycorrhizal Fungi (AMF) Community in Chinese Fir Plantations

Round 1

Reviewer 1 Report

The research reported in this manuscript is good quality, however, it requires better framing (explain what is known and what is not necessarily known, justifying publication in the international journal - the interactions among the different types of slope position, thinning and communities of AM fungi is probably the least known). My main concern is that the main drawback of the paper is that results rely on data of only one year, which strongly affects the robustness of conclusions. On the contrary, no apparent serious flaws are there either in the analytics or the stats, but some points need a better explanation, probably. I have listed specific comments which may guide the authors through revisions.

Comment

Lines 71-72: “Glomeromycota” changed to “Glomeromycotina” according to Spatafora et al. (2016).

You should cite the subphylum when the first time you mention about AM fungi in the Introduction section.

Please, cite the reference for this classification (Spatafora, J. W., Chang, Y., Benny, G. L., Lazarus, K., Smith, M. E., Berbee, M. L., ... & James, T. Y. (2016). A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data. Mycologia 108(5), 1028-1046.).

Actually, you can cite as Glomeromycota after the publication of Tedersoo et al. 2018. (Tedersoo, L., Sánchez-Ramírez, S., Koljalg, U., Bahram, M., Döring, M., Schigel, D., ... & Abarenkov, K. (2018). High-level classification of the Fungi and a tool for evolutionary ecological analyses. Fungal diversity, 90(1), 135-159.).

Comment

Lines 78-79:  “Currently, few studies have been undertaken to examine the effects of slope position

on AMF spore density, root colonization, and abundance using a taxon-based approach [24,26].” Please add the references to improve your paper for making readers given more information about the recent AMF studies in molecular approaches as follows:

Higo et al. (2019) Cover cropping can be a stronger determinant than host crop identity for arbuscular mycorrhizal fungal communities colonizing maize and soybean. PeerJ 7, e6403

Higo et al. (2019) First report of community dynamics of arbuscular mycorrhizal fungi in radiocesium degradation lands after the Fukushima-Daiichi Nuclear disaster in Japan. Sci. Rep. 9, 8240.

Higo et al. (2020) Amplicon sequencing analysis of arbuscular mycorrhizal fungal communities colonizing maize roots in different cover cropping and tillage systems. Sci. Rep. https://doi : 10.1038/s41598-020-58942-3

Comment

Lines 361-363: “Many studies have reported that the colonization was potentially higher in the soils where the soil available phosphorus was lower because host plants need more AMF uptake the nutrients from soil [23,45,73,74].” Please consider adding the reference to improve your paper and provide information you meant for related readers as follows:

Higo et al. (2020) Impact of phosphorus fertilization on tomato growth and arbuscular mycorrhizal fungal communities. Microorganisms, 8, 178.

Comment

Lines 372-374: “In addition, our study also found that AMF colonization was directly positively affected by slope position. The reasons may be with regard to spore density, soil moisture content or other

potential factors [24,73,76].” Please consider adding the reference to improve your paper and provide information you meant for related readers as follows:

Higo et al. (2013) Diversity and vertical distribution of indigenous arbuscular mycorrhizal fungi under two soybean rotational systems. Biol. Fertil. Soils 49, 1085-1096.

Higo et at. (2018a) How are arbuscular mycorrhizal associations related to maize growth performance during short-term cover crop rotation? J. Sci. Food Agri. 98, 1388–1396.

Higo et at. (2018b) Can phosphorus application and cover cropping alter arbuscular mycorrhizal fungal communities and soybean performance after a five-year phosphorus-unfertilized crop rotational system? PeerJ, 6, e4606.

Comment

Did you measure the AMF spore abundance in this study? If you measured them, that would be also good information for understanding the effects of different cover crop types.

Comment

In Figure 8 (a), it is difficult to follow what is each treatment. Please change the shape in each treatment, not enough to change the color in each treatment. For example, US: triangle, MS: square, and  LS: circle. Please try to change the shape of each scatter in Figure 8 (a) to make it clearer for readers. Further, please also try to draw ellipses that represent confidence intervals at 95%, if the grouping in each treatment can be distinct.

Comment

I think that the data in alpha diversity should be analyzed more in-depth. In Figure 3, the authors should analyze alpha phylogenetic diversity (MNTD) to know more detail about alpha diversity in soil fungal diversity (see MNTD and MPD in “picante” R package).

Comment

Lines 401-404: Please consider adding the references to improve your paper for making readers given more information about the recent AMF studies in soil pH as follows:

Higo et al. (2011) Molecular diversity and spore density of indigenous arbuscular mycorrhizal fungi in acid sulfate soil in Thailand. Annals of Microbiology 61(2), 383-389.

Materials and Methods section

I still have trouble seeing a clear general framework of the study. This mainly connects to a lack of clear structure in the Materials and Methods section. Overall, the Materials and Methods section shows a logic structure, however, the methodology to determine DNA analysis and bioinformatics is missing and not enough described.

Comment

Line 158: Please provide more details about how you sampled the roots for DNA extraction. How much g of root samples was used for the extraction of genomic DNA from each composite root sample collected from each plot (for a total of 90 samples)? Or did the authors extract DNA from 2 or 3 subsamples per plot and pool them together for one composite sample per plot (for a total of 90 samples)?

Comment

Lines 174-175: were the primers in the PCR index-tagged? Or how were the samples indexed? Please describe the adaptor sequences of each forward and reverse primer, if you want.

Line 182: In addition, please mention "PCR products (XXX bp) were run on X% agarose gel using gel electrophoresis and visualized using XXXX DNA Stain (company, city, country) on a XXXX (please mention here which UV device was used), if possible.

Comment

Lines 190-198: Taxonomic assignment with BLAST on NCBI needs more details (parameters, database choice, and quality, etc.) for making it clearer to understand your analysis for readers.

Comment

Lines 190-198: With AMF OTU definition, I suggest to perform OTU analyses using the MaarjAM database as in recent papers (see Hontoria et al., 2019, and Parvin et al., 2019).

Comment

Lines 215-216:  About the rarefaction and diversity analysis, did the authors mean that all the analysis for community analysis was performed at the lowest reads per sample or not? Please describe the rarefaction curve in this study in more detail.

Discussion

Comment

I do not understand how amplicon sequencing could be done without potential PCR bias. The authors did not mention anything about the issue. Please mention or discuss possible primer bias of the PCR used here or in the Discussion section.

Comment

Figure 8: This study performed the RDA, which means that the axis lengths were > 4 in the data used by DCA? Your data can show a unimodal by DCA, if so, please add (the axis length > 4) in the explanation of RDA.  If the axis length was bigger than 4, the authors should try to perform CCA to fit a good model in this study.

Author Response

Responses to reviewer 1:

The research reported in this manuscript is good quality, however, it requires better framing (explain what is known and what is not necessarily known, justifying publication in the international journal - the interactions among the different types of slope position, thinning and communities of AM fungi is probably the least known). My main concern is that the main drawback of the paper is that results rely on data of only one year, which strongly affects the robustness of conclusions. On the contrary, no apparent serious flaws are there either in the analytics or the stats, but some points need a better explanation, probably. I have listed specific comments which may guide the authors through revisions.

Response: Thank you for your crucial comments and valuable suggestions. We feel the manuscript has improved greatly with adoption of these suggestions. We have revised our manuscript accordingly. Please note that the responses make reference to the new line numbers in the revised manuscript.

We seriously considered the comment “the main drawback of the paper is that results rely on data of only one year, which strongly affects the robustness of conclusions”. We agree that this is a limitation of this study and continuous observations over several years could provide stronger evidence. Despite this, the results of this research reliably presented the effects of thinning and slope position on AMF at a certain time, which would be instructive for forestry researchers and managers in the management of Chinese fir plantations. Furthermore, inspired by this comment, we are aware of the fact that this is a good emphasis for further research. We have added more statements about this limitation and future needs in the Discussion section (see lines 461 and 467). 

Comment

Lines 71-72: “Glomeromycota” changed to “Glomeromycotina” according to Spatafora et al. (2016). You should cite the subphylum when the first time you mention about AM fungi in the Introduction section.

Please, cite the reference for this classification (Spatafora, J. W., Chang, Y., Benny, G. L., Lazarus, K., Smith, M. E., Berbee, M. L., ... & James, T. Y. (2016). A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data. Mycologia 108(5), 1028-1046.).

Actually, you can cite as Glomeromycota after the publication of Tedersoo et al. 2018. (Tedersoo, L., Sánchez-Ramírez, S., Koljalg, U., Bahram, M., Döring, M., Schigel, D., ... & Abarenkov, K. (2018). High-level classification of the Fungi and a tool for evolutionary ecological analyses. Fungal diversity, 90(1), 135-159.).

Response: We have revised it and cited the reference of Spatafora et al. for this classification (see line 73).

Comment

Lines 78-79: “Currently, few studies have been undertaken to examine the effects of slope position on AMF spore density, root colonization, and abundance using a taxon-based approach [24,26].” Please add the references to improve your paper for making readers given more information about the recent AMF studies in molecular approaches as follows:

Higo et al. (2019) Cover cropping can be a stronger determinant than host crop identity for arbuscular mycorrhizal fungal communities colonizing maize and soybean. PeerJ 7, e6403

Higo et al. (2019) First report of community dynamics of arbuscular mycorrhizal fungi in radiocesium degradation lands after the Fukushima-Daiichi Nuclear disaster in Japan. Sci. Rep. 9, 8240.

Higo et al. (2020) Amplicon sequencing analysis of arbuscular mycorrhizal fungal communities colonizing maize roots in different cover cropping and tillage systems. Sci. Rep. https://doi : 10.1038/s41598-020-58942-3

Response: Thank you for recommending above valuable references. Indeed, they are relevant and are now included in the manuscript (see lines 79-83). However, we failed to find out the last reference online, probably because it is too new. We look forward to consulting it later.

Comment

Lines 361-363: “Many studies have reported that the colonization was potentially higher in the soils where the soil available phosphorus was lower because host plants need more AMF uptake the nutrients from soil [23,45,73,74].” Please consider adding the reference to improve your paper and provide information you meant for related readers as follows:

Higo et al. (2020) Impact of phosphorus fertilization on tomato growth and arbuscular mycorrhizal fungal communities. Microorganisms, 8, 178.

Response: Thank you for your reference, which is again relevant. We have revised this part and added this reference (see lines 400-405).

Comment

Lines 372-374: “In addition, our study also found that AMF colonization was directly positively affected by slope position. The reasons may be with regard to spore density, soil moisture content or other potential factors [24,73,76].” Please consider adding the reference to improve your paper and provide information you meant for related readers as follows:

Higo et al. (2013) Diversity and vertical distribution of indigenous arbuscular mycorrhizal fungi under two soybean rotational systems. Biol. Fertil. Soils 49, 1085-1096.

Higo et at. (2018a) How are arbuscular mycorrhizal associations related to maize growth performance during short-term cover crop rotation? J. Sci. Food Agri. 98, 1388–1396.

Higo et at. (2018b) Can phosphorus application and cover cropping alter arbuscular mycorrhizal fungal communities and soybean performance after a five-year phosphorus-unfertilized crop rotational system? PeerJ, 6, e4606.

Response: Thank you for recommending above valuable references. We took carefully in account these references and our manuscript. We recognized it might be better to cite some references about slope position and AMF here. Despite this, we also found our manuscript benefited from these references. Thus, we added some of above references in other suitable parts of our manuscript (see lines 401, and 425-426).

Comment

Did you measure the AMF spore abundance in this study? If you measured them, that would be also good information for understanding the effects of different cover crop types.

Response: We did not measure the spore abundance in this study (through considered and make reference to other studies), but agree it would be important to do in the further research.

[1] Velázquez María Silvana; Fabisik Juan Carlos; Abarca Camila Lucía; Allegrucci Natalia; Cabello Marta. Colonization dynamics of arbuscular mycorrhizal fungi (AMF) in Ilex paraguariensis crops: Seasonality and influence of management practices. Journal of King Saud University – Science 2018. https://doi.org/10.1016/j.jksus.2018.03.017.

[2] Songachan, L.S.; Lyngdoh, I.; Highland, K. Colonization of arbuscular mycorrhizal fungi in moderately degraded sub-tropical forest stands of Meghalaya, Northeast India. Journal of Agricultural Technology 2011, 6, 1673-1684.

[3] Heinemeyer, A.; Ridgway, K.P.; Edwards, E.J.; Benham, D.G.; Young, J.P.W.; Fitter, A.H. Impact of soil warming and shading on colonization and community structure of arbuscular mycorrhizal fungi in roots of a native grassland community. Global Change Biol 2003, 10, 52-64, doi:10.1046/j.1529-8817.2003.00713.x.

Comment

In Figure 8 (a), it is difficult to follow what is each treatment. Please change the shape in each treatment, not enough to change the color in each treatment. For example, US: triangle, MS: square, and LS: circle. Please try to change the shape of each scatter in Figure 8 (a) to make it clearer for readers. Further, please also try to draw ellipses that represent confidence intervals at 95%, if the grouping in each treatment can be distinct.

Response: We have changed the shape of each scatter in Figure 8 (a). In addition, the grouping (thinning intensity) in each treatment (US, MS and LS) was difficult to distinguish and for this reason we didn’t draw ellipses that represent confidence intervals at 95%.

Comment

I think that the data in alpha diversity should be analyzed more in-depth. In Figure 3, the authors should analyze alpha phylogenetic diversity (MNTD) to know more detail about alpha diversity in soil fungal diversity (see MNTD and MPD in “picante” R package).

Response: We have calculated Faith's phylogenetic diversity (PD), MPD and MNTD using the package of ‘picante’ in R. Considered and referred to other studies, we found that PD would be more robust than other methods (one might use MPD when questions relate to branching occurring deep within a tree versus MNTD for questions related to terminal branching). For this reason, we analyzed PD in this study (see Table 2 and Figure 3), and added more details in the Methods section (see lines 236-237) and Results section (see lines 298-304).

Comment

Lines 401-404: Please consider adding the references to improve your paper for making readers given more information about the recent AMF studies in soil pH as follows:

Higo et al. (2011) Molecular diversity and spore density of indigenous arbuscular mycorrhizal fungi in acid sulfate soil in Thailand. Annals of Microbiology 61(2), 383-389.

Response: We have revised this sentence and added this reference (see lines 441-443).

Materials and Methods section

I still have trouble seeing a clear general framework of the study. This mainly connects to a lack of clear structure in the Materials and Methods section. Overall, the Materials and Methods section shows a logic structure, however, the methodology to determine DNA analysis and bioinformatics is missing and not enough described.

Response: Thank you for your crucial and valuable comments. We have revised Methods section accordingly.

Comment

Line 158: Please provide more details about how you sampled the roots for DNA extraction. How much g of root samples was used for the extraction of genomic DNA from each composite root sample collected from each plot (for a total of 90 samples)? Or did the authors extract DNA from 2 or 3 subsamples per plot and pool them together for one composite sample per plot (for a total of 90 samples)?

Response: Six sample trees were chosen randomly as the research objects in each plot. Then, roots were collected in each sample tree from four directions (east, south, west and north), and then mixed. Thus, we got one composite root sample from each sample tree and a total of 90 root samples from all plots. Finally, we chose 20 root segments (1-cm length) from each composite root sample for the extraction of genomic DNA. We extracted a total of 90 genomic DNA samples in this study. We have added more details in the updated version (see lines 164-167).

Comment

Lines 174-175: were the primers in the PCR index-tagged? Or how were the samples indexed? Please describe the adaptor sequences of each forward and reverse primer, if you want.

Response: Yes, each samples was tagged by a 12 bp unique barcode, which was added at the 5′-end of NS31 in each sample. Therefore, each sample has a unique number. We have added more details about this (see lines 183-184) and a table as supplementary describing the barcode sequences (see Table S1).

Line 182: In addition, please mention "PCR products (XXX bp) were run on X% agarose gel using gel electrophoresis and visualized using XXXX DNA Stain (company, city, country) on a XXXX (please mention here which UV device was used), if possible.

Response: We have added this information in the revised manuscript (see lines 191-193).

Comment

Lines 190-198: Taxonomic assignment with BLAST on NCBI needs more details (parameters, database choice, and quality, etc.) for making it clearer to understand your analysis for readers.

Response: We have rewritten this part to describe these details better (see lines 216-217).

Comment

Lines 190-198: With AMF OTU definition, I suggest to perform OTU analyses using the MaarjAM database as in recent papers (see Hontoria et al., 2019, and Parvin et al., 2019).

Response: We have revised this part to make it clearer and cited above reference (see lines 200-217).

Comment

Lines 215-216: About the rarefaction and diversity analysis, did the authors mean that all the analysis for community analysis was performed at the lowest reads per sample or not? Please describe the rarefaction curve in this study in more detail.

Response: Yes, all the analysis was performed at the lowest reads per sample because the rarefaction curves showed that the amplicon sequencing depths was sufficient to explore the majority of the sequence types. We have revised Figure 2 (including all samples in updated version) and described the rarefaction curves in more detail (see lines 275-284).

Discussion

Comment

I do not understand how amplicon sequencing could be done without potential PCR bias. The authors did not mention anything about the issue. Please mention or discuss possible primer bias of the PCR used here or in the Discussion section.

Response: We have added more description about it (see lines 193-194).

Comment

Figure 8: This study performed the RDA, which means that the axis lengths were > 4 in the data used by DCA? Your data can show a unimodal by DCA, if so, please add (the axis length > 4) in the explanation of RDA. If the axis length was bigger than 4, the authors should try to perform CCA to fit a good model in this study.

Response: Thank you for reminding us of this important point. After DCA, we found that the axis length was bigger than 4 at the OTU level and the axis length was smaller than 4 at the genus level. Thus, we revised Figure 8 (a) and supplemented more details about this (see lines 260-265 and 382-383).

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript titled “ Slope position rather than thinning intensity affects arbuscular mycorrhizal fungi (AMF) in Chinese fir plantations“ is a well-written manuscript. The paper could be very interesting for the reader of the journal Forest. I have some issues with this paper and those should be clarified before the paper would be accepted.

Title

Hard to read, consider writing it more clear.

Abstract

 At line no 18 Statment “: Our research showed that the altitudinal slope position, but not thinning, had significant effects on colonization, diversity, and  community composition of  AMF in Chinese  fir  plantations,  as well  as interaction with thinning intensity on diversity.“ Is not clear to consider rewriting it.

At line no 26 it there should be “ was significantly higher“ instead  “ was significantly higher“

At line no 32 Statment “ These findings suggested that slope position should be considered in the management of Chinese fir plantations,  and both  chemical  fertilization  and  AMF  augmentation should be  undertaken on upper hill slope positions as part of sustainable management practices for Chinese fir plantations“ is too long, consider writing it into two sentences.

Introduction

At line no 68 it should be „the slope“ instead “Slope“

At line no 76 AMF plays a crucial role instead AMF play a crucial role

At line no 85 statement “ Colonization by  AMF can promote nutrient absorption of Chinese fir, thereby improving growth.  Needs to be cited

Material and Method

At the line, no 148 sit should be Zhang et. al. Instead Zhang et al.

At the line, no 148 statement “Therefore, at least 100 intersects were measured to“ needs attention.

At line no 217 it should be  “normal“ not  “normally“

Statical Analysis

How many replications were used for the calculation on ANOVA, it seems it was three, with three replication how did the author get normal distribution, which is the basic condition of ANOVA.

Result and Discussion

Well written with good explanation.

Author Response

The manuscript titled “Slope position rather than thinning intensity affects arbuscular mycorrhizal fungi (AMF) in Chinese fir plantations“ is a well-written manuscript. The paper could be very interesting for the reader of the journal Forest. I have some issues with this paper and those should be clarified before the paper would be accepted.

Response: Thank you for your positive comments and constructive suggestions. We feel the manuscript has improved greatly with adoption of these suggestions. We have revised the manuscript accordingly. Please note that the responses make reference to the new line numbers in the revised manuscript.

Title

Hard to read, consider writing it more clear.

Response: We want the title to be descriptive of the study and its main findings, however we realize that it would provide better clarity to add the word ‘community’ in the title indicating that it is mainly this aspect that was being assessed. Thus, the title now reads “Slope position rather than thinning intensity affects arbuscular mycorrhizal fungi (AMF) community in Chinese fir plantations”.

Abstract

At line no 18 Statement “: Our research showed that the altitudinal slope position, but not thinning, had significant effects on colonization, diversity, and community composition of AMF in Chinese fir plantations, as well as interaction with thinning intensity on diversity.“ Is not clear to consider rewriting it.

Response: We have rewritten it (see lines 24-26)

At line no 26 it there should be “was significantly higher“ instead “ was significantly higher“

Response: We have replaced “were significantly higher“ with “was significantly higher“ (see line 27).

At line no 32 Statement “These findings suggested that slope position should be considered in the management of Chinese fir plantations, and both chemical fertilization and AMF augmentation should be undertaken on upper hill slope positions as part of sustainable management practices for Chinese fir plantations“ is too long, consider writing it into two sentences.

Response: We have fixed it (see line 35).

Introduction

At line no 68 it should be „the slope“ instead “Slope“

Response: We have fixed it (see line 69).

At line no 76 AMF plays a crucial role instead AMF play a crucial role

Response: We reviewed some references and considered it carefully. AMF meant “arbuscular mycorrhizal fungi” in our manuscript. Thus, it would be better to use “play” here.

[1] Hodge, A.; Storer, K. Arbuscular mycorrhiza and nitrogen: implications for individual plants through to ecosystems. Plant Soil 2015, 386, 1-19, doi:10.1007/s11104-014-2162-1.

[2] Kuhn, G., Hijri, M., & Sanders, I. R. Evidence for the evolution of multiple genomes in arbuscular mycorrhizal fungi. Nature 2001, 414(6865), 745–748. doi:10.1038/414745a

At line no 85 statement “Colonization by AMF can promote nutrient absorption of Chinese fir, thereby improving growth”. Needs to be cited

Response: We have added appropriate references to this sentence (see line 92).

Material and Method

At the line, no 148 sit should be Zhang et. al. Instead Zhang et al.

Response: We have fixed it.

At the line, no 148 statement “Therefore, at least 100 intersects were measured to“ needs attention.

Response: This sentence has been reworded (see lines 158-159).

At line no 217 it should be “normal“ not “normally“

Response: We have fixed it.

Statistical Analysis

How many replications were used for the calculation on ANOVA, it seems it was three, with three replications how did the author get normal distribution, which is the basic condition of ANOVA.

Response: We selected six trees randomly as the research objects in each plot, and they were independent from each other. Therefore, there were six repetitions. The data satisfied normal distribution and homogeneity of variance, which satisfied the assumptions of ANOVA.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I appreciate all of your work through the revisions.

Author Response

We are quite grateful for your positive comments and recognition.

Reviewer 2 Report

I am ok with the changes made by the author, so I suggest to accept the manuscript in the present form.

Author Response

We are quite grateful for your positive comments and recognition.

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

The preset study by Xu et al attempts to address the question of what are the impacts of "slope position" and "thinning intensity" in a forest stand of Chinese fir on AMF abundance and community composition. To this end, the authors collected samples in one field trial to conduct microscopic and molecular community analyses of AMF, accompanied by several tree and soil parameters. Overall, the study reads well and the topic might have potential for publication. However, a major design limitation in the experiment, related to inadequate replication, raised critical concerns. I believe that this limitation does not allow the authors to undoubtedly support their claims. However, if the authors manage to incorporate (at least) three independent randomly distributed replicates in each treatment, I would encourage re-submission. My point-by-point comments are presented below:

Line 16: the introduction sentence in the abstract mainly highlights the lack of studies. The authors should briefly provide a rational for why it is important to answer this very specific question.

Lines 20-22: the statement “if and how thinning can result in AMF” is unclear.

Line 44: I think the scientific name of Chinese fir is C. lanceolata, and not C. lanceolate.

Lines 84-85: this statement suggests that the present study will identify the underlying mechanisms of AMF assemblages associated with slope position. However, by reading the manuscript, I cannot find such mechanistic demonstration.

Lines 95-97: The hypothesis is unclear and unexpected. What it means to be closely related to modifying microclimate and altering edaphic factors? In addition, why would the authors expect that thinning intensity would improve the occurrence, diversity, and community compositions of AMF? The underlying rational for such hypothesized effects needs to be better introduced.

Lines 99-102: The aims (1) and (2) seem to be the same. In addition, the aim (3) cannot be conclusively answered by using the current experimental design. To determine the topographical and edaphic effects on AMF community, the authors would need to include proper controls. In addition, see my comments below on SEM.

Both Figure 1 and Table 1 need to be accompanied by detailed self-explanatory legends. The figure showing the plot distribution should contain contour lines. In addition, based on Figure 1, the plots assigned to each treatment are not replicated, nor the plots seems to have a random geographical distribution. Therefore, one cannot discard the impact of confounding factors. This is a major experimental limitation.

Lines 171-172: The authors state that they have used the magnified intersects method, but later state that they have observed 10 fields of view of the microscope for each root segment. How many roots segments were observed for each experimental unit/plot? At least 100 intersects per experimental unit should be measured for an accurate assessment of mycorrhizal colonization of roots.

Line 194 and 201: please provide a supplementary table with primers’ sequences. In addition, refer to the original methods paper for SSU rRNA gene DNA extraction, amplification, and cloning from AM fungi.

Line 206: the authors used Illumina MiSeq platform to sequence DNA fragments of AMF. However, they do not mention any addition of adapters to the ends of the DNA fragments. Was this done? Please clarify. Moreover, I am under the impression that these primers were not specifically developed for Illumina sequencing. How large were the DNA fragments from the PCR products? Were their adequate for illumina sequencing?

Line 218: unless I miss something, the data does not seem to fulfil the assumptions of ANOVA. ANOVA assumes that the observations are random and that the samples are taken from independent replicates. From what is described in Figure 1, neither the plots are randomly distributed nor they seem to be independent from each other, because proximity is dependent from treatment association. Moreover, this design seems to limit the validity of interpretations. For instance, in lines 263-265, the authors conclude that there were significant differences in several variables among the different slope positions, but not among the different thinning intensities. This might just simply occur because the level of replication in the thinning intensity was lower than that of slope position.

Lines 226-228: I disagree with this statement. Although in many sciences, SEMs are used to infer on causal relations, which could not be studied by other means, SEMs are only inference tools. Failure to fit the data to causal assumptions may guide the researcher to diagnose absence of causal effects. However, fitting the data does not provide conclusive prove that causal assumptions are true, it only makes them tentatively more plausible. Therefore, unless the authors add additional experimental evidence with proper controls, they must tone-down the claims on causal effects.

Line 286-293 and Figure 3: Because of the limitation of SEMs, I would suggest the authors to rephrase the interpretation of these results. For instance, in the legend of Figure 3, and when appropriate, instead of stating “Structural equation models (SEM) show the causal effects...” They should state instead “Structural equation models (SEM) allow to infer plausible causal effects…”.

Lines 344-348: the authors found a strong correlation between slope position and tree growth. This raises the question of whether the relation between slope position and AMF community results from a direct effect or instead from an indirect effect caused by the tree growth.

Lines 350-352: because of design limitations, such conclusion cannot be drawn based only on the present experiment. Confounding factors may have played a stronger role that what is admitted. This is an issue that can only be resolved by using a more robust experimental design with sufficient independent replication.

Reviewer 2 Report

The paper from Wei et al. talks about ectomycorrhizal community from soil and roots of Qurcus variabilis. They took into account slope factor as a variable and therefore they analysed the fungal composition in consequence. This is the second revision of this paper for me.

I saw that many improvements have been made. The English is now clear, and some data (as the statistical analyses or the raw reads count) have been updated. But authors decided to completely ignore some interesting advice given by reviewers.

I still feel that the importance of the work is not well presented and stated by the authors. As the AMF fungi play pivotal role in the forest environments, they must introduce the concept of holobiont, wood wide web and explain which ecological services AMF offer to their plant hosts in the specific ecological niche. This would greatly enhance the level of the paper, suggesting to the reader why it is important to study these aspects in wild environments.  

In addition, as I stated in the previous revision I feel that using Qiime 1 (which is no more updated since January 2018) it is not a good idea. Specific tools for fungal community analysis have been developed for Qiime 2, and it would be of great interests to use them. Moreover I would suggest to perform co-occurrence analysis on the whole fungal community. As I already stated in the previous revision, data of the whole fungal community are really interesting: you can assess co-occurrence analysis using your physio-chemical data and assess if relationships between taxa occur! I believe that now a day the mere annotation of amplicons data are insufficient for the description of complex community such as the one you are dealing with. Finally, in line with the previous point, some more attention on the functional aspects can further improve the quality of the paper.

Reviewer 3 Report

Forests 637642

Silvicultural practice, in this case thinning, has the potential to alter tree growth through direct (light, water) and indirect (soil microbial changes) effects. In the current study, the authors evaluated the effects of thinning (accounting for slope position) in the mycorrhizal community composition in Chinese fir plantations. Using DNA fingerprinting, mycorrhizal diversity was estimated and SEM was used to evaluate the relative associations/impacts of soil and environmental variables on community structure. The statistical approach is relatively novel and allows readers to consider the factors driving AM community structure. Maintaining soil microbial diversity in the face of management practices is important is sustainable forestry. This study aids in these considerations, although the authors correctly conclude that the short duration between thinning and measurement of soil properties may obscure more long-terms impacts. English/Writing. Many editorial comments are on the attached pdf file. See also the numbered comments below. The topic, approach, and analyses are suitable for Forests. Study Design/Methodology. The scope is relatively narrow due to the limited sampling undertaken.  The authors acknowledge this, which was good. Ethical Issues. No problems.

Specific comments (numbers on the pdf):

This is your reason statement, but it is very awkward as written. Perhaps change: “However, it is still unclear what impacts thinning management as applied to Chinese fir plantations has on the structure and diversity of soil AMF communities.” “positively affected” – while you used SEM, I am skeptical of the power of assigning these direct responses given the limited degree of your sampling. I would moderate these statements by reversing the association and writing: “AMF community diversity was positively associated with soil Mg and soil temperature”, etc. Again, this last sentence is awkward: “These findings suggest that slope position should be considered in the management of Chinese fir plantations, and both chemical fertilization and AMF diversity augmentation should be undertaken on upper hill slope positions as part of sustainable management practices for Chinese fir plantations.” This sentence is a run-on. Add a period after [1,5,5]. Then: “In addition, these plantations may exhibit other problems associated with monocultures….” “Underlying mechanisms” is extremely strong. You end up with associations by SEM, but I state that these are NOT “mechanisms.”  Instead perhaps: “The results of these studies are far from unequivocal and the environmental factors associated with slope position that influence AMF assemblages have not been identified.” I do not follow how the map positions in Figure 1 and the “southeast slope” in text align. Omit in parentheses. Confusing: Area, plots, sample plots, sites. Clarify perhaps aligning/modifying Figure 1. Same confusion as above, now adding “intensive plots”. Why did you elect to digest samples? Total element concentration does not relate to availability.  You need to justify this somehow.  It would be much stronger if you had done a soil extract instead. “mycorrhizal tips” is usually related to ECM fungi where the short roots are collected. I might simply use “mycorrhizal roots”. You need to re-write this section. You state certain factors are “treatments” but they are co-variates.  Slope and Thinning are your treatments.  “slope and thinning as treatments and x, y, and z as covariates” You have the potential for a two-way ANOVA. You need to re-run these and evaluate interactions. Basically, the upper slope differed from the other two positions and sometimes the lower was the same as upper. You need to be clear when verbalizing Tukey HSD differences. This is not true as written. “AMF colonization was lowest at the upper slope.” The other two are the same. And I do not think you have the data to discuss rainwater movement. I think this paragraph is redundant and should be removed. This section is redundant and should be re-written. Information at 17 could be included up above where you already discussed “low nutrients.” OTHER: Your references need to be manually edited to match journal style. Almost every one has a problem as circled: capitalization, abbreviations, etc.

Comments for author File: Comments.pdf