Variance in Woody Debris Components Is Largely Determined by the Belowground Microbial Phylum-Level Composition

Round 1

Reviewer 1 Report

Dear authors.

Your manuscript "Variance in woody debris components is largely determined by 2 the belowground microbial phylum-level composition" contains original, benefical, interesting and clearly presented results. Methods are apropriate and also clearly described. References are appropriate, actual and correctly cited. I have only few minor remarks (see comments in attached revised manuscript file).

Good luck with your next research.

Reviewer 1.

Comments for author File: Comments.pdf

Author Response

Specific comments

1. L11: Just one correspondence author should be chosen.

Response: Many thanks!

We listed two corresponding authors as they had both greatly contributed to the current study. Without their financial supports, it is impossible to finish this work. Therefore, we hope this article could keep these two authors as corresponding authors.

 

2. L12-13: Repeating information.

Response: Many thanks and done!

We deleted the content as suggested (page 1, L12-13).

 

3. L103: Please state full latin names of species with authors, when they are first mentioned in the manuscript. E.g., Picea jezoensis (Siebold & Zucc.) Carrière.

Response: Many thanks and done!

We’ve revised the contents as suggested (page 3, L106 -110).

 

4. L108: Do you mean coniferous and deciduous forest (described in the Chapter 2.1)?

Response: Many thanks!

We’ve changed revised the content as suggested (page 3, L114-116).

 

5. L158-159: Redundant.

Response: Many thanks and done!

We deleted the content as suggested (page 4, L166-168).

 

6. L251-254: I suggest to divide this sentence to shorter sentences.

Response: Many thanks!

We have split the sentence into two sentences as suggested (page 6, L263-265).

 

7. L325-326: When comparison of values between sets (deciduous vs. conferous, brown rot vs. white rot), sometimes percentages, sometimes "folds higher" or "-fold difference" are used. Please unify it in the whole manuscript. Formualtion with word "increased" inaccurately suggests that the abundances have changed during time. So, I would prefer different formulation, e.g.: "...relative abundance was by 33% and 8% higher..."

Response: Many thanks and done!

Value differences between sets (deciduous vs. coniferous, brown rot vs. white rot) have been uniformly expressed as percentages throughout the text (e.g., page 8, L324, L331, L333, and L336).

We revised the corresponding text to “However, the opposite trends were observed with Acidobacteria (26.48%-35.19%), whose relative abundances were by 33% and 8% higher in brown- than white-rot sets in the deciduous and coniferous forest stands, respectively (Table 1).” (page8, L337-341).

 

8. L380: Latin names of higher level taxons than species and genus should be in normal font, not italics.

Response: Many thanks and done (page10, L394-395).

 

9. L380: Explanations of abbreviations should be in each Table (even if they are the same as in the previous table).

Response: Many thanks and done!

We have added explanations of abbreviations in table 2 (page10 L395-396).

 

10. L397: Some journals are cited by full names, other by abbreviations. Please unify it according to the Forests journal requirements. Please also check publication names. First letters of some words should be CAPITAL.

Response: Many thanks and done!

We uniformly have abbreviated publication names as requested by Forests (e.g., page 11, L415-416, L449, L462, L465, and L471).

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript has an interesting research goal, which is investigated using adequate methods, and the authors support the introduction and discussion with a number of relevant and modern references. However, I have two major comments on the manuscript. The first concerns the structure of the manuscript, where results and discussions are combined, contrary to recommendations from guidelines for authors. This makes it difficult for the reader to easily navigate what the authors' results are and what the cited literature is. I leave it up to the editors to decide how serious violation of the recommended structure this is.

The second comment concerns the authors' approach to the problem. In the text, they state that they are investigating how the wood decay process is determined by the belowground microbial community composition. This insight is also reflected in the title. But this does not correspond to the design of the research, where in each type of forest they sampled already decaying logs (white-rot and brown-rot) and the soil microbiome under them. However, soil microbiome could already be retroactively affected by the decomposition that was taking place above it and the released nutrients. In order to prove what they claim (determination), they would first have to identify (or ideally change) the soil microbiome on selected spots, then place fresh logs on these spots and research the ability to infer from the composition of the microbiome what type of the wood decomposition occur. On the other hand, if they wanted to exclude the reverse effect of decaying logs on soil microbiome, they would have to put already decaying logs (white-rot and brown-rot) on soil with a defined microbiome and monitor its subsequent change. Such an approach could be called experimental. The authors' approach is not experimental in the strict sense of the word, even though they claim so in the conclusion.

However, it is still a very good piece of work, and the chosen methods of data analysis are adequate to the chosen method - they are based on correlations and on multivariate analyzes that are essentially correlational as well, so this is not a problem. I would only ask the authors to tone down expressions like "determined by" to "change with/correlate with" etc.

Less important comments:

L103-L105: Give the Latin names of the species with the names of the authors of the description at first appearance.

L151: Why still use OTU when nowadays it is more modern to use ASV? On the other hand, this does not matter at all if the authors work at the level of phyla. Why don't they work with lower taxonomy as well, for example at least identify what species/genera of fungi were primarily affecting decomposition in their system?

As authors worked with fungal and bacterial DNA amplified by different primers, the relative representation of fungi can only be related to fungi and the relative representation of bacteria only to bacteria. Therefore, I would be very interested in the quantities of both groups in the investigated system. Did the authors try to perform qPCR? Can they tell if there are quantitatively more fungi or bacteria in the soil?

Author Response

General comments

1. … the structure of the manuscript, where results and discussions are combined, contrary to recommendations from guidelines for authors.

Response: Many thanks!

As we prepared this manuscript to Forests, we found Forests are not strictly constraining the structure and some published papers combined their results and discussion section (Wu et al., 2021; Santos et al., 2022; Mao et al., 2022).

Wu, S.S.; Tao, X.; Xu, W., Thermal conductivity of poplar wood veneer impregnated with Graphene/Polyvinyl alcohol. Forests 2021, 12(6), 777.

Santos, L.; Lopes, V.; Baptista, C., MDIR monthly ignition risk maps, an integrated open-source strategy for wildfire prevention. Forests 2022, 13(3), 408.

Mao, J.J.; Wu, Z.H.; Hardwood grain image restoration and enhancement via Gaussian histogram specification and adaptive color adjustment. Forests 2022, 13(6), 863.

 

2. “…1) experimental design ….2) tone down expressions like "determined by" to "change with/correlate with" etc.

Response: Many thanks!

• We apologize for not listing microbial relative abundance data of control soils (1 meter away from the wood rot sets). However, we found no significant difference in the relative abundances of microbial phyla between wood rot sets (underneath the deadwood) and control (far away from the deadwood) (see the Table below).

Table T-test results of relative abundance of soil microbial phyla in deadwood and control

deadwood	Basidiomycota	Ascomycota	Glomeromycota	Rozellomycota	Acidobacteria	Actinobacteria	Armatimonadetes	Nitrospirae
DB	0.1978	0.4342	0.1699	0.9815	0.5419	0.4641	0.9945	0.8514
DW	0.5660	0.7453	0.1118	0.9700	0.7417	0.2829	0.5689	0.9681
CB	0.2059	0.3753	0.0327	0.0965	0.5858	0.2909	0.8231	0.3324
CW	0.4406	0.1000	0.5683	0.9033	0.2686	0.2677	0.4874	0.9999

Significance levels: P < 0.05. DB: Brown-rotten wood in the deciduous forest; DW: White-rot wood in the deciduous forest; CB: Brown-rotten wood in the coniferous forest; CW: White-rot wood in the coniferous forest.

 

• We still prefer using "determined by". Our findings clearly showed that nearly 90% of the variation in the woody debris components was explained by the belowground fungal and bacterial phylum-level compositions. This expression (i.e., determined by) is reasonable as the deadwood decay processes are closely related to soil microbial communities. Such an expression might be interesting to the potential audience.

 

Specific comments

1. L103-L105: Give the Latin names of the species with the names of the authors of the description at first appearance.

Response: Many thanks and done!

We’ve revised the contents as suggested (page 3, L106-110).

 

2. Why still use OTU when nowadays it is more modern to use ASV? On the other hand, this does not matter at all if the authors work at the level of phyla. Why don't they work with lower taxonomy as well, for example at least identify what species/genera of fungi were primarily affecting decomposition in their system?

Response: Many thanks!

The base error rate in OTU can be controlled within 1% according to the UPARSE algorithm. To use OTU is acceptable (Probst et al., 2018; Gómez-Brandón et al., 2020; Ren et al., 2022). We are also very interested in using ASV (e.g., Yang et al., 2022), and we will try in our future studies.

We fully agree with the reviewer that a lower taxonomy would give us a better understanding of microbes and their responses to environmental changes. However, the microbial phylum-level has also been reported in a large number of articles (Buresova et al., 2019; Behnke-Borowczyk et al., 2021; Huang et al., 2022). Most important, the analyses at the phylum-level are enough to clearly explain the effects of microbial communities on wood decay in the current study.

 

Probst, M., Gómez-Brandón, M., Bardelli, T., Egli, M., Insam, H. and Ascher-Jenull, J., Bacterial communities of decaying Norway spruce follow distinct slope exposure and time-dependent trajectories. Environ. Microbiol. 2018. 20, 3657-3670.

Gómez-Brandón, M.; Probst, M.; Siles, J.A.; Peintner, U.; Bardelli, T.; Egli, M.; Insam, H.; Ascher-Jenull, J., Fungal communities and their association with nitrogen-fixing bacteria affect early decomposition of Norway spruce deadwood. Sci. Rep. 2020. 10, 8025.

Ren, W.Z; Penttilae, R.; Kasanen, R.; Asiegbu, F.O., Bacteria community inhabiting heterobasidion fruiting body and associated wood of different decay classes. Front. Microbiol. 2022. 13, 864619.

Yang, S.; Poorter, L.; Kuramae, E.E.; Sass-Klaassen, U.; Leite, M.F.A.; Costa, O.Y.A.; Kowalchuk, G.A.; Cornelissen, J.H.C.; van Hal, J.; Goudzwaard, L.; Hefting, M.M.; van Logtestijn, R.S.P.; Sterck, F.J., Stem traits, compartments and tree species affect fungal communities on decaying wood. Environ. Microbiol. 2022.

Buresova, A.; Kopecky, J.; Hrdinkova, V.; Kamenik, Z.; Omelka, M.; Sagova-Mareckova, M., Succession of microbial decomposers is determined by litter type, but site conditions drive decomposition rates. Appl. Environ. Microb. 2019. 85(24),  e01760-19.

Behnke-Borowczyk, J.; Kwasna, H.; Kartawik, N.; Sijka, B.; Belka, M.; Lakomy, P., Effect of management on fungal communities in dead wood of Scots pine. Forest Ecol. Manag. 2021, 479, 118528.Huang, C.J.; Wu, X.Q.; Liu, X.Y.; Fang, Y.T.; Liu, L.; Wu, C.S., Functional fungal communities dominate wood decomposition and are modified by wood traits in a subtropical forest. Sci. Total. Environ. 2022. 806, 151377.

 

3. As authors worked with fungal and bacterial DNA amplified by different primers, the relative representation of fungi can only be related to fungi and the relative representation of bacteria only to bacteria. Therefore, I would be very interested in the quantities of both groups in the investigated system. Did the authors try to perform qPCR? Can they tell if there are quantitatively more fungi or bacteria in the soil?

Response: Many thanks!

We believe the fungal and bacterial quantities are a very interesting point, but we regret not performing qPCR. Currently, we combined the relative abundances of sequenced data of fungi and bacteria.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The structure of the manuscript: The authors have shown that such combined results and discussion are admissible. If the editors are ok with it, I have no further objections.

Experimental design, determined by,…: You could attach these controls to the manuscript. If the reader is not informed about them, it is as if they do not exist, and then the reader may find your conclusions unconvincing. Please add a short note that you did the controls, that the controls are not significantly different, and include the table you presented to me in the answer, at least in some supplement. You mention controls in: “more independent soil fungal species underneath decaying wood compared to the control (3 meters away from decaying wood) [44]” but since there is a quote attached, I considered that whole sentence to be part of the discussion, not your result. The disadvantage of combining results and discussion into one section.

OTU, qPCR – Yes, I understand it. I also still have some of my data processed in OTU and without qPCR, which I regret. I am very much looking forward to reading some of your other work in which such quantifications will be carried out, because I am interested in the relationship between the actual amount of bacteria and fungi in the sample.

 

Finer taxonomic scale… Yes, you are right that the authors of the mentioned publications mainly work with higher taxonomic units. Nevertheless, at least symbolically, most of them also mention results for lower taxonomic levels: Probst et al., 2018; Gómez-Brandón et al. 2020; Ren et al. 2022 and Behnke-Borowczyk et al. 2021. I don't want to force you, but from my point of view as a reader, it's a shame not to have mentioned some specific genera that could be responsible for the decay.

Author Response

Reviewer #2

1. You could attach these controls to the manuscript. If the reader is not informed about them, it is as if they do not exist, and then the reader may find your conclusions unconvincing. Please add a short note that you did the controls, that the controls are not significantly different, and include the table you presented to me in the answer, at least in some supplement. You mention controls in: “more independent soil fungal species underneath decaying wood compared to the control (3 meters away from decaying wood) [44]” but since there is a quote attached, I considered that whole sentence to be part of the discussion, not your result. The disadvantage of combining results and discussion into one section.

Response: Many thanks and done!

Following the reviewer’s suggestion, we have added the information about the control of this experiment in the Log identification and sample collection session as follows (page3, L120-122): “At the same time, we collected soils located 1 m horizontally from the decaying logs as controls”.

We revised the content related to reference [44] (page 6, L227-230), “As previously reported [44], decaying wood can recruit specific decomposers and may result in a more diversified (i.e., Shannon and Simpson) and independent soil fungal community compared to the control (3 meters away from decaying wood).”

We have also presented the T-test results of relative abundance of soil microbial phyla between deadwood and control in the supplementary materials (page6, L232-234), “However, our results showed that there was no significant difference in the relative abundances of microbial phyla between wood rot sets (underneath the deadwood) and controls (1 m away from the deadwood) (Table S1)”.

 

2. OTU, qPCR – Yes, I understand it. I also still have some of my data processed in OTU and without qPCR, which I regret. I am very much looking forward to reading some of your other work in which such quantifications will be carried out, because I am interested in the relationship between the actual amounts of bacteria and fungi in the sample.

Response: Many thanks!

We regret that we did not perform qPCR. Thanks for your kind advice, and we will pay attention to qPCR in our future study.

 

3. Finer taxonomic scale… Yes, you are right that the authors of the mentioned publications mainly work with higher taxonomic units. Nevertheless, at least symbolically, most of them also mention results for lower taxonomic levels: Probst et al., 2018; Gómez-Brandón et al. 2020; Ren et al. 2022 and Behnke-Borowczyk et al. 2021. I don't want to force you, but from my point of view as a reader, it's a shame not to have mentioned some specific genera that could be responsible for the decay.

Response: Many thanks!

We acknowledge that the readers definitely want to get the information about key specific species involved in wood decay. In fact, this is really an interesting point. Frankly speaking, we wanted to use such those data in our future manuscript but not in the current paper. We sincerely thank your great understanding and not forcing us to use those data in this paper.

Author Response File: Author Response.pdf