Initial Carbon Quality of Newly Shed Foliar Litter in an Alpine Forest from Proximate Analysis and 13C NMR Spectroscopy Perspectives
Round 1
Reviewer 1 Report (Previous Reviewer 2)
Dear Authors
the manuscript could be interesting for the scientific community and would reflect the aims of the journal. The Authors appropriately highlight the multiplicity of factors affecting the litter chemical composition and in turn litter decomposition and C sequestration. Nevertheless, the Authors should revise the manuscript especially in the aims, figures and tables and discussion.
I highly recommend that the Authors engage a native English-speaking technical editorial service or professional colleague to assist them with language editing. In the introductive part of the manuscript some sentences are unclear and some words are used inappropriately.
A fundamental aspect that the Authors must clarify is the definition of litter quality. Often in the text, Authors compare litter quality and litter C fractions. But the C-fractions are included in the litter quality. Another important point is the use of data from Ni et al. (2018), to test the effect of C-fractions on the decomposition. The introduction of these decomposition data must be clarified and justified. The Authors must be more precise, also in the abstract. For example, do Ni and co-workers use the same litter? Are they taken from the same forests?
The Authors should also report the number of laboratory replicates and describe in detail DOC analysis (pre-treated samples).
The captions of tables and figures also need to be revised, they are not always explanatory. For example, the caption of Figures 2 and 3 should state the time course.
Figure 5 is inserted before figure 4 and is not referred to in the text. Furthermore, temperature (MAT) and precipitation (MAP) seem to be missing from the analysis in Figure 5. In the discussion, reference is made to Tables 3 and 5, which are not given in the manuscript.
The discussion should be deeply revised by the Authors who should avoid obvious and commonly accepted concepts or what is already stated in the introduction.
In conclusion, I believe the manuscript needs major revisions before being considered for publication.
Author Response
Individual detailed responses to the Reviewer #1’s comments (forests-1940226)
All critiques and suggestions provided by the reviewer are shown in black, and our responses are shown in blue. Revisions are marked in red in the main text.
Comments from the reviewer #1:
-Reviewer #1
1) the manuscript could be interesting for the scientific community and would reflect the aims of the journal. The Authors appropriately highlight the multiplicity of factors affecting the litter chemical composition and in turn litter decomposition and C sequestration. Nevertheless, the Authors should revise the manuscript especially in the aims, figures and tables and discussion.
Response: Thank you for your positive comments of this paper, which is very important for us. We’ve revised the whole manuscript based on your constructive suggestions.
2) I highly recommend that the Authors engage a native English-speaking technical editorial service or professional colleague to assist them with language editing. In the introductive part of the manuscript some sentences are unclear and some words are used inappropriately.
Response: Thank you for your suggestions, and we engaged a native English-speaking technical editorial service to improve the language editing of our manuscript and make it more readable.
3-1) A fundamental aspect that the Authors must clarify is the definition of litter quality. Often in the text, Authors compare litter quality and litter C fractions. But the C-fractions are included in the litter quality.
Response: Thank you for your suggestions and comments. Indeed, C fractions are included in litter quality. In our study, we mainly explored the initial C quality which includes C fractions detected by sequential extraction technique and functional C groups determined by solid-state 13C NMR measurement. The litter quality mentioned in our manuscript mainly represented other chemical qualities, such as total organic C, nitrogen (N), phosphorus (P), dissolved organic carbon (DOC) and manganese (Mn), etc. In order to determine the impacts of those chemical qualities above on initial C quality, litter quality was thereby further divided into chemical qualities and C quality, and chemical qualities were referred to litter quality except C quality in our study.
3-2) Another important point is the use of data from Ni et al. (2018), to test the effect of C-fractions on the decomposition. The introduction of these decomposition data must be clarified and justified. The Authors must be more precise, also in the abstract. For example, do Ni and co-workers use the same litter? Are they taken from the same forests?
Response: Thanks for your questions and suggestions. Both Ni’s study and our study were conducted at the same research station of alpine forest ecosystem, and we compared the C loss (detected from the 4-year decomposition experiment of Ni) and the initial C quality (detected from our study) of the same foliar litter for fir and willow, and this part was clarified in 2.4 Statistical analysis. Please see the lines 194-196.
4) The Authors should also report the number of laboratory replicates and describe in detail DOC analysis (pre-treated samples).
Response: Thanks for your suggestions. We’ve added the number of laboratory replicates and the method of pre-treated for DOC. Please see the lines 144, 152-155 and 176-179.
5) The captions of tables and figures also need to be revised, they are not always explanatory. For example, the caption of Figures 2 and 3 should state the time course.
Response: Thanks for your suggestions. We’ve revised the captions for figure 1, 2 and 3 into “Monthly dynamics for…” to state the time course, and we didn’t revise any captions of tables because time course was not presented in any tables. Please see the lines 209-211, 238-240 and 250-253.
6) Figure 5 is inserted before figure 4 and is not referred to in the text. Furthermore, temperature (MAT) and precipitation (MAP) seem to be missing from the analysis in Figure 5. In the discussion, reference is made to Tables 3 and 5, which are not given in the manuscript.
Response: Thank for your suggestions, and we added the related content that the figure 5 was referred to in the text, please see the lines 287-290. 13C NMR commonly determined the chemical structure for organic C, and the C structure was less effected by temperature and precipitation than C fractions, such as water-soluble extractives (WSE) and organic soluble extractives (OSE), thus the climatic conditions were not performed in the stepwise regression analyses of functional C groups which was showed in Figure 5, and we’ve deleted the MAT and MAP in the caption of Figure 5, please see the lines 260-265. We have also deleted the Table 3 and Table 5 which were not referred to in the text.
7) The discussion should be deeply revised by the Authors who should avoid obvious and commonly accepted concepts or what is already stated in the introduction.
Response: Thank for your useful suggestions. We’ve revised the Discussion part. Please see the lines 275-365.
8) In conclusion, I believe the manuscript needs major revisions before being considered for publication.
Response: Thank for your suggestions. We’ve reorganized the Conclusions. Please see the lines 367-379.
Author Response File: 
Author Response.docx
Reviewer 2 Report (New Reviewer)
Review of Forests Manuscript. 1094226
First let me applaud the authors on the completeness and use of state of the art methods of analysis. The use of the cross polarization-magic angle spinning variation of C13 NMR analysis avoids the differences in signal strength of C13 atoms in different local environments.
In combination with a “top-down” fractionation scheme “e.g. acid-unhydrolysable residue, avoids excluding more difficult to define forms of carbon structures. These are, however, used only in a correlation approach, as opposed to C13 NMR of each residue, which is a weakness. In addition, analysis of the leaves at different stages of decomposition is a weakness since lignin-like residues can be modified in situ during decomposition, such as a dramatic increase in –COOH groups. Such studies do exist. But let that not take away from the thoroughness of analysis of the initial contents in litterfall.
In addition, analysis of monthly collections of litterfall is unusual, as opposed to simply compositing them by weight by species.
The study is fundamentally descriptive, that is, differences initial chemistry of different species, and a correlative study with long term decomposition. (4 years is a very good time for decomposition). The use of stepwise multiple regression is good since many fractions can be correlated x variables.
I understand the context that species composition of different plots can help predict overall decomposition rates. But, perhaps it would help the scope of the study to better describe the context of the study. For example there is one unconnected statement that the soils tend to be relatively barren. How much is climatological (cold and dry) and how much can be ascribed to species composition. Species composition can interact with climate to exasterbate deficiencies in soil fertility. The dominance of Vaccinium on sandy soils is an example involving acidification.
I believe that this further discussion of the context would help the hypotheses, which now seem to state the obvious. This would also help elevate the scientific “novelty” and “importance ratings.
Author Response
Individual detailed responses to the Reviewer #2’s comments (forests-1940226)
All critiques and suggestions provided by the reviewers are shown in black, and our responses are shown in blue. Revisions are marked in red in the main text.
Comments from the reviewer #2:
-Reviewer #2
1) First let me applaud the authors on the completeness and use of state of the art methods of analysis. The use of the cross polarization-magic angle spinning variation of 13C NMR analysis avoids the differences in signal strength of 13C atoms in different local environments.
Response: Thank you for your positive comments on our study.
2) In combination with a “top-down” fractionation scheme “e.g. acid-unhydrolyzable residue, avoids excluding more difficult to define forms of carbon structures. These are, however, used only in a correlation approach, as opposed to 13C NMR of each residue, which is a weakness. In addition, analysis of the leaves at different stages of decomposition is a weakness since lignin-like residues can be modified in situ during decomposition, such as a dramatic increase in –COOH groups. Such studies do exist. But let that not take away from the thoroughness of analysis of the initial contents in litterfall. In addition, analysis of monthly collections of litterfall is unusual, as opposed to simply compositing them by weight by species
Response: Thank for your comments. Indeed, the alteration of litter quality would lead to a limitation in studying only decomposed litter or newly shed litter, meanwhile, 13C NMR was not performed for decomposed litter in this study. Therefore, it might be restrictive to totally explain the variations in C quality from newly shed foliar litter to decomposed foliar litter, or the effects of C quality in newly shed foliar litter on the C quality in decomposed foliar litter. Based on this, we are planning to conduct an experiment of an approximate decomposition process of newly shed foliar litter that would be systematically detected from “newly shed litter—litterfall in different decomposition stages—soil organic matter (SOM)”, and the aim of this experiment is explore the effects of newly shed litter that go through decomposition process on SOM formation. In combination with temporal sequences and species, this experiment also aims to explore the response of litter decomposition and SOM formation on the temporal sequences of newly shed litter (the temporal sequences of initial litter quality) and species variations, and further indicating the interactions between plants and soils.
3) The study is fundamentally descriptive, that is, differences initial chemistry of different species, and a correlative study with long term decomposition. (4 years is a very good time for decomposition). The use of stepwise multiple regression is good since many fractions can be correlated x variables.
Response: Thank for your positive comments on our study.
4) I understand the context that species composition of different plots can help predict overall decomposition rates. But perhaps it would help the scope of the study to better describe the context of the study. For example, there is one unconnected statement that the soils tend to be relatively barren. How much is climatological (cold and dry) and how much can be ascribed to species composition. Species composition can interact with climate to exacerbate deficiencies in soil fertility. The dominance of Vaccinium on sandy soils is an example involving acidification.
Response: Thank for your useful comments. This study on initial C quality of foliar litter has limitations in explaining soil C sequestration or soil conditions, therefore, we will add the researches related to soil on the basis of this study (dynamic characteristics of initial litter C quality and changes in litter C quality at different decomposition stages) in the future, further discussing the impact of species composition and climatic conditions on soil fertility in combination with climate conditions and relevant soil physical and chemical properties.
5) I believe that this further discussion of the context would help the hypotheses, which now seem to state the obvious. This would also help elevate the scientific “novelty” and “importance ratings.
Response: Thank for your constructive suggestions. We’ve revised the hypotheses based on the Introduction and Discussion which were revised.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report (Previous Reviewer 2)
Manuscript is acceptable for publication
Reviewer 2 Report (New Reviewer)
Accept in current form.
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
Dear authors,
I carefully examined the Manuscript, which you submitted to the Agronomy journal. I find that the MS is relevant for the wide audience of the journal. Your manuscript quality is high, the language is proper and the research work topics are in good reflection with the objectives of the journal, my decision is accepted after a minor language revision. The manuscript still contains a couple of typos that can be confusing in areas that use different dialects. Please put the sentence in Line 100 into the methods chapter or delete it. Please use the World Reference Base for Soil Resources (WRB) for soil qualification in Line 124. If I were to look for the weakness of the manuscript, I would say that there are few references made in the last five years.
Nice work, congratulations!
Reviewer 2 Report
Dear Authors
I think that this paper will be of interest to the scientific community. The manuscript presents a discrete amount of analytical data and the Authors provide useful information on the variation of initial carbon quality of litters for four species dominant in an alpine forest.
The manuscript needs major revisions; the abstract, the aim, the description on the statistical treatments, the results and the conclusion should be reviewed. The aim of the manuscript should be more incisive and the Authors should emphasise the effects of variations in the initial quality of the investigated litter on the C sequestration. Furthermore, although the Authors refer to seasonal variations in the litter chemical composition and the relationship of these changes to climate, they never consider the different litterfall for the four species. The species have different litter production and a different period of maximum litterfall throughout the year. The Authors should completely revise section 2.4; the description of the statistical analysis should be given in detail; why do the Authors use both an one-way and a two-way ANOVA? this is in part a repetition. What do the Authors mean by a linear analysis? Pearson coefficient? why not use Spearman? What is stepwise regression analysis?
In the results, the Authors should first describe the data and then the statistical differences; perhaps it might be useful to reverse tables 1 and 2. Some changes to the figures and tables are also needed. In the figures, the numbers and tests are often too small; even the text of the legends is often difficult to read. The captions of tables and figures also need to be revised, they are not always explanatory. Often the description is too concise. The Authors should added the number of replicates (n=?). In addition they should check the numbering of the figures, particularly for figures 4 and 5. The results in paragraph 3.4 do not correspond to what is shown in the figure.
The “Conclusions” should not only summarise the main findings but be more general and emphasise the importance of these results in the knowledge of processes associated with soil C sequestration in different ecosystems.
Other specific points:
Abstract
The Authors should avoid abbreviations in the abstract.
Line 25: the Authors should explain what they mean by beneficial; has the speed of the C cycle increased? or has soil C sequestration increased?
Keywords
The Authors should modify some keywords , already present in the title.
Introduction
The introduction section is repetitive in some points.
Line 40: the Authors could added the reference:
Sarker Tushar C., Maisto G., De Marco A., Esposito F., Panico S.C., Alam M. F., Mazzoleni S., Bonanomi G., 2018. Explaining trajectories of chemical changes during decomposition of tropical litter by 13C-CPMAS NMR, proximate and nutrients analysis. Plant and Soil, 436, 13-28.
Line 41: the Authors could added the reference:
De Marco A., Spaccini R., Virzo De Santo A., 2021. Differences in nutrients, organic components and decomposition pattern of Phillyrea angustifolia leaf litter across a low maquis. Plant and Soil, 464, 559-578.
Line 43: the Authors could added the references:
De Marco A., Fioretto A., Giordano M., Innangi M., Menta C., Papa S., Virzo De Santo A., 2016. C Stocks in Forest Floor and Mineral Soil of Two Mediterranean Beech Forests. Forests, 7(8), 181, doi.org/10.3390/f7080181.
The Authors could also take into account these papers elsewhere in the introduction and discussion. For example when they refer to Table 3 and compare their results with those of other species in the literature.
Materials and methods
The Authors should be clearer in describing the statistical analyses used and in processing the data.
Lines 130-132: The Authors should also report the number of replicates. On how many replications is the statistical analysis made? only three?
Results
Line 195: replace “C fraction in PA” with “(proximate analysis)”
The comparison with the literature in table 3 can be expanded; table 3 must be formatted (e.g. for coarse and underlined characters).
Reviewer 3 Report
This article describes the study of the general dynamics and characteristics of the transformation of fresh litter fall of the dominant tree and shrubs species of the alpine forest in the Miyaluo Nature Reserve (the eastern Tibetan Plateau). The tree species were as following coniferous fir (Abies faxoniana) and spruce (Picea asperata) and shrubs were willow (Salix paraplesia), rosa (Rosa omeiensis). The amount of foliar litter was counted during August 2015 to April 2016. Litter counts were conducted at the three key sites in twenty-fold replications. At the laboratory litter samples were air-dried, smashed and sieved (0.25-mm) for chemical analysis (total organic C, N, P, Mn concentrations, sequential extracted organic-soluble, water-soluble, acid-soluble extractives and acid-unhydrolyzable residues, 13C nuclear magnetic resonance-NMR). By the literature and authors opinion NMR results are more precise to characterize the plant litter quality in comparison with the sequential extraction method. The results were statistically processed.
The hypotheses (1) the contents of labile and stable C fractions vary with plant species, and more recalcitrant C fractions be accumulated in the warm and rainy seasons and (2) labile C fractions control total fresh litter dynamics (decomposition and accumulation) were tested in the study.
On the whole, the work is interesting and necessary. Although questions arise about some parts of the manuscript. For example, part 3. Results. Table 2. The proportions of initial C fractions and hydrophobicity indexes in each litter species. The plant material from which date of the sampling collections during period from August 2015 to April 2016 was taken as the initial?
As follows from 2.2. Schematic of the experiment and sampling, “new-shed foliar litters were monthly collected during August 2015 to April 2016”. The authors should explain in more detail, were these portions of plant material freshly fallen or lying there since August? As I see it, Table 1 and Figures 1-3 show the results of the analysis of plant material fallen at different times. 3.4. Further in “The relationships between initial functional C groups and litter decomposition” the authors write about “C loss during litter decomposition” and “the third year and the first year of litter decomposition”. However, no descriptions of the litter decomposition experiment are given in “2.2. Experimental design and sample collection”. Perhaps the authors mean the results described in their other article, but it is not clear from the text of this manuscript.
Another obscure point. If the authors believe that initial nitrogen concentration controlled the biochemical process in the early stage of litter decomposition, why was it necessary to determine manganese concentrations in plant materials?
The respected authors can probably find additional examples of inconsistencies in their presentation and refine the manuscript. Interesting results are obtained, but they should be described logically and in more detail.
