Differences in Fine Root Foraging Traits of Two Dominant Tree Species (Cunninghamia lanceolata and Quercus acutissima) in Subtropical Forests

Round 1

Reviewer 1 Report

1.      Xu et al reported differences in fine root foraging traits between two tree species exhibiting distinct mycorrhizal symbioses: Cunninghamia lanceolata (arbuscular mycorrhiza species) and Quercus acutissima (ectomycorrhiza species). Their study showed responses of absorptive fine root versus transport fine root growth, mycorrhizal association, and functional traits. Although the authors supplied mycorrhizal colonization percentages, no accompanying images were provided to substantiate the claim of "contrasting mycorrhizal symbiosis" as indicated in the title.

2.      The Method section requires clarification, particularly regarding the administration of fertiliser treatments (NPK, F), including details on how treatments are applied, their duration, and frequency. In Section 2.2, the sequence of fertilization treatments and root chamber installation is unclear. The author initially introduces fertilization treatments, followed by root chamber installation. To enhance understanding, it is recommended to include images or schematic diagrams illustrating the root chambers, providing a visual representation of the procedure for the readers.

3.      The authors mentioned, "Considering the initial use of chemical fertilizers as base nutrients." Could the author please provide more details?

4.      The authors mentioned an observation period of 4–5 months. Is the data solely derived from one season, or were there any experimental repetitions conducted?

5.      Table 1 typo, it should be fine root biomass for FRB

6.      Please specify that different letters in Figures 3 and 4 represent statistical significance in figure legends.

7.      The study outcome lacks novelty, resembling findings from another study (e.g., https://www.jstor.org/stable/48703612). I suggest enhancing the research by incorporating mycorrhizal species identification and exploring its association with functional traits, which could contribute to a more robust and distinctive contribution to the field.

Overall, the English is good, but there is a need for editing in the Method section, especially in sections 2.1 and 2.2.

Author Response

Dear Ms. Janina Yin and Reviewers,

Thank you for your letter and for the reviewers’ comments concerning our manuscript (Number: forests-2829513). We appreciate these insightful comments. We have studied them carefully and revised our manuscript accordingly. Point by point responses to the reviewers’ comments are as follows. In addition to the reviewers' comments, we have made a number of revisions to sections where the editorial board noted apparent similarity, including the abstract (see lines L18-21), introduction(see lines L80-82, L98, L100-101, L104-105, and L111-113) materials and methods(see lines L196-207). But some of them are fixed interpretations and we can't make changes to them. 

Revised portions are marked in red font in the revised manuscript. Please note that the responses refer to the new line numbers in the revised manuscript. 

With best regards,

Sincerely yours,

Xinying Xu. E-mail: [email protected]

Department of Ecology, College of Ecology and Environment, Nanjing Forestry University,Co-innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu 210037, China

Responses to Reviewer 1:

[Comment 1] Xu et al reported differences in fine root foraging traits between two tree species exhibiting distinct mycorrhizal symbioses: Cunninghamia lanceolata (arbuscular mycorrhiza species) and Quercus acutissima (ectomycorrhiza species). Their study showed responses of absorptive fine root versus transport fine root growth, mycorrhizal association, and functional traits. Although the authors supplied mycorrhizal colonization percentages, no accompanying images were provided to substantiate the claim of "contrasting mycorrhizal symbiosis" as indicated in the title.

[Response 1] Thanks for your positive assessments and valuable comments. Previous researches have demonstrated that Chinese fir (Cunninghamia lanceolata ) is a typical arbuscular mycorrhizal tree species, whereas Quercus acutissima is characterized as a typical ectomycorrhizal tree species. The relevant references to this topic have been cited in the manuscript. (see line L111-113)

Newly added references

39. Xu, X.; Wang, X.; Cleary, M.; Wang, P.; Lu, N.; Sun, Y.; Rönnberg, J. Slope position rather than thinning intensity affects arbuscular mycorrhizal fungi (AMF) community in Chinese fir plantations. Forests 2020, 11, 273.https://doi.org/10.3390/f11030273
40. Li, Y.; Zhang, T.; Zhou, Y.; Zou, X.; Yin, Y.; Li, H.; Liu, L.; Zhang, S. Ectomycorrhizal symbioses increase soil calcium availability and water use efficiency of Quercus acutissimaseedlings under drought stress. European Journal of Forest Research 2021, 140, 1039-1048.https://doi.org/10.1007/s10342-021-01383-y

[Comment 2] The Method section requires clarification, particularly regarding the administration of fertiliser treatments (NPK, F), including details on how treatments are applied, their duration, and frequency. In Section 2.2, the sequence of fertilization treatments and root chamber installation is unclear. The author initially introduces fertilization treatments, followed by root chamber installation. To enhance understanding, it is recommended to include images or schematic diagrams illustrating the root chambers, providing a visual representation of the procedure for the readers.

[Response 2] Done as suggested. (see lines L19-150, 157-158, 161-166 and 177 )

[Comment 3] The authors mentioned, "Considering the initial use of chemical fertilizers as base nutrients." Could the author please provide more details?

[Response 3] Because chemical fertilizers such as urea and calcium superphosphate are commonly applied as a base fertilizer during the planting phase or the early growth stage (typically 1-5 years) for Cunninghamia lanceolata and Quercus acutissima, and the subsequent self-fertilization effect of litter during the later growth stages in the plantation management, so three treatments were applied around each species: control (unfertilized, CK), inorganic nutrient (NPK, Osmocote, slow-release fertilizer containing 15%N-9%P-10%K, duration for 8-9 months), and organic nutrient (F, crushed leaf litter). (see lines L144-147 )

Newly added references

41. Sharma, A.; Chetani, R. A review on the effect of organic and chemical fertilizers on plants. Int. J. Res. Appl. Sci. Eng. Technol 2017, 5, 677-680.https://doi.org/10.22214/IJRASET.2017.2103

[Comment 4] The authors mentioned an observation period of 4–5 months. Is the data solely derived from one season, or were there any experimental repetitions conducted?

[Response 4] The observation period of this study was from the beginning (April) to the end (September) of the growing season and no experimental repetition were conducted.

[Comment 5] Table 1 typo, it should be fine root biomass for FRB

[Response 5]  Done as suggested. (see lines L217)

[Comment 6] Please specify that different letters in Figures 3 and 4 represent statistical significance in figure legends.

[Response 6]  Done as suggested.(see lines L280-282 and L297-298)

[Comment 7] The study outcome lacks novelty, resembling findings from another study (e.g., https://www.jstor.org/stable/48703612). I suggest enhancing the research by incorporating mycorrhizal species identification and exploring its association with functional traits, which could contribute to a more robust and distinctive contribution to the field.

[Response 7] Although the root foraging traits and the categorization of roots based on functional characteristics in our study parallel those described by Han (e.g., Han, 2018, https://www.jstor.org/stable/48703612), the objectives of our research diverge from Han's investigation. Han primarily focused on the influence of understory species diversity on root foraging strategies. In contrast, our study was more concerned with the effects of nutrient addition on root growth and the associated functional traits. Moreover, the soil blocks were selected randomly for root isolation in Han's research, whereas, in our study, the roots with similar initial traits were to select to culture under fertilization addition for a growing season, thereby mitigating the impact of external factors on root development. We also agree with the reviewer' comments on the importance of incorporating mycorrhizal species identification into our research. This will be our further research as suggested in our conclusion.

Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.pdf

Reviewer 2 Report

18 Jan 2024

Overview

This study by Xu et al contrasts the root architecture of two tree species. However, the author paints a narrative of root architecture as influences by mycorrhizae. However, the mycorrhizal-type hypothesis cannot be tested under this experimental design, as the authors lack replication of tree species. The authors need a minimum of three tree species for each mycorrhizal type to evaluate this hypothesis (triplicates).

Major Comments

         I.            This study cannot make assumptions on AM-fungi versus EM-fungi based on one tree species that is associated with AM-fungi (Cunninghamia lanceolata), and one tree species that is associated with EM fungi ( Quercus acutissima). Moreover, you need multiple arbuscular mycorrhizal tree species, to make an assertion  about mycorrhizal type (Malik et al., 2022, Chen et al., 2016). Otherwise, you are just limited difference between tree species. For example, comparing one understory species (e.g., Acer saccharum), to one canopy tree species (e.g., Quercus alba) can tell only us differences that may exist between the two species, but we do not have enough replication to make a generalization about canopy versus understory species. Similarly, you did not include enough trees species that associate with either mycorrhizal type.  In statistics, you usually need three replicates. This means that  you need at least three tree species  that associate with AM-fungi versus three trees that associate with EM-fungi.

       II.            It is important that you remove the AM versus EM contrast from the paper ( title, abstract, introduction, conclusion etc.), as it is erroneous. If you at least sequenced for the AM and EM-fungi, you might have a better argument.

     III.            With respect to the analysis , the F statistics should be cited with DF and p value OR the author should provide 95 percent confident intervals. Also, I would expect Table 2 (ANOVA)  to report F- statistics, sum of squares, and provide degrees of freedom. Instead, the present ANOVA table is an enigma.

    IV.            Methods mention post-hoc analysis, but post-hoc analysis (e.g., Tukey HSD)was not reported in the results

      V.            L 27 it is unclear what is the ‘F treatment’ this early in the manuscript

References

CHEN, W., KOIDE, R. T., ADAMS, T. S., DEFOREST, J. L., CHENG, L. & EISSENSTAT, D. M. 2016. Root morphology and mycorrhizal symbioses together shape nutrient foraging strategies of temperate trees. Proceedings of the National Academy of Sciences, 113, 8741-8746.

MALIK, R. J., BRUNS, M. A. V., BELL, T. H. & EISSENSTAT, D. M. 2022. Phylogenetic Signal, Root Morphology, Mycorrhizal Type, and Macroinvertebrate Exclusion: Exploring Wood Decomposition in Soils Conditioned by 13 Temperate Tree Species. Forests, 13, 536.

Author Response

Dear Ms. Janina Yin and Reviewers,

Thank you for your letter and for the reviewers’ comments concerning our manuscript (Number: forests-2829513). We appreciate these insightful comments. We have studied them carefully and revised our manuscript accordingly. Point by point responses to the reviewers’ comments are as follows. In addition to the reviewers' comments, we have made a number of revisions to sections where the editorial board noted apparent similarity, including the abstract (see lines L18-21), introduction(see lines L80-82, L98 L100-101, L104-105, and L111-113) materials and methods(see lines L196-207). But some of them are fixed interpretations and we can't make changes to them. 

Revised portions are marked in red font in the revised manuscript. Please note that the responses refer to the new line numbers in the revised manuscript. 

With best regards,

Sincerely yours,

Xinying Xu. E-mail: [email protected]

Department of Ecology, College of Ecology and Environment, Nanjing Forestry University,Co-innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu 210037, China

Responses to Reviewer 2:

[Comment 1] This study cannot make assumptions on AM-fungi versus EM-fungi based on one tree species that is associated with AM-fungi (Cunninghamia lanceolata), and one tree species that is associated with EM fungi (Quercus acutissima). Moreover, you need multiple arbuscular mycorrhizal tree species, to make an assertion  about mycorrhizal type (Malik et al., 2022, Chen et al., 2016). Otherwise, you are just limited difference between tree species. For example, comparing one understory species (e.g., Acer saccharum), to one canopy tree species (e.g., Quercus alba) can tell only us differences that may exist between the two species, but we do not have enough replication to make a generalization about canopy versus understory species. Similarly, you did not include enough trees species that associate with either mycorrhizal type.  In statistics, you usually need three replicates. This means that you need at least three tree species  that associate with AM-fungi versus three trees that associate with EM-fungi.

[Response 1] Due to our unclear presentation, the results caused misunderstanding to the reviewer. We have made revisions in the manuscript.(see lines L21, 79-89, 108- 120, and 407) The primary objective of our research was not to compare the relationships between arbuscular mycorrhizal and ectomycorrhizal species. Instead, our aim was to investigate the root growth and functional traits in response to nutrient addition for the two tree species (Cunninghamia lanceolata and Quercus acutissima) with contrasting mycorrhizal associations in China. Given the widespread distribution and cultivation of the two tree species, and their common coexistence in mixed forest management practices in China, our study are help to understand whether and how changes in soil nutrient availability will lead to integrated responses in root traits within and across species and important to plantation management. In light of the reviewer's comments, we have revised some statements to avoid any potential misinterpretation. Furthermore, the issues raised by the reviewer are also areas for future research, as we point out in the conclusion section.(see lines L419-422).

[Comment 2] It is important that you remove the AM versus EM contrast from the paper ( title, abstract, introduction, conclusion etc.), as it is erroneous. If you at least sequenced for the AM and EM-fungi, you might have a better argument.

[Response 2] We have revised the comparative statements concerning the two mycorrhizal species across the abstract, introduction, and conclusion sections, ensuring that they are now accurately reflected and appropriately stated. The modifications are as follows. (see lines L21, 79-89, 108- 120, and 407). 

[Comment 3] With respect to the analysis , the F statistics should be cited with DF and p value OR the author should provide 95 percent confident intervals. Also, I would expect Table 2 (ANOVA)  to report F- statistics, sum of squares, and provide degrees of freedom. Instead, the present ANOVA table is an enigma.

[Response 3] We have added sum of squares, df, mean square and p value. (see lines 256). However, due to the limited layout, we have provided F values and p value for the tables in the manuscript. The sum of squares, df and mean square are shown here.

Table 2. Analysis of variance results for effects of species, root category, treatment, and interactions on fine root traits

*, P < 0.05; ** P < 0.01. Species (S); Root category (R); Treatment (T). Table 1 provides definitions of listed root trait acronyms.

[Comment 4]  Methods mention post-hoc analysis, but post-hoc analysis (e.g., Tukey HSD)was not reported in the results

[Response 4] Done as suggested. (see lines L223-224):

[Comment 5]  L 27 it is unclear what is the ‘F treatment’ this early in the manuscript

[Response 5] Done as suggested. (see lines L26-27).

Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors have addressed all concerns and edited the manuscript; the quality of the manuscript has improved

Author Response

We thank the reviewers for their suggestions to improve the quality of the manuscript.

Reviewer 2 Report

The author did not address the changes.  As mentioned, the design is flawed.  They are a contrasting two tree species.  Thats the basis of the study.  The author does not have enough species replicate to contrast mycorrhizae.  They have 1 EctoMycorrhizal species and 1 arbuscular mycorrhizal species

My original review stands

.

Author Response

Responses to Reviewer 2:

[Comment 1] The author did not address the changes. As mentioned, the design is flawed. They are a contrasting two tree species. Thats the basis of the study. The author does not have enough species replicate to contrast mycorrhizae. They have 1 EctoMycorrhizal species and 1 arbuscular mycorrhizal species.

[Response 1] Following the reviewer's feedback, we have revised the title, abstract, introduction, and conclusion, eliminating the discussion on the types of mycorrhizae. Our study does not intend to use Cunninghamia lanceolata and Quercus acutissima to represent arbuscular and ectomycorrhizal species, respectively. This consideration was taken into account in the early experimental design, hence we did not discuss the differences in fine root foraging between mycorrhizal types in our discussion. The mycorrhizal type between C. lanceolata and Q. acutissima is merely the factor used to explain the differences in nutrient acquisition strategies between the two species. C. lanceolata and Q. acutissima are typical tree species and widely cultivated in the warm temperate and subtropical regions of China, which are the vital sources of timber and bioenergy. Moreover, mixing C. lanceolata and Q. acutissima to form a mixed forest is a common practice in forest management in China. Therefore, understanding the differences in nutrient acquisition of fine roots under nutrient addition is crucial to plantation management.

In the first version of the revised manuscript, we made the following modifications：

The descriptions on effects of mycorrhiza type on nutrient acquisition of fine roots has been removed from the abstract (see line L22) and introduction in the manuscript (see lines L81-93 and L105-118).

On the basis of the first version, we made the following modifications in the second version of the revised manuscript：

The title has been modified to "Differences in fine root foraging traits of two dominant tree species (Cunninghamia lanceolata and Quercus acutissima) in subtropical forests", and the expression on "contrasting mycorrhizal symbiosis" was removed from the title (see line L2-4).

In the revised abstract, the term "mycorrhizal traits" has been excised, with a focus maintained solely on "mycorrhizal colonization" (see lines L19-22).

The expression on "mycorrhiza type (arbuscular mycorrhiza, AM vs. ectomycorrhiza, ECM)" has been intentionally excluded from the introduction(see lines L46, L81 and L94).

In the revised manuscript, we also provide a basic introduction to C. lanceolata and Q. acutissima, as well as to explain the reason for choosing the two tree species (see lines L105-116).

The final conclusion has been modified accordingly (see line L422-427).

Newly added references

38. Wan, X.; Huang, Z.; He, Z.; Hu, Z.; Yang, J.; Yu, Z.; Wang, M. Effects of broadleaf plantation and Chinese fir (Cunninghamia lanceolata) plantation on soil carbon and nitrogen pools.  J. Appl. Ecol. 2013, 24, 345–350.

Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.pdf