PONE-D-24-38030Soil Nutrients Explain Plant Community Species Diversity Changes Better Than Functional Traits Under Short-Term Warming and Nitrogen Deposition in Alpine MeadowsPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study explores the effects of short-term warming and nitrogen deposition on alpine meadow plant communities. Different warming and nitrogen addition treatments were set up at the Chengduo substation in Qinghai, and investigations and analyses were conducted on plant community structure, functional traits, and soil physicochemical properties. The results show that warming, nitrogen deposition, and their interaction have significant impacts on many aspects of the plant community, such as some functional traits, soil physicochemical indicators, and certain aspects of species diversity. The study found that soil nutrients can explain the changes in plant community diversity better than functional traits. Factors such as total soil nitrogen can explain its changes under warming and nitrogen deposition, providing references for related research and grassland management. Previous studies mainly discussed the responses of plant diversity in alpine grasslands on the Qinghai-Tibet Plateau to climate warming and nitrogen addition from the perspectives of environmental temperature and humidity, soil physicochemical properties, etc., while there are fewer studies from the perspective of plant functional traits （e.g., doi: DOI: 10.1007/s00344-016-9595-0; doi:10.1016/j.scitotenv.2023.168878）. Therefore, this study has certain innovation and is very worthy of publication. Nevertheless, before formal publication, the author is advised to consider the following aspects.

1. This study only explored the impact of warming and nitrogen addition on plant species diversity, but did not discuss the changes in plant phylogenetic diversity. Some previous studies have shown that external disturbances such as warming and nitrogen addition have different effects on plant species and phylogenetic diversity (e.g., doi:10.1016/j.ecoleng.2021.106331; doi:10.1016/j.ecolind.2021.108355; doi:10.1016/j.gecco.2023.e02464; doi:10.3389/fevo.2023.1126651; doi:10.3390/plants12173017; doi:10.1016/j.scitotenv.2023.168878). Therefore, it is recommended that the author add relevant discussions in the discussion section.

2. In addition to conventional soil physicochemical properties, soil microbial community diversity is closely related to plant diversity (e.g., doi:10.1016/j.scitotenv.2023.168878; DOI: 10.1016/j.gecco.2022.e02306). However, this study did not involve soil microorganisms. Moreover, in addition to plant functional traits, phyllosphere microorganisms of plants also have a relationship with plant diversity (e.g., doi:10.3390/plants12162891; doi:10.1016/j.jia.2024.06.008), but this study did not involve phyllosphere microorganisms either. Therefore, it is recommended that the author add discussions on the regulation of plant diversity by soil microbial diversity and phyllosphere microbial diversity.

Reviewer #2: The manuscript presents an experimental field study conducted in an alpine meadow in Qinghai-Tibet Plateau. In this study, researchers experimentally simulated the different levels of warming and nitrogen deposition and studied their effect on changes in plant community composition. They focused on the influence of functional traits and soil physicochemical properties on species richness, diversity and evenness under altering warming and nitrogen deposition. The study is interesting although it is conducted only on one alpine meadow, which limits authors to make some more general conclusions. It could be also interesting to observe the effects for longer time and evaluate temporal changes. I understand that multisite and temporal studies are not always possible to do, but it should be at least stated in discussion as study limitations. And this should be also reflected in the title of the paper. You cannot writhe “in Alpine meadows”, when your study was conducted only on one unique site. The topic of the study seems to be nowadays quite popular, but also very often studied and thus not very novel. Nevertheless, it is quite useful to get such information from different places and I also like the comparison of the influence of traits and soil properties. I think, it is quite interesting point of view.

In general, the topic is interesting, and manuscript is written well. Nevertheless, there are some things, I am not very happy with. Especially, the methods should be more appropriate – there are many missing details about experimental design, measurements and formulas. Some conceptual figure showing the experimental design could be helpful. In the whole manuscript, the names of variables and other terms are not unified and thus, it is very difficult to follow the text. There is a chaos in used formulas and the indexes and how you call them through the text. A part of the results is not presented in a well-arranged way. Some results are presented only in the text without any figure or table support, especially one factor results (in figures are results only for interactions, and values of statistical test with degrees of freedom and p values are missing almost everywhere). Arrangements of these values into a table could be helpful. There are more typing errors in the text, which I did not corrected.

Because the text does not have nor line numbers nor page numbers, it is very difficult to refer to something specific (and I have some suggestions). Thus, I am using at least the pdf page numbering, when necessary.

Abstract

“Additionally, Short-term nitrogen deposition affects plant community structure but has no significant impact on species diversity.” – It is not clear for me, what is the difference between community structure and diversity. It is not very clear nor from the further text. For me, community structure could be represented either by species richness or by diversity. It could also be the functional diversity or at least the proportion of functional groups. In any case, it is necessary to specify it more or write directly what do you mean.

Introduction

Page 10:

I do not like very much the first paragraph of the introduction, where you are listed everything together without going in detail (especially the part about functional group and nitrogen deposition). I prefer to describe it separately in individual paragraph with more details.

Page 11:

The paragraph about the nitrogen enrichment is introduced by response of diversity on climate change, but then you are writing only about the nitrogen deposition problematic. Here, the phrases from the previous paragraph about the effect of nitrogen deposition could be more suitable. And the concluding sentence of this paragraph is describing also warming, but it was not discussed here. May be, it could be useful to interconnect the effect of warming and nitrogen deposition. Or reversely, separate these two topics into two paragraphs.

What do you mean by “physiological traits”? Because in following text, you describe both morphological (SLA, leaf nitrogen concentration) and eco-physiological (photosynthetic rate) traits.

“Beyond leaf economics traits” – photosynthetic rate is not LES trait.

Page 12:

“Studies on the Qinghai-Tibet Plateau indicate that soil organic matter, available nitrogen, and phosphorus significantly affect plant diversity in alpine meadows [22].” – You are writing studies, but then you are citing only one study although it is not metanalysis. The same for more cases throughout the whole text (including discussion).

Materials and Methods

Page 14:

Elymus nutans and Poa annua are not forbs but grasses. And forbs (or grasses) are not family, but functional group. And is it Poa annua or P. pratensis. In Fig. 2, you have only P. pratensis.

50 x 50 m2 does not make sense. It is either 50 x 50 m or 2500 m2.

No warming (W0) treatment does mean that there was no enclosure?

Page 15:

I would like more details about soil and air temperature measurement. In which distance from the soil surface was the temperature measured (it is not enough to have it only in the caption of the figure)? How frequently the sensors measured the temperature? How many sensors did you used? One per one OTC or more?

What does “CK” mean?

Fig. 1:

a) and b) show changes in temperature for individual warming treatment. But before, you stated that you had the combination of plots with simulated warming with nitrogen deposition. Does it mean that these values are averages for W0N0, W0N16 and, W0N32, and so as? It should be stated in methodology.

Why there are axis labels in Chinese language?

The colour of columns of different treatment in c) and d) could be the same as line in a) and b). It would increase the readability of the figure.

For c) and d), it should be stated in the caption what do the letters above the columns mean? I expected that it is the result of Tukey test, but it should be stated in the caption of figure and also, this test should be described in methodology appropriately (what was compared with what).

Page 16:

How did you fit three 0.5 x 0.5 m quadrats into circle plot with diameter 0.95 m? Or I do not understand of the experimental design.

Page 17:

What does representative species for measurement of plant height mean? Do you mean five the most abundant species? How many individuals per species did you measure to get the plant height of one species?

What does the first formula before species richness index mean? It is not described anywhere else.

Page 18:

For trait measurement, you selected six from 15 recorded species. What does 15 recorded species mean? Fig. 2 shows that in total, there were 25 recorded species.

Why did you capitalize sedges, grasses and forbs?

You interchanged forbs and grasses. Poa pratensis is grass while the others are forbs.

Fig. 2:

The names of species should be in italic.

Page 19:

The formula for SLA is not correct and does not fit to the description. I think, the description is enough, and formula is not more necessary. On the other hand, there is not described how did you calculate LDMC.

What does subplot mean? Do you mean one 0.5 x 0.5 m quadrat? You did not write about it before as about subplot.

Is not the quadrat for soil sample the same quadrat as for vegetation and trait data? It is not clear for me.

Page 20:

What does importance value mean? It was not described anywhere else. How did you calculate it?

Page 21:

Do you used Tukey test only for interaction of warming and nitrogen deposition or also for these two factors individually? It is not clear from the methodology. From results, it seams that you tested both interaction and individual factors, but the results are stated only in figures only for interactions.

Page 22:

Vegan package should be cited.

Results

Describing the effect of warming and nitrogen deposition on CWM of traits, you did not present any statistical data (no results of statistical tests), only percentages and if it was or not significant. It should be somewhere, for example a table with the values for statistical test, df and p.

Page 23:

You cannot use once leaf dry weight and LDMC at another time. You should be consistent throughout the whole text.

Fig. 3, 4, 5:

You did not state what the values for W, N and W*N mean. What was the test and what are df?

You did not describe what letters mean. It is the result of Tukey but for what? And why did you not compare also the differences between only warming treatment? You describe it in text, but in figures, you have results only for interactions.

In Fig. 4, the y axis for pH (h) is interrupted, but it is not described nor in the caption of figure, nor in methodology.

Page 28:

The final sentence is more for discussion part than for results part.

Page 29:

There is no df for Monte Carlo permutation test (“(F = 2.2, P= 0.016)”).

Fig. 6:

c) Is it necessary to show so many decimals for in legend for Mantel´s r?

Discussion

The first parts of paragraphs contain very often quite introducing information which would fit more into introduction than to the discussion.

Page 32: I am confused about the community height and height measured as trait. It is not appropriately differed and from results, I had the impression that you did not use the community height. But now, it is in discussion, but I did not find nothing about community height in the results.

Page 34:

“Climate warming tends to reduce soil organic carbon and total nitrogen content [54].” This meta-analysis you cited showed that warming significantly increased soil dissolved organic C but did not change soil total organic C and soil total N. So, this does not support your statement.

Page 37:

“Our study indicates that the Shannon-Wiener index initially increases significantly with warming and then decreases…” – This formulation gives the impression that you measured warming in time and it is not true. You only have different levels of warming treatments.

“In the Loess Plateau, low-level warming increased plant community diversity, while high-level warming decreased it [67], suggesting that high-level warming has a more pronounced effect on species diversity [68].” – high-level warming had rather a more negative than more pronounced effect on species diversity.

You are stated that the water availability is a critical limiting factor for plant growth, but you are not discussing it with your results.

Supporting information

In table with data, there are some names of variables only in Chinese. It should be in English.

Reviewer #3: This article assesses the impact of warming and nitrogen deposition on plant traits, soil properties and species community assembly in an experiment carried out on alpine grasslands of the Qinghai-Tibet Plateau. I enjoyed reading this manuscript, the text is well written and the experiment represents a large amount of effort. The discussion section could be improved to link it better to the research questions. The graphs could also be clearer, particularly with reference to the results of the Tukey test. I have provided more details below and made some other suggestions for further improvements.

Materials and methods

In the section ‘Plant Community Structure Survey and Analysis’ the meanings of the letters C and B in the first formula are missing.

Some of the methods mentioned do not have a reference associated with them e.g. the molybdenum antimony anti-colorimetric method, hydrazine sulfate method and the indophenol blue method. Please add appropriate references for these techniques.

As well as increasing the temperature, open top chambers (OTCs) have been shown to alter the soil moisture and also protect plants against wind (see for example Hollister et al. 2023 https://doi.org/10.1139/as-2022-0030). Did you measure soil moisture in the experimental set up, before and after the placement of the OTCs? You mention in the discussion that reduced water availability also has an impact on community composition and growth so it could be this rather than (or in addition to) the warming that explains your results.

Results

Figure 3 – the figure legend does not match all the graphs presented. CWM_LW (leaf width), CWM_LPC (leaf phosphorus concentration) and CWM_LCC (leaf carbon concentration) are listed in the legend but are not part of the graphs. Instead CWM_LC/N and CWM_LC/P are in the graphs but not in the legend. Please correct.

Figures 3, 4 and 5 – it is not clear what the Tukey test letters refer to. Please add a note in the figure legend clarifying the difference between the upper and lower case letters.

Under the section “Effects of Warming and Nitrogen Deposition on Plant Community Species Diversity” the sentence “Nitrogen deposition also significantly affects the importance values of forbs and sedges” is repeated, please delete.

Discussion

At the beginning of the discussion it would be beneficial to have a short summary paragraph of the overall findings before going into the details. The discussion is presented in such a way that seems to highlight previous research findings before talking about your results. I would re-frame this to first emphasise the findings from your study and then explain the results in the context of previously published research.

At the end of the first paragraph of the discussion you mention “intraspecific trait responses to temperature changes vary among different species.”. Did you check for intraspecific variation in traits? It could be that there is a large trait variation within species or that one particular species is driving the changes observed.

Under warming and nitrogen deposition some traits did not change - the community-weighted means of leaf width, leaf phosphorus content, leaf carbon content, and the leaf carbon-to-phosphorus ratio. Do you have any speculation as to why this might be? It would be useful just to add a sentence in the discussion.

You mention that warming increases plant height. Do you think this will have a knock-on effect on herbivory e.g. the taller species will be more targeted by herbivores and so actually be at a disadvantage under warming? My understanding is that herbivory plays an important role in the Qinghai-Tibet Plateau but it is not mentioned in the manuscript. A paragraph about the potential impacts of your findings on herbivores would be interesting.

In the introduction you say “This study seeks to provide a theoretical basis for maintaining the species diversity and sustainability of plant communities in the alpine meadows of the Qinghai-Tibet Plateau under future climate warming and nitrogen deposition scenarios.”. However, I don’t see this mentioned in great detail in the discussion. It would be useful to have a paragraph about the implications of your results for future biodiversity in the Qinghai-Tibet Plateau and also any practical suggestions for how to maintain diversity in the future.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Effects of warming and nitrogen deposition on species and functional diversity of plant communities in the alpine meadow of Qinghai-Tibet Plateau

PONE-D-24-38030R1

Dear Dr. DE,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Francesco Boscutti

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The author has made a large number of revisions and I have no further comments. It is recommended to accept it

Reviewer #2: A revised the manuscript entitled “Effects of warming and nitrogen deposition on species and functional diversity of plant communities in the alpine meadow of Qinghai-Tibet Plateau” is significantly improved compared to the previous version of the manuscript. The authors made a great job on it. I am satisfied with the answers on my questions and with incorporating my suggestions into the text of the manuscript.

I have only few minor suggestions:

While you defined species diversity indices, you did not do it for functional diversity indexes (L 251-253). I think it is not necessary to write the formulas but at least citations of the relevant papers should be there to know what you are referring to.

L 274 – you are firstly mentioned the structural equation models and in following text, you are using the abbreviation SEM. But this abbreviation was not stated before, so it should be there.

The Results part - it would be more comprehensive if you are referring to the Figures and Tables throughout the whole text and not only at the end of each paragraph. You also should refer not only to the whole Figures but also to the individual panels (a, b, c…) of Figures.

Fig.2 – the figure is two time there. In the caption, L 331, you stated “The same bellow”. What does it mean? If you mean that it is the same for next Figures, I think it is not the best solution. I prefer to repeat the proper caption with detail for each Figures (Fig. 3, 4, Table 2). Or another solution is to refer to these Figures in the captions (e.g., abbreviations same as in Fig.2).

L 516-517 – “mainly” used two times is not necessary.

L 555 – two times “leaf length-weighted mean”.

Reviewer #3: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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