Stomatal Characterization of Grasses Present in an Oak-Pine Ecosystem

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of MS: grasses-4173706

 

Major comments

 

Marquez-Godoy et al., present the stomatal characterization of 10 grass species present in an oak-pine ecosystem. While their results are interesting measurements, the lack of ecological analysis (e.g., links with environmental variables) and proper integration of results into existing literature makes for a generally textbook-like read. The goals of the study are not clear (i.e., why were species characterized?) nor is it clear why statistical tests were performed (which hypothesis were the authors trying to answer). While the study has some value, as a characterization of grass species traits, it is currently bloated with textbook like examples that lack proper integration and connection with the study outcomes. As such, it requires a full rework in my opinion.  

 

Regarding the text, overall, many sentences/statements are lacking references. There are many (review) studies describing statements and mechanisms in the intro, and even if they regard general principles, it is useful for readers to find resources and validate what the authors are claiming. Furthermore, the results and discussion sections are not well separated, nor is it clear why some info is mentioned given it does not link back to the actual data analyzed in the study.

 

Minor comments

 

L45-46: ref? Proposed: Zi et al., 2023

L46: ref? Proposed: Reynaert et al., 2021

L49: ref?

L77: The introduction is missing meaningful hypotheses that can be tested using the models. Why would expect certain species to have better/worse traits? Is nothing known about their ecological characteristics (e.g., drought / heat tolerance) from the literature? 

L103-104: How many replicate leaves per individual (pseudo replication, but good to have for plant variability) and per species were taken? Also, how were leaves selected, sun leaves (top of plant) and shade leaves (bottom of plant) may differ greatly in stomatal density/leaf characteristics?  Please mention this here already so it is clear what was sampled and how large sample sizes were.  

L132-133: reference? Also, this type of ‘interpretation’ is usually something for the discussion section. 

L135-141: This is absolutely discussion and already interpretation of results; given the ‘results’ section is somewhat limited, it could be a solution to merge them together in discussion & results (depending on the editor’s agreement obviously). 

L149-L153; could be in the discussion, but since leaf size, photosyntetic efficiency and WUE not actually measured I’m not sure why it is mentioned

L153-L157; this info seems textbook and redundant?

L164-L166: you have 10 species, i.e., 10 data points to do a more formal correlation analysis (ideally non-parametric). While it is a small sample size it is possible, instead of claiming it ‘appears’ without any statistical support.

L179-L184: this is introduction/discussion i.e., textbook info not contributing to the actual results. 

L194 - L206: textbook info, how does this relate directly to your results?

L207-L222: textbook info, how does this relate to your results?

L305: italics

L300-322: This is proper integration of results in existing literature.

References

 

Reynaert S, De Boeck HJ, Verbruggen E, Verlinden M, Flowers N, Nijs I. Risk of short‐term biodiversity loss under more persistent precipitation regimes. Global Change Biology. 2021 Apr;27(8):1614-26.

 

Zi L, Gargallo-Garriga A, Oravec M, Abdelgawad H, Nijs I, De Boeck HJ, Reynaert S, Donnelly C, Li L, Beemster GT, Urban O. Ecometabolomic analysis of the effect of more persistent precipitation regimes reveals common and tolerance related metabolic adjustments in four grassland species. Environmental and experimental botany. 2023 Nov 1;215:105489.

Author Response

We thank the reviewer for these valuable comments. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The submitted manuscript “Stomatal characterization of grasses present in an oak-pine ecosystem” by Jaime Neftalí Márquez-Godoy et al. analyses stomatal parameters of some selected grasses in a particular ecosystem. While I appreciate the work that was put into this research, I think there are some flaws that have to be amended before the manuscript can be published.

General comments

In the introduction, the global needs are addressed sufficiently (stomatal characterisation is valuable), but I think that there is a lack of justification for the selected approach: why did the authors choose this particular ecosystem? Why was it important to choose 10 species, and not 5 or 15? Has this ecosystem been characterised before in terms of the native plant stomatal features? Have any of the grasses described in the present study had their stomatal characteristics researched before? Also, why is it important to evaluate the trichome density (which is not a stomatal characteristic per se) as well in this context?

Fig. 1 shows the sampling location sites, but only for 6 out of 10 grasses. Where are the sites for the other grasses? I failed to find any mention of the sample sizes. How many impressions per leaf were made? How many leaves per species? If a particular grass can be found more or less uniformly throughout the selected area (including different climate types), how did the researchers choose which sites to sample from and how many samples to take?

In the Results section, there is actually very little space devoted to the results themselves. I think that lines 135-162 and 164-184 are more fit to the discussion or to the introduction (to some extent), as they do not describe what the authors have found during their research.

I think it would be very useful for the reader to see scalebars in Fig. 2.

In Table 2, I see that for all the species and all parameters, “average” values are approximately 10 times lower than the corresponding minimum and maximum values, which I presume should not be the case.

I think the authors should expand the results by providing more information (p-values etc.) on their statistical analysis.  I find it hard to believe that there were no differences between leaf surfaces for all grasses and all parameters. For example, DE of B. barbinodis differs by at least 3 times, which I think should be a large enough difference to be significant. Regarding the differences between species – which of the differences were significant? I have doubts whether DE between Ch. submutica and B. anomalus were significantly different. In other words, I believe that all the information showing which values differ from which should be disclosed. I also think that, when comparing stomatal parameters, it is better to focus on the average values rather than the maximum and minimum, since these may be outliers and not representative of the species as a whole.

The authors should carefully reexamine measurement units throughout the article. If I understand correctly, DE, DT, and NC are all counted per mm², so in Table 2 (and throughout the text), the measurement units should be mm^-2, indicating that it is not an area but counts per area.

The Discussion mostly concerns the analysis of why stomata in general and some of their parameters are important. Since there is hardly any discussion involving the results acquired by the authors, it rather feels like a literature review. I think the authors should examine their results and comment on them: are the acquired parameter values as expected? How do they compare with other research? Did the different sampling locations have any influence on the stomatal characteristics? What can the results say about the value/adaptability of these grasses? For example, in the conclusions, the authors state that “stomatal characterization provides a valuable framework for identifying forage species and genotypes with traits associated with improved stress tolerance and resource-use efficiency”. So, can the authors comment on which of the presented grasses show traits associated with improved stress tolerance and resource-use efficiency?

While the abbreviations may be common in Spanish (for example, IE for stomatal index), for English-speaking readers, English-based abbreviations (e.g. SI) would be less confusing.

 

Specific comments:

Lines 43-48.

I think that the statements in these lines that are not justified by [6] or [7], should be justified by one or several citations.

 

Line 86.

A typo: “zonesre”

 

Lines 86-87.

Oak names should not be in parentheses.

 

Line 114:

Wilkinson (1979) should be added to the reference list.

 

Line 124:

I think it should be “normality” and not “normally”

 

Line 173 (Table 2):

I think it should be “species”, not “specie”. Also, not “minimun” and “maximun”, but “minimum” and maximum”.

 

Line 188:

“Bromus anomalus” should be in italic.

 

Line 215:

Trichome density is without measurement units.

 

Line 217:

I failed to find any mention of trichomes in the reference [42].

 

Line 217:

I think “thermal insulation” should be mentioned only once.

 

Line 257-258:

I think “decrease in photosynthesis” is not a precise term, because out of context it can mean many things. Also, I believe that a correlation cannot be used to infer causation. For the sake of an argument, it cannot be disproved that in those particular conditions, Y(II) is so low that the plant defence mechanisms affect stomatal parameters.

 

Line 305:

“S. scoparium” should be in italic.

 

Line 307:

“M. arizonica” should be in italic.

 

Line 329:

I think “Please add” should be removed.

 

Overall, I see value in the present research, but I think that the manuscript must be restructured to a more classical structure: the introduction should provide the scientific problem, its context, and an actionable research question, which would make the readers believe that the experimental approach selected by the authors will answer this question. The methods should provide more details so that the research is, in principle, reproducible. The results section should concern what the authors have found out in detail, and the importance and meaning of these findings should be presented in the discussion. While my concerns are serious, I still think that after recontextualization, these results are worth publishing in Grasses, so I recommend the editor to reconsider publishing this article after a major revision. Hopefully, I have not discouraged the authors, for whom I wish a lot of good luck!

Author Response

We thank the reviewer for these valuable comments. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I read the manuscript which quantify stomatal traits on forage grasses having contrasting adaptation responses, with great interest. The authors assessed stomatal features and related those to the growth environment.  However, I have the following minor comments.

The manuscript report to have carried out non-parameteric test but no indication of KS statistic or p-values were reported.

What is the reason behind the choice of months in the measurement? Did you asses leaf thickness simultanously? That would definetly strengthen the manuscript core proposal to relate the stomatal traits to adaptation response for those grasses as also pointed out on line 169.

The choice of statistics in the Abstract is too detailed and need to be in the methods.

Line 46 needs to be supported by references

Please put a scalebar on Fig. 2. White letter labels on the subpanels are not clearly visible on a print. Could you share the dataset on open data repositories? There seems to be air bubbles on D and F. How was that handled?

Line 129 how is the amphistomy determined? Is there a minimum number of stomata on adaxial side for a leaf to be amphistomatous? 

Table 2 need to be improved. Could bar charts be used instead? Table title should redefine the abbreviations. Show where adaxial and abaxial split in the table. AmfiEs or AnfiEs? Average values well below min and max for a given CV is hard to understand. 

Line 196 doesn't read well. 

The conclusion should be dramatically shortened.

 

Author Response

We thank the reviewer for these valuable comments. Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Review of “STOMATAL CHARACTERIZATION OF GRASSES PRESENT 2 IN AN OAK-PINE ECOSYSTEM “

 

Major comments:

 

The manuscript was somewhat improved but still requires some modifications? The most important relates to interpretation of results regarding the implied correlation with specific traits that is not numerically explored.. 

 

It would be interesting to have an image of what the habitat actually looks like, since a lot of the time the authors talk about it being very open vegetation (but one would not initially associate that with an oak-pine ecosystem). 

 

Minor comments:

 

L50: I presume these changes are globally? Please end sentence with globally if so.

L72: Reference?

L111: Remove ‘a’ between ‘was’ and ‘conducted’ at the start of the sentence.

L117: Should be ‘across’ instead of ‘a cross’.

L151: Don’t yo mean epistomatic instead of epistatic?

L169: How often? In how many of the cases - I already commented on this earlier..

L241: Either choose ‘assessed’ or ‘observed’.

L256: Agreed that an Fv/Fm of 0.33 is generally quite low for most species, but it’s usually in comparison to others that such statements are made. It might be better to rephrase this to ‘downregulated PS II efficiency’ or something more general, given this 0.33 is definitely not universal. 

Comments on the Quality of English Language

Please spell and grammar check the text.

Author Response

We thank the reviewer for these valuable comments. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a review of a revised version of the manuscript “Stomatal characterization of grasses present in an oak-pine ecosystem” by Jaime Neftalí Márquez-Godoy et al. While the authors addressed the majority of my concerns, there are still some areas that, in my opinion, need improvement.

 

While the distribution of the plant selection sites is clearer in the new version of Fig. 1, its legend still only shows 6 species. For example, there is no Chloris submutica.

My comment about adding scalebars in Fig. 2 was not addressed by the authors (by the counting by the authors, it is a comment between Comment 3 and Comment 4).

My comment about statistical analysis was also not addressed (by the counting by the authors, it is a comment between Comment 4 and Comment 5).

The authors changed the abbreviation of stomatal index to IE to SI, but other non-English-based abbreviations, such as DE for stomatal density, were left unchanged.

While the authors agree that the interpretative paragraph consisting of lines 154-160 was more suitable for the Discussion, it remains in the Results. Moreover, it starts with “Conversely”, though it is not clear conversely to what.

Line 151: I think it should be “epistomatic”, not “epistatic”

Table 2:

The authors did not address my observation about “specie” instead of “species” in the top line of the table.

For Bouteloua hirsuta adaxial NC, the average is lower than the minimum.

For Muhlenbergia phleoides adaxial DT, the minimum is higher than both the average and the maximum.

For the same species abaxial NC, the average is lower than the minimum.

The same problem with Panicum bulbosum abaxial DE.

 

Lines 188-189: I think it should be either “stomata per mm²”, or “stomata mm^-2”.

 

Overall, while the majority of my current observations are minor and can be addressed very quickly, I think the lack of reaction to my comment on statistics (and to some extent, continuing problems with the data in Table 2) mean that the manuscript in the current form still has to receive a major revision in order to be published.

Author Response

We thank the reviewer for these valuable comments. Please see the attachment.

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Text has been reworked alright.

Author Response

We thank the reviewer for these valuable comments. 

Reviewer 2 Report

Comments and Suggestions for Authors

To be brisk, the authors have diligently responded to my comments. I have only one remark to add. I am not sure what the reason is, but while the authors did add scale bars to Fig. 2 micrographs, they are extremely blurred so that one might miss them altogether. The resolution must be improved. Also, I am not sure why some of the micrographs were changed, and I am not sure whether this is important.

I think that providing a better quality Fig. 2 is the last adjustment I my ask from the authors. In other words, I believe the article can be accepted after a minor revision.

P.s.

Concerning Fig. 1, it does not have to be corrected here, but, for the future, I think the authors should explore different ways of attributing a property: it does not have to be only colour, since for the reader, it may be hard to discern different shades of, let’s say, blue. It may also seriously impede the information transfer for colour-blind readers. I suggest using different shapes (asterisks, squares and so on) in addition to colour.

P.p.s. Good luck!

Author Response

We thank the reviewer for these valuable comments. Please see the attachment.

Author Response File: Author Response.pdf