Drought Impact on the Morpho-Physiological Parameters of Perennial Rhizomatous Grasses in the Mediterranean Environment
Round 1
Reviewer 1 Report (New Reviewer)
Reviewer
Dear Authors,
The submitted manuscript addresses an important topic that focus on the use of PRGs for bio-energy production for improving plant morpho-physiological indicators in particular under drought conditions. The main objective of this research study was focussed on experimental assessment of the effect of drought on the physi- ology and growth of three bioenergy crops (3 species) in Mediterranean environment. The results were well presented within a good discussion however methodological concept and research hypothesis formulation were moderately presented. In my opinion, the manuscript needs major revision and some clarifications to better fit the research questions of this study with discussion and the main finding.
A/General comments
1. The research hypothesis is poorly developed and not matches well with manuscript title and also within all obtained results. Authors are invited to improve this section in introduction
2. Material and methods section was poorly justified in terms of references that are corresponding to each performed method and analysis. Please try to argument and justify any applied protocol and method.
3. The statistical analysis that was performed on collected data are not clear for reader and they are very complex to understand because authors mentioned both YEAR and SPECIES homogeneous group (test post hoc) for the same colon (Capital and Greck letter). Author could take in consideration of the INTERACTION between these two factor to inspire the homogeneous groups and not to add separately the groups in mean for each studied factor. This very important request in this revision.
4. Discussion section was really limited, as that author could explain and more discuss the interaction between the changes in each measured indicators. Indeed, The mojor finding of the present research was not well discussed. Please try to do this just in the end of discussion section (One paragraph).
B/ Specific comments
5. The title need to be improved: I think that is not necessary to mention the number 3 (bioenergy). Indeed I propose to remore growth and physiology and to add Morpho-physiological indicators.
6. Please add the novelty of study in abstract section just after the background and context. The cited objective in abstract is similar to the title? please rephrase the objective that need to be different to the title of manuscript.
7. Method section in abstract is too long as compared to results section. Please try to make it shorter.
8. Please put the first letter of all key words in upper letter.
9. L37 : please add some examples from literature (that are not involved in this study) of non-food energy to better illustrate the background.
10. L40 : I think that authors could mention perennial rhizomatous grasses in the title to better improve the visibility of the study
11. L57: what do you mean by the loss of biodiversity?
12. L65: steep slopes, please replace this word by another and more generale context (topography...etc).
13. L77: please add the following references to justify and to update more your literature (Kherif et al., 2023: Agriculture; Bouras et al., 2023: Agriculture)
14. L88-90: Author just gives the main objective of the study and which was the same sentence of the manuscript title. Author could rephrase the main objective of the study with more explicit explanation and also give the specific objective of the study as research hypothesis.
15. L119-120: On which base your selected the planting density? Please clarify??
16. L123-124: How many replicates were adopted for each treatment, you mentioned 3 plant? But why you put n= 9? 3 plants for controlled experiment is not really sufficient!
17. Please clarify more the sampling method by which you take-up the plant for measurement? Why you selected the plant from the centre of plot? There are a proper’s methods (ZIGZAG...etc to do this)? Please clarify.
18. Please can you give a preventative and illustrator schema to better you clarify you experimental design?
19. Please add all corresponding references relative to section 2.3
20. L145: Please add the reference of the instrument by which you measured the chlorophyll content (Licore.....chabmbern°....).
21. How you calculated WUE (water use efficiency)? Please clarify this in material and method section.
22. L166-173: Please pu p value in small letter and in italic in all section of the text.
23. Replace this sentence “The drought impact on leaf gas exchange parameter was assessed using a three- factor analysis of variance (ANOVA) and interactions (species, treatment and year) by : The drought impact on leaf gas exchange parameter was assessed using a factorial ANOVA analysis within three- studied factors.
24. Try to interpret well the figure 1 in particular the precipitation during the period before the experiment installation. I have a question about the quantification of soil moisture estimation before the experiment.
25. The statistical analysis As I said in the major comment need to be corrected and try to mention the homogeneous groups among the interaction between the two factors.
26. P values are not mentioned in the results of table 1. Please add all corresponding p values.
27. Please try to change the scale of Y axis in figure 2 E and F, A and B
28. The same comment for Figure 3
29. The same comment for homogeneous groups in figure 3 (comment 25).
30. Please remove the first paragraph of the discussion section and start the discussion with more explicit paragraph that represent the discussion section.
31. L326-329: I am not converted that the decrease of photosynthesis was the as the result of CO2 diffusion limitation due to
the decreased stomatal conductance. In this case you measured stomatal conductance? Please clarify
32. Please try to improve the last section of conclusion in terms of perspective of the study.
33. Check reference 19 and try to complete all DAI in all cited reference
Request just minor correction
Author Response
Response to reviewers
Reviewer 1
The submitted manuscript addresses an important topic that focus on the use of PRGs for bioenergy production for improving plant morpho-physiological indicators in particular under drought conditions. The main objective of this research study was focused on experimental assessment of the effect of drought on the physiology and growth of three bioenergy crops (3 species) in Mediterranean environment. The results were well presented within a good discussion however methodological concept and research hypothesis formulation were moderately presented. In my opinion, the manuscript needs major revision and some clarifications to better fit the research questions of this study with discussion and the main finding.
A/General comments
- The research hypothesis is poorly developed and not matches well with manuscript title and also within all obtained results. Authors are invited to improve this section in Introduction
The research hypothesis has been further developed at the end of the Introduction section.
- Material and methods section was poorly justified in terms of references that are corresponding to each performed method and analysis. Please try to argument and justify any applied protocol and method.
References and justifications have been incorporated into this section.
- The statistical analysis that was performed on collected data are not clear for reader and they are very complex to understand because authors mentioned both YEAR and SPECIES homogeneous group (test post hoc) for the same colon (Capital and Greck letter). Author could take in consideration of the INTERACTION between these two factor to inspire the homogeneous groups and not to add separately the groups in mean for each studied factor. This very important request in this revision.
We thank the reviewer for this comment, but we think ‘year and species’ homogenous groups are clearer for the reader
- Discussion section was really limited, as that author could explain and more discuss the interaction between the changes in each measured indicators. Indeed, the major finding of the present research was not well discussed. Please try to do this just in the end of discussion section (One paragraph).
The paragraph was introduced.
B/ Specific comments
- The title need to be improved: I think that is not necessary to mention the number 3 (bioenergy). Indeed, I propose to remove growth and physiology and to add Morpho-physiological indicators.
A suggested improvement was made to the title, "morpho-physiological parameters" and “perennial rhizomatous grasses” was introduced, and the word "three" was removed:
- Please add the novelty of study in abstract section just after the background and context. The cited objective in abstract is similar to the title? please rephrase the objective that need to be different to the title of manuscript.
The novelty of study was introduced and the objective cited in the summary was replaced by the objective cited at the end of the introduction, which is closely related to the title.
- Method section in abstract is too long as compared to results section. Please try to make it shorter.
The changes were introduced.
- Please put the first letter of all key words in upper letter.
The changes were introduced.
- L37: please add some examples from literature (that are not involved in this study) of non-food energy to better illustrate the background.
L37: agregue algunos ejemplos de la literatura (que no están involucrados en este estudio) de
energía no alimentaria para ilustrar mejor los antecedentes.
- L40: I think that authors could mention perennial rhizomatous grasses in the title to better
improve the visibility of the study
The suggestion was introduced.
- L57: what do you mean by the loss of biodiversity?
The cultivation of the perennial rhizomatous grasses, compared to food crop systems or typical annual crops, in general results in less loss of biodiversity. This means a lower disturbance of landscapes, shelter for birds, plants and small mammals according to Clifton-Brown et al. (2023).
The clarification and the cite were also introduced in the text.
- L65: steep slopes, please replace this word by another and more general context
(topography...etc).
The term steep slopes was replace by a term more general.
- L77: please add the following references to justify and to update more your literature
(Kherif et al., 2023: Agriculture; Bouras et al., 2023: Agriculture)
The references were added.
- L88-90: Author just gives the main objective of the study and which was the same sentence of the manuscript title. Author could rephrase the main objective of the study with more explicit explanation and also give the specific objective of the study as research hypothesis.
Based on the title change, the main objective was reformulated and the research hypothesis was incorporated.
- L119-120: On which base your selected the planting density? Please clarify??
Each plot was 3.24 m2 (1.8 m x 1.8 m); the plant density was 2.7 plants m−2 (0.6 m between rows, 0.6 m between plants). In the manuscript it is expressed in plants per ha because it was previously suggested by another reviewer, but at the same time there was an error in the density value that has been corrected.
The density of plants was chosen based on the bibliography (Cosentino et al., 2006), who used a density of 2.67 plants m−2 (distance between lines of 0.75 m and 0.50 m between plants).
- L123-124: How many replicates were adopted for each treatment, you mentioned 3 plant? But why you put n= 9? 3 plants for controlled experiment is not really sufficient!
The experimental was arranged as a randomized complete block design, replicated three times for each species, that is to say three plots per each species where three plants per plot were measured (n=9).
- Please clarify more the sampling method by which your take-up the plant for measurement? Why you selected the plant from the centre of plot? There are a proper’s
methods (ZIGZAG...etc to do this)? Please clarify.
The total number of plants per plot was 16, no sampling method was used because only 4 plants remained in the center of the plot, of which 3 were chosen.
- Please can you give a preventative and illustrator schema to better you clarify your experimental design?
- Please add all corresponding references relative to section 2.3
The references were incorporated.
- L145: Please add the reference of the instrument by which you measured the chlorophyll
content (Licor chamber).
The informaction was incorporated.
- How you calculated WUE (water use efficiency)? Please clarify this in material and method section.
Intrinsic water use efficiency WUEi was calculated from gas exchange of CO2 and H2O as the ratio of CO2 assimilation over stomatal conductance (WUEi = Asat / gs (µmol CO2 mol H2O-1) under 400 µmol mol-1 CO2, 21% O2 and 1200 μmol m−2 s−1 of PPFD according to Webster et al. (2016).
- L166-173: Please put p value in small letter and in italic in all section of the text.
The changes were introduced.
- Replace this sentence “The drought impact on leaf gas exchange parameter was assessed using a three- factor analysis of variance (ANOVA) and interactions (species, treatment and year) by: The drought impact on leaf gas exchange parameter was assessed using a factorial ANOVA analysis within three- studied factors.
The sentence was replaced.
- Try to interpret well the figure 1 in particular the precipitation during the period before the experiment installation. I have a question about the quantification of soil moisture estimation before the experiment.
Soil moisture was not quantified, only the amount of precipitation that fell was recorded.
- The statistical analysis, as I said in the major comment need to be corrected and try to mention the homogeneous groups among the interaction between the two factors.
Statistical analysis has been corrected.
- P values are not mentioned in the results of table 1. Please add all corresponding p values.
In previous revisions it was requested to decrease the information presented in the table because it contains an overwhelming amount of data, which can be discouraging for readers. That is why the WUE data was removed from table 1 and relocated to table 1 of the supplementary material.
- Please try to change the scale of Y axis in figure 2 E and F, A and B
The changes were introduced.
- The same comment for Figure 3.
The changes were introduced.
- The same comment for homogeneous groups in figure 3 (comment 25).
The changes were introduced.
- Please remove the first paragraph of the discussion section and start the discussion with
more explicit paragraph that represent the discussion section.
The first paragraph was rewritten.
- L326-329: I am not converted that the decrease of photosynthesis was the as the result of CO2 diffusion limitation due to the decreased stomatal conductance. In this case ¿you measured stomatal conductance? Please clarify.
The stomatal conductance (gs) was measured with a Portable Photosynthesis System (Li6400, Li-Cor Inc., Lincoln, NE, USA).
- Please try to improve the last section of conclusion in terms of perspective of the study.
Changes have been made to improve the text.
- Check reference 19 and try to complete all DOI in all cited reference
The reference 19 was reviewed, and all DOI were controlled.
Author Response File: 
Author Response.docx
Reviewer 2 Report (New Reviewer)
To Authors
The publication's strengths lie in addressing the topic of soil drought and bioenergy crops. Additionally, the introduction is a substantive and reliable section. Unfortunately, the Materials and Methods section, along with the presentation of results, requires thorough improvement and clarification of several ambiguities.
- Why were only three out of four months presented in the results? July is missing. In the second year, after low rainfall in June, it could be inferred how quickly the plants recovered. Another piece of information is from August.
- What caused the increase in moisture in August (the 1st), despite a drastic decrease in rainfall? the same situation in the following year in August.
- With 16 plants in the field plot, it is impossible to remove nine plants without causing edge effects.
- "irrigated with 50 mm month-1" - it would be better to provide the watering rate in liters per surface area per time.
- When were the plants planted? Did the plants have any acclimatization time to the new conditions? Why are they stated to be equal in T0 (eg. gas exchange parameters) when this is a field experiment? (the groups were divided from the beginning)?
- In the Results section, it would be better to focus on the months rather than seasons (the months of measurement could also be marked on the graph).
- Table 1 contains an overwhelming number of tests, which can discourage readers. The authors should consider analyzing the obtained results and guide the reader through the interesting findings rather than testing everything against everything.
- It is not specified whether besides the plants for measurement of biomass, the rest of plant is also harvested, making it unclear what is being evaluated in the second year.
-Gas exchange: Detailed information regarding the studied leaf is missing.
If the measurements in June show minimal photosynthesis in miscanthus (it is unfortunate that there are no measurements in July), the question arises: when did the drying occur, in late June, July, or August?(If the climatic conditions improved after T1, yet the plant dried up in August, it can be inferred that the plant condition did not improve after T1, and the drying occurred rapidly.) Does this mean that the following year we are dealing with regrowth from the ground, (despite drying up almost completely in mid-vegetation last year)?
Figure 2: It would be helpful to include the years on the graphs. In Figure 3, there is a lack of consistency with Figure 2, such as species names above the graphs.
Ambiguities in the text: Why do the unirrigated plants show higher relative water content (RWC) than the irrigated A. donax Ta2?
- The mention of biomass in the case of three morphologically different species in the abstract does not contribute much, and providing biomass per plant without reference to a control (e.g., % change) is unclear.
- If the experiment was concluded at a single time point, how was the assessment of dry mass conducted for plants reaching three meters?
The paper contains incorrect page numbering.
Minor editing of English language required.
Author Response
Reviewer 2
The publication's strengths lie in addressing the topic of soil drought and bioenergy crops. Additionally, the introduction is a substantive and reliable section. Unfortunately, the Materials and Methods section, along with the presentation of results, requires thorough improvement and clarification of several ambiguities.
- Why were only three out of four months presented in the results? July is missing.
T2 measuring period is including late July and early August
- In the second year, after low rainfall in June, it could be inferred how quickly the plants recovered. Another piece of information is from August.
We agree with this reviewer comments
- What caused the increase in moisture in August (the 1st), despite a drastic decrease in rainfall? the same situation in the following year in August.
There were no humidity increase during august
- With 16 plants in the field plot, it is impossible to remove nine plants without causing edge effects.
There were 16 plants per plot and three plants from the center were selected to carry out the measurements and thus eliminate the edge effect.
- "irrigated with 50 mm month-1" - it would be better to provide the watering rate in liters per surface area per time.
The suggested change was introduced.
- When were the plants planted? Did the plants have any acclimatization time to the new conditions?
The plants were transplanted in March. Both the plants that were purchased in the nursery and those obtained from germination underwent a period of acclimatization in the greenhouse until the time of transplantation.
- Why are they stated to be equal in T0 (eg. gas exchange parameters) when this is a field experiment? (the groups were divided from the beginning)?
Irrigated (WA) and non-water (NW) plants were quite similar at the beginning of the experiment
- In the Results section, it would be better to focus on the months rather than seasons (the months of measurement could also be marked on the graph).
The suggested change was introduced and it has been indicated with red arrows on the graph (Fig. 1) as suggested.
- Table 1 contains an overwhelming number of tests, which can discourage readers. The authors should consider analyzing the obtained results and guide the reader through the interesting findings rather than testing everything against everything.
The WUE data were removed from Table 1 to reduce the amount of data and have been placed in Supplementary Table 1.
- It is not specified whether besides the plants for measurement of biomass, the rest of plant is also harvested, making it unclear what is being evaluated in the second year.
At the time of harvest, the plants have not yet started the flowering period, so only the weight of stems and leaves were measured. The clarification on the measurement of the dry weight of leaves and stems was incorporated in the M&M section.
The experiment was carried out in a second year since these species, being perennial, usually do not express their full growth potential in the first year of implantation.
- Gas exchange: Detailed information regarding the studied leaf is missing.
Gas exchange measurements were made in the middle area of the last unfolded leaf (clarification was introduced in the text).
- If the measurements in June show minimal photosynthesis in miscanthus (it is unfortunate that there are no measurements in July), the question arises: when did the drying occur, in late June, July, or August? (If the climatic conditions improved after T1, yet the plant dried up in August, it can be inferred that the plant condition did not improve after T1, and the drying occurred rapidly.) Does this mean that the following year we are dealing with regrowth from the ground, (despite drying up almost completely in mid-vegetation last year)?
In the june measurement (T1) the plants already presented severe stress symptoms, mainly chlorosis, and they finished drying in the month of july prior to the rainfall. The plants after the precipitations could not be recovered.
- Figure 2: It would be helpful to include the years on the graphs.
The years were introduced in the graphs.
- In Figure 3, there is a lack of consistency with Figure 2, such as species names above the graphs.
The inconsistency has been checked and modified.
- Ambiguities in the text: Why do the unirrigated plants show higher relative water content (RWC) than the irrigated A. donax T2?
In the first year, the RCW of the three species decreased in T2, being more marked under rainfed conditions, except for Arundo donax, which kept its RWC constant with respect to T1.
- The mention of biomass in the case of three morphologically different species in the abstract does not contribute much, and providing biomass per plant without reference to a control (e.g., % change) is unclear.
The term biomass was changed to candidate as a bioenergy crop to avoid comparison only at the level of biomass production among the three species.
- If the experiment was concluded at a single time point, how was the assessment of dry mass conducted for plants reaching three meters?
The Experimental Field Services of the University of Barcelona have the facilities and equipment to handle this volume of plants. In turn, the leaves were separated from the stems and these were cut into pieces to be able to dry them in the stove.
- The paper contains incorrect page numbering.
Work was done on the journal template but when putting a landscape page (table 1) the numbering had to be changed.
Author Response File: Author Response.docx
Reviewer 3 Report (New Reviewer)
The presented manuscript is focusing on the effect of different water supply on growth and physiology in three bioenergy crops in the Mediterranean environment. The presented manuscript contains well designed experiment with good results and clear discussion.
I have only some minor comments and recommendations.
Introduction
This part is clear and sufficient.
Materials and Methods
I leave it to the authors to decide whether or not to use ”rainfed conditions” for non-watered treatment. Maybe it would be better use “ambient conditions”, because in your case the precipitation are rather low and “rainfed” sounds like well watered.
L117-119: You should write “During period May–August, the plants under the WA treatment were irrigated with 50 mm month–1 supplementary water using droppers…”
Results and Discussion
L179 and 182: Better using “amount of rainfall” instead of “concentration of rainfall”.
L196-293: Use italics for Latin names of plant species.
L321-323: Unify the abbreviations gs – sometimes with a lower index, sometimes without.
Discussion and Conclusions are well written and clear.
Author Response
Reviewer 3
The presented manuscript is focusing on the effect of different water supply on growth and physiology in three bioenergy crops in the Mediterranean environment. The presented manuscript contains well designed experiment with good results and clear discussion. I have only some minor comments and recommendations.
Comments of this reviewer were manly positive
- Introduction
This part is clear and sufficient.
- Materials and Methods
I leave it to the authors to decide whether or not to use “rainfed conditions” for non-watered treatment. Maybe it would be better use “ambient conditions”, because in your case the precipitation is rather low and “rainfed” sounds like well-watered.
Based on previous reviews, we decided to leave the term "rainfed conditions".
L117-119: You should write “During period May–August, the plants under the WA treatment were irrigated with 50 mm month–1 supplementary water using droppers…”
The changes were introduced
- Results and Discussion
L179 and 182: Better using “amount of rainfall” instead of “concentration of rainfall”.
The changes were introduced.
L196-293: Use italics for Latin names of plant species.
Changes were done.
L321-323: Unify the abbreviations gs – sometimes with a lower index, sometimes without.
The abbreviation gs was unified throughout the manuscript.
- Discussion and Conclusions are well written and clear.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report (New Reviewer)
All requested revision was done by author.
Reviewer 2 Report (New Reviewer)
The authors have provided thorough and reliable responses to the questions. The authors have made appropriate changes and clarifications to address the issues raised regarding the Materials and Methods section and the presentation of results. They have provided detailed explanations and incorporated necessary clarifications in response to specific questions from the reviewer. Overall, the authors have demonstrated a commitment to improving the clarity and accuracy of the publication based on the reviewer's feedback.
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
The authors improved the grammar in the manuscript extensively. However, the main issue with the work is the lack of novelty. The authors agreed that they studied the physiology of the species alone. Science has gone beyond publishing results from one area. There is a need to study a few more areas for a better understanding of the crops of interest. Again, I recommend, biochemical, transcriptomic or spectroscopic experiments to see if anything novel would be discovered. It may take some time but it is worth it.
Author Response
Response to reviewers
Reviewer 1
The authors improved the grammar in the manuscript extensively. However, the main issue with the work is the lack of novelty. The authors agreed that they studied the physiology of the species alone. Science has gone beyond publishing results from one area. There is a need to study a few more areas for a better understanding of the crops of interest. Again, I recommend, biochemical, transcriptomic or spectroscopic experiments to see if anything novel would be discovered. It may take some time but it is worth it.
Thanks for your suggestions, however as mentioned before, the main focus of our study is in the physiology and biomass behaviour of theses bioenergy crops. Biochemical, transcriptomic or spectroscopic experiments could be the focus of another study.
This article presents a comparative study of the three bioenergy crops carried out in the field and under rainfed conditions. We consider that the results achieved in this work are original due to most of the literature found addresses the study of these species separately and/or under controlled greenhouse conditions.
Author Response File: Author Response.docx
Reviewer 2 Report
Line 86
Characteristics of Catalonia region ?
Include the deep of the plots precising how deep was the sustrate.
It is unclear some experimental items. What was the experimental unit? What was the replicate (3 or 9?)? How many plots were used?
Specify what accesories and procedures were used for maintaining de 21% O2, and why?
Or what is the level of PPFD that is in that site?
The maximum levels of PPFD used in the measurements are different, which could affect the results. How do you ensure affectation does not occur?
You use plants from seeds, how well developed were they at the measurement time? Were they as adult plants? How can it impact results? Describe it and explain it.
It is necessary explain how you ensure compliance with the independence criteria in measurements of photosynthetic variables? You had 16 plants and you say you measured 9, so you measured the same plant every time, so a repeated measures analysis is more appropriate.
In table 1. You say “Lowercase letters indicate significant differences (P < 0.05) between treatment for the same species, time and year”. But, for example, in Asat for A. donax, values 15.67 and 18.25 are similar because have letter b, but it is not posible because this comparation is for the same time and these values are from different times…, similar situation was observed in comparisson identificated with capital letters. Then it is necessary correct, describe and clirify comparissons in 2.5 Statistical analysis, in table 1 and in table S1. Please use firts letter (A,a,.) for the bighest value in all results (tales and figures)… All the same for Figure 3…
Results indicate came from n=9 but it was stated “three plant plots were measured”… in case analysis was made for the 9 values n=9 is correct but, in case of analyzing for every time “three plan plots were measured” is not.
RWC and SPAD units results lacks of statistical analysis, please include ANAVA and Tukey test parameters in texts and figures. Are there independence in these variables? If not, a repeated measures is needed…
Line 294
The discussion of this paragraph should be enriched. Annual rainfall of P. virgatum includes that of the experiement. C4 species are considered well adapted to dry environments and they should be along gradients, then some “microenvironmental” adaptations occurs. Then explanation of the results could also be due to the position of the species into the gradient, more than to the photosynthesis path. These also must be take into account for the discussion of the paragraph beginning in line 309.
Author Response
Response to reviewers
Reviewer 2
Line 86, Characteristics of Catalonia region ?
Catalonia region was replaced by Mediterranean region.
Include the deep of the plots precising how deep was the substrate.
This information was added in the section of M&M.
It is unclear some experimental items. What was the experimental unit? What was the replicate (3 or 9?)? How many plots were used?
The experimental unit was the plot, three plots per each species were used and the number of replicates was 9 (3 plants per plot).
Changes were introduced in the section of M&M.
Specify what accessories and procedures were used for maintaining de 21% O2, and why?
Oxygen level was maintained at 21% using the Portable Photosynthesis System (Li6400, Li-Cor Inc., Lincoln, NE, USA).
Or what is the level of PPFD that is in that site?
The PPFD level was around 2000 µmol m-2s-1
The maximum levels of PPFD used in the measurements are different, which could affect the results. How do you ensure affectation does not occur?
The A/Ci curves with chlorophyll fluorescence determinations were conducted in fully expanded leaves from each species at a light rate saturated at 1200 µmol mol-1 of PPFD. The A/PPFD curves (light saturating curves) were conducted before the A/Ci curves were done.
You use plants from seeds, how well developed were they at the measurement time? Were they as adult plants? How can it impact results? Describe it and explain it.
Only the species P. virgatum was obtained from seed, the rest were asexually reproduced. All the plants were transplanted to the field with 4 true leaves. At the time (T0) of the measurements the plants were well developed, with a height of around 0.5 to P. virgatum and Miscanthus, and 1.5 m to A. donax (See Figure Supplementary 7). Besides the measurements were made on the last unfolded sheet. Therefore, they were adult plants and the results obtained were not affected by their ontogeny but by the rainfed treatment to which the plants were subjected.
Clarification was introduced in the section of M&M.
It is necessary explain how you ensure compliance with the independence criteria in measurements of photosynthetic variables? You had 16 plants and you say you measured 9, so you measured the same plant every time, so a repeated measures analysis is more appropriate.
The three plants that were selected from each plot were not the same plants at each measurement.
Changes were introduced in the section of M&M.
In table 1. You say “Lowercase letters indicate significant differences (P < 0.05) between treatment for the same species, time and year”. But, for example, in Asat for A. donax, values 15.67 and 18.25 are similar because have letter b, but it is not posible because this comparation is for the same time and these values are from different times…, similar situation was observed in comparisson identificated with capital letters. Then it is necessary correct, describe and clirify comparissons in 2.5 Statistical analysis, in table 1 and in table S1. Please use firts letter (A,a,.) for the bighest value in all results (tales and figures)… All the same for Figure 3…
The suggested changes were introduced in the tables and figure 3.
Results indicate came from n=9 but it was stated “three plant plots were measured” in case analysis was made for the 9 values n=9 is correct but, in case of analyzing for every time “three plan plots were measured” is not.
The number of replicates was 9 (3 plants per plot).
Clarification was introduced in the section of M&M.
RWC and SPAD units results lacks of statistical analysis, please include ANAVA and Tukey test parameters in texts and figures. Is there independence in these variables? If not, a repeated measure is needed.
Yes, if there is independence in these variables. Statistical analysis was added in in the text and figures.
Line 294
The discussion of this paragraph should be enriched. Annual rainfall of P. virgatum includes that of the experiment. C4 species are considered well adapted to dry environments and they should be along gradients, then some “microenvironmental” adaptations occurs. Then explanation of the results could also be due to the position of the species into the gradient, more than to the photosynthesis path. These also must be take into account for the discussion of the paragraph beginning in line 309.
The range of water requirement for P. virgatum species varies between 450-750 mm year-1. Although the range of water requirement in P. virgatum was covered in part by the precipitation of both years, it is important to mention that in this work the Alamo cultivar was used, which corresponds to the lowland ecotype and is characterized by growing better with greater water availability.
Author Response File: Author Response.docx
