Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Article Differentiation of leaf functional traits and driving factors of the allopatric distribution of tetraploid and octaploid Buddleja macrostachya in the Sino-Himalayan region by authors

Weichang Gong, He Li, Hongbo Fu and Chuanming Wang present the results of a study of the anatomical features of Buddleja macrostachya depending on ploidy.

The manuscript is formatted according to the rules and contains all the necessary sections. The literature review contains an extensive list of literature sources.

However, there are a number of problems that require clarification and possible correction.

In the introduction, the authors formulate goals and objectives - they should be highlighted in a separate paragraph. In addition, please note that in the conclusion there is no clear solution to these problems and this is somewhat strange.

So you write: compare ecological strategies used in tetraploid and octaploid B. macrostachya in the introduction, and in the conclusion: Our findings suggested that both tetraploid and octaploid B. macrostachya adopt divergent ecological strategies (you should indicate what exactly the differences are). Otherwise, it turns out that nothing new has been found out. Correct this please.

There are also questions for the materials and methods section. If you characterized the ploidy of the selected plants and this is a key point in the study, the data should be provided. at the same time, you must both provide links to the study where this was established, and prove that these samples correspond to the statement, since plants, even within the same population, may differ.

In Table 2, definitions and abbreviations should be placed below the table rather than in the caption.

Figure 3 requires drilling down and adding missing information. Add the meaning of the axes, what the red line means, as well as a more detailed description of how the data was obtained and an explanation of the symbols. It is also not clear to me whether the significance of reliability is applicable to the data presented in the table, since there is no information whether there were differences between individual plants of each group, or whether these data can be considered correct. If yes, please justify it.

What did you mean by "examine soil stoichiometric characteristics" in section 2.6.2. Correlation between leaf functional traits and environmental factors within B. macrostachya complex.

Figure 2. Why are there only 2 images. What do they refer to? Could it be that the authors confused the images of tetraploid and octoploid forms, since the size of the nuclei and the size of the cell walls indicate the opposite. Perhaps the authors did not place the bar correctly in figure 2 b.

Figures 3, 4 and 5 also lack abbreviations. Their names do not provide complete information sufficient for understanding.

What are "abiotic environmental factors" specifically? Were they the same everywhere? longitude, latitude, altitude are not environmental factors, such as temperature, humidity, pressure, amount of precipitation, soil conditions, etc.

Unfortunately, these comments do not allow us to accept the publication in its present form; the article requires significant revision.

Author Response

Thank you for your comments, and we have listed all our changes as below:

Reviewer 1

Q1:In the introduction, the authors formulate goals and objectives - they should be highlighted in a separate paragraph. In addition, please note that in the conclusion there is no clear solution to these problems and this is somewhat strange.

Reply: We have separated the goals and objectives into a separate paragraph for clarity in the introduction section. Meanwhile, we have also concisely summarized and responded all three objectives in the conclusion section. As follows, “Our results revealed typical differentiation in leaf functional traits between the two cytotypes within B. macrostachya complex. However, RDA analysis indicated that octaploid cytotype has higher SLA, leaf N and P, WUE and LUE compared to the tetraploid plants. These findings suggested that both tetraploid and octaploid B. macrostachya adopt divergent ecological strategies, conservative and acquisitive strategies, respectively. The results of multivariate analysis suggested that the alloptric distribution of B. macrostachya complex is primarily influenced by the abiotic environmental factors in the Sino-Himalayan region. Furthermore, the divergent ecological strategies and high phenotypic plasticity of the key ecological traits collectively enhance species ecological adaptability in B. macrostachya complex. ”. And all the revised sections were highlighted.

 

Q2: So you write: compare ecological strategies used in tetraploid and octaploid B. macrostachya in the introduction, and in the conclusion: Our findings suggested that both tetraploid and octaploid B. macrostachya adopt divergent ecological strategies (you should indicate what exactly the differences are). Otherwise, it turns out that nothing new has been found out. Correct this please.

Reply: In the revised version, we have clearly outlined and explained the ecological strategies disparities between tetraploid and octaploid B. macrostachya. The modifications in the text are as follows: ‘However, RDA analysis indicated that octaploid cytotype has higher SLA, leaf N and P, WUE and LUE compared to the tetraploid plants. These findings suggested that both tetraploid and octaploid B. macrostachya adopt divergent ecological strategies, conservative and acquisitive strategies, respectively.’ We hope our modification on the conclusion section can better highlight the significance of our study.

 

Q3: There are also questions for the materials and methods section. If you characterized the ploidy of the selected plants and this is a key point in the study, the data should be provided. at the same time, you must both provide links to the study where this was established, and prove that these samples correspond to the statement, since plants, even within the same population, may differ.

Reply: In the revised version, we have included a detailed description of ploidy levels of different populations of B. macrostachya complex in Table 1. And all the revised sections were highlighted.

In addition, we also provided the prior study of our team on the ploidy levels of B. macrostachya complex. The statement regarding the the source of materials in the “plant material and methods” section has been updated as follows “The tetraploid and octaploid individuals of B. macrostachya, previously utilized for ploidy level estimation in our prior study [25], were included in the present study.”

 

Q4: In Table 2, definitions and abbreviations should be placed below the table rather than in the caption.

Reply: In Table 2, all definitions and abbreviations have been moved from the caption to the foot note.

 

Q5: Figure 3 requires drilling down and adding missing information. Add the meaning of the axes, what the red line means, as well as a more detailed description of how the data was obtained and an explanation of the symbols. It is also not clear to me whether the significance of reliability is applicable to the data presented in the table, since there is no information whether there were differences between individual plants of each group, or whether these data can be considered correct. If yes, please justify it.

Reply: We appreciate the reviewer's thorough examination of our manuscript and their valuable constructive feedback. Considering that the main text of this part is the comparison of leaf functional traits between these two cytotypes, we have prioritized the description their phenotypic plasticity characteristics (Figure 3) and move the description of the multivariate analysis back by one paragraph.

These mistakes in Figure 3 have been corrected in the revised version. We removed the redundant red line and made corrections to the errors in both the horizontal and vertical axes. In this study, we dominantly compared the differences of functional leaf traits and their plasticity in B. macrostachya at the population level rather than at the individual level. The statement has been refined to provide clarity on the process used to determine the Plasticity Index (PI). In section 2.6.1, it is now specified that “For each cytotype, the PI of leaf functional traits was determined using the minimum and maximum mean values at the population level. ” Additionally, all the sections that have been revised are now clearly highlighted for easy identification.

  

Q6: What did you mean by "examine soil stoichiometric characteristics" in section 2.6.2. Correlation between leaf functional traits and environmental factors within B. macrostachya complex.

Reply: We have refined the pertinent description, and the updated content is presented below. “One-way ANOVA was conducted to compared soil stoichiometric characteristics of distribution regions between two different cytotypes at a significance level of P < 0.05. Data normality, including environmental factors and leaf functional traits between these two cytotypes was tested by using Shapiro-Wilk’ W test.”

 

Q7. Figure 2. Why are there only 2 images. What do they refer to? Could it be that the authors confused the images of tetraploid and octoploid forms, since the size of the nuclei and the size of the cell walls indicate the opposite. Perhaps the authors did not place the bar correctly in figure 2 b.

Reply: In this study, the Figure 2 predominantly showed the leaf anatomical structure of B. macrostachya. Each image distinctly represents the leaf anatomical traits of tetraploid or octoploid plants. In addition, we also confirmed the accuracy of the images and the scale bar in Figure 2. Table 2 supports these findings, indicating that tetraploid plants have larger and thicker leaves compared to octoploid plants.

 

Q8: Figures 3, 4 and 5 also lack abbreviations. Their names do not provide complete information sufficient for understanding.

Reply: In Figure 3, 4 and 5, all referenced abbreviations and definitions have been included within the caption titles.

 

Q9: What are "abiotic environmental factors" specifically? Were they the same everywhere? longitude, latitude, altitude are not environmental factors, such as temperature, humidity, pressure, amount of precipitation, soil conditions, etc.

Reply: In general, environmental factors encompass both abiotic and biotic components. Abiotic environmental factors refer to the non-living components of an ecosystem that influence the growth, development, and distribution of living organisms. These abiotic factors play a crucial role in determining the structure and function of ecosystems. Therein, longitude, latitude and altitude, which significantly affect plant growth development and geographic distribution, were classified as abiotic environmental factors. Moreover, environmental factors exhibit spatial heterogeneity, which is also a key factor in causing the allopatric distribution of plants.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have written a manuscript that compares tetraploid and octoploid Buddleja macrostachya. The manuscript is well-written and provides a detailed methodology. However, it's worth noting that most of the references used in the manuscript are quite old. It would be better to include newer references that cover the last decade, and only a few older ones. Some of the references used in the manuscript are even 20 years old.

Author Response

Q1: The authors have written a manuscript that compares tetraploid and octoploid Buddleja macrostachya. The manuscript is well-written and provides a detailed methodology. However, it's worth noting that most of the references used in the manuscript are quite old. It would be better to include newer references that cover the last decade, and only a few older ones. Some of the references used in the manuscript are even 20 years old.

Reply: We have updated most of the older references, retaining only a few necessary older ones.  

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript give a good differentiation of leaf functional traits and driving factors of the allopatric distribution of tetraploid and octaploid Buddleja macrostachya, data were represented with appropriate statistical analyses and extensive inferential statistics giving a good data presentation and interaction between variables using heatmap and RDA. 

if possible how you checked data normality? if possible clarify in materials and methods the data normality test either shapiro-wilk or Kolmogorov Smirnove , ....etc.

 

Author Response

Q1: The manuscript give a good differentiation of leaf functional traits and driving factors of the allopatric distribution of tetraploid and octaploid Buddleja macrostachya, data were represented with appropriate statistical analyses and extensive inferential statistics giving a good data presentation and interaction between variables using heatmap and RDA. 

if possible how you checked data normality? if possible clarify in materials and methods the data normality test either shapiro-wilk or Kolmogorov Smirnove , ....etc.

Reply: In or study, we employed the Shapiro-Wilk’ W test to rigorously evaluated the normality of the data. In subsection 2.6.2, we have modified and added relevant descriptions, and the updated text is presented below. “One-way ANOVA was conducted to compared soil stoichiometric characteristics of distribution regions between two different cytotypes at a significance level of P < 0.05. Data normality, including environmental factors and leaf functional traits between these two cytotypes was tested by using Shapiro-Wilk’ W test.”

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

Greetings! I appreciate the work. I have included my comments in the attached manuscript file. Please go through it.

Regards

Reviewer

Comments for author File: Comments.pdf

Author Response

Thank you for your recognition of our work. Following the reviewer’s suggestions, we have made modification to certain textual formats and corrected terminological inaccuracies, and also eliminated some redundant language.

Reviewer 5 Report

Comments and Suggestions for Authors

Gong et al. presented work on comparison of leaf functional traits between tetraploid and octaploid populations of   Buddleja macrostachya. I have written a minor correction below.

Table 1. It is advisable to add plant ploidy to geographical names of populations.

Subsection 3.1.

The text duplicates Table 2.

“The leaf areas (LAs) of the tetraploids were 32.67% larger than those of the octaploid plants.” In fact, the LAs of the tetraploids (165.62) were 20.45% larger than those of the octaploid plants (137.5). Check please.

The LAs of the tetraploids are 165.62 ± 12.39 in text but those are 165.62 ± 26.69 in Table 2. Check please.

Pages 2, 5, 11. B. macrostachya should be italicized.

Author Response

Q1: Table 1. It is advisable to add plant ploidy to geographical names of populations.

Reply: The information of ploidy levels of the plants has been added behind each population in Table 1. And all the revised sections were highlighted in the text. 

Q2: Subsection 3.1. The text duplicates Table 2.

Reply: We have further refined our language to ensure that the expression in subsection 3.1 does not simply repeat the information in Table 2. All the revised sections were highlighted in the text.

Q3: “The leaf areas (LAs) of the tetraploids were 32.67% larger than those of the octaploid plants.” In fact, the LAs of the tetraploids (165.62) were 20.45% larger than those of the octaploid plants (137.5). Check please.

Reply: We have corrected the error in that place.

Q4: The LAs of the tetraploids are 165.62 ± 12.39 in text but those are 165.62 ± 26.69 in Table 2. Check please.

Reply: We have corrected the error in that place.

Q5: Pages 2, 5, 11. B. macrostachya should be italicized.

Reply: We have reviewed the entire manuscript and formatted all instances of B. macrostachya in italics. All the revised sections were highlighted in the text.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript Differentiation of leaf functional traits and driving factors of the allopatric distribution of tetraploid and octaploid Buddleja macrostachya in the Sino-Himalayan region by Weichang Gong, He Li, Hongbo Fu and Chuanming Wang has been improved and necessary corrections have been made.

Before publication, I recommend making corrections to the manuscript by enlarging Figures 2 and 4, since the letters are difficult to distinguish.