Inter- and Intraspecific Variation Patterns of Leaf Phenotypic Traits in Two Sympatric Pine Species

Round 1

Reviewer 1 Report

Comments to the Author

In this manuscript, authors systematically analyzed the intra-specific phenotypic variation patterns of the two pine species and their relationships with environmental factors. This work stressed the significance of the intra-specific phenotypic variation in leaf traits in breeding practice. Several issues need to be addressed in order to have greater confidence in this paper.

Major issues are below.

1.     In this paper, authors gave us the detailed information of the possible reasons driving the phenotypic plasticity and their consequence on plants’ evolution or genetic diversity, but you failed to elaborate clearly how these evolutionary or genetic consequences influence the leaf trait variation, or the relationships between evolutionary adaptation or genetic diversity and leaf trait variation.

2.     The Introduction section is very shallow and has to be entirely revised and deepened with the inclusion of the rationale of this study and of the measurements that have been carried out. Authors used large amount of space to be in progress to describing the differences in species evolution and distribution patterns of two pine species, but how such information was related to their leaf trait variation was not mentioned. Moreover, authors need to provide more details about how genetics, environment and their interactions impact intra-specific phenotypic leaf variation. Finally, the relative hypotheses need to be provided.

3.     Such conclusion “For all measured needle traits, the variation within species was generally greater than that among species” or “Most of the variation among populations was smaller than the variation within populations for most of the measured needle traits.” can not be drawn from Figure 4. For example, most of variation in LL and DL occurred among species, and changes in elevation explained most of variation in LDMC.

4.         In the section of Discuss, the presentation of the results is mainly descriptive or simply comparing with others’ results, but lack logical deduction or logical induction. The discussion cannot be followed since no clear scientific question underpins this study. Moreover, some sentences are repeating the results and the contents in Introduction (e.g. L296-302). Importantly, authors need to give us the reasonable explanation of the variation in different leaf traits and between two species, and their main controlling factors (including species differences, climate and soil factors).    

 

Other specific comments are below.

1.     The English should be thoroughly revised. Some sentences are hard to follow.

2.     Figures and tables should be placed under each paragraph, and words in some Figures are too small.

3.     In Material and methods, how many plant individuals per species were sampled?

4.     The ecological significance of SLA is the same as LMA, so it is not necessary to analyze SLA and LMA simultaneously.

5.     Authors did not measure the leaf anatomic traits, so Figure 2 is inappropriate presented here.

6.     The description of Data analysis is too simple. The meteorological and geographical data should be listed as independent sections. More, the soil nutrient factors may act as an important factor for the leaf trait variation, why these factors are not taken into consideration?

7.     L275-277, “Similar findings were obtained in our study (Fig. 3), which demonstrated the components of intra-specific were generally higher than that of inter-specific in the most measured needle traits”, but authors did not compare directly the intra-specific and inter-specific variation in Fig.3

Comments for author File: Comments.docx

Author Response

Dear Professors,

We are really appreciative of the support provided by you and your journal, and also sincerely appreciate the reviewers. According to your suggestions and the reviewers’ comments, we revised the manuscript. We hope that the revised manuscript should be up to meet the standard of your esteemed journal. Now we re-submit the manuscript to your journal.

 All concerns that should be considered were addressed in the revision one by one as follows, please examine. The black words are the reviewers’ comments and the red words are the responses from the authors.

 

Major Issues

1. In this paper, authors gave us the detailed information of the possible reasons driving the phenotypic plasticity and their consequence on plants’ evolution or genetic diversity, but you failed to elaborate clearly how these evolutionary or genetic consequences influence the leaf trait variation, or the relationships between evolutionary adaptation or genetic diversity and leaf trait variation.

 

Response: According to the reviewers’ comments, we have revised the manuscript. Thank you.

 

 

2. The Introduction section is very shallow and has to be entirely revised and deepened with the inclusion of the rationale of this study and of the measurements that have been carried out. Authors used large amount of space to be in progress to describing the differences in species evolution and distribution patterns of two pine species, but how such information was related to their leaf trait variation was not mentioned. Moreover, authors need to provide more details about how genetics, environment and their interactions impact intra-specific phenotypic leaf variation. Finally, the relative hypotheses need to be provided.

 

Response: We are grateful for the suggestion. To be more clearly and in accordance with the reviewer concerns, we have added a more detailed interpretation regarding the Introduction section. More details involved other revised parts were added on page 2, 3, 5, and 10.

 

3. Such conclusion “For all measured needle traits, the variation within species was generally greater than that among species” or “Most of the variation among populations was smaller than the variation within populations for most of the measured needle traits.” can not be drawn from Figure 4. For example, most of variation in LL and DL occurred among species, and changes in elevation explained most of variation in LDMC.

 

Response: Thank you for your suggestion. Based on the nested ANOVA, in this increasing order: individuals, elevations, populations, and species, we decompose the variance to obtain results as shown in Fig. 4 (now Fig. 3). The inter-specific variance components of LL and DL were 61.48 % and 55.73 %, respectively, higher than the intra-specific variance components (16.92 % and 26.37 %), while the remaining traits (TL, LA, SLA, and LDMC) showed opposite results, with LDMC accounting for 63.79 % of the variance in elevation change. As a result, the above conclusions were reached in the manuscript. In addition, we revised the relevant statements in the Abstract section to more accurately present the results.

REVISED SENTENCE: Except for leaf length and leaf width, variation within species was greater than variation among species in needle traits measured.

 

4. In the section of Discuss, the presentation of the results is mainly descriptive or simply comparing with others’ results, but lack logical deduction or logical induction. The discussion cannot be followed since no clear scientific question underpins this study. Moreover, some sentences are repeating the results and the contents in Introduction (e.g. L296-302). Importantly, authors need to give us the reasonable explanation of the variation in different leaf traits and between two species, and their main controlling factors (including species differences, climate and soil factors).

 

Response: Thanks for your good suggestion. As suggested by reviewers, repeated contents has been removed. And we have added more details (in page 10) to explain the variation in different leaf traits and between two species, and their main controlling factors.

 

Other specific comments

1. The English should be thoroughly revised. Some sentences are hard to follow.

 

Response: We are sorry for our poor English to the extent that it is hard to follow. According to the reviewers’ comments, we have revised the English thoroughly.

 

2. Figures and tables should be placed under each paragraph, and words in some Figures are too small.

 

Response: Thanks for your good suggestion. We have adjusted the position of the figures and tables, also the size of the words.

 

3. In Material and methods, how many plant individuals per species were sampled?

 

Response: We apologize for not specifying the number of plant individuals per species sampled. Thus, we added the revised sentence to the beginning of the third paragraph in Material and methods.

REVISED SENTENCE: Needle samples were collected from 60 P. massoniana individuals and 44 P. hwangshanensis individuals.

 

4. The ecological significance of SLA is the same as LMA, so it is not necessary to analyze SLA and LMA simultaneously.

 

Response: We are grateful for the suggestion. Therefore, we reanalyzed the data after LMA or SLA was removed, and the results were changed slightly. According to the reviewers’ comments, we have decided to remove LMA.

 

5. Authors did not measure the leaf anatomic traits, so Figure 2 is inappropriate presented here.

 

Response: We agree with the comment and removed Figure 2 in the revised manuscript. Thank you.

 

6. The description of Data analysis is too simple. The meteorological and geographical data should be listed as independent sections. More, the soil nutrient factors may act as an important factor for the leaf trait variation, why these factors are not taken into consideration?

 

Response: We are grateful for the suggestion, and our reply is as follows:

According to comment, we have added more details about data analysis.

We have readjusted the meteorological and geographical data (Table 1) into independent sections in revised manuscript.

For soil nutrient factors, we collected samples from different locations, where the soil types were relatively complex, and we were worried that the factors would be too many to explain clearly. However, we believed that further exploration of soil factors would be conducted in the future.

 

7. L275-277, “Similar findings were obtained in our study (Fig. 3), which demonstrated the components of intra-specific were generally higher than that of inter-specific in the most measured needle traits”, but authors did not compare directly the intra-specific and inter-specific variation in Fig.3.

 

Response: We are sorry for the error of the description. The figure should have referred to Fig. 4 instead of Fig. 3 in original manuscript, however, after Fig. 2 was deleted, this sentence would still remain as it was.  

Author Response File: Author Response.docx

Reviewer 2 Report

The text was carefully prepared and the results were generalised in an interesting way. The objectives of the work were clear and good presented. The results achieved the stated objectives. Presented draft is interesting and not boring for readers.

I have a few comments on the research methodology:

- the method of sampling pine needles was given in a rather enigmatic way. For the two species studied, sample plots were selected and needles were collected from three trees per plot. The needles were then mixed and randomly sampled for analysis. I could not find information on how many needles were analysed and what age the sample trees were, were they middle-aged or mature trees? (it was only stated that they had a similar dbh).

- I am curious why such locations at the same altitude above sea level were not included in the study for the two species?

- no statistical model was given for the analyses of what factors and in what arrangement were included in the ANOVA and varcomp

- the specification of the variance components is very interesting and adds a lot of information, it will be interesting also include the error in the estimation of the variance component value.

- the description of the types of phenotypic plasticity given in the introduction is not reflected in the objectives of the paper and is poorly highlighted in the results. In the discussion at lines 243-247 the authors address this problem. Because evolutionary diversification through plasticity led varioation is still a controversial topic I propose that a better view in discussion into the physiological mechanisms promoted phenotypic divergence in plasticity.

 

With a few minor corrections, I hope that the text will be published and bring new information on the variation in needle traits of the pine species studied.

Author Response

Dear Professors,

We are really appreciative of the support provided by you and your journal, and also sincerely appreciate the reviewers. According to your suggestions and the reviewers’ comments, we revised the manuscript. We hope that the revised manuscript should be up to meet the standard of your esteemed journal. Now we re-submit the manuscript to your journal.

 All concerns that should be considered were addressed in the revision one by one as follows, please examine. The black words are the reviewers’ comments and the red words are the responses from the authors.

Comments

1. The method of sampling pine needles was given in a rather enigmatic way. For the two species studied, sample plots were selected and needles were collected from three trees per plot. The needles were then mixed and randomly sampled for analysis. I could not find information on how many needles were analyzed and what age the sample trees were, were they middle-aged or mature trees? (it was only stated that they had a similar dbh)

 

Response: Thank you for your comment. It was our fault that some details were not displayed clearly enough. Our original manuscript pointed out that sample trees were mature and ten fascicles of needles selected randomly from each sampled tree were analyzed. Besides, we added more details in Materials and method.

 

2. I am curious why such locations at the same altitude above sea level were not included in the study for the two species?

 

Response: Thank you for your comment. Because we selected the sample sites according to the typical distribution of the two species, and the topography of all the five mountains were so different from each other. Therefore, population locations at the same altitude above sea level were not included in the study for the two species.

 

3. No statistical model was given for the analyses of what factors and in what arrangement were included in the ANOVA and varcomp.

 

Response: Thanks for your good suggestion. According to the comment, we have added more details about the statistical model.

REVISED SENTENCE: We log-transformed the data for all needle traits and performed a nested ANOVA using the lme [43] and varcomp [44] functions in R [45], which is in this increasing order: individuals, elevations, populations, and species. The code was (i.e. LL):

LL< - lme (log10 (LL) ~1, random = ~ 1 | species / population / elevation, data = E, method = "REML")

mLL< - varcomp (LL, TRUE, cum = FALSE)

The same model applied to TL, DL, LA, SLA, LMA, and LDMC, only replacing LL by these traits in the R code. REML referred to a restricted maximum likelihood method in the ‘lme’ function of R (version 4.2.1).

 

4. The specification of the variance components is very interesting and adds a lot of information, it will be interesting also include the error in the estimation of the variance component value.

 

Response: We are grateful for the suggestion. We would like to explain why the error in the estimation of the variance component value was not included. The nested ANOVA was run in R (version 4.2.1) by fitting a general liner model to achieve the variance component. And then we acquired the result from R/REML which is known to be unbiased.

 

5. The description of the types of phenotypic plasticity given in the introduction is not reflected in the objectives of the paper and is poorly highlighted in the results. In the discussion at lines 243-247 the authors address this problem. Because evolutionary diversification through plasticity led variation is still a controversial topic I propose that a better view in discussion into the physiological mechanisms promoted phenotypic divergence in plasticity.

 

Response: We are grateful for your good suggestion. Since we focused on the existence and adaptation meaning of phenotypic plasticity. In addition, according to the comment, we have added more details about the physiological mechanisms on Page 9.

REVISED SENTENCE: Physiological changes, on the other hand, are thought to be central to phenotypic variation, and because of physiology's sensitivity and systemic nature to environmental changes, the division of labor between traits could be regulated by these changes, particularly the endocrine system (Cristina et al., 2021). That is, phenotypic plasticity divergence could result from physio-logical mechanisms with a genetic basis. This allows us to have a more in-depth discussion of phenotypic variation and phenotypic plasticity.

Round 2

Reviewer 1 Report

Compared with the first submission, this version has witnessed a good improvement in scientific writing, but I still have some issues needed to be answered.

1.     The section of Introduction is too long and need to be shorted and refined. Moreover, the hypotheses raised in this ms are unclear, and there is no sufficient evidence to support these hypotheses. For the first hypothesis, it should be better to assume which traits and which pine would have higher variation or plasticity, and need to give us the reasons. For the third hypothesis, authors need to elaborate why you assume that latitude and MAP would be the important factors. Furthermore, authors stressed the geographical difference of two pines in different elevations, but here why you proposed that latitude would be important influencing factor. Also, the latitude or altitude should be geographical factors rather than environmental factors.

2.     Although the title of this ms is “Inter- and Intra-specific Variation Patterns of Leaf Phenotypic 2 Traits in Two Sympatric Pine Species”, the differences of trait variation between two pines are little discussed.

3.     Authors need to discuss deeply how your results are linked with the growth strategies of two pines (L360-367). Also, you’d better include the leaf economic spectrum theory in the Discussion.

4.     There are too many references. About 50 references are appropriate for one piece of paper.

Author Response

Dear Professors,

We are really appreciative of the support provided by you and your journal, and also sincerely appreciate the reviewers. According to your suggestions and the reviewers’ comments, we revised the manuscript. We hope that the revised manuscript should be up to meet the standard of your esteemed journal. Now we re-submit the manuscript to your journal.

 All concerns that should be considered were addressed in the revision one by one as follows, please examine. The black words are the reviewers’ comments and the red words are the responses from the authors.

Comments

1. The section of Introduction is too long and need to be shorted and refined. Moreover, the hypotheses raised in this ms are unclear, and there is no sufficient evidence to support these hypotheses. For the first hypothesis, it should be better to assume which traits and which pine would have higher variation or plasticity, and need to give us the reasons. For the third hypothesis, authors need to elaborate why you assume that latitude and MAP would be the important factors. Furthermore, authors stressed the geographical difference of two pines in different elevations, but here why you proposed that latitude would be important influencing factor. Also, the latitude or altitude should be geographical factors rather than environmental factors.

Response: We are grateful for the suggestion, and our reply is as follows:

According to comment, we have deleted the redundant part of the Introduction.

For the first hypothesis, we have added the assumption which LDMC plasticity being significantly higher in P. hwangshanensis than in P. massoniana, and we gave the reason on page 5 (L353-354 and L359-362).

For the third hypothesis, we have added more details on page 5 (L356-359 and L362-365) to explain why latitude and MAP would be the important factors.

And we have referred to geographical and environmental factors collectively as external factors in revised manuscript.

2. Although the title of this ms is “Inter- and Intra-specific Variation Patterns of Leaf Phenotypic 2 Traits in Two Sympatric Pine Species”, the differences of trait variation between two pines are little discussed.

Response: Thank you for your suggestion. In Results section 3.1 (Page L210-219), we compared the differences between the two pine needle trait variants and summarized the reasons for them in relation to their adaptive significance in Discussion section 4.3 (Page 10, L351-356). We hoped we have presented a clearer picture of the content in revised manuscript.

3. Authors need to discuss deeply how your results are linked with the growth strategies of two pines (L360-367). Also, you’d better include the leaf economic spectrum theory in the Discussion.

Response: Thank you for your good suggestion. We demonstrated the association between the needleleaf trait variations and growth strategies of two pines in Discussion, such as ‘Since P. hwangshanensis populations are distributed at higher elevations, they are subjected to more severe winter stress than P. massoniana populations, which are found at lower elevations. So their needles are thicker and shorter to withstand the stress of strong winds and snow’.

And we have added more details about the leaf economic spectrum theory.

REVISED SENTENCE: Based on ‘leaf economic spectrum theory’, we could conclude that both P. massoniana and P. hwangshanensis were typical ‘slow investment-return’ type species. However, our findings indicated that the resource trade-off strategies of two pine species differed markedly.

4. There are too many references. About 50 references are appropriate for one piece of paper.

Response: We agree with the comment and removed as many references as possible in the revised manuscript. Thank you.

Author Response File: Author Response.docx