Phenotypic Variation and Diversity in Fruit, Leaf, Fatty Acid, and Their Relationships to Geoclimatic Factors in Seven Natural Populations of Malania oleifera Chun et S.K. Lee

Round 1

Reviewer 1 Report

The study assesses the phenotypic variation of Malania oleifera across seven populations. While it was not really clear what the overarching goal of this study for assessing phenotypic diversity was, the implication is that understanding phenotypic variation and diversity is important for genetic improvement.

Overall, the manuscript is well presented, although the English language can be improved to remove awkwardness. The sampling method is adequate and care has been taken in performing rigorous data collection. However, it is not always clear why particular analyses or metrics were used (e.g., CV, Pearson, PCA). What dictated the choice or necessity of these methods and indices? More information would provide more clarity in the Statistical Analysis section. As for the PCA, the individual contributions were rather low. Is there a lot of residuals? Data collinearity? Figure 2 also shows a lot of significant correlations among the 21 traits. I wonder if significantly correlated variables should be removed to reduce redundancy. The discussion can sometimes feel underdeveloped, mostly describing the results without much elaboration. If the phenotypic assessment was done just for the sake of assessing variation, that is fine. But there are implications, for instance, for conservation that can be elaborated (and this species is in the IUCN Red List threatened category). Specific comments are provided in the manuscript.

Comments for author File: Comments.pdf

Author Response

Dear Editor and Reviewer,

We have carefully addressed all the reviewer's concerns. Please see our replies. We hope he/she satisfies with our answers. Changes highlighted in red have been made accordingly in the revised manuscript.

Reviewer 1,

Point 1: Overall, the manuscript is well presented, although the English language can be improved to remove awkwardness. The sampling method is adequate and care has been taken in performing rigorous data collection. However, it is not always clear why particular analyses or metrics were used (e.g., CV, Pearson, PCA). What dictated the choice or necessity of these methods and indices? More information would provide more clarity in the Statistical Analysis section. As for the PCA, the individual contributions were rather low. Is there a lot of residuals? Data collinearity? Figure 2 also shows a lot of significant correlations among the 21 traits. I wonder if significantly correlated variables should be removed to reduce redundancy. The discussion can sometimes feel underdeveloped, mostly describing the results without much elaboration. If the phenotypic assessment was done just for the sake of assessing variation, that is fine. But there are implications, for instance, for conservation that can be elaborated (and this species is in the IUCN Red List threatened category). Specific comments are provided in the manuscript. 
Response 1: Thanks very much for your comments. We have asked Dr. Lai Meng of Jiangxi Agricultural University, who’s a well-established expert, to polish our paper. Please see if the revised version met the English presentation standard. We have rephrased the related part in '2.3. Statistical analysis' to clarify the reason why we used the analyses/indices like CV, Pearson, PCA. The low individual contributions indicated that the variation in phenotypic traits is relatively high and could not be reduced to fewer PCs. PCA analysis is also a typically used method to cluster the records (population in this study). Significant correlations among variables suggested strong data collinearity. We are not going to remove the significantly correlated variables because 1) it won't affect how the results are presented or displayed, 2) the traits considered in this study were specially selected to characterize the diversity of Malania oleifera, and most of their relationship was unclear prior to this study. Based on our results it is safe to say that the redundant and unimportant traits can be exluded in the future. As breeders, we are not good at developing protection strategies, we concentrated more on genetic improvement of nervonic acid. But, as we mentioned in the Introduction, common gardens and trials established in a genetic improvement program are also essential ways for ex situ conservation.

Point 2: L227. Is this causation or correlation? Also, there could be other reasons for the decrease in fatty acid at higher latitudes that may not have anything to do with latitudes.
Response 2: It was correlation. Latitude was negatively correlated with fatty acid. We inferred that it was the colder environment at higher latitudes reduce the fatty acid. A similar result has been reported in another paper (10.1016/j.indcrop.2020.112487). Certainly this result could be caused by other reasons, such as the fruits might be not fully mature. We have rephrased it in the text as "as latitude increased, the colder environment might reduce the fatty acid content".

Point 3: Also, what do you mean by "it"? Are you talking about forest tree species? Or about heritability?
Response 3: L255. It refers to "capture higher genetic gain" and "dissect the underlying genetic architecture". 

All of the other comments have been carefully considered and corrected in the text.

Reviewer 2 Report

 

Dear Editor and Authors,

The paper analyses phenotypic variation of the seven natural populations of Malania oleifera in China. The aim of the study was to perform a morphological and chemical analysis of fruits, kernels, and leaves. In total, author analyses 21 phenotypic traits. I consider it particularly valuable that the authors cover phenotype from two aspects: morphometric and chemical. Although the study includes only a smaller number of populations and individuals, it is valuable as it pertains to a vulnerable and endangered species which is on the IUCN Red List. All in all, I do think that the paper could be interesting, but some things need to be clarified and better explained. Therefore, I suggest a major revision.

The title should be changed as indicated in the enclosed document.

Authorities should be listed in the title, summary and the main document – and only when mentioned for the first time. The same applies to other botanical names mentioned in the paper. With the scientific name for each species mentioned in the paper for the first time you also need to specify authorities.

I think there is no need to emphasize the health aspects of this kind in the paper. Namely, the paper pertains to the morphological variability of populations and its relationship to ecological variables. Therefore, in the introduction one or two sentences can be used to briefly say something about the use of this species in pharmaceuticals. I think that the part describing the use and meaning of nervonic acid is too elaborate for this type of research.

The introduction requires significant modifications. More information should be given on phenotype variability. What does it depend on? What influences it? Provide some examples on woody plants.

In the last paragraph of the Introduction, define the research hypotheses.

A clearer map of the studied area should be generated. It is difficult to see from the map where the studied area is located in the broader context. Provide a map where it is visible where the studied area is located in Asia, and a map clearly indicating the studied populations.

Table 3 is unnecessary. The column with F values should be moved to Table 2.

Please create a biplot for PC analysis.

On Figure 3, please put the axis with distances.

I suggest you rephrase the statement highlighted in green in the first paragraph of the Discussion. Namely, morphological characteristics are greatly influenced by ecological factors so variability can sometimes also be a consequence of phenotypic plasticity, and not solely of plant adaptation to certain habitat circumstances. In view of that, insights into phenotypic variability cannot be the main prerequisite for the preservation of genetic diversity.

In the discussion there is also mention of geographic isolation of populations, so I suggest the authors establish the patterns of isolation by distance and isolation by adaptation. A Mantel test should be performed.

Corrections to minor errors and text editing, and few comments and suggestions are marked in the attached document.

Comments for author File: Comments.pdf

Author Response

Dear Editor and Reviewer,

We have carefully addressed all the reviewer's concerns. Please see our replies. We hope he/she satisfies with our answers. Changes highlighted in red have been made accordingly in the revised manuscript.

Reviewer 2,

Point 1: The title should be changed as indicated in the enclosed document.
Response 1: We thank the reviewer for very important and detailed comments. The title was changed into "Diversity in fruit, leaf, fatty acid, and their relationship to geo-climatic factors in seven natural populations of Malania oleifera Chun et S.K. Lee" as the reviewer's suggestion.

Point 2: Authorities should be listed in the title, summary and the main document – and only when mentioned for the first time. The same applies to other botanical names mentioned in the paper. With the scientific name for each species mentioned in the paper for the first time you also need to specify authorities.
Response 2: We have revised them all in the text and avoid making such mistake in future writing.

Point 3: I think there is no need to emphasize the health aspects of this kind in the paper. Namely, the paper pertains to the morphological variability of populations and its relationship to ecological variables. Therefore, in the introduction one or two sentences can be used to briefly say something about the use of this species in pharmaceuticals. I think that the part describing the use and meaning of nervonic acid is too elaborate for this type of research.
The introduction requires significant modifications. More information should be given on phenotype variability. What does it depend on? What influences it? Provide some examples on woody plants.
In the last paragraph of the Introduction, define the research hypotheses. 
Response 3: We appreciate your constructive comments. The Introduction section has been rewritten. First, the first two paragraphs were shifted around to improve the flow of the context. Second, we removed the use of nervonic acid in pharmaceuticals and condensed the description of the substance. Thirdly, we expanded on phenotypic variability and its influencing variables and provided instances of variation patterns and geo-climatic influences on woody plants. Forth, the final sentence of the Introduction was amended to include implications of our study.

Point 4: A clearer map of the studied area should be generated. It is difficult to see from the map where the studied area is located in the broader context. Provide a map where it is visible where the studied area is located in Asia, and a map clearly indicating the studied populations.
Response 4: We redrew the map, replaced the prior Provincial Map with a China Map, and added population locations to increase visibility of the study area (Fig. 1).

Point 5: Table 3 is unnecessary. The column with F values should be moved to Table 2.
Response 5: We have revised them in the text.

Point 6: Please create a biplot for PC analysis.
Response 6: A biplot for PC analysis was generated (Figure 3A).

Point 7: On Figure 3, please put the axis with distances.
Response 7: The scale bar was replaced with axis with distances

Point 8: I suggest you rephrase the statement highlighted in green in the first paragraph of the Discussion. Namely, morphological characteristics are greatly influenced by ecological factors so variability can sometimes also be a consequence of phenotypic plasticity, and not solely of plant adaptation to certain habitat circumstances. In view of that, insights into phenotypic variability cannot be the main prerequisite for the preservation of genetic diversity.
Response 8: Thanks for pointing this out. We agree with your point of view. Phenotypic plasticity is one of the factors resulting in variation. Evaluation of phenotypic variability is essential for tree selection in genetic improvement programs, and charactering genetic diversity using molecular markers like microsatellites is more reliable in preserving genetic diversity. We have rephrased this part in the text.

Point 9: In the discussion there is also mention of geographic isolation of populations, so I suggest the authors establish the patterns of isolation by distance and isolation by adaptation. A Mantel test should be performed.
Response 9: We performed Mantel tests to determine the correlation between traits and climatic, geographic variables and altitude, and revised the related text.

Point 12: Corrections to minor errors and text editing, and few comments and suggestions are marked in the attached document.
Response 12: We have revised them in the text. 

Round 2

Reviewer 2 Report

I have read the reviewed version of the paper with great interest. Nevertheless, there are still some things that could be improved.

In the last paragraph of the Introduction, please define the research hypotheses.

To test correlations between morphometric (fruit and leaf), chemical, geographic and climatic data, five different matrices should be calculated. Climatic differences should be calculated as the Euclidian distance between the population means for the first three principal components of the principal component analysis. Squared Mahalanobis distances between the populations should be calculated to obtain a matrix of morphometric (fruit and leaf) and chemical distances among the studied populations. Geographic distances should be calculated from the latitude and longitude of the site of sample collection. Finally, to assess isolation by distance (IBD) and isolation by adaptation (IBA), three response matrices (morphological fruit differences; morphological leaf differences; chemical differences) should be compared to the two predictor matrices (climate differences and geographic distance) using simple Mantel tests.

Generally speaking, the results of your research should be discussed better and compared to other studies. In the MDPI group of journals dealing with plant sciences there are many papers that can be used for comparison with the results of your studies and which include morphological characteristics of populations.

Author Response

Dear Editor and Reviewer,

We have carefully addressed all the reviewer's concerns. Please see our replies. We hope he/she satisfies with our answers. Changes highlighted in red have been made accordingly in the revised manuscript.

Point 1: In the last paragraph of the Introduction, please define the research hypotheses.
Response 1: The main objectives, including the research hypotheses, were listed in the last paragraph of the Introduction.

Point 2: To test correlations between morphometric (fruit and leaf), chemical, geographic and climatic data, five different matrices should be calculated. Climatic differences should be calculated as the Euclidian distance between the population means for the first three principal components of the principal component analysis. Squared Mahalanobis distances between the populations should be calculated to obtain a matrix of morphometric (fruit and leaf) and chemical distances among the studied populations. Geographic distances should be calculated from the latitude and longitude of the site of sample collection. Finally, to assess isolation by distance (IBD) and isolation by adaptation (IBA), three response matrices (morphological fruit differences; morphological leaf differences; chemical differences) should be compared to the two predictor matrices (climate differences and geographic distance) using simple Mantel tests. 
Response 2: We have performed Mantel test according to the reviewer's recommondention, then revised the corresponding section in the text.

Point 3: Generally speaking, the results of your research should be discussed better and compared to other studies. In the MDPI group of journals dealing with plant sciences there are many papers that can be used for comparison with the results of your studies and which include morphological characteristics of populations.
Response 3: We have studied some of the related papers published in the MDPI group of journals and the revised the Discussion.

Author Response File: Author Response.docx