Morpho-Anatomical Characteristics and Volatile Profiles of Pinus nigra J.F.Arnold from the Balkan Peninsula and Southern Carpathians

Round 1

Reviewer 1 Report

Dear Editor and authors,

I have read the paper on “Morpho-anatomical characteristics and volatile profiles of Pinus nigra from the Balkan Peninsula and Southern Carpathians” written by Mitić et al. As evident from the title itself, the paper addresses the morpho-anatomical and phytochemical differentiation of Pinus nigra populations in the Balkans and Southern Carpathians. Furthermore, the research deals with taxonomic issues of the studied species, by analyzing populations categorized into four subspecies. The differences between geographic localities and subspecies were studied on plant material from 19 populations, and in total the research included samples from 201 trees. The main strength of this paper is the use of different methods (morphometric, anatomical and chemical) to distinguish between Pinus nigra populations and subspecies. Moreover, it features the first ever analysis of the variability of populations of this species in the Balkan Peninsula, which is home to a large number of its relict populations. Overall, I find the paper interesting and to be generally contributing to better familiarity with the variability of the studied species. Below are some suggestions to improve the paper.

Major comments:

In the introduction, prior to the paragraph on the chemophenetic significance of terpenes in research, the authors should add in the same way a general paragraph on the use of morphometric and anatomical methods. There has been a great deal of research on pines using morphometric and anatomical methods to distinguish between the populations of different pine species: P. sylvestris, P. mugo etc. In the paragraph, the authors should write what the significance of these methods is in these studies and provide some examples.

Before conducting the cluster analysis, the authors should standardize the data, i.e., variables should be standardized to zero mean and unit standard deviation. From the values (distances between the groups) shown in the figures it is evident that this has not been done. The cluster analysis should be repeated. Moreover, I suggest also doing a k-means cluster analysis, i.e. determining the optimum number of groups into which the studied units (trees) can be divided based on morpho-anatomical and chemical data. The obtained results should be shown in Figure 1 or together with the dendrogram.

Minor comments:

The list of references could be updated with some other papers tackling morpho-anatomical and chemical variability of the species populations from the genus Pinus, especially in the introductory part of the paper.

According to the iThenticate report, the amount of wording duplication in the manuscript is 27%. This could be somewhat reduced. In materials and methods, certain parts have been entirely copied from other papers.

Table 1 can be put into additional materials. It is redundant next to Figure 1.

Figure 1 could be simplified. Instead of state borders and names of the countries, the authors should put the borders of ranges of the studied subspecies and their names.

Instead of ssp., it should say subsp. This should be changed throughout the paper.

For the studies species and subspecies, when they are mentioned for the first time in the abstract and main body of the paper, the "authority" should be added, i.e. the standardized abbreviation of the name of the scientist(s) who first published the scientific name of the species or subspecies. The same applies to the title of the paper, i.e., the title should say Pinus nigra J.F.Arnold.

Moderate to minor editing of English language required. Certain sentences in the paper are written strangely.

Author Response

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Reviewer 2 Report

The manuscript is the first report on morpho-anatomical and phytochemical differentiation of 19 native populations representing different Pinus nigra subspecies (banatica, dalmatica, nigra, and pal-lasiana) in the Balkans and Southern Carpathians.

The results might help clarify the issue of phenotypic diversification of P. nigra, especially in the eastern part of its range.Therefore, this article has great innovation and academic value.

How to exclude the influence of habitat differences and sampling time on these indicators when selecting them as classification criteria?

Author Response

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Reviewer 3 Report

all comments in attached file

Comments for author File: Comments.pdf

Some sentences must be reviewed by an English native speaker

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Round 2

Reviewer 1 Report

The paper can be accepted in this form.

Author Response

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Reviewer 3 Report

I maintain the same recommendations made in the original version of the manuscript. Storage at -20 for metabolites, no fixation of biological material, freehand cuts, old references.

The biggest problems are:

1) There are many interesting references that could be cited instead of the low-circulation books. If they were books from reputable publishers with an international reach, it would be another matter, but that is not the case.

2) I insist that biological materials should be promptly analyzed, no more than 15 days after collection, or in permanent contact with liquid nitrogen, at -60 to -80ºC or freeze-dried for the analysis of metabolites. I can't understand metabolism with material stored at -20ºc for 6 months.

3) When I referred to fixing, I was not referring to storing the material. The material was collected and promptly stored in 70% alcohol. This is clear to me, but the plant material should have been fixed in FAA or glutaraldehyde.

4) In my opinion and that of another colleague, also a plant anatomist, freehand cuts should not be used for the purpose proposed in the article as there is no control over thickness.

5) The anatomy images are of much lower quality than what is thought to be in a JCRQ1 magazine. Therefore, I maintain my recommendation to reject the manuscript. I couldn't see any changes that would justify changing my original concept.

Author Response

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