Morpho-Anatomical Properties and Terpene Composition of Picea Omorika (Pančić) Purk. Needles from Bosnia and Herzegovina

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. The Abstract is currently a brief summary of the results obtained by the authors. The authors should have paid attention to the relevance of this work at the beginning of the Abstract, then used the sentence that is currently located on lines 33-35, briefly listed the methods used and the most important results, and made a general conclusion based on the results of the study.
2. In the Introduction, the authors should also indicate the relevance and practical significance of the study. In particular, it should be indicated why the analysis of the terpene composition in Picea omorika is important. Why were these particular parameters chosen for the study - the morpho-anatomical properties of the needles and terpene composition? The introduction should also introduce the reader a little more to the problem being studied. The authors can add a few sentences devoted to the study of the terpene content in Picea, indicate how different species of Picea differ in the content and composition of terpenes.
3. Materials and methods. Sections 2.2-2.5 do not contain references to the literature. This is especially important for sections 2.2 and 2.3. The described methods are used by many researchers. Even if the authors have modified the methods, a reference to the literary source containing the basis of the methods should be provided, and then the modifications made to the method should be indicated.
4. Results. It would be more logical to first describe the morpho-anatomical features of the needles, and then the terpene compositions.
5. The discussion should be significantly expanded. The comparison and discussion of the results obtained by the authors with the results of other researchers is too limited. Section 4.1 can be supplemented with material on the importance of studying the terpene profile in Picea. The role of individual terpenes in the metabolism and physiology of Picea can be described and discussed. Is there a relationship between the morpho-anatomical parameters of the needles in Picea omorika and the content and composition of terpenes? The comparison of the studied characteristics of Picea omorika with similar parameters in other Picea species has also not been carried out sufficiently.
6. The list of references is too limited. In total, the authors used 17 literary sources, and 4 sources are references to the results of their own research. References to studies of the last 5 and 10 years are also insufficient. There are only 3 of them, and 2 are the results of the authors' own research.
7. In the conclusion, it would be necessary to add a description of the significance of the obtained results. For example, to indicate why the studies of differences between different populations of P.omorika are important in general and what new things the authors themselves have obtained. The sentence “Dinarides massive is a geomorphologically complexed own to tectonic disorders in Pliocene and the formation of ecological niches during glaciation in Pleistocene, which are responsible for the presented biodiversity and divergences of some P. omorika populations” on lines 306-308 appears in the Conclusion without any prerequisites. This issue is not considered in the discussion.

Overall, I would like to recommend the authors revise this manuscript, using their own article (reference 7 in this manuscript) as a guide. It has a logically written Introduction, the necessary references in the Materials and Methods section, a serious analysis and comparison of the data obtained by the authors with the results of world research in the Discussion section, a well-founded Conclusion and a summary of the entire study.

Comments for author File: Comments.pdf

Author Response

1. The article entitled "Terpene composition and morpho-anatomical properties of 2 Picea omorika needles from Bosnia and Herzegovina" is a fairly extensive publication of scientific research focusing on the analysis of the composition and morpho-anatomical features of the Picea omorica species in the area of ​​the neighboring countries of the Balkan region.

   Although the article is interesting and brings some novelty to the world of science, it is not emphasized to what extent the research was conducted? There is no deeper explanation of what could have caused the differences within one species in such a close area. The article still requires further refinement.

   • Thank You for Your suggestion. In the end of Discussion section we explained how these differences might be possible, page 13, lines 290-294.

   What is the important - if the analysis of composition and morphological features was determined based on single specimens from a given location, the article should be rejected. Biodiversity within a single species is so great that it is impossible to draw such far-reaching conclusions as the authors did.

   • Thank You for Your suggestion. We are sorry because we didn’t explain precisely method of needle collection. Because of that we inserted one sentence in Material and method section. We added number zero in all three populations in Table 1 (n=150), because we made mistake and present total number of trees instead of total number of samples. Page 4 and 5, line 144-145.

   There are additional several inaccuracies and errors related to the naming of volatile compounds. Below I am including detailed comments worth considering:

   - the first sentence of the abstract requires supplementation at the end with "of P. omorica". Thank You for Your suggestion,

   but Picea omorika is proper latin name of this species.

   - the keywords are poorly chosen, e.g. two-yer-old needles....

   - the keywords should be supplemented with the following terms: Picea omorica, terpene, biodiversity etc..

   . •         Thank You for Your suggestion. New kewords are: Picea omorika, Serbian spruce, population, morpho-anatomy, terpenes, multivariate statistics, page 1, lines 34-35

   - In these experiments, volatile components were not extracted but were isolated simultaneously with hydrodistillation and extraction. Hydrodistillation is a completely different process from extraction! Please correct the wording regarding the method of isolating terpenes throughout the manuscript (extraction into--> isolation)

   • Thank You for Your suggestion. We made corrections in Page 3, lines 85-87.

   - page 3, line 73. The HP-5 MS column is semi-polar - not non-polar !

   • Thank You for Your suggestion. We made correction , Page 3, line 94

   In Table 1, there is no point in giving unidentified components as e.g. n.i.4, n.i.5 etc. Please provide the mass spectrum (3 main peaks + molar mass of the unidentified component) in accordance with the procedure in the journal.

   • Thank You for Your suggestion. We provided the mass spectra for unidentified components and signed them in blue (Table 4, pages 8-10, between lines 207 and 208).

   there are many errors in the names in table 1 and the text. type: treans-2--hexenal should be: trans-hex-2-enal the same problem is occured with: 9-octadecenal (should be ocadec-9-enal). the same with compound no. 15 Compound no. 29, 109 and 110 are gel named in table 1 What is with the description of compound 99? The type of isomerism e or Z or E, E is given in names everywhere, i.e. (E), (Z), (E,E) etc.

   • Thank You for Your suggestion. We made appropriate corrections in Table 4 pages 8-10, between lines 207 and 208), signed in blue.

   - Figure 2 has very bad quality. Text is invisible.

   • Thank You for Your suggestion. We made new Figure (now it is figure 4, page 10, between lines 210-211).

   The first sentence in the discussion line 257-258 is incorrect. First, the compounds were not identified in the needles but in the essential oil from the needles Second, the fact that there are more components in the needles from Bosnia than in the oil from Serbia means that perhaps a larger dose of oil was injected into the chromatographic column or the sensitivity of the apparatus was greater.

   • Thank You for Your suggestion. We rearranged the whole paragraph, page 13, lines 300-304.

   The discussion of the results is rather cursory in contrast to the correctly formulated conclusions in chapter 5.

   • Thank You for Your suggestion. We rearranged chapter 5, Conclusions, with new paragraphs, page 14, 345-349, 354-357.

   Note to reviewers: All corrected words and numbers through the text and tables were signed in blue.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article entitled "Terpene composition and morpho-anatomical properties of 2 Picea omorika needles from Bosnia and Herzegovina" is a fairly extensive publication of scientific research focusing on the analysis of the composition and morpho-anatomical features of the Picea omorica species in the area of ​​the neighboring countries of the Balkan region.

Although the article is interesting and brings some novelty to the world of science, it is not emphasized to what extent the research was conducted?There is no deeper explanation of what could have caused the differences within one species in such a close area. The article still requires further refinement.

What is the important - if the analysis of composition and morphological features was determined based on single specimens from a given location, the article should be rejected.

Biodiversity within a single species is so great that it is impossible to draw such far-reaching conclusions as the authors did.

There are additional several inaccuracies and errors related to the naming of volatile compounds. Below I am including detailed comments worth considering:

- the first sentence of the abstract requires supplementation at the end with "of P. omorica"

- the keywords are poorly chosen, e.g. two-yer-old needles....

- the keywords should be supplemented with the following terms: Picea omorica, terpene, biodiversity etc...

- In these experiments, volatile components were not extracted but were isolated simultaneously with hydrodistillation and extraction. Hydrodistillation is a completely different process from extraction! Please correct the wording regarding the method of isolating terpenes throughout the manuscript (extraction into--> isolation)

- page 3, line 73. The HP-5 MS column is semi-polar - not non-polar !

- In Table 1, there is no point in giving unidentified components as e.g. n.i.4, n.i.5 etc. Please provide the mass spectrum (3 main peaks + molar mass of the unidentified component) in accordance with the procedure in the journal.

there are many errors in the names in table 1 and the text. type: treans-2--hexenal should be: trans-hex-2-enal the same problem is occured with: 9-octadecenal (should be ocadec-9-enal). the same with compound no. 15 Compound no. 29, 109 and 110 are gel named in table 1 What is with the description of compound 99? The type of isomerism e or Z or E, E is given in names everywhere, i.e. (E), (Z), (E,E) etc.

- Figure 2 has very bad quality. Text is invisible

The first sentence in the discussion line 257-258 is incorrect. First, the compounds were not identified in the needles but in the essential oil from the needles Second, the fact that there are more components in the needles from Bosnia than in the oil from Serbia means that perhaps a larger dose of oil was injected into the chromatographic column or the sensitivity of the apparatus was greater.

The discussion of the results is rather cursory in contrast to the correctly formulated conclusions in chapter 5.

Comments for author File: Comments.pdf

Author Response

The article entitled "Terpene composition and morpho-anatomical properties of 2 Picea omorika needles from Bosnia and Herzegovina" is a fairly extensive publication of scientific research focusing on the analysis of the composition and morpho-anatomical features of the Picea omorica species in the area of ​​the neighboring countries of the Balkan region.

Although the article is interesting and brings some novelty to the world of science, it is not emphasized to what extent the research was conducted? There is no deeper explanation of what could have caused the differences within one species in such a close area. The article still requires further refinement.

• Thank You for Your suggestion. In the end of Discussion section we explained how these differences might be possible, page 13, lines 290-294.

What is the important - if the analysis of composition and morphological features was determined based on single specimens from a given location, the article should be rejected. Biodiversity within a single species is so great that it is impossible to draw such far-reaching conclusions as the authors did.

• Thank You for Your suggestion. We are sorry because we didn’t explain precisely method of needle collection. Because of that we inserted one sentence in Material and method section. We added number zero in all three populations in Table 1 (n=150), because we made mistake and present total number of trees instead of total number of samples. Page 4 and 5, line 144-145.

There are additional several inaccuracies and errors related to the naming of volatile compounds. Below I am including detailed comments worth considering:

- the first sentence of the abstract requires supplementation at the end with "of P. omorica". Thank You for Your suggestion,

but Picea omorika is proper latin name of this species.

- the keywords are poorly chosen, e.g. two-yer-old needles....

- the keywords should be supplemented with the following terms: Picea omorica, terpene, biodiversity etc..

. •         Thank You for Your suggestion. New kewords are: Picea omorika, Serbian spruce, population, morpho-anatomy, terpenes, multivariate statistics, page 1, lines 34-35

- In these experiments, volatile components were not extracted but were isolated simultaneously with hydrodistillation and extraction. Hydrodistillation is a completely different process from extraction! Please correct the wording regarding the method of isolating terpenes throughout the manuscript (extraction into--> isolation)

• Thank You for Your suggestion. We made corrections in Page 3, lines 85-87.

- page 3, line 73. The HP-5 MS column is semi-polar - not non-polar !

• Thank You for Your suggestion. We made correction , Page 3, line 94

In Table 1, there is no point in giving unidentified components as e.g. n.i.4, n.i.5 etc. Please provide the mass spectrum (3 main peaks + molar mass of the unidentified component) in accordance with the procedure in the journal.

• Thank You for Your suggestion. We provided the mass spectra for unidentified components and signed them in blue (Table 4, pages 8-10, between lines 207 and 208).

there are many errors in the names in table 1 and the text. type: treans-2--hexenal should be: trans-hex-2-enal the same problem is occured with: 9-octadecenal (should be ocadec-9-enal). the same with compound no. 15 Compound no. 29, 109 and 110 are gel named in table 1 What is with the description of compound 99? The type of isomerism e or Z or E, E is given in names everywhere, i.e. (E), (Z), (E,E) etc.

• Thank You for Your suggestion. We made appropriate corrections in Table 4 pages 8-10, between lines 207 and 208), signed in blue.

- Figure 2 has very bad quality. Text is invisible.

• Thank You for Your suggestion. We made new Figure (now it is figure 4, page 10, between lines 210-211).

The first sentence in the discussion line 257-258 is incorrect. First, the compounds were not identified in the needles but in the essential oil from the needles Second, the fact that there are more components in the needles from Bosnia than in the oil from Serbia means that perhaps a larger dose of oil was injected into the chromatographic column or the sensitivity of the apparatus was greater.

• Thank You for Your suggestion. We rearranged the whole paragraph, page 13, lines 300-304.

The discussion of the results is rather cursory in contrast to the correctly formulated conclusions in chapter 5.

• Thank You for Your suggestion. We rearranged chapter 5, Conclusions, with new paragraphs, page 14, 345-349, 354-357.

Note to reviewers: All corrected words and numbers through the text and tables were signed in blue.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Unfortunately, the authors mixed up the responses to the comments and sent me responses intended for another reviewer. Overall, in the text of the article, I found answers to most of the questions that interested me. However, I would still like to receive from the authors the document itself with direct responses to my questions and comments.

Author Response

1. The Abstract is currently a brief summary of the results obtained by the authors. The authors should have paid attention to the 1. relevance of this work at the beginning of the Abstract, then used the 2. sentence that is currently located on lines 33-35, 3. briefly listed the methods used and the 4. most important results, and made a 5. general conclusion based on the results of the study.

• Thank You very much for Your suggestion. The Abstract is rearranged, see page 1, lines 19-33

1. In the Introduction, the authors should also indicate the relevance and practical significance of the study. In particular, it should be indicated why the analysis of the terpene composition in Picea omorika is important. Why were these particular parameters chosen for the study - the morpho-anatomical properties of the needles and terpene composition? The introduction should also introduce the reader a little more to the problem being studied. The authors can add a few sentences devoted to the study of the terpene content in Picea, indicate how different species of Picea differ in the content and composition of terpenes.

• Thank You very much for Your suggestions. The Introduction is expanded, see page 2, lines 46-61.

1. Materials and methods. Sections 2.2-2.5 do not contain references to the literature. This is especially important for sections 2.2 and 2.3. The described methods are used by many researchers. Even if the authors have modified the methods, a reference to the literary source containing the basis of the methods should be provided, and then the modifications made to the method should be indicated.

• Thank You very much for Your suggestions. We added two references in sections 2.2 and 2.3, page 3, lines 77 and 87.
• Word “extraction” was replaced with word “isolation”, page 3, line 85.
• Word “extracted” was replaced with word “isolated”, page 3, line 86.
• One new sentence was added in section 2.1., page 2, lines 66-67

1. Results. It would be more logical to first describe the morpho-anatomical features of the needles, and then the terpene compositions.

• Thank You very much for Your suggestions. We made changes starting from title to the end of article.

1. The discussion should be significantly expanded. The comparison and discussion of the results obtained by the authors with the results of other researchers is too limited. Section 4.1 can be supplemented with material on the importance of studying the terpene profile in Picea. The role of individual terpenes in the metabolism and physiology of Picea can be described and discussed. Is there a relationship between the morpho-anatomical parameters of the needles in Picea omorika and the content and composition of terpenes? The comparison of the studied characteristics of Picea omorika with similar parameters in other Picea species has also not been carried out sufficiently.

• Thank You very much for Your suggestions.

We added paragraph in 4.2. beginning with “The terpene profile of Picea species….”, Page 13, lines: 297-313.

We significantly expanded Discussion section. We expanded text about the relationships and comparison of P. omorika with other Picea species (lines 290-294).

1. The list of references is too limited. In total, the authors used 17 literary sources, and 4 sources are references to the results of their own research. References to studies of the last 5 and 10 years are also insufficient. There are only 3 of them, and 2 are the results of the authors' own research.

• Thank You very much for Your suggestions. We more than doubled the number of references. We had to change their places in reference list too, since text was seriously scrutinized.

1. In the conclusion, it would be necessary to add a description of the significance of the obtained results. For example, to indicate why the studies of differences between different populations of P.omorika are important in general and what new things the authors themselves have obtained. The sentence “Dinarides massive is a geomorphologically complexed own to tectonic disorders in Pliocene and the formation of ecological niches during glaciation in Pleistocene, which are responsible for the presented biodiversity and divergences of some P. omorika populations” on lines 306-308 appears in the Conclusion without any prerequisites. This issue is not considered in the discussion.

• Thank You very much for Your suggestions. We added two sentences at the beginning of Conclusion section. (page 14, lines 345-349)
• The sentence “Dinarides massive is a geomorphologically complexed own to tectonic disorders in Pliocene and the formation of ecological niches during glaciation in Pleistocene, which are responsible for the presented biodiversity and divergences of some omorika populations” was moved in the new paragraph at the end of Discussion section. (page 14, lines 338-343)

Overall, I would like to recommend the authors revise this manuscript, using their own article (reference 7 in this manuscript) as a guide. It has a logically written Introduction, the necessary references in the Materials and Methods section, a serious analysis and comparison of the data obtained by the authors with the results of world research in the Discussion section, a well-founded Conclusion and a summary of the entire study.

• Thank You very much for Your suggestions. The conclusion and summary are strengthened with new sentences. Page 14, lines 345-349, 354-357.

NOTE TO THE REVIEWER: All changed text is marked in blue.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I accept the manuscript in present form, however there is still small mistke in Table 4 ( the name of compound 2 please write from capital letter).

Author Response

The article entitled "Terpene composition and morpho-anatomical properties of 2 Picea omorika needles from Bosnia and Herzegovina" is a fairly extensive publication of scientific research focusing on the analysis of the composition and morpho-anatomical features of the Picea omorica species in the area of ​​the neighboring countries of the Balkan region.

Although the article is interesting and brings some novelty to the world of science, it is not emphasized to what extent the research was conducted? There is no deeper explanation of what could have caused the differences within one species in such a close area. The article still requires further refinement.

• Thank You for Your suggestion. In the end of Discussion section we explained how these differences might be possible, page 13, lines 290-294.

What is the important - if the analysis of composition and morphological features was determined based on single specimens from a given location, the article should be rejected. Biodiversity within a single species is so great that it is impossible to draw such far-reaching conclusions as the authors did.

• Thank You for Your suggestion. We are sorry because we didn’t explain precisely method of needle collection. Because of that we inserted one sentence in Material and method section. We added number zero in all three populations in Table 1 (n=150), because we made mistake and present total number of trees instead of total number of samples. Page 4 and 5, line 144-145.

There are additional several inaccuracies and errors related to the naming of volatile compounds. Below I am including detailed comments worth considering:

- the first sentence of the abstract requires supplementation at the end with "of P. omorica". Thank You for Your suggestion,

but Picea omorika is proper latin name of this species.

- the keywords are poorly chosen, e.g. two-yer-old needles....

- the keywords should be supplemented with the following terms: Picea omorica, terpene, biodiversity etc..

. •         Thank You for Your suggestion. New kewords are: Picea omorika, Serbian spruce, population, morpho-anatomy, terpenes, multivariate statistics, page 1, lines 34-35

- In these experiments, volatile components were not extracted but were isolated simultaneously with hydrodistillation and extraction. Hydrodistillation is a completely different process from extraction! Please correct the wording regarding the method of isolating terpenes throughout the manuscript (extraction into--> isolation)

• Thank You for Your suggestion. We made corrections in Page 3, lines 85-87.

- page 3, line 73. The HP-5 MS column is semi-polar - not non-polar !

• Thank You for Your suggestion. We made correction , Page 3, line 94

In Table 1, there is no point in giving unidentified components as e.g. n.i.4, n.i.5 etc. Please provide the mass spectrum (3 main peaks + molar mass of the unidentified component) in accordance with the procedure in the journal.

• Thank You for Your suggestion. We provided the mass spectra for unidentified components and signed them in blue (Table 4, pages 8-10, between lines 207 and 208).

there are many errors in the names in table 1 and the text. type: treans-2--hexenal should be: trans-hex-2-enal the same problem is occured with: 9-octadecenal (should be ocadec-9-enal). the same with compound no. 15 Compound no. 29, 109 and 110 are gel named in table 1 What is with the description of compound 99? The type of isomerism e or Z or E, E is given in names everywhere, i.e. (E), (Z), (E,E) etc.

• Thank You for Your suggestion. We made appropriate corrections in Table 4 pages 8-10, between lines 207 and 208), signed in blue.

- Figure 2 has very bad quality. Text is invisible.

• Thank You for Your suggestion. We made new Figure (now it is figure 4, page 10, between lines 210-211).

The first sentence in the discussion line 257-258 is incorrect. First, the compounds were not identified in the needles but in the essential oil from the needles Second, the fact that there are more components in the needles from Bosnia than in the oil from Serbia means that perhaps a larger dose of oil was injected into the chromatographic column or the sensitivity of the apparatus was greater.

• Thank You for Your suggestion. We rearranged the whole paragraph, page 13, lines 300-304.

The discussion of the results is rather cursory in contrast to the correctly formulated conclusions in chapter 5.

• Thank You for Your suggestion. We rearranged chapter 5, Conclusions, with new paragraphs, page 14, 345-349, 354-357.

We corrected compound No 2 in Table 4

Note to reviewers: All corrected words and numbers through the text and tables were signed in blue.