Genetic Diversity and Population Structure of the Endangered Ulmusvillosa in Pakistan Revealed by DNA Barcode Markers

Round 1

Reviewer 1 Report

The study is quite interesting. The barcode marker technology shows potential for practical applications. Further studies could make the technology even more accurate. Overall, the work is very good. 

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

The study is quite interesting. The barcode marker technology shows potential for practical applications. Further studies could make the technology even more accurate. Overall, the work is very good. 

• We are thankful to reviewer 01, for considering the current study worth wile and interesting.

Author Response File: Author Response.pdf

Reviewer 2 Report

The present work entitled “Genetic diversity and population structure of the endangered species Ulmus in Pakistan using Barcode Markers” has represented the comprehensive analysis of Ulmus tree with five barcoding markers. Despite, a detailed phylogenetic and evolutionary analysis, authors failed to represent their findings adequately. Overall the manuscript is fine, but the major drawback of the manuscript is its readability and flow of manuscript. Authors should clearly mention the objective of their study, and the biological relevance of this study. The introduction section needs substantial editing, author should improve introduction section by incorporating relevant information. Authors should provide info about the markers used rbcL, matK XF+5R, matK 390F+1326R, 19 ITS and trnH-psbA (like what is the significance of using these markers). Also, the quality of figures is very poor, author should provide the better-quality figure. Authors should italicise the botanical name eg. Line no 310 Z. serrata and Z. schneideriana, authors should critically check the entire manuscript for such errors.

Author Response

Comments and Suggestions for Authors

The present work entitled “Genetic diversity and population structure of the endangered species Ulmus in Pakistan using Barcode Markers” has represented the comprehensive analysis of Ulmus tree with five barcoding markers. Despite, a detailed phylogenetic and evolutionary analysis, authors failed to represent their findings adequately. Overall, the manuscript is fine, but the major drawback of the manuscript is its readability and flow of manuscript. Authors should clearly mention the objective of their study, and the biological relevance of this study. The introduction section needs substantial editing, author should improve introduction section by incorporating relevant information. Authors should provide info about the markers used rbcL, matK XF+5R, matK 390F+1326R, 19 ITS and trnH-psbA (like what is the significance of using these markers). Also, the quality of figures is very poor, author should provide the better-quality figure. Authors should italicise the botanical name eg. Line no 310 Z. serrata and Z. schneideriana, authors should critically check the entire manuscript for such errors.

• To the best of our abilities, we have revised the whole MS for clarity of the contents and synchronous flow of information.
• Objectives of the study and relevance/importance of the study are mentioned both in the abstract as well as in the introduction section (Lines 21-25, 51-55 and 61-73)
• The introduction section is modified taking into account the Reviewer 2 comments/suggestions and details/significance of the barcoding genes added.
• The data was run once again, and high-quality/resolution figures obtained are added in the MS that replaced the earlier low-resolution figures.
• The MS is critically reviewed, and all scientific/botanical names are italicised in the revised version.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors of the reviewed manuscript examined the genetic diversity and population structure of two species in genus Ulmus sampled from different regions in Pakistan using barcode markers. Although of local interest, this study has the potential to attract readership due to the complex taxonomy of members of the genus Ulmus.

First of all, the authors should take for granted that Ulmus is a genus with several members; it is neither a species nor a tree. The title given to this study should be revised to include the two studied species.

The authors do not provide explicitly the IUCN extinction risk assessments designating a consolidated status for the species in decline dealt with in their study. However, later on in the results section of their manuscript, the authors mention that Ulmus wallichiana is a Critically Endangered species (red listed by IUCN; but unfortunately with no reference which should definitely be added) while no extinction risk status is indicated for the second species under study. The assessment of extinction risk status of species is exactly what designate them as threatened species –namely either Critically Endangered or Endangered or Vulnerable - and these assessments are critical for the purpose of the study presented in this manuscript. Therefore, IUCN extinction risk assessments of the studied species should actually be included in the introduction section and not in the results section, outlining why the studied species are considered threatened and what makes them prone to extinction. In the light of the above-mentioned, the title, the introduction as well as the first lines of the abstract of this manuscript should be revised accordingly. The manuscript will profit greatly from such an amendment.

Keywords: The authors should avoid repeating words that appear in the title.

I am afraid that the introduction part is rather too short and it actually fails to introduce the reader to the necessity or significance of this study. The authors are encouraged to further elaborate the introduction part and to develop a couple of paragraphs outlining in more detail the context of their study.

There are flaws in the material and methods section, especially in 2.1. The authors should clearly refer to the collection of the plant samples in the wild and they should provide more details about their sampling strategy (collection permit used, periods of sampling, selection of target regions with respect to the actual range of these species, sampling design, how many individuals were sampled, how they defined different populations in the wild, how plant taxonomic identification was performed, where they deposited voucher samples, etc.).

The legend of Figure 1 should be further elaborated and should present more details.

Table 1 needs better formatting and legend improvement.

In lines 93-94 of materials and methods, the authors refer to seed germination. However, there are no seed germination experiments in this study; probably this represents a self-plagiarism text from another study.

In the results section as well as in the discussion section, the authors should use past tense in a consistent way when referring to the results of this study.

Some lines from the results section should be transferred to materials and methods where they should be further elaborated (e.g., lines 106-107).

The extant texts in section 3.2 imply that different numbers of samples were used for the selected DNA-Barcoding regions; with no description in materials and methods and no justification in their texts, it is quite unclear why the numbers of samples differed. The authors should refer to exactly how many samples they used for each DNA-Barcoding regions in the material and methods of their manuscript.

Table 2 needs better formatting and legend improvement.

The Figures 2, 3 and 4 need better formatting (full landscape page) and legend improvement to be informative

The authors should try to explain how and why they selected specific threshold values in the analyses of sequences performed.

All Latin names, either genus names or species names, should appear in italics (even in the list of references) while the authorship of a scientific name of a given species should be added at the first mention of a specific taxon.

The names of all genes should be italicized as well.

Lines 205-206 (the last part of the sentence) belong to the discussion part and should be transferred there. The authors should further elaborate on this in their discussion.

Lines 218-219 present ambiguous meaning and should be further elaborated and/or rephrased.

The legends in figures 6-9 need improvement.

The discussion part is comparatively the best part of the reviewed manuscript but again several sentences with ambiguous meaning are detected; these are marked in yellow and are associated with a sticker note indicating the problem encountered (see pdf file).

The conclusions part needs further elaboration and development to support the significance of this study.

The list of references can certainly be enriched if the discussion part is further elaborated.

Last but not least, there are numerous linguistic imperfections that need rephrasing and correction; all these are indicated within the reviewed pdf file (see words or texts highlighted with yellow color without further comment).

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

The authors of the reviewed manuscript examined the genetic diversity and population structure of two species in genus Ulmus sampled from different regions in Pakistan using barcode markers. Although of local interest, this study has the potential to attract readership due to the complex taxonomy of members of the genus Ulmus.

First of all, the authors should take for granted that Ulmus is a genus with several members; it is neither a species nor a tree. The title given to this study should be revised to include the two studied species.

• Correction is made, and throughout the MS Ulmus is mentioned/considered as a genus. Further, the title of the MS is revised and includes the species discussed/studied.

The authors do not provide explicitly the IUCN extinction risk assessments designating a consolidated status for the species in decline dealt with in their study. However, later on in the results section of their manuscript, the authors mention that Ulmus wallichiana is a Critically Endangered species (red listed by IUCN; but unfortunately with no reference which should definitely be added) while no extinction risk status is indicated for the second species under study. The assessment of extinction risk status of species is exactly what designate them as threatened species –namely either Critically Endangered or Endangered or Vulnerable - and these assessments are critical for the purpose of the study presented in this manuscript. Therefore, IUCN extinction risk assessments of the studied species should actually be included in the introduction section and not in the results section, outlining why the studied species are considered threatened and what makes them prone to extinction.

 In the light of the above-mentioned, the title, the introduction as well as the first lines of the abstract of this manuscript should be revised accordingly. The manuscript will profit greatly from such an amendment.

• As per IUCN extinction risk assessment criteria carried out by Khan et al. (2021, DOI: http://dx.doi.org/10.30848/PJB2021-6(31), the species is classified as Endangered and as suggested these details are added in the introduction section (lines 44-46)

• As suggested the title, introduction, as well as abstract sections, have been revised accordingly and indeed this has improved the quality of our MS.

Keywords: The authors should avoid repeating words that appear in the title.

• Keywords are modified.

I am afraid that the introduction part is rather too short and it actually fails to introduce the reader to the necessity or significance of this study. The authors are encouraged to further elaborate the introduction part and to develop a couple of paragraphs outlining in more detail the context of their study.

• As suggested related information central to the scope of the MS are added in the introduction section (see paragraphs 43-55 and 62-73).

There are flaws in the material and methods section, especially in 2.1. The authors should clearly refer to the collection of the plant samples in the wild and they should provide more details about their sampling strategy (collection permit used, periods of sampling, selection of target regions with respect to the actual range of these species, sampling design, how many individuals were sampled, how they defined different populations in the wild, how plant taxonomic identification was performed, where they deposited voucher samples, etc.).

• As suggested details of permission to conduct the study, sampling strategy, individuals sampled, taxonomic identification and where the voucher samples are deposited is added in the M&M section (lines 76-.87)

The legend of Figure 1 should be further elaborated and should present more details.

Table 1 needs better formatting and legend improvement.

• Legend of Figure 1 is elaborated, and that of Table 1 is formatted and improved.

In lines 93-94 of materials and methods, the authors refer to seed germination. However, there are no seed germination experiments in this study; probably this represents a self-plagiarism text from another study.

• Text related to seed germination is deleted.

In the results section as well as in the discussion section, the authors should use past tense in a consistent way when referring to the results of this study.

• Unless present tense was required, past tense is consistently followed/used in both results and discussion sections.

Some lines from the results section should be transferred to materials and methods where they should be further elaborated (e.g., lines 106-107).

• Highlighted/suggested lines transferred into M&M section.

The extant texts in section 3.2 imply that different numbers of samples were used for the selected DNA-Barcoding regions; with no description in materials and methods and no justification in their texts, it is quite unclear why the numbers of samples differed. The authors should refer to exactly how many samples they used for each DNA-Barcoding regions in the material and methods of their manuscript.

• Although, 27 samples were PCR amplified and sent for commercial sequencing with these 04 barcoding regions (each). Sequencing results for all of these regions were not obtained. On the other hand, of the results obtained few sequences were too short to be included in the analyses. This information are discussed and given as lines 138-147.

Table 2 needs better formatting and legend improvement.

• Legend formatted and improved.

The Figures 2, 3 and 4 need better formatting (full landscape page) and legend improvement to be informative

• Figures 2-4 formatted and legend with additional information added.

The authors should try to explain how and why they selected specific threshold values in the analyses of sequences performed.

• The threshold was adopted from the literature survey.

All Latin names, either genus names or species names, should appear in italics (even in the list of references) while the authorship of a scientific name of a given species should be added at the first mention of a specific taxon.

• All scientific names are now italicised, and author citation is added for the species at first mention (lines 18 and 38).

The names of all genes should be italicized as well.

• Names of all genes are italicized

Lines 205-206 (the last part of the sentence) belong to the discussion part and should be transferred there. The authors should further elaborate on this in their discussion.

• Lines 205-205 shifted to discussion section and additional details added.

Lines 218-219 present ambiguous meaning and should be further elaborated and/or rephrased.

• Lines 218-219 are rephrased and now convey a clear message.

The legends in figures 6-9 need improvement.

• Legends of Figs. 6-9 are improved by adding additional information.

The discussion part is comparatively the best part of the reviewed manuscript but again several sentences with ambiguous meaning are detected; these are marked in yellow and are associated with a sticker note indicating the problem encountered (see pdf file).

• The discussion section is revised thoroughly and all elliptical sentences (or that were too long) are rephrased for clarity of the meaning (P13&14: lines 297-391).

The conclusion’s part needs further elaboration and development to support the significance of this study.

• The conclusion section is improved to support the significance of the study.

The list of references can certainly be enriched if the discussion part is further elaborated.

 Last but not least, there are numerous linguistic imperfections that need rephrasing and correction; all these are indicated within the reviewed pdf file (see words or texts highlighted with yellow color without further comment).

• With new/additional information added related references are added to the References section. All suggestions/highlighted texts of reviewer 3 are addressed. We are very much thankful to all the reviewers for their valuable inputs and suggestions that have markedly improved our MS and we are looking forward to a positive decision on our MS.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

The authors have greatly improved the quality of the submitted manuscript following the advice given by the reviewers.

However, there are still many details that need correction, especially scattered linguistic imperfections that require careful checking and editing. Most of these have been highlighted with sticker notes in the attached pdf file.

The abstract needs careful editing to avoid mistakes.

The introduction part needs some changes to be adjusted to the focus of this interesting study. All citation numbers of references should be re-enumerated after the second citation that was newly inserted in the manuscript. Consequently, all references after number 2, should be re-enumerated within the texts. 

The Materials and Methods and the Results sections should be carefully edited to eliminate several scattered mistakes within the texts. All scientific names at their first mention should be followed by the authorship of the scientific name (these are indicated with sticker notes in the pdf file). 

In the discussion part, several improvements have been made and now the texts read better, and the focus of this study is clear. However, since the authors have decided to remove all data regarding U. wallichiana from their manuscript, the claim they provide in lines 309-412 is not further supported. In this way, this part should be rephrased.

All the deletions made in the texts have greatly improved the manuscript, and the new texts inserted in the revised version of the manuscript are all necessary but they suffer from linguistic problems. Most of these have been corrected (see sticker notes).

The reference list needs careful checking and editing following the journal's guidelines. The authors should avoid capitalizing every word in the titles of the studies published in journals (see the journal's guidelines). Italics should be used in all scientific names (genus, species, subspecies) appearing in the titles of the studies mentioned in the reference list.

Despite these problems, the improved manuscript is appreciated. Thus, it may be considered for publication given that the authors will respond positively to the new amendments made in the attached pdf file (74 new comments) and the editorial team will carefully check that all the corrections suggested in the pdf file have been made by the authors.  

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

The authors have greatly improved the quality of the submitted manuscript following the advice given by the reviewers.

However, there are still many details that need correction, especially scattered linguistic imperfections that require careful checking and editing. Most of these have been highlighted with sticker notes in the attached pdf file.

The abstract needs careful editing to avoid mistakes.

The abstract is reviewed thoroughly, and all sticker notes of the pdf have been incorporated. We believe that the abstract is now conveying a much clearer message.

The introduction part needs some changes to be adjusted to the focus of this interesting study. All citation numbers of references should be re-enumerated after the second citation that was newly inserted in the manuscript. Consequently, all references after number 2, should be re-enumerated within the texts. 

The introduction section is reviewed, and only minor changes have been made, all sticker notes and a few typos were found and corrected. The order of references is changed/corrected.

The Materials and Methods and the Results sections should be carefully edited to eliminate several scattered mistakes within the texts. All scientific names at their first mention should be followed by the authorship of the scientific name (these are indicated with sticker notes in the pdf file). 

The M&M, as well as Results sections, are reviewed, and authorities have been added to the scientific names. We are very thankful for all the suggestions/help extended in the pdf file as sticker notes.

In the discussion part, several improvements have been made and now the texts read better, and the focus of this study is clear. However, since the authors have decided to remove all data regarding U. wallichiana from their manuscript, the claim they provide in lines 309-412 is not further supported. In this way, this part should be rephrased.

Information regarding diversity within U. villosa is independently provided and lines 309-412 are now peripheral to the scope of this study and are deleted.

All the deletions made in the texts have greatly improved the manuscript, and the new texts inserted in the revised version of the manuscript are all necessary, but they suffer from linguistic problems. Most of these have been corrected (see sticker notes).

On behalf of all authors, I thank the anonymous Reviewers and Editor/s for improving our MS to this level. Suggestions/corrections on the sticker notes were of tremendous importance and all have been incorporated in the MS.

The reference list needs careful checking and editing following the journal's guidelines. The authors should avoid capitalizing every word in the titles of the studies published in journals (see the journal's guidelines). Italics should be used in all scientific names (genus, species, subspecies) appearing in the titles of the studies mentioned in the reference list.

References have been double-checked, re-enumerated and all scientific names corrected (scientific names are in italics and species name now starts with small letters).

Despite these problems, the improved manuscript is appreciated. Thus, it may be considered for publication given that the authors will respond positively to the new amendments made in the attached pdf file (74 new comments) and the editorial team will carefully check that all the corrections suggested in the pdf file have been made by the authors.  

Thank you for your assessment and appreciation of our manuscript. We made all the necessary changes accordingly as suggested in the pdf file. Hopefully now the readers can easily understand it.

Author Response File: Author Response.pdf