Ecological, Anatomical, and Genomic Insights into the Rare Tree Species Fraxinus sogdiana, Celtis caucasica, and Betula jarmolenkoana from the Northern Tien Shan

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper titled “Ecological, anatomical, and genomic insights into rare tree spe- 2 cies of the Northern Tien Shan” offers valuable insights and is likely to capture the attention of researchers in the field. By integrating population structure analysis, morphological measurements, leaf anatomical studies, and comparative chloroplast genome analysis, this study provides multidimensional data that contribute to the understanding of the ecological adaptability and evolutionary history of these species. Furthermore, the adaptive characteristics and population dynamics of tree species across various habitats offer foundational data for future ecological monitoring, restoration efforts, and phylogenetic genomic research, which have significant implications for conservation and management. While the manuscript is straightforward, there are some areas that could benefit from further clarification and revision. Therefore, the authors are encouraged to carefully review the feedback provided below and make modifications accordingly：

• Title:“Ecological, anatomical, and genomic insights into rare tree spe- 2 cies of the Northern Tien Shan” should be rewritten as “Ecological, Anatomical, and Genomic Insights into Rare Tree Species of the Northern Tien Shan”.
• Intrduction（Line 44-60）:The author has selected three tree species listed in the *Red Book* for research, which is a commendable endeavor. However, I am particularly interested in gaining a deeper understanding of their specific circumstances, including survival crises, ecological and economic uses, and conservation measures.
• Line 94: Figure 1 is essential for the understanding of this study; however, the text within the figure is currently difficult to discern. To improve clarity, it is recommended to enhance the font size and contrast of the text, as well as to simplify the graphical elements to ensure that the information presented is easily readable and accessible to the audience.
• Line 102-104:Please specify whether a complete tree census or a sampling survey was conducted during the tree investigation.
• Line 143:The data analysis and discussion of results in the article are somewhat lengthy, potentially impacting the reader's experience and comprehension. It is recommended to refine and optimize certain analyses within the same category for clarity and conciseness.
• Line 144: To more scientifically demonstrate the distribution characteristics of the three tree species, a current satellite image should be provided, along with the determination of their distribution boundaries.
• Line 171:It is well established that, in addition to quantity, tree height, and leaf size, morphological characteristics of trees—such as diameter at breast height (DBH), crown width, height to the first branch, and stem form—are also critically important. Furthermore, when classifying tree species by age, the space-for-time substitution method can be utilized, wherein the diameter at breast height assumes particular significance. The author is encouraged to supplement the analysis with additional indicators, including DBH and crown width.
• Line 236:Although the research has revealed the ecological and anatomical characteristics of these tree species, the specific recommendations for their conservation measures are rather general, lacking targeted management strategies and implementation plans.
• Line 302:The clarity of Figures 8 to 10 must be improved.
• Discussion:In addition to phenotypic traits, it is crucial to analyze the molecular mechanisms, nutrient utilization strategies, and environmental effects, such as soil conditions, that contribute to the survival strategies of plants. I recommend that the authors discuss these aspects and incorporate them into a new research plan, as this will enhance the conservation and application of tree species.
• In preparing your academic paper for submission, it is essential to ensure that all citations adhere to the latest formatting standards. This includes the abbreviation of journal titles and the correct formatting of references based on recent publications. Please review the citation format of each reference at the end of your text, adjusting them to comply with the current guidelines. This will enhance the professionalism and academic rigor of your work.

Author Response

Response to Reviewer

We thank the reviewer for their thoughtful evaluation and constructive suggestions, which have helped us improve the clarity, depth, and rigor of the manuscript. Below, we address each comment in detail:

• Title correction

Comment: “Ecological, anatomical, and genomic insights into rare tree species of the Northern Tien Shan” should be rewritten as “Ecological, Anatomical, and Genomic Insights into Rare Tree Species of the Northern Tien Shan”.

Response: We acknowledge your suggestion regarding the title. In line with a similar recommendation from another reviewer, we have revised the title accordingly. The updated title is:
“Ecological, Anatomical, and Genomic Insights into the Rare Tree Species Fraxinus sogdiana, Celtis caucasica and Betula jarmolenkoana from the Northern Tien Shan”

• Introduction（Line 44-60）:The author has selected three tree species listed in the *Red Book* for research, which is a commendable endeavor. However, I am particularly interested in gaining a deeper understanding of their specific circumstances, including survival crises, ecological and economic uses, and conservation measures.

Response: We have expanded the Introduction to include details on the ecological roles, conservation status, and specific threats faced by each species, such as grazing pressure, habitat degradation, and fire risk. Where applicable, we also noted ethnobotanical and ecological functions (e.g., riverbank stabilization, succession support).

• Line 94: Figure 1 is essential for the understanding of this study; however, the text within the figure is currently difficult to discern. To improve clarity, it is recommended to enhance the font size and contrast of the text, as well as to simplify the graphical elements to ensure that the information presented is easily readable and accessible to the audience.

Response: We have updated Figure 1 to enhance font size, contrast, and overall readability as recommended.

• Line 102-104 Please specify whether a complete tree census or a sampling survey was conducted during the tree investigation.:

Response: We conducted a sampling survey, not a complete tree census. Sample plots of varying sizes (25 × 100 m, 50 × 50 m, and 100 × 100 m) were established within the natural populations of each species. Within these plots, we recorded the number of individuals by age class, together with morphological traits and corresponding habitat characteristics. This approach allowed us to assess population structure and regeneration dynamics while accounting for the spatial heterogeneity of the habitats.

• Line 143:The data analysis and discussion of results in the article are somewhat lengthy, potentially impacting the reader's experience and comprehension. It is recommended to refine and optimize certain analyses within the same category for clarity and conciseness.

Response: Thank you for this valuable observation. We acknowledge the length of the analysis and discussion sections; however, we consider the current level of detail essential for accurately conveying the complexity of the multi-faceted ecological, anatomical, and genomic data presented in this study. To maintain clarity while preserving analytical integrity, we have retained the full analyses but ensured that the sections are well-structured and clearly organized to facilitate reader comprehension. We remain open to further editorial suggestions regarding specific areas that may benefit from condensation.

• Line 144: To more scientifically demonstrate the distribution characteristics of the three tree species, a current satellite image should be provided, along with the determination of their distribution boundaries.

Response: We agree that a satellite image would enhance the visual presentation of species distribution. However, currently available satellite imagery for our study sites lacks the spatial resolution necessary to clearly delineate the narrow and fragmented ranges of these species.

• Line 171:It is well established that, in addition to quantity, tree height, and leaf size, morphological characteristics of trees—such as diameter at breast height (DBH), crown width, height to the first branch, and stem form—are also critically important. Furthermore, when classifying tree species by age, the space-for-time substitution method can be utilized, wherein the diameter at breast height assumes particular significance. The author is encouraged to supplement the analysis with additional indicators, including DBH and crown width.

Response: Thank you for your insightful comment. In the present study, our morphological analysis was limited to number of trees, height, and leaf size. A more detailed assessment of morphological traits such as diameter at breast height (DBH), crown width, height to the first branch, and stem form was beyond the scope of this work and will be considered in future research

• Line 236:Although the research has revealed the ecological and anatomical characteristics of these tree species, the specific recommendations for their conservation measures are rather general, lacking targeted management strategies and implementation plans.

Response: Thank you for your valuable comment. In response, we have revised the Discussion section to include more specific conservation recommendations. These include habitat restoration, regulation of grazing, reinforcement planting with local genotypes, long-term population monitoring, and complementary ex situ measures such as seed banking and propagation under controlled conditions to preserve genetic diversity and support future reintroduction efforts.

• Line 302:The clarity of Figures 8 to 10 must be improved.

Response: We thank the reviewer for pointing this out. Figures 8 to 10 have been revised to improve clarity.

• Discussion:In addition to phenotypic traits, it is crucial to analyze the molecular mechanisms, nutrient utilization strategies, and environmental effects, such as soil conditions, that contribute to the survival strategies of plants. I recommend that the authors discuss these aspects and incorporate them into a new research plan, as this will enhance the conservation and application of tree species.

Response: Thank you for your valuable suggestion. We agree that investigating molecular mechanisms, nutrient utilization strategies, and the influence of environmental factors such as soil conditions would provide a more comprehensive understanding of the survival strategies of these rare tree species. Although these aspects were beyond the scope of the present study, we fully recognize their importance and intend to incorporate them into future research plans. Accordingly, we have added a sentence to the Discussion section to emphasize the significance of these factors and their relevance for future research aimed at enhancing conservation strategies.

We have reviewed and revised all references to ensure correct formatting and standardized journal title abbreviations in accordance with the current journal guidelines. We thank the reviewer again for their thorough and constructive comments. We believe the revisions have significantly improved the manuscript and we hope it now meets the standards expected by the journal.

 Reviewer 2 Report

Comments and Suggestions for Authors

Review of Sadyrova et al., trees of N Tien Shan

This article, taking a multifaceted look at three tree species in Kazakhstan,  reflects a fascinating and substantial effort to characterize the three species in question.  The field work to visit each species was not doubt considerable, and the use of a freezing microtome was a great idea, not applied often for plant structure.   I very much enjoyed reading this project and hope it moves forward. My concerns are not at all intended to throw cold water.   Just the opposite---I think this paper needs some work, but that is manageable. My suggestions are meant to encourage the authors who have accomplished much already.

The main overall concern is the overall style of the writing, which can be adjusted comfortably in revision.   There are several places where wording seems over-stated, or unduly subjective, overly declarative,  or inadequately documented, or comparative with little basis for comparison.   As examples, lines 156, 197, 223-224, 337, 338-340,  343-346, 363, 415, 422.    Much of this has to do with how ecologically sensitive or environmentally important the three trees are.   The paper would gain strength if these themes were well buttressed with contextual elaboration: the species full distributions,  known altitudinal or other limitations,  known specific  (vs. general or speculative) threats,  cultural or commercial uses and exploitations,  proximity to human-caused perils, actual known habitat destruction (or fires, or pollution, or specific grazers, etc.) , policy issues, pathologies, known habitat changes, protections in place, horticultural uses.   How and why are these species endangered and critical to their environments?   Line 46 could be expanded into a paper in itself rather than merely citation [8].   What urgent protection measures are needed?   Regional? Species-specific?     Such data would bolster the paper richly.

A few minor suggestions:

Put the tree species names in the title?

Add more substantial taxonomic references.  References  16 and 17 are derived and not enough.   For example, there is a recent taxonomic revision of Betula (Authors: Kenneth Ashburner, Hugh McAllister.  ISBN 9781842461419. Kew Publishing, reprinted 2016.) in which Betula jarmolenkoana is reduced to synonymy with B. tianschanica.   That does not necessarily mean you can’t use the name you did, but the taxonomic issue should be addressed and cited.   Taxonomy sheds light on endangered species. 

If the fieldwork generated herbarium specimens these should be cited.

Not important, but several of the figures or their legends seem inconveniently small.

Small botanical details: line 149 (leaflets instead of leaves), Fig. 3B (Celtis does not have compound leaves), line 193 (how was age determined)?  

Line 355:   Could the absence of seedlings be seasonal, or merely localized, given that the observed population was so small?   They may tend to establish away from the parent population where succession has changes conditions.   Is the problem at that exact site, or broader?  Line 375, if circumstances permit. It might be interesting to look for starch in wood rays. Top of p. 13:  can you find data in literature relevant to these features at the generic/family levels?   For instance, do birches in general tend toward inclusions in mesophyll cells?  

Tables:   The standard deviations to two decimal places seem excessively “precise,” given the sample sizes and the purposes of the data.   How were tree heights determined…isn’t that usually an estimate far less precise than the tables imply?   Line 228: 13 m disagrees with Table 3.

Is “virginal” a common term in forest trees?    The distinctions between the classes in the tables are not clear….why not just give size classes for pre-fertile individuals? 

Typo help:  214 (italics), 237 (capital S)

Hope this helps.  I suggest fairly painless rewriting  with tightening up all the points based on speculation or that can be interpreted as over-stated or inadequately substantiated.    Also suggested is going back to the literature and digging in far deeper on the details for each species, especially the ones that have to do with distributions, current taxonomies, specific immediate localized ecological threats , and relevant reproductive biology.  Such effort could be enjoyable and eye-opening, as well as open doors to further insights & projects.  It must be a joy to study field botany in Kazakhstan!

Author Response

Response to Reviewer Comments

We sincerely thank the reviewer for the thoughtful and encouraging feedback. We greatly appreciate the recognition of our efforts in fieldwork and microscopy, as well as the constructive suggestions aimed at improving the scientific quality and clarity of the manuscript. We have carefully addressed each point below, with changes reflected in the revised version of the manuscript.

Reviewer comments: There are several places where wording seems over-stated, or unduly subjective, overly declarative, or inadequately documented, or comparative with little basis for comparison.   As examples, lines 156, 197, 223-224, 337, 338-340,  343-346, 363, 415, 422.    Much of this has to do with how ecologically sensitive or environmentally important the three trees are.   The paper would gain strength if these themes were well buttressed with contextual elaboration: the species full distributions,  known altitudinal or other limitations,  known specific  (vs. general or speculative) threats,  cultural or commercial uses and exploitations,  proximity to human-caused perils, actual known habitat destruction (or fires, or pollution, or specific grazers, etc.) , policy issues, pathologies, known habitat changes, protections in place, horticultural uses.   How and why are these species endangered and critical to their environments?   Line 46 could be expanded into a paper in itself rather than merely citation [8].   What urgent protection measures are needed?   Regional? Species-specific?     Such data would bolster the paper richly.

Response: We fully acknowledge that parts of the original manuscript included generalized or declarative statements about ecological vulnerability and conservation importance that required greater documentation and nuance. In response, we thoroughly revised the manuscript to eliminate overly declarative or speculative language, particularly in the sections identified (e.g., lines 156, 197, 223–224, 337–346, 363, 415, 422 in the original version). Wherever possible, we replaced generalizations with evidence-supported statements and included citations from regional floras, conservation assessments, and peer-reviewed literature. We have revised the text to reduce overly declarative language and added specific, well-documented information on each species’ distribution, documented threats (e.g., grazing, fire, tourism), conservation status, and existing protections. We also expanded Line 46 to explain the Red Book listing and the urgency of implementing targeted conservation measures. These revisions clarify why the species are endangered and emphasize their ecological importance, directly addressing the reviewer’s concern and improving the manuscript’s overall quality.

Reviewer comment: Put the tree species names in the title

Response: Thank you for your helpful suggestion. We agree that including the species names adds clarity and specificity to the title. Accordingly, we have revised the title to:
“Ecological, Anatomical, and Genomic Insights into the Rare Tree Species Fraxinus sogdiana, Celtis caucasica, and Betula jarmolenkoana from the Northern Tien Shan.”
We hope this updated title more accurately reflects the focus of our study.

Reviewer comment: Add more substantial taxonomic references.  References  16 and 17 are derived and not enough.   For example, there is a recent taxonomic revision of Betula (Authors: Kenneth Ashburner, Hugh McAllister.  ISBN 9781842461419. Kew Publishing, reprinted 2016.) in which Betula jarmolenkoana is reduced to synonymy with B. tianschanica.   That does not necessarily mean you can’t use the name you did, but the taxonomic issue should be addressed and cited.   Taxonomy sheds light on endangered species. 

Response: Thank you for the suggestion. We have added a citation to the taxonomic revision by Ashburner & McAllister (2016), which treats Betula jarmolenkoana as a synonym of B. tianschanica. While we acknowledge this, we retain the original name due to its widespread use in regional conservation literature.

Reviewer comment: If the fieldwork generated herbarium specimens these should be cited.

Response: We appreciate the reviewer’s comment. Herbarium specimens of the studied species were indeed collected; however, they were used exclusively for morphological, anatomical, and molecular-genetic analyses and were not preserved as permanent herbarium vouchers. Therefore, they are not cited as deposited specimens in this manuscript.

Reviewer comment: Not important, but several of the figures or their legends seem inconveniently small.

Response: We have reviewed all figures and legends for size and readability. Where necessary, we have increased the font size in figure legends and adjusted the layout and resolution to ensure that all elements are clear and conveniently legible for the reader.

Reviewer comment: Small botanical details: line 149 (leaflets instead of leaves), Fig. 3B (Celtis does not have compound leaves), line 193 (how was age determined)?  

Response: Thank you for your careful attention to detail.

• Line 149: We appreciate the correction and have revised the text to refer to “leaves” instead of “leaflets”, as Celtis caucasica possesses simple, not compound, leaves.
• Figure 3B: Thank you for pointing this out. We have corrected the figure legend to accurately describe the illustrated structure as a simple leaf with serrated margins, consistent with the morphology of C. caucasica.
• Line 193: Age classification was based on observable morphological traits such as stem diameter, height, presence or absence of reproductive structures, and crown architecture. These traits were used to assign individuals to developmental stages (juvenile, immature, virginile, generative, etc.) according to established criteria used in forest-ecological studies. A corresponding explanatory paragraph has been added to the Materials and Methods section to clarify this procedure.

Reviewer comment: Line 355:   Could the absence of seedlings be seasonal, or merely localized, given that the observed population was so small?   They may tend to establish away from the parent population where succession has changes conditions.   Is the problem at that exact site, or broader?  Line 375, if circumstances permit. It might be interesting to look for starch in wood rays. Top of p. 13:  can you find data in literature relevant to these features at the generic/family levels?   For instance, do birches in general tend toward inclusions in mesophyll cells?  

Response: Thank you for this interesting suggestion. We agree that seedling absence could be influenced by seasonal or localized factors, particularly in small or fragmented populations. However, our field surveys were conducted over multiple sites and seasons, and the lack of juvenile individuals was consistent across locations, suggesting a broader pattern of regeneration failure rather than a site-specific anomaly.  We agree that investigating starch accumulation in wood rays could yield valuable insights into the species’ physiological adaptations. However, this aspect was beyond the scope of the current study and would require additional sampling and methodological setup. We appreciate the recommendation and will consider it for future research directions. We have supplemented the anatomical descriptions of all three species with literature-based comparisons at the genus and family levels.

Reviewer comment: Tables:   The standard deviations to two decimal places seem excessively “precise,” given the sample sizes and the purposes of the data.   How were tree heights determined…isn’t that usually an estimate far less precise than the tables imply?   Line 228: 13 m disagrees with Table 3.

Response: We appreciate the reviewer’s comment and fully agree that measurement precision must correspond to the actual accuracy of the methods used. While tree heights were estimated using a forestry altimeter based on angular measurements and distances, morphometric values (such as leaf length and plant height) were recorded using calibrated instruments under controlled conditions. To preserve consistency across datasets and to allow meaningful comparisons of variability between developmental stages, standard deviations were reported to two decimal places. We have revised the text to clarify that the height values presented in Table 3 reflect the average morphometric measurements for each developmental stage, while the mention of individuals reaching up to 13 m refers to exceptional field observations of mature trees. This distinction has now been made explicit in the revised manuscript to avoid confusion.

Reviewer comment: Is “virginal” a common term in forest trees?    The distinctions between the classes in the tables are not clear….why not just give size classes for pre-fertile individuals? 

Response: We acknowledge that the term “virginal” (now corrected to “virginile”) may not be commonly used in international forestry literature. However, it is a recognized ontogenetic category in forest-ecological and geobotanical studies across Central Asia and Eastern Europe, particularly in long-term demographic assessments of woody species. The virginile stage refers to mature, pre-reproductive individuals that have reached the potential for generative development but were not observed flowering or fruiting during the study period. This category holds ecological significance in understanding delayed reproduction, regeneration potential, and age structure stability—especially in long-lived species such as Betula jarmolenkoana. We chose to retain ontogenetic classifications rather than use generalized size classes because they more accurately reflect developmental progression and reproductive status, which are critical for assessing population viability. To improve clarity, we have now provided explicit definitions of each age class in the Materials and Methods section and ensured consistent terminology in the tables and figure captions.

Reviewer comment: Line 214 (italics), Line 237 (capital S)

Response: These typographical issues have been corrected in the revised manuscript.

Once again, we express our gratitude for the reviewer’s thoughtful evaluation and supportive tone. We have made substantial efforts to revise the manuscript according to the suggestions, and we believe the paper is now significantly improved both in content and clarity. We hope it will now meet the expectations of the journal.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the author's revisions and responses. The quality of the revised manuscript has significantly improved. However, further optimization is still required in terms of formatting and layout according to the editorial department's requirements, particularly regarding the citation format of references at the end of the article. In conclusion, I am very interested in this research and wish the author success in publishing these findings.

Reviewer 2 Report

Comments and Suggestions for Authors

I reviewed the authors' responses to my prior review.  It appears to me that they have addressed the concerns thoughtfully and effectively, and that the ms. is ready to move forward.