Mixed Mating System, Dispersal Limitation Shape, and Spatial Genetic Structure of Tamarix chinensis on Isolated Wudi Seashell Island
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsReview of diversity-3548443, Long-distance Gene Flow and Significant Spatial Genetic Structure In A Natural Population of Tamarix chinensis Lour. in the Shell Dike Island region of Shandong, China.
The study by Zhang et al. on Tamarix is an interesting work, using adequate molecular data and both standard and some advanced software analysis packages. The introduction lacks some relevant concepts about the molecular type of data chosen, and the conclusions are poor. It is suggested a Major Revision, addressing specifically the following points:
Introduction. Please add short, informative paragraphs about the markers used, such as “SSR markers are used in population genetic diversity studies because of….”; also, there is no concrete information about the SSR primers, which are usually more difficult to generate or to adapt for use from related taxa. A reference would be necessary. There is also no comment on the software packages Cervus, AIS, and MLTR, which are not so frequent as POPGENE or GenALEx.
Geographic scope. There are some inconsistencies regarding the description of the place where the study has been carried out. In line 68, it is said “…Shell Dike Island and Wetland National Nature Reserve in Wudi County, Binzhou, Shandong Province, extending from Wangzi Island to the coastal area of the Dakou River.” But in the caption to figure 1, line 88, it says “Wudi Seashell Islands in Shandong” (plural, implying many islands). In line 251, it is mentioned “Wudi Shell Island” (a single island). In lines 291 and 311, it says “Shell Dike Islands”. Most probably all those references are pointing to the same place or places, but to non-chinese readers, it could be confusing. Besides, the figure 1 shows islands that are not visible in Google maps.
Material and Methods. It is clear that PCR amplifications result in binary data matrices (presence-absence). It is not clear how the analyses of those two markers ISSR (dominant) and SSR (co-dominant) could be integrated. Please clarify. Related to the paternity analysis, it is also not clear the data on which the Cervus, MLTR, and SPAgeDi analyses were done. Please explain.
It is not clear how “..all Tamarix chinensis individuals..” were sampled (line 78) and these are only 109 individuals in a surface of 28 hectares? (line 79). That would imply very low density, very few plants in such a big surface. Is that correct? Also, data on average height, etc. of plants should be mentioned and discussed not in this section (material and methods) but logically, in results and discussion.
Results. There are subjects that are not mentioned in the introduction, such as breeding, paternity analysis, size and age of plants that are mentioned here. A brief mention in the introduction pointing at the relevance of these for the whole study would be necessary.
The study is supposedly done on Tamarix chinensis (lines 11, 23, 27). However in line 250 it says “.. to investigate the spatial genetic structure of the Tamarix ramosissima population…” . These two are different species. Clarify: T. chinensis or ramosissima? Besides, in lines 262-268, it says “Tamarix”, as implying the genus. Are you expanding the analysis/conclusions to the whole genus? Clarify.
Discussion. The concept of lines 289-291 on lower genetic diversity in isolated islands has no reference supporting it.
You have two sets of results on all genetic diversity indices, and this could provide a more detailed picture of the genetic diversity. However, a proper discussion, comparing you results with similar cases is lacking. Are the two sets of results telling the same evolutionary history?
It is a bit confusing that it is written that maximum distances of dispersal are 948 m for seeds (line 319), and 447 for pollen (line 334), and that this would consequence of insects pollination by aphid flies and bees (line 338). However, a bit further on lines 342-343, it says that the pollen flow is similar to other wind-pollinated species. What is the conclusion?
Conclusions. The concept of lines 366-367 is highly speculative. How do you relate the genetic diversity to the degree of salinity of water? What specific test or analysis has been carried out to test this?
Author Response
Comment 1: Introduction. Please add short, informative paragraphs about the markers used, such as “SSR markers are used in population genetic diversity studies because of….”; also, there is no concrete information about the SSR primers, which are usually more difficult to generate or to adapt for use from related taxa. A reference would be necessary. There is also no comment on the software packages Cervus, AIS, and MLTR, which are not so frequent as POPGENE or GenALEx.
Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the Introduction section to provided a brief introduction for the SSR and ISSR primers used in our study (line 37-45). Additionally, we have supplemented the "Materials and Methods" section with brief explanations of the software packages Cervus, AIS, and MLTR to clarify their functions and justify their use.
Comment 2: Geographic scope. There are some inconsistencies regarding the description of the place where the study has been carried out... "Wudi Shell Island" (a single island), "Shell Dike Islands" (plural), etc. Clarify.
Response 2: Thank you for your careful observation. All the names refer to the same geographic region within the Wudi Shell Dike Island and Wetland National Nature Reserve. We have now standardized the terminology throughout the manuscript to "Wudi Seashell Island" for consistency. Figure 1 caption and all textual mentions have been corrected accordingly.
Comment 3: Material and Methods. Clarify how analyses of ISSR (dominant) and SSR (co-dominant) markers were integrated. Explain the data used for Cervus, MLTR, and SPAGeDi analyses.
Response 3: Thank you for raising this important point. We have clarified in the "Materials and Methods" section that ISSR and SSR data were analyzed separately and not statistically integrated (line 119-167). ISSR was used primarily for genetic diversity assessment, while SSR was employed for both diversity and spatial genetic analyses, including paternity and mating system evaluation. Data for Cervus and MLTR were based on SSR multilocus genotypes of 109 individuals. SPAGeDi analyses used SSR genotypic data to compute pairwise kinship coefficients.In addition, spatial genetic autocorrelation was evaluated using both SSR and ISSR datasets to capture patterns from both codominant and dominant markers.
Comment 4: Sampling. Clarify what "all individuals" refers to, and whether 109 individuals over 28 hectares is correct. Include height data in the results instead of methods.
Response 4: Thank you for the suggestion. We clarified that the 109 individuals represent all accessible and visually distinguishable T. chinensis individuals within continuously distributed patches in the 28-hectare area. We have moved the height, canopy width, and clump data to the Results section and cited it accordingly(Supplyment Table S2).
Comment 5: Results. Some topics like breeding, paternity analysis, and plant size appear only in results. Add brief mention in Introduction.
Response 5: Thank you for your helpful suggestion. We have added a paragraph in the Introduction (53-60) mentioning the relevance of studying the mating system, seed/pollen dispersal, and plant size to better understand fine-scale genetic structure and population resilience.
Comment 6: Clarify species: T. chinensis or T. ramosissima? Are you discussing the genus Tamarix?
Response 6: We appreciate your careful reading. The species studied throughout is Tamarix chinensis. The mention of Tamarix ramosissima on line 250 was a typographical error and has been corrected. Mentions of "Tamarix" (genus level) were used only in general comparisons with related species and have been revised for clarity.
Comment 7: Discussion. Line 289-291 on lower genetic diversity in isolated islands has no reference. Please support it.
Response 7: Thank you. We have added references supporting the general observation that isolated island populations often exhibit lower genetic diversity due to reduced gene flow and population size.
Comment 8: Please provide comparative discussion of the two marker systems. Are they telling the same evolutionary story?
Response 8: Thank you for this excellent suggestion. We added a dedicated paragraph in the Discussion (line 318-320) comparing ISSR and SSR results, noting that both marker systems reveal high diversity but that SSRs provide greater resolution for structure and gene flow analyses due to co-dominance and higher allelic richness.
Comment 9: Clarify inconsistencies about pollen dispersal. Is it insect-pollinated or wind-pollinated? What is the conclusion?
Response 9: Thank you. We clarified that T. chinensis is primarily insect-pollinated, but the sparsity of mates and open habitat may allow pollen to travel further, mimicking wind-dispersed patterns. This is now clearly explained in the Discussion.
Comment 10: Line 366-367 conclusion on salinity and diversity is speculative. Was it tested?
Response 10: We agree and have revised the conclusion to remove unsupported speculation. We now state that while high genetic diversity was observed, its relationship with salinity requires further study.
Reviewer 2 Report
Comments and Suggestions for AuthorsIn the submitted manuscript, Zhang et al. studied the genetic structure of the Tamarix chinensis population using ISSR and SSR molecular markers. The results can be used to study the evolutionary mechanisms and the development of strategies for the sustainable management and conservation of T. chinensis. There are a number of comments on the manuscript.
After the first mention in the text, please write T. chinensis.
L. 11, 14, 20, etc. T. chinensis should be written in italics. Please check the entire text.
L. 40-47. The mechanisms of T. chinensis stress tolerance are not related to the topic of the study.
L. 48-53. The study of T. chinensis populations using molecular markers should be described in detail.
How was the seed dispersal and pollen flow distance estimated (L. 230-244).
L. 250. Tamarix ramosissima was also studied? It is mentioned only once in the manuscript.
L. 223 and L. 248. The two tables are designated as Table 4.
There are no references to Tables 3, 4, 5 in the text.
There is no reference to Fig. 1 in the text.
Fig. 2. What do Figures “a” and “b” mean?
Fig. 3. What do Figures “b” and “c” mean?
Fig. 1 and Fig. 3a are the same. The latter should be deleted.
Ref. 14. The reference should be given in full.
Ref. 21, 22. Authors, volume and page numbers are missing.
Author Response
Comment 1: After the first mention in the text, please write T. chinensis.
Response 1: Thank you. We have checked the entire manuscript and corrected all instances of "T. chinensis" to be in italics where appropriate. The first full mention remains "Tamarix chinensis Lour.", followed by T. chinensis in subsequent mentions(line 28).
Comment 2: L. 11, 14, 20, etc. T. chinensis should be written in italics. Please check the entire text.
Response 2: We appreciate this observation. All occurrences of T. chinensis have been reviewed and corrected to be in italics throughout the manuscript.
Comment 3: L. 40–47. The mechanisms of T. chinensis stress tolerance are not related to the topic of the study.
Response 3: Thank you for the suggestion. We agree that this detail was not essential to the study's focus. This section has now been revised to briefly mention T. chinensis's ecological importance without delving into stress mechanisms.
Comment 4: L. 48–53. The study of T. chinensis populations using molecular markers should be described in detail.
Response 4: Thank you. We have expanded this section to better summarize previous work on the population genetics of T. chinensis using SSR, RAPD, ISSR, and SNP markers, including citation of relevant studies(line 37-45).
Comment 5: How was the seed dispersal and pollen flow distance estimated (L. 230–244).
Response 5: We have added detailed clarification on the methodology used to estimate seed and pollen dispersal in the "Materials and Methods" section (Lines 132–155), including the use of parentage analysis with Cervus and distance measurements based on spatial mapping.
Comment 6: L. 250. Tamarix ramosissima was also studied? It is mentioned only once in the manuscript.
Response 6: This was a typographical error. The species studied was Tamarix chinensis throughout. The mention of T. ramosissima has been corrected.
Comment 7: L. 223 and L. 248. The two tables are designated as Table 4.
Response 7: Thank you for noting this. The tables have been renumbered accordingly to avoid duplication and ensure consistency throughout the manuscript.
Comment 8: There are no references to Tables 3, 4, 5 in the text.
Response 8: We have added appropriate references to Tables 3, 4, and 5 in the Results and Discussion sections where the data is described.
Comment 9: There is no reference to Fig. 1 in the text.
Response 9: We have now referenced Figure 1 in the "Study Area and Sampling" section (line 71) to indicate the location of the study.
Comment 10: Fig. 2. What do Figures “a” and “b” mean?
Response 10: Figure 2 has been revised to include labels and updated captions: (a) Seed Dispersal Distances; (b) Pollen Flow Distances.
Comment 11: Fig. 3. What do Figures “b” and “c” mean?
Response 11: Thank you for the suggestion. In the revised manuscript, the original Figure 3 has been split into two figures (now Figure 3 and Figure 4) to improve clarity. The captions for both figures have been rewritten to provide detailed descriptions of all panels, including the kinship coefficient distance classes, genetic interpolation map, and spatial autocorrelation analysis results. These revisions help readers interpret spatial genetic structure patterns more accurately.
Comment 12: Fig. 1 and Fig. 3a are the same. The latter should be deleted.
Response 12: Thank you for pointing this out. In the revised manuscript, Figure 1 has been redesigned to clearly represent the geographic features of Wudi Seashell Island and its sampling layout. Figure 3a, which shows the spatial kinship coefficient distribution, has been retained as it represents analytical results distinct from the geographic map. Both figures now serve different purposes and have been updated and captioned accordingly to avoid confusion.
Comment 13: Ref. 14. The reference should be given in full.
Response 13: Reference 14 has been corrected and now includes the complete author names, journal title, volume, and page numbers.
Comment 14: Ref. 21, 22. Authors, volume, and page numbers are missing.
Response 14: Thank you. Full bibliographic information for References 21 and 22 has now been provided.
Reviewer 3 Report
Comments and Suggestions for AuthorsIn the reviewed MS a group of scientitsts from China investigated genetical the population of Tamarix chinensis in Shell Dike Island in China. The authors sampled 109 plants, extracted DNA and explored the genetic structure of the targeted plant based on PCR using a series of SSR and ISSR primers. The MS is too long. Some parts of the text needs serious revisions. The figures and caption needs revision. The Discussion is too long, it could be shorter 3 times. The authors are requested to give there genera conclusions and avoid repetition of their results in the section Discussion. There are many statements in the MS which need references. The MS need minor to moderate linguistic polishing. A list of all abbreviations is needed. The authors are also requested to give the photographs of the gels and their row data as supplementary files. Currently there are no Supplement. Some additional remarks are given below.
11,27 Tamarix chinensis Lour. (T. chinensis), - remove repetition
13 This study focuses on … reproductive system of T. chinensis – this was not investigated within the reported study. Please, remove this
19-20 These findings provide critical insights into the genetic resilience of T. chinensis and emphasize the need for conservation strategies to maintain genetic connectivity and adapt to environmental changes. – This statement is very broad and uncertain. Please, say more distinctly, what you mean exactly.
33 as observed in the Laizhou Bay region – redundant
37 such as Ebinur Lake Wetland – redundant. If you want to mention geography, you need to rewrite the text
46 Yellow River Delta – same comment as for 37
50 Phylogeographic analyses further suggest that T. chinensis has a widespread distribution… - this sentence needs revision because widespread distribution of T.chinensis is evident without and phylogeographic studies
57 please, provide references or, if you mean the same papers as cited above, combine these ideas in one sentence and do not disperse them in different part of Introduction
62 evolutionary mechanisms and environmental factors – these two aspects (mechanisms and factors) were not actually investigated; please revise this sentence since it sounds as overgeneralization
63-65 it is not clear what exactly is proposed to do based on the achievements of this study in terms of “strategies for the sustainable management and conservation of T. chinensis”. Please, explain this or remove from the text. Please, also address the fact that your study is based on a very restricted area within the distribution area of T.chinensis. Please, also explain how you plan to generalize your results on other areas.
83 do you give values for adult plants or for seedlings as well? Please, specify this
Figure 1. Please revise the figure in order it is clear that the investigated area is an island. It is ot evident now. Please, also explain better the right part of the Figure, it is also not correlated with the statement about “island”
91 please explain which plant parts were used for DNA extraction
94 please provide data for the total DNA concentrations measured for different samples
104 to amplify 109 Tamarix chinensis individuals(13) – it is not clear what the reference 13 means and why did you need additional primers. Please explain this. Additionally, please give a short data on ISSR and SSR primers and very briefly explain their usage for unprepared readers.
108 conditions followed the literature – revise this
127 Tamarix chinensis – italic, check in the entire text
148 spatial – Spatial
149 SGS – please, give a list of all abbreviations, used in this study
152 6.5(16).Mean – add a space
153 20,75,200,and – add spaces
168 s an excellent software for such analyses. – redundant
206, 208, 225-228 tm – ts – revise (use lower case symbols)
225-228 – include this text in the caption of the Table 4
233-244 please, explain how these distances were calculated/measured. It is not currently clear from the text. Please, add the methodology for this in the section M and Methods
250 Tamarix ramosissima - ???
Figure 3 needs better explanation. Please, provide detailed caption
271-273 please provide a reference for “as indicated by the normal L-shaped allele frequency distribution and the lack of significant deviations under IAM and SMM models.”
276 please provide a reference for “may hint at localized demographic fluctuations or past environmental pressures”
280 “The relatively small effective population size indicates that the population may be susceptible to genetic drift, especially under continued environmental or anthropogenic pressures. However, the absence of significant bottleneck effects suggests that genetic drift has not yet substantially eroded ge-
netic diversity.” – give reference. Do you really need this lecture-like statement here?
289-290 – give a reference
292 relatively high genetic diversity – relative to what? Please, explain this better
306 “Tamarix primarily relies on outcrossing but also has some self-fertilization tendencies” – please specify if this is a new conclusion or previous authors has already shown this
308 “which can lead to higher genetic differentiation and lower genetic diversity” – please, revise this. It is a contradictory statement
309 Tamarix relies on pollinators such as aphid flies and bees for outcrossing – give reference
303-313 this paragraph need complete revision
328 Tamarix seeds can still survive at a salinity of 25% . - reference
330-331 long distance disperse – please explain this better. Do you see this based on your data or this is only a speculation?
Discussion is too long. No need to repeat all results again here.
Comments on the Quality of English Languageminor to moderate
Author Response
Comment 1: 11,27 Tamarix chinensis Lour. (T. chinensis), - remove repetition
Response 1: Thank you for pointing this out. We have removed the repeated full name on line 27 and retained the abbreviated form T. chinensis following the first mention on line 28.
Comment 2: 13 This study focuses on … reproductive system of T. chinensis – this was not investigated within the reported study. Please, remove this.
Response 2: We agree. This sentence has been revised to focus on mating system characteristics that were actually assessed via paternity analysis and outcrossing rate estimation (Abstract line21-23).
Comment 3: 19-20 This statement is very broad and uncertain. Please, say more distinctly, what you mean exactly.
Response 3: We have revised the sentence to specify that the results inform conservation planning by identifying gene flow patterns, levels of genetic diversity, and the impact of spatial isolation.
Comment 4: 33, 37, 46 – These geographical examples are redundant.
Response 4: These examples have been removed and replaced with a brief general statement about the broad distribution of T. chinensis across coastal China.
Comment 5: 50 Phylogeographic analyses sentence needs revision.
Response 5: Revised to: “Phylogeographic studies have suggested that the distribution of T. chinensis has been shaped by historical ecological factors, including glaciation and sea level changes.” (Page 3, Line 7).
Comment 6: 57 please, provide references or consolidate ideas.
Response 6: We have added appropriate references and merged scattered points into a coherent paragraph.
Comment 7: 62 revise overgeneralized sentence on evolutionary mechanisms.
Response 7: Sentence revised to: “This study investigates how spatial isolation and gene dispersal patterns influence local genetic structure and diversity.”
Comment 8: 63-65 clarify contribution to conservation strategy and limitations.
Response 8: We revised this sentence to clarify that our findings support conservation at the local scale and suggest the need for site-specific genetic monitoring. We acknowledge the limited geographic scope and avoid broad generalizations.
Comment 9: 83 Specify whether morphological data applies to adults or seedlings.
Response 9: We have added a clarification stating that the morphological traits were recorded for all individuals (Supplyment Table S2).
Comment 10: Figure 1 unclear about island context and right panel meaning.
Response 10: Figure 1 has been revised to clearly indicate the boundaries of the island, and a new label and description were added to explain the right panel as a detailed spatial mapping of sampled individuals.
Comment 11: 91 specify plant part used for DNA.
Response 11: We have added that healthy young leaves were used for DNA extraction (Line 86).
Comment 12: 94 provide DNA concentration data.
Response 12: We have added a summary table of DNA concentration (Supplementary Table S3) .
Comment 13: 104 explain reference and purpose of SSR primers.
Response 13: Reference [13] has been clarified as the source of primer sequences. We now include a brief description of the use of SSR and ISSR primers and their complementary strengths in capturing codominant and dominant variation.
Comment 14: 108 conditions followed the literature – revise.
Response 14: We now revise that "line 97 Referring to previous study[12]"
Comment 15: 127 italicization of Tamarix chinensis.
Response 15: We have conducted a full-text check and corrected all instances of genus and species names to be italicized.
Comment 16: 148 spatial – Spatial
Response 16: Corrected as suggested.
Comment 17: 149 SGS – list of abbreviations.
Response 17: A "List of Abbreviations" section has been added at supplyment file Table S1.
Comment 18: 152-153 spacing issues.
Response 18: Corrected all spacing inconsistencies throughout the manuscript.
Comment 19: 168 redundant software comment.
Response 19: This sentence has been removed.
Comment 20: 206, 208, 225-228 lowercase formatting for tm – ts.
Response 20: We have corrected all formatting of these parameters to lowercase and clarified their definitions.
Comment 21: Move content on tm – ts to Table 4 caption.
Response 21: We have removed the explanation of mating system parameters.
Comment 22: 233-244 clarify distance estimation.
Response 22: Additional explanation has been added to the Methods (Lines 132–155), detailing the use of spatial coordinates and paternity analysis.
Comment 23: 250 mention of T. ramosissima.
Response 23: This has been corrected. The manuscript exclusively studies T. chinensis.
Comment 24: Figure 3 needs better caption.
Response 24:Thank you for the suggestion. In the revised manuscript, the original Figure 3 has been split into two figures (now Figure 3 and Figure 4) to improve clarity. The captions for both figures have been rewritten to provide detailed descriptions of all panels, including the kinship coefficient distance classes, genetic interpolation map, and spatial autocorrelation analysis results. These revisions help readers interpret spatial genetic structure patterns more accurately.
Comment 25: 271-273, 276, 280 provide references.
Response 25: References have been added for all the statements regarding allele frequency distribution, demographic pressure, and effective population size effects (References 20, 23, and 24).
Comment 26: 289-290 – give reference
Response 26: Added reference to Baldauf et al. (2014) on spatial genetic structure in island populations [25].
Comment 27: 292 clarify "relatively high"
Response 27: Revised to: "higher than expected given the population's small size and isolation, and higher than those reported in other T. chinensis populations."
Comment 28: 306 – specify if outcrossing result is novel.
Response 28: We clarified that this study presents the first empirical outcrossing rate estimate for T. chinensis based on paternity analysis.
Comment 29: 308 contradiction in statement.
Response 29: Sentence revised to: "Clumped distribution may elevate genetic structure, while insect pollination promotes outcrossing, helping to maintain diversity."
Comment 30: 309 give pollination reference.
Response 30: Reference [30] has been added to support the claim about aphid flies and bees.
Comment 31: 303-313 paragraph needs full revision.
Response 31: This paragraph has been substantially rewritten to streamline ideas, improve logic, and reduce redundancy.
Comment 32: 328 add salinity reference.
Response 32: Added reference [29] on seed salinity tolerance.
Comment 33: 330-331 long-distance dispersal clarification.
Response 33: We clarified that long-distance dispersal was inferred from maximum observed parent-offspring distances in spatial data.
Comment 34: Discussion too long, avoid repeating results.
Response 34: The Discussion section has been condensed by approximately 40%, removing redundancies and summarizing findings more concisely.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have included the suggestions made during the first round of review, and therefore the manuscript have improved in clarity.
Author Response
We sincerely thank the reviewer for the positive evaluation and kind recognition of the improvements made in the revised manuscript. We truly appreciate your valuable feedback and constructive suggestions throughout the review process, which have significantly enhanced the clarity and quality of our work.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors carefully revised the MS. The revised version of the MS looks better. I have no major criticism on the MS. Some minor remarks are as follows:
19-20 “However, environmental features such as strong winds, storm surges, and flat terrain may occasionally promote long-distance gene flow.” – this sentence seems inadequate to be included in the Abstract, but it is ok for Discussion
Fig.1 The lines showing geography around Shandong in the left lower part of the plate are very hard to read. Please, revise them
Please, ask the Editors if a space is needed between a word and reference: “word space [reference]” or “word no space [reference]”. Check the entire text.
Table 1,2,3 Please revise the captions and mention the abbreviation. Please explain empty cells in the column PPL (and all others) and explain the abbreviation. Please give a reference to the Supplementary Table S1 and say directly that all abbreviations are explained in TS1.
226 data.( (Table 4) The single-locus – revise
Author Response
We sincerely thank the reviewer for their positive evaluation and constructive suggestions. Please find below our responses to the remaining minor comments:
Comment 1: “However, environmental features such as strong winds, storm surges, and flat terrain may occasionally promote long-distance gene flow.” – this sentence seems inadequate to be included in the Abstract, but it is ok for Discussion.
Response 1: This sentence has been deleted from the Abstract and retained only in the Discussion section as advised.
Comment 2: Fig.1 The lines showing geography around Shandong in the left lower part of the plate are very hard to read. Please, revise them.
Response 2: Figure 1 has been revised. Land areas are now shown in light yellow, and provincial boundaries are clearly marked in green for better visibility.
Comment 3: Please, ask the Editors if a space is needed between a word and reference: “word space [reference]” or “word no space [reference]”. Check the entire text.
Response 3: We have confirmed with the journal that the correct format is “word[reference]” without a space. Line 43 and Line 106 have been corrected accordingly, and the entire manuscript has been reviewed for consistency.
Comment 4: Table 1, 2, 3: Please revise the captions and mention the abbreviation. Please explain empty cells in the column PPL (and all others) and explain the abbreviation. Please give a reference to the Supplementary Table S1 and say directly that all abbreviations are explained in TS1.
Response 4: Captions of Tables 1–3 have been revised to include full explanations of all abbreviations. For Table 1, since PPL (Percentage of Polymorphic Loci) is calculated for the population level only, values for individual loci have been marked with “–”. In Table 3, we added mean values for NE-1P, NE-2P, and NE-PP, and clarified that the “Combined” row represents the joint exclusion probability, not the arithmetic total. All abbreviations are now explicitly referenced as being explained in Supplementary Table S1.
Comment 5: Line 226: “data.( (Table 4)” – revise.
Response 5: This has been corrected to “data (Table 4).”
We appreciate the reviewer’s detailed feedback, which has significantly improved the quality and clarity of the manuscript.
