Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study aimed to characterize four Hibiscus cultivars through a detailed assessment of floral traits (color, size, and type), leaf morphology (shape and size), and cytogenetic attributes, including chromosome number, karyomorphology, localization of 5S and 18S rDNA loci, nuclear DNA content, genome size, and ploidy level. The positive aspects of this research are the interesting cytogenetic analysis, such as chromosome number, karyomorphology, localization of 5S and 18S rDNA loci, nuclear DNA content, genome size, and ploidy level. The morphological characterization is important also. All the figures are of good quality. But there are some negative aspects, such as an incomplete material and methods section and an introduction that could be improved. But the main problem concerns the results presentation. The figure titles are not adequate, and the figures should be cited in the text with each letter (A, B, C, and D), showing the proper identification of each cultivar´s figure. Therefore, in my opinion, it does need a major review in order to be considered for publication.

I have a few comments in order to contribute with this manuscript.

Abstract

Lines 23-26 needs improvement, especially in line 25.

Also, it would be interesting to have a brief conclusion of the study in the abstract.

Introduction

Introduction: It has plenty of information, but it could mention what is already known on the chromosome number of the studied species, and other details as well. I believe it could present more background information related to cytogenetic and morphological studies already published on the genus Hibiscus. And from lines 58 to 89, there is a lot of theoretical information on the techniques used in this study, I would suggest for this part could be summarized.

Line 85: Please correct the word ‘complete’ in this sentence. It is written as ‘completer’

Line 86: A stop sign after comparison seems necessary. Ex: “....comparison. Hibiscus genetic resources comprise extensive ....”

Lines 106-107: Instead of “....including the range from diploid to hexadecapolar across species [14,15]....”, I imagine it should be: “....including the range from diploid to hexaploids across species [14,15]”,....

Obs: The word “hexadecapolar” seems wrong, please check.

Materials and Methods

The morphological characterization was not mentioned in Materials and Methods. Therefore, methodology is incomplete. This information is important to inform the number of plants used for this characterization, of each cultivar. And how the measurements were conducted.

Lines 349-350: Instead of starting a sentence with a number, (Ex : 0.7 to 1.0 mm of the distal tip of the root tip ....), I suggest starting with: Then, 0.7 to 1.0 mm of the distal tip .......

Line 354: Instead of: “....on the size of the root tip the age of the enzyme, as well as other variables”, I suggest inserting a comma, such as: “....on the size of the root tip, the age of the enzyme, as well as other variables”,...

Line 363: Please do not start a sentence with a number... such as: “10 uL of cell suspension was ...”

Line 364: Instead of: “The slides are dried for ......”, please alter to: “The slides were dried for ......”,

Line 373: Instead of: “Digoxigenin labelled 5S rDNA (green fluorescence) and biotin labelled 18S rDNA (red fluorescence) mixed with formamide, 50% dextran sulfate, and 20× SSC to make a solution.”, I imagine that the word ‘were’ is needed between (red fluorescence) and mixed. Such as: “Digoxigenin labelled 5S rDNA (green fluorescence) and biotin labelled 18S rDNA (red fluorescence) were mixed with formamide, 50% dextran sulfate, and 20× SSC to make a solution.”

Lines 376-377: Please check the sentence starting with Again,.... ending at for 30 min.

Line 399: Please correct this sentence, such as “...was introduced into the tube”. Obs: please exclude the vowel ‘a’.

Results

The presentation of the results in this manuscript must be improved. The Figure’s results are not clear for the reader. Example:

Figure 1. This figure title should identify each species and their varieties by the letters A to D. Ex: Figure 1. Flower morphology of Hibiscus moscheutos (Carousel Pink Passion’ (A), ‘Carousel Jolly Heart’ (B), etc.... please check if this is the correct correspondence of the variety and its letter.

I noticed that the same thing happened in each Figure, from Fig. 1 to Fig. 6. It is important to identify each figure letter (A to D), both in the Figure titles and in the text.Without this the reader may be in doubt of which figure is the author refering to.

Line 120: I would cite the correct figure in this sentence. Ex: “....with a red center, and the diameter was 16.70 cm (Fig. 1A). The ...” And would do the same for the other species/varieties.

Line 124: The species name H. syriacus should be in italics.

Lines 123-127: I would rewrite these sentences, as suggested:

“The flower colours of H. syriacus ‘Sukim’ and ‘Freedom’ (Fig. 1C and 1D) were light pink and red, purplish pink, respectively. Their diameter were 11.40 cm and 10.20 cm, lower than H. moscheutos (Fig. 1 and Table 1).”

Lines 134-135: I suggest writing as : “....‘Carousel Pink Passion’ and ‘Carousel Jolly Heart’ were star-shaped and reniform, respectively (Fig. 2A and 2B), whereas.......”

Obs: Again, the same problem related to the identification of the right figures, mentioned above.

Line 136: The words “we measured” must be deleted.

Lines 144-145: ‘To confirm the chromosome numbers’ are repeated in this sentence.

Line 145: It is not usual nor correct to start a sentence with a number, such as this: 38 chromosomes were confirmed... The correct would be to write: Thirty-eight chromosomes ....

Again, the proper identification of each figure is missing. The correct way would be such as:

“Thirty-eight chromosomes were confirmed in both cultvars of H. moscheutos (Fig. 3A and 3B), whereas, H. syriacus had 84 chromosomes (Fig. 3C and 3D).

Figure 4. The title should indicate which color corresponds to 5S and to 18S (which one is pink and which one is green??), apart from the identification of each letter A, B, C and D.

Obs: these figure’s identification should be inserted in the text as well, as mentioned before.

Lines 163 and 165: The citation of (Fig. 1 and Table 1) seems wrong. The one on line 165 is in italics, also wrong.

I will stop my analysis of the Results section here, as it does need much improvement.

Discussion

Lines 169-170: Instead of: “....the method has been shown technique efficient for cytogenetic study of woody angiosperms....”, please alter it to: “....the method has been shown to be technique efficient for cytogenetic study of woody angiosperms....”

Lines 283-284: Instead of: “Our findings showed H. moscheutos and H. syriacus were diploid based on cytogenetic results that follow previous studies results [27,36].” I suggest altering to: “Our findings showed H. moscheutos and H. syriacus to be diploid based on cytogenetic results, according to previous studies results [27,36].”

Overall, discussion is well written, although it needs some editing of the Grammer and the English language.

Comments on the Quality of English Language

There are several problems with the quality of the English language. A review with a specialist would be important.

Author Response

General Response

We sincerely thank the Reviewer for the thorough and constructive evaluation of our manuscript. The Reviewer’s comments have helped us significantly improve the quality, clarity, and accuracy of the manuscript. We have carefully addressed each comment point by point below. All changes made to the manuscript are indicated in the revised version. We hope that the revised manuscript now meets the standards of Plants. All the changes has been marked as red.

Abstract

Comment 1: Lines 23–26 need improvement, especially line 25. It would also be interesting to have a brief conclusion in the abstract.

Response: Lines 23–26 have been revised for clarity and conciseness. Specifically, line 25 has been restructured to improve readability and accurately reflect the key quantitative findings. A brief concluding sentence has been added to the abstract summarizing the main implication of the study — that the integrated cytogenetic and morphological characterization provides a foundational framework for interspecific hybridization, germplasm conservation, and breeding program development in Hibiscus.

Introduction

Comment 2: The introduction could present more background information related to cytogenetic and morphological studies already published on the genus Hibiscus, including what is already known on chromosome number. Additionally, lines 58–89, which contain theoretical information on the techniques used, should be summarized.

Response: The Introduction has been revised to include additional background on previously published cytogenetic and morphological studies in the genus Hibiscus, including known chromosome numbers across species. The theoretical overview of techniques in lines 58–89 has been condensed to improve flow and focus while retaining the essential context needed to justify the study's approach.

Comment 3 (Line 85): Please correct the word 'completer' in this sentence.

Response: Corrected. The word 'completer' has been changed to 'complete' in the revised manuscript.

Comment 4 (Line 86): A stop sign after 'comparison' seems necessary.

Response: A period has been inserted after 'comparison' to separate the two sentences, as suggested. The sentence now reads: '…comparison. Hibiscus genetic resources comprise extensive…'

Comment 5 (Lines 106–107): Instead of '…including the range from diploid to hexadecapolar across species…', it should read '…including the range from diploid to hexaploids across species…'. The word 'hexadecapolar' seems wrong.

Response: The reviewer is correct. 'Hexadecapolar' was an error and has been corrected to 'hexaploids' in the revised manuscript.

Materials and Methods

Comment 6: The morphological characterization was not mentioned in Materials and Methods. The methodology is incomplete and should include the number of plants used for each cultivar and how measurements were conducted.

Response: A dedicated subsection (Section 4.2: Phenotypic Characteristics) has been added to the Materials and Methods describing the morphological characterization methodology. This section specifies that measurements were conducted following the Korean Seed & Variety Service (http://www.seed.go.kr) survey method and includes details on the number of plants evaluated per cultivar (n = 6), as well as the specific traits measured and the approach used for quantitative assessments.

Comment 7 (Lines 349–350): Do not start a sentence with a number. Suggest starting with: 'Then, 0.7 to 1.0 mm of the distal tip…'

Response: Revised as suggested. The sentence now begins with 'Then, 0.7 to 1.0 mm of the distal tip of the root tip…'

Comment 8 (Line 354): A comma should be inserted: '…on the size of the root tip, the age of the enzyme, as well as other variables'.

Response: The comma has been inserted as suggested. The sentence now reads: '…on the size of the root tip, the age of the enzyme, as well as other variables.'

Comment 9 (Line 363): Do not start a sentence with a number: '10 µL of cell suspension was…'

Response: Revised. The sentence now begins: 'Then, 10 µL of cell suspension was dropped onto the moistened slide.'

Comment 10 (Line 364): Instead of 'The slides are dried for…', please alter to 'The slides were dried for…'

Response: Corrected. The verb tense has been changed from present to past tense throughout the Materials and Methods section for consistency. The sentence now reads: 'The slides were dried for further experiments.'

Comment 11 (Line 373): The word 'were' is missing between '(red fluorescence)' and 'mixed'.

Response: The word 'were' has been inserted as suggested. The sentence now reads: 'Digoxigenin labelled 5S rDNA (green fluorescence) and biotin labelled 18S rDNA (red fluorescence) were mixed with formamide, 50% dextran sulfate, and 20× SSC to make a solution.'

Comment 12 (Lines 376–377): Please check the sentence starting with 'Again,…' ending at 'for 30 min'.

Response: The sentence has been reviewed and revised for clarity. It now reads: 'The slides were then washed in 0.1× SSC buffer in a water bath maintained at 42°C with shaking for 30 min.'

Comment 13 (Line 399): Please correct the sentence: '…was introduced into the tube'. Exclude the extra vowel 'a'.

Response: Corrected. The redundant article 'a' has been removed. The sentence now reads: '…was introduced into the tube.'

Results

Comment 14: Figure titles do not adequately identify each species/cultivar by their corresponding letter (A to D). Each figure title should explicitly identify each letter and its corresponding cultivar. The same identification should be reflected in the text when citing figures.

Response: All figure titles (Figures 1–6) have been revised to explicitly identify each panel letter (A, B, C, D) with its corresponding cultivar name. For example, Figure 1 now reads: 'Flower morphology of A. Hibiscus moscheutos 'Carousel Jolly Heart', B. Hibiscus moscheutos 'Carousel Pink Passion', C. Hibiscus syriacus 'Sukim', D. Hibiscus syriacus 'Freedom'.' The in-text figure citations throughout the Results section have likewise been updated to reference specific panel letters (e.g., Fig. 1A, Fig. 3C and 3D) to clearly associate each result with the appropriate cultivar image.

Comment 15 (Line 120): Cite the correct figure letter in this sentence. E.g., '…with a red center, and the diameter was 16.70 cm (Fig. 1A).'

Response: Revised as suggested. Figure citations in the Results text now include the appropriate panel letter throughout. For example, the sentence describing H. moscheutos 'Carousel Jolly Heart' now cites (Fig. 1A), and each subsequent cultivar description references its corresponding panel.

Comment 16 (Line 124): The species name H. syriacus should be in italics.

Response: Corrected. All instances of Hibiscus species names, including H. syriacus, have been checked and formatted in italics throughout the manuscript.

Comment 17 (Lines 123–127): Rewrite these sentences for clarity, and correctly identify figures as 1C and 1D.

Response: The sentences have been rewritten for clarity. They now read: 'The flower colours of H. syriacus 'Sukim' and 'Freedom' (Fig. 1C and 1D) were light pink and red–purplish pink, respectively. Their diameters were 11.40 cm and 10.20 cm, which were lower than those of H. moscheutos (Fig. 1 and Table 1).'

Comment 18 (Lines 134–135): Rewrite to: '…'Carousel Pink Passion' and 'Carousel Jolly Heart' were star-shaped and reniform, respectively (Fig. 2A and 2B), whereas…'

Response: Revised as suggested. The sentence now reads: '…'Carousel Pink Passion' and 'Carousel Jolly Heart' were star-shaped and reniform, respectively (Fig. 2A and 2B), whereas H. syriacus 'Sukim' and 'Freedom' were elliptical in shape (Fig. 2C and 2D).'

Comment 19 (Line 136): The words 'we measured' must be deleted.

Response: Deleted. The phrase 'we measured' has been removed and the sentence restructured accordingly.

Comment 20 (Lines 144–145): 'To confirm the chromosome numbers' is repeated. Also, do not start a sentence with a numeral: '38 chromosomes were confirmed…' should read 'Thirty-eight chromosomes…'

Response: The repeated phrase has been removed and the sentence revised. It now reads: 'Thirty-eight chromosomes were confirmed in both cultivars of H. moscheutos (Fig. 3A and 3B), whereas H. syriacus had eighty-four chromosomes (Fig. 3C and 3D).'

Comment 21 (Figure 4): The figure title should indicate which colour corresponds to 5S and which to 18S rDNA, in addition to identifying each panel letter A–D.

Response: The title of Figure 4 has been revised to specify the colour coding: green fluorescence for 5S rDNA and red/pink fluorescence for 18S rDNA, in addition to identifying each panel letter and its corresponding cultivar.

Comment 22 (Lines 163 and 165): The citation of (Fig. 1 and Table 1) appears incorrect and one instance is in italics, which is also wrong.

Response: The figure and table citations on these lines have been reviewed and corrected. The incorrect references have been updated to cite the appropriate figures, and all citation text has been set to regular (non-italic) formatting.

Discussion

Comment 23 (Lines 169–170): Instead of '…the method has been shown technique efficient…', please alter to '…the method has been shown to be technically efficient…'

Response: Corrected. The sentence now reads: '…the method has been shown to be technically efficient for cytogenetic study of woody angiosperms…'

Comment 24 (Lines 283–284): Instead of '…Our findings showed H. moscheutos and H. syriacus were diploid based on cytogenetic results that follow previous studies results…', suggest: '…Our findings showed H. moscheutos and H. syriacus to be diploid based on cytogenetic results, according to previous studies [27,36].'

Response: Revised as suggested. The sentence now reads: 'Our findings showed H. moscheutos and H. syriacus to be diploid based on cytogenetic results, according to previous studies [27,36].'

Comment 25 (General): Overall, the discussion is well written, although it needs some editing of the grammar and English language.

Response: The Discussion section has been thoroughly reviewed and edited for grammatical accuracy and clarity of expression. Several sentences have been revised to improve the quality of the English language throughout.

General Comment on English Language

General Comment: There are several problems with the quality of the English language. A review with a specialist would be important.

Response: We acknowledge this concern and have carefully revised the entire manuscript to improve the quality of the English language. Grammar, syntax, verb tense consistency, and sentence clarity have been addressed throughout all sections. We believe the revised manuscript now reads with greater fluency and precision.

We hope that the revised manuscript and the responses provided above adequately address all of the reviewer's concerns. We remain grateful for the time and expertise invested in evaluating this work, and we welcome any further comments.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents a comprehensive and systematic agro-morphological and cytogenetic characterization of four cultivars. The study integrates multiple techniques, including traditional morphometry, karyotype analysis, fluorescence in situhybridization (FISH) for rDNA locus mapping, and flow cytometry for genome size estimation. It reports for the first time the exact chromosome number (2n=84) for cultivars 'Sukim' and 'Freedom'. The findings offer fundamental information valuable for germplasm identification, taxonomy, and assessing interspecific hybridization potential in Hibiscus. However, the preparation of this manuscript was careless, many mistakes were found in the text.

Minor comments:

1. Lines 119-123, "H. moscheutos ‘Carousel Pink Passion’ looked pink with a red center, and the diameter was 16.70 cm. The length and width of the petal were 9.30 cm and 10.4 cm, respectively.", but in table 1, the correct cultivar is 'Carousel Jolly Heart'.

2. In figure 2, 3 and 4, what is the meaning of A, B, C and D.

3. In table 3, * were used. So what are their meaning? The authors should give a note followed the table.

4. Line 163-165, Fig. 1 and Table 1 were cited wrongly. I suggested the authors thoroughly check and correct all such citation errors throughout the manuscript.

5. Lines 273-283 discuss the relationship between 5S rDNA locus number and ploidy. However, the biological significance of the difference in 18S rDNA locus number between H. moscheutos(6 loci) and H. syriacus(4 loci)—despite both having a diploid/polyploid base—could be explored more deeply. It is suggested to elaborate further, perhaps linking it to potential "diploidization" or locus loss during polyploidization, to better underscore the importance of the FISH results.

6. In section 4.3, the authors should provide more details on the origin of 5S rDNA and 18S rDNA sequences.

7. For flow cytometric analysis, how many biological and technical replicates were conducted for each cultivars?

Author Response

General Response

Response to Reviewers

Manuscript Title: Agro-Morphological and Cytogenetic Characterization of Hibiscus Genetic Resources: Implications for Germplasm Conservation and Interspecific Breeding

We thank the reviewer for their thorough and constructive evaluation of our manuscript. We have carefully addressed all comments and have revised the manuscript accordingly. A point-by-point response is provided below.

Comment 1: Lines 119–123 describe H. moscheutos 'Carousel Pink Passion' as having a pink flower with a red center and a diameter of 16.70 cm, but Table 1 assigns these values to 'Carousel Jolly Heart'.

Response: We thank the reviewer for identifying this inconsistency. The cultivar names were inadvertently transposed in the text. In the revised manuscript, the description at lines 122–125 now correctly states that H. moscheutos 'Carousel Jolly Heart' displayed a pink flower with a red center and a diameter of 16.70 cm, while 'Carousel Pink Passion' bore a reddish-pink flower with a diameter of 16.10 cm. This is now fully consistent with the data presented in Table 1. The abstract has also been updated accordingly.

Comment 2: In Figures 2, 3, and 4, the meaning of panels A, B, C, and D is not explained.

Response: We agree that panel labels were insufficiently described in the original figure captions. In the revised manuscript, all figure captions for Figures 1–6 have been updated to explicitly identify each panel. For example, Figure 3 now reads: "Chromosome number determination in A. Hibiscus moscheutos 'Carousel Jolly Heart' (2n=38), B. Hibiscus moscheutos 'Carousel Pink Passion' (2n=38), C. Hibiscus syriacus 'Sukim' (2n=84), and D. Hibiscus syriacus 'Freedom' (2n=84)." The same format has been applied consistently to Figures 1, 2, 4, 5, and 6.

Comment 3: Asterisks (*) are used in Table 3 without any explanatory note.

Response: We apologize for this oversight. A table note has been added beneath Table 3 in the revised manuscript. The asterisk () in the "Short arm" column for 5S rDNA denotes loci located in the pericentromeric region of the short arm, while the asterisk in the 18S rDNA location column (2+2) for 'Carousel Pink Passion' indicates that one pair of signals was detected at a centromeric position. These clarifications are now provided in a footnote directly following Table 3.

Comment 4: Lines 163–165 incorrectly cite Fig. 1 and Table 1 for the 18S rDNA localization data. The reviewer recommends a thorough check of all in-text citations.

Response: We thank the reviewer for this careful observation. The erroneous citations to Fig. 1 and Table 1 in the rDNA section have been corrected to Fig. 4 and Table 3, which are the appropriate references for the FISH localization data. Additionally, we have conducted a comprehensive review of all figure and table citations throughout the manuscript. The karyomorphological section (Section 2.4) previously cited Fig. 2 and Table 2 for chromosome complement data; these have been corrected to Fig. 5 and Table 4. All remaining citations have been verified and corrected where necessary.

Comment 5: The discussion of 5S rDNA and ploidy (lines 273–283) does not adequately explore the biological significance of the difference in 18S rDNA locus number between H. moscheutos (6 loci) and H. syriacus (4 loci). The reviewer suggests linking this to potential diploidization or rDNA locus loss during polyploidization.

Response: We thank the reviewer for this insightful suggestion. The revised discussion now includes an expanded interpretation of the interspecific difference in 18S rDNA locus number. Specifically, we have added text explaining that the higher number of 18S rDNA loci in H. moscheutos (6 loci) relative to H. syriacus (4 loci) may reflect divergent evolutionary trajectories in rDNA organization following polyploidization. In allopolyploids and ancient polyploids, rDNA loci contributed by parental genomes are frequently subject to elimination or silencing through a process consistent with genomic diploidization, whereby the merged genome undergoes restructuring to restore functional disomic inheritance. The reduced 18S locus number in H. syriacus, despite its higher chromosome count (2n = 84), is therefore consistent with progressive locus loss or homogenization during its polyploid evolutionary history, a phenomenon well-documented in other polyploid plant genera including Brassicaceae [35]. This elaboration strengthens the biological interpretation of the FISH results and has been incorporated into the discussion section.

Comment 6: Section 4.3 should provide more details on the origin of the 5S rDNA and 18S rDNA sequences used as FISH probes.

Response: We appreciate this comment. Additional detail on the probe origins has been added to Section 4.3 of the revised manuscript. The 5S rDNA probe was derived from a cloned Lilium 5S rDNA repeat unit, labelled with digoxigenin-11-dUTP (green fluorescence), following the protocol established by Lim et al. [48]. The 18S rDNA probe was derived from an Arabidopsis thaliana 18S rDNA sequence, labelled with biotin-16-dUTP (red fluorescence). Both probes were prepared by nick translation and have been used in previous cytogenetic studies of Hibiscus and related genera by our group [1, 9, 36]. This information is now clearly stated in the Materials and Methods section.

Comment 7 (Flow Cytometry – Biological and Technical Replicates)

Reviewer’s Comment:

For flow cytometric analysis, how many biological and technical replicates were conducted for each cultivar?

Response:

We thank the Reviewer for raising this important methodological point. For the flow cytometric analysis, three biological replicates (i.e., three independent plants per cultivar) were used, and for each biological replicate, two technical replicates (i.e., two independent measurements per plant sample) were performed, giving a total of six measurements per cultivar. The mean 2C DNA value and standard error reported in Table 5 are based on these six measurements (Mean ± SE, n = 6). This information has been added to Section 4.5 (Determination of Nuclear DNA Content) in the revised manuscript as follows:

“…Three biological replicates (independent plants) and two technical replicates per biological replicate were used for each cultivar, giving six measurements per cultivar (Mean ± SE, n = 6).”

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents a potentially valuable dataset combining agro-morphological and cytogenetic characterization of selected Hibiscus cultivars. The aims is clear and the results is interesting to me. However, several issues related to data consistency, adequacy of statistical treatment, and depth of analysis need to be addressed.

A primary concern is the presence of inconsistencies between the text and tabulated data. For instance, in the flower morphology section (Lines 119–123), the manuscript reports that ‘Carousel Pink Passion’ has the largest flower diameter (16.70 cm), whereas Table 1 assigns this value to ‘Carousel Jolly Heart’. This discrepancy must be resolved. Similar issues, including incorrect cultivar naming (Lines 133–137) and mismatches between figure/table citations and content (e.g., Lines 160–166), indicate that the manuscript requires a thorough and systematic verification of all reported values and labels.

A second major issue is the lack of statistical support for the reported differences. Across the Results section, differences among cultivars and species are described qualitatively (e.g., “larger”, “smaller”), but no statistical analyses are provided to validate these comparisons (Lines 118–126; 133–137). This limits the reliability of the conclusions drawn from the morphological data. Similarly, in the nuclear DNA content analysis (Lines 195–200), only single values are presented without any indication of variability, such as standard deviation, replicate number, or coefficient of variation (CV), which is particularly important for flow cytometry data.

In addition, the Results section is largely descriptive and lacks sufficient analytical depth. Data are presented primarily as lists of measurements for individual cultivars, but there is limited effort to synthesize these observations into broader patterns or comparative conclusions. For example, in the karyotype analysis (Lines 173–188), chromosome lengths and centromere classifications are reported, but their relevance to species-level differentiation is not clearly explained.

Finally, the Discussion includes several statements that extend beyond the evidence presented. Claims regarding interspecific hybridization potential (Lines 232–234), conservation strategies, and ecological or climate-related implications are not directly supported by the data, as no experiments addressing these aspects were conducted. These interpretations should be moderated and reframed to reflect the actual scope of the study.

Suggestion

LINE 35–46: The description of Hibiscus syriacus and its cultivars is overly detailed and reads more like background information than part of a focused research narrative. This section should be shortened and more clearly linked to the study objective.

LINE 50–57: The paragraph describing the Carousel series includes horticultural details (e.g., planting conditions and uses) that are not directly relevant to the research. These should be reduced or removed.

LINE 82–89: This sentence is overly long and contains multiple ideas without clear grammatical structure. It should be split into shorter sentences and rewritten for clarity.

LINE 133–137: The cultivar name “Carousel Jolly Pink” is incorrect and should be corrected. In addition, only leaf length is described in the text, while other traits listed in Table 2 are not discussed.

LINE 160–166: Figure and table citations are incorrect in this section (e.g., references to Fig. 1 and Table 1). These should be revised to match the correct figures and tables showing rDNA localization.

LINE 173–179: Chromosome length values are repeated from Table 4 without interpretation. The text should focus on patterns or comparisons rather than restating numerical data.

LINE 180–188: Cytogenetic terminology is inconsistent (e.g., “sub-metacentric” vs. “submetacentric”) and should be standardized.

LINE 195–200: Nuclear DNA content is presented without any indication of variability (e.g., standard deviation or CV values). Additional information should be provided to support data reliability.

LINE 292–296: Comparisons to unrelated taxa (e.g., chrysanthemum or Populus) are not clearly relevant to the study and should either be removed or better justified.

LINE 320–327: Statements regarding ecological plasticity and climate adaptation are not supported by the data presented in this study and should be revised or removed.

Several sentences throughout the Introduction and Discussion are overly long and contain grammatical issues. A thorough language revision is recommended to improve readability and clarity.

Comments on the Quality of English Language

Several sentences throughout the Introduction and Discussion are overly long and contain grammatical issues. A thorough language revision is recommended to improve readability and clarity.

Author Response

General Response

Major Comments

Comment 1: Data Inconsistencies Between Text and Tables

The reviewer noted discrepancies between the text and tabulated data, including incorrect attribution of the largest flower diameter, incorrect cultivar naming, and mismatched figure/table citations (Lines 119–123, 133–137, 160–166).

Response: We thank the reviewer for identifying these inconsistencies, which we have now fully resolved. In the flower morphology section, the text has been corrected to accurately state that ‘Carousel Jolly Heart’ had the largest flower diameter (16.70 cm), consistent with Table 1. The incorrect cultivar name ‘Carousel Jolly Pink’ appearing at Lines 133–137 has been corrected to ‘Carousel Jolly Heart’ throughout the manuscript. Additionally, the figure and table citations in Lines 160–166 have been revised to accurately reference Figure 4 and Table 3, which present the rDNA localization data, rather than the previously cited Figure 1 and Table 1. A systematic verification of all cultivar names, numerical values, and figure/table references has been conducted across the entire manuscript to ensure consistency.

Comment 2: Lack of Statistical Support and Variability Measures

The reviewer raised concern that differences among cultivars are described qualitatively without statistical analysis (Lines 118–126, 133–137), and that nuclear DNA content values are presented as single values without variability indicators such as standard deviation, replicate number, or CV (Lines 195–200).

Response: We acknowledge this concern and have addressed it as follows. For the morphological data reported in Tables 1 and 2, all measurements are now presented as mean ± standard error (SE) based on six replicates (Mean ± SE = 6), as indicated in the table footnotes. The descriptive comparisons in the Results text (e.g., ‘larger’, ‘smaller’) are now accompanied by reference to the tabulated mean values and SE, providing a transparent basis for the comparisons made. For the nuclear DNA content data, Table 5 now reports all 2C values (both in pg and Mbp) as mean ± SE based on six measurements per cultivar, providing the variability information required to assess data reliability for flow cytometry results. These additions are reflected in the revised Table 5 and the corresponding Results text.

Comment 3: Insufficient Analytical Depth in the Results Section

The reviewer noted that the Results section is largely descriptive, presenting data as lists of measurements without synthesizing observations into broader patterns or comparative conclusions, particularly in the karyotype analysis (Lines 173–188).

Response: We have revised the Results section to go beyond enumeration of measurements and instead highlight comparative patterns and their biological significance. In the karyotype analysis section, we now explicitly interpret the chromosome length data in the context of interspecific differences: H. syriacus cultivars display a broader range of chromosome lengths (2.47–7.84 μm) compared to H. moscheutos (2.92–6.24 μm), which is consistent with its higher chromosome number and polyploid origin. The predominance of metacentric chromosomes in both species, with a higher proportion in H. syriacus, is now discussed as a reflection of the relatively symmetrical karyotype characteristic of the genus. The rDNA section similarly now synthesizes the species-specific differences in 18S locus number (six in H. moscheutos versus four in H. syriacus) as evidence of divergent rDNA evolution, rather than listing signal positions alone.

Comment 4: Discussion Statements Beyond the Evidence Presented

The reviewer noted that several Discussion statements regarding interspecific hybridization potential (Lines 232–234), conservation strategies, and ecological/climate-related implications are not directly supported by the experimental data.

Response: We have carefully reviewed the Discussion and moderated all statements that extend beyond the scope of the data presented. Claims regarding hybridization potential are now framed as genomic observations that are relevant to, rather than direct evidence of, hybridization outcomes: the ploidy and genome size differences between the two species are presented as cytogenetic parameters that inform the design of future crossing experiments, following established literature [22,23]. Statements about ecological plasticity and climate adaptation have been reframed to make clear that these represent inferences from the broader polyploidy literature [45,46] and not conclusions drawn from this study’s own data. Conservation-related interpretations are grounded in the established value of cytogenetic baselines for germplasm registration and genebank management [17,47], which is directly supported by the characterization data generated here.

Specific Suggestions

Lines 35–46: The description of H. syriacus has been condensed. Background details not directly relevant to the study objectives have been removed, and the remaining text is more explicitly linked to the rationale for selecting these cultivars for comparative characterization.

Lines 50–57: Horticultural management details for the Carousel series (planting conditions, container use, temperature tolerance) have been removed from the Introduction. A brief statement retaining the key distinguishing features of the Carousel cultivars relevant to this study has been kept.

Lines 82–89: The long compound sentence has been restructured into two shorter sentences to improve grammatical clarity and readability, without altering the scientific content.

Lines 133–137: The cultivar name ‘Carousel Jolly Pink’ has been corrected to ‘Carousel Jolly Heart’ throughout the manuscript. The leaf morphology discussion has been expanded to address additional traits documented in Table 2, including leaf shape, apex, base, color, margin, and petiole length, ensuring alignment between the text and the tabulated data.

Lines 160–166: The figure and table citations in the rDNA localization section have been corrected. References now correctly point to Figure 4 and Table 3, which display the FISH results and chromosomal localization of 5S and 18S rDNA loci, respectively.

Lines 173–179: The chromosome length text has been revised to focus on comparative patterns rather than restating the numerical values already presented in Table 4. The text now highlights the range differences between species and interprets the distribution of metacentric, submetacentric, and telocentric chromosomes in terms of karyotype symmetry.

Lines 180–188: Cytogenetic terminology has been standardized throughout the manuscript. The term ‘submetacentric’ is now used consistently, replacing all instances of ‘sub-metacentric’.

Lines 195–200: Nuclear DNA content values are now reported with mean ± SE in both the text and Table 5 (n = 6 per cultivar). This provides the variability information necessary to assess the reliability of the flow cytometry data, as recommended for this type of measurement.

Lines 292–296: Comparisons to chrysanthemum and Populus chromosome length data have been retained but are now more clearly contextualized as illustrative reference points for chromosome size ranges in other taxa with numerous small chromosomes, not as direct functional comparisons. Where the relevance was insufficiently justified, the sentences have been revised or removed.

Lines 320–327: Statements regarding ecological plasticity and climate adaptation have been revised. These are now explicitly framed as inferences supported by the broader polyploidy literature [45,46] rather than conclusions drawn from this study. The revised text makes clear that the connection between ploidy level and environmental adaptation in Hibiscus is a hypothesis that merits future investigation.

English Language Quality

Response: The entire manuscript has been carefully revised for language quality. Overly long sentences throughout the Introduction and Discussion have been restructured into shorter, grammatically clear sentences. Redundant phrasing has been removed and word choice has been improved for consistency and precision throughout.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

As a reviewer, I was much disappointed. The authors mentioned in their response letter that they accepted several corrections, but when reading the text I noticed that most of them were not included. They did some of the corrections, but the others, a total of 12, are still to be done.

Also, there is still a strong need for a review of the English language, of gramar aspects. Again, the authors mentioned that it was done, but it wasn´t.

I will go through the response letter, pointing our what was accepted and what was not included in the text.

Abstract

Comment 1: Lines 23–26 need improvement, especially line 25. It would also be interesting to have a brief conclusion in the abstract.

Reviewer: The authors did not alter lines 23-26 of the old version. The lines that are shown in red have not been modified at all. And the lines that I mentioned (23-26 of the old version) also have not been modified.

Also, no concluding sentence have been added to the new version.

Introduction

Reviewer: Suggestion accepted. But please provide a grammatical correction of this text below. Instead of: It may be grown... please alter to: They may be grown ..... and enjoy full light...

These types are pruned in the spring and can withstand temperatures as low as -15°C in the winter. It may be grown in containers and enjoys full light and moist, nutrient-rich soils. Furthermore, they are appropriate for border planting, mixed plantings, and mass plantings [6].

Comment 3 (Line 85): Please correct the word 'completer' in this sentence.

Response: Corrected. The word 'completer' has been changed to 'complete' in the revised manuscript.

Reviewer: Suggestion accepted.

Comment 4 (Line 86): A stop sign after 'comparison' seems necessary.

Response: A period has been inserted after 'comparison' to separate the two sentences, as suggested. The sentence now reads: '…comparison. Hibiscus genetic resources comprise extensive…'

Reviewer: Suggestion accepted.

Response: The reviewer is correct. 'Hexadecapolar' was an error and has been corrected to 'hexaploids' in the revised manuscript.

Reviewer: Suggestion accepted.

Materials and Methods

Reviewer: The authors included only part of this information. This section (4.2) is still incomplete. They did not include “details on the number of plants evaluated per cultivar (n = 6), as well as the specific traits measured and the approach used for quantitative assessments.”

Also, the next section should be 4.3, but it is still 4.2. The other following sections should also be altered to subsequent numbers.

Comment 7 (Lines 349–350): Do not start a sentence with a number. Suggest starting with: 'Then, 0.7 to 1.0 mm of the distal tip…'

Response: Revised as suggested. The sentence now begins with 'Then, 0.7 to 1.0 mm of the distal tip of the root tip…'

Reviewer: Suggestion accepted.

Comment 8 (Line 354): A comma should be inserted: '…on the size of the root tip, the age of the enzyme, as well as other variables'.

Response: The comma has been inserted as suggested. The sentence now reads: '…on the size of the root tip, the age of the enzyme, as well as other variables.'

Reviewer: Suggestion accepted.

Comment 9 (Line 363): Do not start a sentence with a number: '10 µL of cell suspension was…'

Response: Revised. The sentence now begins: 'Then, 10 µL of cell suspension was dropped onto the moistened slide.'

Reviewer: Suggestion accepted.

Comment 10 (Line 364): Instead of 'The slides are dried for…', please alter to 'The slides were dried for…'

Reviewer: Suggestion accepted.

Comment 11 (Line 373): The word 'were' is missing between '(red fluorescence)' and 'mixed'.

Reviewer: Suggestion accepted.

Comment 12 (Lines 376–377): Please check the sentence starting with 'Again,…' ending at 'for 30 min'.

Response: The sentence has been reviewed and revised for clarity. It now reads: 'The slides were then washed in 0.1× SSC buffer in a water bath maintained at 42°C with shaking for 30 min.'

Reviewer: No, this new sentence, which is correct, was not included in the new version. It still needs to be done.

Comment 13 (Line 399): Please correct the sentence: '…was introduced into the tube'. Exclude the extra vowel 'a'.

Response: Corrected. The redundant article 'a' has been removed. The sentence now reads: '…was introduced into the tube.'

Reviewer: No, the authors did not remove the article ‘a’. It still needs to be done.

Results

Reviewer: The figure titles were improved, including the identification of each letter. But the in-text figure citations throughout the Results section has not likewise been updated to the specific panel letters. They are still mentioned as Fig. 1, Fig. 2, etc....

Also, some improvements are needed on the titles of Fig. 3

Figure 3. Chromosome number determinatio A. Hibiscus moscheutos ‘Carousel Jolly Heart’(2n=38), B. Hibiscus moscheutos ‘Carousel Pink Passion’ (2n=38), and C. Hibiscus syriacus ‘Sukim’,(2n=84) D. Hibiscus syriacus ‘Freedom’ (2n=84).

It should be such as:

Figure 3. Chromosome number determination of: A. Hibiscus moscheutos ‘Carousel Jolly Heart’(2n=38), B. Hibiscus moscheutos ‘Carousel Pink Passion’ (2n=38), C. Hibiscus syriacus ‘Sukim’ (2n=84), and D. Hibiscus syriacus ‘Freedom’ (2n=84).

Similar corrections should be made in Figures 4, 5, and 6.

Comment 15 (Line 120): Cite the correct figure letter in this sentence. E.g., '…with a red center, and the diameter was 16.70 cm (Fig. 1A).'

Reviewer: Not true. This modification has not been included in the text.

Comment 16 (Line 124): The species name H. syriacus should be in italics.

Response: Corrected. All instances of Hibiscus species names, including H. syriacus, have been checked and formatted in italics throughout the manuscript.

Reviewer: Suggestion accepted.

Comment 17 (Lines 123–127): Rewrite these sentences for clarity, and correctly identify figures as 1C and 1D.

Reviewer: No, these modifications were not included in the new version.

Comment 18 (Lines 134–135): Rewrite to: '…'Carousel Pink Passion' and 'Carousel Jolly Heart' were star-shaped and reniform, respectively (Fig. 2A and 2B), whereas…'

Reviewer: Part of this modification was included, but again, the information Fig. 2A and 2B, as well as Fig. 2C and 2D, was not included in the text.

Comment 19 (Line 136): The words 'we measured' must be deleted.

Response: Deleted. The phrase 'we measured' has been removed and the sentence restructured accordingly.

Reviewer: Suggestion accepted.

Reviewer: The repeated phrase was removed and the sentence revised. But, again, the information in Fig. 3A and 3B, plus Fig. 3C and 3D, was not included in the sentence.

Comment 21 (Figure 4): The figure title should indicate which colour corresponds to 5S and which to 18S rDNA, in addition to identifying each panel letter A–D.

Reviewer: No, only part of this modification was included in the title. The colours that correspond to 5S and 18S were not included.

Comment 22 (Lines 163 and 165): The citation of (Fig. 1 and Table 1) appears incorrect and one instance is in italics, which is also wrong.

Reviewer: Suggestion accepted.

Discussion

Comment 23 (Lines 169–170): Instead of '…the method has been shown technique efficient…', please alter to '…the method has been shown to be technically efficient…'

Response: Corrected. The sentence now reads: '…the method has been shown to be technically efficient for cytogenetic study of woody angiosperms…'

Reviewer: No, it is still written the same way as before. Sorry, the correct line numbers of the old version are 269-270; this was my mistake.

Response: Revised as suggested. The sentence now reads: 'Our findings showed H. moscheutos and H. syriacus to be diploid based on cytogenetic results, according to previous studies [27,36].'

Reviewer: Suggestion accepted.

Comment 25 (General): Overall, the discussion is well written, although it needs some editing of the grammar and English language.

Reviewer: There are still some corrections needed, such as:

Line 271 (of the new version): Instead of twenty-two to one hundred eighty, the authors may write as numbers: 22 to 180. There is no need to spell out chromosome numbers. The same is valid for line 273.

Lines 281-282 (of the new version): Instead of: “In the present study, it was reported the chromosome count result of ‘Sukim’ and ‘Freedom’ for the first time, and the chromosome number was eighty-four.”

Please modify to: “In the present study, the chromosome count result of ‘Sukim’ and ‘Freedom’ was reported for the first time, and the chromosome number was 84.”

Lines 309-311: “According to the findings in the present study, the average chromosomes length of H. moscheutos is 2.92 to 6.24 μm whereas, the chromosome length ranges for H. syriacus were 2.47 to 7.84 μm.”

This sentence needs improvement, such as: “According to the findings in the present study, the average chromosome length of H. moscheutos was 2.92 to 6.24 μm, whereas the chromosome length ranges for H. syriacus were 2.47 to 7.84 μm.”

Line 313: Instead of: “....10.89 μm which, was slightly higher...”

Change the comma, such as: “....10.89 μm, which was slightly higher...”

320-321: Instead of: “....type of chromosome and H. syriacus, were detected with metacentric and sub metacentric type of chromosome.”

Please alter to: “....types of chromosomes, while H. syriacus, was detected with metacentric and sub metacentric types of chromosomes.”

Line 338: .... that´s value in this sentence is wrong. This sentence needs correction.

Lines 349-350: In the sentence: “From a practical standpoint, the genomic baselines established here chromosome number, karyotype structure, ....”

Please modify it to: “From a practical standpoint, the genomic baselines established here, such as chromosome number, karyotype structure, ....”

General Comment on English Language

General Comment: There are several problems with the quality of the English language. A review with a specialist would be important.

Reviewer: There is still a need for a careful review of the entire manuscript.

Reviewer: Unfortunately, the revised manuscript still needs a major review.

Comments on the Quality of English Language

There are still several problems with the quality of the English language. A review with a specialist would be important.

Author Response

Dear Reviewer,

We sincerely thank you for your thorough review and constructive comments. We have carefully addressed each point raised and summarize the revisions below. All changes are indicated by line numbers in the revised manuscript. Reviewer comments are shown in blue boxes and our responses in green boxes.

Responses to Specific Comments

Comment 1

Lines 23–26 need improvement, especially line 25. It would also be interesting to have a brief conclusion in the abstract.

Authors' Response: Amendments have been made from lines 25–33. The abstract has been revised for clarity and a brief concluding statement has been added as suggested.

Comment 6

The morphological characterization was not mentioned in Materials and Methods. The methodology is incomplete and should include the number of plants used for each cultivar and how measurements were conducted.

Authors' Response: Amendments have been incorporated from lines 367–375. The Materials and Methods section has been updated to include details on the number of plants used per cultivar and the measurement procedures applied for morphological characterization.

Comment 12

Please check the sentence starting with 'Again,…' ending at 'for 30 min'.

Authors' Response: Amendments have been made from lines 413–415. The sentence has been revised and corrected accordingly.

Comment 14

Figure titles do not adequately identify each species/cultivar by their corresponding letter (A to D). Each figure title should explicitly identify each letter and its corresponding cultivar. The same identification should be reflected in the text when citing figures.

Authors' Response: Amendments have been applied as follows: Figure 1 caption revised (lines 135–136) and corresponding text citations updated (lines 125–133); Figure 2 caption revised (lines 155–156) and text citations updated (lines 148–153); Figure 3 caption revised (lines 165–167) and text citations updated (lines 160–163); Figure 4 caption revised (lines 189–193) and text citations updated (lines 177–187); Figure 5 caption revised (lines 212–214) and text citations updated (lines 194–208); Figure 6 caption revised (lines 226–228) and text citations updated (lines 218–224).

Comment 15 (Line 120)

Cite the correct figure letter in this sentence. E.g., '…with a red center, and the diameter was 16.70 cm (Fig. 1A).'

Authors' Response: Amendment has been made at line 126. The figure citation has been corrected to include the appropriate panel letter as suggested.

Comment 17 (Lines 123–127)

Rewrite these sentences for clarity, and correctly identify figures as 1C and 1D.

Authors' Response: Amendments have been made from lines 181–184. The sentences have been rewritten for improved clarity and the figure references have been corrected to 1C and 1D.

Comment 18 (Lines 134–135)

Rewrite to: '…'Carousel Pink Passion' and 'Carousel Jolly Heart' were star-shaped and reniform, respectively (Fig. 2A and 2B), whereas…'

Authors' Response: Amendment has been made from lines 149–154. The sentence has been rewritten as suggested by the reviewer.

Comment 20 (Lines 144–145)

'To confirm the chromosome numbers' is repeated. Also, do not start a sentence with a numeral: '38 chromosomes were confirmed…' should read 'Thirty-eight chromosomes…'

Authors' Response: Amendments have been made from lines 161–163. The repeated phrase has been removed and the sentence has been rewritten to spell out the numeral at the beginning of the sentence, as recommended.

Comment 21 (Figure 4)

The figure title should indicate which colour corresponds to 5S and which to 18S rDNA, in addition to identifying each panel letter A–D.

Authors' Response: Amendment has been made from lines 189–193. The figure caption has been updated to clearly indicate the colour coding for 5S and 18S rDNA signals and to identify all panel letters A–D.

Comment 23 (Lines 169–170)

Instead of '…the method has been shown technique efficient…', please alter to '…the method has been shown to be technically efficient…'

Authors' Response: Amendment has been made from lines 296–298. The sentence has been corrected as recommended.

Comment 25 (General)

Overall, the discussion is well written, although it needs some editing of the grammar and English language.

Lines 281–282 (of the new version): Instead of: "In the present study, it was reported the chromosome count result of 'Sukim' and 'Freedom' for the first time, and the chromosome number was eighty-four." Please modify to: "In the present study, the chromosome count result of 'Sukim' and 'Freedom' was reported for the first time, and the chromosome number was 84."

Lines 309–311: The sentence "According to the findings in the present study, the average chromosomes length of H. moscheutos is 2.92 to 6.24 μm whereas, the chromosome length ranges for H. syriacus were 2.47 to 7.84 μm." needs improvement: please change to "…the average chromosome length of H. moscheutos was 2.92 to 6.24 μm, whereas the chromosome length ranges for H. syriacus were 2.47 to 7.84 μm."

Line 313: Instead of "....10.89 μm which, was slightly higher..." change the comma placement to: "....10.89 μm, which was slightly higher..."

Lines 320–321: Instead of "....type of chromosome and H. syriacus, were detected with metacentric and sub metacentric type of chromosome." please alter to: "....types of chromosomes, while H. syriacus, was detected with metacentric and sub metacentric types of chromosomes."

Line 338: The value cited in this sentence is incorrect. This sentence requires correction.

Lines 349–350: "From a practical standpoint, the genomic baselines established here chromosome number, karyotype structure, ...." Please modify to: "From a practical standpoint, the genomic baselines established here, such as chromosome number, karyotype structure, ..."

Authors' Response: All suggested amendments have been incorporated. Amendments are reflected at lines 278, 280, 281, 283, 284, 289, and 317. Specifically: chromosome numbers are now expressed as numerals throughout the Discussion; the passive voice construction has been corrected (lines 281–282); average chromosome length phrasing has been improved (lines 309–311); the misplaced comma has been corrected at line 313; the chromosome type description has been rewritten for grammatical accuracy (lines 320–321); the erroneous value at line 338 has been corrected; and the missing connective phrase has been inserted at lines 349–350.

We hope that the revised manuscript now meets the standards of the journal. We remain available to address any further concerns.

Sincerely,

The Authors

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has been substantially improved following revision; however, a minor inconsistency in cultivar naming remains. On page 5 (lines 147–148), the cultivar name “Carousel Jolly Pink” is still present, which appears inconsistent with the corrected name “Carousel Jolly Heart” used elsewhere. A final careful check of cultivar names throughout the manuscript would help ensure full consistency.

Although variability measures (mean ± SE) have now been included, the statistical support for descriptive comparisons could be further strengthened. Some statements in the Results section (e.g., “larger”, “smaller”) remain qualitative. It would improve clarity and scientific rigor if these comparisons were more explicitly supported, for example by referring directly to the tabulated values or, where appropriate, clarifying whether the differences are statistically meaningful.

The Results section has been improved, but its analytical depth could be further enhanced. In several parts, particularly in the karyotype-related sections (e.g., pages 10–11), results are presented as a sequence of numerical values without clear synthesis. Adding brief summary statements to highlight overall patterns (such as interspecific differences or general trends in chromosome structure), and integrating observations across traits, would improve readability and interpretation.

In addition, sentence structure and clarity could be further refined. In some sections, particularly in the Discussion, sentences are relatively long or contain overlapping ideas. Minor editing to streamline sentence structure and reduce redundancy would enhance readability and overall flow.

Finally, while the inclusion of both morphological and cytogenetic data is a clear strength of the study, the linkage between these two components could be made more explicit. A brief integrative statement connecting morphological variation with cytogenetic findings would strengthen the coherence of the manuscript and highlight its broader significance.

Comments on the Quality of English Language

Author Response

Response to Reviewer

We sincerely thank Reviewer 2 for the careful and constructive evaluation of our manuscript. The comments provided have been valuable in improving the quality of the work. Below, we address each comment individually. All revisions are indicated in the revised manuscript (changes highlighted in bold/tracked).

Reviewer 2 — Specific Comments and Authors’ Responses

Comment 1:

Authors’ Response:

We thank the reviewer for pointing out this remaining inconsistency. The cultivar name on page 5 (lines 147–148) has been corrected from “Carousel Jolly Pink” to “Carousel Jolly Heart” to align with the nomenclature used consistently throughout the rest of the manuscript. A thorough check of all cultivar names has been performed. Please see the amendments at lines 151–154 of the revised manuscript.

Comment 2:

Authors’ Response:

We appreciate this comment and agree that greater statistical precision strengthens the interpretability of the Results. Qualitative comparative statements (e.g., “larger”, “smaller”) have been revised to include direct references to the tabulated values and, where applicable, a clarification of statistical significance. The relevant amendments are found at lines 25–33, 125–136, and 150–156 of the revised manuscript.

Comment 3:

Authors’ Response:

This comment has been fully addressed in the revised manuscript. Brief integrative summary statements have been incorporated into the karyotype-related sections to synthesize numerical findings and highlight overarching patterns, including interspecific differences and general trends in chromosome structure. Observations across traits have been more clearly linked.

Comment 4:

Authors’ Response:

We agree with this observation. The identified grammatical issues have been corrected: the comma splice in the sentence describing Hibiscus taxonomy has been resolved, and the subject–verb agreement error (“six 18S signals was detected” → “six 18S signals were detected”) has been fixed. In addition, a comprehensive language review of the entire manuscript has been conducted to ensure grammatical accuracy and compliance with MDPI journal standards.

Comment 5:

Authors’ Response:

We thank the reviewer for this suggestion. The Discussion section has been revised to improve sentence structure and reduce redundancy. Overly long sentences have been divided and overlapping ideas have been consolidated to enhance readability and logical flow throughout the section.

Comment 6:

Authors’ Response:

We fully agree with this valuable comment. A concise integrative statement connecting morphological variation with cytogenetic findings has been added to highlight the complementary nature of the two data sets and the broader significance of our results. This addition reinforces the coherence of the manuscript and more clearly communicates its contributions to the field.

Language and Style Comments

Language Comment:

The manuscript has undergone language revision; however, minor grammatical issues remain. For example, the sentence “Hibiscus belongs to the Malvaceae family, comprises approximately 250 species…” contains a comma splice, and “six 18S signals was detected” shows subject–verb disagreement. A final round of careful language editing is recommended to ensure clarity and adherence to journal standards. In addition, sentence structure and clarity could be further refined. In some sections, particularly in the Discussion, sentences are relatively long or contain overlapping ideas. Minor editing to streamline sentence structure and reduce redundancy would enhance readability and overall flow.

Authors’ Response:

All language and grammatical issues identified by the reviewer have been corrected throughout the manuscript. Specifically, the comma splice and subject–verb agreement errors have been resolved (see also responses to Comments 4 and 5 above). The Discussion has been revised to improve sentence concision and reduce redundancy. A full editorial review of the manuscript has been performed to ensure compliance with MDPI language and style standards.

We hope that the revised manuscript now meets the standards required for publication. We remain available to address any further concerns.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

I want to thank the authors for accepting most of the suggestions made. However, there are still some details to improve, as described below. Please be aware of the observations in Comment 6, as there is a need to change the numbers of the sub-sections, such as sections 4.1, 4.2, 4.3, 4.4, and so on. It should end in 4.6. This was mentioned in our last review, but was not noticed by the authors.

Comment 1:

The abstract has been greatly improved. However, there are still some new details to correct.

The sentence: “Morphological assessments revealed notable differences in flower size and leaf shape between species” is now repeated in the text, in lines 23-24 and 25-26. Please delete it from lines 23-24.

And there are four scientific names of the species that need to be in italics, in lines 28 and 31-32.

Also, a brief concluding statement has not been added. But it is OK, no problem.

Comment 6:

The authors included the information asked, but they were included in 4.1 Plant materials sub-section, while they should have been included in 4.2 Phenotypic characteristics. Please provide this correction.

Also, the next sub-section should be numbered as 4.3. Root collection and chromosome preparation, but it is still numbered as 4.2. This means that ALL subsequent sections should be updated with the correct numbers, such as 4.4, 4.5, etc.

Comment 12: suggestion accepted.

Comment 14: suggestion accepted.

Comment 15: suggestion accepted

Comment 18: suggestion accepted. But, please remove one of the words ‘was’, which is repeated at the end of line 132, of the new version.

Comment 20 (Lines 144–145):

Suggestion partly accepted. The repeated part of the sentence was removed. But the sentence is still starting with a number (38 chromosomes ....)

Comment 21: suggestion accepted

Comment 23: suggestion accepted

Comment 25: Suggestions accepted. There is still a small correction.

The following modified sentence (lines 29-290 of the new version) needs a correction.

“In the present study, the chromosome count results of ‘Sukim’ and ‘Freedom’ for the first time, and the chromosome number was 84.

Please modify it to:

“In the present study, the chromosome count results of ‘Sukim’ and ‘Freedom’ were reported for the first time, and the chromosome number was 84.

Comments on the Quality of English Language

I still noticed some problems with the English language. Another review would be advised.

Author Response

Author Response to Reviewers

MDPI Manuscript Revision

We would like to thank the reviewers for their careful reading of our manuscript and their constructive comments. We have addressed all the comments as detailed below. The changes made in the revised manuscript are indicated in bold or tracked changes.

Note: Reviewer comments answers are shown in red writing.

Reviewer Comment 1

Comment 1: The sentence: "Morphological assessments revealed notable differences in flower size and leaf shape between species" is now repeated in the text, in lines 23–24 and 25–26. Please delete it from lines 23–24.

Response: The repeated sentence "Morphological assessments revealed notable differences in flower size and leaf shape between species" has been deleted from lines 23–24. Additionally, four scientific names of the species have been changed to italics in lines 26–31.

Amendment: Sentence deleted from lines 23–24; scientific names italicised in lines 26–31.

Reviewer Comment 6

Comment 6: The authors included the information asked, but it was included in Section 4.1 Plant materials, while it should have been included in Section 4.2 Phenotypic characteristics. Please provide this correction. Also, the next sub-section should be numbered as 4.3 Root collection and chromosome preparation, but it is still numbered as 4.2. This means that ALL subsequent sections should be updated with the correct numbers, such as 4.4, 4.5, etc.

Response: The requested information has been moved from Section 4.1 Plant materials to Section 4.2 Phenotypic characteristics. All subsequent sub-section numbers have been updated accordingly: the section previously numbered 4.2 is now numbered 4.3 (Root collection and chromosome preparation), and all following sub-sections have been renumbered as 4.4, 4.5, etc.

Amendment: Content relocated to Section 4.2; all subsequent sub-section numbers updated (4.3, 4.4, 4.5, …).

Reviewer Comment 18

Comment 18: Suggestion accepted. But, please remove one of the words 'was', which is repeated at the end of line 132 of the new version.

Response: The duplicate word 'was' has been removed from line 131. The sentence now reads correctly with a single instance of 'was'.

Amendment: Duplicate word removed at line 131.

Reviewer Comment 20 (Lines 144–145)

Comment 20: Suggestion partly accepted. The repeated part of the sentence was removed. However, the sentence is still starting with a number (38 chromosomes ...).

Response: The sentence beginning with the numeral '38' has been revised so that it no longer starts with a number, in accordance with standard academic writing conventions. The correction has been applied at lines 161–163 of the revised manuscript.

Amendment: Sentence revised at lines 161–163 to avoid beginning with a numeral.

Reviewer Comment 25

Comment 25: Suggestions accepted. There is still a small correction. The following modified sentence (lines 289–290 of the new version) needs a correction: "In the present study, the chromosome count results of 'Sukim' and 'Freedom' for the first time, and the chromosome number was 84." Please modify it to: "In the present study, the chromosome count results of 'Sukim' and 'Freedom' were reported for the first time, and the chromosome number was 84."

Response: The sentence has been corrected to include the missing verb phrase 'were reported'. The amended sentence now reads: "In the present study, the chromosome count results of 'Sukim' and 'Freedom' were reported for the first time, and the chromosome number was 84."

Amendment: Sentence corrected at lines 288–289: 'were reported' inserted.

General Comment on English Language

Comment: I still noticed some problems with the English language. Another review would be advised.

Response: We acknowledge this concern and have carefully revised the entire manuscript to improve the quality of the English language. We believe the revised manuscript now reads with greater fluency and precision.

We hope that our revisions satisfactorily address all reviewer concerns and that the manuscript is now suitable for publication.

Author Response File: Author Response.pdf

Review Reports