Natural Diversity of Crataegus monogyna Jacq. in Northeastern Türkiye Encompassing Morphological, Biochemical, and Molecular Features

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

See the attachment for specific comments

Comments for author File: Comments.pdf

Author Response

Responses to Comments of Reviewer 1

General Response:

First, we thank the potential reviewer for her/his valuable comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. All revised parts have been highlighted in yellow. We hope the revised version of the manuscript gets positive feedback from you and will be acceptable for publication in the Horticulturae journal.

Sincerely.

Comments

Comments 1# The manuscript clearly defines its objective to evaluate the genetic diversity in hawthorn genotypes using morphological, biochemical, and molecular markers. However, the study could further emphasize the significance of its findings in relation to the conservation and breeding programs for this species.

Response to Comment 1# Thank you for your feedback. Based on their feedback, improvements have been made to the all manuscript.

Comments 2# The methods used for genetic diversity assessment (ISSR markers), phenolic content analysis, and other biochemical measurements are robust. However, it would be beneficial to include more details on the experimental controls, particularly in the biochemical assays, to ensure reproducibility.

Response to Comment 2# Based on your feedback, improvements have been made to the manuscript. Analyzes of biochemical properties are given in detail. Individual phenolic compound analysis is detailed.

Comments 3# The manuscript mentions 21 genotypes; however, the justification for selecting this specific number of samples could be expanded. How does this sample size compare to previous studies, and does it provide enough statistical power for the diversity analysis?

Response to Comment 3# Based on your feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence " The genus Crataegus has a well-deserved reputation for taxonomic complexity. Extensive hybridization and introgression between closely related species have resulted in the formation of hybrid, polyploid and agamic complexes (Christensen, 1992, Martinelli et al., 2021). This is particularly relevant when closely related species grow in the vicinity of each other, e. g., C. monogyna hybridises easily with C. laevigata (Thomas et al., 2021). For this reason, genotypes were selected from areas where we thought there was no hybridization occurring. The narrowness of these areas in the region caused the number of genotypes to be low. There are studies on similar genotype numbers in the literature (such as Alirezalu et al., 2020; Yıldız et al., 2023)" has been added to the plant material section.

Comments 4# The presentation of data in the tables and figures is effective, but some visualizations, such as the PCA and heatmaps, could benefit from clearer labeling and legends. A more in-depth discussion of the PCA results could enhance the reader's understanding of the genotypic clustering and its biological significance.

Response to Comment 4# Thank you for your feedback. With these revisions, we have significantly improved the manuscript and produced a study that is better integrated with the literature, with a more comprehensive interpretation of the analysis results. Your feedback has been very valuable in improving the quality of our study. In line with your suggestions, PCA and heatmap analyses are explained in more depth in the discussion section.

Comments 5# While the ISSR markers reveal substantial genetic polymorphism, the manuscript could clarify how these results correlate with previous studies. For example, comparison with other ISSR or SSR-based studies on hawthorn could add depth to the discussion.

Response to Comment 5# Based on your feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence "One of the reliable markers in determining the genetic relationships of entire plant populations is molecular markers (Güney et al. 2018). During the last decade several novel DNA-based markers have been rapidly developed for characterizing the Crataegus genome and to study genetic diversity within and among wild landraces of this species. Such marker techniques include randomly amplified polymorphic DNA (RAPD) (Erfani-Moghadam et al. 2016), inter-simple sequence repeats (ISSRs) (Emami et al. 2018), and simple sequence repeats (SSRs) (Khiari et al. 2015), SSR, ISSR and SCoT markers (Mohammed et al., 2025)" has been added to the introduction section.

In addition, the molecular analysis section was enriched in the discussion section of the article. However, since the aim of the study was to evaluate morphological, biochemical and molecular markers together, not much literature information about molecular markers was included.

Comments 6# It would be useful to discuss the potential ecological factors influencing genetic diversity in the genotypes studied, such as altitude, climate, or soil type. A brief exploration of how environmental conditions might contribute to the observed genetic and biochemical diversity could add further insight.

Response to Comment 6# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence "It is known that the biochemical contents of fruits can be affected by genetic factors, environmental factors and post-harvest processes (Lorenza and Munekata, 2016). In addition, the amount of biochemical contents varies according to species and even genetic varieties within the same species (Wang et al., 2009)" has been added to the introduction section.

Comments 7# The phenolic compounds and antioxidant content analysis is comprehensive, but it would be beneficial to link the biochemical data more directly to the potential health benefits or industrial applications of these genotypes, as this would enhance the manuscript's relevance to both academic and practical audiences.

Response to Comment 7# Thank you for your feedback. With these revisions, we have significantly improved the manuscript and produced a study that is better integrated with the literature, with a more comprehensive interpretation of the analysis results. Your feedback has been very valuable in improving the quality of our study. Based on your suggestion, the discussion of biochemical properties has been rewritten by linking biochemical data to potential health benefits.

Comments 8# The manuscript does a good job of comparing its findings with previous studies. However, the comparison could be more robust by discussing more recent literature on Crataegus monogyna diversity studies, particularly in terms of molecular analysis and its applications in breeding and conservation.

Response to Comment 8# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. Care was taken to ensure that the resources covered within the scope of the study were as up-to-date as possible.

Comments 9# While the conclusions drawn are informative, the authors could expand on the potential practical applications of this research. Suggestions for future research directions, especially concerning the integration of genetic and phenotypic data in breeding programs or functional food development, would strengthen the impact of this study.

Response to Comment 9# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. The conclusion section of the article has been reviewed.

Reviewer 2 Report

Comments and Suggestions for Authors

This study provides valuable information on the genetic diversity of Crataegus monogyna genotypes in Türkiye and however, at present it is not suitable for publication yet as it lacks of several key issues, therefore, I recommend author to address the following points:

Important comments:

• Could authors clarify the rationale for selecting only 21 genotypes? Given the wide genetic diversity of Crataegus monogyna, a larger or more geographically diverse sample might provide a more comprehensive representation. If this sample size is a limitation, a brief justification would be helpful.
• This study would benefit from a deeper discussion on how environmental factors influence genetic and biochemical variations. Since these factors can play a crucial role, addressing them in more detail could add valuable context to the findings.
• Using ISSR markers to assess genetic diversity is a strong approach, but integrating functional analysis focused on specific phenotypic traits or biochemical properties would enhance the study’s impact. If feasible, validating the ISSR results with complementary methods like SSR, SNPs, or sequencing could strengthen the reliability of the conclusions.
• Comparing the findings with studies from other regions or related species would provide a broader perspective and help contextualize the results. Incorporating such comparisons would significantly enrich the discussion.
• While PCA and correlation analyses are insightful, exploring additional multivariate techniques such as hierarchical clustering, discriminant analysis, or regression models could offer deeper insights into genotype-trait relationships. These methods might help uncover patterns that are not immediately apparent.
• Lastly, the conclusion could be expanded to discuss the practical applications of these findings, particularly in breeding programs or commercial use. Highlighting how this research can be applied in real-world contexts would enhance its relevance and impact.

General comments:

• The abstract should include an introductory statement.
• Keywords are too lengthy. Please make them concise or choose more appropriate words.
• In Table 1, under the Region column, if possible, the author should mention the direction of the region, such as south, north, west, etc.
• Since this study dealt with genetics, is three replications sufficient? Is this replication for determination or sampling?
• Section 2.3 should have subheadings for each determination. Line 168 - use phenolic profile.
• In Table 2, statistical letters should be superscripted. A similar recommendation applies to all the other tables as well.
• Why does the data not contain standard deviation (SD) values? It should be included for every data set.
• Figures 1-5 are of poor resolution and should be replaced with high-quality figures. Furthermore, Figure 3 should be in the tables.

Author Response

Responses to Comments of Reviewer 2

General Response:

First, we thank the potential reviewer for her/his valuable comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. All revised parts have been highlighted in green. We hope the revised version of the manuscript gets positive feedback from you and will be acceptable for publication in the Horticulturae journal.

Sincerely.

Comments

Comments 1# Could authors clarify the rationale for selecting only 21 genotypes? Given the wide genetic diversity of Crataegus monogyna, a larger or more geographically diverse sample might provide a more comprehensive representation. If this sample size is a limitation, a brief justification would be helpful.

Response to Comment 1# Based on your feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence " The genus Crataegus has a well-deserved reputation for taxonomic complexity. Extensive hybridization and introgression between closely related species have resulted in the formation of hybrid, polyploid and agamic complexes (Christensen, 1992, Martinelli et al., 2021). This is particularly relevant when closely related species grow in the vicinity of each other, e. g., C. monogyna hybridises easily with C. laevigata (Thomas et al., 2021). For this reason, genotypes were selected from areas where we thought there was no hybridization occurring. The narrowness of these areas in the region caused the number of genotypes to be low. There are studies on similar genotype numbers in the literature (such as Alirezalu et al., 2020; Yıldız et al., 2023)" has been added to the plant material section.

Comments 2# This study would benefit from a deeper discussion on how environmental factors influence genetic and biochemical variations. Since these factors can play a crucial role, addressing them in more detail could add valuable context to the findings.

Response to Comment 2# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence "It is known that the biochemical contents of fruits can be affected by genetic factors, environmental factors and post-harvest processes (Lorenza and Munekata, 2016). In addition, the amount of biochemical contents varies according to species and even genetic varieties within the same species (Wang et al., 2009)" has been added to the introduction section.

Additionally, the effects of environmental conditions are discussed in the discussion section of the article.

Comments 3# Using ISSR markers to assess genetic diversity is a strong approach, but integrating functional analysis focused on specific phenotypic traits or biochemical properties would enhance the study’s impact. If feasible, validating the ISSR results with complementary methods like SSR, SNPs, or sequencing could strengthen the reliability of the conclusions.

Response to Comment 3# Thank you for your feedback. We definitely agree with your opinions. However, within the scope of the study, it was aimed to evaluate morphological, biochemical, individual phenolics and molecular markers together. Since genotypes were addressed with more than one marker, important literature information was included in the article. In addition, ISSR results were compared with different marker results. ISSR markers are widely used in genetic studies due to their ability to reveal high levels of polymorphism, which makes them valuable for assessing genetic diversity and population structure (Pradeep Reddy et al., 2002). The ISSR marker has been recognized as a reliable marker system in many plant species in recent years in the MDPI Horticulturae journal. (https://doi.org/10.3390/horticulturae10121364 - https://doi.org/10.3390/horticulturae10111146 - https://doi.org/10.3390/cimb46110700).

Comments 4# Comparing the findings with studies from other regions or related species would provide a broader perspective and help contextualize the results. Incorporating such comparisons would significantly enrich the discussion.

Response to Comment 4# Thank you for your feedback. We definitely agree with your opinions. Based on their feedback, improvements have been made to the manuscript. In line with your suggestion, the findings were enriched with the results of studies conducted in different regions.

Comments 5# Lastly, the conclusion could be expanded to discuss the practical applications of these findings, particularly in breeding programs or commercial use. Highlighting how this research can be applied in real-world contexts would enhance its relevance and impact.

Response to Comment 5# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. The conclusion section of the article has been reviewed.

General comments:

Comments 1# The abstract should include an introductory statement.

Response to Comment 1# Thank you for your feedback. In line with your suggestion, the sentence "Hawthorn (Cratageus monogyna), one of the important wild fruit species in Turkey, is an important fruit species in many countries with its use in traditional medicine, landscaping studies, and the food and beverage industry" has been added to the abstract section.

Comments 2# Keywords are too lengthy. Please make them concise or choose more appropriate words.

Response to Comment 2# Thank you for your feedback. According to your suggestion, keywords are abbreviated as “Genetic diversity; hawthorn; biochemical content; multivariate analysis; ISSR marker”.

Comments 3# In Table 1, under the Region column, if possible, the author should mention the direction of the region, such as south, north, west, etc.

Response to Comment 3# Thank you for your feedback. Based on their feedback, improvements have been made to the table.

Comments 4# Since this study dealt with genetics, is three replications sufficient? Is this replication for determination or sampling?

Response to Comment 4# Thank you for your feedback. In order to clarify this issue, the necessary corrections have been made in line with your suggestions. In line with your suggestion, the sentence "In the study, fruit and leaf analyses were performed for each of the naturally growing hawthorn genotypes in three replications. In each replication, the necessary measurements were performed on 20 randomly selected fruits and leaves. Fruit samples were collected at the maturity stage in late September and early October" has been added to the Fruit and leaf characterization section.

Comments 5# Section 2.3 should have subheadings for each determination. Line 168 - use phenolic profile.

Response to Comment 5# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript.

Comments 6# In Table 2, statistical letters should be superscripted. A similar recommendation applies to all the other tables as well.

Response to Comment 6# Thank you for your feedback. Based on their feedback, improvements have been made to the table.

Comments 7# Why does the data not contain standard deviation (SD) values? It should be included for every data set.

Response to Comment 7# Thank you for your feedback. Based on their feedback, improvements have been made to the table.

Comments 8# Figures 1-5 are of poor resolution and should be replaced with high-quality figures. Furthermore, Figure 3 should be in the tables.

Response to Comment 8# Thank you for your feedback. Based on their feedback, improvements have been made to the table. The resolution of the figures has been increased.

Reviewer 3 Report

Comments and Suggestions for Authors

Please re-number the subchapters in the paper. After 3.2, 3.1 appears twice.

ABSTRACT

In the biochemistry section, the total flavonoid and phenol content have the same minimum and maximum values, which seems suspicious. Is this an error or a coincidence?

            Some values are difficult for readers to interpret:

- Ex: "Antioxidant activity (29.67% to 29.20%)" → decreasing instead of increasing?

- Is it a correct range or a drafting error?

1. It is mentioned that these findings are useful for conservation and reproduction, but it is not clear how the results will be applied. For example:

- Genotypes with the highest content of bioactive compounds are recommended for selection?

- Are there any genotypes with exceptional potential for the pharmaceutical or food industry?

- Which genotypes are more valuable and why?

2. You mention 55 polymorphic bands out of 62, but do not specify what type of ISSR markers were used (e.g. 3' or 5' anchors, number of primers). The Jaccard coefficient is also reported, but it is not clearly stated whether the clustering analysis was confirmed by an additional statistical method (e.g. bootstrap).

INTRODUCTION

1. Rather than simply listing the properties of hawthorn, the introduction should emphasize why the genetic diversity of this species should be specifically investigated in the Kelkit Valley.

2. Why is this study important in the wider context of research on Crataegus monogyna?

3. There are overlaps between the different paragraphs dealing with the bioactive components and importance of hawthorn. The first part discusses the benefits of phenolics, and the same information on flavonoids and bioactive compounds is repeated below. Unifying this information would make the introduction clearer and more coherent.

4. Genetic diversity is mentioned as influencing biochemical properties, but it is not clear whether previous studies have already demonstrated this or whether it is just a hypothesis. It also talks about practical use in conservation and breeding, but does not specify how this information can guide such programs.

MATERIALS AND METHOD

1. A more detailed description of the criteria used for the selection of the 21 genotypes would be useful, not just mentioning differences in traits.
2. Avoid repetition! For example, in the section on measurements, you should mention only once that all measurements were made with specific equipment, and then only detail the parameters measured.
3. Biochemical analysis has many subsections, which can make it difficult to read. A clearer grouping (e.g. phenolics, flavonoids, anthocyanins) with a short introduction might help.
4. The Minolta CR-300 chromometer is mentioned, but a brief explanation of the advantages of the CIE Lab method over other methods would be helpful.
5. PCR parameters could be justified by a reference or an explanation of primer selection.
6. The paper states, "They were measured... using a digital caliper". Please explain how the accuracy of the measurements (e.g. number of measurements per leaf) was ensured?
7. For statistical and genetic analysis XLSTAT and R are mentioned, but it would be useful to specify the software versions and packages used in R for reproducibility.
8. Avoid excessive repetition of "Later" at the beginning of sentences.

RESULTS and DISCUSSIONS

1. The clarity of transitions between sections can be improved. For example, the discussion of morphological traits (fruits, leaves) can be more clearly linked to the biochemical analysis so that the reader better understands how these traits relate to each other and why they are important to the study.
2. It would be helpful to more clearly emphasize the importance of the reported results, especially when discrepancies between your study and previous studies are noted. For example, you could explain in more detail the possible reasons for these differences (e.g. variability of genotypes, experimental conditions, techniques used).
3. In the section dealing with "statistically significant differences", it would be useful to include more details about the statistical tests used (type of test, p-value, etc.) and explain the significance of these differences for the context of the study. This would help readers better understand the relevance of these results.
4. In the sections where you compare your results with the existing literature, you could add some additional comments that clarify why some discrepancies may be significant. For example, a few considerations about whether environmental conditions, data processing techniques, or accessibility of genetic material may explain variations between your studies and previous ones might be useful.
5. You mentioned the biplot plot and heat maps, but did not discuss much about the implications of these visualizations. It would be useful to provide a detailed interpretation of how traits influence the distribution of genotypes and which groupings are most relevant for selection and improvement.
6. Try to structure your ideas so that each section builds on the previous one. For example, the discussion of correlations could be linked more closely to the PCA analysis and heat maps to make a clear connection between morphological and biochemical data.

CONCLUSIONS

1. It would be useful to elaborate a little more on the practical implications of the results for the agricultural and medicinal fields. For example, what does it mean in practice to identify a 'promising' genotype for breeding? How could these genotypes influence the hawthorn breeding process in practical terms?
2. Emphasize further how your study extends or complements previous knowledge about hawthorn, especially in the context of genetics and phytochemistry.
3. In your conclusions, it would be interesting to include some recommendations for future research. For example, what other methods could be applied to further explore the medicinal potential of hawthorn? How could conservation strategies evolve based on your findings?
4. In the last sentences of the conclusions, "unusual and unusual genotypes" is repeated. A more fluid variant could be "rare and distinct genotypes" to avoid redundancy.

Author Response

Responses to Comments of Reviewer 3

General Response:

First, we thank the potential reviewer for her/his valuable comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. All revised parts have been highlighted in blue. We hope the revised version of the manuscript gets positive feedback from you and will be acceptable for publication in the Horticulturae journal.

Sincerely.

Comments

Comments 1# Please re-number the subchapters in the paper. After 3.2, 3.1 appears twice.

Response to Comment 1# Thank you for your feedback. Based on their feedback, improvements have been made to re-number the subchapters.

ABSTRACT

Comments 1# In the biochemistry section, the total flavonoid and phenol content have the same minimum and maximum values, which seems suspicious. Is this an error or a coincidence?

Response to Comment 1# Thank you for your feedback. This situation was the result of an error. Based on their feedback, improvements have been made to the abstract.

Comments 2# Some values are difficult for readers to interpret:- Ex: "Antioxidant activity (29.67% to 29.20%)" → decreasing instead of increasing?

Response to Comment 2# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript.

Comments 3# It is mentioned that these findings are useful for conservation and reproduction, but it is not clear how the results will be applied. For example:- Genotypes with the highest content of bioactive compounds are recommended for selection?- Are there any genotypes with exceptional potential for the pharmaceutical or food industry?- Which genotypes are more valuable and why?

Response to Comment 3# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript.

Comments 4# You mention 55 polymorphic bands out of 62, but do not specify what type of ISSR markers were used (e.g. 3' or 5' anchors, number of primers). The Jaccard coefficient is also reported, but it is not clearly stated whether the clustering analysis was confirmed by an additional statistical method (e.g. bootstrap).

Response to Comment 4# Thank you for your feedback. The number of ISSR primers used and primer sequences are detailed in Table 1.

INTRODUCTION

Comments 1# Rather than simply listing the properties of hawthorn, the introduction should emphasize why the genetic diversity of this species should be specifically investigated in the Kelkit Valley.

Response to Comment 1# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence "The Kelkit Valley is a geographical transition region between the Black Sea region and the Central Anatolia region. The region has both Black Sea climate and continental climate. Kelkit Valley is very rich in terms of natural resources and biodiversity and the climate and soil conditions are extremely suitable for agricultural production. The Kelkit Valley is at the intersection of the European-Siberian and Iran-Turanian floristic regions. It is also seen in plants belonging to the Mediterranean floristic region (Karaer and Kılınç 2001). Kelkit Valley is located within grid squares A6 and A7 in the grid system created by Davis (Davis, 1965-1988). In a study carried out in the region, 2800 plants were collected. It was determined that the collected plants consisted of 519 genera and 1316 species and subspecific taxa belonging to 105 families, and 132 of these taxa were endemic  (Karaer ve Kılınç, 2001). Hawthorn has been growing naturally for hundreds of years in the Kelkit Valley, which is rich in both climate and plant diversity. For this reason, the study area is very rich in hawthorn genetic resources" has been added to the introduction section.

Comments 2# Why is this study important in the wider context of research on Crataegus monogyna?

Response to Comment 2# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence "It is possible to see hawthorn trees in almost every region of Turkey due to its low water requirement and its ability to grow naturally in mountainous and rocky areas (Çalışkan et al., 2022). Polymorphism and hybridization in hawthorns are very high and genetic diversity extends from Turkey to Iran (Donmez, 2007). Hawthorns, which have grown spontaneously and openly in nature for many years, are propagated by seed (Tadeo et al., 2017). This situation creates a high level of genetic diversity in nature through hybridization of different species and genotypes over the years. Although many hawthorn species are grown for their edible fruits in Asia, Central America and the Mediterranean countries, the species commonly grown in the Mediterranean countries, including Turkey, is Crataegus monogyna Jacq (Çalışkan, 2018)" has been added to the introduction section.

Comments 3# There are overlaps between the different paragraphs dealing with the bioactive components and importance of hawthorn. The first part discusses the benefits of phenolics, and the same information on flavonoids and bioactive compounds is repeated below. Unifying this information would make the introduction clearer and more coherent.

Response to Comment 3# Thank you for your feedback. We agree with your opinion. Based on their feedback, improvements have been made to the manuscript. The sections on bioactive compounds in the introduction of the manuscript have been shortened.

Comments 4# Genetic diversity is mentioned as influencing biochemical properties, but it is not clear whether previous studies have already demonstrated this or whether it is just a hypothesis. It also talks about practical use in conservation and breeding, but does not specify how this information can guide such programs.

Response to Comment 4# Thank you for your feedback. We agree with your opinion. Based on their feedback, improvements have been made to the manuscript. Throughout the article, information on how this information will guide you is included in line with your suggestion. In addition, information on the factors affecting biochemical properties is provided in the discussion section.

MATERIALS AND METHOD

Comments 1# A more detailed description of the criteria used for the selection of the 21 genotypes would be useful, not just mentioning differences in traits.

Response to Comment 1# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence "Fruit and seed weights were measured with a digital scale with a sensitivity of 0.01 g. Dimensional measurements of fruit and leaves were measured with a digital caliper with a sensitivity of 0.01 mm. The number of seeds was expressed by numerically determining the seeds coming out of each fruit" has been added to the plant material section.

Comments 2# Biochemical analysis has many subsections, which can make it difficult to read. A clearer grouping (e.g. phenolics, flavonoids, anthocyanins) with a short introduction might help.

Response to Comment 2# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence "Biochemical measurements were performed on a repetitive basis for each genotype. After the fruits were washed with water and dried, the seeds of the fruits were removed. The fruits separated from their seeds were taken to a food processor and turned into a homogeneous puree. The pureed fruits were stored at -20 °C until biochemical analyses were performed. After the samples were thawed at room conditions for analysis, they were centrifuged at 12,000 x g for 35 minutes at 4 °C. The filtrate obtained was used to determine the total phenolic content, total flavonoid content, antioxidant activity (DPPH) and anthocyanin content of the samples. UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan) was used to determine the biochemical compounds" has been added to the biochemical analysis section.

Comments 3# The Minolta CR-300 chromometer is mentioned, but a brief explanation of the advantages of the CIE Lab method over other methods would be helpful.

Response to Comment 3# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence "The CIE Lab color space is based on color difference formulas. This method provides a lot of information in evaluating color differences (Hill et al., 1997). The CIE Lab method provides simpler, more reliable and closer to reality color tones compared to other color methods (Corbala et al., 2002; Recky and Leberl, 2010). For this reason, the CIE Lab method was preferred in the study" has been added to the fruit and leaf characterization section.

Comments 4# PCR parameters could be justified by a reference or an explanation of primer selection.

Response to Comment 4# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence "In total, 20 different ISSR primers were screened for polymorphism using eight randomly picked hawhorn genotypes. Consequently, only 7 of the most polymorphic primers with clear and visible banding patterns were chosen for the genetic diversity assessment of all genotypes" has been added to the molecular analaysis section.

Comments 5# The paper states, "They were measured... using a digital caliper". Please explain how the accuracy of the measurements (e.g. number of measurements per leaf) was ensured?

Response to Comment 5# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence "In the study, fruit and leaf analyses were performed for each of the naturally growing hawthorn genotypes in three replications. In each replication, the necessary measurements were performed on 20 randomly selected fruits and leaves. Fruit samples were collected at the maturity stage in late September and early October.  Fruit and seed weights were measured with a digital scale with a sensitivity of 0.01 g. Dimensional measurements of fruit and leaves were measured with a digital caliper with a sensitivity of 0.01 mm. The number of seeds was expressed by numerically determining the seeds coming out of each fruit." has been added to the fruit and leaf characterization section.

Comments 6# For statistical and genetic analysis XLSTAT and R are mentioned, but it would be useful to specify the software versions and packages used in R for reproducibility.

Response to Comment 5# Thank you for your feedback. Based on their feedback, improvements have been made to the statistical and genetic analysis.

Comments 7# Avoid excessive repetition of "Later" at the beginning of sentences.

Response to Comment 5# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript.

RESULTS and DISCUSSIONS

Comments 1#The clarity of transitions between sections can be improved. For example, the discussion of morphological traits (fruits, leaves) can be more clearly linked to the biochemical analysis so that the reader better understands how these traits relate to each other and why they are important to the study.

Response to Comment 1# Thank you for your feedback. We agree with your opinion. Based on their feedback, improvements have been made to the manuscript. The discussion section has been enriched according to your suggestion.

Comments 2#It would be helpful to more clearly emphasize the importance of the reported results, especially when discrepancies between your study and previous studies are noted. For example, you could explain in more detail the possible reasons for these differences (e.g. variability of genotypes, experimental conditions, techniques used).

Response to Comment 2# Thank you for your feedback. We agree with your opinion. Based on their feedback, improvements have been made to the manuscript. The discussion section has been enriched according to your suggestion.

Comments 3#In the section dealing with "statistically significant differences", it would be useful to include more details about the statistical tests used (type of test, p-value, etc.) and explain the significance of these differences for the context of the study. This would help readers better understand the relevance of these results.

Response to Comment 3# Thank you for your feedback. Based on their feedback, improvements have been made to the table. In line with your suggestion, the sentence " ¹The difference between the averages indicated by different letters letter in the same column is significant (p≤0.05, p≤0.01, p≤0.001, ns: non significant).”has been added to the tables.

Comments 4#In the sections where you compare your results with the existing literature, you could add some additional comments that clarify why some discrepancies may be significant. For example, a few considerations about whether environmental conditions, data processing techniques, or accessibility of genetic material may explain variations between your studies and previous ones might be useful.

Response to Comment 4#Thank you for your feedback. We agree with your opinion. Based on their feedback, improvements have been made to the manuscript. The discussion section has been enriched according to your suggestion.

Comments 5#You mentioned the biplot plot and heat maps, but did not discuss much about the implications of these visualizations. It would be useful to provide a detailed interpretation of how traits influence the distribution of genotypes and which groupings are most relevant for selection and improvement.

Response to Comment 5#Thank you for your feedback. We agree with your opinion. Based on their feedback, improvements have been made to the manuscript. The discussion section has been enriched according to your suggestion.

Comments 6#Try to structure your ideas so that each section builds on the previous one. For example, the discussion of correlations could be linked more closely to the PCA analysis and heat maps to make a clear connection between morphological and biochemical data.

Response to Comment 6# Thank you for your feedback. We agree with your opinion. Based on their feedback, improvements have been made to the manuscript. The discussion section has been enriched according to your suggestion. In line with your suggestion, the sentence " It is known that there is a positive correlation between narrow-angle features and a negative correlation between wide-angle features in the principal component analysis graph (Garazhian et al., 2020). Accordingly, the correlation analysis results coincide with the two-dimensional PCA graph results”has been added to the disscusion.

CONCLUSIONS

Comments 1# 1.         It would be useful to elaborate a little more on the practical implications of the results for the agricultural and medicinal fields. For example, what does it mean in practice to identify a 'promising' genotype for breeding? How could these genotypes influence the hawthorn breeding process in practical terms?

2.Emphasize further how your study extends or complements previous knowledge about hawthorn, especially in the context of genetics and phytochemistry.

3.In your conclusions, it would be interesting to include some recommendations for future research. For example, what other methods could be applied to further explore the medicinal potential of hawthorn? How could conservation strategies evolve based on your findings?

4.In the last sentences of the conclusions, "unusual and unusual genotypes" is repeated. A more fluid variant could be "rare and distinct genotypes" to avoid redundancy.

Response to Comment 1# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. The conclusion section of the article has been reviewed.

Reviewer 4 Report

Comments and Suggestions for Authors

Determination of genetic diversity in Crataegus monogyna L. species in Türkiye in terms of morphological, biochemical, individual phenolics and molecular

By Bora Erkek et al.

REVIEW

As stated in the title, this article analyses biological, or natural, diversity of the medicinal plant species, Crataegus monogyna L., growing in the wild in the northcentral-eastern Türkiye. The paper is well-written and covers a variety of methods used to evaluate morphological, biochemical and molecular-genetic diversity of the species by studying 21 genotypes occurring in the valley of Kelkit river. Nevertheless, there are some points to be considered before proceeding with the paper.

The genus Crataegus has a well-deserved reputation for taxonomic complexity. Extensive hybridization and introgression between closely related species have resulted in the formation of hybrid, polyploid and agamic complexes (see Christensen, K. I.: The structure of some Crataegus (Rosaceae) populations in Greece. – Willdenowia 22: 65-79. 1992). This is particularly relevant when closely related species grow in the vicinity of each other, e. g., C. monogyna hybridises easily with C. laevigata (Thomas et al., 2021). The authors should have considered this point in advance. Hopefully this is not the case, but it would be very appropriate to discuss this question in the Results and Discussion. Below are my minor comments and suggestions.

I suggest modifying the title to, e. g., “Natural diversity of Crataegus monogyna L. in northeastern Türkiye encompassing morphological, biochemical, and molecular features”.

In Abstract, lines 31–32: Indicate dimensions of phenolics determined, e. g., mg/100 g of fresh or dry weight.

Line 51 (and some other places): “phenolics and antioxidants” – bear in mind that phenolics are considered antioxidants too.

Line 127: “C.I.E. L*a*b method” – Explain please, what is this method.

Line 213: Replace “Hawthorn“ with „hawthorn“.

Table 2: What does it mean double asterisk (**) at each value in the bottom line?

In the text, there is no reference to Table 3. The abbreviations in Table 3 – L*, a*, b* – are not explained.

Tables 2 through 4 are quite overloaded with numbers and letters, particularly Table 4. Authors may consider using bar chart instead.

Line 307: Replace “Colorimetric and biochemical properties“ with „Fruit colorimetric and biochemical properties“.

Before Line 368: Missing reference in the text to Table 4.

Line 368: Replace “Individual phenolic compounds contents” with “Contents of individual phenolic compounds in fruit”.

Line 438: Replace “traits” with “traits of fruit”.

Line 462: Replace “Table 1” with “Table 5”.

Page 10: missing page number and then restarting from 1 again.

Figure 1: Missing reference to this Figure in the text.

See attached manuscript pdf for these comments.

Comments for author File: Comments.pdf

Author Response

Responses to Comments of Reviewer 4

General Response:

First, we thank the potential reviewer for her/his valuable comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. All revised parts have been highlighted in purple. We hope the revised version of the manuscript gets positive feedback from you and will be acceptable for publication in the Horticulturae journal.

Sincerely.

Comments

Comments 1# The genus Crataegus has a well-deserved reputation for taxonomic complexity. Extensive hybridization and introgression between closely related species have resulted in the formation of hybrid, polyploid and agamic complexes (see Christensen, K. I.: The structure of some Crataegus (Rosaceae) populations in Greece. – Willdenowia 22: 65-79. 1992). This is particularly relevant when closely related species grow in the vicinity of each other, e. g., C. monogyna hybridises easily with C. laevigata (Thomas et al., 2021). The authors should have considered this point in advance. Hopefully this is not the case, but it would be very appropriate to discuss this question in the Results and Discussion.

Response to Comment 1# Thank you for your feedback. Based on your feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence " The genus Crataegus has a well-deserved reputation for taxonomic complexity. Extensive hybridization and introgression between closely related species have resulted in the formation of hybrid, polyploid and agamic complexes (Christensen, 1992, Martinelli et al., 2021). This is particularly relevant when closely related species grow in the vicinity of each other, e. g., C. monogyna hybridises easily with C. laevigata (Thomas et al., 2021). For this reason, genotypes were selected from areas where we thought there was no hybridization occurring. The narrowness of these areas in the region caused the number of genotypes to be low. There are studies on similar genotype numbers in the literature (such as Alirezalu et al., 2020; Yıldız et al., 2023)" has been added to the plant material section.

Comments 2# I suggest modifying the title to, e. g., “Natural diversity of Crataegus monogyna L. in northeastern Türkiye encompassing morphological, biochemical, and molecular features”.

Response to Comment 2# Thank you for your feedback. Based on your feedback, improvements have been made to the manuscript. Your suggestion has been accepted and the title has been changed.

Comments 3# In Abstract, lines 31–32: Indicate dimensions of phenolics determined, e. g., mg/100 g of fresh or dry weight.

Response to Comment 3# Thank you for your feedback. Based on your feedback, improvements have been made to the manuscript.

Comments 4# Line 51 (and some other places): “phenolics and antioxidants” – bear in mind that phenolics are considered antioxidants too.

Response to Comment 4# Your suggestion has been accepted and the necessary arrangements have been made.

Comments 5# Line 127: “C.I.E. L*a*b method” – Explain please, what is this method.

Response to Comment 5# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript. In line with your suggestion, the sentence "The CIE Lab color space is based on color difference formulas. This method provides a lot of information in evaluating color differences (Hill et al., 1997). The CIE Lab method provides simpler, more reliable and closer to reality color tones compared to other color methods (Corbala et al., 2002; Recky and Leberl, 2010). For this reason, the CIE Lab method was preferred in the study" has been added to the fruit and leaf characterization section.

Comments 6#Line 213: Replace “Hawthorn“ with „hawthorn“.

Response to Comment 6# Your suggestion has been accepted and the necessary arrangements have been made.

Comments7#Table 2: What does it mean double asterisk (**) at each value in the bottom line?

Response to Comment 7# Thank you for your feedback. Based on their feedback, improvements have been made to the table. In line with your suggestion, the sentence " ¹The difference between the averages indicated by different letters letter in the same column is significant (p≤0.05, p≤0.01, p≤0.001, ns: non significant)”has been added to the tables.

Comments 8#In the text, there is no reference to Table 3. The abbreviations in Table 3 – L*, a*, b* – are not explained.

Response to Comment 8# Thank you for your feedback. Based on their feedback, improvements have been made to the manuscript and table. In line with your suggestion, the sentence " The letter L* represents the change in the brightness of the color (L; 0 is black, 100 is white), the letter a* represents the color change from green to red (positive values ​​are red, negative values ​​are green), the letter b* represents the color change from yellow to blue (positive values ​​are yellow, negative values ​​are blue)” has been added to the tables.

Comments 9#Tables 2 through 4 are quite overloaded with numbers and letters, particularly Table 4. Authors may consider using bar chart instead.

Response to Comment 9# Thank you for your feedback. In line with your suggestion, table 4 is presented as a graphical supplementary file.

Comments 8#Line 307: Replace “Colorimetric and biochemical properties“ with „Fruit colorimetric and biochemical properties“.

Response to Comment 8# Your suggestion has been accepted and the necessary arrangements have been made.

Comments 9#Before Line 368: Missing reference in the text to Table 4.

Response to Comment 9# Your suggestion has been accepted and the necessary arrangements have been made.

Comments 10#Line 368: Replace “Individual phenolic compounds contents” with “Contents of individual phenolic compounds in fruit”

Response to Comment 10# Your suggestion has been accepted and the necessary arrangements have been made

Comments 11#Line 438: Replace “traits” with “traits of fruit”.

Response to Comment 11# Your suggestion has been accepted and the necessary arrangements have been made

Comments 12#Line 462: Replace “Table 1” with “Table 5”.

Response to Comment 12# Your suggestion has been accepted and the necessary arrangements have been made

Comments 13#Page 10: missing page number and then restarting from 1 again.

Response to Comment 13# Your suggestion has been accepted and the necessary arrangements have been made

Comments 14#Figure 1: Missing reference to this Figure in the text.

Response to Comment 14# Your suggestion has been accepted and the necessary arrangements have been made

Comments 15#Replace with 'Jacq.'

Response to Comment 15# Your suggestion has been accepted and the necessary arrangements have been made

Comments 16#Looks like incomplete title. I would add a word 'features' or 'indicator

Response to Comment 16# Thank you for your feedback. Based on your feedback, improvements have been made to the manuscript. Your suggestion has been accepted and the title has been changed.

Comments 17#Replace with "In this study"

Response to Comment 17# Your suggestion has been accepted and the necessary arrangements have been made

Comments 18#Are these amounts in mg/100 g of fruit fresh or dry weight? State, please.

Response to Comment 18# Your suggestion has been accepted and the necessary arrangements have been made

Comments 19#Altitude, m

Response to Comment 19# Your suggestion has been accepted and the necessary arrangements have been made

Comments 20#Missing reference in the text to Table 4.

Response to Comment 20# Your suggestion has been accepted and the necessary arrangements have been made

Comments 21#Missing reference to this Figure in the text.

Response to Comment 21# Your suggestion has been accepted and the necessary arrangements have been made

Comments 22#This should be Table 5 (?)

Response to Comment 22# Your suggestion has been accepted and the necessary arrangements have been made

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept in present form.

Reviewer 2 Report

Comments and Suggestions for Authors

Authors have addressed all the given comments sufficiently, this paper can be accepted in its present form.

Comments on the Quality of English Language

none

Reviewer 3 Report

Comments and Suggestions for Authors

Congratulations for all your research work and for writing this paper!