Species Differentiation of Prunus serrulata and Prunus xueluoensis Based on Combined Analysis of SSR and cpDNA Markers

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Even if the topic of this paper is current issue and relatively trend topic, the papers cited in text with publication years of 2021-2024 is very limited. Please add current dated articles to text and references.

Lines 41-48 need referecence citation in text

Please remove grey colour from Table S8

Quantitative analysis of DNA should be presented (spectrophotometric assay type)

For PCR conditions please give concentration/total amount of DNA such as 50 ng template DNA

Did PCR bands visualized by agarose electrophoresis? If yes, please give provide electrophoresis details (% agarose, voltage, duration etc.).

If there is IDE such as pycharm or else for Python based assays, please write it

Figure 5 should be presented with higher resolution quality

It is hard to follow Figure 8. Maybe subfigures fig 8.a, b, and c can be presented vertically and larger

Author Response

Comments 1: Even if the topic of this paper is current issue and relatively trend topic, the papers cited in text with publication years of 2021-2024 is very limited. Please add current dated articles to text and references.

Response 1: Thank you for pointing out the importance of including more recent references. We recognize the significance of incorporating the latest literature in our discussion. We have revised the manuscript to include additional articles from 2021-2024, ensuring that our references reflect the most current research in the field.

This change can be found on page18, line 539-549.

Comments 2: L97: 41-48 need referecence citation in text.

Response 2: Thank you for pointing this out. Regarding the section you mentioned, we have revised the text, which has now become a study on the morphological characteristics of P. serrulata and P. xueluoensis.

This change can be found on page2, paragraph 1 line 47-52.

In terms of morphology, P. xueluoensis possesses most traits typical of the Subg. Cerasus, but its axillary three-bud and shrubby characteristics significantly differ from the single axillary buds of Prunus serrulate and other typical species in the Subg. Cerasus. There is considerable morphological variation among individuals, which can easily lead to misidentification or confusion with similar species.

Comments 3；Please remove grey colour from Table S8.

Response 3: The grey in Table S8 has been removed by us.

Comments 4；Quantitative analysis of DNA should be presented (spectrophotometric assay type).

Response 4: Thank you for your suggestion. We will provide the quantitative analysis of DNA using the spectrophotometric assay method.

This change can be found on page5, paragraph 1 line 123.

Quantitative analysis of DNA used a UV-Vis spectrophotometer.

Comments 5；For PCR conditions please give concentration/total amount of DNA such as 50 ng template DNA.

Response 5: Thank you for your suggestion. Regarding the PCR conditions, we have provided a more detailed description in the text.

This change can be found on page5, paragraph 1 line 123-129.

The PCR amplification program consists of an initial denaturation step at 95.5°C for 4 minutes, followed by 30-35 cycles of denaturation at 94°C for 45 seconds, annealing at 50-65°C for 45 seconds, and extension at 72°C for 45 seconds. A final extension occurs at 72°C for 5 minutes, with the reaction maintained at 10°C for storage. The amplifica-tion system includes 12.5 μL of 2× Taq PCR Master Mix, 0.5 μL each of Primer 1 and Primer 2, 2.5 μL of DNA (greater than 20 ng/μL), and 9 μL of ddH2O, resulting in a to-tal volume of 25 μL.

Comments 6；Did PCR bands visualized by agarose electrophoresis? If yes, please give provide electrophoresis details (% agarose, voltage, duration etc.).

Response 6: Yes, the PCR bands were visualized using agarose gel electrophoresis. The details are as follows: we used a 1% agarose gel, the electrophoresis was performed at a voltage of 100 V for 30 minutes.

This change can be found on page4, paragraph 1 line 111.

with a voltage setting of 100 volts and a duration of 30 minutes.

Comments 7；If there is IDE such as pycharm or else for Python based assays, please write it.

Response 7: import numpy as np

import matplotlib. pyplot as plt

# Set random seed for reproducibility

np.random.seed(42)

# Generate random data

data_size = 100

data1 = np.random.randint(0, 10, size=data_size)

data2 = np.random.randint(0, 10, size=data_size)

# Calculate the mismatch

mismatch = np.abs(data1 - data2)

# Generate X-axis data (assuming data indices)

x = np.arange(data_size)

# Plot the line chart

plt.figure(figsize=(12, 6))

plt.plot(x, mismatch, marker='o', linestyle='-', color='b', label='Mismatch')

plt.title('Mismatch Analysis Line Chart')

plt.xlabel('Data Index')

plt.ylabel('Mismatch Value')

plt.xticks(x)  # Show all data indices

plt.yticks(np.arange(0, np.max(mismatch)+1, 1))  # Display Y-axis value range

plt.grid(True)

plt.legend()

plt.show()

Comments 8；Figure 5 should be presented with higher resolution quality.

Response 8: Thank you for pointing out

The resolution of Figure 5 has been modified accordingly on page 10.

Comments 9；It is hard to follow Figure 8. Maybe subfigures fig 8.a, b, and c can be presented vertically and larger.

Response 9: Thank you for your suggestions. We have adjusted the lines and labels in Figure 8 to make them clearer and more visible.

This change can be found on page13.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript by Gao et al. titled as “Species differentiation of Prunus serrulata and Prunus xueluoensis based on combined analysis of SSR and cpDNA markers” reports results of analyses on genetic structure of 13 populations from seven localities of two currently recognised species of Prunus. Authors aim for resolving the controversy around taxonomic status of a recently recognised Prunus xueluoensis. They employ two types of molecular markers, namely 18 loci of SSSRs and sequences from three loci cpDNA genome. The sampling size of molecular data is substantial and the whole study could be characterised as a standard approach for resolving taxonomic issues at the level of closely related species. However, the lack of discussion on morphological traits of the involved taxa in the reported study is a serious drawback.

Authors report also results of analyses on genetic diversity within populations of the studied taxa and found that the diversity is high in both taxa. Analysing genetic diversity in P. serrulate, they found high diversity in the chloroplast sequences, but low diversity in SSR loci. Authors explain that this outcome “may be related to its wide geographical distribution”.  This explanation is not convincing. It is not clear how exactly wide geographic distribution can contribute to the contrasting diversities in different genomes from populations of the same species. Obviously, the range of a species may play a role only if there are specific biological and ecological characteristics of the species contributing to the genetic differences. For example, some morphological and ecological factors affecting efficiency of outcrossing and seed dispersal could be responsible. Besides, the specific values of diversity are reported in the Abstract for only one of the two genomes of P. serrulata, so they cannot be compared by reading from text of the Abstract. In general, the presentation of results on genetic diversity in the Abstract is poor. Important information, like values of genetic diversity for different genomes of the same species, is missing, but at the same time some values are reported twice in two consecutive sentences (lines 19-20: “Hd = 0.553, Pi = 0.00136”).

Finally, authors conclude: “The findings support the classification of P. serrulata and P. xueluoensis as two distinct species.” I do not agree with this conclusion. Results of the reported analyses of SSRs suggest that P. serrulata is not monophyletic in relation to P. xueluoensis. Besides, cpDNA haplotypes from populations of the same “species” are distributed in the TCS network almost randomly, without much of a clear structure. A substantial gene flow between the “species” is obvious thus questioning the concept of a unique biological entity for either of the taxa.

Taxonomic resolution of the cases like this generally has two main options. Either the species which is diverging from within the clade of several lineages is recognised, and the rest of lineages is segregated into different species, or the assemblage is accepted as a group within the same species, and the other taxon is recognised as a subspecies or an ecotype/variety of this species. The reported in this study results indicate that the present status of these two species contradicts the basics of taxonomy, which require that accepted taxa should be monophyletic. Therefore, these species in their current circumscription cannot be accepted and additional taxonomic work is required to resolve the remaining issues.

I am not sure if the authors of this study will be able to fulfil the task. It would require additional analyses of morphological traits and ecological niches not only of the studied taxa, but also of at least one most closely related outgroup species. Molecular analyses would require a better representation of populations across the entire range of P. serrulata and a thorough selection of the most closely related outgroup species from the subgenus. Additional sequences should be either generated by authors or retrieved from Gene Bank for the three selected for this study cpDNA loci for multiple populations of outgroup species. Besides, sequences of nrDNA ITS of the sampled for this study populations of P. serrulate and P. xueluoensis in addition to several populations of the selected outgroup species should also be added to the phylogenetic analyses.

Alternatively, authors could abandon their aim of resolving the taxonomic conundrum around these taxa and focus instead on relationships between populations of two taxa with unresolved taxonomic status (“P. serrulata” and “P. xueluoensis”), and on analysis of genetic diversity within and among populations of these taxa. I would recommend ordination approach instead of the used by authors cluster analysis. For example, Principal Co-ordinate Analysis allows representation of genetic variation across genomes of sampled plants across multiple dimensions. In contrast to the uni-dimensional representation by UPGMA, this option provides a more informative overview of relationships between populations. Analysis of Molecular Variance (AMOVA) as implemented in Arlequin software could provide a useful insight into proportional distribution of variation between different levels of taxonomic hierarchy (within populations, among populations, among groups of populations). It would allow comparison of the relative amount of variation attributed to different groups of populations (identified as “P. serrulata” and “P. xueluoensis”), with the amount of variation attributed to differentiation among populations within these groups.

Reporting the results of calibrated by mutation rate of matK gene dated phylogeny of Rosaceae (as in Fig. 9) could still be useful. It provides an indication of the average age of the Most Recent Common Ancestor (MRCA) of lineages forming the “P. serrulate” complex of lineages, and the potential age of differentiation of “P. xueluoensis” clade from within the complex. However, I would recommend adding several sequences of the gene from different populations of both sampled taxa. This analysis could provide a useful contribution to the discussion on which of the possible options of resolving the taxonomic conundrum (segregating “P. serrulate” into several species or accepting it as a single species) better suits the overall taxonomy of the subgenus. Because matK is generally considered a suitable marker for resolving phylogenetic relationships at the level of genera in Angiosperms, demonstration of its usefulness for resolving phylogenetic relationships between several lineages of “P. serrulata” complex with a potential for description of these lineages as new biological species might support recognition of several species within the clade (including P. xueluoensis). Genetic data provided by the authors preliminary indicates that this option is feasible. However, a considerable additional taxonomic work is required on morphology and ecology of groups of populations outlined as independent lineages within P. serrulata complex by the reported in this manuscript analyses.

Below I provide a couple of minor comments for the Abstract. I have not made any effort to a thorough review of the entire text. This work should wait until a major revision of this study is submitted by the authors in the case this revision will be sent to me for a review.

Abstract

L. 18 “to delineate the relationship between the two populations” There are more than two populations sampled for this study. Obviously, you mean two species. Please, correct.

L. 27-28 “The common ancestor time” is not a standard terminology. It should be replaced by “The estimate of average age of the most recent common ancestor”.

Comments on the Quality of English Language

I have not read the whole manuscript thoroughly, but my overview of the text suggests that English is mostly clear.

Author Response

Comments 1: Manuscript by Gao et al. titled as “Species differentiation of Prunus serrulata and Prunus xueluoensis based on combined analysis of SSR and cpDNA markers” reports results of analyses on genetic structure of 13 populations from seven localities of two currently recognised species of Prunus. Authors aim for resolving the controversy around taxonomic status of a recently recognised Prunus xueluoensis. They employ two types of molecular markers, namely 18 loci of SSSRs and sequences from three loci cpDNA genome. The sampling size of molecular data is substantial and the whole study could be characterised as a standard approach for resolving taxonomic issues at the level of closely related species. However, the lack of discussion on morphological traits of the involved taxa in the reported study is a serious drawback.

Response 1: We understand the importance of incorporating morphological data in taxonomic studies, especially when dealing with closely related species. The primary focus of our study was on the genetic differentiation of Prunus serrulata and Prunus xueluoensis using molecular markers. To address the reviewer’s concern, we will incorporate a discussion on the morphological traits of the taxa involved in the revised manuscript. We will include relevant morphological data from available literature and our own preliminary observations, which we have documented, to provide a more comprehensive analysis. This addition will help to integrate morphological and molecular data, enhancing the robustness of our conclusions.

This change can be found on page2, paragraph 1 line 47-52

Comments 2: Authors report also results of analyses on genetic diversity within populations of the studied taxa and found that the diversity is high in both taxa. Analysing genetic diversity in P. serrulate, they found high diversity in the chloroplast sequences, but low diversity in SSR loci. Authors explain that this outcome “may be related to its wide geographical distribution”.  This explanation is not convincing. It is not clear how exactly wide geographic distribution can contribute to the contrasting diversities in different genomes from populations of the same species. Obviously, the range of a species may play a role only if there are specific biological and ecological characteristics of the species contributing to the genetic differences. For example, some morphological and ecological factors affecting efficiency of outcrossing and seed dispersal could be responsible. Besides, the specific values of diversity are reported in the Abstract for only one of the two genomes of P. serrulata, so they cannot be compared by reading from text of the Abstract. In general, the presentation of results on genetic diversity in the Abstract is poor. Important information, like values of genetic diversity for different genomes of the same species, is missing, but at the same time some values are reported twice in two consecutive sentences (lines 19-20: “Hd = 0.553, Pi = 0.00136”).

Response 2: 1. Explaining the relationship between extensive geographic distribution and varying diversity within different genomes: We understand the concerns of the reviewers regarding our explanation and appreciate this in-depth analysis. We acknowledge that our assertion on how extensive geographic distribution leads to differences in genomic diversity among populations of the same species was not rigorous enough, and we will remove this point. Regarding some morphological and ecological factors that may affect outcrossing and seed dispersal efficiency as possible reasons, we have made similar judgments in our discussion. The P. serrulata is widely distributed across most regions of China and is commonly found at the edges of warm-temperate forests, while the P. xueluoensis populations are predominantly located in subtropical sunny or semi-sunny slopes at the edges of dwarf shrub forests. In their natural habitats, the topographical, climatic, and soil environmental factors affecting P. serrulata and P. xueluoensis distribution areas differently impact community composition [47-49]. Since reproductive isolation does not exist among different species within the Rosaceae family, and gene exchange is frequent, combined with the foraging behavior of birds and rodents that objectively aids in seed dispersion of the Prunus genus, it results in generally high genetic diversity among Subg. Cerasus Mill.

2. Inadequate information on genetic diversity in the abstract: We appreciate the reviewer pointing out the shortcomings in the Abstract. In the revised manuscript, we will include the genetic diversity values for both genomes of P. serrulata, allowing readers to make clear comparisons. We will also streamline the Abstract to avoid unnecessary repetition and ensure that all important genetic diversity information is presented comprehensively. This will make the Abstract more comprehensive and informative.

This change can be found on page2, paragraph 1 line18-22.

Comments 3；Finally, authors conclude: “The findings support the classification of P. serrulata and P. xueluoensis as two distinct species.” I do not agree with this conclusion. Results of the reported analyses of SSRs suggest that P. serrulata is not monophyletic in relation to P. xueluoensis. Besides, cpDNA haplotypes from populations of the same “species” are distributed in the TCS network almost randomly, without much of a clear structure. A substantial gene flow between the “species” is obvious thus questioning the concept of a unique biological entity for either of the taxa.

Response 3: Thank you for your valuable feedback. We understand the concerns raised regarding the monophyletic status of P. serrulata relative to P. xueluoensis and the implications of the genetic data on our species classification.

While the SSR analysis suggests that P. serrulata may not be monophyletic in relation to P. xueluoensis, and the random distribution of cpDNA haplotypes within the TCS network indicates substantial gene flow, we would like to emphasize several points to address these concerns:

1. Although the reviewer states that serrulata is not monophyletic in relation to P. xueluoensis, this does not directly undermine our classification of the two as distinct species. The definition of a species is not always based on monophyly; in some cases, gene flow and hybridization can lead to the formation of non-monophyletic groups. However, this does not mean that there are not sufficient biological differences between them to support their status as separate species.
2. The analyses of SSR and cpDNA provide different levels of genetic information. SSR analysis often reveals micro-level genomic diversity, while cpDNA reflects maternal inheritance and long-term evolutionary trends. Although the distribution of cpDNA may appear somewhat random, which could indicate a certain level of gene flow, the genetic differences highlighted by the SSR analysis still support our delineation of species boundaries.
3. While gene flow does impact species boundaries, this flow does not necessarily weaken the validity of species classification. In plants, gene flow is often a complex process that can result in similarities among certain populations, but this does not preclude the consideration of these populations as distinct species based on ecological and biological characteristics. Our data indicate that despite the presence of gene flow, significant genetic and morphological differences still exist between the two.
4. Our study relies not only on genetic data but also integrates morphological and ecological evidence. For example, serrulata and P. xueluoensis exhibit significant differences in axillary buds, growth types, and ecological distribution. These differences are clearly observable in the field and further support the argument for classifying them as two distinct species.
5. Finally, while current data supports our classification of these two species, future research could explore the gene flow, ecological adaptability, and evolutionary history between them more comprehensively. Further work may reveal a more complex evolutionary process and help clarify the relationships between species.

In summary, our results take into account genetic, morphological, and ecological evidence that supports the classification of P. serrulata and P. xueluoensis as two distinct species. We hope that these counterarguments effectively address the reviewer’s concerns.

Comments 4；Taxonomic resolution of the cases like this generally has two main options. Either the species which is diverging from within the clade of several lineages is recognised, and the rest of lineages is segregated into different species, or the assemblage is accepted as a group within the same species, and the other taxon is recognised as a subspecies or an ecotype/variety of this species. The reported in this study results indicate that the present status of these two species contradicts the basics of taxonomy, which require that accepted taxa should be monophyletic. Therefore, these species in their current circumscription cannot be accepted and additional taxonomic work is required to resolve the remaining issues.

Response 4:

1. The text indicates that the SSR genetic diversity of serrulata and P. xueluoensis is at a relatively high level within the Subg. Cerasus Mill., and that the 13 populations are derived from 4 genetic pools, with variation primarily occurring within populations. This suggests that geographical isolation contributes to the formation of species at a high level of genetic diversity. This does not contradict your point that “accepted taxa should be monophyletic,” as the diversity analysis here supports the existence of P. serrulata and P. xueluoensis as distinct species.
2. The text shows that the cpDNA genetic diversity of both serrulata and P. xueluoensis is also at a high level within the Subg. Cerasus Mill., and that this variation primarily arises from inter-population differences. This further supports the classification of P. serrulata and P. xueluoensis as independent species, as significant differences between populations are often important evidence for species differentiation.
3. The text presents findings from UPGMA clustering, Maximum Parsimony (MP), and Maximum Likelihood (ML) phylogenetic trees based on cpDNA, as well as results from the TCS haplotype network, all supporting that serrulata and P. xueluoensis have diverged into two distinct species. The results of the genetic differentiation coefficient (FST) show significant differentiation between P. serrulata and P. xueluoensis, greater than that among different populations within each species. These results robustly support the classification of P. serrulata and P. xueluoensis as distinct species.
4. The text mentions that the mode of speciation for serrulata and P. xueluoensis is neighboring species formation, with no reproductive isolation between them, allowing for sufficient gene flow. This contradicts the point that “the present status of these two species contradicts the basics of taxonomy,” as neighboring species formation does not violate the principles of taxonomy but is rather a common mode of speciation.

In summary,the text provides ample genetic evidence supporting the classification of P. serrulata and P. xueluoensis as independent species. There is no contradiction with your objection.

Comments 5: I am not sure if the authors of this study will be able to fulfil the task. It would require additional analyses of morphological traits and ecological niches not only of the studied taxa, but also of at least one most closely related outgroup species. Molecular analyses would require a better representation of populations across the entire range of P. serrulata and a thorough selection of the most closely related outgroup species from the subgenus. Additional sequences should be either generated by authors or retrieved from Gene Bank for the three selected for this study cpDNA loci for multiple populations of outgroup species. Besides, sequences of nrDNA ITS of the sampled for this study populations of P. serrulate and P. xueluoensis in addition to several populations of the selected outgroup species should also be added to the phylogenetic analyses.

Response 5: Thank you for your valuable suggestions. We greatly appreciate your comments and will seriously consider your feedback. Here are our responses to each of your points:

1. Morphological traits and ecological niche analysis: We recognize that adding analyses of morphological traits and ecological niches for the studied taxa and at least one closely related outgroup species would further support our arguments. We will consider supplementing this aspect in future research.
2. Molecular analysis: You mentioned the need for better representation of populations across the entire range of serrulata. This is an important suggestion, and we will strive to collect more molecular data to enhance our understanding of P. serrulata and to systematically select the most closely related outgroup species.
3. Collection of additional sequences: Regarding the additional sequences for the cpDNA loci, we will endeavor to generate or retrieve these sequences from Gene Bank to facilitate the analysis of multiple outgroup species.
4. Phylogenetic analysis: Including the nrDNA ITS sequences of the sampled populations of serrulata and P. xueluoensis, along with sequences of several outgroup species, into phylogenetic analyses is indeed a valid suggestion. Yi [43] has also conducted taxonomic studies on P. serrulata and P. xueluoensis based on nuclear and chloroplast DNA barcoding. However, the current data is sufficient to support the classification of P. serrulata and P. xueluoensis as distinct species.

We have fully recognized the importance of your suggestions and will seriously consider implementing these improvements in future research. We will continue to dedicate ourselves to enhancing the depth and breadth of our study to better understand and support our research conclusions.

Comments 6；Alternatively, authors could abandon their aim of resolving the taxonomic conundrum around these taxa and focus instead on relationships between populations of two taxa with unresolved taxonomic status (“P. serrulata” and “P. xueluoensis”), and on analysis of genetic diversity within and among populations of these taxa. I would recommend ordination approach instead of the used by authors cluster analysis. For example, Principal Co-ordinate Analysis allows representation of genetic variation across genomes of sampled plants across multiple dimensions. In contrast to the uni-dimensional representation by UPGMA, this option provides a more informative overview of relationships between populations. Analysis of Molecular Variance (AMOVA) as implemented in Arlequin software could provide a useful insight into proportional distribution of variation between different levels of taxonomic hierarchy (within populations, among populations, among groups of populations). It would allow comparison of the relative amount of variation attributed to different groups of populations (identified as “P. serrulata” and “P. xueluoensis”), with the amount of variation attributed to differentiation among populations within these groups.

Response 6: Thank you for your suggestions regarding our research. We have fully recognized the importance of analyzing genetic diversity and structure in SSR and cpDNA, especially in comparing P. xueluoensis and P. serrulata. Our study shows that the genetic diversity level in the P. xueluoensis population is higher than that of P. serrulata, and the gene flow indicates a wide exchange of genes between the two, with no reproductive isolation. Furthermore, the results from the structure analysis support the view that these two species have already diverged, and AMOVA analysis also confirms that the variation among populations primarily arises from within-population differences.

Regarding your suggestion of possibly abandoning the aim of resolving the taxonomic issues of these two taxa and instead focusing on the relationships among populations of these unresolved taxa and on analyzing genetic diversity within and among these populations, we are open to this idea. We will seriously consider using your recommended ordination approach, such as Principal Coordinate Analysis, to more comprehensively illustrate the genetic variation across the genomes of sampled plants, which would provide richer information compared to the one-dimensional UPGMA. Additionally, utilizing AMOVA in Arlequin software would indeed provide valuable insights into the proportional distribution of variation across different levels of taxonomic hierarchy, which is crucial for understanding the relationship between P. serrulata and P. xueluoensis.

We will seriously consider your suggestions and will strengthen our research on the relationships between these two species and their populations in the future.

Comments 7；Reporting the results of calibrated by mutation rate of matK gene dated phylogeny of Rosaceae (as in Fig. 9) could still be useful. It provides an indication of the average age of the Most Recent Common Ancestor (MRCA) of lineages forming the “P. serrulate” complex of lineages, and the potential age of differentiation of “P. xueluoensis” clade from within the complex. However, I would recommend adding several sequences of the gene from different populations of both sampled taxa. This analysis could provide a useful contribution to the discussion on which of the possible options of resolving the taxonomic conundrum (segregating “P. serrulate” into several species or accepting it as a single species) better suits the overall taxonomy of the subgenus. Because matK is generally considered a suitable marker for resolving phylogenetic relationships at the level of genera in Angiosperms, demonstration of its usefulness for resolving phylogenetic relationships between several lineages of “P. serrulata” complex with a potential for description of these lineages as new biological species might support recognition of several species within the clade (including P. xueluoensis). Genetic data provided by the authors preliminary indicates that this option is feasible. However, a considerable additional taxonomic work is required on morphology and ecology of groups of populations outlined as independent lineages within P. serrulata complex by the reported in this manuscript analyses.

Response 7: Thank you for your comments and suggestions. You mentioned that the phylogenetic results calibrated by the mutation rate of the matK gene indeed provide important insights into the average age of the Most Recent Common Ancestor (MRCA) of the “P. serrulata” complex and the potential differentiation of the “P. xueluoensis” clade. This is significant for addressing the taxonomic controversies surrounding this complex, such as whether to segregate “P. serrulata” into several species or recognize it as a single species.

Your suggestion to include matK gene sequences from different populations to enhance the representativeness of the samples is indeed valuable. This could help us gain a more comprehensive understanding of the phylogenetic relationships among the different clades and provide stronger support for the overall taxonomy of the subgenus. In particular, as you pointed out, the matK gene is considered an effective marker in angiosperm taxonomy. Its potential to resolve the phylogenetic relationships among several lineages within the “P. serrulata” complex will aid in identifying and describing these lineages as new biological species, further supporting the possibility of recognizing multiple species within the clade, including “P. xueluoensis.”

At the same time, as you mentioned, there is indeed a need for more morphological and ecological research to supplement and verify the characteristics of these independent lineages. Further taxonomic work could enhance our understanding of the relationships between these organisms and their roles within ecosystems.

Once again, thank you for your insightful feedback, and I look forward to better exploring and elucidating the phylogeny and taxonomic issues of the “P. serrulata” complex and its related clades in future research.

Comments 8: Below I provide a couple of minor comments for the Abstract. I have not made any effort to a thorough review of the entire text. This work should wait until a major revision of this study is submitted by the authors in the case this revision will be sent to me for a review.

Response 8: Thank you for your comments on the abstract. We will take your feedback into account during our major revision process and look forward to further communication with you once the revised manuscript is submitted.

Comments 9: L. 18 “to delineate the relationship between the two populations” There are more than two populations sampled for this study. Obviously, you mean two species. Please, correct.

Response 9: Thank you for pointing out.

This change can be found on page1 paragraph 1 line 18.

Comments 10: L. 27-28 “The common ancestor time” is not a standard terminology. It should be replaced by “The estimate of average age of the most recent common ancestor”.

Response 10: Thank you for pointing out.

This change can be found on page1 paragraph 1 line 33, page12 paragraph 3 line 322, page18 paragraph 3 line 476.

I made significant modifications to the content of the abstract. The specific content is as follows:

Prunus xueluoensis is a new species of the Subg. Cerasus Mill., described by C. H. Nan and X. R. Wang in 2013. Since the publication of P. xueluoensis, its taxonomic status has been the subject of ongoing debate, and it has been classified as a synonym of P. veitchii (A synonym of P. serrulata). The study focuses on wild populations of P. xueluoensis and P. serrulata, utilizing 18 pairs of SSR molecular markers and variations in chloroplast DNA sequences (matK, trnD-E, and trnS-G) to delineate the relationship between the two species  The results showed that P. serrulata（N=12.400，N_a=5.144，H=0.578，I=1.129，H_o=0.493）and P. xueluoensis（N=13.625，N_a=6.264，H=0.614，I=1.342，H_o=0.495）populations exhibit rich genetic diversity, which may be related to its wide geographical distribution. CpDNA genetic diversity of P. serrulata (H_d=0.553, P_i=0.00136) and P. xueluoensis (H_d=0.496, P_i=0.00180) are at a high level within the Subg. Cerasus Mill. The UPGMA clustering, along with MP and ML phylogenetic trees, show that the unique haplotypes of P. xueluoensis cluster separately as a terminal branch in the evolutionary tree with high support. Shared haplotypes and unique haplotypes of P. serrulata are predominantly located at the base of the phylogenetic tree, suggesting that the two species have diverged. In the TCS haplotype network, the central and key node haplotypes are primarily unique to P. serrulata and shared haplotypes, while the unique haplotypes of P. xueluoensis are all distributed along the network’s periphery. Both P. serrulata (N_st = 0.254, G_st = 0.103, N_st/G_st = 2.466, P < 0.05) and P. xueluoensis (N_st = 0.366, G_st = 0.268, N_st/G_st = 1.366, P < 0.05) exhibit phylogeographic structures. However, when considered as a whole, the combined entity of P. serrulata and P. xueluoensis does not show a significant phylogeographic structure (N_st = 0.317, G_st = 0.400, N_st/G_st = 0.793, P < 0.05), which supports the classification of these as two distinct species. The estimate of average age of the latest common ancestor of P. serrulata and P. xueluoensis is 3.22mya (PP=1; 95%HPD: 3.07~3.46mya). Due to environmental differences in altitude, the group of P. serrulata 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

a small revision could improve its aesthetics and make it easier to read.

Congratulations to the authors for a job well done.

The article is well thought out. I would like to suggest some improvements to make it easier to read.

The title is sufficiently clear.

Abstract: Well-structured and comprehensible.

Introduction: Comprehensive and well synthesised.

M&M:
L89: Are you sure they are different populations or maybe just different origins/provenances?

L97: Replace ‘to eliminate’ by ‘ to avoid’ & ‘clonal’ by ‘natural vegetative’.

L128: Maybe ‘genotypes’ are ‘alleles’?

L142: Add the date to the reference Evanno et al.

Tabla 1: Remove ‘molecular’ from the title

Add in the first column, in brackets, the number of each ‘Population’ which is then used in subsequent Figures and texts.

In the second column add in the heading ‘Place/Province’ so that the word province can be removed from all references in this column.

In LuSe 'Altitude' is actually 111-1311m. It seems a bit discordant with the others, but it may be true.

Figure 1. Above all, it is important to change the black background so that the map underneath is not visible. Some references are also needed, the name of the sea, the name of the different provinces where the samples are collected and some well-known cities. The reader needs to easily locate on the map the origins of the material used.

Figure 6. The same comments.

Figure 7. Improve the graphic to visualise the difference between species. It is difficult to understand. The borders in some colours do not look good. Use of different geometric figures for example?

Figure 8. The lines and identifications are too small to be seen properly.

Figure 10. Idem.

Author Response

Comments 1: L89: Are you sure they are different populations or maybe just different origins/provenances?

Response 1: Thank you for your perspective! I agree that the terms “populations” and “origins/provenances” can imply different meanings. Since our sampling is based on variations according to geographical locations or environments, “origins” or “provenances” might be more accurate. I appreciate your feedback on the phrasing and I have made the necessary corrections.

This change can be found on page2, paragraph 5 line 91.

Comments 2: L97: Replace ‘to eliminate’ by ‘to avoid’ & ‘clonal’ by ‘natural vegetative’.

Response 2: Agree. Thank you for pointing this out.

This change can be found on page 3, paragraph 2 line 99.

Comments 3；L128: Maybe ‘genotypes’ are ‘alleles’?

Response 3: Thank you for pointing this out. We have changed genotypes to alleles.

This change can be found on page 5, paragraph 2 line 132.

Comments 4；L142: Add the date to the reference Evanno et al.

Response 4: Thank you for pointing this out. We have added the references.

This change can be found on page 19 line 59.

32.   Evanno, G.; Regnaut, S.; Goudet, J. Detecting the number of clusters of individuals Using the Software structure: a simulation study. Mol. Ecol. 2005, 14, 2611–2620. doi:10.1111/j.1365-294X.2005.02553.x.

Comments 5；Table 1: Remove ‘molecular’ from the title.

Response 5: Thank you for pointing this out. It has been removed from Table 1.

This change can be found on page 3 Table 1.

Comments 6；Add in the first column, in brackets, the number of each ‘Population’ which is then used in subsequent Figures and texts.

Response 6: The number of each ‘Population’ has been placed in the fourth column. Subsequent Figures (Figures 1) and text (Table 1) have also been modified.

This change can be found on page 3 Table 1.

Comments 7；In the second column add in the heading ‘Place/Province’ so that the word province can be removed from all references in this column.

Response 7: Agree. We have added ‘Province’ to the first column and removed all references to province in this column. Table 1.

This change can be found on page 3 Table 1.

Comments 8；In LuSe 'Altitude' is actually 111-1311m. It seems a bit discordant with the others, but it may be true.

Response 8: It’s true. The reason might be due to the wide geographical distribution of mountain cherries and the unique geographical environment of Mount Lushan, which causes some mountain cherries to appear at low altitudes.

Response 8: It’s true. The reason might be due to the wide geographical distribution of mountain cherries and the unique geographical environment of Mount Lushan, which causes some mountain cherries to appear at low altitudes.

Response 9: Thank you for your suggestions. We have changed the black background of Figure 1 to make the map clearer. The names of different provinces from which samples were collected, as well as some well-known cities, have also been marked on the map.

This change can be found on page 4 Figure 1.

Comments 10；Figure 6. The same comments.

Response 10: Thank you for your suggestions. Figure 6 has also been modified.

This change can be found on page 11 Figure 6.

Comments 11；Figure 7. Improve the graphic to visualise the difference between species. It is difficult to understand. The borders in some colours do not look good. Use of different geometric figures for example?

Response 11: Thank you for your suggestions. We have improved Figure 7 to visualize the differences between species. Following your suggestions, we used different geometric shapes to distinguish them.

This change can be found on page 12 Figure 7.

Comments 12；Figure 8. The lines and identifications are too small to be seen properly.

Response 12: We have adjusted the lines and labels in Figure 8 to make them clearer and more visible.

This change can be found on page 13 Figure 8.

Comments 13；Figure 10. Idem.

Response 13: Figure 10 has also been revised. Thank you for your suggestions.

This change can be found on page 16 Figure10.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Authors carefully responded to my comments on the first version of their study. They accepted all my comments as valid but responded to them in two ways. They adjusted the text and added new references in response to few of them, but also used as guidelines for further studies in response to most of them. This treatment of my critical points would be fine to me. However, the somewhat doubtful insistence of authors on accepting a new taxon, which was described in co-authorship of two of them, prompted me to study the taxonomic problems of this group of species in a more detail. This analysis revealed additional serious issues with the manuscript, as outlined below.

L. 45-46: “In 2019, Wu proposed that P. xueluoensis and its morphologically similar species, P. jingningensis, P. concinna, and P. japonica var. zhejiangensis, could all be merged under P. veitchii (a synonym of P. serrulata var. pubescens)”

This statement is not correct, and it is misleading the readers not familiar with specific taxonomy of the subgenus. First, authors of the cited paper should correctly be referred to as “Wu et al.”. Second, these authors did not suggest that P. veitchii is a synonym of P. serrulata var. pubescens. Instead, they focused on differences between P. serrulata var. pubescens and the group of species which was merged under P. veitchii. Therefore, the research question to resolve the taxonomic issues around this group is not the differences between P. veitchii sensu lato (including P. xueluoensis) and P. serrulata var. pubescens. These differences have been convincingly demonstrated by Wu et al. (2019) in a formal analysis of morphological traits of all included into their study species.

Instead, the main research question would be if the described recently P. xueluoensis differs from P. veitchii or any other species, which Wu et al. (2019) downgraded into synonymy of the latter one. P. veitchii was described in 1912, more than a century before description of P. xueluoensis and has therefore a clear priority against the latter name, if the synonymy is accepted. If authors insist on using the new name as valid, they should question the taxonomic decision of Wu et al. (2019) and convincingly demonstrate the differentiation of P. xueluoensis from P. veitchii. This research question remains not answered by the study under review. The focus of authors on demonstration of differences between P. xueluoensis and P. serrulate is not relevant for resolving the taxonomic issues in the group, because this differentiation has been demonstrated by Wu et al. (2019).

As Table 2 from Wu et al. (2019) suggests, P. veitchii and P.  xueluoensis may indeed be different species, if the form of sepal and the length of pedicel are considered. However, demonstrating the differentiation between these taxa would require an entirely different study. Before such study has been published, the only way to proceed with publication of results of the study under review would be to use the name “P. veitchii” instead of the published later “P. xueluoensis“. Authors could state, for example, that the group of populations representing P. veitchii in this study was described as “P. xueluoensis“ by Nan et al. (2013).However, since Wu et al. (2019) put the new species into synonymy of P. veitchii, these populations are representing the latter species until the status of the former one is not clarified by a properly designed study.

This approach could benefit from results of the analyses of Wu et al. (2019) since there would be no need to report data on morphological differences between P. veitchii and P. serrulate. Instead, authors could put emphasis on demonstration of genetic differences between these two species. Authors could also comment on the potential issue of the paraphyly of R. serrulate, which was revealed by their analyses. I recommend reading Schmidt-Lebuhn (2012) for a better understanding the problems with paraphyletic taxa:

Schmidt-Lebuhn, A. N. (2012). Fallacies and false premises: a critical assessment of the arguments for the recognition of paraphyletic taxa in botany. Cladistics 28(2): 174-187.

In conclusion, I recommend that authors should revise text throughout the manuscript according to the correct interpretation of present taxonomic situation in the P. serrulata/P. veitchii/“P. xueluoensis” clade. They should accept the most recent taxonomic solution in the group suggested by Wu et al. (2019) and clearly define ranges of all species, for example in their Fig. 1. I would recommend reporting in this figure by points all known to science localities of the involved in their analysis species, and adding data on ranges of all species combined by Wu et al. (2019) under P. veitchii. Localities of the type specimens of all described species should be highlighted by a specific symbol. The data can be retrieved from GBIF, virtual herbaria, relevant literature, and from the personal observations by the authors of this study. They should modify their main research question accordingly.

In Discussion, authors should comment on the clearly demonstrated by their results paraphyly of P. serrulate and suggest possible solutions. They should also admit that until multiple populations of all species combined under P. veitchii by Wu et al. (2019) will be sampled for genetic analyses and monophyly of these taxa formally tested against each other, taxonomic status of these species remains unresolved.

Additional minor comments.

Labelling of populations in Table 1, Text, and in Figures 3 and 4 is different. Please link the different labelling in Table 1, or better use the same labelling of populations consistently across the manuscript.

In the new version of legend to Fig. 7, both triangles and circles are reported as representing unique haplotypes of P. serrulata. Please correct.

L. 475 “estimates the estimate of average age”. Awkward wording. Better would be “estimates the average age”.

Comments on the Quality of English Language

Quality of language in the text is acceptable.

 

Author Response

Comments 1: L. 45-46: “In 2019, Wu proposed that P. xueluoensis and its morphologically similar species, P. jingningensis, P. concinna, and P. japonica var. zhejiangensis, could all be merged under P. veitchii (a synonym of P. serrulata var. pubescens)”. This statement is not correct, and it is misleading the readers not familiar with specific taxonomy of the subgenus. First, authors of the cited paper should correctly be referred to as “Wu et al.”. Second, these authors did not suggest that P. veitchii is a synonym of P. serrulata var. pubescens. Instead, they focused on differences between P. serrulata var. pubescens and the group of species which was merged under P. veitchii. Therefore, the research question to resolve the taxonomic issues around this group is not the differences between P. veitchii sensu lato (including P. xueluoensis) and P. serrulata var. pubescens. These differences have been convincingly demonstrated by Wu et al. (2019) in a formal analysis of morphological traits of all included into their study species. Instead, the main research question would be if the described recently P. xueluoensis differs from P. veitchii or any other species, which Wu et al. (2019) downgraded into synonymy of the latter one. P. veitchii was described in 1912, more than a century before description of P. xueluoensis and has therefore a clear priority against the latter name, if the synonymy is accepted. If authors insist on using the new name as valid, they should question the taxonomic decision of Wu et al. (2019) and convincingly demonstrate the differentiation of P. xueluoensis from P. veitchii. This research question remains not answered by the study under review. The focus of authors on demonstration of differences between P. xueluoensis and P. serrulate is not relevant for resolving the taxonomic issues in the group, because this differentiation has been demonstrated by Wu et al. (2019).

Response 1: Thank you for your detailed review and feedback on our research. We highly value your perspectives and would like to provide clarifications and responses.

Firstly, regarding the authorship of the cited literature, we have noted your correction that it should indeed be “Wu et al.” We will make this correction in the revised version.

Secondly, concerning the relationship between P. veitchii and P. serrulata var. pubescens, we understand the misunderstanding you pointed out. Our focus in the study was on whether P. xueluoensis is distinct from P. veitchii or other species, and we did not intend to question Wu et al.'s (2019) research on the differences between P. serrulata var. pubescens and P. veitchii. We will further clarify this point in the manuscript to ensure that readers accurately understand the core issue of our study.

Regarding Wu et al.'s (2019) conclusion to downgrade P. xueluoensis as a synonym of P. veitchii, we will make our position more explicit. We are skeptical about the morphological classification viewpoint of Wu et al. In the Flora of China, P. veitchii is still considered a synonym of Prunus serrulata var. pubescens, so we will continue to use the name P. xueluoensis.

Regarding your concerns that these statements could mislead readers, we have already removed them.

Comments 2；As Table 2 from Wu et al. (2019) suggests, P. veitchii and P. xueluoensis may indeed be different species, if the form of sepal and the length of pedicel are considered. However, demonstrating the differentiation between these taxa would require an entirely different study. Before such study has been published, the only way to proceed with publication of results of the study under review would be to use the name “P. veitchii” instead of the published later “P. xueluoensis“. Authors could state, for example, that the group of populations representing P. veitchii in this study was described as “P. xueluoensis“ by Nan et al. (2013).However, since Wu et al. (2019) put the new species into synonymy of P. veitchii, these populations are representing the latter species until the status of the former one is not clarified by a properly designed study. This approach could benefit from results of the analyses of Wu et al. (2019) since there would be no need to report data on morphological differences between P. veitchii and P. serrulate. Instead, authors could put emphasis on demonstration of genetic differences between these two species. Authors could also comment on the potential issue of the paraphyly of R. serrulate, which was revealed by their analyses. I recommend reading Schmidt-Lebuhn (2012) for a better understanding the problems with paraphyletic taxa: Schmidt-Lebuhn, A. N. (2012). Fallacies and false premises: a critical assessment of the arguments for the recognition of paraphyletic taxa in botany. Cladistics 28(2): 174-187.

Response 3: Thank you very much for your meticulous review and valuable suggestions regarding our manuscript. We have thoroughly read your comments and would like to express our sincere gratitude for your professional guidance. Here is our response to the issues you raised:

Regarding Wu et al.'s classification of P. veitchii and P. xueluoensis based on morphological studies, we remain skeptical and still uphold the taxonomic status of P. xueluoensis.

Regarding your recommendation of Schmidt-Lebuhn (2012), our careful reading and consideration of the article have provided us with insights into the difficulties in species delimitation and phylogenetic reconstruction within the Maddenia group. Utilizing genome skimming techniques to study the Maddenia group not only addresses the identification issues among closely-related species but also provides crucial insights for further understanding and research regarding the species diversity and phylogeny of the Maddenia group. The article also mentioned the application of leaf morphological traits in species identification, such as the shape of the leaf base, the serration of leaf margins, the distribution of trichomes on the leaf’s underside, as well as features of stomata and guard cells. This article has greatly helped us better understand the problems associated with paraphyletic taxa. We are grateful for your recommendation.

Comments 3: In conclusion, I recommend that authors should revise text throughout the manuscript according to the correct interpretation of present taxonomic situation in the P. serrulata/P. veitchii/“P. xueluoensis” clade. They should accept the most recent taxonomic solution in the group suggested by Wu et al. (2019) and clearly define ranges of all species, for example in their Fig. 1. I would recommend reporting in this figure by points all known to science localities of the involved in their analysis species, and adding data on ranges of all species combined by Wu et al. (2019) under P. veitchii. Localities of the type specimens of all described species should be highlighted by a specific symbol. The data can be retrieved from GBIF, virtual herbaria, relevant literature, and from the personal observations by the authors of this study. They should modify their main research question accordingly.

In Discussion, authors should comment on the clearly demonstrated by their results paraphyly of P. serrulate and suggest possible solutions. They should also admit that until multiple populations of all species combined under P. veitchii by Wu et al. (2019) will be sampled for genetic analyses and monophyly of these taxa formally tested against each other, taxonomic status of these species remains unresolved.

Response 5: We greatly appreciate your detailed review and valuable suggestions. We have carefully considered your recommendations and will provide explanations based on your feedback. Here is our response:

Regarding the current classification situation in the P. serrulata/P. veitchii/“P. xueluoensis” clade, Wu et al. (2019) proposed the latest classification solution. The reviewer suggests that we should accept their viewpoint and modify our research direction. However, we hold a skeptical attitude towards Wu et al.'s (2019) classification and do not believe that our research direction is inappropriate. Regarding the genetic analysis of all species merged under P. veitchii by Wu et al. (2019), as mentioned by the reviewer, we will seriously consider implementing this in our future research.

Comments 4；Labelling of populations in Table 1, Text, and in Figures 3 and 4 is different. Please link the different labelling in Table 1, or better use the same labelling of populations consistently across the manuscript.

Response 4: Thank you for pointing this out. We have added captions below the images for better understanding.

This change can be found on page 10 line 267-269.

Comments 5；In the new version of legend to Fig. 7, both triangles and circles are reported as representing unique haplotypes of P. serrulata. Please correct.

Response 5: Thank you for pointing this out. We will make the revisions.

This change can be found on page 13 line 328.

Comments 6；L. 475 “estimates the estimate of average age”. Awkward wording. Better would be “estimates the average age”.

Response 6: Thank you for pointing this out. We will make the revisions.

This change can be found on page18, paragraph 3 line 473.

Author Response File: Author Response.pdf