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The authors have addressed all the reviewers' comments.
Before publishing, please check on line 317 "P. zhennan" should be italicized. Just ensure all species names are italicized consistently.
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
**PeerJ Staff Note:** Although the Academic and Section Editors are happy to accept your article as being scientifically sound, a final check of the manuscript shows that it would benefit from further editing. Therefore, please identify necessary edits and address these while in proof stage.
After the last round of revisions, I no longer have any changes to suggest to the authors.
All of the changes suggested by the reviewers in the previous rounds of revision were considered by the authors, and the manuscript has been improved.
For me, the paper can be accepted for publication. My worry were the references, but the authors performed an extensive review and now everything appears to be in order.
Nothing to add.
Nothing to add.
Nothing to add.
Clear and unambiguous.
Original primary research within Aims and Scope of the journal
Underlying data has been provided; they yare robust, statistically sound, and controlled.
All the comments given are addressed.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
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Line number 12, 44 - Phoebe Zhennan S. K. Lee & F. N. Weiis (Check the spelling of Weiis or Weiss)
Line number 259 - Change to -A total of 218 alleles were identified across 16 individuals using the 20 selected SSR primers
Line number 314 - Change to -An AMOVA of the 20 loci in Guizhou P. zhennan populations
Throughout the text, the authors should check for spaces and spelling.
After reading the revised manuscript, I found that it has been much improved, following the previous round of revisions. The introduction and results have been supplemented with additional information, clarifying some existing doubts related to demographic, ecological issues of the species, and the use of genetic tools in this work, which enriches the manuscript. The quality of some figures has been improved, and the titles have become more self-explicatory. For that, I congratulate the authors. I believe that it has become closer to a version that can potentially be accepted for publication. However, there are still some issues that need to be addressed.
Some issues with spacing and punctuation persist, especially in the introduction and results sections. I advise to give it a look throughout the text.
For me, the greatest issue is still the use of citations in the text. Whilst I appreciate the work put on by the authors, who added several citations in the needed places of the manuscript, the citations in the text are almost all wrong. In the reference list, the authors’ last names are used, but then in the manuscript text, the authors used the authors’ first names to cited their works, which really confused me (and took me a while to understand why, for example, GenAlex was cited using Rod & Peter, but then I could not find Rod & Peter on the reference list. Then I found that you have used the first names of Peakall and Smouse (2012, as it is in the reference list). That way, it will be impossible to find and connect the works cited to the ones in the reference list.
I urge the authors to conduct a thorough review of the literature cited throughout the text, to correct the names of the authors, using their last names instead of their first names (e.g., Leilton Willians et al., 2023 should be Luna et al., 2023; Rod & Peter, 2012 should be Peakall and Smouse, 2012; Dharminder and Gandadeep Singh, 2022 should be cited as Bhatia and Banga, 2022; Xiaxia et al., 2022 is Liu et al., 2022; and there are a lot more examples!). Additionally, I think there are works cited in the reference list that are no longer in the manuscript and vice versa.
In the discussion, please revise the sentence between lines 287-290: Why are the names of the species repeated? It feels a bit confusing (…it is believed that P. bournei has a closer kinship with P. chekiangensis, P. bournei, P.chekiangensis, and P. sheareri…). It probably is a formatting error or lack of punctuation. Please correct the sentence.
I see no flaws in the experimental design.
All data is provided, either in the text or in figures, tables, or supplementary data, and it has statistical robustness.
The discussion is well articulated around the results obtained, and the conclusions are well stated.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
The language is good and understandable. It was massively improved following an initial round of revisions. However, some parts could still be simplified, and some spacing and punctuation could be improved to allow an easier reading experience.
Figures and tables appear ok, but at least two figures, namely Fig. 1 and 3 need to be improved in terms of quality, which at the moment is poor.
The introduction provides enough background information, and the references are adequate.
The supplementary Tables lack the titles. I also think that the titles of Tables 2 and 3 and the titles of the images could be improved to become more informative.
The sequences generated are available in genomic repositories.
The work is interesting and is according to the Aims and Scope of the journal. I have not found any scientific flaws in it. The research question is relevant and fills a gap in the knowledge that is acknowledged in the state of the art.
Sample harvesting, and methodologies appear in conformity with the technical and ethical standards, and are referred by the authors in the manuscript.
Moreover, the methodology used feels correct and sound, and the descriptions of the techniques used allow replication of the work. However, something that bothered me in the methods was the lack of citations to the methodologies used and the authors should really address this issue. I give plenty of examples and in most cases I provide, in my revision, the missing references, for the authors to complete the manuscript.
The study brings new and important insight about an endemic, endangered tree species of the Chinese forests, and the newly designed SSR markers can be important both to the conservation of the species and also in future studies that address the taxonomic circumscription of the Pheobe genus.
The results are coherent with the research goals, and support the subsequent discussion and conclusions, focusing on the strengths of the marker system used here, and discussing the implications of the GD and structure of P. zhennan populations, in terms of its evolution and conservation. However, there are parts in the results that should be moved into the discussion, and the discussion itself needs to be better structured and, if possibly shortened, to make it less repetitive and more cohesive.
Overall, I think that if the authors positively address the questions raised, the research can eventually be published in PeerJ.
Specific comments:
Line 43: The full name of the species (not the abbreviated genus), followed by the family, should be provided at the beginning of the first sentence of the introduction.
I have found some minor formatting errors, such as absence of spacing or punctuation in several places (e.g., Abstract, line 15 (…) Investigating the genetic diversity(GD) (…), lacks a space; Line 54, absence of space between markers:DNA; Line 58 (…) species identification(Christian 2004) – here lacks a space and a comma, between the author and year of publication; Line 61 (…genetic structures(Changming et al.), and 63 (…relied on EST(expressed…) again. I would advise the authors to review the introduction and discussion, where I found some issues with the missing spaces and punctuation.
Line71: When referring to a species for the first time (Pheobe bournei), please provide species authorities.
Line 72: Maybe improve the sentence regarding your aims: “ Therefore, in this study, we aim to develop molecular markers and investigate the GD within the Phoebe genus. This is essential for enhancing…”
Line 93: Please add the authorities of Lindera megaphylla, and P. chakiangensis.
Line 131-132: Please simplify: “Sequence assembly was assembled using…” Instead write: “Sequences were assembled using…”
Line 139: Pheobe chinensis should be in italics. Please also add species’ authorities when referring to a species for the first time, in the manuscript.
Line 152: In the section Data analysis, I find that several references are missing, especially when referring the softwares used to analyse the genetic diversity and population structure. I advise the authors to provide the citations in the appropriate places. For example:
GenAlEx 6.51 software (Peakall and Smouse, 2012; Peakall, R. O. D., & Smouse, P. E. (2006). GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular ecology notes, 6(1), 288-295.).
STRUCTURE (Pritchard et al., 2000; Pritchard, J. K., Stephens, M., & Donnelly, P. (2000). Inference of population structure using multilocus genotype data. Genetics, 155(2), 945-959.).
Arlequin 3.5.2.2. (Excoffier and Lischer, 2010; Excofer L, Lischer HEL (2010) Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10:564–567).
Structure Harvester (Earl, D.A. and von Holdt, B. M. (2011). STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genetics Resources DOI: 10.1007/s12686-011-9548-7)
PowerMarker (Liu K., Muse, S. V. (2005). PowerMarker: an integrated analysis environment for genetic marker analysis, Bioinformatics, Volume 21, Issue 9, Pages 2128–2129, https://doi.org/10.1093/bioinformatics/bti282)
iTOL: Letunic and Bork (2024) Nucleic Acids Res doi: 10.1093/nar/gkae268
Please add the citations in their appropriate places in the manuscript, and add them to the references.
Line 166. In the beginning of the results section, you have mentioned the results of the SSR-enriched library using MISA software. I apologize if I am wrong, but I have read the methods section again and again and have not found a reference to the use of this software there. I believe that the software that you’re referring to its MISA – MIcroSAtellite Identification Tool, by Thiel et al. (2003) - Thiel T, Michalek W, Varshney RK, Graner A. (2003) Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.).Theor Appl Genet.,106(3):411-422.
I ask you to add this information to its appropriate subsection in the methods, using the appropriate citation.
**PeerJ Staff Note:** It is PeerJ policy that additional references suggested during the peer-review process should only be included if the authors are in agreement that they are relevant and useful.
Lines 181-192. I believe that this paragraph should belong to the methods subsection 2.2.4, as I find it mostly descriptive of the methodology employed on the preliminary screening. It should be moved to complement the information on the methods.
I think that in the results, it is enough to mention that: To verify the 108 synthesized SSR primer pairs, 16 P. zhennan samples from different provenances of Guizhou were used for preliminary analysis. Based on the level of polymorphism, polymorphism information content (PIC) and capillary electrophoresis results (Fig. S1), 20 pairs of primers were selected for further screening, in the subsequent analysis (Table S2).
Line 200: Something is missing in: “could be effectively used for assessing the GD of ? - P. zhennan ?
Line 202: Maybe erase the first sentence, and start directly on : The analysis results revealed that…
Line 208: By referring to the populations as groups, I think that might generate some confusion to the readers. You can either refer to them as populations or just cite the populations’ abbreviations (e.g., The Ho values ranged from 0.406 to 0.823, with the maximum value observed in JK and the minimum in DZ).
Line 210: I suggest rewrite this sentence to a simpler form: “In addition, the Ho values for the P. zhennan populations were smaller than the He values, except in DJ, JH, and MT, suggesting the possibility of inbreeding within these populations”.
Line 211: P. zhennan should be in italics.
Line 215: GD (Table 4) – space is missing.
Table 4 – I would advise to improve the title of table 4. Genetic diversity parameters in 20 populations of P. zhennan, and GD comparison with four other related species.
Lines 226-227: the sentence: “the Ho values for all populations, except the P. chekiangensis, were lower than the He values which may indicate inbreeding or could be attributed to sample size or environmental factors” belongs in the discussion.
Line 232-235: From: “When Fis, the inbreeding….pure heterozygotes” – this should be in the discussion. In the results, maintain only a description of your results.
The quality of images 1 and 3 needs to be improved. Please refer to the manuscript guidelines – Figures and tables.
Line 263-264: Please move this sentence to the discussion, where is probably fits better: “ This indicates that the Guizhou, 264 Sichuan, Chongqing, and Hunan populations share a certain degree of genetic relatedness”.
Line 268-269. Again, the first sentence will probably fit better when discussing this part of the results.
Line 272-273: Please, simplify: and the third group included all P. zhennan populations.
Line 277: rewrite: “and the fifth group was composed by the remaining populations of P. zhennan”.
Lines 278-283: The authors should carefully reassess and distinguish what should be on the results (description of the results you have achieved) and what should be moved into the discussion (compare, discuss and interpret your results considering the state of art and other works’ findings). For instances, the population of Yahne County was clustered into a separate group (that is your result), which might be another species of the Phoebe genus (that is discussion of your results, and it could be further explored in the discussion, with examples).
Line 289: Discussion, overall ideas – Throughout the manuscript, the authors did a good job on portioning the data into several subcategories, especially in the methods and results. Thus, a similar approach should be used in the discussion, as in its current form, the discussion is overlong, sometimes repetitive and confusing. By dividing the discussion into subcategories (e.g., Characterisation and transferability of the newly developed SSR markers; Genetic diversity; Genetic structure; Impacts in the conservation of Pheobe Shennan) would contribute to lighten and shorten the discussion, making it more concise and easier to understand.
Line 309: Please, cite the work that obtained the transcriptome data.
Line 311: Again, please cite the works that used traditional EST-SSRs approaches, which you are using to make comparisons regarding the polymorphism level and efficacy.
Line 332: Please correct, the value of Ho was lower, not greater than the expected He value.
Line 336: Please, add the authorities of the species, and put the species names in italics.
Line 351-352. I think you can delete this sentence, as it is repeated below, in lines 372-373.
Line 388: Please cite some works about fruit dispersal in Lauraceae.
Line 413: Please refer the works that have used ISSR, SRAP and AFLP marker systems with Phoebe species.
Line 421: Phoebe fortune in italics and with authorities.
Line 436-437: You can delete the names of the populations within parenthesis, as they are referred in Table 1.
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See attachment
**PeerJ Staff Note:** Please ensure that all review and editorial comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
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The manuscript deals with the development of SSR markers through enrichment technique and use of these markers to assess the genetic diversity of Phoebe species distributed in certain locations. Detailed study on genetic diversity and population structure of Phobe zhennan is carried out. Though the title says Phoebe zhennan but the authors used few more species of Phobe. More clarifications are essential in this part and title may be changed, if required.
Experimental procedures listed comprehensively except under the plant materials. List of species used for the study may be included. Corrections suggested in the document.
Results are reported systematically. Few suggestions are provided in the document.
The manuscript targets an important work which facilitates conservation of rare genetic resources. New genomic SSR markers generated for the target species. Experimental procedures and statistical analysis are well structured. However, authors need to revise the manuscript to bring in more clarity. Suggestions provided in the document.
Example:
In results - The YH Nanmu group from Guizhou formed a separate branch, suggesting that it may represent a different species of Phoebe.
In Discussion - The YH group was morphologically identified as Lindera megaphylla
Such discrepancies need to be addressed.
Improvements are needed in the English used throughout the manuscript. The tone is too informal for an international scientific study. However, the manuscript has significant potential.
The experimental design needs further more explanation
- clearly state the purpose of sample collection and its relevance to the study (e.g., assessing genetic diversity, developing markers).
- organize collection sites and populations more systematically, preferably by geographic region or in a table format..in order to be much easier to fallow
- It’s unclear how many individuals were collected at each site.
-the method is described as “adapted CTAB”, but ..what was adapted?
- PCR conditions are missing
- SSR library construction is vague - was any size selection performed?
- data scoring and reliability-were error rates tests performed?
The Validity of the findings could be improved in the discussion section
Dear Doctor,
Congratulation for the research ,,Development of gene-SSR molecular markers and analysis of
genetic diversity in Phoebe zhennan from Guizhou.
I strongly advise investing more effort in the article, as its foundation is solid and I see great potential for significant improvement.
As a general remark, the tone of the English is too informal for an international scientific article. Please modify the English expressions throughout the article to ensure a more formal and academic style
Also please correct typo and Formatting -
As recommendation :
Abstract & Introductions section :
- i suggest to avoid superlative observations like: exceptional (paragraph 13) and other...
- (reformulate, paragraph 53) Should be something like this: ,,Several molecular markers have proven to be more effective than ...
- Final statement of the introduction – please underline the objectives of the study: (1), (2) … . I highly recommend not to jump to the conclusions of the study at the end of the introduction section
Material and Method
- latitude and ongitude - (87 -88) - Longitude
-“Three Nanmu germplasm species” → better to say "three additional Phoebe species" and clarify their purpose (e.g., for comparison or genetic diversity analysis) (99-104)
- clearly state the purpose of sample collection and its relevance to the study (e.g., assessing genetic diversity, developing markers).
- organize collection sites and populations more systematically, preferably by geographic region or in a table format..in order to be much easier to fallow
- It’s unclear how many individuals were collected at each site.
-the method is described as “adapted CTAB”, but ..what was adapted?
- PCR conditions are missing
- SSR library construction is vague - was any size selection performed?
- data scoring and reliability-were error rates tests performed?
Results
Expand on polymorphism analysis (3.2)
-Indicate why only 33 primers amplified successfully out of 108—was it due to non-specific bands, failed PCR, or lack of amplification?
-clarify conclusions from UPGMA and PCA (3.6)
Disscusion
-clarify the novelty of SSR marker development ?
Are they cross-transferable across Phoebe species? -novelty, reproducibility, and potential for use in breeding/conservation programs...
- discuss why those populations may have higher or lower diversity (e.g., elevation, habitat fragmentation, population size
- introduce the limitations of the study
- practical implications - describe more the use of the results obtained in the study. Field verification of species? Marker-assisted conservation?
-add a concise summary paragraph at the end of the discussion
- include prespectives and new studies
Thank you,
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