PONE-D-21-20639

Comparative Chromosome Painting in the Black-Hawk-Eagle (Spizaetus tyrannus) and Gallus gallus with the use of Macro and Microchromosome Probes.

PLOS ONE

Dear Dr. de Oliveira,

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All reviewers suggest that the introduction should be more informative, and I agree with them. I also agree that the short title is too long and should be reduced.

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The work from Carvalho et al brings and interesting analysis in the in the Black-Hawk-Eagle (Spizaetus tyrannus) using BAC and WCP experiments, both derived from Gallus gallus. The results are very well represented in high-quality figures and the and brings new pieces of information for the big puzzle that avian karyotype evolution represents. Below I indicate some points to be adjusted and put special attention to one suggestion for discussion: The work would profit too much if the analyses were done under a phylogenetic context. An updated phylogeny with the special focus on the phylogenetic position and relation between Chicken and Spizaetus would bring more light to the general results found.

Abstract

I missed some introduction about BIRDs cytogenetic in the beginning of Abstract. The authors go too direct about Accipitriformes and most readers do not have such phylogenetic information to which group we are dealing with.

Why are the authors aiming to investigate homologies with chicken? (Please see comments below). Abstract also brings some previous data on NNI that were not originated from this work. I suggest removing. The terms STY and NNI are used without any previous information/explanation about, causing misunderstandings.

A final conclusion is missing in the abstract.

Introduction

Page3 line3: Present an unusual (delete with)

Page 3 line 12: The accurate identification of the chromosomal pairs involved.… if we aim to identify synapomophies….

Introduction miss a clear motivation for the study. Why using GGA probes? I missed some info (Figure would be better) about the phylogenetic position and relation between Chicken and Spizaetus. Moreover, inform here as well thar STY corresponds to Spizaetus tyrannus on its first mention.

Results

Subtitle Karyotypes before the first paragraph

Figures

Figure 3: I did not understand why using the metaphases present in right column? The signals and chromosomal morphology are clearly visible in the FISH images.

Scale bars are missing in all figures.

Reviewer #2: In this study, the authors used comparative chromosome painting to reveal homology of chromosomal segments between Spizaetus tyrannus (the Black-Hawk-Eagle) and Gallus gallus. The study is conceptually and methodically motivated. The karyotype of S. tyrannus was previously studied only using conventional chromosome staining technique that showed 2n=68 (32m/sm+8st+18a+8m+ZW), and this happened for the first time that the karyotype of this bird of prey was studied by comparative chromosome painting. As a result, the study provides a novel insight into the cytogenetics of the Black-Hawk-Eagle. Both whole chromosome-specific G. gallus probes of the 1st-10th pairs and chromosome-specific G. gallus BAC probes from 11 pairs of microchromosomes were used. The study evidenced 29 evolutionary fission-fusion events that happened in the evolution of S. tyrannus and identified the particular chromosome pairs in the referenced G. gallus karyotype, which have undergone restructuring or have remained unchanged. Another sufficient result concerns the comparison between S. tyrannus and Nisaetus nipalensis orientalis, which is its only close relative studied so far by comparative chromosome painting. The comparison showed both similarities and differences between the species. The MS is well illustrated by tables and pictures of good quality.

In general, the work is interesting and deserves publication in the journal.

However, there are some shortcomings in the work. Main disadvantages are (1) almost complete absence of basic data on the karyotypes of the discussed species, which makes it difficult to adequately assess the results obtained, and (2) ignorance of the taxonomic component.

All other comments that I have are mainly suggestions for improving the article (unfortunately, there is no line numbering in the MS, which makes it difficult for the reviewer to work).

1. Running title. It is too long, should be shorter, e.g., “Comparative Chromosome Painting in Spizaetus tyrannus and Gallus gallus“.

2. Abstract. Please, decipher GGA, should be Gallus gallus (GGA), as done above for Spizaetus tyrannus (STY)

3. Introduction.

Page 3. Paragraph 1. Please, provide a putative avian ancestral karyotype, with 2n and the number of microchromosomes.

Page 3. Paragraph 1. Here (or further, in Discussion), provide the G. gallus karyotype to make further reasoning clearer.

Page 3. Paragraph 3. Unify way of citing – in other places you use numbering.

Page 4. Paragraph 4. Please, specify the species studied, Spizaetus tyrannus

4. Discussion.

Page 6, Paragraph 1. Please, expand the statement “We report slight differences in chromosome morphology….” by clarifying what Tagliarini et al. (2007) have reported. Describe the differences, give for comparison one and the other karyotypes (otherwise, you only have a declaration).

Page 7. Paragraph 3. The Japanese mountain hawk-eagle Nisaetus nipalensis orientalis (Temminck & Schlegel, 1844), not the Hodgson's hawk-eagle Nisaetus nipalensis Hodgson, 1836.

Other remarks can be found in the MS attached.

Reviewer #3: This study provides a cytogenetic mapping of the Black-Hawk-Eagle (Spizaetus tyrannus) using whole-chromosome paints and BAC probes of Gallus gallus. Using this cytogenetic mapping, the authors investigate the chromosome homologies between Spizaetus tyrannus and Gallus gallus. Overall the manuscript presents an advance in cytogenetic of Accipitriformes. The manuscript is written in understandable English, but some procedures require more details. Please see below my

comments that could help to further enhance the quality of the study.

1) This version is without line numbers to make it easier for reviewers to comment on the text.

2) Abstract: “chicken (Gallus gallus – GGA)”.

3) Introduction and discussion: The manuscript is written in a manner suitable for a more specialized journal such as Cytogenetic and Genome Research or Comparative Cytogenetics, but not ideally for a general journal such as PlosOne. The introduction and discussion are rather limited to the focus of the analyses. This is a shame as I think the authors did a good job to gather nice cytogenetic results. Thus, the authors should consider redrafting these sections on a broad context.

The introduction could be more informative about the study as a whole. For example, the authors can give information about the contribution of this kind of work to karyotypic evolution and chromosomal organization of Accipitriformes. In this line, the last paragraph of introduction can be better written showing the importance of this study, and not merely comment “This study presents the cytogenetic mapping of a species…”. Moreover, other intriguing topic is about the origin of microchromosomes. And maybe the authors could comment a little bit about this topic in the introduction and also discussion. Below is a reference related to this topic.

- Waters, P. D., Patel, H. R., Ruíz-Herrera, A., Álvarez-González, L., Lister, N. C., Simakov, O., ... & Graves, J. A. M. (2021). Microchromosomes are building blocks of bird, reptile and mammal chromosomes. bioRxiv.

4) Results, paragraph 1: The authors commented that they detected “four pairs of microchromosomes” but they indicated five (29, 30, 31, 32, and 33). Considering the Figure 1B the pair 29 apparently is not a microchromosome.

5) Results, paragraph 2: “The most extreme examples are the fission of GGA1 into six pairs in STY, and GGA3 into three distinct pairs”. It seems that for GGA3 are four pairs (13, 16, 19, and 20) (Figure 4), right?

6) Discussion, paragraph 3: “…karyotype of STY when compared to Gallus”; “…karyotype of STY when compared to Gallus gallus”.

7) Discussion, paragraph 4: “…microchromosomes (STY4, 7 and 9; NNI2, 4 and 9), none of them”; “microchromosomes (STY: pairs 4, 7 and 9; NNI: pairs 2, 4 and 9), none of them”.

“…with microchromosomes (GGA: pairs 18, 19, 24 and 25)”.

8) Discussion, paragraph 5: “These results show that they are morphologically similar species that until the last decade were part of the same genus [14].”. I think the authors results did not show an association of chromosomal data with morphological similarity and this should be corrected.

9) Conclusion: “…of S.tyrannus should be…”; “…of S. tyrannus should be…”.

10) Methods, paragraph 1: What the name and country of the Zoos?

11) Methods, paragraph 2: “…and labelled directly by FTIC”; “and labelled directly by fluorescein isothiocyanate (FITC)”.

12) Methods: the results of this study are based on FISH with GGA probes. However, FISH procedure is not sufficiently described. I would appreciate if at least main/important steps of the FISH procedure are described. This would also avoid any doubt about the accuracy of the results.

13) Figure 3: “FITC”.

14) Please, give a scale bar information for all figures. It is very important, specially considering the presence of macrochromosomes and microchromosomes.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: PONE-D-21-20639_reviewer.pdf

PONE-D-21-20639R1Comparative Chromosome Painting in the Black-Hawk-Eagle (Spizaetus tyrannus) and Gallus gallus with the use of Macro and Microchromosome Probes.PLOS ONE

Dear Dr. de Oliveira,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Dec 11 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Maykon Passos Cristiano, D. Sc.

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

I return with another round of review. Two reviewers (2 and 3) make small suggestions to improve the quality of the manuscript. Also, the Plos one does not submit proof before publication, for this reason, I suggest special care in this review.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: N/A

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The manuscript has been extensively revised. Generally, all my comments were addressed and I am satisfied with the revision. However, minor typos remain (listed below and marked in the attached PDF), which can be corrected while editorial processing and preparing the manuscript for publication.

Title

1. Comparative Chromosome Painting in the Black-Hawk-Eagle

(Spizaetus tyrannus) and Gallus gallus with the use of Macro

and Microchromosome Probes.

It is better to use only Latin names in both cases, e.g.

Comparative Chromosome Painting in Spizaetus tyrannus and Gallus gallus with the use of Macro-and Microchromosome Probes.

Then, Macro-and Microchromosome Probes should be written with a hyphen.

Abstract

1. Here and elsewhere (Introduction, Results), should not be duplicated diploid number and 2n, as for example in the case “diploid number around 2n=80”. Should be either diploid number around 80 or 2n around 80

2. The family Accipitridae contains 14 subfamilies, not only Aquilinae, then, the phrase “the Accipitridae family is the most diverse and includes the subfamily Aquilinae…” needs to be revised (see my suggestion in the PDF text).

Results

1. Table 3. Please, explain in the title the abbreviation “q”

-

Reviewer #3: Carvalho et al. 2021 – Resubmission “Comparative Chromosome Painting in the Black-Hawk-Eagle (Spizaetus tyrannus) and Gallus gallus with the use of Macro and Microchromosome Probes”. This is a re-review of the manuscript for PlosOne. The new version of the manuscript is improved, and I do think the results overall are publishable. I only have a few suggestions that can be considered during the production process.

- Please alphabetic order: “…In fact, rearrangements involving microchromosomes were detected in few orders: Psittaciformes, Cuculiformes, Suliformes, Caprimulgiformes and the Accipitriformes [13-15].”

-“Gallus gallus probes used in the fluorescent in situ hybridization (FISH) experiments produced…”.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: PONE-D-21-20639_R1_rev.pdf

Comparative Chromosome Painting in Spizaetus tyrannus) and Gallus gallus with the use of Macro and Microchromosome Probes.

PONE-D-21-20639R2

Dear Dr. de Oliveira,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Maykon Passos Cristiano, D. Sc.

Academic Editor

PLOS ONE