Dear Dr. Mazzoni Zerbinato A Silva,

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Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors demonstrated that the four B variants of three Psalidodon species contain retrotransposed copies of the sbno2 and simc1 genes. These gene copies were acquired in a common ancestor and have been maintained across speciation events. The findings are supported by bioinformatic analysis of gDNA and RNA reads, PCR, and FISH. The absence of introns in both genes suggests that retrotransposition caused the transfer of these gene fragments. Interestingly, it appears that both pseudogenes may influence the expression of their counterparts located on the A chromosomes. The quality of the results is high, and the findings are logically discussed in the context of current knowledge.

Line 45

Please consider clarifying that B chromosome drive mechanisms can occur pre-meiotically and post-meiotically, not just during meiosis, depending on the species.

Line 75 and others (e.g. line 201, )

Please avoid using the term “A genome” and instead use “A chromosomes.” The term “A genome” is typically reserved for distinguishing subgenomes in allopolyploid species, which could cause confusion.

Line 301

Please indicate where in the manuscript the quick protocol for identifying B-chromosome-bearing individuals is shown.

Figure 3.

Please add in the figure “A chromosome-derived retrotransposition of….”

Reviewer #2: Manuscript ID: PONE-D-25-45744

Title: B chromosome retrotransposed sequences persist through speciation, contributing to genomic and regulatory innovations in the fish genus Psalidodon (Characiformes, Acestrorhamphidae)

Authors: Lucas F. Lasmar, Mateus R. Vidal, Pamela C. F. Nadai, Rodrigo Zeni dos Santos, Raquel da Costa Machado, Ricardo Utsunomia, Francisco J. Ruiz-Ruano, Alexander Suh, Claudio Oliveira, Duilio M. Z. A. Silva, Fausto Foresti

The goal of the presente paper was to investigate the evolutionary history of two pseudogenes, sbno2-B and simc1-B, on the B chromosomes in Psalidodon species, as well as their potential effects on gene expression. The authors demonstrate that four B chromosome variants of three Psalidodon species have retrotransposed copies of the sbno2 and simc1 genes that were acquired in a common ancestor and maintained across speciation events.

B chromosomes and retrotransposons are distinct genetic elements that share a remarkable ability to selfishly take advantage of biological processes, thereby increasing their transmission to future generations through different molecular mechanisms. B chromosomes are supernumerary genetic elements that primarily cheat by preferentially being incorporated into generative cells during meiosis. In contrast, retrotransposons are genomic sequences that over-replicate during cell cycles.

The work is of great interest, primarily because it addresses two distinct and intriguing genetic elements. In this study, the authors demonstrated that retrotransposons have activity on B chromosomes, generating genomic diversity. Furthermore, Psalidodon spp. can be considered “model species” for the study of B chromosomes.

I recommend your publication.

One correction is pointed out:

Page 2, Line 58: “The genus Psalidodon Eigenmann, 1911 within the family Characiformes includes about 30 species,…”

within the Family Acestrorhamphidae or order Characiformes

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Reviewer #1: No

Reviewer #2: No

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B chromosome retrotransposed sequences persist through speciation, contributing to genomic and regulatory innovations in the fish genus Psalidodon (Characiformes, Acestrorhamphidae)

PONE-D-25-45744R1

Dear Dr. Mazzoni Zerbinato A Silva,

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,

James Lee Crainey, Ph.D.

Academic Editor

PLOS One

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