PONE-D-24-38914Gut microbiota signatures of the three Mexican primate species, including hybrid populationsPLOS ONE

Dear Dr. Vázquez-Domínguez,

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“This project was funded by Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica-Dirección General de Asuntos del Personal Académico (PAPIIT-DGAPA, UNAM; Projects IN202122 and IN202819) awarded to E.V.D., including a Postdoctoral scholarship from DGAPA to B.S.G. D.Z. was supported by a graduate scholarship from CONAHCyT (1146867).”

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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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: No

Reviewer #2: No

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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

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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: 

Summary: The study presented in “Gut microbiota signatures of the three Mexican primate species, including hybrid populations” by Zubillaga and colleagues examines the gut microbiome composition of wild populations of the three Mexican primates and hybrid populations. Their results revealed distinct microbiome compositions in the 3 NHP species strongly shaped by host specific factors.

While I believe this is an important work and is a good fit for the PLOS ONE journal, there are number of minor concerns I would like the authors to address.

Minor Concerns:

Sample size: A total sample size of 40 with 9-11 samples might reduce the power the statistical analysis. Did the authors do a power analysis to determine if these samples sizes are sufficient?

Rarefaction: This is a crucial step in microbiome studies which addresses biases that arise from unequal sampling depths allowing for fair comparisons between samples. Given that all 40 samples had more than 20,000 reads, why did the authors choose not to perform rarefaction. With such high number of reads per sample, they would not have to drop any samples even with rarefaction. Could the authors check how rarefaction would impact the results?

Reviewer #2: 

In this brief report the authors provide results from a study of gut microbiota from three wild primate populations of black and mantled howler monkeys (Alouatta palliata and Alouatta pigra, respectively) and spider monkeys (Ateles geoffroyi) from different regions in Mexico. They also examined hybrid offspring between the groups. The methodology used was genomic DNA extraction, followed by 16S rRNA gene amplification and sequencing of the V4 region using Illumina MiSeq platform. The resulting reads were processed into ASVs with QIIME2, followed by SILVA classification and alignment with MAFFT and FastTree, phyloseq, rarefaction, and PCoA analysis. The authors examined the interspecific and intraspecific gut microbial diversity, abundance, and composition profiles of 40 individuals from the three populations. They found that interspecific differences of gut microbial diversity were higher than intraspecific differences. They found that hybrids had microbial diversities in between that of their parental species, but also showed some unique microbial taxa. They also found evidence that microbial compositions were affected by environment, geographic distribution, and host genetics. Overall, the results are interesting and so long as the authors make the sequences available to the community through public sequence databases the data will contribute to our understanding of the microbial communities of primates. However, there some issues that if adequately addressed would significantly improve this manuscript:

1. There does not appear to have been any power analysis performed to assess the statistical significance of the results. Is a total sample size of 40 individuals with 3 species groups and several hybrid groups provide sufficient power? This is particularly a concern considering that the authors mention their results differ from other reported studies and that it might be due to sample size differences or due to temporal differences in sampling. Not knowing the more likely contributor to the differences lowers the overall impact of the study.

2. Line 253 – since the cited study is in preparation (not published), this data should be either included here in this manuscript or excluded completely. There is limited way for a reviewer to make an adequate assessment of the quality of the results described without that data.

3. Figure 3 is not very useful. A more comprehensive analysis of the connection of the taxa in each of the samples with the nature of the environment, location, food sources at time of sample collection, dietary preferences, and metabolic implications regarding the taxa present would have been more meaningful. For example, what taxa are shared between the hybrids and their parent groups? Does this make sense with the environmental factors and their diets or locations? More focus on the differences observed in taxa based on some of the demographics would be more informative.

4. The study design was limited with regard to the resolution of taxonomic classification based on PCR amplification of the V4 region. The authors should include information regarding the number of reads that were discarded during processing due to chimeras etc.

5. The authors note the similarities and differences among the samples, but they do not offer much in terms of explanation for the observed differences. Also, there is little comparison to what was previously reported for these primates.

6. It does not appear that the authors have submitted their sequencing data to a public database to share with the research community. This should be done.

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Reviewer #1: No

Reviewer #2: No

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Gut microbiota signatures of the three Mexican primate species, including hybrid populations

PONE-D-24-38914R1

Dear Dr. Vázquez-Domínguez,

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,

Brenda A Wilson, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

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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: The authors addressed my concerns. I don't have any more concerns at this time. The paper can be accepted

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

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