Dear Dr Aylward,

Thank you for submitting your manuscript entitled "A Phylogenomic Framework for Charting the Diversity and Evolution of Giant Viruses" for consideration as a Methods and Resources by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I'm writing to let you know that we would like to send your submission out for external peer review. Please accept my apologies for the delay incurred while we sought external advice.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Please re-submit your manuscript within two working days, i.e. by Jun 28 2021 11:59PM.

Login to Editorial Manager here: https://www.editorialmanager.com/pbiology

During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

Given the disruptions resulting from the ongoing COVID-19 pandemic, please expect delays in the editorial process. We apologise in advance for any inconvenience caused and will do our best to minimize impact as far as possible.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Roli Roberts

Roland G Roberts PhD

Senior Editor

PLOS Biology

rroberts@plos.org

on behalf of

Paula Jauregui, PhD

Editor

PLOS Biology

pjaureguionieva@plos.org

Dear Dr. Aylward,

Thank you very much for submitting your manuscript "A Phylogenomic Framework for Charting the Diversity and Evolution of Giant Viruses" for consideration as a Methods and Resources at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by several independent reviewers.

In light of the reviews (below), we are pleased to offer you the opportunity to address the comments from the reviewers in a revised version that we anticipate should not take you very long. We will then assess your revised manuscript and your response to the reviewers' comments and we may consult the reviewers again. Please also address the formatting and editorial policy requirements.

In particular, reviewer #1 wants you to provide the number of currently recognized viral genera in the phylum Nucleocytoviricota, and to explain “megataxonomy”, disagrees with the attribution the low TC value for the MCP tree to the existence of paralogs, says that the choice of ortholog is needed, has questions about the root of the phylogenetic tree, suggests you to describe your standpoint about Klosneuvirinae, wants you to add a few words about the enrichment of Translation genes in the PM_09, thinks that you should distinguish the transfer from medusavirus and eukaryotes, wants you to explain the classification of Pimascovirales and Pokkesviricetes, and asks how the genes with introns were treated. Reviewer #2 wants you to rewrite some sentences for clarification, says that you should provide information about the source of the genomes and about the viruses contributing to each branch of the tree, asks whether there are 8 or 9 or 7 marker gene sets, thinks that you should differentiate branches containing viruses derived from culturable isolates vs. viruses assembled from metagenomic data, and wants you to add a supplementary table that provides the complete set of RED values along with the identification of the nodes/clades associated with each value. Reviewer #3 thinks that you should add gene names to the supplemental figures S1-25 and wants to see the trees associated with the combined marker genes in Fig.1. This reviewer also thinks that it would be helpful for communication if names were given at the lower levels too when possible.

DATA POLICY:

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it: Figures 1AB, 3A, S26.

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

We expect to receive your revised manuscript within 1 month.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point by point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Related" file type.

*Resubmission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this resubmission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Paula

---

Paula Jauregui, PhD

Associate Editor

PLOS Biology

pjaureguionieva@plos.org

*****************************************************

REVIEWS:

Reviewer #1: Hiroyuki Ogata. Evolution and ecology of Giant viruses.

Reviewer #2: Virus taxonomy.

Reviewer #3: Giant viruses in soil.

Reviewer #1: Viruses of the phylum Nucleocytoviricota are known for their large genomes, particles, and intriguing biological traits and potential ecological roles. The ICTV taxonomy of this viral group had been incorrect in many aspects for long time until when the recent viral megataxonomy was established in 2020, by a group of leading virologists/taxonomists including one of the authors of this manuscript. However, recent metagenomics has started to produce hundreds to thousands of additional environmental genomes of the Nucleocytoviricota. Consequently, the need for updated phylogenomic studies have re-emerged in order to establish common languages on the taxonomies within the phylum including those uncultivated. As authors correctly pointed out in their manuscript, this updated taxonomic framework is important for ecological and evolutionary analyses of this group of viruses in connection with their relationships with the evolution of cellular organisms. This is very timely and useful work.

The authors compiled 1380 Nucleocytoviricota viral genomes (both isolated and uncultured ones), identified 8863 protein families (named GVOGs), of which they identified 25 GVOGs that are represented widely in the genomes. I have appreciated the next steps that authors took to identify 7 marker genes that are considered as the best choice for phylogenomic analyses, and to objectively delineate clades (class, order, family, and genus level). The choice of marker genes for the phylogenomics is based on the Tree Certainty (TC) scores, while the taxonomic delineation has been performed using a procedure based on the Relative Evolutionary Distance (RAD). To my opinion, this is the best plausible strategy to delineate the taxonomic clades of this viral group. With these methods, the authors partitioned 2 classes, 6 orders, 32 families (this high number is appropriate), and 344 genera within the phylum. The following descriptions of the compositions of each order are easy to read and well-balanced with previous taxonomic proposals. I have nevertheless a few comments that I hope would help to further clarify some points in the current version of the manuscript.

L.33: It would be useful to provide the number of currently recognized viral genera in the phylum Nucleocytoviricota.

LL.48-54: The citation of the references 9,15,20 appear twice in this part, which makes the logical flow awkward. Probably, the first citation of "9,15,20" would not be needed.

L.75: The definition of the word "megataxonomy" may be unclear to some readers (although it is a title of well-cited paper). A few words explaining this jargon would be useful.

L.103: "this is likely the case because … multiple copies of MCP, … orthologs from paralogs (Fig. 1a)." To my understanding, for tree reconstructions, the authors chose one sequence for multicopy genes from each genome based on the score against respective HMMs. If it is the case, one MCP at most was chosen from each genome. If this is how data was processed, then, I do not agree to attribute the low TC value for the MCP tree to the existence of paralogs. Because the choice of paralogs or orthologs does not affect the reliability of their tree (if paralogs are mixed, then the tree would not be a species tree but still a gene tree). I guess it is more due to the variable nature of MCPs. Related to this, LL.365-366 for the chose of sequence dataset for individual families is somehow unclear. Does this include only the best hits, or multiple hits if they pass the threshold? A few additional words may help readers to completely understand the procedure.

L.119: The fact that the addition of RNAPS led to the decreased TC may be due to the existence of paralog. In this case, because the goal is to gain the consistency among species trees, the choice of ortholog is needed. RNAPS paralogs was described in https://journals.asm.org/doi/10.1128/JVI.00230-17.

L.140: The computation of RED requires the root of the whole tree to my understanding. The authors mentioned that Pox-Asfar group was used to root the tree. However, this would not give the root of the whole phylogenetic tree. How was the root defined? Mid-point? This should be described in the Method section.

L.154: "including 213 genera with a single representative". Just above this statement, the authors state that clades with two or one members were left as singletons, and for the family count (n=32), the authors excluded the singletons. Consistent counting scheme would be easy to follow.

L.165: The citation of ref-22 should be ref-47.

LL.173-192: The proposed classification scheme does not refer to the Klosneuvirinae, which are also used in several published papers. I suggest the authors to describe their standpoint about this taxonomic group, so that the readers can judge if they wish to continue using this taxonomic group name.

L.199-202: Citations of the papers that described the discoveries of individual genes would be needed. For actin, myosin, and kinesin, the citations seem to be https://doi.org/10.1101/2020.06.16.150565, https://doi.org/10.3389/fmicb.2021.683294, doi: 10.1128/mSystems.00293-21.

Fig.4: The enrichment of Translation genes in the PM_09 is striking but this is not mentioned in the text. A few words on this may be useful for readers.

L.240-241: In the ref-45, the PolB of medusavirus was proposed to be transferred to eukaryotes, whereas the authors assume a transfer from eukaryotes to medusavirus. These should be distinguished.

L.263-272: The authors cite two genomes in the Pimascovirales and three genomes in the Pokkesviricetes. First of all, the branches that correspond to these five genomes should be clearly indicated in the Fig.2. In addition, if the two genomes in the Pimascovirales correspond to the two branches below the Mininucleoviridae, then they look like a family level clade (within the Pimascovirales) instead of an order level clade. If these two genomes form a new order, Mininucleoviridae should be also placed at the order level too. Additional explanation is needed.

L.333: The author used Prodigal for gene call. How were genes with introns treated? This should be clear as some of the genomes contain many introns.

Reviewer #2: The work described in this manuscript to develop a framework for studying the diversity and evolution of large DNA viruses, provides an extremely useful and valuable set of resources (the giant virus orthologous group database along with associated alignments and trees) and data that help to further classify these viruses with members that have either been physically isolated or identified via metagenomic sequencing of environmental samples. As such, it represents an important contribution to the literature. The manuscript will benefit from a careful revision to correct a number of problems, including those detailed below. Major problems include failure to include Table 1, and a misnumbering of tables in the text. In addition, it will be extremely useful to have more of the original data generated in support of this work, available to its readers.

Lines 54-55: The initial wording of the sentence beginning "The uncertainty of the phylogenetic structure and taxonomy Nucleocytoviricota…" needs rewriting.

Line 83: Table S1 is not a list of genomes. This reference should most likely be to Table S2. Table S2 should provide traditional GenBank accession numbers for all genome sequences when available. When GenBank accessions are not available, the source of the indicated genome sequence (and the provided genome_id) should be identified. I have no idea what some of the provided genome_ids refer to. (I would recommend not concatenating different IDs together.)

Line 89: There is no Table 1 provided with the manuscript.

Lines 311,312: Four sequences are mentioned that are not in an NCBI database. If these sequences are not readily available, they should not be included in this analysis.

Line 90: Table S1 does not provide, as indicated in the text, a "descriptions of all GVOGs".

Line 377: There is no Table 1

Line 387: Are there eight or nine marker gene sets?

Line 401: Now we are at (the final) 7 marker sets. The number of marker gene sets varies according to the analysis providing in figure 3. But in this section of the Methods, it is not clear why these numbers vary from 9 to 8 to 7.

In the supplementary phylogenetic trees for the set of 25 gene sets, it is very difficult to determine from the provided figures the characterizations of the sets provided in Table S1. For example, for GVOGs 115 and 152, the table indicates that these GVOGS are not represented in viruses belonging to the order Asfuvirales. In the figures for these two GVOG trees, there is also no indication of the presence of poxviruses (order Chitovirales) in the tree. GVOG 152 also appears to not have a representative from the pandoraviruses.

In all text and figures, the proposed order pandoravirales should not be capitalized or italicized since it is only proposed and is not an official taxon approved by the ICTV.

Figure 2 and corresponding text: All proposed taxa should, as indicated above, not be capitalized or italicized since they are not official taxa approved by the ICTV.

Figure 2: It would be useful to be able to identify the viruses contributing to each branch of the tree, especially for unlabeled, single genome branches. It is not clear if this information is available or where it might be.

Figure 2: It would be useful to differentiate branches containing viruses derived from culturable isolates vs. viruses assembled from metagenomic data.

Figure 3a: A supplementary table should be available that provides the complete set of RED values along with, importantly, the identification of the nodes/clades associated with each value. Along with Table S2, this will provide the data that supports the creation of the proposed new species and families.

Figure 4. Panels A and B should be labeled on the figure.

Reviewer #3: Recently several metadata studies (including one by these authors) have identified thousands of new genomes in the Nucleocytoviricota from environmental samples. This current work provides a phylogenetic and more importantly a taxonomic context to interpret and communicate this data. The research uses appropriate state of the art methods. An important aspect is the used of RED values to normalize taxonomic levels to ranges of genetic distances. The manuscript is very well written and will be an important contribution to the field. I only have minor concerns.

In browsing the 25 GVOG trees is the supplemental data S1-25, it was clear that different trees gave very different results. It would be nice to add gene names to the supplemental figures S1-25.

It would be good to see the trees associated with the combined marker genes in Fig.1 (maybe I missed these?). How did they vary? This would give a sense of how robust the taxonomy is to any individual gene.

Reference in the manuscript the itol tree https://itol.embl.de/tree/1281731864487941620067021 on the github site.

The authors only suggested names at the phylum, class and order level. All families and genera were given numbers. It would be helpful for communication if names were given at these lower levels too when possible or is the expectation that these will need to be dealt with separately as many genera will need to be renamed as species.

Dear Dr. Aylward,

Thank you for submitting your revised Methods and Resources entitled "A Phylogenomic Framework for Charting the Diversity and Evolution of Giant Viruses" for publication in PLOS Biology. I have now discussed your revision with the Academic Editor. 

We will probably accept this manuscript for publication, provided you satisfactorily address the following data and other policy-related requests.

DATA POLICY:

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it: Figures 1AB, 3A, S26.

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

As you address these items, please take this last chance to 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 cover letter that accompanies your revised manuscript.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

-  a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

-  a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable)

-  a track-changes file indicating any changes that you have made to the manuscript. 

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information  

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Early Version*

Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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

Please do not hesitate to contact me should you have any questions.

Sincerely,

Paula 

---

Paula Jauregui, PhD,

Associate Editor,

pjaureguionieva@plos.org,

PLOS Biology

Dear Dr Aylward,

I'm handling your paper temporarily while my colleague Dr Paula Jauregui is out of the office. On behalf of my colleagues and the Academic Editor, Curtis Suttle, I'm pleased to say that we can in principle offer to publish your Methods and Resources "A Phylogenomic Framework for Charting the Diversity and Evolution of Giant Viruses" in PLOS Biology, provided you address any remaining formatting and reporting issues. These will be detailed in an email that will follow this letter and that you will usually receive within 2-3 business days, during which time no action is required from you. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have made the required changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS: We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Roli Roberts

Roland G Roberts PhD

Senior Editor

PLOS Biology

rroberts@plos.org

on behalf of

Paula Jauregui, PhD 

Associate Editor 

PLOS Biology

pjaureguionieva@plos.org