Host species is linked to pathogen genotype for the amphibian chytrid fungus (Batrachochytrium dendrobatidis)

Allison Q. Byrne; Anthony W. Waddle; Veronica Saenz; Michel Ohmer; Jef R. Jaeger; Corinne L. Richards-Zawacki; Jamie Voyles; Erica Bree Rosenblum

doi:10.1371/journal.pone.0261047

Peer Review History

Original SubmissionNovember 22, 2021
5 Jan 2022 Decision Letter - Louise A. Rollins-Smith, Editor PONE-D-21-37079Host Species is Linked to Pathogen Genotype for the Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis) PLOS ONE Dear Dr. Byrne and Co-authors, 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. ============================== I apologize for the delay in a decision on you paper. For reasons that are unclear to me, I found it difficult to secure good reviewers. I contacted many potential reviewers, and many of them declined to review. Your manuscript has now been reviewed by two knowledgeable reviewers, and both are generally positive. Thus, I recommend minor revisions to address any reviewer concerns. Please carefully read their comments and respond with a revised manuscript that addresses as many of the comments as possible. Provide your responses in a point-by-point Response to Reviewers. ============================== Please submit your revised manuscript by Feb 19 2022 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, Louise A. Rollins-Smith Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. 2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why. 3. To comply with PLOS ONE submissions requirements, please provide methods of sacrifice in the Methods section of your manuscript. 4. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. 5. We note that Figure 1 in your submission contain map/satellite images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: a) You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license. We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text: “I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.” Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].” b) If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. The following resources for replacing copyrighted map figures may be helpful: USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/ The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/ Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/ Landsat: http://landsat.visibleearth.nasa.gov/ USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/# Natural Earth (public domain): http://www.naturalearthdata.com/ 6. 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. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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 ****** 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 ****** 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: Byrne et al. examine the population genetic structure of a fungal pathogen of amphibians from two regions of North America using a SNP genotyping method (Fluidigm). They sampled multiple species from a handful of sites in both Pennsylvania and Nevada. All of the samples are of the global panzootic lineage (GPL), and the authors are able to divide the genotypes into GPL-1, GPL-2 or “unassigned”. Unassigned genotypes are either a coinfection by both a GPL-1 & GPL-2 or a hybrid genotype. A major finding is that genotypes associated with bullfrogs are more often GPL-1, and genotypes from green frogs are more often “unassigned”. This leads to the conclusion that there is some level of specificity between host and pathogen genotypes and possibly coevolution. Overall this paper is technically sound and presents a lot of new data. It also is a nice investigation of population structure and may change the way people think about the importance and role of Bd genotype in local disease dynamics. Most of my comments are about clarifications and interpretations. Suggestions: 1. I think it is worth explaining what the basis for the GPL-1 vs. GPL-2 split is in the introduction and possibly discussion sections. GPL is really a clonal lineage that has fixed heterozygosity of some markers. GPL-1 is a version that has not had some consistent loss of heterozygosity (LOH) events that define GPL-2. A lot of these differences can be obscured by independent LOH events, and there may be intermediate genotypes that have only had some but not all of the LOH events. This paper is interesting in that it clearly picks up the two major groups and the intermediate. Ultimately it will be really fascinating if the unassigned genotypes in this study are sexually produced hybrids of these two sublineages. One thing worth noting is that European genotypes have largely been ignored in many pop gen studies, and I think it is important to consider that Europe may actually be the original location from which GPL dispersed across the world. See the phylogeny in Rosenblum 2013 which shows European strains at the base of the GPL clade. 2. There are a few places where terms like “potential ancestral population of GPL” are used, such as line 36 of the abstract. O’Hanlon and Byrne have shown that Asia has the highest amount of lineage diversity and is the most likely source of the GPL for that reason. At this point there is no evidence of any genotypes other than GPL in North America, but it is unclear when GPL might have colonized the region. Perhaps the authors are thinking about the source population of the MRCA of the known GPL strains in the world. As stated in point 1, I think we don’t know but I also think that you have to carefully consider European samples and collect more GPL genotypes from Asia before concluding that the GPL MRCA was North American. 3. I mentioned fixed heterozygosity in point 1. Do the fluidigm markers allow heterozygosity estimation? If so, it would be interesting to see average observed heterozygosity across GPL-1, GPL-2 and unassigned. If the markers allow phasing and haplotype estimation shouldn’t it be possible that some of the markers in the unassigned would show more than 2 haplotypes under coinfection and never more than 2 for the hybrid hypothesis? I realize the authors do mention parasexual reproduction in the Discussion, and that could lead to more than 2 alleles at an individual locus. I think it’s fine to mention parasexual reproduction, but it is perhaps worth mentioning that the hybrid genotypes reported in Schloegel and Jenkinson have never had more than 2 alleles per locus. 4. Line 47, some wording seems weird to me. Increased virulence will never be advantageous for a host. I think it may be more appropriate to say that strong host-specificity should favor intermediate virulence for that host. 5. Line 69, delete “remains untested” 6. Line 86, what kind of gloves? 7. Line 141, what is “(N=13; 13,25)” 8. Line 198-199, I think this should be clarified because as written it’s not clear why GPL-2 isolates would be heterozygous. 9. Line 247-248. Please explain what the test for within individual variation means. Variation is significantly structured by individuals? 10. Figure S2, please explain what the X axis represents. 11. The associations in the PA population are interesting. These data really suggest future experimental studies. It might be worth expressing how you might test what is happening here with bullfrog GPL-1 association experimentally. 12. Line 382, suggest “possible” instead of “likely”. In most senses parasexual and sexual reproduction yield a pretty similar outcome. It could be hard to distinguish the two without observing intermediates or complete genome sequences. 13. Line 386-387. This sentence was hard for me to follow. The use of parasexuality in nature is to present complex gene sets? Reviewer #2: This study describes the genetic variation of a batrachochytrid fungus lineage (Bd-GPL) across several amphibian hosts in two regions of North America to explore pathogen diversification concerning host species. While the manuscript tackles an important topic, is well written, and provides detailed methods, in my opinion, the results are a little bit misleading, not providing enough support for the authors’ conclusions. My main concern is that the sub-lineage category is ignored in the AMOVA analyses. Sub-lineage information might affect and confuse the relative contribution of host species to the genetic variance. In the data reduction analysis, the authors find that the sub-lineage explains the highest variation among samples (58.4%, Figure 1C), but this information is ignored in the subsequent analyses. Also, the PCA analysis clusters together samples from the two studied locations (Nevada and Pennsylvania), which, in turn, could change the interpretation of the results. A phylogenetic analysis integrating both regions could also improve result interpretation. Likewise, when describing the phylogenetic clades would be important to better characterize them. For instance, genetic diversity statistics (e.g., nucleotide diversity) could be computed between each sub-lineage to compare regions. Moreover, the coevolutionary hypothesis should be supported with further analysis of co-phylogeny congruence between pathogen and host. Another of my concerns is that Pennsylvania sampling sites could be dependent due to the geographic proximity. I recommend exploring the correlation between genetic and geographic distances (Mantel test). Finally, I think that one overlooked difference between Pennsylvania and Nevada sites that affects the pathogen diversification is the amphibian community structure. It would be worthwhile to characterize and integrate amphibian community metrics (e.g., richness per site) in this study. ****** 6. 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: Yes:** Tim James Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0261047.r001
Revision 1
28 Feb 2022 Author Response Note that the line numbers listed below refer to the track changes version of the manuscript. Editor Comments: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. � The manuscript has been carefully reformatted to match the PLOS ONE style guide. 2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why. � Corrected as suggested. We now include specific information about the permits granted for this work (Lines 131-135). 3. To comply with PLOS ONE submissions requirements, please provide methods of sacrifice in the Methods section of your manuscript. � We added this additional detail to the methods section (Lines 152-153) 4. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. � Corrected as suggested. We now include additional details about the ethics approvals received from each institution in the methods section (Lines 126-131) 5. We note that Figure 1 in your submission contain map/satellite images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: a) You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license. We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text: “I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.” Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].” b) If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. The following resources for replacing copyrighted map figures may be helpful: USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/ The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/ Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/ Landsat: http://landsat.visibleearth.nasa.gov/ USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/# Natural Earth (public domain): http://www.naturalearthdata.com/ � We changed the imagery used in Figure 1, sourcing the satellite images from the Gateway to Astronaut Photography of Earth. We thank the editor for pointing us to this public domain resource. We added an acknowledgement for the photos in the Figure 1 legend (Lines 307-308). 6. 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. � We reviewed the references and edited them to match PLOS ONE’s guidelines (Lines 585-1343) Reviewer Comments Reviewer #1: Byrne et al. examine the population genetic structure of a fungal pathogen of amphibians from two regions of North America using a SNP genotyping method (Fluidigm). They sampled multiple species from a handful of sites in both Pennsylvania and Nevada. All of the samples are of the global panzootic lineage (GPL), and the authors are able to divide the genotypes into GPL-1, GPL-2 or “unassigned”. Unassigned genotypes are either a coinfection by both a GPL-1 & GPL-2 or a hybrid genotype. A major finding is that genotypes associated with bullfrogs are more often GPL-1, and genotypes from green frogs are more often “unassigned”. This leads to the conclusion that there is some level of specificity between host and pathogen genotypes and possibly coevolution. Overall this paper is technically sound and presents a lot of new data. It also is a nice investigation of population structure and may change the way people think about the importance and role of Bd genotype in local disease dynamics. Most of my comments are about clarifications and interpretations. Suggestions: 1. I think it is worth explaining what the basis for the GPL-1 vs. GPL-2 split is in the introduction and possibly discussion sections. GPL is really a clonal lineage that has fixed heterozygosity of some markers. GPL-1 is a version that has not had some consistent loss of heterozygosity (LOH) events that define GPL-2. A lot of these differences can be obscured by independent LOH events, and there may be intermediate genotypes that have only had some but not all of the LOH events. This paper is interesting in that it clearly picks up the two major groups and the intermediate. Ultimately it will be really fascinating if the unassigned genotypes in this study are sexually produced hybrids of these two sublineages. One thing worth noting is that European genotypes have largely been ignored in many pop gen studies, and I think it is important to consider that Europe may actually be the original location from which GPL dispersed across the world. See the phylogeny in Rosenblum 2013 which shows European strains at the base of the GPL clade. � We added an additional sentence explaining the original genomic characterizations of GPL-1 and GPL-2 in the introduction (Lines 91-95). Additionally, we added a discussion of Europe as an important site of high GPL genetic diversity (Lines 500-504). 2. There are a few places where terms like “potential ancestral population of GPL” are used, such as line 36 of the abstract. O’Hanlon and Byrne have shown that Asia has the highest amount of lineage diversity and is the most likely source of the GPL for that reason. At this point there is no evidence of any genotypes other than GPL in North America, but it is unclear when GPL might have colonized the region. Perhaps the authors are thinking about the source population of the MRCA of the known GPL strains in the world. As stated in point 1, I think we don’t know but I also think that you have to carefully consider European samples and collect more GPL genotypes from Asia before concluding that the GPL MRCA was North American. � We thank this reviewer for pointing out that Europe may also be an “ancestral population” for the Bd-GPL. We added a note to our discussion including this important possibility. (Lines 500-504). Additionally, we changed some of the wording in our abstract (Lines 48,58) and discussion (Lines 484, 486, 490, 502, 566) to better describe what we mean by “ancestral population of Bd-GPL”, which is “the source population of the most recent common ancestor of modern Bd-GPL lineages.” 3. I mentioned fixed heterozygosity in point 1. Do the fluidigm markers allow heterozygosity estimation? If so, it would be interesting to see average observed heterozygosity across GPL-1, GPL-2 and unassigned. If the markers allow phasing and haplotype estimation shouldn’t it be possible that some of the markers in the unassigned would show more than 2 haplotypes under coinfection and never more than 2 for the hybrid hypothesis? I realize the authors do mention parasexual reproduction in the Discussion, and that could lead to more than 2 alleles at an individual locus. I think it’s fine to mention parasexual reproduction, but it is perhaps worth mentioning that the hybrid genotypes reported in Schloegel and Jenkinson have never had more than 2 alleles per locus. � We thank the reviewer for this suggestion. We now include a supplemental figure showing observed heterozygosity per individual for Bd-GPL1, Bd-GPL2 and the unassigned genotypes (Figure S6). We also now mention the fact that previously known hybrids have never had more than two alleles to the discussion (Line 544-545). In our data we find that even the isolates sometimes have more than 2 alleles at certain loci, perhaps due to copy number variation. We are currently working on applying new analytical methods to try to better address this question, but do not feel confident discriminating between coinfections and hybrids for this study. Future work isolating Bd at the PA site is ongoing and we hope to know more about the frequency of hybrid lineages at these sites in the near future. 4. Line 47, some wording seems weird to me. Increased virulence will never be advantageous for a host. I think it may be more appropriate to say that strong host-specificity should favor intermediate virulence for that host. � Corrected as suggested. We edited this sentence for clarity (Lines 71-72). 5. Line 69, delete “remains untested” � Corrected as suggested (Line 100). 6. Line 86, what kind of gloves? � Added “nitrile” to this description (Line 136). 7. Line 141, what is “(N=13; 13,25)” � Edited this for clarity. Now it just includes the in-text citation and not the sample size. (Line 200) 8. Line 198-199, I think this should be clarified because as written it’s not clear why GPL-2 isolates would be heterozygous. � We edited this sentence for clarity (Line 271-272). 9. Line 247-248. Please explain what the test for within individual variation means. Variation is significantly structured by individuals? � The test for within-individual variation is included as a null hypothesis for the AMOVA. If most of the variation were to be attributed to within individual variation (rather than between sites or between species within a site), it would indicate a panmictic population. We included a sentence in our methods to explain this reasoning (Lines 244-247). 10. Figure S2, please explain what the X axis represents. � In Figure S2 the x-axis represents the component of covariance for the observed data (black line) and for the randomly permuted sample matrices (gray bars). We added this detail the figure legend for Figure S4 (previously Figure S2) (Line 1379) and labeled the axis on the figure itself. 11. The associations in the PA population are interesting. These data really suggest future experimental studies. It might be worth expressing how you might test what is happening here with bullfrog GPL-1 association experimentally. � We agree that this is an exciting opportunity for future research. We added a paragraph to the discussion that mentions some potential future research approaches (Lines 468-483). 12. Line 382, suggest “possible” instead of “likely”. In most senses parasexual and sexual reproduction yield a pretty similar outcome. It could be hard to distinguish the two without observing intermediates or complete genome sequences. � Corrected as suggested (Line 539). 13. Line 386-387. This sentence was hard for me to follow. The use of parasexuality in nature is to present complex gene sets? � We edited this sentence for clarity (Line 543-545). Reviewer #2: This study describes the genetic variation of a batrachochytrid fungus lineage (Bd-GPL) across several amphibian hosts in two regions of North America to explore pathogen diversification concerning host species. While the manuscript tackles an important topic, is well written, and provides detailed methods, in my opinion, the results are a little bit misleading, not providing enough support for the authors’ conclusions. My main concern is that the sub-lineage category is ignored in the AMOVA analyses. Sub-lineage information might affect and confuse the relative contribution of host species to the genetic variance. In the data reduction analysis, the authors find that the sub-lineage explains the highest variation among samples (58.4%, Figure 1C), but this information is ignored in the subsequent analyses. Also, the PCA analysis clusters together samples from the two studied locations (Nevada and Pennsylvania), which, in turn, could change the interpretation of the results. A phylogenetic analysis integrating both regions could also improve result interpretation. � We thank the reviewer for their thoughtful suggestions. In this study, we used an AMOVA as a complimentary analysis to the DAPC clustering analysis. Each of these analyses use the raw genetic data and test for structure either based solely on the genetic patterns (as in the DAPC) or based on predefined, ecological categories (as in the AMOVA). The sub-lineage information is not included as a category in the AMOVA because we know for a fact that the variance in the molecular data is related to these sub-lineage categories (as the sub-lineages were calculated from the genetic data to describe the clusters within). The purpose of the AMOVA is to test our hypotheses that the variation in the genetic data is structured by either sampling locality or host species. While Figure 1 includes a PCA containing samples from both PA and NV, we note that Figures 2 and 3 include PCAs calculated from each separate sample group. We agree with the suggestion of including a combined phylogeny for all samples used in this study. We now include this figure in the supplement (Figure S3) and added a note in the methods to reflect this addition (Line 197-198). Likewise, when describing the phylogenetic clades would be important to better characterize them. For instance, genetic diversity statistics (e.g., nucleotide diversity) could be computed between each sub-lineage to compare regions. Moreover, the coevolutionary hypothesis should be supported with further analysis of co-phylogeny congruence between pathogen and host. � We added a new supplementary figure (Figure S6) and related that compares heterozygosity among the different sub-lineages described in the paper. We feel that our general hypotheses of coevolution are ripe for further studies, including cophylogeny analyses, but that this analysis is outside the scope of this particular study. We added a new paragraph to the discussion that mentions some potential future research approaches, including cophylogenetic studies (Lines 468-483). Another of my concerns is that Pennsylvania sampling sites could be dependent due to the geographic proximity. I recommend exploring the correlation between genetic and geographic distances (Mantel test). � We thank the reviewer for the recommendation and now include a mantel test (Figure S5) to explore the correlation of genetic and geographic distance for our samples. We found that there is a statistically significant, yet weak positive correlation between genetic and geographic distance for Pennsylvania. This is now mentioned in the paper (Line 350-352). Finally, I think that one overlooked difference between Pennsylvania and Nevada sites that affects the pathogen diversification is the amphibian community structure. It would be worthwhile to characterize and integrate amphibian community metrics (e.g., richness per site) in this study. � We added more information about the amphibian species richness to the site descriptions (Lines 104-106, 108-109). We think this is an important point and added text to discuss the possible impacts that amphibian community diversity may have on Bd (Lines 462-464). Attachments Attachment Submitted filename: plost_one_rebuttal_letter_final_feb2022.pdf https://doi.org/10.1371/journal.pone.0261047.r002
2 Mar 2022 Decision Letter - Louise A. Rollins-Smith, Editor Host species is linked to pathogen genotype for the amphibian chytrid fungus (Batrachochytrium dendrobatidis) PONE-D-21-37079R1 Dear Dr. Byrne, 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. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Louise A. Rollins-Smith Academic Editor PLOS ONE I have looked carefully at your response to reviewers and your revised manuscript and figures. It is my view that you have done a very good job of addressing the reviewer comments. Thus, I am happy to recommend acceptance of the manuscript. https://doi.org/10.1371/journal.pone.0261047.r003
Formally Accepted
4 Mar 2022 Acceptance Letter - Louise A. Rollins-Smith, Editor PONE-D-21-37079R1 Host species is linked to pathogen genotype for the amphibian chytrid fungus (Batrachochytrium dendrobatidis) Dear Dr. Byrne: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Louise A. Rollins-Smith Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0261047.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .