PONE-D-23-28975Diversity of major histocompatibility complex of II B geneand mate choice in a monogamous and long-lived seabird, the little auk (Alle alle)PLOS ONE

Dear Dr. Wojczulanis-Jakubas,

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2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s) among others, which needs to be addressed:

Minias, Piotr & Pikus, Ewa & Anderwald, Dariusz. (2019). Allelic diversity and selection at the MHC class I and class II in a bottlenecked bird of prey, the White-tailed Eagle. BMC Evolutionary Biology. 19. 10.1186/s12862-018-1338-3. 

Juola Frans A. and Dearborn Donald C. 2012Sequence-based evidence for major histocompatibility complex-disassortative mating in a colonial seabirdProc. R. Soc. B.279153–162 http://doi.org/10.1098/rspb.2011.0562

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Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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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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: Wojczulanis-Jakubas and colleagues present a study on MHC variation and its impact on behavior in little auks. The study is novel in its choice of system, as the species represents a rarity in avian MHC studies that are typically restricted to easier-to-study taxa and model organisms. This species is also of interest to MHC biology for being long-lived, monogamous, and, for a portion of the year, subject to the pathogen stress from extremely high population density. The authors found high diversity at the portion of the MHC they genotyped, consistent with this high pathogen-mediated selective pressure.

The portion of the paper dedicated to MHC-based mating effects is the area with the least confidence, but that is inescapable. Although the authors of the study found no evidence for disassortative mating, this may be explained (as noted by the authors) by the auks' large effective population size and extremely high MHC polymorphism, findings that will be of interest beyond the study system. The authors did, however, find evidence of significant MHC assortative mating, but concerns about distinguishing duplicated loci reduced confidence in this result. This is a weakness of the paper, but not one that diminishes all interest in the results.

The introduction does a solid job of introducing a reader to the evolution of MHC, and why little auks in particular are of interest. Perhaps this addition would make the study overly topical, but I think mentioning highly pathogenic avian influenza (HPAI), which has been found in little auks and linked to migrating seabirds, would help non-specialists appreciate the importance of studying immune system evolution in this species.

I have concerns about the methods used to distinguish technical artifacts from real sequence variation. If these were addressed (in many cases by just providing more detail), it would increase confidence in results.

- At line 185-186, what kind of sequencing is being performed? Sanger sequencing? More detail is needed on the method.

- Can you provide more information on how you distinguished sequencing artifacts and real variation? Was there a similarity cutoff or did you have a cutoff based on coverage? Line 197: "We identified and removed two classes of artefacts created during PCR (point mutation and chimeras) and during next-generation sequencing (substitution and inserts)"

- The duplication of exon 2 in some individuals (but not all) was a point of surprise for me. Two papers (refs. 67 and 68) are given as evidence of duplication of this locus in other alcids. However, are there other auk populations with polymorphism in copy number within a population? I worry it could be a technical artifact, but I would be happy to stand corrected.

- How well did the MHC alleles sequenced via RNA match those sequenced from DNA? That is, did it look like all alleles were expressed? Did you have DNA and RNA collected from the same individual to be able to do this check?

Minor suggestions:

- I would clarify at the start of the paragraph beginning on Line 108 to make it obvious that these samples are separate from the above and represent only one population. Perhaps the following would be suitable: "To evaluate the expression status of MHC class II sequences and to identify potential pseudogenes obtained with tested primers, we [additionally] amplified and sequenced cDNA [from the spleens of 10 individuals from one population.]"

- The "Molecular sexing" paragraph occurs before any mention of DNA extraction. Please consider reordering.

- At line 141, I'm not sure what a "bench" of PCR primers is. Perhaps a "panel" would be better?

- I was surprised to see vectorette PCR used, since I thought it was not a very common technique, but I think the rationale was to design more specific primers for the species of interest? Please clarify the rationale. The paper cited as a model (ref. 50) is a Drosophila phylogeny paper from 2003. Could you explain the rationale for the selection of the method and perhaps note and cite other MHC papers that have used the same technique?

- You note ultimately the following: "We used the pair of primers: AukLz1 and AukRz3 for further MHC II B second exon genotyping. We also designed a separate set of primers (AukR0709, AukL0709 and AukL0809, AukR0809) to obtain longer sequences of the second and partial third exon of the MHC class II gene. However, the generated fragments were too long to use freely with Ion Torrent technology." So does this mean that you ended up only using the previously-published primers and that the specific primers you designed via vectorette PCR were not ultimately used? If so, please consider cutting the mention of vectorette PCR since it makes the methods section hard to understand. If you feel those primers are still worth publishing, perhaps the relevant methods can be put into a supplement and only briefly mentioned in the main text.

- At Line 196, Bioconductor is spelled incorrectly.

Reviewer #2: This paper describes a study of MHC class IIb diversity in the little auk. The authors make a good case for evaluating immunogenetic diversity in typically under-studied taxa, such as seabirds. It is important to study MHC evolution across a wide range of species so that we can get a more complete picture of the way these genes evolve across vertebrates. The methods and analyses are sound. The conclusions reached were reasonable, and the authors were careful to note the limitations of the study. I think this paper is an appropriate fit for the journal. I only have minor comments, which I will list here. Provided that the authors address all reviewer feedback, I recommend this for publication.

60: Wouldn't this scenario select for assortative *or* disassortative mate choice depending on whether the female had the desirable genotype herself?

103: should "mist-nest" be "mist-net"?

141: "was" designed

183: delete "with"

188: "amplicon"

190: "barcode"

191: delete "of"

241: delete the comma

242: What was also the case? This is vague wording. Please clarify.

246: "remaining 26 alleles"

273-275: I don't know if you need to summarize these numbers again, since you already did so in the results.

285: "such as"

314: delete "as a matter of fact"

316: What about the other species you mentioned? Do they cover the breeding grounds extensively?

321: "that are certainly worth exploring further"

Fig. 4: I'm not sure this figure needs to be in the main body of the paper because it doesn't show a significant result. Might want to add it to the supplementals.

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Reviewer #2: No

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PONE-D-23-28975R1Diversity of major histocompatibility complex of II B geneand mate choice in a monogamous and long-lived seabird, the little auk (Alle alle)PLOS ONE

Dear Dr. Wojczulanis-Jakubas,

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 Feb 16 2024 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'.
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• 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,

Tzen-Yuh Chiang

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.

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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 #3: All comments have been addressed

Reviewer #4: (No Response)

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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 #3: Yes

Reviewer #4: Yes

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

Reviewer #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: No

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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 #3: The manuscript studies the diversity of MHC loci in little auks and hypothesize that disassortative mating may be present in the populations studied. Such observation would confirm a mechanism of genetic diversity maintenance for this loci complex as has been observed in other species. The authors establish the aims and background of the study in a complete and clear manner. They provide resources for future studies on this species and related. The previous comments by other reviewers have been addressed and the paper is very close to completion and acceptance for publication.

I only have one concern with the paper and it is the distinction between assortative mating and mate choice. Assortative (or disassortative) mating refers in the literature to the correlation in pairs concerning a particular trait, in this case the MHC genotypes. Mate choice, or preference, refers to the actual behaviour, males, females or both may exhibit when pairing. Correlation does not mean causation and this mean that the observation of assortative or disassortative mating does not necessarily mean there is mate choice. I suggest the authors to clarify this. For example, stating that whatever pattern of assortative or disassortative mating observed, may serve as proxy to identify the mate choice behaviour. The authors may find clarification and more in depth explanations in papers from the Emilio Rolán-Alvarez group (10.3389/fmars.2020.614237 and references therein for example).

Reviewer #4: This manuscript presents original scientific work. It deals with a very interesting and important subject of diversity of MHC class II B gene in little auk, which is the first such investigation in the species.

Although the writing is generally quite comprehensible, some parts of the manuscript are written quite unclear and sloppy, and should be rewritten. I would also suggest English editing to correct some errors and improve readability (some corrections I suggest in my comments). Additionally, there are some inconsistencies and in different sections of the manuscript (see my comments and suggestions).

Here are some comments and suggestions to improve the manuscript:

Introduction

-line 53: With this context – change to In this context, but as the next sentence has the same beginning, re-write it (use synonyms)

-line 68: As a taxon – seabirds are a taxonomically varied group (comprising of at least 4 taxonomic orders) – so this should be changed or deleted

-the authors should be consistent in writing scientific names of the species, e.g. line 77 – write Alle alle in parenthesis (and should check throughout the text)

-line 81 – delete „also“

-line 84: pathenogenic selection – should be pathogenic selection?!

-Was one of the aims to compare MHC diversity of the three sampled breeding populations? If not – why are the samples divided in three groups? If yes, I would expect more results focusing on comparing them.

Materials and methods

-the authors should write more clearly what they mean by „small blood samples“ (line 96) – I suppose they collected small volumes of blood … further, the authors should add a description of the blood collecting procedure (how did they collect blood samples from auks)

-the authors write that they collected bird samples „across most of their breeding range“ (line 97), but in the discussion section, they write that „examined individuals … do not cover the whole breeding range of the species“ (line 316) – so I find the first sentence (line 97) a bit misleading – I suggest the authors to change the first sentence

-line 108 – „To evaluate the expression status of MHC class II sequences and to identify potential pseudogenes obtained with tested primers …“ – however I did not find any results regarding to that – please add those results in the manuscript (there is mention on cDNA in discussion section, but not in the results)

-the authors further describe in detail the procedure for cDNA analysis (lines 124-137) – but it ends quite abruptly (reverse transcriptase was inactivated …) – but what succeeding analysis they did with the mixture? The authors come back to cDNA at line 178 – but it is quite difficult to follow the procedures as they are discontinuously described (maybe the authors could firstly describe primer development)

-further, it is not clear what the authors mean by (line 178) „Based on received cDNA, we performed PCR amplification in order to receive targeted sequence of MHC.“? Please, clarify.

-further, the authors mention primer aukRz1 (line 131) – here it is not clear what primer is that – at least cite Table 1 (and describe a primer – e.g. it is degenerate primer designed to anneal to ….)

-please correct to „according to manufacturer's protocol“ (lines 132, 136)

-line 140: … we used two approaches – I think it would be better to write two phases, or two steps, to procedures … as those „approaches“ followed one after another, and they were not different approaches – it is a slight difference, but I think is important to be precise to avoid misleading the reader

-line 147: … to design specific, degenerate primers …“ – I suppose they are those in Table 1 (although not all primers in Table 1 are degenerate), but it should be written more precisely … further, it is not clear why are all those primers in Table 1 important (maybe the authors could write in the text the 2 primers used for genotyping, and the rest of primers move to supplementary table) … further, use the term „degenerate primer“ instead of „degenerated primer“ throughout the text

-my suggestion would be to completely move a description of vectorette PCR to supplementary materials, so that the main text is not interrupted with such detailed description, and consequently, re-writing of that part of Mat and methods section accordingly

(it seems to me that this section is quite difficult to follow, so I suggest the authors re-write it in a clearer and more intelligible way)

-the authors should check citations in the text carefully, e.g. „Ko et al. 2003“ (lines 163, 169) – I suppose it should be citation 50

-lines 175-177: I am not sure what is the point of this sentence – is it important for this research? If it is, it should be re-written to clarify it.

-lines 185-186: what does it mean „ … in order to confirm receiving sequence of targeted MHC“?... further, the authors should write what sequencing method was used in sequencing service Genomed – and describe a bit how the sequencing was performed (e.g. primers used for sequencing)

-in relation to that – MHC genotyping (lines 188-189) is actually sequencing again – the authors should write it

-line 206: Conserved MHC class II exon 2 motifs were considered as functional alleles. – the authors should explain this

-the authors should explain better mate-choice analysis - they should explain each „criteria“ (by the way – is that appropriate expression? Later, the authors use the term „metric“, line 226), and/or cite the source for the method. Further, they only cite ii) criteria – MHC-band sharing coefficients – but the reference cites research using DNA fingerprinting (Wetton JH, Carter RE, Parkin DT, Walters D. Demographic study of a wild house sparrow population by DNA fingerprinting. Nature. 1987;327: 147–149) – so, at least the authors should explain a bit, give more details (and is the term band sharing appropriate?). Additive amino acid and maximum amino acid differences should also be explained. Further, the authors should explain how they estimated heterozygosity as they were not able to assign alleles to each of the two loci.

-lines 224-225: „We assessed amino acid substitutions for both PBR regions and total exon sequences.“ – It is not clearly written, should be explained better, re-written

-lastly, I think the authors should specify somewhere (either in Mat and methods section or in the Results) the number of individuals of each sex

Results

-line 239: … 267 bp in length fragment – earlier the length of the fragment is written as 298 bp (e.g. line 205) – it is important that this discrepancy is explained in the text (further – better to write xx bp – long fragment instead of xx bp in length fragment)

-the authors should be consistent with the naming of populations, e.g. line 247 – omit Hornsund, write only Spitsbergen – the name written on map in Fig. 1 (as for the other two populations)

-line 240: … indicating at least 2 loci (by the way – use words for numbers 1-9) and the presence of a duplicated MHC II gene fragment … - is that not the same thing? So I suggest writing „i.e.“ instead of „and“

-line 241: „On average two alleles, per individual were recorded …“ (by the way, delete the comma in this part of sentence) … but in line 244 „… 56 individuals had 2 alleles (40%)…“ – so, what does it mean „on average“ in the first sentence? Further, this is repetition … should be re-written.

-the authors should be consistent with the names of alleles (Fig 1. and Fig. 2 – allele 1, allele 2 etc., Table S2 – there is AS allele name and Allele name – both are different from those in Figure 1) – the authors should unify the names throughout the manuscript

-lines 247-251 – I would suggest that the authors write (again) the number of samples for each population, and discuss in discussion section number of alleles and number of private alleles identified per population in relation to the number of individuals analysed

-line 253: correct Table 1 to Table 2 (but if you accept my previous suggestion to move Table 1 to supplement, then this will indeed be Table 1 – of course, in that case, the authors should carefully check and correct all the tables numbers in the text)

-Table 2 – the authors should define „aa seqs“

-further, as the authors did not report basic polymorphism statistics separately for each population, I do not see the point in reporting them in the table as all those values could be written in a sentence.

-Fig 4 caption (line 706) - the authors should not repeat the explanation, they should just write what the figures depict (the explanation should be incorporated in the text of Results section)

-the authors should be consistent with writing the species name (in potential use of capital letters), e.g. in Table 2 they write Little auks, in line 172 they write Little Auk, but mostly it is written little auk – the authors should check and correct accordingly throughout the text, figures and tables

-lines 259-260: … while Tajimas D analysis revealed sites with a significant excess of high-frequency segregating sites.“ – Which sites? Where those results can be found?

-lines 261-262: First sentence is redundant.

-lines 267: Subsequent analysis suggests … - what the authors mean by „subsequent analysis“? Did they perform some subsequent analyses? If yes, should be clearly written and explained.

-line 703 (Fig 3 caption): „negatively selection“ should be corrected to „negative selection“

Discussion

-lines 281-282: Was the aim of using cDNA to confirm putative alleles? Please, refer to my previous comments on cDNA – it should be clearly noted why cDNA were used, and mentioned appropriately.

-line 285: „The high similarity between alleles, as is shown in the little auk …“ – Where is that shown in the results? It should be cited again (e.g. table yy, or by repeating the values from the results). Additionally, I suppose you meant high similarity among alleles (or you had in mind two particular alleles that were very similar)? Further, are there any other explanations for the high similarity between/among alleles?

-lines 291-292: when making comparisons or comments, the authors should explicitly specify the values that they are commenting, like here: … can reach very high number – so please specify the numbers (like they did for non-passerine birds in the next sentence). Also, specify the MHC class that you are referring to in your discussion, at least at the beginning of the sentence.

-line 295-296: the sentence is not clear, even conflicting (the little auk polymorphism is lower, but then one of the highest?) – should be re-written … further, I think the authors should put those comparisons in context of number of analysed individuals, or at least make some comment on that

-line 303: what does „then“ stand for in this sentence? What does it signify?

-line 307: „compare“ should be changed to „compared“

-line 309: „This suggests blue petrels are colonial species …“ – Does the maintenance of many MHC loci class I (but not class II) really suggest this? I think the authors should be much more attentive on the meaning of their sentences, throughout the manuscript.

-line 317: … in frequencies of the shared alleles … should be changed to …. frequencies of the most common share alleles.

-line 328: … locus -specific MHC II B genotyping was outside the focus of this study … why is it so? Could the authors comment on that in discussion?

-lines 335-336: „While we did not investigate gene expression in this study, our evidence supports the idea that the identified alleles are functional and expressed.“ Firstly, what about cDNA analysis that you wrote you performed? Secondly, what evidence supports that identified alleles are functional? You wrote „our evidence“ so the reader would expect that it is the evidence from this investigation, but then you cite some previously published papers.

-line 339: „… MHC genotypes, they are likely to be important components of little auk immune systems.“ – It is very well established that MHC genotypes are important to immunity. Is there something else the authors wanted to suggest?

-line 345: The lack of such a pattern suggests several possibilities. – This is one example of poor English writing (as far as I can tell), so the authors should consult a native speaker … maybe: There could be several reasons for the absence of such a pattern.

-lines 351-354: The sentence starts with „… random mating….“ and ends „… little auks may select mates …“ – it is quite in conflict – should be rewritten for more clarity

-lines 361-365: this is quite confusing part, it is not clearly written … „effect of the same mechanism“ – which mechanism does the authors refer to?

References

There are some errors in references, here are some of them that I noticed:

-references 33 and 34 are very similar, but cited in a different way – the authors should check them (additionally ref 34 has repetition)

-reference 38 – repetition, no journal name

-reference 39 – please check

-all references should be carefully checked once again

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Reviewer #3: No

Reviewer #4: No

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PONE-D-23-28975R2Diversity of major histocompatibility complex of II B gene and mate choice in a monogamous and long-lived seabird, the little auk (Alle alle)PLOS ONE

Dear Dr. Wojczulanis-Jakubas,

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.

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Reviewer #3: (No Response)

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Reviewer #3: Yes

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

Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: No

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6. Review Comments to the Author

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Reviewer #3: MAJOR

First, I apologize to the authors for not being more careful in the first review, I placed my effort in what I have experience with (this is assortative mating and mate choice), and I did not pay enough attention to the materials and methods section. That is why my second review is considerably more extensive. Otherwise, I really like this study: the introduction, Results and Discussion parts are well written and are compelling. The methodology part, and specially the primer development section, albeit being confusing, show a lot of thought and careful consideration (as the authors showcase in the first paragraph of the discussion).

After reading the comments from the second reviewer, who seems to have confused the manuscript versions, I did pay more attention to the methodology section. The part that covers from the cDNA to the primer design is confusing. I hope that my comments can help the authors to simplify the text, make it easier to read, and give speed in addressing the issues. For this last part I have added text alternatives for the authors to consider.

#Introduction

No comments, the introduction is good.

#Materials and methods

The previous sections to the following look good to me, with perhaps the exception of the cDNA samples section (see below).

-Primer development and Ion-torrent sequencing

I sympathize with the authors concerning the inconsistency on the materials and methods section. In general, I consider the terms oligo, barcode, and primer to be used in very broad and confusing manners in the genetics research world. I think we would all beneficiate from being more descriptive in defining these.

I feel that the text must be more structured, and names used in a consistent manner. Preferably, following the same names as the literature that started using the Vectorette PCR approach (e.g. https://doi.org/10.1007/s00239-003-2510-x).

What I mean for more structure is to start with a paragraph which overviews all the steps required for the design of primers. Alternatively or additionally, the authors could draw a schematic figure which describes the steps. As an example of the first paragraph, you could use the following:

“The second exon of the MHC class II B genes, which is the … [52], was sequenced… . In order to do that, and since only one primer sequence is available for this genomic region (ref?), we used a vectorette PCR approach (see ref) to obtain adequate primers specific to the little auk. We use this approach given its succesful use in MHC research (refs). This procedure entails (1) to ligate a designed vectorette to the sticky ends of restriction enzyme digested DNA, (2) to perform a nested PCR where specific and vectorette primers are used and (3) to sanger-sequence resulting amplicons”

At this point I would like to ask why was it nested? Perhaps you should clarify that as well.

After that first paragraph you could first explain how you get the vectorette, the specific primer and the vectorette primer and then, and only then, describe the laboratory procedure. By the way, you do not seem to describe where are you getting the vectorette libraries from, and the ligation process of the vectorette to the digested cDNA is lacking. It could also be good to clarify how many different vectorette libraries you constructed.

At the end of the primer development section, I am afraid I do not understand the aim of the last paragraph. If you already have the primers that you need to amplify DNA and do ion-torrent sequencing, why are you making an extra amplification and using Sanger sequencing?

I am assuming that the genomic DNA used for the little auk primer discovery is the cDNA obtained for individuals described in the cDNA samples section. Is this correct? In that case, don’t say that they were only used as controls, it is a fundamental step in the primer discovery. The fact you can use them as controls as well is additionally. Be clear. You may have wanted to do so but in Lines 160 to 161 that quote does not make sense, were used to what?

I had to go through the literature to understand the process (I read the steps undertaken in https://doi.org/10.1007/s00239-003-2510-x), I would ask you, and specially for this kind of journal, not to assume that the readers are familiar with the concepts, and name and define things clearly and consistently through the text. For instance, if I am not getting it wrong, the primers you test from related species would be the specific primers (the primers that are known for the region of interest that you want to statistically analyze). You seem to be using interchangeably “specific”, “degenerate” and “internal”. If I do not get this wrong either, the universal primers would be what in the paper by Ko et al. (2003) are called vectorette primers (which is not the same as the vectorette itself from what I can understand), and in your case would be C20 and B21. Also, I am assuming that the digested genome you are using for primer discovery is the cDNA obtained from reverse transcription (see on the comments I made previously).

I believe that the authors have been very thorough to ensure good data quality and that the steps to reach it have been considered carefully and fully. Nonetheless, the text does not show that so well. If this section gets clearer, there is to my knowledge little else needed.

-Data filtering

No comments, this looks good.

-Genetic analysis

No comments, this looks good.

-Mate-choice analysis

This looks very good, well done.

-Results

No comments, looks good.

-Discussion

Line 379-382. I agree, very good point. And in general, very good discussion.

MINOR

Line 106. Vein not vain

Line 180. Afterwards not afterward

Line 184. You cannot start a new paragraph with “For this purpose”, the paragraph should be new to the previous one.

Line 184. I do not think that is the right reference, I looked it up and there was no mentioning of vectorette PCR.

I imagine that figures were sent separately and in better resolution. The ones present in the submitted revision do not look good.

Line 203-204. Again, consistency, are you sure they can be called vectorette primers? The vectorette primers you are using are C20 and B21, readers can get confused and assume that they are the same.

Line 360. Repeated “then”

Line 377. Nest instead of Next

Line 421-423. Can you clarify some more what you mean here?

Line 423-426. Good, but can you provide some reference for that minor correlation?

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Reviewer #3: No

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Attachments

Attachment

Submitted filename: PONE-D-23-28975_R2_Revision2_comments.docx

Diversity of major histocompatibility complex of II B gene and mate choice in a monogamous and long-lived seabird, the little auk (Alle alle)

PONE-D-23-28975R3

Dear Dr. Wojczulanis-Jakubas,

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.

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Kind regards,

Tzen-Yuh Chiang

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 #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 #3: Yes

**********

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

Reviewer #3: Yes

**********

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 #3: Yes

**********

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 #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 #3: The article has achieved the requirements from review. The methods are now clear to follow and the results are of interest for the journal readers.

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Reviewer #3: Yes: Daniel Estévez-Barcia

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