PONE-D-23-01131Genotype and growth rate influence female mate preference in Xiphophorus multilineatus: potential selection to optimize mortality-growth rate tradeoffPLOS ONE

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Dear authors,

First, let me apologize for the delay in getting this decision finalized; it was partly my doing and also an inability to located suitable reviewers. That said, we now have a decision based on two reviews of your manuscript "Genotype and growth rate influence female mate preference in Xiphophorus

multilineatus: potential selection to optimize mortality-growth rate tradeoff". Both reviewers indicated the manuscript could be suitable for publication, but it will require major revisions. Please find the detailed comments from both reviewers below.

In particular, consider how the results are explained, including consideration of alternate hypotheses; this was identified by both reviewers.

Sincerely, Quenton M. Tuckett

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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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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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: This MS deals with a study of a model system that has been detailed studied in the past for mate choice, the live-bearing fish Xiphophorus multilineatus. Previous studies have shown that males have genetically-based alternative reproductive tactics (ARTs): one male type is called COURTER (C type; having higher size and growth rate, later sexual maturity and displaying extreme courtship behaviour) while the other is called SNEAKER (S type; having smaller size, lower growth rate and earlier sexual maturity, with earlier courtship and coercive force copulation). Natural populations show a possible protected polymorphism in ARTs via presumably frequency dependent selection. Although much is known, the details of the mechanism acting as well as the different interactions between traits (size, growth rate, mate choice tactic) are basically unknown. Therefore, the study provides two new experiments trying to improve the knowledge on the system. The first experiment, a female choice design, try to test if the alternative reproductive genotypes (i.e. genotypes) show a pattern of assortative mating or not. The dependent variables were preference (female time expend with C – female time expend with S) and choosiness (female time expend with C + female time expend with S). The studied treatment were three genotypes (C; S and both included) in (non-virgin) females and two (C and S) in males. As the genotypes cannot be morphologically identified, they produced them by rearing specimens of each phenotype for 4-5 generations in mesocosms. The results show a trend consistent with negative assortative mating (a mechanism able to cause negative frequency-dependent sexual selection). The second experiment tested the mating preferences of 10 pure (F2) female genotypes with males of both genotypes as virgin females, then they got some mate experience (maintained 4 weeks in presence of another female plus two males) and them tested again for mating preference as before. As females were measured at both dates they could calculate female growth rate as well. This second experiment is important for understanding the results of the first experiment as well as identifying trait interaction contributing to mate choice. The preference showed a significant interaction between genotypes and growth rate (positive relationship in C and negative in S) a trend which do not support any of the former hypothesis used in this context. The authors discuss and speculate why they got these results as well as their possible interpretation. This study adds a useful piece of information to the study of mate choice in this system, it is reasonable well designed and analysed and so its merits publication. However, I found several potential drawbacks that should be discussed or even mentioned/discussed in the text as potential limitations of the study. I list them below

1. The analyses were done using several fishes repeated in different trials. For example, in experiment 1 they used 10 C and 10 S males, for an overall 87 trials. I understand that they tried to use different combinations of males in different trials (50 new, and so at least 40% of data was not independent). Actually, perhaps it is not bad to use the same males for different female genotypes, providing that they behaviour (of the males) do not change with experience (but see second experiment). I do not think that this invalidates the whole experiment but the potential consequences and limitations of this should be discussed. The same happened in experiment 2.

2. The authors claim that they got a results that supports negative assortative mating (lines 266-269). Actually I think that they got complex results. I will discuss results for both experiments.

2.1. Although in experiment 1 S female prefers to mate the other type, it did not happen with the C females. Notice that a pure negative assortative mating trend will require of both genotypes favouring the other or alternative that the four combinations of possible mates show a sexual isolation index that was negative, or similar. So the authors should be more caution with this result, I think.

2.2. Similarly, in the second experiment, S type female showed a trend consistent with growth rate favouring disassortative mating, as claimed in the text (lines 369-372). Again this was far from being so clear, I think, as the relationship is so in the S genotype but it is the opposite in the C one. Again the authors should be caution with the interpretation regarding assortative mating as they got complex results.

3. One interesting result has not achieved enough author attention, I think. They got that ART trait (their genotypes) show actually a plastic characteristic, as the trait needs of certain previous experience in order to produce certain aspects of the mate choice, as the preference for particular genotype needs of previous experience. I do not know if this has been observed before in other organism, and perhaps merits a particular discussion about the interaction between genetic determination and phenotypic plasticity, aspects considered two alternatives but which could also be two sides of the same coin.

4. The authors and too worried about to produce an evolutionary hypothesis/mechanism that explain their results, which can be understandable, but their new hypothesis seems to me too speculative, perhaps is too soon to advance new hypothesis and just more clear and compressive data is just needed. In my opinion, they should try to reduce the importance of the speculative ideas in discussion, and emphasize the new findings in relation to the previous ones, without any need to present a closed interpretation.

5. In addition, I have a few comments on the text

. Lines 17-19. They only describe the results of one genotype, say that the other showed the opposite at least.

. Lines 63-64. Too frequent repeat “females with courter sires” Once you explained how you got these females they are “courter females”. I think that some sentences could be simplified in the text.

. Line 64-65. It should be explained the exact characteristic of the mate choice relationship in guppy

. Line 81-82. Perhaps this explanation is obvious for the authors or specialized readers, but I think that general reader needs a better explanation about how such hypothesis works and produces its predictions.

. Lines 85-87. Actually several theoreticians claim that disassortative mating may directly cause negative frequency-dependent sexual selection, and so disassortative mating would be the cause of the selection not the consequence (Pusey and wolf 1996. TREE, 11: 201-206; Hedrick several papers; for example 2016; Evolution 70: 757-766). Any fast search on the WEB should give a few more useful references to discuss this if interested.

. Lines 132-134. I personally do not see clear the test group factor used, for me repeated measurements are typically analysed as a nested factor instead of a new factor, as you are not interested per se in such factor just to know if the main effects are true irrespective of such variation.

. Legend of Figure 2. The P > 0.05, I assume that actually means <

I enjoined the MS and so hope that authors may be able to produce an improved version.

Reviewer #2: The authors of “Genotype and growth rate influence female mate preference in Xiphophorus

multilineatus: potential selection to optimize mortality-growth rate tradeoff” report on two experiments used to identify the factors affecting female choice for males exhibiting alternative reproductive tactics. Overall, I enjoyed the manuscript; it wasn’t overly long and rarely strayed from the main objectives. There are some writing issues throughout, but always minor. I do note one issue that should be mentioned alongside their chosen hypotheses: the potential for sperm storage and quality to affect the results. See below for one specific issue (sperm storage) and some specific comments listed line by line.

Specific Issues

1. Somewhere the authors should discuss sperm storage and competition might affect the results of this experiment and also how it might be his might be related to their hypothesis on 272-275. For example, there has been studies on X. nigrensis showing that male tactic is related to traits related to sperm competition (e.g., sneakers might have sperm with greater viability and longevity compared to courters; doi: 10.1098/rsbl.2011.0286). I also think a discussion of sperm storage (in general) would be warranted early on.

Specific Comments

L36: comma after physiology

L93: “not hosing”

L93-94: I am curious about how these sterile environments might affect mate choice experiments

L95: any water quality testing?

L104: remove “a”

L102-: regarding the dichotomous choice tank; this is a bit unclear without a diagram. In doing an image search, these tanks will seemingly vary

L124: “mesocosms”

L141: lowercase females

L141: 22.7-L aquaria

L144: delete “number of”

L146: more important than reporting the range for both males and females would be to examine if there are differences

L156: delete “number of”

L157: once again; differences between tested populations would be more informative, even if expected (e.g., sneaker and courter)

L161: “experience males”?

L208: how was effect size calculated? Was this presented in the methods?

L218: sometimes test statistics and df are reported and at other times not

L228: delete “from”

L239: some variation in significant figures

L257-260: that’s a mouthful

L297: comma after “growth rates”

L299-312: not quite convinced by this explanation and not quite sure why it was included. Some other explanations might be more fruitful. For example, why not mention sperm traits and alternative reproductive tactics? This seems like a missed opportunity, perhaps even something that should be discussed.

L315-316: is this reference comparable to a swordtail that exhibits sperm storage? I’m not sure; I am also not sure how much or how little sperm storage affects the costs of assessing and choosing mates. Presumably, if females can store the sperm from multiple males, there could be arguments for both an increase and decrease in costs.

L367: a possible place for sperm quality as well?

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Reviewer #1: Yes: Emilio Rolán-Alvarez (Universidade de Vigo, Spain)

Reviewer #2: No

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Genotype and growth rate influence female mate preference in Xiphophorus multilineatus: potential selection to optimize mortality-growth rate tradeoff

PONE-D-23-01131R1

Dear Fitschen-Brown,

Thank you for this thorough revision, which has now been reexamined by myself and one of the reviewers. We both found the manuscript to be be suitable for publication. We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Quenton M. Tuckett

Academic Editor

PLOS ONE