PONE-D-19-21985

Sexual dichromatism in the neotropical genus Mannophryne (Anura: Aromobatidae)

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: No

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: I found there a lot to be interested in here both empirically (the new data presented) as well as the information reviewed and summarized from the literature. However, the “package” needs better selling. The authors give good reasons for the work they did to demonstrate dynamic and ontogenetic dichromatism but less of a good job justifying the data they collected on variation in throat color, escape performance, etc. I think there is a very interesting story in here and a lot of work has been done to uncover that story; I think there just needs to be better packaging in the Intro and Discussion to really make it clear to the reader why these questions need answering and how they are all related to one another. Below are detailed comments.

Detailed Comments

Line 84 “tree frog example”, does this need to be here?

Line 98-99 For the uninitiated, it may be beneficial to define Bd as a fungal pathogen.

Lines 119-120 I wonder if using Generalized Linear Mixed Models (GLMMs) might be a more powerful and flexible approach to take here. The main advantage of GLMMs is that they can be used with data that are not continuous and/or do not conform to the standard parametric assumptions.

Line 145 Just one ‘l’ in caliper or is this an American/British English thing? (also 156)

Lines 177 There might be some multiple comparisons issues here with the various measures of escape performance. It seems likely that these are not independent of one another but if one uses a alpha of 0.05 for every analysis performed then they are be treated that way. Perhaps a correction for multiple comparisons is necessary. See Table S2.

Line 181-183 Is this section needed?

Line 193 Checked what?

Lines 269-287 Why no graphs illustrating the escape performance test results?

Lines 299-309 this seems more like background information but it is placed in the Results section.

Line 340 focussed

Lines 332-339 This seems repetitive from the Intro; maybe just start at line 340.

Line 390 Perhaps briefly describe how you envision Zahavi’s principle being at work here.

Lines 445-47 Is escape performance necessarily related to mate attraction and reproductive success? It is a proxy, but is it a good proxy for who wins and who loses in territorial encounters? Maybe larger females have larger R:G ratios (on average, demonstrated in Fig. 2) and larger females also can jump farther (stated in line 287-289). Maybe jump performance only is driven by size (but since size also influences color, it makes it appear as though this is also important. A statistical analysis that clearly separates out the influence of SVL versus color on jump performance is needed. And I think they authors already attempted it, with the two-way ANOVA referred to in line 289 but the full results of this or another test (the influence of color on jump performance after the effect of SVL has been removed) is needed. Maybe an ANCOVA with SVL as the covariate?

Line 475 Perhaps suggesting some effective tests of the proposed hypothesis here. What would be really nice is to have data on actual reproductive success of females with different throat colors, though I realize how difficult this would be to get in the real world.

Table 2 Use n instead of samples and other abbreviations to tighten up this table a bit.

Reviewer #2: This is a study of coloration in Mannyphryne frogs from Trinidad and Tobago. The study 1) analyzes a large data set of photographs of female M. trinitatis to document variation in yellow throat coloration and relate this to body size, 2) documents sex-specific throat color development, 3) supports the hypothesis that throat coloration is related to quality by showing females with yellower throats have farther escape jumps, 4) documents sexual dichromatism in a close relative, M. olmonae, and 5) reviews literature which suggest that ontogenetic and dynamic dichromatism are widespread in this genus.

In general, I think this is a really valuable study, with nice, big sample sizes and sampling across many populations. I also really like the escape response experiments to link this variation in coloration with some measure of quality or condition. The behavioral experiments and the across-site sampling are strengths to the study.

I have no major concerns other than wanting more details about the photography protocol and how the authors ensured even lighting across photographs. I think this is simply an issue of more detail being needed in the methods. Otherwise, I have a bunch of minor comments where I think more details are needed or things could be clarified. I’m confident the authors can address these concerns.

Line 34: Suggestion: Delete “It is well established that” from the beginning of this sentence. And include a general reference for this first sentence. Wells (2007) or Gerhardt and Huber (2002) are good ones.

Gerhardt, H. C., and Huber, F. (2002). Acoustic Commmunication in Insects and Anurans. Chicago: University of Chicago Press.

Wells, K. D. (2007). The Ecology and Behavior of Amphibians. Chicago: University of Chicago Press.

Line 35: should be “acoustic signals”

Lines 47-48: A recent paper has documented both in an Aromobatid frog, Anomaloglossus beebei (Engelbrecht‐Wiggans and Tumulty, 2019).

Engelbrecht‐Wiggans, E., and Tumulty, J. P. (2019). “Reverse” sexual dichromatism in a Neotropical frog. Ethology. doi:10.1111/eth.12942.

Lines 50-50: I recommend breaking up this sentence, it is kind of hard to follow the logic here. If a color signal is not useful for nocturnal breeders, why later in the sentence do you say that it is?

Lines 49-53: Probably worth also discussing the potential for temporary color change to function as a sex recognition signal for explosive breeders. This seems to be the case in some species (e.g., Sztatecsny et al., 2012) and this hypothesis is support by the comparative analysis of (Bell et al., 2017).

Sztatecsny, M., Preininger, D., Freudmann, A., Loretto, M. C., Maier, F., and Hödl, W. (2012). Don’t get the blues: Conspicuous nuptial colouration of male moor frogs (Rana arvalis) supports visual mate recognition during scramble competition in large breeding aggregations. Behav. Ecol. Sociobiol. 66, 1587–1593. doi:10.1007/s00265-012-1412-6.

Bell, R. C., Webster, G. N., and Whiting, M. J. (2017). Breeding biology and the evolution of dynamic sexual dichromatism in frogs. J. Evol. Biol. 30, 2104–2115. doi:10.1111/jeb.13170.

Line 86: Please articulate the hypothesis of this experiment. Also, I don’t think “fitness” is the right word here. I think this is more a question of whether the signals indicate something about “condition” or “quality”. If acquiring and putting carotenoids into skin is somehow costly, then individuals in better condition may be able to invest more of the limited carotenoids to coloration. Escape response could be a metric of condition or quality, but I think it’s a step too far to say it is linked with fitness (e.g., maybe bolder individuals have higher fitness and are less likely to escape?). Whether having increased yellow coloration is linked with fitness is another question that will take a few more pieces of evidence to link to. So, I recommend being more cautious with word choice here.

Line 98: Please provide slightly more detail on “returned to their collection sites”. Approximately how large is a “site”? Poison frogs can sometimes have very small home ranges, so, ensuring that the frogs were within ~10-20m of their capture points or putting each frog back at its exact capture location is very different than dumping all the frogs back in the same general area where they were caught.

Lines 105-114: I’m a bit unclear on the photography protocol. Were Colorchecker color standards included in each photographs or photographed separately? If separately, the lighting conditions need to be identical across photographs. Please provide more details on what a color profile is and how it ensures standard lighting conditions across photographs.

Line 114: What was the areas of interest? Please be precise.

Lines 114-115: Why only R and G, and not B? Also please provide some justification for using the R:B ratio as your measurement.

Lines 115-117: I’m confused by this sentence, why does combining R:G and SVL allow for comparisons between sites?

Lines 121-122: How was presence of colour posterior to the collar measured? This is the first we are hearing about this in the methods.

Lines 126-127: The fact that 60 tadpoles were collected suggests that these tadpoles were from, at least, five or more different families (assuming a clutch size of around 10-12 eggs?), but please provide some justification for this. If they were all collected from one small pool, how representative is this sample of the population as a whole? Do many different parents deposit tadpoles in the same pool?

Lines 146-147: Please try to be more precise here. The four-week intervals part is confusing me. Based on this sentence it sounds like the froglets were photographed approximately once every 4 weeks for approximately 16 weeks, is this right?

Line 157: Check grammar here, missing subject?

Line 158: If you give a time for night photography, you should also give one for morning photography.

Line 159: Please describe lighting conditions. This is especially important to be able to compare the results of this photography to the descriptive statistics of the throat color variation earlier.

Line 162: Need comma after “ratio”?

Lines 181-183: Please provide a bit more detail on observations. For example, did you conduct focal observations of individuals in a systematic way or only if you observed something interesting or unusual?

Line 192: Perhaps change this to “We performed a literature review on…”

Figure 3. Need clarification on what, exactly, the y-axis is showing. What is “SVLxR:G” and how was it calculated?

Lines 224-229: Unclear if these are all quantitative results or sometimes qualitative. I think this relates to my earlier question about what the regions of interest were. I was surprised to read hear about quantitative results about presence of yellow posterior to the color because I didn’t see anything about measuring this in the methods section.

Figure 5. Same issue of unclear y-axis as figure 3. Also, is “SVLxR:G” the same thing as “SVL*R:G”? If so, why use different characters for the interaction? If not, need a lot more clarification on what is being measured. This figure legend also needs details on the Collar Yes vs. No key. No explanation for this key is provided.

Line 245: Recommend changing “classed” to “classified”.

Lines 252-254: I think these results are really interesting (i.e., that yellow coloration shows up prior to sexual maturation) and I would appreciate seeing more data to support this conclusion. It would appear that you probably have data on adult size and size of juveniles through development. Perhaps some sort of graph showing SVL on the y-axis over time on the x-axis, and a line or range showing the SVL/time when yellow coloration first shows up?

Figure 6. Could you add the details about age and SVL to the figure itself? Perhaps as small inset details on the bottom of each image? This would make it a lot easier on readers instead of having to look back and forth between the figure and the figure legend for this information.

Table 2. I don’t think this table is all that informative given the goal of the experiment. Are we really that interest in the overall mean? Why not show graphs of the relationships of interest? i.e., the relationships between R:G ratio and measures of jump performance?

Lines 300-309: I’m confused why this background information is presented in the results. Shouldn’t it be in the Introduction?

Lines 317-320: Could you add photos of the female throat of M. olmonae too? Might be a nice addition.

Lines 324-30: I really think this Table S3 should be in the main text and not the supplementary. Otherwise, why even devote a results subsection to it? And I also think more summary details should be included in this results sub-section. For example, for how many species has female yellow throat coloration been observed? Or male color change?

Line 340: “focused”

Lines 340-346: Please also provide a clear statement that your results supported the hypothesis that female throat coloration is related to female quality (i.e., yellower females jumped farther).

Lines 332-346: A suggestion: I think this discussion could be a lot stronger if you provide clearer statements of what your study accomplished and what the key results were right in the beginning, without making the reader search for it later on.

Line 365: La Marca reference?

Line 390. Seems odd to suggest Zahavi as an afterthought with no real explanation. If this is a reasonable hypothesis, please provide a more clear explanation to how turning black when calling could be a “handicap”.

Line 398. Please include the full genus name before the first Mannyphryne species listed, and “M.” before each subsequent species of the same genus.

Lines 401-407: I find this explanation based on phylogeny unclear. Are you saying that based on the presence of this trait among clades that is likely ancestral to the genus?

Lines 412-414: Long sentence. I recommend moving reed frogs to the beginning so it doesn’t take so long to get to the subject.

Line 429: Change to “no one”

Lines 465-467: Is this true? Allobates femoralis have been studied in quite some detail and males typically have several clutches in their territories and may take 1-2 days to transport tadpoles (round trip), if I recall correctly.

Lines 451-475: I was a bit surprised to hear the only concluding hypothesis is one about mutual mate choice (i.e., suggesting males may choose females based on yellow throat coloration). Didn’t Wells observe contests between females that seemed to be resolved with these yellow throat signals? Given these natural history observations, isn’t intrasexual communication of dominance associated with territory defense also a reasonable hypothesis for the function of these signals?

Table S1. Please specify that the data presented are sample sizes (assuming that’s what they are).

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Reviewer #1: No

Reviewer #2: Yes: James Tumulty

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PONE-D-19-21985R1

Sexual dichromatism in the neotropical genus Mannophryne (Anura: Aromobatidae)

PLOS ONE

Dear Mr Greener,

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.

One of the original reviewers has reviewed the revised manuscript and found it to be improved. There are still some concerns regarding the figure and statistics that need to be addressed.

We would appreciate receiving your revised manuscript by May 15 2020 11:59PM. When you are 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.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #2: (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 #2: Yes

**********

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

Reviewer #2: 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 #2: (No Response)

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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 #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Overall, I’m happy with the edits that the authors have made to the manuscript to address my concerns, I just have three minor comments.

Lines 54-61: The edits to this section actually don’t include the function I suggested and appears to misrepresent the conclusions of the cited work. The conclusion of the Sztatecsny et al. (2012) study, along with several others that investigate explosive breeders, was that color change may function in sex recognition, not necessarily female choice – e.g., in the case of Rana arvalis, that turning blue allows males to be recognized as males by other males, so that they aren’t recipients of mistaken amplexus. This is different than color change having a function in female choice. The Sztatecsny et al. (2012) actually examined the potential for blue color to be attractive to females and found no evidence that females prefer blue males. This is an important distinction that is not made in the text.

Lines 128-129: The edits to the photograph protocol above are helpful, but in applying the color profile to other photographs, I’m assuming the profile was only applied to other photographs within a batch, and that each batch had it’s own color profile? By saying you applied the color profile to all photographs, this detail isn’t clear. Please clarify.

Lines 134-137: I agree that the linear mixed models are the better way to go, but please also state what tests were used to evaluate statistical significance, since these vary when implemented in R (e.g., wald chi-square tests, likelihood ratio tests? etc.)

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Reviewer #2: No

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Sexual dichromatism in the neotropical genus Mannophryne (Anura: Aromobatidae)

PONE-D-19-21985R2

Dear Dr. Greener,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Section Editor

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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 #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: (No Response)

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

Reviewer #2: (No Response)

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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 #2: (No Response)

**********

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 #2: (No Response)

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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 #2: (No Response)

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Reviewer #2: No