PONE-D-20-27518

Amplitude of circadian rhythms becomes weaker in the north, but there is no cline in the period of rhythm in a beetle

PLOS ONE

Dear Dr. Miyatake,

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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In the discussion please elaborate on: What is the seasonal variation in photoperiod at the northern vs. southern end of the range? What are the typical temperature ranges? How does this compare with other studies in which circadian clines were or weren't found? 

Fix issues with the Methods as indicated by reviewer 2.

Discuss the Helfrich-Foerster and Costa studies on weaker fly circadian rhythms in northern latitudes mentioned by reviewer 1. 

Consider adding phase shift response data. 

Discuss (small) effect sizes.

==============================

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PLOS ONE

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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: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: No

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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 is an interesting ms concerning the geographical variability of the circadian rhythm phenotype in T castaneum. The literature is mixed on this subject with classical Pittendrigh experiments in Drosophila species in Japan suggesting that the amplitude of the rhythm is stronger at higher latitudes because it has to counterbalance the effects of extreme photoperiods which would tend to make animals arrhythmic. There is not much molecular evidence to back this up, indeed recent studies from Helfrich-Foerster suggest the opposite, in that a weaker circadian rhythm in northern latitudes is more flexible for adaptation of circadian behavior to extreme photoperiodic conditions. Costa's group suggests that the circadian light response in D. melanogaster should be reduced at higher latitudes - this pair of papers (Science, 2007???) is not cited. Consequently it would have been nice if the study had also included a phase shift response to light as that would have been perhaps more relevant at these latitudes. Nevertheless, this study comes out in favour of the latter hypothesis. The results reflect a lot of work and the ms is clear to read and understand. My one quibble is that the effects are tiny and require a huge N to be observed. Also the variation in latitude is only 8oC which doesn't help to dissect out an effect. Furthermore the power statistic used is not very sophisticated and there are better methods available, but I suspect the result would be the same. The Discussion is a bit too long considering the brevity and conciseness of the results, so perhaps a little less speculation would be in order. Otherwise, the study is OK.

Reviewer #2: The authors conducted a study on the effects of geographic (primarily latitudinal) variation on circadian rhythmicity in the red flour beetle. Strengths of the study included the large sample size and rearing the populations for 2 generations in the lab to separate out maternal/environmental effects. While the English was professionally edited, I often found the ideas to be communicated in an imprecise or unclear manner.

1. It was completely unclear why the authors repeatedly referred to longitude early in the manuscript (e.g., L29, L40...a key word!, L47, L53, L78). The study is primarily designed to detect latitudinal clines given the much larger latitudinal range sampled. The authors state that longitudinal clines are also known (L47) but don't give a citation. Based on their titles, all the references cited on L54 (refs 8-12) refer to latitudinal clines. I can understand why they would want to test for this possibility within their data, but I don't know that there is any a priori reason to expect a longitudinal cline. This was also very confusing to me because they referred to altitudinal clines (L26 of abstract) making me think that maybe these would be investigated, but think they were ever discussed in the manuscript. A third point of confusion is that the authors said they were analyzing effects of temperature (L130), but I don't think they ever did.

2. The discussion on L61-69 is very unclear. The authors seem to be mixing points related to "where flies live" and latitude. By "where flies live" do they mean geographic features that might be distinct from latitude? If so what are they? L67 "pioneering"? Some of the studies cited here are quite old ("pioneering"). Is there a consensus emerging in the newer literature? The discussion overall was hard to follow.

3. The predominant finding of the paper was that there is an effect of latitude on amplitude/power of the circadian rhythm. This finding would be more useful if it were put into some kind of context. What is the seasonal variation in photoperiod at the northern vs. southern end of the range? What are the typical temperature ranges? How does this compare with other studies in which circadian clines were or weren't found?

4. While the sample size was apparently quite large, it was described in a very confusing way. For example, L109-110 makes it sound like only one beetle was measured per population. Maybe one was measured per population in each trial and several were monitored at once? L117/TableS1 is confusing because it sounds like it would be the number of beetles measured per population, but I think this is the starting population that was collected and used for breeding (??).

5. L113 the mention of "a clear Petri dish" again confused me...these are the same petri dishes referred to in L110, right? How many petri dishes were monitored at a time? Was there one animal in each dish? I was really confused.

6. L151-152. This description was inadequate. In what direction was the relationship, and how did it compare with the latitudinal effect on amplitude/power? I think this is important because total activity may affect the power to detect a change in amplitude or period (if animals aren't moving much it's harder to detect a change). Are these trends strong enough that such an indirect effect might be important?

7. L158 this statement is really vague. Are the authors talking about clines or just inter-individual variation? What are they trying to say?

8. L166-167 The transition to this sentence is really rough. "On the other hand" would usually indicate a contrast with the previous statement, which isn't the case here.

9. L176-177 This sentence seemed like an odd way to end the paragraph. And it is a sort of contrast to the opening sentence (L168-9). Why not combine them into one strong sentence at the beginning. Apparently, a limited number of studies have identified this trend. It's fine to just say that (once) and would be even better if the authors could put it in context. Are there just a few because it's only occasionally studied? Because most studies don't have sufficient power, or because it seems to be rare?

10. L188-194 This section again convoluted. L188-190 is a wordy sentence that should be streamlined/clarified. The examples should be clearly organized to indicate which support wide dispersal and which don't. My sense is that both Drury (33) and Arnold (34) suggest limited dispersal. This is obscured by the use of "on the other hand" which implies a contrast. On top of this the text about Semeao's study is repeated twice with very similar text (L195-197 and L233-237).

11. L252 I would argue that not only do they need molecular studies of the circadian rhythmicity (e.g., clines in alleles of specific circadian genes), but also to couple genetic studies (i.e. population assignment) more directly with phenotypic studies.

Minor

L51 "indicators of circadian rhythms" is vague. These are characteristics of rhythms, and variations in these characteristics could indicate adaptation etc.

L55 "Additionally" here doesn't make sense. The topic sentence makes it sound like they are switching to talking about "insects other than Drosophila" but the preceding paragraph is also mostly about insects and there is no mention of Drosophila.

L58-59 a positive relationship between "latitude and circadian rhythm" doesn't make sense. I think the authors might mean a positive relationship between latitude and period.

L75 The syntax is hard to follow. I suggest "This species is found throughout most of Japan, except for the northern island of Hokkaido, meaning..."

L89-91 Please write the coordinates in a more standard way.

L101-103 These two sentences almost completely repeat each other.

L106 "To assess circadian rhythmicity" or "To assess characteristics of circadian rhythms" or "To assess circadian phenotypes"

L123 periodogram is misspelled.

L137 power of the rhythm

L138-139 this description was confusing. It made it sound like they were only considering one (presumably latitude), but in fact they considered each of them in separate analyses.

L218 "if without human cleaning" doesn't make any sense. I kind of understand that humans cleaning out the pests would disrupt the colonization/selection/evolution, but that's not explained.

L236 I might misunderstand, but wouldn't lack of isolation suggest higher gene flow?

L242 suggest "is not occurring on the spatial scale that we examined"

L246 significantly smaller than what? I think the authors just mean significant p values.

L253 I don't know what the authors mean by different phenotypic phenomena than other insect species.

Table 1 was misaligned in my version.

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PONE-D-20-27518R1

Amplitude of circadian rhythms becomes weaker in the north, but there is no cline in the period of rhythm in a beetle

PLOS ONE

Dear Dr. Miyatake,

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 fix all minor issues raised by the reviewers.

Further, it is highly recommended to get support from a native speaker or a professional editing service to improve the English of the manuscript. 

==============================

Please submit your revised manuscript by Jan 15 2021 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:

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• 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Henrik Oster, Ph.D.

Academic Editor

PLOS ONE

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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 #1: All comments have been addressed

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 #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: 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 #1: No

Reviewer #2: 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 #1: The ms is interesting but the English really needs to be improved before publication so some editorial assistance is required. Also the organisation of the Discussion could be better. Here are a few further comments.

L66-68. Incorrect statement. The long TIM isoform, not the short one, is negatively correlated with latitude ie higher frequency at lower latitudes.

L203-205 Again the timeless statement is not correct.

‘The frequency of a clock gene ( timeless ) of D. melanogaster studied from 40 to 65 degrees in latitude was negatively phased with latitude, but in populations col lected in southern

Europe by other years , this negative trend seemed to be unclear [21] .’

If the author wishes to use the timeless story, which I think has an important relevant message for this ms. which the authors do touch upon in their discussion of local adaptations and bottlenecks later on, perhaps they might use the paragraph below. I suggest the timeless part be moved to the latter part of the discussion.

‘The frequency of the timeless allele encoding a long TIM isoform of D. melanogaster initially studied from 40 to 65oN was initially negatively correlated with latitude, but in populations collected in extreme southern Europe (32-35oN) this trend was reversed [21] . It turned out that the latitudinal cline was actually caused by natural selection spreading the recently derived tim allele in all directions from a point of origin in southern Italy, thereby generating a distance rather than a latitudinal cline. This ‘distance cline’ was confirmed by studying the tim allele in the Iberian Peninsula (reference Zonato et al 2018 PMID: 29183263) where the cline was reversed. These studies reveal that the history of the genes that encode clinal characteristics also need to be considered when attempting to interpret spatial distributions. Such historical perspectives may account for some of the contradictory results that have been generated in the geographical analyses of biological rhythm phenotypes.

L221 the power of a rhythm is not necessarily the amplitude. It is the general sinusoidal shape of the rhythm. Thus a very sinusoidal but low amplitude rhythms could still have considerable power.

L229 ‘rear flying’????? explain

L291-293. Suggest ‘ Furthermore, given the seasonal differences in light exposure at different latitudes, studying the phase shift of the locomotor rhythm to brief light pulses might also provide a relevant phenotype that might discriminate between northern and southern population, as was the case for Drosophila timeless variants (21)

Reviewer #2: L25 missing word "these traits are" (?)

L55-59 It takes a little "mental gymnastics" for a reader to compare a trend in "higher latitudes" with "a positive relationship with latitude". I suggest adding something to indicate that the two studies are showing trends in the same direction. Like "Similarly, a positive relationship..."

L66 I'm not quite sure what is meant by "frequency of a clock gene". I think the authors mean something like period/frequency of expression. Also not quite sure what is meant by "negatively phased" (negatively correlated?)

L69 I'm not really sure what's meant by "The other context of circadian rhythm". It's not really clear how this is really different from the previous paragraphs where the authors were also talking about relationships with latitude and rhythms. Seasonal variation is mentioned but not really explained.

L71 pioneering

L74 wording doesn't seem right, and it seems trivial to say that the results will be discussed in the context of the literature. Perhaps eliminate this sentence, especially since the next sentence acknowledges the discrepancies.

L82 The authors know more about this than I do, but I usually see this name written as "Hokkaido" in English. Confirm that the spelling is as desired.

L101 this sentence seems to repeat the sentence starting on L91.

L107 Suggest deleting this sentence as it is essentially repeated in the sentence beginning on L109. The sentence beginning on L109 seems better because the association with grain is the part most similar to natural conditions. The temperature and photoperiod presumably vary among sites.

L152-154 I don't understand what the authors are saying here. If you are trying to analyze the relationship between two things (geographical area and power) you need to include them in the same model (?) I think they perhaps mean that effects of latitude and sex were analyzed separately? Or maybe they mean 3 things: latitude, longitude and sex?

L198-199 this is misleading sounds like it spans a range of 32 degrees. Suggest something like "in a range of approximately 8 degrees latitude (from about 32 to 41°N).

L199 Since the authors make it a point that there are no effects of longitude, the longitudinal range (about 10 degrees) should also be given.

L199 The authors say "Previous studies have often" but only cite one study. Preferably cite several examples or refer to the reference in a different way like "(e.g., [21])" (to indicate that it is an example) or ([21] and references therein) if you are citing the reference as a sort of review.

L203 see my comments about similar text within the introduction

L206 what does "this negative trend seemed to be unclear" mean? A negative trend wasn't observed? Or maybe not significant?

L207 it is possible that a cline could be detected

L211 I don't really understand what the authors mean by "survey with closely related species". Do they mean extend the geographic range by pooling results from two species? This idea is unclear.

L211 "In examining the clock cline between different species" is awkward wording. Maybe "In comparing clock clines among speices"

L225-227 wording unclear...kind of repeats "suggest that this beetle disperses very well [35] suggested very high levels of active dispersal" (??) I think the second part of the sentence is meant to be an example. Maybe authors mean "....very well, e.g., Ridley et al. [35] inferred very high levels of active dispersal..."

L229 I don't know what "including rear flying" means

L232; as follows

L248 I'm not sure what the authors mean by ideal environment. I think they mean "ideal" for large effects of bottlenecks and local adaptation. It's not written clearly.

L272 suggest "a cline in circadian amplitude"

L277-279 and L282-286 These two sentences say the same thing. Can they be consolidated?

L288 This line doesn't make sense.

L469 indicate threshold (p-value) for significance.

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

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.]

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Amplitude of circadian rhythms becomes weaken in the north, but there is no cline in the period of rhythm in a beetle

PONE-D-20-27518R2

Dear Dr. Miyatake,

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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Academic Editor

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Additional Editor Comments (optional):

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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 #1: 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 #1: Yes

**********

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

Reviewer #1: 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 #1: 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 #1: Yes

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6. Review Comments to the Author

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Reviewer #1: The ms is much improved although the English still needs some work. L217-218 is redundant so cut. the effects on power are small but significant and fit with other findings on other species. Given the Results section is so short and concise, could the authors not similarly reduce the length of the Discussion which is a little repetitive and overlong and a little rambling.

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Reviewer #1: No