PGENETICS-D-26-00009

Convergent latitudinal erosion of circadian systems in a rapidly diversifying order of fishes

PLOS Genetics

Dear Dr. Daane,

Thank you for submitting your manuscript to PLOS Genetics. After careful consideration, we feel that it has merit but does not fully meet PLOS Genetics'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.

As you will see, the reviewers, all experts in the field, agreed that this is a strong study that will be of general interest. They also value the combination of large-scale phylogenomic analyses and detailed physiological studies. However, they also have a series of concerns and requests that should be addressed before acceptance.

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Josep M Comeron

Academic Editor

PLOS Genetics

Monica Colaiácovo

Section Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Daniel Wright, Yangfan Zhang, and Jacob Daane. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

Reviewer #1:

In this manuscript, Wright et al explore an interesting hypothesis, that Perciformes fish species which have adapted to deep sea or high lattitude environments with no or little circadian rhythm would have lost genes related to circadian clock regulation. For this, they characterise orthologs of core circadian clock genes in perciform genomes. They indeed find loss of circadian genes with high lattitude, but surprisingly not with deep sea environment.

I appreciated the interesting question, and the detailed analysis of gene loss rather than a simple "no Blast hit", which provides insight into the process of pseudogeneisation. The authors also nicely confirm the loss of physiological circadian rhythm in high lattitude notothenioids, in relation to gene loss.

Major comments:

1- Orthology results are central to the work, yet only one orthology method is used. Although it is a good method, and well adapted, I suggest confirming the robustness of results with at least one protein similarity graph method, and reporting orthology results from public databases such as Ensembl Compara, EggNOGG, OrthoDB or OMA. In addition to limiting bias from the choice of method, this would limit bias due to the choice of two specific reference genomes.

2- I have several comments on the comparison between species and phylogenetic reasoning. Gene loss in the ancestor of a species clade is one single event, which mostly cannot be reversed. Thus:

2a- Gene losses should not be counted in number of genomes without a gene, but in number of monophyletic clades without a gene, which correspond to the number of events of loss. This notably applies to Fig. 2, and to the paragraph starting line 155.

2b- For Fig. 4, what biology are the authors representing with a model "which allows for backward and forward transitions"? A more straighforward interpretation of the tree is that each clade with only losses corresponds to one loss event, and every clade with a gene present in even one species implies that the gene was present in the ancestor of this clade. It would be more informative to map on the tree of Fig 4 the number of circadian genes infered lost on each branch.

2c- The randomisation around line 198 doesn't take into account phylogenetic non-independence. Instead of randomising genomes, the authors should randomise branches on the tree on which losses can occur.

2d- The authors could use CAFE5 (https://doi.org/10.1093/bioinformatics/btaa1022) to evaluate significant patterns of gain and loss.

3- Potential losses of function can sometimes be solved with gene expression. For example stickleback Per1b has expression in the database Bgee (https://doi.org/10.1093/nar/gkae1118). The authors should consider checking these potential losses in expression datasets or databases.

4- The first paragraph of the Discussion is ambiguous about drift or adaptation ("likely a key component of their success"). Unless there is evidence of adaptation, the default assumption should be that these genes were lost by drift, in the absence of purifying selection maintaining their function in these environments.

5- While the comparison to cave species is interesting, the authors should note that these species have evolved for much shorter times in their extreme environments than the high lattitude clades have, and with much smaller population sizes.

6- Fig. 2 shows indentical one-sided and two-sided p-values, which is impossible. Moreover, there is no strong reason to use one-sided tests, i.e. an excess in the other direction would also have been of interest. The authors should use only two-sided tests. See e.g. https://doi.org/10.1111/j.1442-9993.2009.01946.x, https://doi.org/10.1111/j.2041-210X.2010.00014.x.

7- Please provide the MCMCglmm model diagnostic plots and autocorrelation plots.

8a- In Table S2, please provide BUSCO or equivalent scores for the genomes, which is important for this type of study of gene loss.

8b- Statistics are missing for H. otakii.

Minor comments:

9- It is worth noting that Ref 42 Toloza-Villalobos et al which is discussed several times did not consider all of the genes which are studied here, e.g. they don't consider Bmal.

10- It would be informative to show transitions to high lattitude or deep sea on the branches of trees of figs. 1 and 4.

Reviewer #2: This manuscript provides a large-scale analysis of evolved changes in circadian clock genes across Perciforms. The broader question about whether clock genes are lost is particularly interesting. Circadian clocks are widely believe to be nearly ubiquitous. This manuscript examines a relatively comprehensive list of circadian genes across 96 Perciform species. The paper is of significance and of broad interest, given that it provides the most throrough analysis to date of clock-gene loss. The paper also presents ambitious metabolic profiling of ice fish, showing a functional loss of rhythms. The manuscript is well-written and statistically sound. Below are a number of suggestions that would help increase the accessibility to a wider audience, particularly the circadian community.

1. The paper would benefit from greater discussion the role of clock genes outside of Zeitgeber regulation (non-circadian). Many have functions that differ from their role in in daily rhythms. Presumably loss of clock-genes has wide-ranging effects on physiological and cellular function.

2. Why would a species lose seven circadian genes? Are there shared or partial functions? Presumably loss of one, or a few, key genes can fully disable the clock. If there is a way to extend the analysis in 2E,F to discuss the broader functions of the circadian clock? Could the analysis be extended to compare core-clock genes vs output-associated genes?

3. Some reorganization and clarification of jargon would make this more accessible to a generalist audience. For example, see paragraph at line 225.

4. Could the authors discuss on changes in circadian genes in highly divergent subterranean animals with loss of circadian genes including mole rates and cavefish?

5. It would be helpful to discuss why loss of clock genes might be advantageous, and particularly how the loss of specific Zeitgebers may push adaptive pressure towards loss of the clock.

Reviewer #3: Please see attachment.

Reviewer #4: This manuscript explores the loss of circadian related genes in numerous perciform fishes . This manuscript combines comparative genomics of circadian genes and metabolic phenotyping of 4 species (2 polar, 1 nearly polar, and 1 non-polar). This manuscript does a nice job of combining a detailed comparative genomics study with data on several species. I enjoyed reading this manuscript. My apologies for the delay in returning the review.

The figures 1-4 seem to have somewhat redundant information. The information is displayed nicely but it’s unclear to me how different the information is that is being displayed in each figure.

The maximum percentage of intact coding sequence across all transcript isoforms does not strike me as a relevant metric as early frameshifts can render a transcript non-functional and the amount of functional sequence does not translate to functional protein domains. I recommend removing this portion of the main and supplementary figures and only showing the mutational variants across the genes. There are also numerous tools to quantify the impacts of variants that could be used to quantify the impacts of frameshifts that don’t result in premature stop codons but may remove functionally relevant regions.

It should be possible to jointly analyze depth and latitude…

Figure 3D and 3F has several genes that are colored different colors (e.g. below dbx1 in pink), those genes are not defined anywhere and it’s not clear why they are below dbx1 if they are not that gene. There should be another column for other genes with their gene names. If space is an issue, the assembly scaffold names could be provided in a supplementary table instead of within the figure.

I would argue that the manuscript does not provide evidence that “reduced dependence on circadian systems may have contributed to their success as one of the most species-rich vertebrate orders”. I recognize that this is a very general statement and includes "may". I agree that there is evidence that the losses “occurred early in their [the lineages] diversification”.

It is possible to measure relaxed selection, for example using RELAX or other dn/ds methods which would add substantial analyses to this manuscript to quantify whether other (non-lost) circadian genes show relaxation of selection in deep sea lineages.

There does not seem to be directionality in TOGA for where mutations are outside of the middle 80% of a coding sequence, however mutations in the first 10% are likely to have a much larger impact than those at the final 10%.

The light/dark cycles for the metabolic study are not reported. Was the light/dark the fall conditions or was there any artificial light (or none) provided during the study?

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics  data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No:  The data for the metabolic study do not appear to be provided in the supporting information.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Attachments

Attachment

Submitted filename: Wright et al.pdf

Dear Dr Daane,

Thank you for submitting a revised version of the study that has addressed the reviewers' comments and concerns. We are pleased to inform you that your manuscript entitled "Convergent latitudinal erosion of circadian systems in a rapidly diversifying order of fishes" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Monica Colaiácovo

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Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: I thank the authors for the excellent job they did in this revision. The revised manuscript is a pleasure to read.

One small comment: p. 5, it should probably be "expected mean" in the parentheses rather than just "mean".

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics  data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

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Reviewer #1: No

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