Cortex cis -regulatory switches establish scale colour identity and pattern diversity in Heliconius

  1. Luca Livraghi
  2. Joseph J. Hanly
  3. Steven M. Van Belleghem
  4. Gabriela Montejo-Kovacevich
  5. Eva S. M. van der Heijden
  6. Ling Sheng Loh
  7. Anna Ren
  8. Ian A. Warren
  9. James J. Lewis
  10. Carolina Concha
  11. Laura H. López
  12. Charlotte Wright
  13. Jonah M. Walker
  14. Jessica Foley
  15. Zachary H. Goldberg
  16. Henry Arenas-Castro
  17. Michael W. Perry
  18. Riccardo Papa
  19. Arnaud Martin
  20. W. Owen McMillan
  21. Chris D. Jiggins

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    This paper shows complex gene regulation for a simple binary switch phenotype. Cis-regulatory elements that control expression from the promoter of the gene cortex are shown to be evolutionary targets for changing cortex-dependent scale types in a discrete region of the Heliconius hindwing. Multiple approaches are used to identify the likely causal genetic variation in the cortex locus that is responsible for the presence/absence of the yellow band. It is an interesting case showing how a gene with a homogeneous expression pattern across the wing (during the pupal stage) can still have "hidden" modular regulatory regions that drive unique functions (albeit not expression) is specific regions of the wing.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

In Heliconius butterflies, wing pattern diversity is controlled by a few genes of large effect that regulate colour pattern switches between morphs and species across a large mimetic radiation. One of these genes, cortex , has been repeatedly associated with colour pattern evolution in butterflies. Here we carried out CRISPR knock-outs in multiple Heliconius species and show that cortex is a major determinant of scale cell identity. Chromatin accessibility profiling and introgression scans identified cis -regulatory regions associated with discrete phenotypic switches. CRISPR perturbation of these regions in black hindwing genotypes recreated a yellow bar, revealing their spatially limited activity. In the H. melpomene/timareta lineage, the candidate CRE from yellow-barred phenotype morphs is interrupted by a transposable element, suggesting that cis -regulatory structural variation underlies these mimetic adaptations. Our work shows that cortex functionally controls scale colour fate and that its cis -regulatory regions control a phenotypic switch in a modular and pattern-specific fashion.

Article activity feed

  1. eLife

    Evaluation Summary:

    This paper shows complex gene regulation for a simple binary switch phenotype. Cis-regulatory elements that control expression from the promoter of the gene cortex are shown to be evolutionary targets for changing cortex-dependent scale types in a discrete region of the Heliconius hindwing. Multiple approaches are used to identify the likely causal genetic variation in the cortex locus that is responsible for the presence/absence of the yellow band. It is an interesting case showing how a gene with a homogeneous expression pattern across the wing (during the pupal stage) can still have "hidden" modular regulatory regions that drive unique functions (albeit not expression) is specific regions of the wing.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. eLife

    Reviewer #1 (Public Review):

    In this paper the authors associate genetic variation in regulatory sequences of the gene cortex with the presence/absence of a yellow band of color in the wings of two species of Heliconius butterflies. They show that cortex is spatially regulated in larval wings, but the expression of this gene does not correlate with the presence or absence of the yellow band. Then they show that the gene is expressed in the nuclei of all cells of the pupal wing. By disrupting cortex they show that black cells (Type II) become white or yellow (Type I), and red scales (Type III) become paler across the whole wing.

    By examining open regions of chromatin around cortex, they discover that at least in one of the species, the insertion of two transposable elements in an open region of chromatin associates with the presence of the yellow band. They show that disrupting this regulatory region in a race of butterflies that does not contain the yellow band, nor the TE insertions, leads to the loss of the black color in a band-like shape, and the appearance of yellow scales in that region of the wing. They identify a different region of open-chromatin in the other Heliconius species that when disrupted also leads to the transformation of black scales into yellow scales in a band-like pattern.

    The authors achieved their aims and the results support their conclusions.

    The strength of this manuscript lies in the use of multiple approaches to identify the likely causal genetic variation in the cortex locus that is responsible for the presence/absence of the yellow band. The only weakness (if I can call it that) is that it is still not clear how cortex, which is also expressed in the nuclei of the yellow scales in races that supposedly have the TE insertion and closed chromatin in that enhancer region, fail to develop black scales in that region of the wing.

    This is one of the first few papers that examines the function of specific open regions of chromatin in the DNA of butterfly species using CRISPR-Cas9. The main novelty of this paper is in identifying how a gene with a homogeneous expression pattern across the wing (during the pupal stage) can still have "hidden" modular regulatory regions that drive unique functions (albeit not expression) is specific regions of the wing.

    This work reminds me of the regulation of the vestigial gene in the wings of Drosophila. vestigial also has homogeneous expression across the wing pouch but it achieves this homogeneous expression via two separate enhancers that have complementary expression patterns.

  3. eLife

    Reviewer #2 (Public Review):

    The gene cortex was reported to control mimicry and crypsis in butterflies (Nadeau et al. 2016). This study finds cortex function to be essential for Heliconius wing scale type determination at the transition from scale type I to type II / III. This is shown by genetic loss of function assays and characterization of scale structure by scanning electron microscopy. In particular, the authors show that cortex function is essential for scale type determination throughout the wings that mainly contain type II/III scales in Heliconius butterflies. This is revealed by a series of CRISPR/Cas9 derived somatic mosaic mutants in diverse genetic backgrounds and species. Expression of a specific yellow (type I scale) hind-wing stripe in some Heliconius melpomene and H. erato morphs was found to depend on molecular tinkering and malfunction of a discrete cortex cis-regulatory element (CRE). The authors identify distinct CRE's in both species by ATAC-seq open chromating mapping and narrow down candidate regions by genetic association to the yellow stripe. Hi-C assays were used to verify that the elements indeed interact with the cortex promoter. However, a possible regulation of other genes cannot be excluded. Tinkering of these elements appears to be a natural mechanism in wing colour pattern evolution, since a yellow stripe morph is associated with an insertion of a transposable element in the corresponding region in the morph H. melpomene timareta. Expression of cortex was investigated at different developmental stages by in-situ hybridization and immuno-staining techniques. Cortex transcripts reveal complex expression pattern that do not seem to be associated with the yellow hindwing stripe in corresponding morphs. Cortex protein is localized in the cell nucleus throughout wing cells and future studies must resolve how cortex regulatory elements determine such specific stripe-pattern. This article contrasts the widespread expression of cortex with a complex transcriptional regulation of this gene and scale type transition in discrete wing domains. The authors argue that cortex is a prime target for wing pattern evolution, acting as "input-output" module, whereby complex spatio-temporal information is translated to determine scale type and colour.

  4. Version published to 10.1101/2020.05.26.116533v3 on bioRxiv
  5. eLife

    ###Reviewer #3:

    PREreview of "The gene cortex controls scale colour identity in Heliconius" Authored by Luca Livraghi et al. and posted on bioRxiv DOI: 10.1101/2020.05.26.116533

    Review authors in alphabetical order of last name: Monica Granados, Vinodh IlangovanORCiD, Katrina Murphy, Aaron Pomerantz

    This review is the result of a virtual, live-streamed preprint journal club organized and hosted by PREreview and eLife. The discussion was joined by 17 people in total, including researchers from several regions of the world.

    Overview and take-home message:

    In this preprint, Livraghi et al. present noteworthy advances in evolutionary biology by characterizing the role of cortex gene in multiple Heliconius butterfly species, which is responsible for the wing patterns: yellow bar or the Type I scale cell fates (white/yellow). The authors identified cortex gene’s major role in sympatric speciation, the modulation of convergent wing patterns, and the regulation of scale identity in multiple Heliconius species, which naturally have different niches to help explain different co-mimetic morphology. Livragi’s team provides strong evidence for the cortex gene as one of the earliest regulators and its ability to set the differentiation of scale cells in a molecular switch fashion from yellow to red/black at a particular development stage through distal localization. This important discovery on the role of cortex gene fills a gap in our existing knowledge about the gene’s ability to control scale cell identity and wing color patterns. Since this work is of significant interest in evolutionary biology, we outlined some concerns below that could be addressed in the next version.

    Positive feedback:

    1. We strongly recommend this preprint to others/for peer review. In addition, we recommend this article to trainees as educational material to learn evolutionary developmental biology through interactive tutorials.

    2. The authors have provided a good amount of novel results and have utilized current tools to address their questions.

    3. This research fills a gap in our understanding of wing patterning in Heliconius while doing so in a very comprehensive way across multiple species and using techniques that systematically detail the association between gene expression and phenotype.

    4. It was interesting to learn that the cortex gene doesn’t follow the typical pattern gene paradigm. We do not have many examples of integrator genes like cortex, which give binary outputs from a network of genes and integrate elements to produce a singular output.

    5. This is a textbook example and is important for evolutionary development and mimicry studies. It is hard to find and/or work with a developmentally important gene that is amenable for genetic modification and still be able to work with viable offspring and have it be relevant for evolution.

    6. The current cortex protein data as seen in Figure 6 adds novel data to the manuscript.

    7. Thanks to the authors for setting a great example of showing modeling information. The graphics are visually appealing and convey complex information well.

    8. This preprint sets up a good next step of how cortex evolved in a more broad context. We know the cortex gene is potentially implicated in wing pattern evolution in other distantly related butterflies and moths (e.g. peppered moth Biston betularia) and in possible roles of evolution/speciation by pattern changes due to genomic inversions at cortex locus.

    9. The authors did a good job of creating a well-composed manuscript. Yellow bar with one species had a contradiction but did reconcile with further research questions.

    10. Definitely, [the results are likely to lead to future research] especially with understanding how a cell cycle regulator affects developmental cell fates in terms of these scale colors and structures.

    11. Antibodies can open up future research. This research team figured out three elements and there are possibly more to explore. Future research might investigate how cortex possibly regulates endocycling and what this means for color identity determination.

    Major concerns:

    1. The use of the term “race” to define butterflies with specific phenotypes needs to be revised to clines or strains or variants. “Race” is a social construct and not a biological reality and we strongly suggest revising this term.

    2. The authors state that cortex and dome/wash genes are controlled by inversion (see Line 375, page 19). Does the strain they engineered have/carry the inversion ?

    -We are aware that inversion for species is complex - strains, genetic background - starting material for inversion.

    -Inversion events occurred millions of years ago in the loci contributing to the wing pattern. Authors describe the first generation of CRIPSR knock-outs in Heliconius sp. and hence we suggest to include further information.

    1. We strongly suggest the authors elaborate on their qRT-PCR analysis pipeline. Did the authors follow MIQE guidelines in their quantitative real time PCR assays?

    2. More explanation could be provided for cortex protein experiments. Figure 6 could explicitly say what developmental stage/time after pupation (they report this in the Methods section) and the rationale behind presenting data for this stage in development.

    -If a systematic developmental time series of cortex protein expression is observed using immunostaining, we suggest adding the data. Otherwise we request the authors to comment on the rationale behind selecting this particular stage of development.

    1. We recommend the authors mention institutional or local animal care ethical approval and safety regulations in the field working on Heliconius sp. for setting best practice reporting standards.

    2. We suggest to clarify the lack of a clear correlation between in situ stains and the mutational effects of cortex CRISPR knock-outs.

    3. Please add statistical analyses in figure legends, e.g. Figure 2 lacks statistical analysis information. Which test was performed and why? A statistical analysis subsection under the Methods section could be useful.

    4. Could a sized-down Figure S10 be added to Figure 6 in the manuscript to provide more information about the nuclear ploidy and cortex antibody signal? Even no association is informative and helps the reader think about the connection between color/endopolyploidy.

    Minor concerns:

    General

    1. We request authors to revise the introduction section allowing an easy to comprehend information on gene regulatory complex affecting each patterning region.

    2. We strongly recommend minor rephrasing of the on/off switch to guide non-experts in evo-devo biology.

    Figures

    1. Figure S10 has a couple of typos - ‘localisation’ and ‘punctae’ in the first sentence of the figure caption.

    2. It will be helpful to guide the readers, if a high-level phylogenetic tree mapping the related Heliconius’ evolution is presented in Figure 1. We suggest a compass guide to be added in the map of Figure 1b.

    3. The scale bar is missing in Figures 6a and 7a.

    4. In Figure 4, some of the mosaic KOs are very apparent and others are not especially for researchers unfamiliar with butterfly CRISPR, e.g. H. charithonia. I might suggest highlighting or using arrows to indicate the mKO regions.

    5. We request the authors to consider reflecting on the distribution of samples in qPCR data superimposed on box-whisker plots .

    Sufficient Detail

    1. More information about the genes would be helpful, such as accession numbers and annotated gene information rather than the complete genome data.

    -Might not be able to repeat CRISPR from the details in the Methods section. If the gene information is not well annotated as a model system then it is difficult. What about Heliconius? It might be helpful to report the scores for low off-targets.

    -Non-standard genetic model systems present a challenge particularly to create genomic resources.

    1. Multiple people mentioned not able to repeat in situ hybridization methods from the available information on methods. The hybridization conditions for thicker whole mounts were not fully explained.

    2. Please provide more information about the number of animals.

    Data Accessibility

    1. We appreciate the authors adding supplemental information as figures and we request to report data files associated with the manuscript.

    2. R code was used for morphometric analysis - this is difficult to track from pay walled reference mentioned and thus a problem. We request to make this analysis information/pipeline available openly.

    3. Please include supplemental information on the microscope settings and metadata of images used for analysis explicitly.

    4. High-resolution images of the CRISPR mutants could be provided in a supplemental/data repository.

    5. Providing gene sequences used in this study will be very helpful rather than the SRA repository, especially probes used for in situ and sequences targeted for CRISPR.

    Acknowledgments:

    We thank all participants for attending this preprint journal club. We especially thank those that engaged in the discussion. Their participation contributed to both a constructive and lively discussion.

    Below are the names of participants who wanted to be recognized publicly for their contribution to the discussion:

    -Monica Granados | PREreview | Leadership Team | Ottawa, ON

    -Vinodh Ilangovan | Labdemic - Founder |Postdoc | @I_Vinodh

    -Katrina Murphy | PREreview | Project Manager | Portland, OR

    -Aaron Pomerantz | UC Berkeley/Marine Biological Laboratory | Ph.D. Candidate | Berkeley, CA/Woods Hole, MA

  6. eLife

    ###Reviewer #2:

    This manuscript explores the role of the gene cortex in the specification of wing scales in the butterfly genus Heliconius. Species of Heliconius butterflies are notorious for their reciprocal mimicry of wing color patterns. Several genes are known to control variation of specific color pattern elements within and between species, cortex is one of them. The authors combine RNAseq analysis across wing development, in situ hybridizations, antibody stainings and analysis of crispr somatic mutations to dissect the role of cortex in the specification of scales. Their main claim is that cortex imparts scale identity (color, morphology), namely type II and type III identity.

    Although this paper includes a substantial amount of work and a number of interesting observations, I am not sure what can really be concluded in the end, and several results would need follow-up experiments to reach a stable conclusion.

    The strongest part, in my opinion, is the analysis of somatic mutant clones of cortex in the wings of different species. The authors show that the lack of cortex consistently results in the conversion of type II and type III scales into type I scales, and thereby demonstrate the necessity of this gene for type II & III identity. This is solid, interesting, but not a novel concept from a genetic or developmental biology point of view. There are countless examples in the 1990s literature of genes whose mutations results in such shifts in cell identity (e.g., poxn and cut in the peripheral nervous system of flies).

    From this result, two questions emerge: how and when does cortex assign this identity during development? And how does cortex explain the variation in color pattern among Heliconius morphs and species? Although the paper discusses these two questions, I find the answers unclear and the results confusing.

    The authors first examine the expression dynamics of cortex. They re-annotated the 47-gene genomic interval where cortex maps and analyzed the differential expression of all genes in the interval, across developmental stages, across species and morphs and also compared wing compartments. Their main conclusion is that cortex is the most likely candidate in this interval to explain color pattern variation. I am not sure why the authors did this. I thought this was already clearly established from a previous paper (Nadeau et al. 2016, Nature). Moreover, the explanations of the differential gene expression (DGE) analysis are often too shallow to really understand what the authors really did, including the method description. The figures are poorly annotated and it's difficult to understand if there are replicates in the RNA-seq analysis (see minor comments). One striking result from this part, is that the DGE suggests that cortex is differentially expressed in the the 5th instar larvae between 2 morphs of Heliconius erato and 2 morphs of Heliconius melpomene, but the differential expression goes into opposite directions between this 2 species. How could the same phenotypic variation between morphs of 2 species be caused by opposite DGE? They authors note that it is interesting but do not comment or analyze further.

    They pursue their investigation with in situ hybridization on 5th larval instar wings and mitigate the notion of a spatial correlation between cortex transcripts spatial distribution and color patten elements proposed by Nadeau et al., 2016. Here again, the figure would benefit from better annotation. The authors indicate subtle differences in the local distribution of cortex transcripts between morphs but do not really conclude anything from their observation. They also give no indication of sample size or replicates, which I find unsettling given the noise associated with this experiment. I am not sure what this figure really adds to the published work, or to the present manuscript.

    Finally, the authors examine the distribution of Cortex protein in late (2-day pupa) developing wings with a polyclonal antibody. They find, surprisingly, that the protein is distributed more or less uniformly in the wing epithelium and localizes to the cell nuclei. While this is very different from the patterned transcript distribution, it is consistent with the somatic mutant clone analysis that showed that any mutated cell at any position of the wing displayed a phenotype. But this opens many questions: what is the origin of the apparent difference in expression between protein and transcripts? Is cortex secreted and it diffuses across the wing? Or is the transcript expression spatially dynamic and the protein distribution revealed by the authors reflects the temporal integration of this expression? And if Cortex is present and functional across the wing, how does it produce discrete pattern elements?

    The authors conclude their paper with a figure suggesting that cortex specifies typeII/III scale identity early during wing disc development and that the distinction between type II and type III is subsequently governed by the gene optix at a later stage. But what substantiates the idea that cortex imparts cell type identity early on? What does Cortex larval (5th instar) distribution look like? Is it as uniform as that of later stages? The data presented here do not offer the temporal or functional resolution to support this conclusion.

    In conclusion, this paper shows that the mutation of the gene cortex results in scale type transformation, but fails to explain or suggest how this may happen during development. It also does not suggest how cortex may control the "fantastically diverse" pattern variation in Heliconius.

  7. eLife

    ###Reviewer #1:

    This is an interesting but complex study that examines the role of a few genes in a previously mapped interval in being the "switch" gene that regulates the presence or absence of a yellow band in the wings of Heliconius butterflies. The study first examines whether there is a correlation between expression level of several (47) genes in the mapped interval in developing wings (or parts of wings) in two separate species of Heliconius each having a race with the yellow band and a race without the yellow band. This part of the study highlights three genes (among others) that show some pattern of differential regulation but shows that there is no simple correlation between the expression level of these three genes in either larval or pupal wings and the presence of the yellow band. The authors then examine the function of one of the genes in the interval, cortex, in scale color development by using CRISPR. They find that cortex crispant individuals display color changes across the whole wing, not just in the region of the yellow band. In particular the black scales (Type II) become white or yellow (Type I), and the red scales (Type III) also become white or yellow (although this last transformation is not documented at the SEM level). The authors examine, once again, the expression domain of the cortex gene, this time during pupal development with an antibody, and they find that the gene is expressed across the whole wing, supporting its functional effects also across the whole wing. They observe that cortex is expressed in multiple punctate domains in the nuclei of scale building cells, which are polyploid cells, and in a single punctate domain in the nucleus of non-scale building epidermal cells, which are not polyploid. They then test whether perhaps there are more of these punctate nuclei in the region of the yellow band, but they find no such correlation.

    In the end the authors try to argue that either 1) cortex is the yellow-band switch gene they are after but that the switch is not in the form of a typical spatially expressed gene (in the shape of the yellow band) but perhaps in the form of some threshold or heterochronic mechanism (not clearly explained), or that 2) another gene in the mapped interval, not examined for function in this study, is instead the switch genes they are after, and which may (or may not) interact with cortex in the differentiation of the yellow band.

    I believe the authors are trying hard to implicate cortex in some way, as the yellow band switch locus, but the data just does not support this. Instead the authors implicate cortex in scale color identity (the title of the manuscript). However, given that cortex (alone) cannot control a specific color either, because the effect of cortex on color is different in different parts of the wing, their model for how cortex acts is too simple and does not fit their data. A combinatorial genetic code for both scale color and morphology (see below), where cortex is simply one of the players (rather than a major switch/homeotic gene) is required to explain the data in this manuscript.

    Furthermore there are several data missing from the manuscript that need to be added to support some of the conclusions drawn, and several other data that would be important to add for purposes of data replication across labs.

    1. The authors claim that cortex converts Type II (black) scales into Type I (white/yellow) scales but their SEM data and scale morphological measurements presented in the supplement don't fully support this conclusion. These transformations vary from species to species (e.g. H. melpomene and H. erato show different degrees of transformation) and only some features of the scale are actually transformed (e.g., cross rib periodicity in both species, and scale width and length and ridge periodicity in H. melpomene). The remainder of the measurements show that cortex is not sufficient to convert scale Type II into scale Type I.

    2. I suggest that the definition of the scale types presented should be made more explicit. What are scale types I, II and III really? In line 87 it is mentioned that these scale types are based on scale color and on scale morphology but what follows is just a description of the pigments found in each scale and not their morphology. Furthermore, the data presented in the manuscript suggests that color and morphology can be uncoupled with genetic perturbations of cortex, so is it even useful to stick to this scale type nomenclature going forward? Something to consider.

    3. There is a need for a new figure showing how the scale morphological measurements were actually conducted. There is no scale bar in the SEM images of yellow and black scales and this should be added. The SEM images used to represent a typical yellow WT scale and a transformed yellow scale of H. melpomene (in Figure 7) show very different densities of cross-ribs (but I am not even sure what exactly is being considered a cross-rib), yet the graph indicates that there is no difference between these scale types. This is confusing and needs clarification. Make sure you look up scale morphology nomenclature in Ghiradella 1991 (Applied Optics) to make sure you designate ribs (crossribs) and microribs appropriately. There seem to be quite a lot of differences in microrib density across Wt scales and transformed yellow scales in H. melpomene.

    4. The authors claim that cortex converts Type III (red) scales into Type I (white) but they only describe conversions of Type II (black) into Type I (yellow) scales at the SEM level and don't provide any SEM images or quantitative data for the red to yellow, red to white, and black to white scale transformation. Adding these data is important to support the conclusions of the study.

    5. I suggest the authors remove the dome-t and dome/washout gene data from the manuscript as 1) nothing about these genes is mentioned in the abstract; 2) the expression of these genes doesn't correlate with presence of the yellow band; 3) the genes are not investigated at the functional level; 4) the whole gene duplication issues surrounding these genes make the whole manuscript more difficult to read and does not, in the end, contribute to the main story that yielded results - which is the function of cortex in scale development. The function of these genes might still be worthy of investigating using CRISPR at a later date, and perhaps it would be useful to include the expression pattern data in that subsequent paper. This is merely a suggestion that I believe will make this manuscript less heavy and easier to read by focusing the reader's attention on the main points of this story.

    6. Pigmentation and scale morphology is most likely controlled at the pupal stages of wing development and by measuring RNA levels of candidate switch genes at just two time points during pupal development (36hrs and 60-70 hrs after pupation) you may not have sampled the correct time window for yellow band differentiation. Several genes are expressed only during the first 16-30 hrs of pupal development, in species that need 7 days for pupal development (see Monteiro et al. 2006 for genes such as Wg, pMad and Sal) so sampling wings (for RNA-seq and antibody stains) at 36hrs and 60-70 hours may not be an ideal sampling strategy going forward.

    7. The authors mention that because cortex causes changes in both scale color and morphology this suggests "that cortex acts during early stages of scale cell fate specification rather than during the deployment of effector genes". This conclusion needs more discussion. Matsuoka and Monteiro (2018) showed that knockout of the gene yellow, an effector gene at the end of a gene regulatory network for melanin pigment production, also led to both changes in scale color and morphology. These authors proposed instead that absence of certain pigments on the wing, such as dopa melanin, caused chitin to polymerize differently and form an extra lamina that prevent the windows from forming in the scales (just as seen in cortex mutants). The authors should consider and evaluate this alternative explanation in their discussion.

    8. Did the authors examine whether there were protein coding changes between the 47 genes in the mapped interval between the yellow and black races? Please mention whether this was done. Please also upload the sequences of the genes that were studied and provide accession numbers for these sequences.

  8. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    The topic of your work is timely and intriguing, but the reviewers raise several issues with the study. For example, the reviewers propose that the major conclusions of the manuscript are not supported by the data presented, and that a full set of SEM data across all scale type color transformations should be presented. Given the results presented, the relationship between cortex expression and the actual pigmentation remains unclear, and the sole phenotypic analysis is insufficient to make conclusions about the role of a gene in producing pigmentation pattern variation.

  9. Version published to 10.1101/2020.05.26.116533v2 on bioRxiv
  10. Version published to 10.1101/2020.05.26.116533v1 on bioRxiv