Colour polymorphism associated with a gene duplication in male wood tiger moths
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eLife assessment
This study combines different approaches to unravel the genetic basis of a pigmentation polymorphism in natural populations of a fascinating study system with well-studied ecology. The paper has the potential to be of general interest to biologists curious about the genetic basis of adaptive variation, which is especially relevant to evolutionary biologists and ecologists. The study reports substantial data and makes a strong case for the contribution of a duplication-derived gene acquiring a morph-specific function. Further information is required to implicate valkea in pigmentation morph formation and for diagnosing the duplicated segment as a supergene (associated with low recombination).
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Abstract
Colour is often used as an aposematic warning signal, with predator learning expected to lead to a single colour pattern within a population. However, there are many puzzling cases where aposematic signals are also polymorphic. The wood tiger moth, Arctia plantaginis , displays bright hindwing colours associated with unpalatability, and males have discrete colour morphs which vary in frequency between localities. In Finland, both white and yellow morphs can be found, and these colour morphs also differ in behavioural and life-history traits. Here, we show that male colour is linked to an extra copy of a yellow family gene that is only present in the white morphs. This white-specific duplication, which we name valkea, is highly upregulated during wing development. CRISPR targeting valkea resulted in editing of both valkea and its paralog, yellow-e, and led to the production of yellow wings. We also characterise the pigments responsible for yellow, white, and black colouration, showing that yellow is partly produced by pheomelanins, while black is dopamine-derived eumelanin. Our results add to a growing number of studies on the genetic architecture of complex and seemingly paradoxical polymorphisms, and the role of gene duplications and structural variation in adaptive evolution.
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eLife assessment
This study combines different approaches to unravel the genetic basis of a pigmentation polymorphism in natural populations of a fascinating study system with well-studied ecology. The paper has the potential to be of general interest to biologists curious about the genetic basis of adaptive variation, which is especially relevant to evolutionary biologists and ecologists. The study reports substantial data and makes a strong case for the contribution of a duplication-derived gene acquiring a morph-specific function. Further information is required to implicate valkea in pigmentation morph formation and for diagnosing the duplicated segment as a supergene (associated with low recombination).
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Reviewer #1 (Public Review):
The authors present a nice collection of analyses identifying the likely causal locus and pigmentation basis underlying color polymorphism in a model aposematic moth system. In general, the writing and presentation are very clear. There are several areas of the text, however, that could benefit from more clarity and attention to detail. Those changes should be very simple for the authors to make.
My primary concern however is the interpretation of their findings, in light of the lack of analysis of recombination, as well as the flanking region of their identified gene duplication. Specifically, while the authors do an OK job characterizing the genomic region 3' of their identified novel insertion/duplication associated with white coloration, I could not find an analysis of the 5' region, in which there could …
Reviewer #1 (Public Review):
The authors present a nice collection of analyses identifying the likely causal locus and pigmentation basis underlying color polymorphism in a model aposematic moth system. In general, the writing and presentation are very clear. There are several areas of the text, however, that could benefit from more clarity and attention to detail. Those changes should be very simple for the authors to make.
My primary concern however is the interpretation of their findings, in light of the lack of analysis of recombination, as well as the flanking region of their identified gene duplication. Specifically, while the authors do an OK job characterizing the genomic region 3' of their identified novel insertion/duplication associated with white coloration, I could not find an analysis of the 5' region, in which there could be other functional elements that could give rise to their "complex polymorphism". Additionally, the authors discuss their findings and the potential of their duplicated region to "provide a region of reduced recombination" (lines 249-251). However, they need to be much more clear with the reader that this is a hypothesis that they have not measured (even though they have WGS data from a sufficient number of individuals estimating LD, which I find strange).
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Reviewer #2 (Public Review):
The authors set out to characterize the genetic architecture for aposematic color polymorphism in a species of tiger moths. It was previously known that the color polymorphism showed a non-sex-linked Mendelian inheritance pattern, and was thus likely controlled by an allelic change at a single autosomal locus. Based on observations in other species that traits with a similar simple inheritance pattern of polymorphic aposematic colors often involve supergenes, which refers to a tightly linked cluster of co-adapted loci, the authors tested the hypotheses that a supergene may be involved here in tiger moth polymorphism. To test this hypothesis, they used a combination of QTL mapping, GWAS, and RNA-seq approaches to identify regions of the genome that showed an association with the color pattern polymorphism. …
Reviewer #2 (Public Review):
The authors set out to characterize the genetic architecture for aposematic color polymorphism in a species of tiger moths. It was previously known that the color polymorphism showed a non-sex-linked Mendelian inheritance pattern, and was thus likely controlled by an allelic change at a single autosomal locus. Based on observations in other species that traits with a similar simple inheritance pattern of polymorphic aposematic colors often involve supergenes, which refers to a tightly linked cluster of co-adapted loci, the authors tested the hypotheses that a supergene may be involved here in tiger moth polymorphism. To test this hypothesis, they used a combination of QTL mapping, GWAS, and RNA-seq approaches to identify regions of the genome that showed an association with the color pattern polymorphism. The genetic mapping approaches identified a candidate genomic region that contained >20 genes, including the genes yellow-e, and its paralog, valkea. The RNA-seq data showed these genes to be expressed differently in the developing wings of the different color morphs. The valkea paralog is associated with a duplicated chromosomal region that appears to only be present in the genomes of yellow-colored morphs. A phylogenetic-weighting approach was also used to attempt to distinguish the strength of associations of the yellow-e and valkea genes with the color polymorphism and found evidence suggesting valkea was the likely genetic switch for the color polymorphism. Lastly, the authors provide evidence that the differences in coloration involve a change in melanins, through chemical characterization of pigments extracts. Collectively, the authors provide a comprehensive examination of the color pattern genetics and compelling evidence that the polymorphism in pigmentation is controlled by an allelic change at a single autosomal locus that includes the yellow-e/valkea genes that show different expression patterns in the differently colored morphs.
Strengths:
This study provides a comprehensive mapping effort to identify a locus responsible for modulating adaptive variation in natural populations of the tiger moth. This is an ideal trait and system to study the genetic basis of adaptive variation, as the trait variation has clear impacts on fitness and is under strong selection in natural populations. The genomes of Lepidoptera and their amenability for laboratory research and molecular methods make them well-suited for such mapping efforts. The authors used an impressive number of offspring from genetic crosses to conduct QTL mapping, which was nicely complemented with a population genomic GWAS approach to further narrow the candidate locus. The addition of the RNA-seq provides compelling evidence that genes at this locus are clearly involved in differences in wing pattern development.The greatest strength of this study is perhaps its finding of "something new, using something old". I am referring to the finding of a novel duplication of the yellow gene being involved in pigment variation. Yellow is well-known to be involved in color pattern development in Drosophila and butterflies, but its role in the tiger moths is completely novel. A recent duplication of yellow being involved in adaptive variation is completely new and quite exciting. With other recent examples of gene duplications being involved in differences in butterfly color pattern development, there are now numerous cases of the rapid evolution of gene duplicates involved in generating wing pattern variation. Thus, the findings here should be of broad interest to those interested in the genetic changes involved in generating adaptive variation in natural populations.
Another strength of the study is the characterization of the melanic pigment changes involved in the polymorphism. Such detailed phenotypic analyses can offer critical insights into how the genetic differences found to be associated with color pattern variation, may function and influence wing pattern development.
Weaknesses:
Despite narrowing the locus to a small number of genes through mapping efforts, the study falls short in identifying the genetic switch and sufficient evidence to confirm valkea's role in the color polymorphism.The mapping efforts identified a narrow locus covering multiple genes from the yellow gene family and RNA-seq data clearly identified valkea and yellow-e as being differentially expressed between color morphs thereby implicating their involvement in differences in wing color pattern development. However, the type and number of genetic changes at this locus involved in generating the color polymorphism remain unresolved. Tree topology provides only suggestive evidence that genotypes at valkea show a stronger association with color pattern differences than at the other nearby yellow genes, and offers limited further resolution as the where the genetic switch may be (e.g. within coding or non-coding regions across the locus).
I am unconvinced that framing this study as a test for the role of a supergene, or "to test whether the polymorphism is associated with large structural rearrangements controlling multiple phenotypic elements, or the result of a single gene mutation" is most appropriate or strengthens the study. The alternative hypotheses of "large structural rearrangements" versus "single gene mutation" do not necessarily reflect the possible, or most likely hypotheses, and neither are not necessarily clearly supported by the results of the study. In other studies of wing color pattern polymorphisms in butterflies, the genetic changes controlling the variation have been non-coding mutations in putative cis-regulatory elements (CREs) that control the expression of a nearby gene involved in wing pattern development (see examples from Heliconius butterflies). These would be considered changes in CREs, not "single gene mutations". There are instances in which such changes impacting color pattern variation have been captured within structural rearrangements, such as polymorphic inversions of Heliconius numata, the single gene or CRE mutation and structural rearrangements both being involved are not mutually exclusive, thus it is difficult to frame this study as testing them as alternative hypotheses. The data presented in the study celery implicate a genomic region with multiple genes differentially expressed (DE) between color morphs, with one of the DE genes residing within a structural variation (insertion/deletion polymorphism). However, the study is unable to resolve if the large structural rearrangement is involved, or if a single versus multiple genes or CRE changes may be involved. Thus, I find it challenging and perhaps a weakness of the paper to frame the study as a test of these alternative hypotheses that are not necessarily mutually exclusive or able to be distinguished using the data in the study. I have similar concerns with the focus on supergenes (i.e. co-adapted gene complex) being a weakness for the paper, as the results of the study don't directly test for the presence or role of a co-adapted gene complex at the locus identified.
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Reviewer #3 (Public Review):
This article aims at investigating the genetic and developmental basis underlying colour pattern polymorphism in the wood tiger moth. It combines GWAS and QTL data pointing at a candidate gene from the yellow gene family. The pattern of gene expression during wing disk development is then consistent with a potential role of this gene in the control of colour pattern variation but functional validation is lacking. The pigment analyses reveal the presence of pheomelanin on the wings, whose synthesis is known to be controlled by a pathway regulated by genes from the yellow family. The identification of these pigments suggests that variations in the colours of the wings in this species could indeed be caused by the regulation of the yellow pathway. Although functional validation establishing the exact role of …
Reviewer #3 (Public Review):
This article aims at investigating the genetic and developmental basis underlying colour pattern polymorphism in the wood tiger moth. It combines GWAS and QTL data pointing at a candidate gene from the yellow gene family. The pattern of gene expression during wing disk development is then consistent with a potential role of this gene in the control of colour pattern variation but functional validation is lacking. The pigment analyses reveal the presence of pheomelanin on the wings, whose synthesis is known to be controlled by a pathway regulated by genes from the yellow family. The identification of these pigments suggests that variations in the colours of the wings in this species could indeed be caused by the regulation of the yellow pathway. Although functional validation establishing the exact role of the valkea gene is lacking, the data provided are in line with a pleiotropic effect of controlled by a small region of the genome enabling the series of phenotypic variations associated with the white coloration. The duplication event restricted to a single haplotype also provides a convincing mechanism for the restriction of recombination in this genomic region. However, the fact that the valkea gene is truncated questions its functionality. It remains possible that the developmental switch could be rather caused by the variations detected in the non-coding part of the duplicated region, causing differential patterns of expression in different genes, including yellow-e. Some deeper discussion is needed on the putative role of the valkea gene vs. of the regulatory regions in controlling the developmental switch between yellow and white morphs.
Altogether, this interesting study provides original and important results on the genetic architecture underlying balanced polymorphism in the wild.
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