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Colour polymorphism associated with a gene duplication in male wood tiger moths

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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 , uses bright hindwing colours as a signal of unpalatability, and males have discrete colour morphs which vary in frequency geographically. In Finland, both white and yellow morphs can be found, and these colour morphs also differ in behavioural and life-history traits. Complex polymorphisms such as these are often explained by supergenes. 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, and could act to reduce recombination, thus potentially representing a supergene. 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. The yellow family genes have been linked to melanin synthesis and behavioural traits in other insect species. Our results add to only a few examples of seemingly paradoxical and complex polymorphisms which are associated with single genes.
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Colour polymorphism associated with a gene duplication in male wood tiger moths
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Melanie N. Brien1*!, Anna Orteu2*, Eugenie C. Yen2, Juan A. Galarza1,3, Jimi Kirvesoja3,
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Hannu Pakkanen4, Kazumasa Wakamatsu5, Chris D. Jiggins2**, Johanna Mappes1,3**.
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1Organismal and Evolutionary Biology Research Program, Faculty of Biological and
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Environmental Sciences, University of Helsinki, Finland.
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2Department of Zoology, University of Cambridge, United Kingdom.
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3Department of Biological and Environmental Science, University of Jyväskylä, Finland.
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4Department of Chemistry, University of Jyväskylä, Finland.
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5Institue for Melanin Chemistry, Fujita Health University, Toyoake, Japan.
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*These authors contributed equally.
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**Co-senior authors.
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!Correspondence: mnbrien1@gmail.com
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Abstract
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Colour is often used as an aposematic warning signal, with predator learning expected to lead
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to a single colour pattern within a population. However, there are many puzzling cases where
17
aposematic signals are also polymorphic. The wood tiger moth, Arctia plantaginis, uses
18
bright hindwing colours as a signal of unpalatability, and males have discrete colour morphs
19
which vary in frequency geographically. In Finland, both white and yellow morphs can be
20
found, and these colour morphs also differ in behavioural and life-history traits. Complex
21
polymorphisms such as these are often explained by supergenes. Here, we show that male
22
colour is linked to an extra copy of a yellow family gene that is only present in the white
23
morphs. This white-specific duplication, which we name valkea, is highly upregulated during
24
wing development, and could act to reduce recombination, thus potentially representing a
25
supergene. We also characterise the pigments responsible for yellow, white and black
26
colouration, showing that yellow is partly produced by pheomelanins, while black is
27
dopamine-derived eumelanin. The yellow family genes have been linked to melanin synthesis
28
and behavioural traits in other insect species. Our results add to only a few examples of
29
seemingly paradoxical and complex polymorphisms which are associated with single genes.
30
31
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Introduction
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Colour polymorphisms, defined as the presence of multiple discrete colour phenotypes within
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a population (Huxley, 1955), provide an ideal trait to study natural and sexual selection.
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Colour phenotypes are linked to fitness in many contexts including camouflage, mimicry and
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mating success. Colour is often associated with aposematism, warning predators of
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unpalatability (Cott, 1940; Cuthill et al., 2017), and in such cases, predator learning should
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favour the most common colour pattern, leading to positive frequency dependent selection
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(Endler, 1988). Despite this, aposematic polymorphisms can be stable when selection is
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context-dependent (Briolat et al., 2019), especially where genetic correlations between colour
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phenotypes and other traits lead to complex fitness landscapes (reviewed by McKinnon and
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Pierotti, 2010).
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A variety of genetic mechanisms can underpin these types of complex polymorphisms
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involving multiple associated traits (Orteu and Jiggins, 2020). In many cases, such complex
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polymorphisms are controlled by ‘supergenes’ in which divergent alleles at several linked
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genes are maintained in strong linkage disequilibrium by reduced recombination. The most
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common mechanism for locally reduced recombination are inversions, which range from
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single inversions involving a small number of genes, to multiple nested inversions covering
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large genomic regions (Wang et al., 2013; Küpper et al., 2016; Tuttle et al., 2016).
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Nonetheless, other mechanisms for reducing recombination, such as centromeres or large
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genomic deletions, may also play a role. An alternative mechanism is that a single regulatory
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gene could act in the same way as a supergene, by controlling variation via multiple
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downstream effects (Thompson and Jiggins, 2014). While there are fewer instances in which
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multiple phenotypes seem to be controlled by a single gene, one potential example is the
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common wall lizard, where colour genes have pleiotropic effects on behavioural and
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reproductive traits (Andrade et al., 2019). Multiple mutations within a single gene can also
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lead to variation in multiple traits (Linnen et al., 2013).
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The wood tiger moth, Arctia plantaginis, has a complex polymorphism that has been well
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studied in an ecological context. Males show polymorphic aposematic hindwing colouration
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with discrete yellow, white or red hindwing colour morphs found at varying frequencies in
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different geographic locations. In Finland, for example, both yellow and white morphs can be
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found, with white morphs varying in frequency from 40 to 75% (Galarza et al., 2014). In
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Estonia, white morphs make up 97% of the population, while yellows morphs form a
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completely monomorphic population in Scotland (Hegna, Galarza and Mappes, 2015) (Figure
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1). Long term breeding studies of these moths have shown that male hindwing colour is a
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Mendelian trait controlled by a single locus with two alleles (Suomalainen, 1938; Nokelainen
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et al., 2022). White alleles (W) are dominant over the yellow (y). These colour genotypes
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also covary with behavioural and life-history traits, contributing to the maintenance of this
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polymorphism. Yellow males are subject to lower levels of predation in the wild (Nokelainen
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et al., 2012, 2014), while white males have a positive frequency-dependent mating advantage
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(Gordon et al., 2015). Yellow morphs have stronger chemical defences (Rojas et al., 2017),
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but show reduced flight activity compared to white males, although yellows may fly at more
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selective times, i.e. at peak female calling periods (Rojas, Gordon and Mappes, 2015). In
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summary, there is a trade-off between natural selection through predation and reproductive
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success, which contributes to the maintenance of this polymorphism (Rönkä et al., 2020).
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Despite the large body of research on A. plantaginis colour morphs, the genetic basis of this
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polymorphism is unknown. Here, we explore the genetic basis of male hindwing colour using
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wild populations and lab crosses to test whether the polymorphism is associated with large
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structural rearrangements controlling multiple phenotypic elements, or the result of a single
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gene mutation. We then narrow down the candidate genes using a differential gene
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expression analysis and identify the pigments producing yellow, white and black colouration
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on the wings of male A. plantaginis.
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Figure 1: (A) Sampling locations and frequencies of yellow and white Arctia plantaginis in Finland,
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Scotland and Estonia. (B) Males of the white and yellow colour morphs (Credit: Samuel Waldron).
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Results
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A narrow genomic region is associated with hindwing colour
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To investigate the genetic basis of male hindwing colouration in A. plantaginis, we carried
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out a quantitative trait locus (QTL) mapping analysis using crosses between heterozygous
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Wy males and homozygous yy females. We used RADseq data aligned to the yellow A.
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plantaginis reference genome from 172 male offspring (90 white and 82 yellow) from four
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families. The QTL analysis identified a single marker associated with male hindwing colour
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(Figure 2). This marker was found on scaffold YY_tarseq_206_arrow at position 9,887,968bp
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(95% confidence intervals 9,349,978-9,888,009bp) and had a LOD score of 32.8 (p<0.001).
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The significant marker explains around 75% of the phenotypic variation and, with one
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exception, yellow individuals all had a homozygous yy genotype at this marker. This is
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consistent with tight linkage of this marker to the causal SNP.
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To further narrow down this region, we ran a genome wide association study (GWAS) using
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whole genome sequences of males from four populations: Southern Finland (5 white, 5
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yellow) and Central Finland (10 w, 10 y), where male morphs are either white or yellow,
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Estonia (4 w), where males are mostly white, and Scotland (12 y), where all males are
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yellow. This identified a region of associated SNPs also on scaffold 206 (Figure 2). Two
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SNPs, 137bp apart (at positions 9,885,384 and 9,885,521), were significant above a strict
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Bonferroni corrected threshold. 162 SNPs were over the threshold of p<0.0001 and, of these,
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155 are within a 99kb region on scaffold 206 (9,833,387-9,932,264bp). The top SNPs are
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within 2.5kb from the top QTL marker, and the SNP at this marker has a p-value <0.0001.
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The 538Kb QTL interval contains 21 genes (Table S1) which were annotated with reference
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to D. melanogaster. Of these genes, four are part of the yellow gene family. The top two
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SNPs from the GWAS, and the top marker from the QTL, fall in a non-coding region
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upstream of the gene, yellow-e, and are also close to an additional yellow gene, yellow-g.
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Identifying structural variation in this region
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The trio binning method used by Yen et al. (2020) to assemble the A. plantaginis reference
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genome produced two reference sequences, one for a white allele and one for a yellow allele.
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We extracted the region containing the QTL interval from the yellow reference and aligned it
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against the white reference. The alignment showed a duplicated region approximately 117kb
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long on scaffold 419 of the white reference from around 6,941,000-7,058,000bp (Figure S1).
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The yellow-e gene and its flanking regions are within this sequence and are therefore
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duplicated in the white reference (Figure 2). One copy of the gene (named jg1310 in the W
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annotation) has 7 exons and is similar to the yellow-e gene in the yellow reference (99.7%
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identity in coding sequences). The second copy unique to the white scaffold (jg1308) has
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only the first 5 exons (81.8% identical to the gene in the yellow reference), possibly due to a
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stop codon mutation in the 5th exon. For clarity, we named this duplicated white-specific
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copy valkea, in reference to a Finnish term for ‘white’. While all white samples had
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consistent coverage of reads across the duplicated region, coverage was patchy in yellow
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samples, with many regions having no coverage in yellow samples (Figure 2). Those reads
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that map in the valkea region in yellow samples are likely to be mapping errors.
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To confirm that both of these gene copies are related to yellow-e, we compared them to
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yellow-e orthologs found in Bombyx mori, Heliconius melpomene and Drosophila
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melanogaster, along with other yellow genes from A. plantaginis and B. mori. Both of the
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tiger moth genes were most closely related to the H. melpomene yellow-e (Figure S2).
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Between valkea and yellow-e there is an additional gene which showed highest similarity to
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Drosophila yellow-g2 (when extracted from both the white and yellow references). This gene
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is not part of the duplicated sequence and is present as a single copy in both morphs.
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Coverage across yellow-g and yellow-e genome regions in wild samples is similar in both
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morphs (Figure 2).
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Figure 2: (A) QTL analysis reveals a 500kb region significant on scaffold 206, part of linkage group
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9. (B) A GWAS of wild samples showed SNPs associated with hindwing colour along the same
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scaffold. The dotted line shows the Bonferroni corrected significance threshold. (C) An insertion in
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the white reference sequence contains a copy of the yellow-e gene, which we named valkea, in
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addition to the yellow-e present in both white and yellow morphs. (D) Read depth across the candidate
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region in all Finnish white and yellow samples.
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Valkea is differentially expressed between morphs
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To pinpoint which of these candidate genes is associated with male wing polymorphism in A.
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plantaginis, we next performed gene expression analyses across several developmental
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stages. Based on knowledge of the expression patterns of yellow genes and other colour
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pattern genes such as optix (Reed et al., 2011) and cortex (Nadeau et al., 2016) in
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Lepidoptera, we hypothesised that changes in gene regulation that control the development of
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wing colour morphs in the tiger moth most likely occur during pupal development. Pupal
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development in the wood tiger moth lasts for approximately 8 days, and no colour is present
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in the wings until day 7, when the yellow pigment appears. A few hours later, black melanin
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pigmentation is deposited. We sampled two stages early in development when no colouration
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is present in the wings (72 hours post-pupation, and 5 day old pupae), and two stages later in
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development: the point when yellow appears in yy morphs (Pre-mel, 7 day old pupae) and the
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other after black melanin has also been deposited (Mel, 7-8 days old). Five individuals per
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genotype and stage were sampled.
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We analysed differential gene expression using the W reference genome, which contains the
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duplicated region that includes valkea. Developmental stage was the main factor explaining
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most of the genome-wide variation in gene expression between samples (Figure S3). Such a
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pattern would be expected as many genes are involved in development and thus are likely to
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be differentially expressed (DE) between developmental stages. No apparent clustering can
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be observed among samples of the same colour morph.
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We next compared gene expression between yy and WW individuals at each of the
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developmental stages. From the 22 genes identified in the GWAS and QTL analysis, only
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two, yellow-e and valkea, were differentially expressed in any of the comparisons. Valkea
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was overexpressed in white individuals in the pre-melanin stage with a Log Fold Change of
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10.32 and a p-value of 2.18e-06. As valkea is only fully present in the W genome, it is not
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expected to be expressed at all in the Y genome. Yellow-e was also overexpressed in white
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individuals with a Log Fold Change of 3.86 and adjusted p-value of 5.62e-06. In other
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developmental stages, neither valkea nor yellow-e showed differences in expression between
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morphs (Figure 3).
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The GWAS and QTL peaks of association are situated in scaffold 419 of the WW reference
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assembly, which in a WW linkage map forms a linkage group along with 6 more scaffolds
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(472, 487, 515, 531, 540 and 609). We found that 12 genes present in this linkage group were
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differentially expressed, including valkea and yellow-e (Table S2), and identified their
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orthologues in Drosophila melanogaster.
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We next conducted a genome-wide analysis of the RNA-seq data. Lowly expressed genes
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were filtered out with 10,920 genes retained. Overall, 99 genes were differentially expressed
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(FDR < 0.05) between the two morphs. Of these, 49 were upregulated in the yy morph, while
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the remaining 50 were upregulated in WW individuals. The earliest developmental stage, 72-
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hours, was the stage with the highest number of DE genes (n = 48), while the 5-day old stage
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had the fewest (n=7). One gene which encodes a C2H2 zinc finger transcription factor in D.
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melanogaster, ‘jg15945’, was over-expressed in yy in the first three stages (Figure S4).
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Figure 3: (A) RNA-seq of developing wings shows 99 genes that are differentially expressed between
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morphs. (B) Valkea is highly expressed in the white morph at the pre-melanin stage. Expression of
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valkea in yellow morphs is around 0.
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Yellow-e is not associated with colour
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We next used a tree topology approach to explore the relationships between alleles around the
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duplicated sites among the populations. For four populations (Finnish white and yellow,
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Estonian and Scottish populations), three topologies are possible; grouping populations by
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colour, by geography and one grouping neither by colour nor geography. At yellow-e, the
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highest weighting is towards the geography grouping, suggesting that this gene is not
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associated with the colour switch (Figure S5).
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Pigment analysis
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Since the yellow gene family, to which valkea is related, is known to be responsible for the
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production of melanin pigments, we further investigated the identity of the wing
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pigmentation. First, we ruled out the presence of several non-melanin pigment types in the
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hindwings, including pterins and carotenoids. Pterins are commonly found in insects and,
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along with purine derivatives, papiliochromes and flavonoids, are soluble in strong acids and
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bases or in organic solvents (Umebachi, 1975; Kayser, 1985; Shamim et al., 2014). We
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placed wing samples from each morph in NaOH overnight, then measured the absorbance of
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the supernatant using a spectrophotometer. We also left wings in methanol overnight before
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measuring the supernatant. The spectra did not show any peaks indicative of any pigment
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dissolved in the sample. Similarly, we found no evidence for carotenoid pigments after
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dissolving in a hexane:tert-butyl methyl ether solution (Figure S6). Wings did not fluoresce
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under UV light, providing further evidence for the lack of fluorescent pigments including
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pterins, flavonoids, flavins and papiliochromes (Umebachi, 1975; Kayser, 1985).
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Ommochromes are red and yellow pigments; high performance liquid chromatography
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(HPLC) ruled out the presence of these pigments on the moth wings, which we compared to
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data from ommochrome-containing Heliconius wings and a Xanthurenic acid standard
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(Figure S7).
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HPLC analysis showed peaks characteristic of pheomelanin (Figure S8). Pheomelanins
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produce red-brown colour in grasshoppers and wasps (Galván et al., 2015; Jorge García,
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Polidori and Nieves-Aldrey, 2016), and orange-red colours in ants and bumblebees (Hines et
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al., 2017; Polidori, Jorge and Ornosa, 2017). Insects generally have dopamine-derived
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pheomelanin and a breakdown product of this is 4-Amino-3-hydroxyphenylethylamine (4-
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AHPEA) (Barek et al., 2018). Yellow wings showed large peaks for 4-AHPEA. White wings
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had around 27% of the 4-AHPEA levels seen in yellow wings, and black sections of the
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wings had 16%. Hydrogen iodide hydrolysis of wings produced the isomer 3-AHPEA, which
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may come from 3-nitrotyramine originating from the decarboxylation of 3-nitrotyrosine.
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Reduction of 3-nitrotyrosine produces 3-AHP, another marker of pheomelanin (Wakamatsu,
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Ito and Rees, 2002).
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Analysis of the black portions of the wing found pyrrole-2,3-dicarboxylic acid (PDCA) and
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pyrrole-2,3,5-tricarboxylic acid (PTCA) (Figure S9). Both are components of eumelanin
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(Barek et al., 2018), suggesting that the black colouration seen in the wood tiger moth is
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predominantly eumelanin derived from dopamine. This is common in producing black
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colouration and providing structural components of the exoskeleton. In addition, dopamine is
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acylated to both N-β-alanyldopamine (NBAD) and N-acetyldopamine (NADA) sclerotins.
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NADA sclerotins are colourless and likely to be present on the white wings. This analysis of
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pigmentation is therefore consistent with a role for yellow family genes in regulating the
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colour polymorphism.
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Discussion
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Hindwing colouration of male Arctia plantaginis is polymorphic and these colour morphs
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vary in multiple behavioural and life-history traits, providing an example of a complex
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polymorphism. Here, we have shown that variation in male hindwing colour is associated
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with a narrow region in the genome, characterised by a duplicated sequence found only in
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white morphs and containing a gene from the yellow gene family. The white-specific copy,
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valkea, is highly expressed during pupal development, consistent with genetic dominance of
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the white allele. The presence of only one gene is in contrast to ‘classical’ supergenes which
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often explain the genetic architecture of complex polymorphisms (Thompson and Jiggins,
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2014) and involve chromosomal rearrangements containing multiple genes that allow the
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maintenance of co-adapted haplotypes (Joron et al., 2006; Lowry and Willis, 2010; Küpper et
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al., 2016; Funk et al., 2021).
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Although we did not find evidence for a supergene involving multiple loci, this morph-
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specific duplication still provides a region of reduced recombination between morphs. This is
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similar to the genetic architecture of the Primula supergene controlling heterostyly, which
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involves a large duplication containing five genes (Huu et al., 2020). The duplicated region is
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effectively hemizygous and cannot recombine except in homozygote genotypes, which could
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contribute to the maintenance of the complex polymorphism and the linkage of multiple
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traits. In polymorphic Papilio dardanus, one colour pattern morph is associated with a
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duplicated region, again providing physical constraints on recombination (Timmermans et
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al., 2014). Nonetheless, in the case of the wood tiger moth, it remains unclear how a single
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gene, such as valkea, can control the development of a broad array of phenotypic traits.
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One possible mechanism is that there is a regulatory element along the scaffold which is
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controlling colour via the valkea gene, but also regulating other genes to control different
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phenotypic traits. We found the most significant markers and SNPs located in a non-coding
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region close to the yellow genes, which likely contains a cis-regulatory element (CRE)
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controlling transcription of valkea. In cichlids, for example, a CRE at the gene encoding
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agouti-related peptide 2 controls variation in strip patterning in two closely related species
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(Kratochwil et al., 2018).
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Differential expression of other genes on the same chromosome controlled by the CRE could
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explain variation in covarying traits. The over-expression of another gene, possibly encoding
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a zinc transcription factor, in yellow individuals in the early pupal stages suggests that there
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is differential expression of unlinked genes as a result of the polymorphism, although since
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this gene is on a different chromosome to valkea it is unlikely to be directly controlled by the
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CRE. Another hypothesis is that somehow valkea itself regulates other genes. However,
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yellow family genes are not known to regulate transcription of other genes, unlike for
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example doublesex, a transcription factor that undergoes alternative splicing and female
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mimetic wing pattern polymorphism in Papilio polytes (Kunte et al., 2014; Nishikawa et al.,
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2015).
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The yellow family genes are highly conserved throughout insects (Ferguson et al., 2011).
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They have been widely linked to colouration (Wittkopp, Vaccaro and Carroll, 2002;
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Miyazaki et al., 2014; Zhang et al., 2017; Zhang, Mazo-Vargas and Reed, 2017), as well as
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behaviour, sex-specific phenotypes and reproductive maturation (Wilson et al., 1976). These
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genes share a common origin with the major royal jelly protein (MRJP) genes (Drapeau et
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al., 2006) which are crucial in caste development in honeybees. Like the MRJP genes, yellow
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genes in honeybees have diverse spatial and temporal expression patterns. As our focus in A.
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plantaginis has been on wing tissue, we are missing expression of genes in other tissues that
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could be linked to other traits. Thus, it is not impossible to imagine that a yellow gene could
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have a similar function to a MRJP in regulating the development of a complex phenotype.
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Recent work with Bicyclus anynana showed that yellow functions as a repressor of male
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courtship (Connahs et al., 2022). On the other hand, sex specific behavioural phenotypes of
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yellow mutants in Drosophila were found to be due to pigmentation effects (Massey et al.,
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2019), so more evidence is needed to suggest a functional role for yellow genes outside of
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pigmentation.
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The duplication of yellow-e and surrounding regions in the white morphs suggests that the
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yellow morph is the ancestral form. Valkea could have evolved in a stepwise fashion, first as
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13
a tandem duplication then with a stop codon mutation altering the gene structure. Gene
293
duplications can facilitate adaptation and, in some examples, lead to polymorphism. Although
294
both valkea and yellow-e are overexpressed in the white morph, the topology weighting
295
suggests that yellow-e is not closely associated with the colour change. Valkea likely
296
represents an example of neofunctionalization, where the duplicated gene gains a different
297
function to the original gene copy.
298
Contrary to previous work that attributed red and yellow colours to pterins in another tiger
299
moth species (Gawne and Nijhout, 2019), we found high levels of 4-AHPEA in the yellow
300
wings confirming the presence of pheomelanins. These pigments have been widely associated
301
with red and yellow colours in mammals (e.g. Mcgraw and Wakamatsu, 2004), but only
302
relatively recently described in insects and likely to be more widespread than previously
303
thought. Yellow colours can also be produced by NBAD sclerotins which are sclerotizing
304
precursor molecules made from dopamine and these have an important role in the
305
sclerotization pathway for hardening the insect cuticle (Andersen, 2007; Barek, Evans and
306
Sugumaran, 2017) before becoming involved in melanisation (Barek et al., 2018). Thus, we
307
suggest that the yellow colour arises partly from the NBAD sclerotins and partly from the
308
presence of pheomelanin pigments, which has been proposed in other Lepidoptera (Matsuoka
309
and Monteiro, 2018). While some 4-AHPEA also occurred in white wings, this may be due to
310
its role in production of cross-linking cuticular proteins and chitin during sclerotization
311
(Sugumaran, 2010). Upregulation of genes on the white allele could be acting as a repressor
312
of the generation of yellow colour. We suspect that yellow family genes play multiple roles
313
within the melanin production pathway. In the wood tiger moth, yellow affects the conversion
314
of DOPA into black dopamine melanin (Galarza, 2021). Yellow-e in particular has been
315
linked to larval colouration in Bombyx mori (Ito et al., 2010) and adult colour in beetles
316
(Wang et al., 2022), while another gene, yellow-f, has a role in eumelanin production (Barek
317
et al., 2018).
318
In summary, we identified a structural variant containing a previously undescribed gene,
319
valkea, which is only present in white morphs of A. plantaginis. While functional studies are
320
needed to determine the exact function of this locus, the presence of a regulatory element
321
controlling wing colour and other traits via multiple downstream effects could explain how
322
multiple traits are linked to wing colouration. This complex polymorphism allows multiple
323
beneficial phenotypes to be inherited together, whereas recombination would separate
324
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14
multiple loci leading to maladapted individuals. Our results provide the basis for further
325
exploration of the genetic basis of covarying behavioural and life-history traits, and offer an
326
intriguing example of how complex polymorphisms are maintained.
327
Methods
328
Sampling
329
Homozygous lines of white (WW) and yellow (yy) Arctia plantaginis moths were created
330
from Finnish populations at the University of Jyväskylä, Finland. Larvae were fed with wild
331
dandelion (Taraxacum sp.) and reared under natural light conditions, with an average day
332
temperature of 25oC and night temperature between 15-20oC. For the crosses, a heterozygous
333
male, created from crossing a heterozygous male with a homozygous yy female, was
334
backcrossed with a yy female. This was repeated to obtain four families totalling 172
335
offspring and 8 parents (Table S3). Samples from wild populations were caught in Southern
336
Finland (n=10) and Central Finland (n=20), where male morphs are either white or yellow,
337
Estonia (n=4), where males are mostly white, and Scotland (n=4), where males are yellow
338
(Table S4). In addition, we included 8 samples which are F1 offspring of wild Scottish
339
samples. Forty pupae with known genotypes from lab populations (20 WW and 20 yy) were
340
used for the RNA extractions.
341
DNA extraction and sequencing
342
For the lab crosses, DNA was extracted from two legs crushed with sterilised PVC pestles
343
using a Qiagen DNeasy Blood & Tissue kit, following the manufacturer’s instructions.
344
Library preparation and GBS sequencing were performed by BGI Genomics on an Illumina
345
HiSeq X Ten. For the wild samples, DNA was extracted from the thoraces also with a Qiagen
346
kit. Library preparation and sequencing were performed by Novogene (Hong Kong, China).
347
150bp paired-end reads were sequenced on an Illumina NovaSeq 6000 platform.
348
Linkage mapping analysis
349
FASTQ reads were mapped using bowtie v2.3.2 (Langmead and Salzberg, 2012) to the
350
yellow Arctia plantaginis scaffold-level genome assembly (Yen et al., 2020). BAM files
351
were sorted and indexed using SAMtools v.1.9 (Li et al., 2009) and duplicates removed using
352
PicardTools MarkDuplicates (broadinstitute.github.io/picard). Twelve samples which had
353
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15
aligned <30% were removed. Reads of the remaining samples had an average alignment of
354
94%. SNPs were called using SAMtools mpileup with minimum mapping quality set to 20
355
and bcftools call function. Lep-MAP3 (Rastas, 2017) was used for linkage map construction
356
and we ran the following modules: ParentCall2 which called 105,622 markers, Filtering2,
357
SeparateChromosomes2 with lodLimit=5 and sizeLimit=100, JoinSingles2All and
358
OrderMarkers2 with recombination2=0 to denote the lack of female recombination.
359
Genotypes were phased using the map2genotypes.awk script included with Lep-MAP3.
360
Markers were named based on the genomic positions of the SNPs in the reference genome
361
and the map.awk script, and this was used to further order the markers within the linkage
362
groups. This resolved 30 linkage groups. Although we expect that there are 31 chromosomes
363
in the moth genome, we suspect that the sex chromosome is missing in this assembly as the
364
yy individual used in the genome assembly was female (Yen et al., 2020). A small number of
365
markers which caused long gaps at the beginning or end of linkage groups were manually
366
removed, leaving the final map 948.7cM long with 19,803 markers. Markers were well
367
distributed so we began the first analyses with this map. A linkage map was also assembled
368
using sequences aligned to the white reference and this separated into 31 linkage groups.
369
The QTL analysis was carried out in R/qtl (Broman et al., 2003). Genotype probabilities were
370
calculated before running a genome scan using the scanone function with the Haley-Knott
371
method and binary model parameters, and including family as an additive covariate. The
372
phenotype was labelled as either 0 (Wy) or 1 (yy). We ran 5000 permutations to determine
373
the significance level for the QTL LOD scores. The bayesint function calculated the 95%
374
Bayesian confidence intervals around the significant marker.
375
Analysis of whole genome sequences
376
FASTQ reads were mapped to the yellow A. plantaginis genome assembly (Yen et al., 2020)
377
using BWA-MEM v7.17 (Li, 2013). As before, BAM files were sorted and indexed, and
378
duplicates were removed. Genotyping and variant calling was carried out with the Genome
379
Analysis Toolkit (GATK) (McKenna et al., 2010). Variants were called using
380
HaplotypeCaller (v.3.7) in GVCF mode, then gVCFs combined with GenomicsDBImport
381
(v.4.0). Joint genotyping was run with GenotypeGVCFs, set with a heterozygosity of 0.01,
382
and SNPs were called using SelectVariants. Finally, the set of 20,787,772 raw SNPs were
383
filtered using VariantFiltration and thresholds: quality by depth (QD > 2.0), root mean square
384
mapping quality (MQ > 50.0), mapping quality rank sum test (MQRankSum > −12.5), read
385
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16
position rank sum test (ReadPosRankSum > −8.0), Fisher strand bias (FS < 60.0), and strand
386
odds ratio (SOR < 3.0). A set of 5,227,288 SNPs passed the filtering.
387
We carried out a Genome Wide Association Study (GWAS) using the R package GenABEL
388
v.1.8 (Aulchenko et al., 2007). The set of filtered SNPs were converted to BED format with
389
PLINK2, keeping only biallelic SNPs (cog-genomics.org/plink/2.0/). Sites which were not in
390
Hardy-Weinberg equilibrium (p<0.01), or had a call rate of <0.5, were excluded. Following
391
this, 381,266 sites were retained across 40 individuals (out of 57). To account for population
392
stratification, we performed multidimensional scaling on kinship and identity-by-state (IBS)
393
information estimated from the data, and included this as a covariate in the association test.
394
Significance levels were calculated using Bonferroni corrected thresholds to account for
395
multiple testing. Central and Southern Finnish populations were pooled for this analysis,
396
based on a previous principal component analysis of these samples (Yen et al., 2020).
397
In Yen et al. (2020), many of these samples were processed in the same way but aligned to
398
the white genome assembly (based on a white individual). We used the VCF file from this
399
study for a Twisst analysis (Martin and Van Belleghem, 2017) which included samples from
400
the Finnish, Estonian and Scottish populations. A minor allele frequency of 3 was applied to
401
the VCF with VCFtools, and this was phased using Beagle v5.1 (Browning, Zhou and
402
Browning, 2018). We generated neighbour joining trees with a window size of 50 using
403
PhyML and ran Twisst using scripts from github.com/simonhmartin/twisst. Read depth of the
404
W-mapped samples was calculated in 1kb windows across the candidate region using
405
BEDtools (v.2.20.1) multicov (Quinlan and Hall, 2010).
406
For analysis of structural variants, sequences from the white and yellow genome assemblies
407
were aligned using MAFFT v7.450 (Katoh and Standley, 2013) and viewed with Geneious.
408
Our focal sequence, scaffold 419 in the white genome, is the reverse complement of scaffold
409
206 in the yellow genome.
410
Identification of candidate genes and tree construction
411
To identify candidate genes in the QTL interval and GWAS region, we ran a protein
412
BLASTP v.2.4.0 search to identify Heliconius melpomene (Hmel2.5) proteins homologous to
413
predicted A. plantaginis proteins in the region from the genome annotation. Informative gene
414
names were obtained by performing a BLASTP search with the H. melpomene proteins
415
against all Drosophila melanogaster proteins in FlyBase v.FB2020_01 (flybase.org/blast).
416
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17
For the yellow gene tree, we used Lepbase (Challi et al., 2016) to search for yellow genes in
417
Bombyx mori (ASM15162v1). We identified yellow-e in H. melpomene by searching for
418
major royal jelly proteins, then comparing protein sequences of these against Drosophila
419
proteins in Flybase. The sequence for Dmel yellow-e was downloaded from Flybase. To make
420
the tree, coding sequences of all genes were aligned in Geneious using MAFFT v7.450
421
(Katoh and Standley, 2013), then the tree was constructed with PhyML using 10 bootstraps
422
(Guindon et al., 2010).
423
Differential gene expression
424
We dissected the wings out of the pupae in Cambridge, UK. Pupae and larvae were sent to
425
Cambridge from Jyväskylä and were kept between 22 and 30˚C. Pupae were sexed and only
426
males were used. Dissections were made at 4 different stages: 72 hours after pupation (72h),
427
5 days after pupation (5d; counting 0-24 first hours after pupation as day 1), pre melanin
428
deposition (Pre-mel) and post melanin deposition (Mel). We sampled 5 individuals per stage
429
and genotype. Hindwings and forewings were stored separately in RNA-later (Sigma-
430
Aldrich) at 4 ˚C for 2 weeks and later transferred to -20 ˚C, while the rest of the body was
431
stored in pure ethanol.
432
Total RNA was extracted from hindwing tissue using a standard hybrid protocol. First, we
433
transferred the wing tissue into Trizol Reagent (Invitrogen) and homogenised it using dounce
434
tissue grinders (Sigma-Aldrich). Then, we performed a chloroform phase extraction, followed
435
by DNase treatment (Ambion) for 30 mins at 37˚C. We measured the concentration of total
436
RNA using Qubit Fluorometric Quantitation (Thermofisher) and performed a quality check
437
using an Agilent 4200 TapeStation (Agilent). The extracted total RNA was stored at -20˚C
438
before being sent to Novogene UK for sequencing.
439
We performed quality control and low-quality base and adapter trimming of the sequence
440
data using TrimGalore! We then mapped the trimmed reads to the two A. plantaginis
441
genomes using STAR. We performed a second round of mapping (2pass) including as input
442
the output splice junctions from the first round. The A. plantaginis genome annotations WW
443
and YY were included in each round of mapping respectively. We then used FeatureCounts
444
to count the mapped reads. Finally, we used DESeq2 to analyse the counts and perform the
445
DE analysis.
446
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18
To identify the gene or genes controlling the development of wing colour in A. plantaginis,
447
we performed a genome wide differential expression analysis using limma-voom (Ritchie et
448
al., 2015). First, we defined a categorical variable, ‘GenStage’, with 8 levels containing the
449
genotype and stage information of every individual sample (e.g. YY72h, WW72h, YY5days,
450
etc.). Then, we built the design matrix fitting a model with GenStage as the only fixed effect
451
factor contributing to the variance in gene expression and included family as a random effect
452
factor (gene expression ~ 0 + GenStage + (1|Family)). We then filtered lowly expressed
453
genes, which resulted in a reduction of the number of tested genes from 17,615 to 11,330
454
genes in the Y-mapped analysis and 17,930 to 10,920 in the W-mapped one. Finally, the list
455
of genes that are differentially expressed in each stage were extracted using the Benjamini-
456
Hochberg procedure to correct for multiple testing.
457
Orthology assignment
458
To infer genome wide orthology between A. plantaginis and D. melanogaster, we used
459
OrthoFinder (v2.5.4) (Emms and Kelly, 2019). We used proteomes from 6 Lepidoptera
460
species, Plutela xylostella (GCA_905116875_2), Bombyx mori (GCF_014905235_1),
461
Spodoptera frugiperda (GCF_011064685_1), Parnassius apollo (GCA_907164705_1),
462
Pieris macdunnoughi (GCA_905332375_1) amd Pararge aegeria (GCF_905163445_1) and
463
Drosophila melanogaster (GCF_000001215_4). We ran the primary_transcript.py utility
464
from OrthoFinder to extract only one transcript per protein, and then ran OrthoFinder with
465
default settings.
466
Pigment analysis
467
Solubility and fluorescence tests
468
Five hindwings from each morph were placed in two separate solvents (0.1M NaOH and 90%
469
MeOH) and left overnight. The supernatant was analysed with an Agilent Cary 8454 UV-
470
Visible spectrophotometer and the spectra compared to known spectra for various pigments.
471
A UV lamp (Philips TL8W/08 F8T5/BLB) used to test for fluorescence on the wings. The
472
presence of carotenoids was tested by placing wings into 1ml of pyridine and leaving at 95oC
473
for 4 hours (McGraw et al., 2002). To these we added 1ml of 1:1 hexane:tert-butyl methyl
474
ether and 2ml of water before shaking and leaving overnight. Again, the supernatant was
475
measured with the spectrometer.
476
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19
High-Performance Liquid Chromatography (HPLC) to test to eumelanin and ommochrome
477
pigments
478
To determine the type of melanin producing the black colour on the wings, we cut out
479
approximately 5mg of the black sections of the wings, from both females and males.
480
Eumelanin analysis was carried out according to (Borges et al., 2001). Each sample was
481
added to a tube containing 820μl 0.5 M NaOH, 80μl 3% H2O2 and an internal standard
482
(48nmol phthalic acid) and heated in a boiling water bath for 20 minutes. Once cool, 20μl of
483
10% Na2SO3 and 250μl of 6M HCl was added. Samples were then extracted twice with 7ml
484
of ethyl acetate. Ethyl acetate was dried at 45 °C under a stream of nitrogen. The residue was
485
dissolved into 0.5ml of 0.1% formic acid.
486
We carried out high-performance liquid chromatography on an Agilent 1100 HPLC. 20μl of
487
the sample was injected into a Waters Atlantis T3, 100 x 3.0mm i.d. analytical column
488
(Waters, Milford, MA, USA). The column was set to 25°C and analytes were detected at
489
wavelength 280nm. The HPLC mobile phase consisted of two eluents: UHQ-water/MeOH
490
(98/2; v/v) with 0.1% formic acid and UHQ-water/MeOH (40/60; v/v) with 0.1% formic acid.
491
Flow rate was 0.4 ml/min and the used gradient started with 100% of eluent A and ramped
492
evenly from time 0 to 15 minutes to 40:60 (A:B; v/v), held at 40:60 for 6 minutes and ramped
493
evenly back to initial eluent composition (100% A) over 5 minutes. We compared
494
chromatograms obtained from the samples to the chromatograms obtained from synthetic
495
melanin, ink from sepia officinalis and black human hair.
496
HPLC was also applied to observe the possible presence of ommochrome pigments. Injection
497
volume was 10µl and for the separation we used the same Waters Atlantis T3 column (100 x
498
3.0 mm i.d.) set to 30°C. Solvent A was UHQ-water and B was acetonitrile (ACN), both
499
containing 0.1% formic acid. Flow rate was 0.4ml/min and the used gradient was as follows:
500
Initial flow ratio was 98/2 water/ACN (v/v) ramping then evenly from time 1 to 15 min to
501
30:70 water:ACN (v/v), held for 1.5 min and then ramped evenly back to initial eluent
502
composition over 0.5 minutes. The column was stabilised for 7 minutes before a new run.
503
Pheomelanin analysis
504
Samples were analysed for pheomelanin content according to the method of Kolb et al.
505
(1997) with modifications. A 2mg sample was placed in a screw-capped tube with 100μl
506
water, 500μl ~55-58% hydrogen iodide (HI), and 20μl 50% hypophosphorous acid (H3PO2).
507
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20
Samples were capped tightly and hydrolysed for 20 hours at 130°. After cooling, samples
508
were evaporated under nitrogen flow, then dissolved in 1ml of 0.1M HCl and purified with
509
solid phase extraction. Strata SCX cartridges were preconditioned with 2ml of methanol, 3ml
510
of water and 1ml of 0.1M HCl. Sample was then applied to the cartridge, washed with 1ml of
511
0.1M HCl, and finally eluted with 1ml of methanol (MeOH): 0.5M ammonium acetate
512
(NH4CH3CO2) (20:80 v/v).
513
Hydrogen Iodide hydrolysis products were determined by a Dionex high-performance liquid
514
chromatograph equipped with pulsed amperometric detection (HPLC/PAD). A Phenomenex
515
Kinetex C18 column (150 x 4.6 mm i.d.; 5µm particle size) with a gradient elution (Table S5)
516
at a flow rate of 0.9 ml min- 1 with the eluents: (A) sodium citrate buffer (Hines et al., 2017)
517
in ultra-high-quality water (internal resistance ≥ 18.2 MΩ cm; Milli-Q Plus; Millipore,
518
Bedford, MA, USA) and (B) methanol were used for the separation. Dionex ED-50 pulsed
519
amperometric detector (Dionex, Sunnyvale, CA, USA) equipped with a disposable working
520
electrode by using a Dionex waveform A with potentials presented in Table S6 was used for
521
detection. 4-AHP, 3-AHPEA and 4-AHPEA standards used in calibration were kindly offered
522
from the laboratory of Emeritus Professor Wakamatsu and the preparation method is
523
described in (Wakamatsu et al., 2014).
524
Acknowledgements
525
We thank Kaisa Suisto and the greenhouse staff for insect rearing, and Elisa Salmivirta and
526
Sari Viinikainen for lab assistance. Thanks to Emeritus Prof. Shosuke Ito for providing
527
pheomelanin standards and to Bodo Wilts for advice on the pigment analyses.
528
This work was supported by Academy of Finland grants to MB (#343356) and JM (projects
529
345091 and 328474).
530
Data availability
531
All the scripts used for mapping and DE analysis will be uploaded to an online repository.
532
Scripts and data for the QTL and GWAS analyses can currently be found at
533
github.com/mnbrien/Aplantaginis-male-colour. Raw sequencing data of wild samples has
534
previously been deposited in ENA, study accession No. PRJEB36595.
535
536
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21
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... The wood tiger moth (Arctia plantaginis) represents a compelling study species to investigate how different selective pressures can act on a single color locus and maintain within-population trait variation. In this system, male hindwing coloration is determined by a simple genetic basis (Suomalainen 1938;Nokelainen et al., 2022b;Brien et al., 2022): a one locustwo allele polymorphism (dominant W allele and recessive y allele), which translates into white (genotype: WW, Wy) and yellow (genotype: yy) males. Because this is an aposematic moth species, the color trait is not only used for intraspecific communication (i.e., sexual selection) but also to advertise their unpalatability to predators (i.e., interspecific communication). ...
... The male coloration in the wood tiger moth is likely regulated by a yellow-family gene (Brien et al., 2022), which is conserved across insects (Ferguson et al., 2011) and has wellknown functions in the melanin production pathway (Wittkopp et al., 2002). Yellow genes have also been shown to have pleiotropic effects on life-history and behavioral traits (Bastock, 1956;Massey et al., 2019;Connahs et al., 2021). ...
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The involvement of yellow genes y-b, y-c, y-e, and y-h in cuticle tanning has poorly been clarified. In the present paper, six putative yellow (y-y, y-b, y-c, y-e y-f, and y-h) genes were identified in Henosepilachna vigintioctopunctata. Hvy-b, Hvy-c, Hvy-e, and Hvy-h were abundantly transcribed at early larval and late pupal stages, especially in the epidermis. Accordingly, RNA interference (RNAi) experiments were performed by an injection of dsy-b, dsy-c, dsy-e, or dsy-h into the second instar larvae and 1-day-old pupae. The head capsule, scoli and strumae, and legs in the fourth-instar larvae became blacker; the blackish spots in the pupae were darkened and widened after RNAi of Hvy-b, compared with those of dsegfp-treated controls. Depletion of Hvy-b at the 1-day-old pupal stage expanded two pair of black markings on the sternum of the metathorax, and darkened the black patched on the sterna of the abdomen segments I-VI in the resultant adults. Depletion of Hvy-e caused darker pigmented adult body and elytral cuticles than those of dsegfp-introduced controls. However, there was no obvious difference in pigmentation of the black markings. Hvy-h-deficient larvae displayed dark yellow body color, whereas the body color of the dsegfp-injected control was pale yellow. There was no obvious difference in coloration of larval specific-black markings or pupal cuticle between dsHvy-h- and dsegfp-treated animals. Moreover, silence of Hvy-c at the second instar larval stage lightened black markings in the resulting larvae and pupae, but had no influence on pale yellow body color. Our results demonstrated their different roles of the four yellow genes during body pigmentation: HvY-b and HvY-c, respectively, inhibit and facilitate the coloration within dark markings, whereas HvY-e and HvY-h, respectively, repress the pigmentation in adult and larval body cuticles outside the black patches in H. vigintioctopunctata.
Article
The carotenoid-pigmented bill of Zebra Finches (Taeniopygia guttata) has received much recent attention as a sexually selected signal of quality, but these birds also display several sexually dichromatic plumage traits, including rust-colored cheek patches, a black breast band, and brown flanks. Black, brown, and earth-toned features in animals are thought to be produced by melanin pigments, but few studies have identified the melanin content of such colors in bird feathers. We used a series of biochemical techniques to investigate the pigmentary basis of these plumage colors in male Zebra Finches. All three feather traits contained melanin pigments, but varied in the amounts of the two basic forms of melanin (eumelanin and phaeomelanin). Black breast feathers contained predominantly eumelanin, whereas cheek and flank feathers contained extraordinarily high concentrations of phaeomelanin. Conventional methods of carotenoid analysis detected no carotenoids in either the cheek or flank feathers. Coloración Basada en Melaninas en las Plumas Ornamentales de los Machos de Taeniopygia guttata Resumen. El pico pigmentado con carotenoides de Taeniopygia guttata ha sido destacado recientemente como una señal de calidad seleccionada sexualmente, pero estas aves también presentan varios caracteres de plumaje sexualmente dicromáticos, incluyendo parches en las mejillas de color óxido, una faja pectoral negra y flancos de color café. Se cree que las tonalidades negras, cafés y color tierra son producidas por melaninas en los animales, pero existen pocos estudios que hayan identificado el contenido de melanina de dichos colores en las plumas de las aves. En este estudio empleamos una serie de técnicas bioquímicas para investigar la base pigmentaria de estos colores del plumaje en machos de T. guttata. Los tres caracteres de las plumas contaron con pigmentos melánicos, pero variaron en las cantidades de las dos formas básicas de melanina (eumelanina y feomelanina). Las plumas negras del pecho presentaron principalmente eumelanina, mientras que las de las mejillas y los flancos presentaron concentraciones extraordinariamente altas de feomelanina. Los métodos tradicionales de análisis de carotenoides no detectaron este tipo de pigmentos en las plumas de las mejillas y los flancos.
Article
The nymphalid groundplan allows for the identification of homologous characters across species, making this archetype an invaluable tool for research on the development and evolutionary diversification of butterfly wing patterns. However, whether the groundplan applies to moths remains unknown. To better understand the domain of applicability of the nymphalid groundplan, we attempted to identify homologous elements in the dorsal forewing patterns of the hyper-variable arctiid moth Utetheisa ornatrix. Using trait correlation analyses, we located homologues such as the basal and central symmetry systems. However, we also identified two unique characters that are not found in modern renditions of the groundplan, which suggests that comparative research on arctiid wing patterns might require a return to earlier versions of the archetype that were not specifically tailored to research on butterflies. We also analysed the chemical basis underlying the conspicuous coloration of U. ornatrix, and found that non-melanic portions of the colour pattern are attributable to three pterin pigments: drosopterin, erythropterin and leucopterin. Given its pigmentary and morphological simplicity, the forewing pattern of this species could represent a primitive character state. If this is the case, developmental studies of U. ornatrix wing patterning could yield novel insights into pattern evolution in the Lepidoptera. Keywords: arctiid moths-nymphalid groundplan-pterin pigments-Utetheisa ornatrix-wing patterning.