Symbionts that distort their host's sex ratio by favouring the production and survival of females are common in arthropods. Their presence produces intense Fisherian selection to return the sex ratio to parity, typified by the rapid spread of host ‘suppressor’ loci that restore male survival/development. In this study, we investigated the genomic impact of a selective event of this kind in the butterfly Hypolimnas bolina. Through linkage mapping, we first identified a genomic region that was necessary for males to survive Wolbachia-induced male-killing. We then investigated the genomic impact of the rapid spread of suppression, which converted the Samoan population of this butterfly from a 100∶1 female-biased sex ratio in 2001 to a 1∶1 sex ratio by 2006. Models of this process revealed the potential for a chromosome-wide effect. To measure the impact of this episode of selection directly, the pattern of genetic variation before and after the spread of suppression was compared. Changes in allele frequencies were observed over a 25 cM region surrounding the suppressor locus, with a reduction in overall diversity observed at loci that co-segregate with the suppressor. These changes exceeded those expected from drift and occurred alongside the generation of linkage disequilibrium. The presence of novel allelic variants in 2006 suggests that the suppressor was likely to have been introduced via immigration rather than through de novo mutation. In addition, further sampling in 2010 indicated that many of the introduced variants were lost or had declined in frequency since 2006. We hypothesize that this loss may have resulted from a period of purifying selection, removing deleterious material that introgressed during the initial sweep. Our observations of the impact of suppression of sex ratio distorting activity reveal a very wide genomic imprint, reflecting its status as one of the strongest selective forces in nature.

The rise of dark (melanic) forms of many species of moth in heavily coal-polluted areas of nineteenth- and twentieth-century Britain, and their post-1970s fall, point to a common selective pressure (camouflage against bird predators) acting at the community level. The extent to which this convergent phenotypic response relied on similar genetic and developmental mechanisms is unknown. We examine this problem by testing the hypothesis that the locus controlling melanism in Phigalia pilosaria and Odontopera bidentata, two species of geometrid moth that showed strong associations between melanism and coal pollution, is the same as that controlling melanism in Biston betularia, previously identified as the gene cortex. Comparative linkage mapping using family material supports the hypothesis for both species, indicating a deeply conserved developmental mechanism for melanism involving cortex. However, in contrast to the strong selective sweep signature seen in British B. betularia, no significant association was detected between cortex-region markers and melanic morphs in wild-caught samples of P. pilosaria and O. bidentata, implying much older, or diverse, origins of melanic morph alleles in these latter species.

Background Sex ratio distorting agents (maternally inherited symbionts and meiotically-driving sex chromosomes) are common in insects. When these agents rise to high frequencies they create strong population sex ratio bias and selection then favours mutations that act to restore the rare sex. Despite this strong selection pressure, the evolution of mutations that suppress sex ratio distorting elements appears to be constrained in many cases, where sex-biased populations persist for many generations. This scenario has been observed in the butterfly Hypolimnas bolina, where Wolbachia-mediated male killing endured for 800–1,000 generations across multiple populations before the evolution of suppression. Here we test the hypothesis that this evolutionary lag is the result of suppression being a multilocus trait requiring multiple mutations. Methods We developed genetic markers, based on conservation of synteny, for each H. bolina chromosome and verified coverage using recombinational mapping. We then used a Wolbachia-infected mapping family to assess each chromosome for the presence of loci required for male survival, as determined by the presence of markers in all surviving sons. Results Informative markers were obtained for each of the 31 chromosomes in H. bolina. The only marker that cosegregated with suppression was located on chromosome 25. A genomic region necessary for suppression has previously been located on this chromosome. We therefore conclude that a single genomic region of the H. bolina genome is necessary for male-killing suppression. Discussion The evolutionary lag observed in our system is not caused by a need for changes at multiple genomic locations. The findings favour hypotheses in which either multiple mutations are required within a single genomic region, or the suppressor mutation is a singularly rare event.

22Symbionts that distort their host's sex ratio by favouring the production and survival of females are 23 common in arthropods. Their presence produces intense Fisherian selection to return the sex ratio to 24 parity, typified by the rapid spread of host 'suppressor' loci that restore male survival/development. 25 In this study, we investigated the genomic impact of a selective event of this kind in the butterfly 26Hypolimnas bolina. Through linkage mapping we first identified a genomic region that was 27 necessary for males to survive Wolbachia-induced killing. We then investigated the genomic impact 28 of the rapid spread of suppression that converted the Samoan population of this butterfly from a 29 100:1 female-biased sex ratio in 2001, to a 1:1 sex ratio by 2006. Models of this process revealed 30 the potential for a chromosome-wide selective sweep. To measure the impact directly, the pattern of 31 genetic variation before and after the episode of selection was compared. Significant changes in 32 allele frequencies were observed over a 25cM region surrounding the suppressor locus, alongside 33 generation of linkage disequilibrium. The presence of novel allelic variants in 2006 suggests that 34 the suppressor was introduced via immigration rather than through de novo mutation. In addition, 35 further sampling in 2010 indicated that many of the introduced variants were lost or had reduced in 36 frequency since 2006. We hypothesise that this loss may have resulted from a period of purifying 37 selection -removing deleterious material that introgressed during the initial sweep. Our 38 observations of the impact of suppression of sex ratio distorting activity reveal an extraordinarily 39 wide genomic imprint, reflecting its status as one of the strongest selective forces in nature. 40 41 42 ! 3! Author summary 43The sex ratio produced by an individual can be an evolutionary battleground. In many arthropod 44 species, maternally inherited microbes selectively kill male hosts, and the host may then in turn 45 evolve strategies to restore the production or survival of males. When males are rare, the intensity 46 of selection on the host may be extreme. We recently observed one such episode, in which the 47 population sex ratio of the butterfly Hypolimnas bolina shifted from 100 females per male to near 48 parity through evolution of a suppressor gene. In our current study, we investigate the hypothesis 49 that the strength of selection was so strong in this case that the genomic impact would go well 50 beyond the suppressor gene itself. Following location of the suppressor within the genome of H. 51 bolina, we examined changes in genetic variation at sites on the same chromosome as the 52 suppressor. We show that an extraordinarily wide area of the genome was affected by the spread of 53 the suppressor. Our data also suggest that the selection may have been sufficiently strong to 54 introduce deleterious material to the population that was later purged by selection. 55 56 ! 4! ! 6! through immigration, and that...

Moths and butterflies (Lepidoptera) have a heterogametic sex chromosome system with females carrying ZW chromosomes and males ZZ. The lack of W chromosomes in early diverging lepidopteran lineages has led to the suggestion of an ancestral Z0 system in this clade and a B chromosome origin of the W. This contrasts with the canonical model of W chromosome evolution in which the W would have originated from the same homologous autosomal pair as the Z chromosome. Despite the distinct models proposed, the rapid evolution of the W chromosome has hindered the elucidation of its origin. Here, we present high-quality, chromosome-level genome assemblies of two Hypolimnas species (Hypolimnas bolina and Hypolimnas misippus) and use the H. misippus assembly to explore the evolution of W chromosomes in butterflies and moths. We show that in H. misippus the W chromosome has higher similarity to the Z chromosome than any other chromosome, which could suggest a possible origin from the same homologous autosome pair as the Z chromosome. However, using genome assemblies of closely related species (ditrysian lineages) containing assembled W chromosomes, we present contrasting evidence suggesting that the W chromosome might have evolved from a B chromosome instead. Crucially, by using a synteny analysis to infer homology, we show that W chromosomes are likely to share a common evolutionary origin in Lepidoptera. This study highlights the difficulty of studying the evolution of W chromosomes and contributes to better understanding its evolutionary origins.