Sex-limited polymorphism is widespread in animals, including a variety of human traits and diseases, yet we lack a general functional understanding of sex-limited polymorphism in any organism. Supergene mimicry in the swallowtail butterfly Papilio polytes stands out as a particularly striking example of sex-limited polymorphism and one that is amenable to functional characterization. While much theoretical work has explored the evolutionary dynamics of supergene mimicry, its molecular and developmental basis is virtually unexplored. We propose to investigate the functional basis of supergene mimicry in P. polytes by integrating genomics, functional genetics, molecular and developmental biology, and behavior, providing the single most comprehensive investigation of its kind. In so doing, this work will greatly expand the known role of the sexual determination pathway, and generate general insights into molecular and cellular causes of sexual differentiation, dimorphism, and sex-limited polymorphism. Specifically, we will: Aim 1: Identify the genetic basis of supergene mimicry, both in terms of the specific gene and the mutational origin of alternative mimicry alleles, using a comprehensive association mapping approach combined with structural variant detection. We will use a genomic enrichment method and DNA sequencing to generate high coverage sequence data for the target genomic interval from multiple individuals of each wing pattern phenotype. We will then assemble these data against a targeted reference assembly and examine statistical associations among polymorphisms and wing pattern phenotype to characterize the functional genetic basis for supergene mimicry at a fine scale. Aim 2: Characterize tissue-specific patterns of candidate gene expression throughout the process of wing development. We will examine wing pattern associated expression patterns by using qRT-PCR to run a complete time series across development, analyzing expression of all genes in our critical interval, on forewings, hindwings, and non-wing tissue, for males and females with various mimicry genotypes. We will then follow-up using in situ hybridization, to examine spatial patterns of expression at critical time points identified from the qRT-PCR time series, and RNA-seq, to characterize the downstream targets of the mimicry supergene. Aim 3: Functionally test the genes and inferred molecular mechanisms responsible for mimetic polymorphism and examine potential pleiotropic effects on mate choice behavior. We will functionally test our candidate mimicry gene(s) using RNAi to knock-down expression during wing development. Furthermore, we will follow-up on preliminary data that suggest the mimicry supergene has pleiotropic effects on mate choice behavior by preference testing large samples of butterflies of all mimicry genotypes.