Pigmentation is involved in a wide array of biological functions across insect orders, including body patterning, thermoregulation, and immunity. The melanin pathway, in particular, has been characterized in several species. However, molecular evolution of the genes involved in this pathway is poorly explored. We traced the molecular evolution of six melanin pathway genes in 53 species of Lepidoptera covering butterflies and moths, and representing over 100 million years of diversification. We compared the rates of synonymous and non-synonymous substitutions within and between these genes to study the signatures of selection at the level of individual sites, genes, and branches of the gene tree. We found that molecular evolution of all six genes was governed by strong purifying selection. Yet, a number of sites showed signs of being under positive selection, including in the highly conserved domain regions of three genes. Further, we traced the expression of these genes across developmental stages, tissues, and sexes in the Papilio polytes butterfly using a developmental transcriptome dataset. We observed that the expression patterns of the genes in P. polytes largely reflected their known tissue-specific function in other species. The expression of sequentially acting genes in the melanin pathway was correlated. Interestingly, melanin pathway genes also showed a sexually dimorphic pattern of developmental heterochrony, i.e., females showed prominent upregulation of melanin pathway genes in pre-pupal stage compared to males, while males showed prominent upregulation in 9-day pupal wings compared to females. Our evolutionary and developmental analyses suggest that the vast diversity of wing patterning and pigmentation in Lepidoptera may have evolved despite largely constrained sequence evolution, with potential contribution from differential developmental expression of genes in a highly conserved pathway.

Gene duplication is a vital process for evolutionary innovation. Functional diversification of duplicated genes is best explored in multicopy gene families such as histones, hemoglobin, and opsins. Rhodopsins are photo-sensitive proteins that respond to different wavelengths of light and contribute to diverse visual adaptations across insects. While there are several instances of gene duplications in opsin lineages, the functional diversification of duplicated copies and their ecological significance is properly characterised only in a few insect groups. We examined molecular and structural evolution that underlies diversification and sub-functionalisation of four opsin genes and their duplicated copies across 132 species of the diverse insect order-Lepidoptera. Opsins have largely evolved under purifying selection with few residues showing signs of episodic and pervasive diversifying selection. Although these do not affect overall protein structures of opsins, substitutions in key amino acids in the chromophore-binding pocket of duplicated copies might cause spectral sensitivity shifts leading to sub-functionalisation or neofunctionalisation. Duplicated copies of opsins also exhibit developmental stage-specific expression in Papilio polytes, suggesting functional partitioning during development. Together, altered spectral sensitivities owing to key substitutions and differential expression of duplicated copies across developmental stages might enable enhanced colour perception and improved discrimination across wavelengths in this highly visual insect group.

Pigmentation is involved in a wide array of biological functions across insect 13 orders, including body patterning, thermoregulation, and immunity. The melanin pathway, in 14 particular, has been characterized in several species. However, molecular evolution of the genes 15 involved in this pathway is poorly characterized, and their roles in pigmentation of early 16 developmental stages are just beginning to be explored in non-model organisms. We traced the 17 molecular evolution of six melanin pathway genes in 53 species of Lepidoptera covering butterflies 18 and moths, and representing over 100 million years of diversification. We compared the rates of 19 synonymous and nonsynonymous substitutions within and between these genes to study signatures 20 of selection at the level of individual sites, genes, and branches of the gene tree. We found that 21 molecular evolution of all six genes was governed by strong purifying selection. Yet, a number of 22 sites showed signs of being under positive selection, including in the highly conserved domain 23 regions of three genes. Further, we traced the expression of these genes across developmental 24 stages, tissues, and sexes in the Papilio polytes butterfly using a developmental transcriptome 25 dataset. We observed that the expression patterns of the genes in P. polytes largely reflected their 26 known tissue-specific function in other species. The expression of sequentially acting genes in the 27 melanin pathway was correlated. Interestingly, four out of six melanin pathway genes (ebony, pale, 28 aaNAT, and DDC) showed a sexually dimorphic pattern of developmental heterochrony; i.e., 29 females showed peak activity much earlier in pupal development compared to that of males. Our 30 evolutionary and developmental analyses suggest that the vast diversity of wing patterning and 31 pigmentation in Lepidoptera may have been aided largely by differential developmental regulation 32 of genes in a highly conserved pathway, in which the sequence evolution of individual genes is 33 highly constrained. 34 35