The gain, loss or modification of morphological traits is generally associated with changes in gene regulation during development. However, the molecular bases underlying these evolutionary changes have remained elusive. Here we identify one of the molecular mechanisms that contributes to the evolutionary gain of a male-specific wing pigmentation spot in Drosophila biarmipes, a species closely related to Drosophila melanogaster. We show that the evolution of this spot involved modifications of an ancestral cis-regulatory element of the yellow pigmentation gene. This element has gained multiple binding sites for transcription factors that are deeply conserved components of the regulatory landscape controlling wing development, including the selector protein Engrailed. The evolutionary stability of components of regulatory landscapes, which can be co-opted by chance mutations in cis-regulatory elements, might explain the repeated evolution of similar morphological patterns, such as wing pigmentation patterns in flies.

The independent evolution of morphological similarities is widespread. For simple traits, such as overall body colour, repeated transitions by means of mutations in the same gene may be common. However, for more complex traits, the possible genetic paths may be more numerous; the molecular mechanisms underlying their independent origins and the extent to which they are constrained to follow certain genetic paths are largely unknown. Here we show that a male wing pigmentation pattern involved in courtship display has been gained and lost multiple times in a Drosophila clade. Each of the cases we have analysed (two gains and two losses) involved regulatory changes at the pleiotropic pigmentation gene yellow. Losses involved the parallel inactivation of the same cis-regulatory element (CRE), with changes at a few nucleotides sufficient to account for the functional divergence of one element between two sibling species. Surprisingly, two independent gains of wing spots resulted from the co-option of distinct ancestral CREs. These results demonstrate how the functional diversification of the modular CREs of pleiotropic genes contributes to evolutionary novelty and the independent evolution of morphological similarities.

The evolution of cis regulatory elements (enhancers) of developmentally regulated genes plays a large role in the evolution of animal morphology. However, the mutational path of enhancer evolution-the number, origin, effect, and order of mutations that alter enhancer function-has not been elucidated. Here, we localized a suite of substitutions in a modular enhancer of the ebony locus responsible for adaptive melanism in a Ugandan Drosophila population. We show that at least five mutations with varied effects arose recently from a combination of standing variation and new mutations and combined to create an allele of large phenotypic effect. We underscore how enhancers are distinct macromolecular entities, subject to fundamentally different, and generally more relaxed, functional constraints relative to protein sequences.Three major challenges for understanding the genetic and molecular bases of morphological evolution are to identify loci underlying trait divergence, to pinpoint functional changes within these loci, and to trace the origin of functional variation in populations. The evolution of animal morphological diversity is generally associated with changes in the spatial expression of genes that govern development (1,2). The divergence of particular morphological traits has been linked to changes in specific enhancers of individual loci (3-9). Mutations in individual, modular enhancers are thought to circumvent the potentially pleiotropic effects of mutations in coding sequences of genes that participate in many developmental processes (10-12).Nonetheless, there is relatively little detailed knowledge of how enhancer sequences evolve, of the genetic path of enhancer evolution. In most instances, functional mutations have not been identified, so their individual effects and origins have not been traced. In contrast, the evolutionary paths of several proteins have been traced and revealed that many trajectories, including reversals, are not allowed because of structural constraints (13-15). To decipher the mode and tempo of regulatory sequence evolution, we must determine the following: