Gene expression variation is a major contributor to phenotypic diversity within species and is thought to play an important role in adaptation. However, examples of adaptive regulatory polymorphism are rare, especially those that have been characterized at both the molecular genetic level and the organismal level. In this study, we perform a functional analysis of the Drosophila melanogaster CG9509 enhancer, a cis -regulatory element that shows evidence of adaptive evolution in populations outside the species’ ancestral range in sub-Saharan Africa. Using site-directed mutagenesis and transgenic reporter gene assays, we determined that 3 single nucleotide polymorphisms are responsible for the difference in CG9509 expression that is observed between sub-Saharan African and cosmopolitan populations. Interestingly, while 2 of these variants appear to have been the targets of a selective sweep outside of sub-Saharan Africa, the variant with the largest effect on expression remains polymorphic in cosmopolitan populations, suggesting it may be subject to a different mode of selection. To elucidate the function of CG9509 , we performed a series of functional and tolerance assays on flies in which CG9509 expression was disrupted. We found that CG9509 plays a role in larval growth and influences adult body and wing size, as well as wing loading. Furthermore, variation in several of these traits was associated with variation within the CG9509 enhancer. The effect on growth appears to result from a modulation of active ecdysone levels and expression of growth factors. Taken together, our findings suggest that selection acted on 3 sites within the CG9509 enhancer to increase CG9509 expression and, as a result, reduce wing loading as D . melanogaster expanded out of sub-Saharan Africa. Author summary Much of the phenotypic variation that is observed within species is thought to be caused by variation in gene expression. Variants within cis -regulatory elements, which affect the expression of nearby genes within the same DNA strand, are thought to be an abundant resource upon which natural selection can act. Understanding the functional consequences of adaptive cis -regulatory changes is important, as it can help elucidate the mechanisms underlying phenotypic evolution in general and provide insight into the development and maintenance of biodiversity. However, functional analyses of these types of changes remain rare. Here we present a functional analysis of an adaptively evolving enhancer element of a D . melanogaster gene called CG9509 , of previously unknown function. We show that 3 single nucleotide polymorphisms located within the enhancer of this gene are responsible for an increase in CG9509 expression in cosmopolitan populations (outside of south and central Africa) relative to sub-Saharan populations, which include ancestral populations. We further show that CG9509 is involved in the regulation of growth rate and body size determination and propose that the CG9509 enhancer underwent positive selection to reduce wing loading as the species expanded out of sub-Saharan Africa.
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