Background: Changes in gene regulation are thought to be crucial for the adaptation of organisms to their environment. Transcriptome analyses can be used to identify candidate genes for ecological adaptation, but can be complicated by variation in gene expression between tissues, sexes, or individuals. Here we use high-throughput RNA sequencing of a singleDrosophila melanogastertissue to detect brain-specific differences in gene expression between the sexes and between two populations, one from the ancestral species range in sub-Saharan Africa and one from the recently colonized species range in Europe.Results: Relatively few genes (<100) displayed sexually dimorphic expression in the brain, but there was an enrichment of sex-biased genes, especially male-biased genes, on the X chromosome. Over 340 genes differed in brain expression between flies from the African and European populations, with the inter-population divergence being highly correlated between males and females. The differentially expressed genes included those involved in stress response, olfaction, and detoxification. Expression differences were associated with transposable element insertions at two genes implicated in insecticide resistance (Cyp6g1andCHKov1).Conclusions: Analysis of the brain transcriptome revealed many genes differing in expression between populations that were not detected in previous studies using whole flies. There was little evidence for sex-specific regulatory adaptation in the brain, as most expression differences between populations were observed in both males and females. The enrichment of genes with sexually dimorphic expression on the X chromosome is consistent with dosage compensation mechanisms affecting sex-biased expression in somatic tissues.

Insertions and deletions (indels) are a major source of genetic variation within species and may result in functional changes to coding or regulatory sequences. In this study we report that an indel polymorphism in the 3’ untranslated region (UTR) of the metallothionein gene MtnA is associated with gene expression variation in natural populations of Drosophila melanogaster. A derived allele of MtnA with a 49-bp deletion in the 3' UTR segregates at high frequency in populations outside of sub-Saharan Africa. The frequency of the deletion increases with latitude across multiple continents and approaches 100% in northern Europe. Flies with the deletion have more than 4-fold higher MtnA expression than flies with the ancestral sequence. Using reporter gene constructs in transgenic flies, we show that the 3' UTR deletion significantly contributes to the observed expression difference. Population genetic analyses uncovered signatures of a selective sweep in the MtnA region within populations from northern Europe. We also find that the 3’ UTR deletion is associated with increased oxidative stress tolerance. These results suggest that the 3' UTR deletion has been a target of selection for its ability to confer increased levels of MtnA expression in northern European populations, likely due to a local adaptive advantage of increased oxidative stress tolerance. Although molecular variation is abundant in natural populations, understanding how this variation affects organismal phenotypes that are subject to natural selection remains a major challenge in the field of evolutionary genetics. Here we show that a deletion mutation in a noncoding region of the Drosophila melanogaster Metallothionein A gene leads to a significant increase in gene expression and increases survival under oxidative stress. The deletion is in high frequency in three distinct geographic regions: in northern European populations, in northern populations along the east coast of North America, and in southern populations along the east coast of Australia. In northern European populations the deletion shows population genetic signatures of recent positive selection. Thus, we provide evidence for a regulatory polymorphism that underlies local adaptation in natural populations.

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. 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.