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PLOS Genetics


The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting “enhancer” elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pigment phenotypes among geographically disparate Japanese populations of Drosophila auraria, we discovered a remarkable degree of variation in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, we found that phenotypes uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony’s transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain alleles, we localized function-altering mutations to a conserved silencer that mediates a male-specific pattern of ebony repression. This suggests that the light allele was derived through the loss of this silencer’s activity. Furthermore, examination of the ebony gene of D. serrata, a close relative of D. auraria which secondarily lost male-specific pigmentation revealed the parallel loss of this silencer element. These results demonstrate how loss-of-function mutations in a silencer element resulted in increased gene expression. We propose that the mutational inactivation of silencer elements may represent a favored path to evolve gene expression, impacting morphological traits.

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S1_Fig.jpg (236 kB)
Intraspecific variation in D. auraria pigmentation does not follow a traditional geographical cline

S1_Table.docx (39 kB)
Additional strains used in this study

S2_Fig.jpg (130 kB)
Yellow is expressed similarly in lines that contain the light or dark phenotype

S2_Table.docx (47 kB)
Primers for in situ hybridization probes

S3_Fig.jpg (101 kB)
Tan is expressed similarly in lines that exhibit the light or dark phenotype

S3_Table.docx (56 kB)
Primers for constructing reporter constructs, including chimeric reporters

S4_Fig.jpg (98 kB)
Variable ebony expression in lines that contain a mixture of light and dark pigmentation phenotypes

S5_Fig.jpg (37 kB)
Isolation of the ebony upstream region from montium subgroup species

S6_Fig.jpg (496 kB)
Chimeric constructs localize causative changes to the conserved promoter-proximal silencer region

S7_Fig.jpg (178 kB)
The expansion of ebony expression is fixed among males in D. serrata strains

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