Lack of knowledge about how regulatory regions evolve in relation to their 
structure-function may limit the utility of comparative sequence analysis in 
deciphering cis-regulatory sequences. To address this we applied reverse 
genetics to carry out a functional genetic complementation analysis of a 
eukaryotic cis-regulatory module-the even-skipped stripe 2 enhancer-from four 
Drosophila species. The evolution of this enhancer is non-clock-like, with 
important functional differences between closely related species and functional 
convergence between distantly related species. Functional divergence is 
attributable to differences in activation levels rather than spatiotemporal 
control of gene expression. Our findings have implications for understanding 
enhancer structure-function, mechanisms of speciation and computational 
identification of regulatory modules.