Natural selection has played an important role in establishing various functions in nature, but molecular mechanisms of such adaptive events are not well understood. The slow progress is a consequence of mutagenesis experiments using present-day molecules and the limited scope of statistical methods used to detect adaptation. To fully appreciate functional differentiation, the precise roles of critical mutations should be understood by engineering and manipulating ancestral molecules. Experimental and quantum chemical analyses of dim-light and color vision reveal some surprising results and provide a foundation for a productive study of actual functional differentiations.