A Direct Experimental Test of Ohno’s Hypothesis

Gene duplication drives evolution by providing raw material for proteins with novel functions. The oldest and historically most influential hypothesis about the evolutionary fate and potential of duplicated genes has been proposed by Susumu Ohno in 1970. This hypothesis essentially posits that gene duplication can help genes tolerate new mutations and thus facilitates the evolution of new phenotypes Competing hypotheses argue that deleterious mutations will usually inactivate gene duplicates too rapidly for Ohno’s hypothesis to work. Here, we provide a first direct experimental test of Ohno’s hypothesis. Specifically, we evolved one or two genes encoding a fluorescent protein in Escherichia coli through multiple rounds of mutagenesis and selection. We then analyzed the genotypic and phenotypic evolutionary dynamics of the evolving populations through high-throughput DNA sequencing, biochemical assays, and engineering of selected variants. In support of Ohno’s hypothesis, populations carrying two gene copies displayed higher mutational robustness than those carrying a single gene copy. As a consequence, the double-copy populations experienced relaxed purifying selection, evolved higher phenotypic and genetic diversity, carried more mutations and accumulated combinations of key beneficial mutations earlier. However, their phenotypic evolution was not accelerated, possibly because one gene copy rapidly became inactivated by deleterious mutations. Our work provides an experimental platform to test models of evolution by gene duplication, and it supports alternatives to Ohno’s hypothesis.### Competing Interest StatementThe authors have declared no competing interest.