Adaptation in an RNA fitness landscape is constrained by indirect paths

Fitness landscapes provide a way to quantitatively understand the outcomes of evolution from natural and laboratory settings. Real fitness landscapes typically contain more sequences than can be experimentally studied in a comprehensive fashion. The vast majority of experimentally constructed fitness landscapes have analyzed subsets of sequence space with only two mutations at each variable position because these combinations of mutations represent the parsimonious pathways between ancestral and evolved sequences. However, it has been proposed that these parsimonious “direct” pathways between sequences overemphasize the adaptive challenge caused by local fitness peaks, and that more circuitous “indirect” pathways available in higher-dimensional sequence space can facilitate adaptation. Here, we present evidence in contrast to this hypothesis. We generated an experimental fitness landscape of an RNA enzyme (class III Ligase ribozyme) and compared direct and indirect pathways to higher fitness on this landscape. We generated all nucleotide combinations (genotypes) at seven positions and measured RNA fitness as the enrichment rate of each unique genotype during a single round of a laboratory selection experiment. Several analysis approaches revealed that the landscape is more rugged when indirect pathways are considered. We confirmed that this ruggedness translates to slower adaptation using repeated stochastic evolutionary simulations to replay the tape of evolution allowing a comparison of rates of adaptation when either direct or indirect pathways were available. The majority of the simulations showed that population fitness increased at the same rate or slower when indirect pathways were allowed. In 36% of the simulations, the increased ruggedness of the indirect landscape caused slower adaptation as populations more frequently encountered local fitness optima (peaks). Increased dimensionality was not a clear advantage in the simulation-based comparison of these RNA fitness landscapes suggesting that in certain evolutionary situations the potential to circumnavigate fitness valleys can be overwhelmed by the increased ruggedness of high-dimensional indirect pathways.