Reductive Evolution Can Prevent Populations from Taking Simple Adaptive Paths to High Fitness

New functions requiring multiple mutations are thought to be evolutionarily feasible if they can be achieved by means of adaptive paths-successions of simple adaptations each involving a single mutation.  The presence or absence of these adaptive paths to new function therefore constrains what can evolve.  But since emerging functions may require costly over-expression to improve fitness, it is also possible for reductive (i.e., cost-cutting) mutations that eliminate over-expression to be adaptive.  Consequently, the relative abundance of these kinds of adaptive paths--constructive paths leading to new function versus reductive paths that increase metabolic efficiency--is an important evolutionary constraint.  To study the impact of this constraint, we observed the paths actually taken during long-term laboratory evolution of an Escherichia coli strain carrying a doubly mutated trpA gene. The presence of these two mutations prevents tryptophan biosynthesis.  One of the mutations is partially inactivating, while the other is fully inactivating, thus permitting a two-step adaptive path to full tryptophan biosynthesis. Despite the theoretical existence of this short adaptive path to high fitness, multiple independent lines grown in tryptophan-limiting liquid culture failed to take it.  Instead, cells consistently acquired mutations that reduced expression of the double-mutant trpA gene.  Our results show that competition between reductive and constructive paths may significantly decrease the likelihood that a particular constructive path will be taken. This finding has particular significance for models of gene recruitment, since weak new functions are likely to require costly over-expression in order to improve fitness. If reductive, cost-cutting mutations are more abundant than mutations that convert or improve function, recruitment may be unlikely even in cases where a short adaptive path to a new function exists.