(A) Selection for access to oxygen allows wrinkly spreader (WS) mutants to invade the ancestral smooth (SM) population in static microcosms. WS mutants form a mat at the air-liquid interface through increased expression of the main structural component, cellulose, encoded by the wss operon. Expression of cellulose is controlled by the second messenger c-di-GMP, which is produced by diguanylate cyclases (DGCs). Mutations in the wsp, aws and mws operons, that activate their respective DGCs (WspR, AwsR, MwsR), are the primary mutational pathways to WS. (B) When a reporter construct connecting expression of the wss operon to resistance to kanamycin is used under shaken non-selective conditions, WS mutants can be isolated without the biasing influence of natural selection. This allows estimation of the mutation rate to WS and an unbiased spectrum of mutations defining the mutational target. Fitness can then be assayed in competition with a common reference strain.