Epistatic interactions determine the mutational pathways and coexistence of lineages in clonal Escherichia coli populations

Understanding how diversity emerges in a single niche is not fully understood. Rugged fitness landscapes and epistasis between beneficial mutations could explain coexistence amongst emerging lineages. To provide an experimental test of this notion, we investigated epistasis amongst four pleiotropic mutations in rpoS, mglD, malT and hfq present in two coexisting lineages that repeatedly fixed in experimental populations of Escherichia coli. The mutations were transferred into the ancestral background individually or in combination of double or triple alleles. The combined competitive fitness of two or three beneficial mutations from the same lineage was consistently lower than the sum of the competitive fitness of single mutants – a clear indication of negative epistasis within lineages. We also found sign epistasis (i.e. the combined fitness of two beneficial mutations lower than the ancestor), not only from two different lineages (i.e. hfq and rpoS) but also from the same lineage (i.e....