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Fig. S1. Stability of evolved phenotypes. The five evolved isolates of GE3 from the LE1 (α1–5) and LE2 (A1–5) selection environments were grown under batch conditions, first, overnight (day 0) then followed by two rounds of subculture and 24 h incubation (day 1 and day 2). Samples were taken at day 0, day 1 and day 2, diluted and spot-plated onto Congo Red plates to assess rdar morphology (A), glutamate plates (B) and serine plates (C) to assess ability to grow on these amino acids. In panels D and E, the evolved strain GE3.A2 was dilution-plated to monitor population heterogeneity on rdar and glutamate plates.

Fig. S2. Biolog analysis of replicate GE3 isolates from five populations. See the legend to Fig. 6 for details.

Fig. S3. Growth on glutamate and serine. The extent of growth on glutamate plates relative to ancestor (grey bars) and serine relative to ancestor (black bars) are shown in the panels for: (A) GE3, (B) MEM, (C) PAR, (D) WAT, E. MG1655. In each graph, the ancestor strain is Anc, and the evolved isolates from the LE1 selection condition are α 1–5; evolved isolates from the LE2 selection condition are A 1–5; and the LE3 selection evolved isolate is S. Values are the average of at least five repeats with SD error bars.

Fig. S4. Growth yields of ancestors and evolved clones in PYE medium. Twenty-four hour growth measurements of strains grown in PYE. Panels show strains: (A) GE3, (B) MEM, (C) PAR, (D) WAT, (E) MG1655. Each graph shows the ancestor strain (labelled Anc, hatched shading), five isolates from the LE1 selection condition (population α 1–5, grey shading), five isolates from the LE2 selection condition (population A , 1–5, black) and the LE3 selection condition (S, no shading). Values are the average of at least three biological repeats with SD error bars.

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