Fig. 4

Confirmed pyoverdine phenotypes in selected clones. Evolved clones with putatively altered pyoverdine production levels, according to the initial screen shown in Fig. 3, were re-tested to confirm their phenotype. The different panels show clones evolved from the pvdS_gene ancestor with putatively decreased (a) or increased (b) pyoverdine production; and clones evolved from the pvdS_prom ancestor with putatively decreased (c) or increased (d) pyoverdine production. While the phenotype of evolved clones with putatively decreased pyoverdine production could be mostly confirmed, confirmation rate for the clones with putatively increased pyoverdine production was low. Y axes show pyoverdine-specific fluorescence divided by growth (optical density at 600 nm) after 24 h of incubation in iron-limited medium. X axes show clone ID and replicate ID together with the iron availability experienced during experimental evolution: lo (red bars) = low iron with iron chelator only; me (green bars) = intermediate iron with iron chelator + 1 μM FeCl₃; hi (blue bars) = high iron with iron chelator + 40 μM FeCl₃. Bars represent mean values of three replicates per evolved clone. Error bars denote standard error of the mean. The black line represents the average wildtype production level, while the blue line denotes the average production level of the respective low-producing ancestors pvdS_gene and pvdS_prom. We used one-way ANOVAs with Tukey’s post-hoc test for comparisons between evolved clones and the low-producing ancestor. Asterisks indicate significant differences (p < 0.05)