a, Expansion speeds and growth rates of ancestor, strain B, and strain D (see Supplementary Table 2). b, Calibrated fluorescence intensity profiles of singly grown strains 12 h after inoculation at the center of 0.25 % TB agar plates. Unless noted otherwise, the fluorescence intensity in this study is normalized according to the calibration curve shown in Extended Data Fig. 3c; the relative value 1 refers to 5×107 cells/ml. c, Relative fluorescence intensities obtained at various times for the fluorescent mutant strains BG (orange) and DG (cyan) and the ancestor AncG (purple), each grown singly on TB plate. As can be seen, the faster strain has higher fitness everywhere. d, Raw photographs (upper row) and fluorescence intensity profiles (lower row) before and after merging of a representative two-strain-competition between the fluorescent derivatives of the ancestor and strain D 12 h after initial equal inoculation. We used plasmids GFP and NFP in this study to distinguish the two strains from each other in the head-to-head competition, since there is no systematic influence caused by the expression of GFP or NFP (see Extended Data Fig. 3). Upper row (from left to right): the competition between DG and AncN, the competition between DN and AncG, and the merged photograph in pseudocolor (DG, cyan; AncG, purple). Lower row: the corresponding relative fluorescence intensity profiles. e, The same as panel d, only that strain D was replaced by strain B. Evolved strains and the ancestor with the GFP/NFP were cultured to log phase separately. Two types of the combined mixed strains were prepared, the evolved strain with GFP was mixed with the AncN while the evolved strain with NFP was mixed with the AncG in a 1:1 ratio. Later, 2 μl of the two types of the combined mixture were inoculated onto the center of pre-warmed semi-solid agar plates separately and allowed to expand at 37°C for up to 24 h. The photograph and fluorescent intensity of the evolved strains/ancestor with the GFP reporter from these two plates after the expansion were taken at various times and merged. See Methods for details. f, The relative fitness WB obtained as a ratio of direct cell count of the fluorescent derivatives of the ancestor and strain B, inoculated at the center of agar plate. From left to right: the competition between BG and AncN, the competition between BN and AncG, and the averaged curve of the left two ones, giving WB (shown as panel m). The competition experiments are the same as those shown in panel e. The initial and final ratio of the two competitors were measured by cell counting with a flow cytometer, see Methods for details. The spatiotemporal development of the bacterial density profiles, indicated by fluorescence intensities, are shown for the competition between the ancestor (purple) and strain D (cyan) (g) and between the ancestor and strain B (orange) (i) at various times during the 24h course of their competition. Beyond the initial period (~6 h), the crossover distance could be clearly defined (vertical dashed line) and was practically time-independent, with the ancestor gaining advantage in the interior and strain D gaining advantage in the exterior. The relative fitness values, WD and WB, taken as the ratio of the fluorescence intensities (mutant:ancestor) at various distances, are shown for the two competition runs in panels h and j, respectively, for time = 6 h and beyond. The vertical dashed lines indicate the crossover distance d× where the densities of the two competing strains are equal, i.e., Wi = 1. Thus, the crossover distance was frozen in shortly after the initial period. Fluorescence intensity profiles (k, l), and relative fitness W (m) of representative two-strain competitions between the ancestor and evolution isolates. The black solid line indicates the ancestor. The data were taken 12 h (k, m) or 24 h (l) after co-inoculation of equal initial mixture of the two competing strains at the center of 0.25% TB agar plates, showing that the slower strain spatially outcompetes the faster strain within the crossover distance d× (dashed lines). From left to right, the evolution isolates are A (red), B (orange), C (green), D (cyan), and E (blue), respectively. See Supplementary Table 2 for strain information. Experiments in a-l were repeated independently 3 times, obtaining similar results. In m, mean±s.d. of n=3 biologically independent repeats is shown.