a–d, Vertical distribution is reported for a population grown under a diel light cycle (a), for regrown cells (b), for a monoclonal population (c) and for a starved population (d). a, Upward bias for exponential-phase HA452 cells grown under a diel light cycle (14 h light: 10 h dark), showing the characteristic split after 100 flips (blue, n = 844 cells; red curve is control, n = 910). A similar split was observed for cells cultured under constant illumination (Fig. 1g). b, Cells regrown from those collected from the bottom of the chamber after 100 flips. Although cells collected from the bottom were positively gravitactic (swimming downwards; blue curve in a), cells regrown from these are negatively gravitactic (swimming upwards; pink curve in b, n = 411). Upon exposure to 100 flips, these regrown cells again exhibited the population split (cyan curve, n = 391). a, b, The solid lines represent the mean of the equilibrium vertical distribution over time (a: mean of 92 frames; b: mean of 78 frames), and the shaded regions represent ± 1 s.d. from the mean. In the insets, the error bars of the upward bias, r, are extracted from the cells’ vertical distribution. c, The population split also occurs in a monoclonal population of HA452. The inset shows the upward bias (mean ± s.d. of three replicates) calculated from the relative distribution of the cells at equilibrium, after being exposed to 100 flips (blue, total number of cells n = 2,985) and for the control (red, n = 2,490). The star indicates statistical significance in the difference between treatment and control (two-sided t-test, t4 = 4.79, P = 0.009). d, A nutrient-starved HA452 population does not split upon flipping. Nutrient-starved cells were harvested at stationary phase (350 h after inoculation). The inset shows the upward bias (mean ± s.d. of three replicates, two-sided t-test, t4 = 0.91, P = 0.42) of the cells after being exposed to 100 flips (blue, n = 743) and for the control (red, n = 1,011). c, d, The solid lines represent the mean of the equilibrium vertical distribution over three replicates, and the shaded regions represent ± 1 s.d. from the mean.