Figure 2. Division time asymmetry as a function of selection and current glucose concentration. Effect on the median index of cell division time asymmetry of current glucose concentration (10mg/ ml and 0.1 mg/ml) and selection for 1000 and 2000 generations under high (H) and low (L) nutrient concentrations. A, B and C show results of strain 1, 2 and 3 respectively. Trends in means were similar. On prolonged selection H lineages develop consistently higher value of the index and lose sensitivity to the immediate nutritional environment. On the other hand, two of the three L lineages appear to be locked into lower asymmetry and lose responsiveness to environment. doi:10.1371/journal.pone.0014516.g002 generations of selection H1l2000 did not differ significantly from H1l1000 but the difference between H1l and H1h became non- significant. On the other hand asymmetry index of L1h2000 decreased significantly from L1h1000 (p,0.0001) and became indistinguishable from L1l2000. At 2000 generations, H1h was significantly greater than L1h (p,0.0001) and L1l (p,0.0001). Also L1l was significantly lower than H1l (p,0.0001) (Figure 2A). This indicates that although the wild type showed different asymmetry indices under h and l, the selected strains retained their symmetry or asymmetry even after changing current nutrient concentrations. In other words, H1 became committed to asymmetric division and L1 became committed to symmetric division with selection. In a pair- wise comparison at two substrate concentrations and two selection durations, all L strains had lower median asymmetry indices than their respective H counterparts (paired t = 4.287, p = 0.024). Bacterial Division Symmetry The pattern of changes with selection was similar in strain 2. At 1000 generations, H2h divided significantly more asymmetrically than L2l (p,0.0001) and W2h (p = 0.03). The mean index of asymmetry of L2h was found to be significantly greater than L2l (p,0.0001) but H2h and H2l did not differ significantly in their asymmetry indices (Figure 2B). After 2000 generations H2l2000 did not differ significantly from H2l1000 and remained significantly greater than L2l (p = 0.001). L2h2000 decreased significantly than L2h1000 (p = 0.016) and showed significantly more symmetric division than H2h (p = 0.005) (Figure 2B). There was no significant difference in H2h, H2L and L2l between 1000 and 2000 generations. This indicates loss of plasticity with selection and commitment to symmetric division in L2 and asymmetric division in H2. In a pair-wise comparison at two substrate concentrations and two selection durations, all L strains had lower median asymmetry indices than their respective H counterparts (paired t = 3.332, p = 0.044). In strain 3 after 1000 generations L3l showed significantly lower asymmetry index than L3h (p,0.0001) but contrary to our expectations L3h showed significantly greater asymmetry than H3h (p = 0.049). No other differences were significant (Figure 2C). At 2000 generations, L3h (p = 0.004) and H3l (p,0.0001) showed significantly higher asymmetry than L3l (Figure 2C). There was no significant difference in asymmetries of L3h, H3h and H3l. Thus after prolonged selection H3 appears to have committed to asymmetric cell division but L3 unlike strain 1 and 2 did not commit to symmetric cell division (Table S1 and Dataset S1). In a pair-wise comparison at two substrate concentrations and two selection durations, unlike strains 1 and 2 L3 strains did not differ significantly than their respective H3 counterparts (paired t = 0.663, p = 0.554). Pooling data on all the three strains, the L lineages had significantly lower median asymmetry indices than their H counterparts (paired t = 3.345, p = 0.006). Taking the median indices of all the three strains at both selection conditions, selection durations and current nutrient concentrations we performed a four factor ANOVA without replication which revealed that only the effect of selection was statistically significant (p = 0.05) whereas strain, duration of selection and current nutrient concentration did not affect asymmetry index significantly. In the wild type, the effect of current nutrient concentration was highly significant whereas after selection it appears to have lost its significance in most of the pairs. This indicates that selection led to commitment to one type of division and the plasticity of the response was significantly reduced. This change was evident by 1000 generations itself but appeared to become more pronounced by 2000 generations with the only exception of L3. The proportion of cells that become exceedingly slow are represented as outliers in the distribution of asymmetry index. Some of the cells failed to divide until the end of the observation period, owing to which an index of asymmetry for such pairs could not be calculated. However their index can be assumed to be greater than the one calculated taking the end of observation period as the division time. Extreme outliers in the distribution, including the cells that failed to divide were assumed to be old or dead cells. Since the frequency of such outliers is small, comparisons according to selection-treatment-strain wise catego- ries was not statistically meaningful. Therefore we compared the frequency of outliers by pooling data. Taking a cut off asymmetry index of 0.667 (corresponding to old cell having at least double the division time of the young sibling) to define old cells, all H lineages pooled together had significantly greater proportion of old cells (0.051) than all wild types pooled (0.019) and all L lineages pooled (0.005) (Z test for comparison of proportions, one-tailed, p = 0.01 PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e14516