Phenotypic analysis of the Pseudomonas population
Growth data were obtained for a total of 95 carbon substrates included in the Biolog GN2 MicroPlate. To ensure that the phenotypic and genotypic data could be compared, the raw A 660values were used to create a distance matrix for further analysis. The results clearly indicate the presence of geographic structure, which is almost identical to the above-described genotypic variation by location (ANOSIM, r = 0.454, P < 0.001 ). Principal component analysis (PCA) indicated that 62% of the phenotypic variability can be accounted for by the principal component, whereas only 29% of the genotypic variability can be accounted for by the principal component (Fig. 5). There was significant correlation between the phenotypic and genotypic principal components (R2 = 0.5303).
Further Canonical variate analysis separated the Pseudomonasisolates into discrete populations, which matched well with the ancestral genotypes identified by STRUCTURE (Fig. 6A). There was a significant correlation between carbon source utilization with the canonical axes CAP1 and CAP2 (Fig. 6A). The following eight substrates had an r value > 0.8 (location in GN2 MicoPlate shown in parenthesis): γ-hydroxybutyric acid (D12), xylitol (C10), D-galactose (B4), inosine (H2), urocanic acid (H1), L-arabinose (A10), D-glucosaminic acid (D8) and m-inositol (B7).
The roles of individual carbon substrate in separating thePseudomonas populations by location are summarized in Figure 6B. Of particular note are histidine (his) and urocanic acid (urocanate, uro) whose genetic basis in Pseudomonas has been well characterized (Zhang & Rainey, 2007). Both substrates are co-catabolised with the involvement of only one additional enzyme histidase (HutC) catalysing the conversion from histidine and urocanate. Interestingly, there was a strong genotypic association with urocanate utilization (ANOSIM, r = 0.758, P < 0.001), but not with histidine utilization (ANOSIM, r = 0.066, P = 0.145). Further analysis indicated a clear linkage between urocanate utilization and location (location, PERMANOVA P = 0.0001; uro, PERMANOVA P = 0.0002; location x uro, PERMANOVA P = 0.0146). Figure 7 clearly shows that the Oxford and Auckland populations are well separated by their ability to grow on urocanate (His-, Uro+ versus His- Uro-).
Finally, it should be noted that minor but significant differences were detected between genotypes for strains isolated from young vs. mature leaves (ANOSIM, r = 0.103, P = 0.01), and also for young vs. old leaves (r = 0.075, P = 0.01), but not for mature vs. old leaves (r = 0.015, P = 0=7.4). Similar results were found in terms of growth phenotypes on histidine and urocanate: young vs. mature leave (r = 0.185, P = 0.01), young vs. old leaves (r = 0.136, P = 0.01), and mature vs. old leaves (r = 0.05, P = 1.7). No significant differences were detected for the potential effects of plants and plots.