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.