Phylogeny of Competence Table 4. Clade with most frequent presence in topologies from each dataset (indicated by bold text). CLADE WITH HIGHEST SUPPORT 1207 and PittHH R2866 and PittII PittAA and 3655 MLST DATASET 5 of 7 topologies 1 of 7 topologies 1 of 7 topologies GENOME DATASET Strain 1207 not in dataset 56 of 101 topologies 41 of 101 topologies UPTAKE/TRANSFORMATION DATASET Strain 1207 not in dataset 11 of 30 topologies 14 of 30 topologies The support of each clade in other datasets is listed for comparison. doi:10.1371/journal.pone.0005854.t004 relationships in this tree differed from those in the BUCKy tree, many of the more recent relationships were in agreement. Transformational recombination between strains may have been an important contributor to their recombinational histories. Because lab cultures of some strains exhibited much more recombination than others, we asked whether these strains’ relationships were more difficult to resolve, using Mesquite to measure the stability of each strains’ evolutionary relationships [34]. In this analysis strains that often have different close relatives in different trees are given high instability scores (see Table 1 for instability values). However highly transformable strains were not significantly less stable, as we found no significant relationship between these two properties in any of the three datasets (r2MLST = 0.002, P = 0.8; r 2 genome = 0.04, P = 0.53; r2uptake/transformation = 0.007, P = 0.8). This lack of relationship between instability and transformation does not rule out a contribution of transformation to allelic exchange, but may simply indicate that the majority of recombination between strains occurs via conjugation and/or transduction. Tracing the evolution of competence The extensive variation we found in amounts of DNA taken up and recombined [16] must reflect changes in competence and transformability as lineages diverged from their common ancestor. We inferred the relationships of strains using multiple methods, but many of these relationships had low support or were not consistent between datasets or inference methods. Because the trees were inconsistent between different datasets and methods, we traced the evolution of DNA uptake and transformation separately on each of four trees: the MLST BUCKy tree (Figure 4A), the MLST ClonalFrame tree (Figure 6), the Genome BUCKy tree (Figure 4B), and the Uptake/transformation BUCKy tree (Figure 4C). Parsimony methods implemented in Mesquite were used to reconstruct ancestral states to infer whether DNA uptake and/or transformation have changed since each strain diverged from its most recent ancestor. For each strain, we compared the results between trees and discuss only increases or decreases that were consistent between all available trees. For each strain and for each tree, the change in DNA uptake and transformation since divergence from its ancestor is shown in Table 5. As visual examples of competence evolution, DNA uptake and transforma- tion mapped onto the MLST ClonalFrame topology are shown in Figure 7. DNA uptake. The amount of DNA taken up by strains varied from 0.0013 to 1.6; these numbers indicate the percent of radio- labeled DNA taken up by cells, normalized by the optical density of culture [16]. Tracing the evolution of DNA uptake on each of the four trees showed that 28 of 35 strains took up different amounts of DNA than were predicted for their most recent ancestors. Thirteen of these changes were increases and fifteen were decreases. Increases ranged from 0.00075 to 1.58 in strains 1181 and Rd, respectively, and decreases ranged from 0.00035 to 0.07 in strains 1209 and 1124, respectively. Strains with the most pronounced changes in uptake all had increases; these were strains 375, PittDD, R2866, Rd, and RM6169. Transformation. The transformation frequencies of strains varied from 1.161028 to 0.01 [16]. Tracing changes in transformation frequency on each of the four trees indicated that all strains with a change in uptake had a change in transformation. Because DNA uptake is a prerequisite for transformation, we discuss these candidate changes in the context of the changes in DNA uptake inferred above. Eight strains with increased uptake had increased transformation whereas five strains with increased uptake had decreased transformation. Similarly, ten strains with decreased uptake had decreased transformation whereas five strains with decreased uptake had increased transformation. Changes in transformation that are in the same direction as changes in uptake are best interpreted as consequences of changes in DNA uptake. However, in ten strains uptake and Figure 2. A putative recombination breakpoint identified using RDP. Only polymorphic sites are shown, corresponding to 3,095 bp of the original 7,014 bp in the aligned block. Grey squares indicate regions where other strains are identical to strain Rd and white squares indicate where strains are different. The black line indicates the approximate breakpoint. Strains without recombination in this region have been shaded. doi:10.1371/journal.pone.0005854.g002 PLoS ONE | www.plosone.org 5 June 2009 | Volume 4 | Issue 6 | e5854