Phylogeny of Competence Figure 6. Extended majority rule consensus topology from three replicate ClonalFrame runs with the MLST dataset. The number next to each node indicates the number of replicate runs (out of three) that support that node. doi:10.1371/journal.pone.0005854.g006 What do these results tell us about the evolution of competence? Under the model proposed by Redfield et al. [36], ancestral H. influenzae was naturally competent but its descendants frequently lost competence by mutation. Although such losses might be transiently neutral or beneficial, the resulting lack of either nucleotides or recombination would be detrimental in the long term. Non-competent clades would then rarely persist for long periods of time, either because non-competent clades went extinct or because subsequent mutations restored competence. This model’s prediction that most changes in competence should be recent events is consistent with the recent changes inferred above, but the poor support for basal clades and disagreement between the four trees makes it difficult to determine whether some strains have persisted despite ancestrally low competence. The strongest candidate is strain PittEE; although different trees assigned it different close relatives, it was always most closely related to a strain of low competence (strain 22.4-21 in the MLST trees, strain 86028NP in the Genome tree, and strain R2846 in the Uptake/ transformation tree). Clades of lowly competent strains are not unique to H. influenzae as several clades of entirely non- transformable strains exist in Pseudomonas stutzeri [13]. The poor resolution of strain relationships in H. influenzae prevented identification of non-competent lineages that have persisted for longer periods of time, indicating that phylogenetic methods may not be well suited for tracing competence evolution in H. influenzae. As an alternative, the sequences of competence genes can provide evidence of the timing of loss of competence. For example, a strain with multiple inactivating mutations in several competence genes is likely to have lost competence long before a strain with a single inactivating mutation in one competence gene. None of the 13 sequenced genomes fall in the former category [16], so those that are not competent likely lost competence recently. Because phenotypic variation can reveal differences in how selection has acted, understanding the molecular causes of competence differences may help shed light on the relative importance of its potential benefits. The best approach may be to compare the variation in competence with that in other bacterial phenotypes whose functions are undisputed, such as nutrient acquisition and DNA repair. Unfortunately, variation in even these straightforward phenotypes has not been rigorously documented. Although many pathways for nutrient acquisition appear to vary across different niches, consistent with well- PLoS ONE | www.plosone.org 9 June 2009 | Volume 4 | Issue 6 | e5854