Rapid Evolution of Mycoplasma gallisepticum Figure 3. Phylogeny of Mycoplasma gallisepticum isolates collected at time points 1994–2007 following a host shift from poultry to House Finches. The basic topology and branch lengths of the tree come from the output for the BEAST analysis made while estimating evolutionary rates. From this tree we collapsed branches with less than 0.6 posterior probability or if there were no phylogenetically informative SNPs supporting that branch. Several strains are shown as polytomies because their genomic histories are shaped by recombination. Within the House Finch MG clade, branch lengths are proportional to time. Major genomic events are indicated on appropriate branches. The numbers of diagnostic SNPs indicated on various branches are minima. The numbers of CRISPR changes shown are only those that can be constructed with reasonable support (Figure 5); one possible reconstruction is presented. doi:10.1371/journal.pgen.1002511.g003 epizootic. We estimated a similar substitution rate when considering only the four-gene multistrain alignment use to identify poultry strains for sequencing (Text S2). We verified that our estimate of substitution rate is robust to different protocols for SNP identification, statistical models and data sets (Figure 4; Text S7). Altogether we estimated the substitution rate within a coalescent framework on 34 combinations of SNP calling and model assumptions and found consistent estimates throughout (Text S1, Figure 4, Figure S6). In addition, we achieved a similar estimate using a Poisson regression approach as well as a root-to- tip regression (Text S7 and Figure 4). A possible mutator strains in House Finch MG In addition to a high estimated substitution rate in MG, we found a mutation in the gene-encoding UvrB that could elevate this rate yet further. UvrB is an essential part of the nucleotide excision repair system, which has been posited to be the most important pathway for maintaining genomic integrity in Mycoplasma [38]. The mutation truncates the UvrB protein by three amino acids (Table S10) and raises the possibility of the origin of a mutator strain in House Finch MG [39] as the C-terminal of this protein is essential for its function [40]. Consistent with this idea, we found 14 instances of adjacent SNPs among the 12 House Finch isolates, a notable excess in an alignment with only 412 variable sites (Table S11). Moreover, 12 of these 14 are CCRTT double substitutions, which are normally repaired by the UVR system (Table S10). For 13 of the 14 doublets, both sites are inferred to have mutated on the same branch of the tree, suggesting single mutational events, and the proportion of doublet mutations involving the same base was drastically higher (92.8%) in lineages with the UvrB mutation as compared to those without (p,0.0001; Table S10). Nonetheless, these doublet mutations are not required to achieve the high rate of substitution that we measured. They account for less than 7% of the segregating variation and removal of these doublet sites does not affect the high estimated substitution rate. The UvrB mutation is found in all of our House Finch MG strains as well as the turkey strain TK_2001, but not in the ancestral chicken strains or the reference chicken strain. Thus, the mutation appears to have arisen on the lineage leading to the House Finch. PLoS Genetics | www.plosgenetics.org 5 February 2012 | Volume 8 | Issue 2 | e1002511