Rapid Evolution of Mycoplasma gallisepticum Figure 2. Patterns of polymorphism among Mycoplasma gallisepticum isolates collected from House Finches. a) Comparison of nucleotide diversity between historical chicken MG strains and serially sampled House Finch MG isolates for a 1.3 kb region [28]. b) Expansion of House Finch nucleotide diversity measured across the whole-genome alignment (approximately 738 kb when considering only the 12 House Finch isolates). c) Patterns of synonymous and nonsynonymous substitution for all MG isolates sequenced in this study as well as the reference. The values in this histogram reflect estimates of v = dn/ds across a tree including all House Finch isolates and the poultry Rlow reference. For a full list of patterns of substitution for each gene, see Data S1 (Estimates of omega.xls). d) Bayesian skyline plot estimated from the alignment of 12 of house finch Mycoplasma strains. Although the upper and lower 95% confidence limits (gray lines) on the skyline plot are substantial, the overall trend (black line) is indicative of population growth approximately 17 years before 2007, or 1990, placing the spread of MG somewhat earlier than the first field observations in 1994. Note that time is reversed so that time proceeds from left (past) to right (most recent time of sampling). doi:10.1371/journal.pgen.1002511.g002 strains except AL_2001_17 clearly formed well defined clades based on 85 and 28 SNPs, respectively, establishing the phylogenetic relationships for the other 5 House Finch MG strains exclusively via SNPs was not possible (Text S6, Figure 3). Although a total of 16 SNPs were phylogenetically informative for the placement of these five strains, the largest cluster of SNPs that were phylogenetically consistent was seven, and overall, 13 different trees were supported by at least 3 SNPs each. Similarly, substantial homoplasy was found among the four newly sequenced poultry strains and the Rlow reference. Although 6,152 SNPs were parsimony informative for these five strains, the unrooted tree with the best support was in conflict with 4,619 (75%) of these SNPs. These patterns are expected if sites are being shuffled by recombination or horizontal gene transfer (HGT) among isolates, and analysis of the entire data set found strong support for this (Text S4, Figures S3, S4, S5). Using the pairwise homoplasy index test [34] revealed a statistically significant signal of recombination (p,1029). This signal comes predominantly from the four newly sequenced poultry strains because there is not enough genetic variation to make this test significant when only the House Finch strains are considered. However if we apply to the House Finch MG strains the homoplasy test by Maynard-Smith and Smith [35], which is found to perform well in situations of low nucleotide diversity [36], we again obtain a significant signal for recombina- tion (p,1026). We conclude that, despite a significant signal for recombination in both the poultry and House Finch strains, the House Finch MG cluster as a whole is a distinct and easily identifiable phylogenetic lineage with a long branch separating it from the poultry strains (Figure 3). Substitution rate and robustness to model assumptions Coalescent analysis [32] of the 12 House Finch isolates sampled at different dates suggested an extraordinary point substitution rate of 1.0261025 substitutions per site per year (95% HPD 7.9561026 to 1.236 1025 (Text S2), consistent with earlier suggestions that Mycoplasma may be among the fastest evolving bacteria [37]. This rate of point substitution is not restricted to House Finch MG strains but was also found in the poultry strains when analyzed separately (Text S2), suggesting that rapid evolution was characteristic of MG prior to the House Finch PLoS Genetics | www.plosgenetics.org 4 February 2012 | Volume 8 | Issue 2 | e1002511