strains newly sequenced in this study to yield a Large Sample Multiple Sequence Alignment (LS-MSA) 1,363 bp in length (Figure S2). We estimated nucleotide diversity and the standard deviation of this estimate within and among subgroups of these sequences using DNAsp version 4.10.9 [64] (Table S5). In estimating diversity of MG strains sampled from chickens and turkeys, we restricted analysis to those strains sampled during 1994–1996 for comparison with our earliest House Finch strains sampled in a similar time interval. Strain selection and genome sequencing Twelve strains of MG isolated from House Finches in the Southeastern US were sequenced with the Roche 454 Gene Sequencer. The average coverage level was 9.4X (Table S1). Additionally, four MG strains isolated from poultry hosts and selected based on their positions in the multistrain phylogenetic tree were sequenced with the Illumina sequencing platform to an average coverage of ,410 X (Tables S2, S3, S4, Text S1, Figure S2). Inference of substitutions rates, times to common ancestry, and population dynamics Using a coalescent model and a Bayesian framework as implement in BEAST v1.52 [32] we estimated the mutation rate and times to common ancestry from a 13-taxon alignment composed of the reference MG genome and all of the House Finch MG strains whose genomes were sequenced in this study (Text S2). We also ensured that the conclusions from this inference were not sensitive to the SNP calling procedures or the choice of substitution models (Text S2, S7, Figure S6). In order to compare the mutation rate between the poultry and House Finch MG populations, these quantities were similarly estimated from the 82 taxon LS-MSA after removing nine laboratory strains from the alignment that likely experienced different population dynamics than the wild strains and had unknown sampling dates. A Poisson regression model was also used to estimate substitution rates by counting mutations along a single lineage assumed to span the dates of sampling for each strain (Text S7). Transposon movements, recombination, and lateral gene flow We catalogued IS elements using BLAST and the ISFinder database [65, Text S4]. We tested for evidence of genetic recombination between MG strains using the genome sequences from our 4 poultry and 2 House Finch strains using the pairwise homoplasy index test [34] as implement in splitstree4 [66], and the homoplasy test by Maynard-Smith and Smith [35]. Further evidence for the presence of recombination and the number of nonrecombining blocks was provided by other methods (Text S6, Figures S3, S4, S5). Supporting Information Figure S1 To understand the broad phylogenetic diversity of House Finch and poultry MG strains, guide our choice of poultry strains for genomic sequencing and compare mutation rates in the HF and poultry MG population, we used DNA sequence data from Ferguson et al. [28] to generate a multisequence alignment for 82 MG strains collected from four host species (Turkey, Chicken, House Finch and Gold Finch). This data, henceforth the Large Sample Multiple Sequence Alignment, LS-MSA) was composed of four gene fragments (from pvpA, mgc2, gapA and an unnamed surface lipoprotein) that when concatenated yielded approximately 1.9 kb of sequence data per strain (with the exact Rapid Evolution of Mycoplasma gallisepticum length of each strain varying due to small indels). We added to this dataset sequences for 8 of the 12 House Finch MG strains sequenced in this study that had complete coverage for these gene fragments. The four strains from this study not incorporated into the dataset (TN_1996, GA_1995, AL_2001_53 and AL_2007_05) were excluded because there was not enough sequencing data to accurately assemble the relevant fragments. We also excluded 3 strains from the original work[28] where we could not identify the host-animal species, leaving 82 strains in the final multiple sequence alignment. In this alignment, all the House Finch haplotypes were identical, except for the 2007 strains that differed from the others at two adjacent nucleotide positions. Certain sections of the gene fragments in the LS-MSA were polymorphic due to insertions/deletions of tandem repeats, and because there is no clear criteria by which to assign the locations of these repeats in an alignment for phylogenetic purposes, for analysis purposes we reduced the ,1.9kb of sequence down to1,36 bp that could be confidently aligned. The tree shown is a phylogeny of 82 avian MG strains inferred from four concatenated gene-segments, totaling 1,363 bp, using Neighbor-joining in PHYLIP. Due to recombination in Mycoplasma gallisepticum, this single tree may not be completely representative of the organismal history of the strains from which the gene segments were sampled. However, the pattern showing poultry hosts interspersed amongst the leaves of the tree and high diversity within the MG population is also present in neighbor-joining trees separately inferred for each individual gene fragment, consistent with frequent host-shifts by MG. Strain K4366GF97_10 is from an American Goldfinch (Carduelis tristis), also a songbird and the chicken reference strain used to obtain the reference genome is R63_44. (EPS) Figure S2 Cross Validation of the 454 Sequencing Data with the Illumina Sequencing Data. Our dataset provides an opportunity to validate the SNP calls made with our 4X-19X coverage 454 data for the House Finch MG isolates by using the SNP calls made with the 294X coverage Illumina data that was generated for TK_2001. TK_2001 and the House Finch MG isolates (partic- ularly the pre-2001 isolates) are nearly genetically identical, and SNPs for both strains were called relative to the much more distantly related strain that was used to generate the reference genome. As outlined with the unrooted tree shown in this figure. This means that most of the SNPs called for each of the House Finch isolates should also be called for the TK_2001 strain, with any unmatched SNPs likely due to either genetic divergence between the two strains or SNP calling errors. The results of this comparison are shown in Table S4. For our most stringent threshold, of the up to 6,461 SNPs that were called in our pre- 2001 House Finch isolates, 99.7% of the SNPs called with the 454 data were also called with the Illumina data. This bounds the false positive rate for SNP calls in the 454 stringent data at 0.3%. However, we believe that this unmatched 0.3% is due to true genetic divergence between the strains and not sequencing errors, as these SNPs are very well supported. For example, all 21 SNPs in VA_1994 that did not match TK_2001 were supported by at least 9 reads that contained the variant, and often many more. Table S4 documents the robustness of our population genetic estimates on variations in SNP calling protocol, leading only to minor variations (,1%) in the false positive rate for our SNP datasets. This shows that almost all of the uncertainty in estimating the mutation rate from these genomes is due to the inherent sampling variability that naturally results from the stochastic process that generated them and is not due to any variability that comes from calling SNPs in these genomes. Additionally the ratio of PLoS Genetics | www.plosgenetics.org 9 February 2012 | Volume 8 | Issue 2 | e1002511