Author Summary Documenting the evolutionary changes occurring in pathogens when they switch hosts is important for understanding mechanisms of adaptation and rates of evolution. We took advantage of a novel host–pathogen system involving a bacterial pathogen (Mycoplasma gallisepticum, or MG) and a songbird host, the House Finch, to study genome-wide changes during a host-shift. Around 1994, biologists noticed that House Finches were contracting conjunctivitis and MG from poultry was discovered to be the cause. The resulting epizootic was one of the best documented for a wildlife species, partly as a result of thousands of citizen science observers. We sequenced the genomes of 12 House Finch MG strains sampled throughout the epizootic, from 1994–2007, as well as four additional putatively ancestral poultry MG strains. Using this serial sample, we estimate a remarkably high rate of substitution, consistent with past implications that mycoplasmas are among the fastest evolving bacteria. We also find that an array of likely phage-derived sequences known as CRISPRs has degraded and ceased to recruit new repeats in the House Finch MG strains, as compared to the poultry strains in which it is diverse and rapidly evolving. This suggests that phage dynamics might be important in the dynamics of MG infection. series beginning immediately after the host switch, as well as an opportunity to directly measure the tempo and mode of evolution in a natural bacterial population whose genome is as yet uncharacterized. To characterize patterns of genomic change during its host switch between distantly related avian species, we sequenced whole genomes of 12 House Finch MG isolates from this 13-year time series, with four samples each from the beginning (1994– 1996), middle (2001) and recent (2007) periods (Table S1). In addition, to identify putative source strains as well to determine if differences between the House Finch MG strains and the ,1 Mb published reference Rlow strain from chicken [23] were ancestral or derived, we sequenced four additional strains from chicken and turkey based on phylogenetic analysis of a smaller multistrain data set (Figure S1). Our sequence, SNP filtering and between-platform cross-validation protocols yielded a high quality 756,552 bp alignment encompassing 612 genes (Tables S2, S3, S4, Text S1, Figure S2), and allowed us to monitor point substitutions, genomic indels, IS element insertions, and other changes across the entire genome (Figure 1), including the entire array of clustered regularly interspaced short palindromic repeats (CRISPR) of all 17 strains (finch and poultry isolates). Results Phylogenomic diversity of House Finch and poultry MG All House Finch MG samples were collected in the southeastern U.S. (Table S1), with an emphasis on the well studied population in Alabama [24,25]. The population structure of Eastern House Finches before the epizootic was virtually panmictic [26], suggesting that there is likely to be little geographic structuring of MG in the east, a hypothesis that could be tested with additional data. The 12 House Finch strains from the three time periods spanned the known temporal and phylogenetic diversity of this lineage, and included strains that have been used to study host response to pathogen infection in House Finches [27]. To determine genetic diversity and phylogenetic identity of putative source populations of the House Finch MG strains, and to aid in Rapid Evolution of Mycoplasma gallisepticum sampling chicken and turkey strains for sequencing, we first analyzed a previously published data set [28]. Phylogenetic analysis of 1,363 bp obtained from four genomic regions for a large sample (n = 82) of MG strains suggests that turkeys rather than chickens were the source of House Finch MG and that the MG lineage colonizing House Finches first passed multiple times among chickens and turkeys (Figure S2). Although this analysis suggests frequent host switches between chickens and turkeys, which diverged 28–40 MYA [29,30], it also suggests a single switch to the House Finch, a songbird species diverged from chickens by ,80 MYA [31]. The whole genome alignment contained strong signals of a founder event as a result of colonization of House Finches. The total nucleotide diversity (p) in the House Finch strains for the four-gene region was only 3.1% of the diversity in circulating poultry strains prior to the epizootic, and only 2.3% of the poultry diversity when considering the entire House Finch MG genome [28] (Figure 2 and Table S5). In agreement with the four-gene analysis, our whole genome sequencing showed that the four sequenced poultry isolates were much more genetically diverse than the 12 House Finch isolates, possessing a total of 13,175 SNPs as compared to only 412 SNPs among the House Finch isolates (Table S2). The House Finch MG diversity corresponds to p = 0.00014, or roughly 1 SNP every 1,800 bp. Consistent with purifying selection acting over the longer time period encompass- ing the divergence of House Finch and poultry MG strains (as opposed to acting after the host-switch among House Finch strains alone), there was a stronger bias against non-synonymous substitutions among the more diverged poultry strains than among the recently diverged House Finch MG strains (Table S6). Across the entire genome, only 147 (35%) of the SNPs among the House Finch isolates were phylogenetically informative; the majority (265 or 64%) appeared as singletons. To further quantify House Finch MG demography, we used a statistical model, the Bayesian skyline plot implemented with BEAST, that utilizes information on dates of sampling to estimate changes in genetic diversity through time [32,33] (Text S2). The analysis is broadly consistent with field observations suggesting a mid-1990s origin followed by rapid population expansion, though it estimates that the House Finch MG lineages coalesced roughly in 1988, several years prior to the observation of sick birds in the field (estimated MRCA of the House Finch MG strains is 19.2 years prior to 2007 [95% HPD 16.9 – 21.7]; Figure 2d). Discrepancies between coalescence times and observed outbreaks in host populations have been observed for other pathogens, and could possibly be due to selective or demographic effects, or in our case low sample size [12]. Phylogenetic analysis suggests substantial turnover in the standing SNP variation between sampling intervals, with strong clustering of the 2007 strains, which are distinguished from other House Finch strains by 85 diagnostic SNPs (Figure 3). We found that one of the sequenced turkey strains, TK_2001, was highly similar in sequence to the House Finch strains and shares a number of genomic deletions and transposon insertions as well as duplications and losses of CRISPR spacers (see below) with the House Finch MG strains. This turkey strain may represent a poultry lineage close to the source lineage for House Finch MG (Figure 3). In addition to SNPs in House Finch MG we found five large genomic deletions that occurred by 2007 and amounted to ,42, 245 bp and encompassing 34 genes relative to the chicken Rlow strain (Figure 1 and Figure 3, Table S7). Three of these deletions are phylogenetically informative among the 17 MG strains (Table S7), but their conflicting phylogenetic distribution underscores the presence of recombination (see next section). Two deletions PLoS Genetics | www.plosgenetics.org 2 February 2012 | Volume 8 | Issue 2 | e1002511