Degredation and apparent functional loss of CRISPR loci in House Finch MG In some bacterial systems, CRISPRs have a well-recognized function in bacterial immunity and defense against phage, although they may possess additional functions, such as gene regulation [41–44]. We extensively catalogued CRISPR repeats in the House Finch and ancestral poultry strains (Figure 5, Text S8, Table S12). In so doing we observed drastic changes in the CRISPR system between House Finch and poultry strains (Figure 5) [45–48]. The House Finch MG strains from 1994–96 contain up to 50 unique spacers, none of which is shared with the four divergent poultry genomes, which each contained a unique set of 36 to 147 spacer regions consistent with a high rate of turnover for a population actively acquiring new spacer sequences. We found that less than 1% of the 302 unique spacer sequences had similarity to any sequences in the House Finch MG genomes and that none of the remaining spacers had any similarity to sequences in Genbank, indicating an external source for these sequences (Text S8). Surprisingly, no novel spacer elements are present in any of the House Finch MG samples or TK_2001, indicating that the CRISPR array ceased recruiting additional spacers around the time of host switch into the House Finch. In fact, over the 13-year period of the epizootic, the number of unique spacers present in the CRISPR array of the samples decreased to 28 (Figure 5). Further evidence for degradation of the CRISPR locus following the host switch is the complete loss of the four CRISPR-associated (i.e. ‘‘CAS’’) genes in all of the 2007 isolates, a loss that likely renders the CRISPR system in House Finch MG non-functional [45]. Rapid Evolution of Mycoplasma gallisepticum Discussion Rapid substitution rate We conducted whole-genome sequencing on a unique 13-year serial sample of Mycoplasma strains circulating in wild House Finches to characterize genomic changes accompanying a host shift from poultry in the mid-1990s as well as to obtain a very high substitution rate for this avian pathogen. Previous estimates using serial samples and/or the known timing of events presumably tied to the divergence of bacterial strains have generally found much lower rates. An estimate of 2.061026 was obtained for Staphyloccous aureus [12], 1.161027 for Buchnera [19], 7.4261027 in Yersinia pestis and 1.461026 in Heliobacter pylori [14]. Disentangling the effects of recombination and point substitution can be challenging and some previously published substitution rates are likely to be upper bounds rather than point estimates [12]. Our estimate appears to be among the highest reported for a bacterium, and is consistent with other reports of exceptionally high substitution rates in mycoplasmas [37]. Estimates of substitution rates can be influenced by the interval over which sequences are sampled, with estimates taken from short time intervals often exceeding those taken on biogeographic or geological time scales [49]. However the small number of SNPs that we detected segregating in House Finch MG populations suggest negligible effects of multiple hits on our estimate, and our use of a coalescent model suggests that effects of ancestral polymorphism on substitution rate estimates should be adequately accounted for [32,50]. Additionally, our estimates of substitution rate were robust to many potential complicating factors, including Figure 5. Evolution of the CRISPR locus in Mycoplasma gallisepticum isolates collected from House Finches, chickens, and turkeys. Numbers by each strain indicate the number of repeats in each CRISPR array. The ancestral 71-repeat CRISPR array of the chicken MG strain is shown in simplified form at bottom. Diagnostic CRISPR repeats for House Finch MG isolates are indicated in repeat-specific patterns. The black ovals signify the cluster of four CRISP-associated (CAS) genes, which are deleted in the 2007 strains. The tree at left is broadly consistent with the tree based on SNPs (Figure 3) but emphasizes strain clusters indicated by rare genomic changes and CRISPR deletions; it was constructed as described in Text S3. doi:10.1371/journal.pgen.1002511.g005 PLoS Genetics | www.plosgenetics.org 7 February 2012 | Volume 8 | Issue 2 | e1002511