SNP calling protocol and whether poultry or House Finches were used as the host for sampled sequences. Given the history and genetic isolation of the House Finch MG strains, the influence of recombination or lateral gene transfer on our estimate of substitution rate is likely also minimized (Text S7). Rapid evolution and degradation of CRISPRs The CRISPR dynamics we observed in House Finch MG differ from that seen in other pathogen and bacterial populations. A recent study of Y. pestis CRISPR arrays from 131 strains [51] indicated a slower pace of CRISPR evolution than observed in MG and pattern of evolution in which acquisition of novel sequences does not play a prominent role. This study found that in Y. pestis the first part of the CRISPR arrays were conserved and that over 76% of all spacer sequences derived from within the Y. pestis genome. Similarly, a recent study of E. coli and Salmonella genomes found that strains within 0.02% divergence typically have identical CRISPR loci [52] and that spacer sequences were often matched to elements of the E. coli genome. Additionally, some spacer sequences were shared between strains within a species exhibiting over 1% sequence divergence. These observations and an estimated substitution rate on the order of 10210 per site per year suggested that E. coli strains that had diverged for 1,000 years sometimes shared identical CRISPR loci, suggesting patterns of evolution different from that expected for a rapidly changing adaptive immune system primed to combat phages, a conclusion that was supported by later work [53]. By contrast to the pattern seen in these c-proteobacteria, none of the House Finch MG strains in this study have the same CRISPR locus despite differing at only 0.01–0.02% of sites and likely having last shared a common ancestor less than 20 years ago. Our serial sampling suggests that the loss of spacer sequences and the CRISPR system itself can take place on very short time scales in Mycoplasma. Unlike the patterns seen in E. coli, Y. pestis, and Salmonella, the poultry MG strains in our study did not share any spacer sequences, even though they differed by ,1%. These strains had very large CRISPR arrays and 99% of all spacer sequences did not match any known sequence in their genome or in the databases. Therefore the MG CRISPR loci studied here differ from the those observed in some c-proteobacteria, a group for which CRISPR dynamics can appear functionally unrelated to ecology or immunity [53–55]. Instead, our finding of rapid evolution and degradation of the CRISPR loci more closely resembles patterns found in other bacterial groups, particularly those in which CRISPR is involved in phage defense [56]. CRISPRs are found in only 40% of sequenced bacteria investigated thus far, and often have major roles in bacterial immunity in several lineages investigated in detail [45]. We were surprised to find a gradual degradation and ultimate apparent functional loss of the CRISPR system in House Finch MG after the host switch and a shift in CRISPR dynamics appears to be a major correlate of host switch in this system. One possible explanation for this pattern is that MG experienced release from its ancestral phage parasite community (or other mobile genetic elements such as plasmids) following introduction into the House Finch. Loss of traits upon removal of the agent of selection is a common evolutionary response, as are population expansions of animals and plants when introduced into novel habitats unaccompanied by their parasites [57]. Despite the large amount of ecological research focusing on this host-pathogen system [3–7], at present nothing is known about phages that infect MG or their role in its evolutionary dynamics. Therefore the hypothesis of parasite release as a driver of CRISPR loss is purely speculative. We know of no phage known to infect the Rapid Evolution of Mycoplasma gallisepticum Pneumoniae phylogenetic group of mycoplasmas and the few phages known to infect Mycoplasma have proven difficult to characterize [58]. We might expect Mycoplasma bacteriophages to be host-specific given that they seem to be unusual in their ability to bind to a bacterium with no cell wall and a diverse assortment of surface proteins [58]. However, we are not aware of even basic data on the degree to which Mycoplasma might be susceptible to the many bacteriophages that they presumably encounter in their environ- ment. Although phage represent one possible source for these novel ,30 bp sequences, another possible explanation for the source of the spacer sequences is that they derive from plasmids. Although unprecedented (we know of no examples of a naturally occurring plasmid in the Pneumoniae mycoplasmas), such a scenario could raise the possibility of easier genetic manipulations in MG where development of such tools has been challenging [59]. Of the many other possibilities that could explain the observed degradation of the CRISPR loci, we can at least rule out self-interference as an explanation in derived MG strains, given that there is only a single CRISPR cluster in House Finch MG [54]. Measurement of costs, possible advantages and consequences of CRISPR loss, as well as functional and evolutionary assays and surveys of phage diversity will help determine if the rapid and deadly spread of Mycoplasma following their expansion into the House Finch was facilitated by a lack of phage predation, a short-term advantage of CRISPR degradation or some other, possibly neutral, mechanism. Although our sequence data is suggestive, explicit functional studies will also be required to demonstrate CRISPR functionality or lack thereof in poultry and House Finch MG and its role, if any, in phage defense. Pseudogenization and possible mutator strains Genome evolution of MG during its host-switch from poultry to House Finches adds to a growing list of host-switches that are successful in the complete absence of novel genes [21,60,61] and bacterial lineages exhibiting high rates of point substitution [14]. Mycoplasmas are some of the fastest evolving organisms on earth [62] having lost many of the repair mechanisms present in other bacteria [38] and this high mutation rate could help introduce deleterious mutations and contribute to the substantial level of pseudogenization that was observed in this study. The high basal substitution rate in MG may well be elevated yet further by UvrB mutation that we detected, a mutation that could have consequences for the long term genomic integrity of this MG lineage, particularly if it remains genetically distinct from and unable to exchange genes with the poultry MG lineages with a functional UvrB. Alternatively, given the short (3 amino acid) truncation of this gene in the House Finch strains, another explanation for the greatly increased number of doublet mutations in the lineage carrying the UvrB truncation is that selection has not had enough time to remove them as it has for poultry strains without this mutation. Although mutator strains are known to have a selective advantage in rapidly evolving laboratory and natural populations [39,63], additional functional and experimen- tal work will be required to determine the selective and functional effect of the mutation we have detected in UvrB, and over what time scales such selective effects might persist. For this and other endeavors, serial sampling of additional bacterial populations in nature will further clarify the rate at which genomes are remolded during host switches in the wild. Materials and Methods Sampling of House Finch and poultry MG strain diversity DNA sequence data for 4 gene fragments collected from 74 strains in Ferguson et. al. [28], was combined with data from 8 PLoS Genetics | www.plosgenetics.org 8 February 2012 | Volume 8 | Issue 2 | e1002511