Ultrafast Evolution and Loss of CRISPRs Following a Host Shift in a Novel Wildlife Pathogen, Mycoplasma gallisepticum Nigel F. Delaney1, Susan Balenger2, Camille Bonneaud1¤a, Christopher J. Marx1, Geoffrey E. Hill2, Naola Ferguson-Noel3, Peter Tsai4, Allen Rodrigo4¤b, Scott V. Edwards1* 1 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, United States of America, 2 Department of Biological Sciences, Auburn University, Auburn, Alabama, United States of America, 3 Poultry Diagnostic and Research Center, University of Georgia, Athens, Georgia, United States of America, 4 Bioinformatics Institute, University of Auckland, Auckland, New Zealand Abstract Measureable rates of genome evolution are well documented in human pathogens but are less well understood in bacterial pathogens in the wild, particularly during and after host switches. Mycoplasma gallisepticum (MG) is a pathogenic bacterium that has evolved predominantly in poultry and recently jumped to wild house finches (Carpodacus mexicanus), a common North American songbird. For the first time we characterize the genome and measure rates of genome evolution in House Finch isolates of MG, as well as in poultry outgroups. Using whole-genome sequences of 12 House Finch isolates across a 13-year serial sample and an additional four newly sequenced poultry strains, we estimate a nucleotide diversity in House Finch isolates of only ,2% of ancestral poultry strains and a nucleotide substitution rate of 0.821.261025 per site per year both in poultry and in House Finches, an exceptionally fast rate rivaling some of the highest estimates reported thus far for bacteria. We also found high diversity and complete turnover of CRISPR arrays in poultry MG strains prior to the switch to the House Finch host, but after the invasion of House Finches there is progressive loss of CRISPR repeat diversity, and recruitment of novel CRISPR repeats ceases. Recent (2007) House Finch MG strains retain only ,50% of the CRISPR repertoire founding (1994–95) strains and have lost the CRISPR–associated genes required for CRISPR function. Our results suggest that genome evolution in bacterial pathogens of wild birds can be extremely rapid and in this case is accompanied by apparent functional loss of CRISPRs. Citation: Delaney NF, Balenger S, Bonneaud C, Marx CJ, Hill GE, et al. (2012) Ultrafast Evolution and Loss of CRISPRs Following a Host Shift in a Novel Wildlife Pathogen, Mycoplasma gallisepticum. PLoS Genet 8(2): e1002511. doi:10.1371/journal.pgen.1002511 Editor: David S. Guttman, University of Toronto, Canada Received August 13, 2011; Accepted December 9, 2011; Published February 9, 2012 Copyright: 2012 Delaney et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Science Foundation (http://www.nsf.gov/) grant DEB-0923088 to GEH and SVE, a NSF graduate fellowship to NFD, and a Milton Grant through Harvard University to SVE. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sedwards@fas.harvard.edu ¤a Current address: Station d’Ecologie Expe´rimentale du CNRS a Moulis USR 2936 Moulis, Saint-Girons, France ¤b Current address: National Evolutionary Synthesis Center, Durham, North Carolina, United States of America Introduction Populations of animals are under constant threat from bacterial pathogens, which can be particularly destructive following a switch to a new host or the evolution of novel virulence mechanisms. Understanding the rate and process of evolutionary change in pathogens is thus important to assessing the risks of pandemics and developing means to predict and avoid such catastrophic events. In 1994, a strain of Mycoplasma gallisepticum (MG) was identified as the causative agent of an emerging epizootic in House Finches, a wild songbird inhabiting Eastern North America [1]. This bacterial pathogen frequently causes disease in commercial chicken and turkey flocks, but it had never been reported in House Finches or any songbird, leading to the suggestion that the epidemic began when MG expanded its host range from poultry to this phylogenetically distant songbird. MG prevalence reached 60% in some areas, and killed an estimated 225 million finches in the first three years after detection [2]. The early detection of the epizootic allowed research and citizen-science teams to track its rapid spread throughout eastern North America in exceptional detail, making it one of the best documented wildlife pathogen outbreaks [3–7]. Although previous genome-wide studies have clarified rates of measurable evolution in viral pathogens [8,9] and in bacterial populations evolving under laboratory conditions or as human pathogens [10–18], less is known about rates of genetic change in bacterial pathogens of non-mammalian vertebrates, particularly on short evolutionary time scales. Genome-wide and gene-specific estimates of point substitution in bacterial lineages measured over centuries [19] to millions of years [20] suggest maximum substitution rates on the order of 1027 to 1029 per site per year. Although recent work suggests the rate may be even faster for several bacterial species [12,14,19], the number of studies documenting whole-genome changes in bacteria during host switches is still small, particularly for wildlife pathogens [21,22]. Asx part of ongoing surveillance, field isolates of MG obtained from infected finches were sampled at multiple time points from the start of the epidemic in 1994 to 2007, providing a genetic time PLoS Genetics | www.plosgenetics.org 1 February 2012 | Volume 8 | Issue 2 | e1002511