Selection at a Single Locus Leads to Widespread Expansion of Toxoplasma gondii Lineages That Are Virulent in Mice Asis Khan1., Sonya Taylor1., James W. Ajioka2, Benjamin M. Rosenthal3, L. David Sibley1* 1 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America, 2 Department of Pathology, University of Cambridge, Cambridge, United Kingdom, 3 Animal Parasitic Disease Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland, United States of America Abstract Pathogenicity differences among laboratory isolates of the dominant clonal North American and European lineages of Toxoplasma gondii are largely controlled by polymorphisms and expression differences in rhoptry secretory proteins (ROPs). However, the extent to which such differences control virulence in natural isolates of T. gondii, including those from more diverse genetic backgrounds, is uncertain. We elucidated the evolutionary history and functional consequences of diversification in the serine/threonine kinase ROP18, a major virulence determinant in the mouse model. We characterized the extent of sequence polymorphism and the evolutionary forces acting on ROP18 and several antigen-encoding genes within a large collection of natural isolates, comparing them to housekeeping genes and introns. Surprisingly, despite substantial genetic diversity between lineages, we identified just three principal alleles of ROP18, which had very ancient ancestry compared to other sampled loci. Expression and allelic differences between these three alleles of ROP18 accounted for much of the variation in acute mouse virulence among natural isolates. While the avirulent type III allele was the most ancient, intermediate virulent (type II) and highly virulent (type I) lineages predominated and showed evidence of strong selective pressure. Out-group comparison indicated that historical loss of an upstream regulatory element increased ROP18 expression, exposing it to newfound diversifying selection, resulting in greatly enhanced virulence in the mouse model and expansion of new lineages. Population sweeps are evident in many genomes, yet their causes and evolutionary histories are rarely known. Our results establish that up-regulation of expression and selection at ROP18 in T. gondii has resulted in three distinct alleles with widely different levels of acute virulence in the mouse model. Preservation of all three alleles in the wild indicates they are likely adaptations for different niches. Our findings demonstrate that sweeping changes in population structure can result from alterations in a single gene. Citation: Khan A, Taylor S, Ajioka JW, Rosenthal BM, Sibley LD (2009) Selection at a Single Locus Leads to Widespread Expansion of Toxoplasma gondii Lineages That Are Virulent in Mice. PLoS Genet 5(3): e1000404. doi:10.1371/journal.pgen.1000404 Editor: Takashi Gojobori, National Institute of Genetics, Japan Received November 5, 2008; Accepted February 3, 2009; Published March 6, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: Supported by grants from the NIH (AI036629, AI059176 to L.D.S) and ARS project 1265-140-11 (to BMR). The funders had no role in the 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: sibley@borcim.wsutl.edu . These authors contributed equally to this work. Introduction Toxoplasma gondii provides a valuable model for studying the evolution of pathogens because it is widespread and infects virtually all warm-blooded vertebrates, including companion and agricultural animals. This remarkably successful parasite is easily acquired by ingesting either oocysts (excreted by cats) or tissue cysts (in undercooked meat) [1]. These different modes of transmission allow efficient spread via water or food borne ingestion and result in very high prevalence rates of chronic infection in animals and humans [1]. Despite the presence of a sexual cycle, which occurs only in cats, the population structure can be remarkably clonal, with just three highly similar lineages predominating in North America and Europe [2–4]. These lineages were previously referred to as types I, II, and III (or groups 1, 2, and 3) and are referred to here as ‘‘the clonal lineages’’. Estimates of the common ancestry of these clonal lineages indicates that a dramatic genetic sweep lead to the successful expansion of these lineages during the past ,10,000 years [5]. In contrast, distinct strains from South America are more genetically variable, reflecting both asexual and sexual propagation. Phylogenetic and population genetic analyses indicate that northern and southern strains diversified during geographic separation over the past several million years [6,7]. The three clonal lineages in the North share a monomorphic version of chromosome Ia, which appears to have arisen coincident with their origin, and which has more recently penetrated into the South [7,8]. Comparison of interstrain polymorphisms suggests the clonal lineages originated from only a few genetic crosses between highly related parents (1–2% divergence), engendering an unusual pattern of biallelism at most loci [9]. Within these otherwise highly homogeneous genomes, a small number of polymorphic genes encode variable surface antigens (SAGs) and antigenic proteins secreted from rhoptries (ROP) or dense granules (GRA) [10–13]. Despite their genetic similarities, the northern clonal PLoS Genetics | www.plosgenetics.org 1 March 2009 | Volume 5 | Issue 3 | e1000404