Population Strucure and Virulence of T. gondii Figure 1. Diversity among T. gondii genes varies by category of coding function. A) Sequence diversity of genes and intronic sequences between representatives of type I (GT1), type II (ME49) and type III (VEG) strains of T. gondii. Loci listed at the bottom in rank order, annotations in Table S1. B) Ratio of pNS/pS as an indication of positive selection. Pairwise comparisons of sequences between strains as indicated. C) Neighbor joining trees reconstructed from variation in the five most divergent genes. Scale = substitutions per site. doi:10.1371/journal.pgen.1000404.g001 lineages of T. gondii [26,27], yet its role in the pathogenesis of natural isolates remains unknown. To compare the genetic diversity of ROP18 in T. gondii, we characterized 25 representative isolates representing the 11 previously defined haplogroups, sampled from animals and humans in North America, Europe, and South America (Table S2). Genetic diversity in ROP18 was compared within eight introns from five unlinked loci, two surface antigen genes (i.e. SAG1 and SAG2), the secreted protein GRA3, and two house keeping genes (i.e. actin and b-tubulin). Phylogenetic analysis of the intron sequences revealed 11 main haplogroups, which occupy particular geographic ranges (Figure 2A), consistent with previous findings [7]. Although individual gene tress reconstructed from the less variable SAG1 and SAG2 sequences resolved fewer lineages, these were generally concordant with the topology established from intron sequences (Figure 2A). GRA3, which was one of the most highly polymorphic genes identified in Figure 1, showed greater divergence than the SAGs (Figure 2B). The several identified alleles of GRA3 did not correlate with either geographic distribution or virulence in murine infections (Figure 2B). The extent and distribution of variation evident in ROP18 alleles contrasts sharply with the patterns seen in other genes. Firstly, only three major lineages of ROP18 are evident, each typifying one of the three clonal lineages that predominate in North America and Europe (designated as ROP18I*, ROP18II*, and ROP18III* alleles) (Figure 2C). Secondly, diversity in ROP18 is an order of magnitude greater than that of SAG1 and SAG2, which themselves may experience diversifying selection [11]. Finally, the vast majority of South American isolates share the type I* allele characteristic of the highly virulent North American lineage. In total, members of 8 different haplogroups were found to express ROP18I* alleles. Collectively, these findings reveal that the history of ROP18 is very different from that of the rest of the genome, resulting in the survival of only three, highly divergent allelic types. Polymorphism in ROP18 Is Asymmetrically Distributed Divergence between the clonal lineages extends for approxi- mately 30 kilobase pairs (kb) surrounding ROP18, the peak divergence occurring in the coding sequence (Figure S1). To better PLoS Genetics | www.plosgenetics.org 3 March 2009 | Volume 5 | Issue 3 | e1000404