Network analysis of the ROP18 genes from the complete set of strains confirmed that the sequences cluster as three discrete nodes (Figure 7A). To estimate when the major ROP18 alleles began diverging, we employed a model based on the accumulation of synonymous mutations and estimated the time since a common ancestry based on several assumptions for an average neutral mutation rate. These analyses revealed that the ROP18 III* allele shared a common ancestor ,10 million years ago, while ROP18I* and ROP18II* diverged ,1 million years ago (Figure 7A, Table S5). Although these age estimates are based on synonymous sites that are not expected to be under direct selection, it is still possible that diversifying selection acting on nearby nonsynonymous sites could have inflated the coalescence estimate. Despite uncertainty about the absolute ages of these alleles, two periods diversification are nonetheless evident. Over a relatively long initial period, primarily neutral substitutions accumulated between ROP18 III* and the common ancestor of ROP18I* and ROP18II*. A later period of diversifying selection drove expansion of ROP18I* and ROP18II* to differ markedly in their amino acid composition Population Strucure and Virulence of T. gondii (pNS/pS ratio = 4.66) (Figure 7A, Table S3). The majority of extant haplogroups (8 of 11 studied here) are typified by ROP18I*, demonstrating that this allele has successfully expanded since its relatively recent origin. A Model for Evolution of Virulence by Alterations at a Single Locus Our data are consistent with a model that the common ancestor of the more closely related ROP18I* and ROP18II* alleles lost the expression-suppressing UPS that occurs in ROP18III* and in the out-group homologue, ROP18Nc-1 (Figure 7B). The resulting DNA deletion or rearrangement lead to upregulation of ROP18 expression in ROP18I* and ROP18II* alleles. Subsequent expansion of these alleles substantially shaped the population structure of T. gondii. The highly virulent ROP18I* allele predominates among known haplogroups, especially those in South America, whereas the type II* allele (conferring interme- diate virulence) typifies most human and animal infections in North America and Europe. Each of the three highly divergent Figure 7. Evolutionary history of ROP18: changes in expression levels of ROP18 correlate with acute virulence. A) Network analysis of ROP18 reveals two different periods of evolution. A long period of nearly neutral evolution differentiated type III* from the ancestor of types I* and II*. More recently, the types I* and II* diverged under a period of strong selection. Acute virulence is associated with the successful expansion of the type I* alleles. Table includes proportion of nonsynonymous (pNS)/synonymous (pS) substitutions for ROP18 (see also Table S3 for additional ratios). Age estimates are based on synonymous changes in ROP18 from the data in Table S5. B) Out-group rooting using the ROP18 homologue in Neospora caninum confirms that the type III* lineage has undergone a longer period of independent evolution than have types I* and II*, which shared a more recent common ancestor. Neighbor joining analysis based on the coding regions of ROP18. Bootstrap values based on 1000 replicates. Color bars in the ROP18 models for each strain type denote the positions of allele-specific SNPs. doi:10.1371/journal.pgen.1000404.g007 PLoS Genetics | www.plosgenetics.org 10 March 2009 | Volume 5 | Issue 3 | e1000404