Population Strucure and Virulence of T. gondii in ROP18 than in surface antigens (SAGs), GRA3 or housekeeping genes (Figure 3A). Such mutations were distributed throughout the ROP18 gene and were not due to changes in the usage of nucleotides or codons (data not shown). The ratio of pNS/pS substitutions, which indirectly measures the selective advantage of mutations incurring amino acid substitutions, was greater in ROP18 (,2.5 average pairwise comparison for I* vs. II*, I* vs. III*, and II* vs. III*)) than in SAG1 (,1.29 average pairwise comparison). This was especially evident when comparing ROP18I* and ROP18II* (3.54) while a higher proportion of S changes in ROP18III* resulted in lower ratios (average value of pNS/pS = 1.32 for ROP18III* vs. ROP18I* and ROP18III* vs. ROP18II*, (Table S3)). Phylogenic trees drawn based on S vs. NS differences illustrate the marked deficiency of synonymous changes differentiating ROP18I* and ROP18II* from each other (Figure 3B). These data imply markedly different selective pressures have shaped the divergence among these three ROP18 alleles over time. Deep divergence also characterizes other members of the ROP2 family, which contains a number of predicted pseudokinases of unknown function [31]. Although the ancestry described here for ROP18 may reflect that of the ROP2 family more generally, ROP18 is notable among this group for showing evidence of strong diversifying selection. ROP18 is also the only member of this group known to be catalytically active and directly implicated in virulence, hence it is reasonable to assume that selection on this locus had important functional consequences. ROP18I* Alleles Confer Acute Virulence in the Murine Model Previous studies have shown that ROP18 is under expressed in the type III lineage, providing a suitable background for testing Figure 3. Polymorphism of ROP18 between major alleles. A) Cumulative nonsynonymous (NS) and synonymous (S) polymorphisms in ROP18 are significantly higher than surface antigens, GRA3, or housekeeping genes. B) Phylogenetic analysis of ROP18 analyzed separately for nonsynonymous changes (left) and synonymous changes (right) reveals a strongly asymmetric pattern. The major difference between types I* and II* is due to nonsynonymous changes. Scales = substitutions/site. doi:10.1371/journal.pgen.1000404.g003 PLoS Genetics | www.plosgenetics.org 5 March 2009 | Volume 5 | Issue 3 | e1000404