Population Strucure and Virulence of T. gondii Figure 2. Phylogenetic analysis reveals ROP18 is highly divergent relative to other loci. A) Combined analysis of 8 introns from 25 strains defines 11 major haplogroups of T. gondii that show strong geographic segregation. Separate gene trees for the antigens SAG1 and SAG2 show overall lower diversity of alleles but approximate the same level of divergence seen in the combined intron tree. B) The highly polymorphic gene GRA3 shows deeper branching of multiple major alleles, which are not associated with geographic region or virulence. C) In contrast the much higher genetic divergence of ROP18 is partitioned into only three major alleles (denoted by I*, II*, and III*). Neighbor-joining analysis with 1000 bootstraps (values shown at major nodes). Color-coded by continent of origin: North America-Europe (blue), South America (red), widespread (denoted as Pan, black). doi:10.1371/journal.pgen.1000404.g002 understand whether alleles of ROP18 have experienced atypical evolutionary pressures, we classified the extent of synonymous (S) vs. nonsynonymous (NS) mutations at this and several other genes, including housekeeping genes (i.e. actin and b-tubulin), the surface antigens (i.e. SAG1, SAG2), and GRA3. This comparison was based not only on the clonal lineages, but on all strains that were grouped by their ROP18 alleles into major types as defined in Figure 2B. Both S and NS substitutions were much more frequent PLoS Genetics | www.plosgenetics.org 4 March 2009 | Volume 5 | Issue 3 | e1000404