alleles of ROP18, conferring markedly different virulence levels in laboratory mice, have been maintained over long periods of time, suggesting that they confer selective advantages in particular environmental circumstances. Strains harboring ROP18I* have clearly enjoyed recent evolutionary success, as they characterize 8 of 11 haplogroups studied here, including a number of recently derived subgroups that appear to be expanding. However, it is doubtful that the acute virulence as expressed in the laboratory mouse model would be adaptive in natural hosts. Laboratory mice, while a convenient model, are likely more susceptible than many species that are natural hosts of T. gondii. For example, other rodents (Peromyscus spp., Rattus spp.) survive infections with type I strains of T. gondii and develop chronic infection [34,35], promoting eventual transmission to other hosts. Thus, a reasonable hypothesis is that while ROP18I* leads to mortality in especially vulnerable hosts, such as the laboratory mouse, it instead facilitates the establish- ment of infection in resistant hosts, thus enhancing transmission. By contrast, type II strains are intermediate in virulence in laboratory mice, express a proclivity for developing into tissue cysts [36], and modulate host immune responses [25,37,38]. Collec- tively, such adaptations may promote the establishment of chronic infection (although this genotype has also been associated with immunopathology in several models [39,40]). Clearly enhanced pathogenicity is not always advantageous to parasite fitness. Adaptation to an alternative niche may explain the long-term persistence of lineages expressing ROP18III*. While the ROP18III* allele is expressed at very low levels, it is not a pseudogene and despite its very high divergence, it preserves all of the essential residues in the S/T kinase domain necessary for activity (data not shown). This suggests that low levels of expression may be an adaptation for infection in some particular hosts, potentially those that are highly susceptible to infection. Defining the precise advantages of each of these alleles will require further studies of the transmission between natural hosts and a better understanding of the population genetic structure in the wild. Mathematical modeling studies predict that pathogens may evolve greater virulence provided that doing so does not reduce their potential for transmission [41]. A clear relationship between enhanced growth, transmission, and virulence has previously been demonstrated in other pathogens, including malaria [42]. Typically the underlying molecular mechanisms for such adaptations are not known. Our findings indicate that changes in the expression of a single gene, that has undergone strong diversifying selection, has markedly increased virulence as monitored in laboratory mice. Understating pathogenic determinants of T. gondii in model systems, will facilitate future studies to more precisely evaluate the contribution of parasite genotype and specific genes, to transmission in natural hosts and potentially for human disease. Materials and Methods Comparison of T. gondii Gene Diversity Sequences of 32 loci from representative genomes of clonal lineages type I, II and III, were downloaded from the T. gondii genome database (www.toxodb.org). These 32 loci include surface antigens, secretory proteins such as rhoptries, and dense granules, as well as housekeeping genes and introns that are presumed not to be undergoing positive (diversifying) selection (Gene ids given in Table S1). Clustal X/W [43] was used to align the sequences to calculate the percentage diversity, expressed as changes per 100 bp. The proportion of synonymous and nonsynonymous changes in coding regions were calculated using the Synonymous Nonsynonymous Analysis Program (SNAP) [44]. Neighbor- Population Strucure and Virulence of T. gondii Joining phylogenetic trees were constructed using Molecular Evolutionary Genetic Analysis (MEGA) Version 3.1 [45]. Sequencing and Phylogenetic Analysis of T. gondii Strains Twenty five strains representing the 11 previously described haplogroups of T. gondii found in North America, Europe and South America were chosen for analysis [7]. These isolates were selected from more than 300 that have been previously genotyped based on the following criteria: known source (host and geography), passage history, mutlilocus genotyping and assignment to haplogroups, phenotypic data in animals. Parasites were propagated as tachyzoites in monolayers of human foreskin fibroblasts (HFF) [5] and lysates were made for PCR-based sequencing [7]. The frequency of single nucleotide polymorphisms (SNPs) were determined by sequencing three classes of loci: 1) noncoding regions consisting of 8 introns from 5 unlinked loci, 2) the housekeeping genes b-TUB and TgActin and 3) the surface antigen genes SAG1 and SAG2 in addition to coding regions of ROP18 (Gene ids given in Table S1). Sequencing was conducted on PCR amplified templates from each strain using BigDye cycle sequencing (Applied Biosystems, Foster City, CA). Sequences were aligned with Clustal X/W [43] and phylogenetic trees were derived using Neighbor-Joining in Molecular Evolutionary Genetics Analysis (MEGA), Version 3.1 [45]. P-distances were calculated for sequence pairs after removal of insertions and deletions in 1000 bootstrap replicates. Consensus trees were drawn with an arbitrary root and a scale length of 0.0005 substitutions/site. Cumulative Values of Synonymous and Nonsynonymous Changes Coding sequences were analyzed for cumulative values of polymorphisms using the Synonymous Nonsynonymous Analysis Program (SNAP) [44], based on the method of Nei and Gojobori 1986 [46] for conducting all pairwise comparisons of sequences in an alignment, and incorporating a statistic developed by Ota and Nei 1994 [47]. Ambiguous codons and insertions were excluded. Phylograms of ROP18 based only on either synonymous or nonsynonymous substitutions were performed using maximum likelihood analysis in HyPhy [48] to infer rates of heterogeneity and positive selection. These analyses were conducted assuming the codon frequency matrix MG94xHKY85_3x4 and using default setting for other values. Cloning of ROP18 Alleles The ROP18 gene was PCR amplified from genomic DNA of the T. gondii strains RUB [21], CAST (ATCC# 50868) and MAS (ATCC# 50870) and epitpoe tagged at the C-terminus using the Ty-epitope tag, detected by mAb BB2 [49]. Plasmids expressing C-terminal Ty-tagged ROP18 alleles were expressed under the control of the TUB promoter in the pTUB-Ty vector described previously [27]. The CTG strain (type III) was electroporated with 100 mg of pROP18I* plasmid DNAs and 10 mg of pSAG1/ble/ SAG1 in cytomix buffer, as described previously [50]. Clones were isolated by single cell cloning in 96-well plates containing HFF cells following two rounds of selection with phleomycin [50]. Individual clones were screened for expression of the Ty tag by immunofluorescence and western blotting. A parallel transfection containing only the backbone plasmid was used to isolate a control transformant (denoted as CTG-Ble). Expression Analyses of ROP18 Alleles ROP18 was localized in transgenic parasites by immunofluore- sence labeling using the mAb BB2 to the Ty tag [49] followed by PLoS Genetics | www.plosgenetics.org 11 March 2009 | Volume 5 | Issue 3 | e1000404