different lineages given in Table S5. We grouped the strains into allele types in order to analyze the age of each ROP18 allele type separately (i.e. I*, II*, and III*). Additionally we compared the alleles pairwise to estimate their divergence. Since we were interested in determining the common ancestry between alleles, we included all synonymous mutations including those that define biallelic poly- morphisms between the alleles, as described previously [5]. The number of SNPs for the calculations was determined using equations presented in Table S5. Briefly, to determine ancestral vs. derived polymorphisms, we compared the T. gondii alleles to the coding region of ROP18 from the N. caninum out-group. Alleles shared with N. caninum were considered to represent the ancestral state. Where N. caninum has diverged from T. gondii (no shared allele) we used a majority rule (i.e. the ancestral allele was considered to the be the allele shared by two strain types). MRCA calculations were performed using the formula: t = S/ (maSnili+mbSnimi), where n is the number of lineages examined at the ith locus, li and mi are the number of 4-fold and 2-fold synonymous sites, S is the number of polymorphisms and ma and mb are the neutral mutation rates. Two estimates of the neutral mutation rate from the closely related parasite Plasmodium falciparum were used for calculating the MRCA, as described previously [53,54]. Analysis of ROP18 in Neospora caninum BLAST analysis identified a sequence 68% identical to the coding sequence of T. gondii ROP18 in the N. caninum genome database (http://www.sanger.ac.uk/sequencing/Neospora/cani- num/). The ROP18 coding and upstream flanking sequences from the Nc-1 strain of N. caninum (ATCC# 50843) were confirmed by manual sequencing as described above. ClustalX/W was used to align the sequences using default settings. Phylogenetic compar- isons were conducted on the coding regions of ROP18 under the criterion of minimum evolution in PAUP*4.0b [55] and by using the BioNeighbor-Joining algorithm. A consensus tree was drawn according to bootstrap 50% majority rule with Nc-1 as the root. Comparison of ROP18 and Flanking Regions from N. caninum and T. gondii Strains Genome sequence of N. caninum was obtained from The Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/ sequencing/Neospora/caninum/) by blasting the T. gondii ROP18 (ToxoDB gene id 20.m03896) and 2 flanking genes (20.m03897 and 20.m03895) (located on VIIa from1475116 to 1487487 bp) from ToxoDB (www.toxodb.org) [56]. This identified a ,30 kb syntenic region from N. caninum. Pairwise comparisons of T. gondii and N. caninum sequences from this 40 kb region were carried out using AlignX (a component of Vector NTI Suite 9.0.0) (InforMax, Invitrogen Life Science Software, USA). Similarity values (in a 0–1 range) were assigned to each residue at a given alignment position in References 1. Dubey JP, Beattie CP (1988) Toxoplasmosis of animals and man. Boca Raton: CRC Press. 220 p. 2. Howe DK, Sibley LD (1995) Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J Infect Dis 172: 1561–1566. 3. Darde´ ML, Bouteille B, Pestre-Alexandre M (1992) Isoenzyme analysis of 35 Toxoplasma gondii isolates and the biological and epidemiological implications. J Parasitol 78: 786–794. 4. Ajzenberg D, Cogne’ N, Paris L, Bessieres MH, Thulliez P, et al. (2002) Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis and correlation with clinical findings. J Infect Dis 186: 684–689. 5. Su C, Evans D, Cole RH, Kissinger JC, Ajioka JW, et al. (2003) Recent expansion of Toxoplasma through enhanced oral transmission. Science 299: 414–416. Population Strucure and Virulence of T. gondii each aligned sequence, depending on whether the residue was identical, similar, weakly similar, or different. Supporting Information Figure S1 Distribution of SNPs surrounding ROP18. Elevated levels of type III-specific single nucleotide polymorphisms (SNPs) occur within a 30 kb region of ROP18. A) Screen capture of the Genome browser view of ROP18 (20.03896) and flanking regions from position 1449000 to 1489000 bp on chromosome VIIa (http://www.toxodb.org/). Annotated genes are shown in colored rectangles with corresponding gene ids. SNPs were identified by NUCmer alignments of GT1 (type I) and VEG (type III) to ME49 (type II) whole genome sequences and corresponding predicted coding regions. Red, green and blue color diamonds indicate the type I, II, and III specific SNPs, respectively. B) Graphical representation of type I (red), II (green), and III (blue) specific SNPs (in 1 kb windows) present on 40 kb region surrounding the ROP18 gene. Type III-specific SNPs are elevated throughout but show a strong peak for ROP18. Found at: doi:10.1371/journal.pgen.1000404.s001 (0.39 MB PDF) Table S1 List of loci used for diversity comparison of T. gondii strains. Found at: doi:10.1371/journal.pgen.1000404.s002 (0.08 MB PDF) Table S2 Strains used in this study. Found at: doi:10.1371/journal.pgen.1000404.s003 (0.07 MB PDF) Table S3 Comparison of pNS and pS ratios for ROP18 alleles. Found at: doi:10.1371/journal.pgen.1000404.s004 (0.07 MB PDF) Table S4 Substitution rates and estimates of most recent common ancestor (MRCA) of the clonal lineages. Found at: doi:10.1371/journal.pgen.1000404.s005 (0.08 MB PDF) Table S5 Estimating the age of ROP18 based on synonymous substitutions. Found at: doi:10.1371/journal.pgen.1000404.s006 (0.08 MB PDF) Acknowledgments We thank John Wootton, J.P. Dubey, John Boothroyd, Henry Huang, and Michael White for helpful discussions, and Blima Fux, Keliang Tang and Julie Nawas for expert technical assistance. Preliminary sequence data were obtained from http://ToxoDB.org (for T. gondii strains) and from the Wellcome Trust Sanger Institute (for N. caninum). A complete summary of the haplotypes based on genes sequences here can be found at: http:// toxohapmap.wustl.edu/. Author Contributions Conceived and designed the experiments: AK ST LDS. Performed the experiments: AK ST. Analyzed the data: AK ST JWA BMR LDS. Contributed reagents/materials/analysis tools: JWA BMR. Wrote the paper: BMR LDS. 6. Lehmann T, Marcet PL, Graham DH, Dahl ER, Dubey JP (2006) Globalization and the population structure of Toxoplasma gondii. Proc Natl Acad Sci (USA) 103: 11423–11428. 7. Khan A, Fux B, Su C, Dubey JP, Darde ML, et al. (2007) Recent transcontinental sweep of Toxoplasma gondii driven by a single monomorphic chromosome. Proc Natl Acad Sci (USA) 104: 14872–14877. 8. Khan A, Bohme U, Kelly KA, Adlem E, Brooks K, et al. (2006) Common inheritance of chromosome Ia associated with clonal expansion of Toxoplasma gondii. Gen Res 16: 1119–1125. 9. Boyle JP, Rajasekar B, Saeij JPJ, Ajioka JW, Berriman M, et al. (2006) Just one cross appears capable of dramatically altering the population biology of a eukaryotic pathogen like Toxoplasma gondii. Proc Natl Acad Sci (USA) 103: 10514–10519. PLoS Genetics | www.plosgenetics.org 13 March 2009 | Volume 5 | Issue 3 | e1000404