Analysis of DNA Polymorphisms. Sequencing of introns was conducted on PCR-amplified templates from 46 representative strains by using BigDye cycle sequencing (Applied Biosystems, Foster City, CA) (performed by SeqWright DNA Technology Services, Houston, TX). Primers used for sequencing are listed in SI Table 1; primary data are summarized at www.toxohapmap.wustl.edu. Clustal (21) was used to align the sequences that were analyzed by using PAUP*4.0B (22). Unrooted phylogenetic comparisons were conducted with distance and parsimony methods. The conditions were set to distance (mean character difference, minimum evolution, negative branches = 0), and 1,000 bootstrap replicates were performed by using the BioNeighbor-Joining algorithm. Alternatively, parsimony analysis was conducted by heuristic stepwise searching, with bootstrapping for >1,000 replicates. Consensus trees were drawn with an arbitrary root according to the bootstrap 50% majority rule. Pairwise FST values between NA, E, and SA T. gondii strains were calculated in Arlequin Ver 3.01 with 10,000 permutations (23).
LD. Intron sequences from different loci were combined in a single file and analyzed for LD across the entire composite sequence. Haplotype blocks and LD plots were constructed by using DnaSP 4.0 (24). D' was calculated for all pairs of sites. Both the two-tailed Fisher's exact test and the 2 test were computed to determine significance of the associations between polymorphic sites. The average LD was estimated by using the Zns statistic (13), which averages LD over all pairwise comparisons for S polymorphisms in N sequences.
Analysis of Populations. Genotype data for unlinked markers (introns) were used to infer ancestral population patterns by using the program STRUCTURE 2.0 (25) run under the assumption of linkage with independent allele frequencies. Population sizes (K) of 2–10 were tested by five runs each using 105 burn in iterations followed by 106 iterations using the Markov Chain Monte Carlo method. The highest-probability runs were selected and compared for log likelihood and lambda, the allele frequency, to estimate the most probable model.
Sequence Analysis of Chr1a Blocks. Twelve scattered blocks of 800–900 bp were chosen from a group of 26 previously characterized regions of ChrIa (8). Each region was amplified with gene-specific primers and sequenced from 30 representative strains (primary data are summarized at: www.toxohapmap.wustl.edu). Sequences were aligned in Clustal and classified based on SNP profile.
Analysis of Apicoplast Regions. Sequences were determined from three loci within the circular apicoplast genome (NC_001799 [GenBank] ) from the segments called AP1, AP2, and AP3 for a total of 1,685 bp per strain for 35 strains. Primers used for sequencing are listed in SI Table 1; primary data are summarized at www.toxohapmap.wustl.edu. Sequences were aligned by using Clustal, and the output nexus file was analyzed by using TCS 1.21 (26) to determine the probability of parsimony for pairwise differences. A graphical output file of the resulting network was generated with missing intermediates and displayed by using the VGJ 1.0.3 drawing tool (http://www.eng.auburn.edu/department/cse/research/graph_drawing/graph_drawing.html).
Age Calculations. Estimates of the time to MRCA were measured from the rates of polymorphism between strains by using several estimates of the neutral mutation rate from the closely related parasite Plasmodium falciparum, as described (7). For estimating the common ancestry between northern and southern strains, we compared all strains by using the biallelic polymorphisms present in each of these regions. For estimating the ancestry of the clonal lineages, we pooled and compared those haplogroups that were dominant in the North vs. those in the South and removed the ancestral biallelic SNPs.
Animal Infections. Virulence of T. gondii strains was monitored after i.p. inoculation of parasites grown in culture as described (16). Oral transmission of bradyzoites from tissue cysts was monitored as described (7). Further details are found in SI Table 2.