Toxoplasma gondii is an obligate intracellular protozoan parasite with a haploid genome. Toxoplasma has the capacity to infect any warm-blooded animal and infects between 30-80% of humans and 47% of wildlife surveyed globally. Although this parasite possesses an exceedingly flexible replication strategy, with both a prevalent asexual replication cycle in any intermediate host, and a fecund sexual cycle capable of producing in excess of 100 million infectious oocysts exclusively in its definitive felid host; the extent to which each replication method is utilized to transmit this generalist parasite in nature has long been under debate. Sexual recombination in Toxoplasma is an advantageous trait that allows the parasite to admix population genetic diversity to generate new biological potential, such as the ability to invade new hosts, evade host immunity, or cause outbreaks. However, despite its fecund sexual cycle, Toxoplasma has a genetically limited population structure that is dominated by just a few clonal lines that circulate predominantly in North America and Europe. Two theories exist to explain this clonal population structure, the first suggests that the clonal lines are highly adapted for oral transmission among intermediate hosts and are expanding exclusively asexually within this population of hosts, the second states that the clonal lines are particularly fit clones that are being expanded in cats by uniparental self-mating due to Toxoplasma’s lack of mating types and its ability to undergo unisexual expansion via single clone infection in the definitive host. In support of the latter theory, fungal pathogens have been shown previously to cryptically expand certain clones when sexual recombination occurs between closely related strains. The recombinant progeny produced are typically indistinguishable from each other due to the use of limited, low-resolution markers that fail to capture the genetic differences between the two mating clones. During the tenure of this thesis, studies have been published that chart Toxoplasma global population genetic diversity, but no study has examined intra-typic (i.e., within a clonal lineage) population genetics at whole genome resolution. Work shown here interrogates the most prevalent clonal lineage characterized worldwide, Type II, and the most prevalent clonal lineage in North America, Type X. Work here aims to determine at whole genome resolution the intratypic genetic relationship within two previously reported clonal lineages: Type II and Type X (also referred to as HG12), and whether strains within these two clonal groups isolated across different geographies and animal host species, are evolving via sexual recombination or genetic drift. Strains studied in this thesis were examined by limited sequencing at established Toxoplasma genotyping markers, as well as by whole genome sequencing, which has only recently come into use to interrogate genetic diversity and determine the extent to which recombination is occurring within the genome of this parasite. Type II strains were identified to have undergone limited mitotic drift within the lineage, and the polymorphism detected was generally correlated with the region of the strains’ geographic isolation. However, distinct haploblocks of Type II sequences bearing the hallmarks of different geographic regions were identified in a subset of Type II isolates, and these admixtures suggested that intra-typic recombination (unisexual mating) was occurring between Type II strains of distinct geographies. Proof of unisexual mating between two highly homologous, but independent clones, was identified phylogenetically by an incongruence between nuclear and maternally inherited organellar genomes among these Type II strains. Importantly, genotyping using just ten low resolution PCR-RFLP markers generally predicted that the strain belonged to the Type II lineage, because relatively few admixtures between Type II and other genetic backgrounds were identified after increasing the resolution to WGS. Although at first glance this appears to support a model whereby Type II strains bypass their sexual cycle, and are expanding largely asexually, closer examination of the haploblocks of geographically shared polymorphism identified a number of Type II strains that possessed different admixtures of these Type II, geographically derived haploblocks. This observation was parsimonious with recombination among cladespecific genotypes and supports expansion by unisexual mating. The other clonotype supported by low-resolution PCR-RFLP analyses, and interrogated here, Type X, is the most prevalent genotype infecting wildlife in North America. This thesis performed an unprecedented, longitudinal study of isolates collected over a 7-year period from a single host species, Southern sea otters, in a localized geographic region. In contrast to the Type II lineage, outcrossing was especially pronounced among the Type X strains examined. In fact, Type X was determined to be a clade of sexually-related progeny from at least one sexual cross between a Type II ancestral strain and a previously unknown strain of mosaic ancestry, referred to as g/d. When infected in mice, Type X strains displayed a range of virulence phenotypes from highly virulent to avirulent, like a Type II strain. Because these natural isolates resembled recombinant progeny, the Type X strains were utilized as if they were true F1 progeny from a sexual cross and a QTL was performed to identify genes conferring mouse virulence. The analysis identified a novel virulence gene, ROP33. Evidence from whole genome sequencing demonstrated that while Type X strains resembled closely related progeny, mitotic drift had occurred within these strains prior to their infection in sea otters. However, the observed mitotic drift, in combination with the clear and limited number of crossover break points in Type X, supports a model in which only a limited number of crosses between Type II and a strain of distinct ancestry occurred to generate the Type X strains, but that the Type II strain that crossed with the g/d parent was divergent. Two mouse virulent Type X strains were engineered to be drug resistant and crossed with a cat-competent Type II ME49 strain to perform a forward genetic screen to identify the murine virulence loci. Rather than selecting progeny that were double-drug resistant, progeny from these crosses were individually isolated prior to drug selection and then tested to establish whether they were recombinant. Progeny isolated in this unbiased manner allowed for a more accurate determination of the degree to which clones derived from meiosis undergo outcrossing versus self-mating. In four independent genetic crosses, the relative recombination rate was low (approximately 1-3%), rather than the expected 25% rate indicating that in all instances, self-mating was favored. Taken together, the evidence from the Type II and Type X population genetic analyses, as well as from several independent sexual crosses between these two clonal lineages, established that sexual outcrossing is more prevalent than previously envisaged, but that the majority of clones derived from sexual replication closely resemble a single parental type that was amplified by self-mating. And in geographies with predominantly clonal population genetic structures, it is clear that unisexual mating between closely-related strains will be underestimated and not considered as a major contributor to maintaining the clonal population genetic structure. As a consequence, whole genome typing is required to resolve differences between strains within a clonal group, which are preferentially admixing with each other.
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The Role of Sexual Recombination in the Evolution of the Protozoan Parasite, Toxoplasma gondii
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