An improved understanding of how a parasite species exploits its genetic repertoire to colonize novel hosts and environmental niches is crucial to establish the epidemiological risk associated with emergent pathogenic genotypes. Trypanosoma cruzi, a genetically heterogeneous, multi-host zoonosis, provides an ideal system to examine the sylvatic diversification of parasitic protozoa. In Bolivia, T. cruzi I, the oldest and most widespread genetic lineage, is pervasive across a range of ecological clines. High-resolution nuclear (26 loci) and mitochondrial (10 loci) genotyping of 199 contemporaneous sylvatic TcI clones was undertaken to provide insights into the biogeographical basis of T. cruzi evolution. Three distinct sylvatic parasite transmission cycles were identified: one highland population among terrestrial rodent and triatomine species, composed of genetically homogenous strains (Ar = 2.95; PA/L = 0.61; DAS = 0.151), and two highly diverse, parasite assemblages circulating among predominantly arboreal mammals and vectors in the lowlands (Ar = 3.40 and 3.93; PA/L = 1.12 and 0.60; DAS = 0.425 and 0.311, respectively). Very limited gene flow between neighbouring terrestrial highland and arboreal lowland areas (distance ~220 km; FST = 0.42 and 0.35) but strong connectivity between ecologically similar but geographically disparate terrestrial highland ecotopes (distance >465 km; FST = 0.016–0.084) strongly supports ecological host fitting as the predominant mechanism of parasite diversification. Dissimilar heterozygosity estimates (excess in highlands, deficit in lowlands) and mitochondrial introgression among lowland strains may indicate fundamental differences in mating strategies between populations. Finally, accelerated parasite dissemination between densely populated, highland areas, compared to uninhabited lowland foci, likely reflects passive, long-range anthroponotic dispersal. The impact of humans on the risk of epizootic Chagas disease transmission in Bolivia is discussed.

Table S1: Panel of Bolivian T. cruzi TcI biological clonesPanel of Bolivian T. cruzi biological clones assembled for analysis.Table_S1_Panel_of_Bolivian_T_cruzi_TcI_biological_clones.docxTable S2: Panel of microsatellite loci and primersPanel of microsatellite loci and primers employed in this study.Table_S2_Panel_of_microsatellite_loci_and_primers.docxConcatenated maxicircle sequence alignmentTen mitochondrial gene fragments sequenced and concatenated from 78 T. cruzi TcI biological clones.Mitochondrial maximum-likelihood phylogenyMaximum-likelihood tree constructed from concatenated maxicircle sequences for 78 sylvatic Bolivian TcI clones and 24 additional TcI isolates from across the Americas.Microsatellite neighbour-joining phylogenyUnrooted Neighbour-Joining tree based on DAS values between MLGs generated from 199 sylvatic Bolivian TcI clones.Supplementary File S1 T cruzi microsatellite alleles at 26 lociMicrosatellite allele sizes amplified at 26 loci across 199 TcI biological clones.