Analyses of 32 Loci Clarify Phylogenetic Relationships among Trypanosoma cruzi Lineages and Support a Single Hybridization prior to Human Contact

Machado CA (2011) Analyses of 32 Loci Clarify Phylogenetic Relationships among Trypanosoma cruzi Lineages and Support a Single Hybridization prior to Human Contact. PLoS Negl Trop Dis 5(8): e1272. doi:10.1371/journal.pntd.0001272 Analyses of 32 Loci Clarify Phylogenetic Relationships among Trypanosoma cruzi Lineages and Support a Single Hybridization prior to Human Contact Carlos A. Flores-Lo pez 0 Carlos A. Machado 0 Peter J. Myler, SBRI, United States America 0 Department of Biology, University of Maryland , College Park, Maryland , United States of America Background: The genetic diversity of Trypanosoma cruzi, the etiological agent of Chagas disease, has been traditionally divided in two major groups, T. cruzi I and II, corresponding to discrete typing units TcI and TcII-VI under a recently proposed nomenclature. The two major groups of T. cruzi seem to differ in important biological characteristics, and are thus thought to represent a natural division relevant for epidemiological studies and development of prophylaxis. To understand the potential connection between the different manifestations of Chagas disease and variability of T. cruzi strains, it is essential to have a correct reconstruction of the evolutionary history of T. cruzi. Methodology/Principal Findings: Nucleotide sequences from 32 unlinked loci (.26 Kilobases of aligned sequence) were used to reconstruct the evolutionary history of strains representing the known genetic variability of T. cruzi. Thorough phylogenetic analyses show that the original classification of T. cruzi in two major lineages does not reflect its evolutionary history and that there is only strong evidence for one major and recent hybridization event in the history of this species. Furthermore, estimates of divergence times using Bayesian methods show that current extant lineages of T. cruzi diverged very recently, within the last 3 million years, and that the major hybridization event leading to hybrid lineages TcV and TcVI occurred less than 1 million years ago, well before the contact of T. cruzi with humans in South America. Conclusions/Significance: The described phylogenetic relationships among the six major genetic subdivisions of T. cruzi should serve as guidelines for targeted epidemiological and prophylaxis studies. We suggest that it is important to reconsider conclusions from previous studies that have attempted to uncover important biological differences between the two originally defined major lineages of T. cruzi especially if those conclusions were obtained from single or few strains. - Funding: This work was conducted using setup funds from the University of Maryland. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Trypanosoma cruzi is the etiological agent of American Trypanosomiasis, also known as Chagas disease. Recent estimates suggest that about 15 million people in Latin America are infected with this parasite, and 12 to 20 thousand people die every year of the disease [1]. In nature, the parasite has two different cycles: a sylvatic cycle in which T. cruzi cycles between triatomines and wild mammalian reservoirs (e.g. opossums, raccoons, armadillos), and a domestic cycle in which T. cruzi infects humans through domiciliated triatomines [2,3]. Since the 1980s the genetic variability and population structure of T. cruzi have been extensively characterized with a wide array of genetic markers [4,5,6,7,8,9,10,11,12,13,14]. Three main conclusions have been drawn from these studies: 1) T. cruzi has a mainly clonal mode of reproduction [5,6,12], although historical and experimental evidence of sporadic genetic exchange has been uncovered [9,10,15,16,17,18,19,20,21,22,23,24]. 2) The genetic variability of T. cruzi can be divided in two major groups [7,25,26,27,28,29], originally termed T. cruzi I and T. cruzi II [30]. T. cruzi II was additionally divided in 5 distinct subgroups or stable discrete typing units (DTUs IIa-IIe) [8,31]. 3) DTUs IId and IIe are hybrids, the result of recent genetic exchange between ancestors of lineages IIb and IIc [10,19]. Although a new intraspecific nomenclature was recently proposed [32], renaming the six major T. cruzi DTUs (I, IIa-IIe) as TcI-TcVI, no changes in the inferred division of T. cruzi in the two major evolutionary groups T. cruzi I (DTU TcI) and T. cruzi II (DTUs TcII-VI)) were implied or proposed. The two major groups of T. cruzi seem to differ in important biological characteristics (e.g. pathogenicity in mice, doubling time of epimastigotes in vivo, susceptibility to drugs), and thus are thought to represent a natural division relevant for epidemiological studies and development of prophylaxis [33,34,35]. For instance, in the southern region of South America, where Chagas disease is most devastating, it has been observed that T. cruzi II strains (TcIIVI) are usually responsible for human infections, whereas T. cruzi I strains (TcI) are usually associated with the sylvatic cycle [36,37,38,39,40,41]. Further, in regions north of the Amazon basin T. cruzi I strains are the main cause of Chagas disease, although the most acute manifestations of the disease are Trypanosoma cruzi is the protozoan parasite that causes Chagas disease, a major health problem in Latin America. The genetic diversity of this parasite has been traditionally divided in two major groups: T. cruzi I and II, which can be further divided in six major genetic subdivisions (subgroups TcI-TcVI). T. cruzi I and II seem to differ in important biological characteristics, and are thought to represent a natural division relevant for epidemiological studies and development of prophylaxis. Having a correct reconstruction of the evolutionary history of T. cruzi is essential for understanding the potential connection between the genetic and phenotypic variability of T. cruzi with the different manifestations of Chagas disease. Here we present results from a comprehensive phylogenetic analysis of T. cruzi using more than 26 Kb of aligned sequence data. We show strong evidence that T. cruzi II (TcII-VI) is not a natural evolutionary group but a paraphyletic lineage and that all major lineages of T. cruzi evolved recently (,3 million years ago [mya]). Furthermore, the sequence data is consistent with one major hybridization event having occurred in this species recently (, 1 mya) but well before T. cruzi entered in contact with humans in South America. seemingly less common than in the southern cone of South America where most research on the disease has been conducted [22,38,42]. Thus, the current consensus is that T. cruzi II strains (TcII-VI) are more pathogenic to humans than T. cruzi I strains (TcI), although at least one author has clearly stated that the six DTUs (TcI-VI) should be considered the only relevant units of analyses for epidemiol (...truncated)