New Insights on Taxonomy, Phylogeny and Population Genetics of Leishmania (Viannia) Parasites Based on Multilocus Sequence Analysis

Abstract

The Leishmania genus comprises up to 35 species, some with status still under discussion. The multilocus sequence typing (MLST)—extensively used for bacteria—has been proposed for pathogenic trypanosomatids. For Leishmania, however, a detailed analysis and revision on the taxonomy is still required. We have partially sequenced four housekeeping genes—glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), mannose phosphate isomerase (MPI) and isocitrate dehydrogenase (ICD)—from 96 Leishmania (Viannia) strains and assessed their discriminatory typing capacity. The fragments had different degrees of diversity, and are thus suitable to be used in combination for intra- and inter-specific inferences. Species-specific single nucleotide polymorphisms were detected, but not for all species; ambiguous sites indicating heterozygosis were observed, as well as the putative homozygous donor. A large number of haplotypes were detected for each marker; for 6PGD a possible ancestral allele for L. (Viannia) was found. Maximum parsimony-based haplotype networks were built. Strains of different species, as identified by multilocus enzyme electrophoresis (MLEE), formed separated clusters in each network, with exceptions. NeighborNet of concatenated sequences confirmed species-specific clusters, suggesting recombination occurring in L. braziliensis and L. guyanensis. Phylogenetic analysis indicates L. lainsoni and L. naiffi as the most divergent species and does not support L. shawi as a distinct species, placing it in the L. guyanensis cluster. BURST analysis resulted in six clonal complexes (CC), corresponding to distinct species. The L. braziliensis strains evaluated correspond to one widely geographically distributed CC and another restricted to one endemic area. This study demonstrates the value of systematic multilocus sequence analysis (MLSA) for determining intra- and inter-species relationships and presents an approach to validate the species status of some entities. Furthermore, it contributes to the phylogeny of L. (Viannia) and might be helpful for epidemiological and population genetics analysis based on haplotype/diplotype determinations and inferences.

Author Summary

Leishmania is a protozoan genus comprising many species, some associated with a human neglected disease called leishmaniasis. This parasite is found worldwide and is transmitted by sand flies, having numerous domestic and sylvatic animals as reservoirs. Leishmania is genetically and ecologically diverse and it has been argued that this has an impact on the epidemiology of the disease. Many typing methods have been proposed for the study of this diversity, although a generally agreed methodology is still required. Also, there is still a lack of consensus on the validity of some species. Multilocus sequence typing (MLST) is a method for studying the population structure and diversity of pathogens, but before an MLST scheme can be proposed it is essential to undertake a detailed analysis and selection of the sequences that are to be included in the system. Here, we sequenced four gene fragments of 96 L. ( Viannia) strains, representing most species from this subgenus. Our results showed a good agreement between the current species assignment and the multilocus sequence analysis. Evidence of genetic recombination was found and the phylogenetic relationships were determined. Overall the results point to the feasibility of an MLST scheme for Leishmania and indicate that the four gene fragments analyzed could form part of this typing system. This will certainly be a valuable approach for taxonomy, population genetics, and epidemiological studies of this pathogen.