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.
Highlights
Leishmania are the causative agents of leishmaniasis, which can present in different forms, from simple cutaneous to the deadly visceral disease, and are found in most tropical and sub-tropical regions
Our data support monophyly of all but one Brazilian L. (Viannia) species analyzed here and highlight the close relationships between L. braziliensis and L. guyanensis and the recombination events occurring in both species
The taxonomic validity of L. shawi has been questioned and the markers studied here suggested that L. shawi isolates were closely related to or were part of the L. guyanensis group
Summary
Leishmania are the causative agents of leishmaniasis, which can present in different forms, from simple cutaneous to the deadly visceral disease, and are found in most tropical and sub-tropical regions. DNA sequencing and PCR-RFLP of hsp genes have been shown to be promising for the identification of Leishmania parasites [5,6], too conserved for intra-specific diversity studies. Polymorphic markers, such as microsatellites, perform poorly at taxonomic levels higher than species, whilst most other genotyping methods rely on multicopy genes that are more difficult to analyze
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