New insights on taxonomy, phylogeny and population genetics of Leishmania (Viannia) parasites based on multilocus sequence analysis.

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.publishersversionpublishe

Genetic diversity in the Leishmania donovani complex.

The Leishmania donovani complex comprises four described species: L. donovani, L. archibaldi, L. infantum and L. chagasi. L. chagasi is the only New World species and has been considered similar to L. infantum, although some authors insist on maintenance of its independent species status. L. donovani has at least two major epidemiological subgroups whose relationships are poorly understood. In this thesis, molecular biological techniques were used to investigate the taxonomy and phylogenetic relationships within the L. donovani complex, with isoenzyme analysis (lEA) as reference technique. Random amplification of polymorphic DNA (RAPD) was used to provide anonymous genetic markers which allowed overall comparisons of genomes. Selected target genes and intergenic regions were also amplified by the polymerase chain reaction (PCR), namely the major surface protease (msp or gp63), the mini-exon and the ribosomal internal transcribed spacer (ITS). PCR products of intergenic regions between msp genes (ITG/CS and ITG/L), mini-exon and ITS were analysed by restriction fragment length polymorphism (RFLP). Phylogenies generated from each of the methods were compared with that of IEA. L. infantum and L. chagasi were found to be synonymous, whilst L. donovani was found to be more polymorphic than L. infantum and a fourth possible species in the complex, L. archibaldi, was not supported. Six genetic groups of strains were identified in the L. donovani complex, based on all DNA based analyses, which agreed with IEA typing. Pooled data from RFLP and RAPD analyses generated robust phylogenies which were congruent with ITG/CS RFLP and msp DNA sequence based phylogenies, but not with lEA phylogenies. The evolutionary history of the L. donovani complex is analysed in the light of the present results. The diverse typing methods were also evaluated and genetic markers suggested, that are applicable to classification and typing of L. donovani species and strains