Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy

Abstract

Leishmaniasis is a geographically widespread severe disease, with an increasing incidence of two million cases per year and 350 million people from 88 countries at risk. The causative agents are species of Leishmania, a protozoan flagellate. Visceral leishmaniasis, the most severe form of the disease, lethal if untreated, is caused by species of the Leishmania donovani complex. These species are morphologically indistinguishable but have been identified by molecular methods, predominantly multilocus enzyme electrophoresis. We have conducted a multifactorial genetic analysis that includes DNA sequences of protein-coding genes as well as noncoding segments, microsatellites, restriction-fragment length polymorphisms, and randomly amplified polymorphic DNAs, for a total of approximately 18,000 characters for each of 25 geographically representative strains. Genotype is strongly correlated with geographical (continental) origin, but not with current taxonomy or clinical outcome. We propose a new taxonomy, in which Leishmania infantum and L. donovani are the only recognized species of the L. donovani complex, and we present an evolutionary hypothesis for the origin and dispersal of the species. The genus Leishmania may have originated in South America, but diversified after migration into Asia. L. donovani and L. infantum diverged approximately 1 Mya, with further divergence of infraspecific genetic groups between 0.4 and 0.8 Mya. The prevailing mode of reproduction is clonal, but there is evidence of genetic exchange between strains, particularly in Africa.

Fig. 1.

Unrooted maximum-parsimony tree (one of 16 equally parsimonious trees) inferred from the combined data set of DNA sequences, microsatellites, and RFLP data (17,831 characters, 287 parsimony informative characters). All characters are given equal weight; numbers above branches are percent bootstrap values based on 1,000 replicates. Length of the tree is 773 steps. VL, visceral leishmaniasis; CL, cutaneous leishmaniasis; PKDL, post-kala azar dermal leishmaniasis. (Scale bar, 10 steps.)

Fig. 2.

Statistical parsimony network (TCS software, Version 1.21) based on a concatenated data set of 10 enzyme-coding genes (12,694 nt, 37 parsimony informative characters). Circles with strain designation represent different haplotypes. The short crosslines represent mutational steps between haplotypes. Heterozygous positions were not considered for the network construction.

Fig. 3.

Maximum-likelihood γ-corrected tree constructed under the clock model, used for divergence time estimates (Mya). Numbers at nodes denote age inferred by the clock-like Langley-Fitch method (r8s software) with calibration points inferred from the gapdh and rpoII data sets (upper and lower values).

Fig. 4.

Origin and dispersal of Leishmania. A predecessor of L. donovani group and L. major would have evolved from monoxenous parasites of insects in South America ≈46–36 Mya and moved to Asia via the Bering land bridge (yellow line). The ancestor of the L. donovani complex diverged from other Leishmania species ≈14–24 Mya (red line). This predecessor arrived in central Asia and ≈1 Mya diverged into European L. infantum, African L. donovani and Indian/Kenyan L. donovani. L. infantum was later introduced in South America by European settlers, whereas L. donovani, represented here by strain LG16, would have been transferred by immigrants/slaves from India to Kenya and/or vice versa. Time estimates based on our data are in gray; those in white are taken from published literature.