Leishmania parasites cause a spectrum of clinical pathology in humans ranging from disfiguring cutaneous lesions to fatal visceral leishmaniasis. We have generated a reference genome for Leishmania mexicana and refined the reference genomes for Leishmania major, Leishmania infantum, and Leishmania braziliensis. This has allowed the identification of a remarkably low number of genes or paralog groups (2, 14, 19, and 67, respectively) unique to one species. These were found to be conserved in additional isolates of the same species. We have predicted allelic variation and find that in these isolates, L. major and L. infantum have a surprisingly low number of predicted heterozygous SNPs compared with L. braziliensis and L. mexicana. We used short read coverage to infer ploidy and gene copy numbers, identifying large copy number variations between species, with 200 tandem gene arrays in L. major and 132 in L. mexicana. Chromosome copy number also varied significantly between species, with nine supernumerary chromosomes in L. infantum, four in L. mexicana, two in L. braziliensis, and one in L. major. A significant bias against gene arrays on supernumerary chromosomes was shown to exist, indicating that duplication events occur more frequently on disomic chromosomes. Taken together, our data demonstrate that there is little variation in unique gene content across Leishmania species, but large-scale genetic heterogeneity can result through gene amplification on disomic chromosomes and variation in chromosome number. Increased gene copy number due to chromosome amplification may contribute to alterations in gene expression in response to environmental conditions in the host, providing a genetic basis for disease tropism.

Visceral leishmaniasis is a potentially fatal disease endemic to large parts of Asia and Africa, primarily caused by the protozoan parasite Leishmania donovani. Here, we report a high-quality reference genome sequence for a strain of L. donovani from Nepal, and use this sequence to study variation in a set of 16 related clinical lines, isolated from visceral leishmaniasis patients from the same region, which also differ in their response to in vitro drug susceptibility. We show that wholegenome sequence data reveals genetic structure within these lines not shown by multilocus typing, and suggests that drug resistance has emerged multiple times in this closely related set of lines. Sequence comparisons with other Leishmania species and analysis of single-nucleotide diversity within our sample showed evidence of selection acting in a range of surface-and transport-related genes, including genes associated with drug resistance. Against a background of relative genetic homogeneity, we found extensive variation in chromosome copy number between our lines. Other forms of structural variation were significantly associated with drug resistance, notably including gene dosage and the copy number of an experimentally verified circular episome present in all lines and described here for the first time. This study provides a basis for more powerful molecular profiling of visceral leishmaniasis, providing additional power to track the drug resistance and epidemiology of an important human pathogen.[Supplemental material is available for this article.]Leishmaniases are a complex of diseases that range from self-curing lesions to gross disfigurations and potentially deadly visceral disease. The diseases are caused by protozoan parasites that are transmitted by sandflies in 88 countries and infect an estimated 12 million people (www.who.int/leishmaniasis/en/). Parasites of the Leishmania genus are remarkably biologically, clinically, and epidemiologically diverse and present enormous differences in disease tropism. The mildest form is cutaneous leishmaniasis, which is caused by Leishmania major and other species, and is largely limited to lesions around the area of a sandfly bite-though a diffuse form can also occur. Disfiguring mucocutaneous leishmaniasis is due to the destruction of nasopharyngeal tissue by parasites such as L. braziliensis. More significantly, visceral leishmaniasis is caused by parasites of the L. donovani species complex that can spread to internal organs and cause death.In 2005, sequencing the genome of L. major identified 8311 protein-coding genes and provided a framework for future comparative genomic studies (Ivens et al. 2005). The genome elucidated the full structural architecture of Leishmania chromosomes, which includes an unusual pattern of genes distributed in large directional clusters. Subsequently, the genomes of L. braziliensis and L. infantum were described-the latter is a member of the L. donovani complex (Peacock et al. 2007). A detailed comparison of these first three Leishmania genomes re...

Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.

Leishmania donovani causes visceral leishmaniasis (VL), the second most deadly vector-borne parasitic disease. A recent epidemic in the Indian subcontinent (ISC) caused up to 80% of global VL and over 30,000 deaths per year. Resistance against antimonial drugs has probably been a contributing factor in the persistence of this epidemic. Here we use whole genome sequences from 204 clinical isolates to track the evolution and epidemiology of L. donovani from the ISC. We identify independent radiations that have emerged since a bottleneck coincident with 1960s DDT spraying campaigns. A genetically distinct population frequently resistant to antimonials has a two base-pair insertion in the aquaglyceroporin gene LdAQP1 that prevents the transport of trivalent antimonials. We find evidence of genetic exchange between ISC populations, and show that the mutation in LdAQP1 has spread by recombination. Our results reveal the complexity of L. donovani evolution in the ISC in response to drug treatment.DOI: http://dx.doi.org/10.7554/eLife.12613.001

Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.