High-quality genome assemblies and comparative genomics of Leishmania (Viannia) species

Leishmaniasis affects over 350 million people worldwide, caused by Leishmania parasites including the Viannia subgenus comprising nine Americas-endemic species causing cutaneous and mucocutaneous disease. High-quality genomic resources remain limited for most Viannia species, hindering evolutionary and epidemiological studies.

This study generated chromosome-level genome assemblies for eight Viannia strains and five additional Leishmania species using Oxford Nanopore Technologies long-read sequencing with Illumina polishing. The assembly pipeline employed NECAT, followed by multi-stage polishing using Medaka, Racon, and Pilon. Comparative genomics across 63 trypanosomatid species used OrthoFinder for ortholog identification and phylogenetic reconstruction. Genomic ancestry was analyzed using ADMIXTURE with 21,530 genome-wide variants, focusing on putative hybrid strain MHOM/VE/1991/PM-H197. Strains were sourced from a biological collection; eight underwent a promastigote–amastigote–promastigote differentiation process with five passages prior to DNA extraction, while the remainder were analysed from pre-extracted DNA of unrecorded passage history.

Generated assemblies substantially improved upon existing references, achieving N50 values of 563-1,144 kb and >90% BUSCO completeness for euglenozoan gene sets. Comparative analysis identified 16,683 orthogroups, revealing minimal core genome conservation (5.06%) and extensive accessory genome diversity (53.76%). Phylogenetic analysis confirmed four major Leishmania lineages supporting subgeneric classifications.

Most significantly, strain PM-H197 exhibited 80% L. mexicana complex ancestry and 20% Viannia ancestry, representing the first genomic characterization of intersubgeneric Leishmania hybridization. However, Oxford Nanopore coverage analysis revealed contrasting 2:1 Viannia genomic dominance, creating an 8-fold discrepancy with ancestry predictions. Ultra-long read competitive mapping demonstrated 96% Viannia preference, supporting unbalanced hybrid or mixed infection scenarios.

This work provides the most comprehensive Viannia genomic dataset to date, establishing new parasitic genome assembly benchmarks. The discovery of intersubgeneric genetic exchange challenges current understanding of Leishmania reproductive barriers with important implications for population genetics, epidemiology, and diagnostics. These resources provide essential foundations for future leishmaniasis research and therapeutic development.