Genomic Flexibility Through Extrachromosomal Amplifications: A <i>Leishmania</i> Survival Strategy

Abstract Leishmania parasites modulate gene copy number through extrachromosomal DNA (ecDNA) amplification, enabling adaptation to environmental stress. Under drug pressure, both linear and circular ecDNA amplifications (amplicons) carrying resistance genes emerge. However, how these ecDNA structures form, diversify, and coexist remains poorly understood. Here, using experimental evolution and Oxford Nanopore long-read sequencing, we show that a single clonal population of drug resistant Leishmania produces a variety of linear and circular amplicons. As antimonial pressure increases, linear amplicons transition into circular forms, with high-stress conditions favoring circular amplicons carrying at least two copies of the resistance gene. Using the Nanopore long reads, we map recombination events driving linear and circular amplicon formation. Our model suggests that gene duplication in the amplicons originates from inter-chromatid homologous recombination, leading to an intermediate intra-chromosomal duplication, followed by a second homologous recombination event. Additionally, different Leishmania species exhibited distinct biases toward linear or circular amplification under identical drug conditions, suggesting species-specific adaptive strategies. Together, these findings define recombination-driven ecDNA dynamics as a central axis of genomic plasticity in Leishmania and underscore the potential for targeting ecDNA in therapeutic and diagnostic strategies against Leishmania and related pathogens.