Cryptic sex in Leishmania depends on SPO11 paralogs.

Genetic exchange in Leishmania is established, yet the molecular mechanisms enabling hybrid formation in sand flies remain poorly defined. In Leishmania, as in plants and several protists, two paralogs of the conserved meiotic endonuclease SPO11 are present, but their contribution to hybridization is unknown. Here, we dissect the roles of SPO11-1 and SPO11-2 during in vivo sand fly infections using targeted gene deletions, catalytically-dead mutants, expression analyses, and genome-wide characterization of hybrid progeny. We show that both SPO11 paralogs are essential for efficient hybridization: deletion of either paralog in both parents abolishes hybrid recovery, and catalytic inactivation fails to rescue mating. When only one parent lacks SPO11-1 or SPO11-2, hybrid formation is reduced in a strain-dependent manner, revealing asymmetric requirements for each paralog. Genomic analysis of hybrids from SPO11-deficient crosses reveals polyploidy and altered parental genome contributions, including unbalanced and near-balanced triploid configurations, indicating disrupted reductional processes. Together, these results establish SPO11-dependent DNA break formation as a core requirement for Leishmania hybridization and define distinct, strain-specific roles for the two SPO11 paralogs.