Telomeric assemblies of Paracoccidioides genomes

Paracoccidioides is a genus of dimorphic fungal pathogens endemic to Latin America. We generated long-read de novo assemblies for 11 isolates representing four species of the brasiliensis complex (P. brasiliensis, P. americana, P. restrepiensis, P. venezuelensis) and P. lutzii. These include the first complete telomere-to-telomere assemblies for P. brasiliensis (Pb18) and P. americana (Pb03), each with five chromosomes. Comparative analyses revealed chromosomal fusion and fission events distinguishing P. brasiliensis and P. americana, and a 90 kb tandem duplication in P. americana containing siderophore biosynthesis genes (sid1, sid3, sid4), a cluster of putative virulence factors. Mitochondrial genomes showed conserved gene order but a phylogenetic topology inconsistent with the nuclear tree, suggesting mitochondrial introgression between P. lutzii and P. venezuelensis. RNA transposable elements were enriched near telomeres, correlated with genome size, and most abundant in P. lutzii. These assemblies provide key resources for understanding genome evolution and introgression in Paracoccidioides. SIGNIFICANCESpecies of Paracoccidioides cause paracoccidioidomycosis, a systemic mycosis that remains a major public health problem in Latin America. Despite their clinical importance, genome evolution across the genus is poorly understood owing to the lack of complete reference assemblies. Here, we present the first telomere-to-telomere reference genomes for P. brasiliensis and P. americana, enabling a comprehensive comparison of chromosomal structure across the genus. Our analyses reveal that the nuclear genome is highly dynamic and shaped by large-scale rearrangements and structural variants, including the duplication of a siderophore biosynthesis-related gene cluster linked to virulence. In contrast, the mitochondrial genome is structurally conserved but shows introgression between species, revealing hidden evolutionary exchange. Together, these genomic resources redefine our understanding of Paracoccidioides evolution and provide a foundation for advances in molecular diagnostics, epidemiological surveillance, and studies of fungal pathogenicity.