High-quality genome assemblies provide new insights into the genomic architecture, adaptation, and virulence of neurotropic dematiaceous fungi implicated in cerebral phaeohyphomycosis.

Neurotropic dematiaceous fungi are primary agents of cerebral phaeohyphomycosis, a life-threatening brain infection with high mortality. However, the genomic basis underlying their virulence, stress tolerance, and antifungal resistance is poorly understood. In this study, we present high-quality hybrid genome assemblies of three major neurotropic dematiaceous fungi, Cladophialophora bantiana, Fonsecaea monophora, and Cladosporium cladosporioides, using Nanopore long-read and Illumina short-read sequencing platforms. The assembled genomes ranged from 31.5 to 39.9 Mb, with high completeness (>98.9%). Functional annotation revealed diverse coding and non-coding elements associated with stress responses, iron metabolism, and antifungal resistance. The Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses uncovered metabolic versatility, enriched xenobiotic degradation pathways, and lineage-specific functional divergence. Notably, C. bantiana and F. monophora exhibited greater genomic plasticity, higher transposable element content, and broader repertoires of virulence factors, extracellular peptidases, and secondary metabolite biosynthetic gene clusters, suggesting enhanced pathogenic potential. All three genera harbored conserved stress tolerance mechanisms, melanin biosynthesis pathways, and pathogenicity-related genes linked to immune evasion and neuroinvasion. Additionally, we identified distinct multidrug efflux transporter families linked to antifungal resistance. Orthology analysis revealed a shared core proteome alongside genus-specific adaptations likely underpinning niche specialization. While the findings highlight critical genomic features driving fungal resilience and neurotropism, functional validation through transcriptomics and phenotypic assays remains essential. Despite current limitations in experimental tractability, this work provides a foundational resource for understanding the molecular basis of fungal pathogenesis and offers valuable targets for future diagnostic and therapeutic strategies against cerebral phaeohyphomycosis and related infections.