Comparative Phylogenomics Reveals Conserved Protoilludene Synthase Neighbourhoods and Unevenly Distributed Biosynthetic Gene Cluster Families in Armillaria.

Fungal biosynthetic gene clusters (BGCs) offer a genomic entry point for natural-product discovery, but predicted regions alone neither establish pathway activity nor identify metabolites. Armillaria is a useful model because chemically rich, forest-associated species span pathogenic and saprotrophic lifestyles. We analysed nine assemblies in a role-aware design: seven independent taxa with high completeness assessed using Benchmarking Universal Single-Copy Orthologs (BUSCO), an A. mellea BUSCO duplication-sensitivity sample and an A. solidipes intraspecific strain control for A. ostoyae. The analysis resolved a terpene-rich repertoire containing a shared but edge-robust gene cluster family (GCF) component alongside unevenly distributed candidate families. Targeted neighbourhood analysis recovered protoilludene synthase (PRO1) orthologs in all assemblies (92.2-100.0% amino-acid identity); the core neighbourhood was retained across the seven primary taxa, whereas flanking tailoring-associated genes varied. Notably, the A. gallica PRO1 ortholog lay 23,614 bp outside the nearest antiSMASH region. Thus, whole-region prediction can obscure pathway-centred conservation in basidiomycetes. The resulting framework prioritises reproducible candidates while reserving chemical identity, expression and ecological function for direct validation.