Phylogenetically diverse Mucorales–Mycetohabitans endosymbiotic interactions identified from whole-genome sequencing using a targeted metagenomic assembly pipeline

Endosymbiotic bacteria of the genus Mycetohabitans are obligate intracellular associates of Mucorales fungi, yet the understanding of their diversity, distribution and evolutionary dynamics is in its infancy. By screening 1,696 public sequencing datasets from Mucorales fungi, we detected Mycetohabitans in 46 fungal accessions spanning 5 host taxa across the fungal genera Rhizopus and Apophysomyces . These included 13 previously unreported associations. Genome reconstruction yielded 38 Mycetohabitans metagenome-assembled genomes (MAGs), of which 34 were of high quality. Incorporating these MAGs into genome-based species delimitation expanded known Mycetohabitans diversity from four to nine species-level clusters, including novel host-associated lineages. Re-examination of fungal host identities revealed frequent misidentification of isolates in fungal collection catalogues and/or misannotation in GenBank, with nearly a quarter of positive datasets requiring correction through internal transcribed spacer and genome-scale verification. Host–symbiont associations were non-random under this revised framework, with significant structure detected by contingency analysis and ParaFit. MAG-focused pangenome analysis revealed an open pangenome and mosaic lineage-associated functional traits, including variation in metabolism, secretion, cell-envelope systems, metal resistance, antimicrobial-resistance-associated functions and mobile elements. The most distinctive lineage comprised two Apophysomyces -associated MAGs, provisionally named M. apophysomyceticola , which showed pronounced genome reduction compared with other sampled Mycetohabitans spp. and loss of multiple central metabolic, nutrient assimilation, cofactor biosynthesis, catabolic, stress-response and defence pathways, consistent with reduced metabolic flexibility and increased host dependence. Together, these results show that Mycetohabitans symbioses are more geographically widespread, taxonomically diverse and functionally differentiated than previously recognized. More broadly, this work demonstrates the value of public sequencing repositories for uncovering hidden fungal–bacterial symbioses, while emphasizing that repository-derived patterns must be interpreted considering host misidentification, uneven sampling and incomplete metadata. Overall, our work establishes a global framework for Mycetohabitans diversity and function, with implications for fungal ecology, evolution and clinical mycology.