Lineage - specific endosymbiosis in Mucorales: restriction of Mycetohabitans to the genus Rhizopus

• Screening of 578 Mucorales isolates from clinical and fermented food indicated that endosymbiotic bacteria occur exclusively in Rhizopus species. • 16S rDNA sequencing and FISH analyses confirmed Mycetohabitans as the predominant bacterial symbiont detected in pathogenic Rhizopus strains. • A Rhizopus homothallicus isolate was found to harbor Mycetohabitans sp. showing 98.37% 16S rRNA similarity to M. rhizoxinica , suggesting a potential novel species-level lineage. • Ecological and geographic data compilation illustrated that Mucorales - bacteria associations span multiple environments and continents. Endosymbiotic bacteria have been reported in mucoralean fungi, yet their taxonomic distribution, range of ecological niches, and host specificity remain incompletely understood. Clarifying the occurrence of these bacterial partners across clinical and environmental Mucorales is essential for understanding their evolutionary and biological significance. In this study, we screened 578 isolates of Mucorales from both clinical and fermented food sources, including 360 from mucormycosis patients in the Netherlands, 40 from COVID-19-associated mucormycosis (CAM) patients in India, and 178 foodborne isolates from fermented soybean foods in China. Although 16S rDNA gene amplification revealed the presence of bacteria in sixteen mucoralean isolates, fluorescence in situ hybridization (FISH) demonstrated intracellular localization in only five of them. In all five cases, the endobacteria were identified as Mycetohabitans , and all corresponding fungal hosts belonged to Rhizopus species, suggesting that bacterial endosymbiosis within Mucorales is primarily restricted to this genus. Notably, a Rhizopus homothallicus isolate was found to harbor Mycetohabitans sp., with 98.37% 16S rRNA similarity to the type of M. rhizoxinica , forming a separate phylogenetic clade and potentially representing a novel lineage. Endosymbionts were not detected in foodborne Mucorales from China, consistent with the predominance of Mucor species in these samples. Together, these results demonstrate a lineage - specific association between Mycetohabitans and Rhizopus species and highlight a lineage - dependent pattern across ecological niches. This study provides a systematic approach to evaluating fungal - bacterial symbiosis and offers a basis for future investigations into the functional and ecological roles of endosymbiotic bacteria in Mucorales.