Mucoromycota representatives are known to harbor two types of endohyphal bacteria (EHB)-Burkholderia-related endobacteria (BRE) and Mycoplasma-related endobacteria (MRE). While both BRE and MRE occur in fungi representing all subphyla of Mucoromycota, their distribution is not well studied. Therefore, it is difficult to resolve the evolutionary history of these associations in favor of one of the following two alternative hypotheses explaining their origin: "early invasion" and "late invasion." Our main goal was to fill this knowledge gap by surveying Mucoromycota fungi for the presence of EHB. We screened 196 fungal strains from 16 genera using a PCR-based approach to detect bacterial 16S rRNA genes, complemented with fluorescence in situ hybridization (FISH) imaging to confirm the presence of bacteria within the hyphae. We detected Burkholderiaceae in ca. 20% of fungal strains. Some of these bacteria clustered phylogenetically with previously described BRE clades, whereas others grouped with free-living Paraburkholderia Importantly, the latter were detected in Umbelopsidales, which previously were not known to harbor endobacteria. Our results suggest that this group of EHB is recruited from the environment, supporting the late invasion scenario. This pattern complements the early invasion scenario apparent in the BRE clade of EHB.IMPORTANCE Bacteria living within fungal hyphae present an example of one of the most intimate relationships between fungi and bacteria. Even though there are several well-described examples of such partnerships, their prevalence within the fungal kingdom remains unknown. Our study focused on early divergent terrestrial fungi in the phylum Mucoromycota. We found that ca. 20% of the strains tested harbored bacteria from the family Burkholderiaceae Not only did we confirm the presence of bacteria from previously described endosymbiont clades, we also identified a new group of endohyphal Burkholderiaceae representing the genus Paraburkholderia We established that more than half of the screened Umbelopsis strains were positive for bacteria from this new group. We also determined that, while previously described BRE codiverged with their fungal hosts, Paraburkholderia symbionts did not.

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