Fulfilling Koch-like postulates for animal fungal mutualists: gallery and mycangial colonization by Xyleborus ambrosia symbiotic fungi

Fungal-animal mutualisms remain significantly understudied, yet they represent some of the most successful partnerships known in nature. Fungal farming ambrosia beetles cultivate a consortium of fungal partners that include obligate filamentous members and yeasts. These fungi are maintained in highly specialized insect organs, termed mycangia, and are cultivated as food along the beetle galleries elaborated within host trees. Here, we isolated fungi from the mycangia of Xyleborus affinis ambrosia beetles using both standard ethanol-wash and ethanol-free protocols. Omitting the ethanol wash significantly increased fungal recovery and diversity. We identify two previously described filamentous species, Raffaelea arxii and R. fusca , and the yeast, Ambrosiozyma monospora , together with two new filamentous fungi, Neocosmospora affinis and Graphium ambrosium , and two novel yeasts, Alloascoidea xylebori and Wickerhamomyces ambrosius , from both gallery walls and beetle mycangia. To meet Koch-like postulates, using mycangial colonization assays, we demonstrate that all seven fungal species were individually competent at colonizing aposymbiotic X. affinis mycangia, demonstrating each as viable symbionts. Our results show that ethanol-protocols can bias recovery of mycangial fungi, leading to underestimation of fungal diversity associated with ambrosia beetles. These findings provide a framework for improved characterization of ambrosia beetle-fungal mutualisms and experimental validation of fungal symbionts.