Masoudi, A.; Valdiviezo, M. J.; Tirmizi, E.; Joseph, R. A.; Keyhani, N. O.

Fungal-animal mutualisms remain significantly understudied, yet they represent some of the most successful partnerships known in nature. Fungal farming ambrosia beetles of the Xyleborus genus 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. While isolation and genomics approaches have identified numerous beetle-fungal partners, confirming mycangial colonization, and thus fulfilling Koch-like postulates for mutualism, has been lacking. Here we identify two previously described filamentous species, Raffaelea arxii and R. fusca, and the yeast, Ambrosiozyma monospora, as well as two new filamentous fungal species, Neocosmospora affinis and Graphium ambrosium, and two novel yeasts, designated Alloascoidea xylebori and Wickerhamomyces ambrosius, from beetle gallery walls. We demonstrate that isolation of contents from ambrosia beetle mycangia, which often includes treatment with ethanol to eliminate non-mycangial microbes, biases fungal partner recovery. Using a revised protocol, we show significantly increased content enumeration and recovery of all seven fungal taxa listed above from the mycangia. To meet Koch-like postulates, we further demonstrate that all seven fungal species were individually competent at colonizing aposymbiotic X. affinis mycangia, thus demonstrating each as a viable mycangial mutualist. These data highlight methodological considerations that overcome previous limitations in mycangial content characterization, resulting in the discovery of new ambrosia beetle fungal partners. We further validate the fungal-animal mutualism by demonstrating specific colonization of the mycangial organ by potential fungal partners.