Mycobiomes of two distinct clades of ambrosia gall midges (Diptera: Cecidomyiidae) are species-specific in larvae but similar in nutritive mycelia

Ambrosia gall midges (AGMs) are mostly host plant-specific. In their galls, they harbor fungal symbionts on which they feed. Therefore, they represent unique steps in the evolution of the gall-forming Cecidomyiidae (Diptera). Gall-associated fungi have been studied predominantly by cultivations, and potential larval endosymbionts have been completely neglected. Using ITS2 rRNA metabarcoding, we characterized the mycobiomes of individual gall compartments (gall surface, gall interior, and larva) of six species from two phylogenetically separated tribes (Asphondyliini and Lasiopterini). Compared to the gall surface and interior, the larvae harbored significantly higher fungal richness and taxonomic diversity, and a larger pool of indicator taxa. Larval mycobiome composition was more species-specific; however, the fungal genera Fusarium, Filobasidium, Tilletiopsis, Alternaria, and Aureobasidium were indicator taxa shared among species. Overall, the larvae harbored 29% of unique taxa that can play a functional role in the host (e.g., initiation of gall development or selection of the mycelia composition). The mycobiome of the gall interior was assembled least stochastically, and its composition was the least species-specific, being dominated by Botryosphaeria dothidea (except for Lasioptera arundinis). Therefore, the interior of ambrosia galls offers a unique environment that supports the growth of similar fungi, regardless of the host plant species and the phylogenetic distance between the AGM tribes. Our study illustrates a range of fungal microorganisms indicative of individual gall compartments, but their potential function, especially in larvae, remains to be solved.