species associated with in South Africa, including two novel species.

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) are small wood-boring insects that live in an obligate symbiosis with fungi, which serve as their primary food source. Beetles residing in the genus have evolved a unique association with a clade of that falls within the aptly named Ambrosia Clade (AFC). The discovery of the invasive polyphagous shot hole borer, , in South Africa, has heightened awareness of ambrosia beetles and their symbionts in the country. In this study, we investigated the symbionts of three species of in South Africa, with a specific focus on those associated with . Isolations of strains from both living and dissected beetles yielded nearly 100 isolates. Using multigene phylogenetic analyses, these isolates were identified as six different species. and have previously been reported from South Africa. and AF-6 are new records for the country. The remaining two species are new to science and are described here as sp. nov. and sp. nov. Targeted fungal isolation from specific beetle body parts revealed that the AFC species collected were typically associated with the dissected beetle heads and helped us identify the likely nutritional symbiont of . This study highlights the understudied diversity of fungal associates of ambrosia beetles present in South Africa.