Detection of a conserved bacterial symbiosis in non-frugivorous Australian fruit flies (Diptera, Tephritidae, Tephritinae) supports its widespread association

Several insect lineages, including some fruit flies, have evolved mutualistic associations with primary symbiotic bacteria. Some species of Tephritinae, the most specialized subfamily of fruit flies (Diptera, Tephritidae) predominantly infesting flowerheads of Asteraceae, harbour co-evolved, vertically transmitted and non-culturable bacterial symbionts in their midgut, known as Candidatus Stammerula spp. (Enterobacteriaceae). While such associations have previously been reported in the Palearctic and Hawaiian Archipelago, their occurrence in Australasia had not been investigated.In this study, eight Australian species from six genera belonging to the Tephritini tribe were analysed using bacterial (16S rRNA gene) and mitochondrial (16S rRNA and COI–tRNALeu–COII genes) markers. We detected the presence of specific symbiotic bacteria in all sampled species. Phylogenetic analyses showed that, with one exception, all Australian symbionts clustered in a well-supported monophyletic clade with Stammerula detected in Palearctic and Hawaiian Tephritini. Distinct Stammerula lineages were identified in several taxa, while two species, Trupanea prolata and Spathulina acroleuca shared identical symbiont sequences and the same host plant. Notably, Australian and Palearctic Sphenella spp. harboured closely related symbionts. The cophylogenetic analysis revealed a substantial congruence between host and symbiont tree, supporting a history of cospeciation and suggesting biogeographic links between Australasian and Palearctic taxa. Overall, the results expand the geographic knowledge of Tephritini- Stammerula association and highlight a global pattern of co-diversification.