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) 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 we assessed the genetic diversity of eight Australian fruit fly's species from six genera belonging to the Tephritini tribe using mitochondrial markers (16 S rRNA and COI-tRNALeu-COII genes) and compared their bacterial diversity using the 16 S rRNA gene. We detected the presence of specific symbiotic bacteria in all sampled species. Analysis of bacterial 16 S rRNA showed that, with one exception, all Australian symbionts clustered in a well-supported monophyletic clade with Ca. 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-Ca. Stammerula association and highlight a global pattern of co-diversification.