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 50 primary symbiotic bacteria. Some species of Tephritinae, the most specialized subfamily of 51 fruit flies (Diptera, Tephritidae) harbour co-evolved, vertically transmitted and non-culturable 52 bacterial symbionts in their midgut, known as Candidatus Stammerula spp. 53 (Enterobacteriaceae). While such associations have previously been reported in the Palearctic 54 and Hawaiian Archipelago, their occurrence in Australasia had not been investigated. 55 In this study we assessed the genetic diversity of eight Australian fruit fly’s species from six 56 genera belonging to the Tephritini tribe using mitochondrial markers (16S rRNA and COI57 tRNALeu–COII genes) and compared their bacterial diversity using the 16S rRNA gene. 58 We detected the presence of specific symbiotic bacteria in all sampled species. Analysis of 59 bacterial 16S rRNA showed that, with one exception, all Australian symbionts clustered in a 60 well-supported monophyletic clade with Ca. Stammerula detected in Palearctic and Hawaiian 61 Tephritini. Distinct Stammerula lineages were identified in several taxa, while two species, 62 Trupanea prolata and Spathulina acroleuca shared identical symbiont sequences and the same 63 host plant. Notably, Australian and Palearctic Sphenella spp. harboured closely related 64 symbionts. The cophylogenetic analysis revealed a substantial congruence between host and 65 symbiont tree, supporting a history of cospeciation and suggesting biogeographic links between 66 Australasian and Palearctic taxa. Overall, the results expand the geographic knowledge of 67 Tephritini-Ca. Stammerula association and highlight a global pattern of co-diversification.