Spiroplasma (Class Mollicutes) is a diverse wall-less bacterial genus whose members are strictly dependent on eukaryotic hosts (mostly arthropods and plants), with which they engage in pathogenic to mutualistic interactions. Spiroplasma are generally fastidious to culture in vitro, especially those that are vertically transmitted by their hosts, which include flies in the genus Drosophila. Drosophila has been invaded by at least three independent clades of Spiroplasma: Poulsonii (the best studied; contains reproductive manipulators and defensive mutualists associated two major clades of Drosophila; and has among the highest substitution rates within bacteria); Citri (restricted to the repleta group of Drosophila); and Ixodetis. We report the first genome drafts of Drosophila-associated Citri Clade Spiroplasma: strain sMoj from D. mojavensis; strain sAld-Tx from D. aldrichi from Texas (newly discovered; also associated with D. mulleri); and strain sHy2 from D. hydei (the only Drosophila species known to naturally also harbor a Poulsonii clade strain, thereby providing an arena for horizontal gene transfer). Compared to their Poulsonii clade counterparts, we infer that the three Citri clade strains have: (1) equal or worse DNA repair abilities; (b) more limited metabolic capacities, which may underlie their comparatively lower titers and transmission efficiency; and (c) similar content of toxin domains, including at least one ribosome inactivating protein (RIP), which are implicated in the Poulsonii-conferred defense against natural enemies. As a byproduct of our phylogenomic analyses and exhaustive search for certain toxin domains in public databases, we document the toxin repertoire in close relatives of Drosophila-associated Spiroplasma, and in a very divergent newly discovered lineage (i.e., "clade X"). Phylogenies of toxin-encoding genes or domains imply substantial exchanges between closely and distantly related strains. Surprisingly, despite encoding several toxin genes and achieving relatively high prevalences in certain natural populations (sAld-Tx in this study; sMoj in prior work), fitness assays of sMoj (this study) and sAld-Tx (prior work) in the context of wasp parasitism fail to detect a beneficial effect to their hosts. Thus, how Citri clade strains persist in their Drosophila host populations remains elusive.
