Arsenophonus is a widespread insect symbiont with life strategies that vary from parasitism to obligate mutualism. In insects living exclusively on vertebrate blood, mutualistic Arsenophonus strains are presumed to provide B vitamins missing in the insect host diet. Hippoboscidae, obligate blood feeders related to tsetse flies, have been previously suggested to have acquired Arsenophonus symbionts in several independent events. Based on comparative genomic analyses of 11 Hippoboscidae-associated strains, 9 of them newly assembled, we reveal a wide range of their genomic characteristics and phylogenetic affiliations. Phylogenetic patterns and genomic traits split the strains into two different types. Seven strains display characteristics of obligate mutualists with significantly reduced genomes and long phylogenetic branches. The remaining four strains cluster on short branches, and their genomes resemble those of free-living bacteria or facultative symbionts. Both phylogenetic positions and genomic traits indicate that evolutionary history of the Hippoboscidae-Arsenophonus associations is a mixture of short-term coevolutions with at least four independent origins. The comparative approach to a reconstruction of B vitamin pathways across the available Arsenophonus genomes has produced two kinds of patterns. On one hand, it indicates the different importance of individual B vitamins in the host-symbiont interaction. While some (riboflavin, pantothenate, and folate) seem to be synthesized by all Hippoboscidae-associated obligate symbionts, pathways for others (thiamine, nicotinamide, and cobalamin) are mostly missing. On the other hand, the broad comparison has produced patterns that can serve as bases for further assessments of the pathways' completeness and functionality. IMPORTANCE Insects that live exclusively on vertebrate blood utilize symbiotic bacteria as a source of essential compounds, e.g., B vitamins. In louse flies, the most frequent symbiont originated in genus Arsenophonus, known from a wide range of insects. Here, we analyze genomic traits, phylogenetic origins, and metabolic capacities of 11 Arsenophonus strains associated with louse flies. We show that in louse flies, Arsenophonus established symbiosis in at least four independent events, reaching different stages of symbiogenesis. This allowed for comparative genomic analysis, including convergence of metabolic capacities. The significance of the results is twofold. First, based on a comparison of independently originated Arsenophonus symbioses, it determines the importance of individual B vitamins for the insect host. This expands our theoretical insight into insect-bacteria symbiosis. The second outcome is of methodological significance. We show that the comparative approach reveals artifacts that would be difficult to identify based on a single-genome analysis.
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