Phylogenomics of flavobacterial insect nutritional endosymbionts with implications for Auchenorrhyncha phylogeny

"Candidatus Sulcia muelleri" (Sulcia) is a diverse lineage of intracellular nutritional endosymbiotic bacteria strictly associated with auchenorrhynchous hemipteran insects. Sulcia has undergone long-term codiversification with its insect hosts but the phylogeny of these endosymbionts, their relationships to other bacteria, and the extent of their occurrence within various groups of Auchenorrhyncha remain inadequately explored. Comprehensive phylogenetic analyses of Sulcia and related bacteria were performed to elucidate its position relative to other members of Phylum Bacteroidetes and the degree of congruence to the phylogeny of its auchenorrhynchous hosts. Maximum likelihood (ML) and maximum parsimony (MP) analyses of Flavobacteriales based on genomic data from 182 bacterial strains recover a monophyletic Sulcia within a larger clade of flavobacterial insect endosymbionts, closely related to Weeksellaceae. Molecular divergence time analysis of Sulcia dates the origin of Sulcia at approximately 339.95 million years ago (Myr) and the initial divergence within Sulcia at approximately 256.91 Myr but these are considered underestimates due to the tendency for endosymbionts to evolve at higher rates compared to their free-living relatives. Screening of 96 recently sequenced hemipteran transcriptomes revealed that 73 of these species, all Auchenorrhyncha, harbored Sulcia. Phylogenetic analysis of 131 orthologous genes plus 16S rRNA for 101 Sulcia strains, representing six fulgoroid families and all the families of Cicadomorpha except Tettigarctidae, recover largely congruent phylogenies between Sulcia and Auchenorrhyncha. The phylogeny of Sulcia strongly supports the superfamily relationships Fulgoroidea + (Cicadoidea + (Cercopoidea + Membracoidea)). Relationships within individual superfamilies are also largely concordant, with the few areas of apparent incongruence between Sulcia and insect genes attributable to low branch support in one or both datasets. These results suggest that analysis of Sulcia phylogeny may contribute to resolution of contentious aspects of Auchenorrhyncha phylogeny.