Small, smaller, smallest: The origins and evolution of ancient dual-obligate symbioses in Macrosteles quadrilineatus (Cicadellidae: Deltocephalinae)

Many insects rely on bacterial symbionts with tiny genomes specialized for provisioning nutrients deficient in host diets. Xylem sap and phloem sap are both deficient as complete diets for insects, but differ dramatically in nutrient content, potentially affecting symbiont genome evolution. For sap-feeding insects, sequenced symbiont genomes are available only for phloem-feeding examples from the suborder Sternorrhyncha and xylem-feeding examples from the suborder Auchenorrhyncha, confounding comparisons. We sequenced genomes of the obligate symbionts, Sulcia muelleri and Nasuia deltocephalinicola, of the phloem-feeding pest insect, Macrosteles quadrilineatus (Auchenorrhyncha: Cicadellidae). Our results reveal that Nasuia-ALF has the smallest bacterial genome yet sequenced, and that the Sulcia-ALF genome is smaller than that of Sulcia in other insect lineages. Together, these symbionts retain the capability to synthesize the 10 required essential amino acids. Nasuia retains genes enabling synthesis of two amino acids, DNA replication, transcription, and translation. Both symbionts have lost genes underlying ATP synthesis through oxidative phosphorylation, possibly as a result of the enriched sugar content of phloem. Shared genomic features, including reassignment of the UGA codon from Stop to Trp, and phylogenetic results suggest that Nasuia-ALF is most closely related to Zinderia, the betaproteobacterial symbiont of spittlebugs. Thus, Nasuia/Zinderia and Sulcia likely represent ancient associates that have co-resided in hosts since the divergence of the Cicadomorpha >200 million years ago (Ma), and possibly since the origin of the Auchenorrhyncha, >260 Ma.