Evolution of multi-partner symbiotic systems in the Cerataphidini tribe: genome reduction of Buchnera and frequent turnover of companion symbionts

Abstract How multi-partner symbiotic systems originate and diversify within a lineage remains poorly understood. To address this, we investigated the evolutionary dynamics of bacterial symbionts across eight species of the aphid subfamily Hormaphidinae, with detailed molecular, genomic, and microscopic analyses focused on two Cerataphidini species, Ceratovacuna nekoashi and Pseudoregma panicola. Our 16S ribosomal RNA gene amplicon sequencing analysis revealed that Cerataphidini species consistently harbor companion symbionts alongside Buchnera, whereas the examined Hormaphidini and Nipponaphidini species harbor only Buchnera. Notably, Arsenophonus in C. nekoashi and C. japonica have distinct phylogenetic origins, and P. panicola has acquired Symbiopectobacterium instead of Arsenophonus. Microscopic analyses demonstrated that these companion symbionts are maternally transmitted and occupy distinct cell types from those harboring Buchnera. Genome sequencing revealed extreme reduction in Buchnera genomes to ∼0.4 Mbp in both C. nekoashi and P. panicola, comparable to C. japonica but significantly smaller than Buchnera genomes of ∼0.6 Mbp in mono-symbioses. The companion symbiont genomes retain complete riboflavin biosynthesis pathways absent in Buchnera. Our findings suggest that Buchnera genome reduction and companion symbiont acquisition occurred in the common ancestor of Cerataphidini, followed by multiple companion symbiont replacements, demonstrating that ancient obligate symbionts can remain evolutionarily stable while companion symbionts undergo frequent turnover. Importance Many insects depend on multiple bacterial symbionts to survive on nutritionally limited diets, yet how such multi-partner symbiotic systems originate and change over evolutionary time is poorly understood. By examining the aphid subfamily Hormaphidinae, particularly Ceratovacuna nekoashi and Pseudoregma panicola, we show that a dramatic reduction in the genome of the ancient symbiont Buchnera and the acquisition of a companion symbiont occurred together in the common ancestor of the tribe Cerataphidini. Strikingly, while Buchnera has been stably maintained across all examined species, the companion symbiont has been repeatedly replaced by phylogenetically diverse bacteria, even among closely related species. These findings reveal an evolutionary asymmetry between ancient and recently acquired symbionts and suggest that companion symbiont turnover is a common consequence in insects harboring genomically degraded obligate symbionts. Competing Interest Statement The authors have declared no competing interest. Footnotes Microscopy data substantially expanded. The manuscript was revised to reflect updated data.