Rouil, J.; Manzano-marin, A.; Clamens, A.-L.; Tavoillot, J.; Cruaud, c.; Barbe, V.; Coeur d'acier, A.; Jousselin, E.

Many insects depend on obligate endosymbionts for their nutrition. Yet, endosymbiotic lifestyle leads to genomic instability and the history of insects-symbiont associations often entails the addition of new symbionts that replace or complement an ancient one and maintain mutualistic functions. Aphids typically rely on Buchnera aphidicola to synthesize nutrients lacking in their diet, but several lineages have acquired an additional obligate symbiont that fulfills essential functions lost by Buchnera, the most common being Serratia symbiotica. However, the diversification pattern and pace of evolution of this endosymbiont once integrated as a co-obligate partner has not been investigated. Here using bacterial metagenomic data from thirteen aphid species of a clade that harbours Serratia as co-obligate partner of Buchnera, we assemble Buchnera, Serratia and host mitochondrial genome sequences. Phylogenomic analyses reveal strict cospeciation between aphids and both endosymbionts, and similar substitution rates in the two bacterial lineages. Analyses of endosymbiont genome variations within and between aphid species indicate strong purifying selection acting on both symbionts. There is no evidence of relaxed selection on protein-coding genes that are shared between co-occurring symbionts, suggesting that the presence of Serratia does not exacerbate genome erosion in Buchnera even on redundant metabolic genes. Finally, fluorescence in-situ hybridization reveals that Serratia and Buchnera are compartmentalized in distinct bacteriocytes within the aphid bacteriome. These results show that Buchnera and Serratia have coevolved under comparable selective constraints and demographic events. Once established in aphid di-symbiotic systems, both ancient and newly acquired symbiont evolution is strictly parallel with no further acceleration in genome evolution.