Secondary (γ- <i>Proteobacteria</i> ) Endosymbionts Infect the Primary (β- <i>Proteobacteria</i> ) Endosymbionts of Mealybugs Multiple Times and Coevolve with Their Hosts

Mealybugs (Hemiptera, Coccoidea, Pseudococcidae) are plant sap-sucking insects that have within their body cavities specialized cells containing prokaryotic primary endosymbionts (P-endosymbionts). The P-endosymbionts have the unusual property of containing within their cytoplasm prokaryotic secondary endosymbionts (S-endosymbionts) [C. D. von Dohlen, S. Kohler, S. T. Alsop, and W. R. McManus, Nature (London) 412:433-436, 2001]. Four-kilobase fragments containing 16S-23S ribosomal DNA (rDNA) were obtained from the P-endosymbionts of 22 mealybug species and the S-endosymbionts of 12 representative species. Phylogenetic analyses of the P-endosymbionts indicated that they have a monophyletic origin and are members of the beta-subdivision of the Proteobacteria; these organisms were subdivided into five different clusters. The S-endosymbionts were members of the gamma-subdivision of the Proteobacteria and were grouped into clusters similar to those observed with the P-endosymbionts. The S-endosymbiont clusters were distinct from each other and from other insect-associated bacteria. The similarity of the clusters formed by the P- and S-endosymbionts suggests that the P-endosymbionts of mealybugs were infected multiple times with different precursors of the S-endosymbionts and once the association was established, the P- and S-endosymbionts were transmitted together. The lineage consisting of the P-endosymbionts of mealybugs was given the designation "Candidatus Tremblaya" gen. nov., with a single species, "Candidatus Tremblaya princeps" sp. nov. The results of phylogenetic analyses of mitochondrial DNA fragments encoding cytochrome oxidase subunits I and II from four representative mealybug species were in agreement with the results of 16S-23S rDNA analyses, suggesting that relationships among strains of "Candidatus T. princeps" are useful in inferring the phylogeny of their mealybug hosts.