A novel secondary intracellular symbiotic bacterium from aphids of the genus Yamatocallis (subfamily Drepanosiphinae) was characterized by using molecular phylogenetic analysis, in situ hybridization, and diagnostic PCR detection. In the aphid tissues, this bacterium (tentatively designated YSMS [Yamatocallis secondary mycetocyte symbiont]) was found specifically in large cells surrounded by primary mycetocytes harboring Buchnera cells. Of nine drepanosiphine aphids examined, YSMS was detected in only two species of the same genus, Yamatocallis tokyoensis and Yamatocallis hirayamae. In natural populations of these aphids, YSMS was present in 100% of the individuals. Phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences demonstrated that YSMS of Y. tokyoensis and Y. hirayamae constitute a distinct and isolated clade in the γ subdivision of the class Proteobacteria. No 16S rDNA sequences of secondary endosymbionts characterized so far from other aphids showed phylogenetic affinity to YSMS. Based on these results, I suggest that YSMS was acquired by an ancestor of the genus Yamatocallis and has been conserved throughout the evolution of the lineage. By using the nucleotide substitution rate for 16S rDNA of Buchnera spp., the time of acquisition of YSMS was estimated to be about 13 to 26 million years ago, in the Miocene epoch of the Tertiary period
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