Stable endosymbiosis of a bacterium into a host cell promotes cellular and genomic complexity. The mealybug Planococcus citri has two bacterial endosymbionts with an unusual nested arrangement: the γ-proteobacterium Moranella endobia lives in the cytoplasm of the β-proteobacterium Tremblaya princeps These two bacteria, along with genes horizontally transferred from other bacteria to the P. citri genome, encode gene sets that form an interdependent metabolic patchwork. Here, we test the stability of this three-way symbiosis by sequencing host and symbiont genomes for five diverse mealybug species and find marked fluidity over evolutionary time. Although Tremblaya is the result of a single infection in the ancestor of mealybugs, the γ-proteobacterial symbionts result from multiple replacements of inferred different ages from related but distinct bacterial lineages. Our data show that symbiont replacement can happen even in the most intricate symbiotic arrangements and that preexisting horizontally transferred genes can remain stable on genomes in the face of extensive symbiont turnover.

Division of Biological Sciences University of Montana Missoula MT 59812; Canadian Institute for Advanced Research Program in Integrated Microbial Biodiversity Toronto ON Canada M5G 1Z8

Division of Biological Sciences University of Montana Missoula MT 59812; Institute of Parasitology Biology Centre of the Czech Academy of Sciences Ceske Budejovice 37005 Czech Republic; Faculty of Science University of South Bohemia Ceske Budejovice 37005 Czech Republic;

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