Abstract
Microbial pangenomes vary across species; their size and structure are determined by genetic diversity within the population and by gene loss and horizontal gene transfer (HGT). Many bacteria are associated with eukaryotic hosts where the host colonization dynamics may impact bacterial genome evolution. Host-associated lifestyle has been recognized as a barrier to HGT in parentally transmitted bacteria. However, pangenome evolution of environmentally acquired symbionts remains understudied, often due to limitations in symbiont cultivation. Using high-resolution metagenomics, here we study pangenome evolution of two co-occurring endosymbionts inhabiting Bathymodiolus brooksi mussels from a single cold seep. The symbionts, sulfur-oxidizing (SOX) and methane-oxidizing (MOX) gamma-proteobacteria, are environmentally acquired at an early developmental stage and individual mussels may harbor multiple strains of each symbiont species. We found differences in the accessory gene content of both symbionts across individual mussels, which are reflected by differences in symbiont strain composition. Compared with core genes, accessory genes are enriched in genome plasticity functions. We found no evidence for recent HGT between both symbionts. A comparison between the symbiont pangenomes revealed that the MOX population is less diverged and contains fewer accessory genes, supporting that the MOX association with B. brooksi is more recent in comparison to that of SOX. Our results show that the pangenomes of both symbionts evolved mainly by vertical inheritance. We conclude that genome evolution of environmentally transmitted symbionts that associate with individual hosts over their lifetime is affected by a narrow symbiosis where the frequency of HGT is constrained.