Background: Chemolithoautotrophic primary production sustains dense invertebrate communities at deep-sea hydrothermal vents and hydrocarbon seeps. Symbiotic bacteria that oxidize dissolved sulfur, methane, and hydrogen gases nourish bathymodiolin mussels that thrive in these environments worldwide. The mussel symbionts are newly acquired in each generation via infection by free-living forms. This study examined geographical subdivision of the thiotrophic endosymbionts hosted by Bathymodiolus mussels living along the eastern Pacific hydrothermal vents. High-throughput sequencing data of 16S ribosomal RNA encoding gene and fragments of six protein-coding genes of symbionts were examined in the samples collected from nine vent localities at the East Pacific Rise, Galápagos Rift, and Pacific-Antarctic Ridge. Results: Both of the parapatric sister-species, B. thermophilus and B. antarcticus, hosted the same numerically dominant phylotype of thiotrophic Gammaproteobacteria. However, sequences from six protein-coding genes revealed highly divergent symbiont lineages living north and south of the Easter Microplate and hosted by these two Bathymodiolus mussel species. High heterogeneity of symbiont haplotypes among host individuals sampled from the same location suggested that stochasticity associated with initial infections was amplified as symbionts proliferated within the host individuals. The mussel species presently contact one another and hybridize along the Easter Microplate, but the northern and southern symbionts appear to be completely isolated. Vicariance associated with orogeny of the Easter Microplate region, 2.5–5.3 million years ago, may have initiated isolation of the symbiont and host populations. Estimates of synonymous substitution rates for the protein-coding bacterial genes examined in this study were 0.77–1.62%/nucleotide/million years. Conclusions: Our present study reports the most comprehensive population genetic analyses of the chemosynthetic endosymbiotic bacteria based on high-throughput genetic data and extensive geographical sampling to date, and demonstrates the role of the geographical features, the Easter Microplate and geographical distance, in the intraspecific divergence of this bacterial species along the mid-ocean ridge axes in the eastern Pacific. Altogether, our results provide insights into extrinsic and intrinsic factors affecting the dispersal and evolution of chemosynthetic symbiotic partners in the hydrothermal vents along the eastern Pacific Ocean.

Geographical structure of endosymbiotic bacteria hosted by Bathymodiolus mussels at eastern Pacific hydrothermal ventsSampling region of Bathymodiolus thermophilus and B. antarticus sulfur-oxidizing gammaproteobacteria EPR, East Pacific Rise GAR, Galapagos Rift PAR, Pacific-Antartic Ridge Information of population and geographic groups population group (HA) 11N EPR (HB) 9N EPR (HI) GAR EPR (HC) 7S EPR (HD) 11S EPR (HE) 17S EPR (HF) 23S EPR (HG) 32S PAR (HH) 38S PAR Folder and file information: FASTA folder - 2 folders containing multi loci DNA sequences of all the sampled individuals from 9 populations sorted by Population and Subpopulation (individual host mussel) Arlequin input files (16S) folder - input files used for calculation molecular indices of 16S gene at two levels of Population and Subpopulation Arlequin input files (Funtional genes, Subpopulation) folder - input files used for calculation molecular indices of functional genes at level of Subpopulation (individual host mussel) Input files (Functional genetic data, Population) folder - input files used for calculation of molecular indices of functional genes at level of population, synonymous/nonsynonymous substitutions estimation, symbiont isolation-by-distance test, and nucleotide substitution rate, generating haplotype networks of functional genes Polymorphic tables.xlsx - Polymorphic table of 16S and functional genes used in this studyDryad.zipNetworks with OTUs labelSequence networks of six protein-coding genes of Bathymodiolus bacterial symbionts labeling with OTUs.