Pyrosequencing analysis of endosymbiont population structure: co-occurrence of divergent symbiont lineages in a single vesicomyid host clam.

Bacteria-eukaryote endosymbioses are perhaps the most pervasive co-evolutionary associations in nature. Here, intracellular chemosynthetic symbionts of deep-sea clams (Vesicomyidae) were analysed by amplicon pyrosequencing to explore how symbiont transmission mode affects the genetic diversity of the within-host symbiont population. Vesicomyid symbionts (Gammaproteobacteria) are presumed to be obligately intracellular, to undergo nearly strict vertical transmission between host generations, and to be clonal within a host. However, recent data show that vesicomyid symbionts can be acquired laterally via horizontal transfer between hosts or uptake from the environment, potentially creating opportunities for multiple symbiont strains to occupy the same host. Here, genotype-specific PCR and direct sequencing of the bacterial internal transcribed spacer initially demonstrated the co-occurrence of two symbiont strains, symA and symB (93.5% nt identity), in 8 of 118 Vesicomya sp. clams from 3 of 7 hydrothermal vent sites on the Juan de Fuca Ridge. To confirm multiple strains within individual clams, amplicon pyrosequencing of two symbiont loci was used to obtain deep-coverage measurements (mean: approximately 1500x coverage per locus per clam) of symbiont population structure. Pyrosequencing confirmed symA-symB co-occurrence for two individuals, showing the presence of both genotypes in amplicon pools. However, in the majority of clams, the endosymbiont population was remarkably homogenous, with > 99.5% of sequences collapsing into a single symbiont genotype in each clam. These results support the hypothesis that a predominantly vertical transmission strategy leads to the fixation of a single symbiont strain in most hosts. However, mixed symbiont populations do occur in vesicomyids, potentially facilitating the exchange of genetic material between divergent symbiont lineages.