Pyrosequencing analysis of endosymbiont populationstructure: co-occurrence of divergent symbiontlineages in a single vesicomyid host clam

摘要

Bacteria—eukaryote endosymbioses are perhapsthe most pervasive co-evolutionary associations innature. Here, intracellular chemosynthetic symbiontsof deep-sea clams (Vesicomyidae) were analysed byamplicon pyrosequencing to explore how symbionttransmission mode affects the genetic diversity of thewithin-host symbiont population. Vesicomyid sym-bionts (Gammaproteobacteria) are presumed to beobligately intracellular, to undergo nearly strict verti-cal transmission between host generations, and to beclonal within a host. However, recent data show thatvesicomyid symbionts can be acquired laterally viahorizontal transfer between hosts or uptake from theenvironment, potentially creating opportunities formultiple symbiont strains to occupy the same host.Here, genotype-specific PCR and direct sequencingof the bacterial internal transcribed spacer initiallydemonstrated the co-occurrence of two symbiontstrains, symA and symB (93.5% nt identity), in 8 of118 Vesicomya sp. clams from 3 of 7 hydrothermalvent sites on the Juan de Fuca Ridge. To confirmmultiple strains within individual clams, ampliconpyrosequencing of two symbiont loci was used toobtain deep-coverage measurements (mean: —1500xcoverage per locus per clam) of symbiont populationstructure. Pyrosequencing confirmed symA—symBco-occurrence for two individuals, showing the pres-ence of both genotypes in amplicon pools. However,in the majority of clams, the endosymbiont popu-lation was remarkably homogenous, with > 99.5%of sequences collapsing into a single symbiont