Vestimentiferan tubeworms thriving in sulfidic deep-sea hydrothermal vents and cold seeps are constrained by their nutritional reliance on chemoautotrophic endosymbionts. In a recent phylogenetic study using 16S ribosomal DNA, we found that endosymbionts from vent and seep habitats form two distinct clades with little variation within each clade. In the present study, we used two different approaches to assess the genetic variation among biogeographically distinct vestimentiferan symbionts. DNA sequences were obtained for the noncoding, internal transcribed spacer (ITS) regions of the rRNA operons of symbionts associated with six different genera of vestimentiferan tubeworms. ITS sequences from endosymbionts of host genera collected from different habitats and widely distributed vent sites were surprisingly conserved. Because the ITS region was not sufficient for distinguishing endosymbionts from different habitats or locations, we used a DNA fingerprinting technique, repetitive-extragenic-palindrome PCR (REP-PCR), to reveal differences in the distribution of repetitive sequences in the genomes of the bacterial endosymbionts. Most of the endosymbionts displayed unique REP-PCR patterns. A cladogram generated from these fingerprints reflected relationships that may be influenced by a variety of factors, including host genera, geographic location, and bottom type.Invertebrates that are endemic to the highly sulfidic, reducing environments at deep-sea hydrothermal vents and cold seeps are commonly associated with chemosynthetic endosymbiotic bacteria (7,18,19). These bacteria oxidize the reduced sulfur compounds that are abundant in hydrothermal fluid (8,36,46). The resultant energy produced by the endosymbiont is coupled to the production of carbon sources that support the growth and maintenance of the invertebrate hosts (6,8,37,48). The metabolic link between the invertebrate hosts and their endosymbionts has obvious implications for the dispersal and colonization strategies employed by the host organisms.Vent endemic host organisms may employ one of three different mechanisms for transmission of their endosymbionts to the next generation. Previous studies have shown that the Vesicomyid bivalves form species-specific associations with endosymbiotic bacteria (12,13,16). Host specificity is maintained in these associations by vertical transmission of the bacteria through the egg from the parent to the offspring (3, 35). The endosymbiont and host phylogenies are congruent, which is consistent with a vertical mode of symbiont transmission (3,4,12,35). In contrast, results from several studies suggest that endosymbiont transmission in vestimentiferan tubeworms occurs horizontally (5, 17), possibly through ingestion of bacteria upon larval settlement (28, 42). It is also possible that horizontal transmission in vestimentiferan-bacterial symbioses could occur through infection of new recruits with bacteria previously associated with established congeners (20), although potential mechanisms for this transfer have not been...

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