Reference: Biol. Bull. 194: 116-119. (April. 1998) Vestimentiferan on a Whale Fall ROBERT A. FELDMAN' *t. TIMOTHY M. SHANK'. MICHAEL B. BLACK', AMY R. BACO-. CRAIG R. SMITHS AND ROBERT C. VRIJENHOEK' ' Centerfor Theoretical and Applied Genetics. Institute ofMarine and Coastal Sciences, Cook College. Rutgers University. New Brunswick. New Jersey 08903-0231, and 'Biological Oceanography. Department ofOceanography. School ofOcean and Earth Science and Technology. Unirersit}' ofHawaii. 1000 Pope Road. Honolulu. Hawaii 96822 Discovery of chemosynthetic communities associated sp.), vesicomyid clams (Vesicomya/Calyptogena spp.), with whale bones led to the hypothesis that whalefalls mytilids {Idas sp.), limpets (Pyropelta spp., Coccidina mayserveasstepping-stonesforfaunaldispersalbetween sp.), snails (Mitrella sp.), and polynoid polychaetes (Ba- disjunct hxdrothermal vents and cold seeps on the ocean thykurila sp.). We also recovered a single vestimentiferan floor(1). The initialobsenation wasfollowedbyafaunal tubeworm growing near the squamosal bone ofthe whale inventory that revealeda diverse assemblage ofmicrobes skeleton (Fig. 1). The tubeworm was collected and pre- and invertebrates, supported by chemoautotrophic pro- served in 95% ethanol once aboard the support ship. The duction, living in close proximity to whale remains (2. worm was initially identified as Escarpia spicata on the 3). To date, the conspicuous absencefrom whalefalls of basis ofdiagnostic morphological features (6), most nota- vestimentiferan tuheworms ia predominant constituent of bly the central, slightly curved spikelike structure on the eastern Pacific vent and seep habitats) has been a major obturacular face. objection to the stepping-stone hypothesis (4-5). We re- DNA sequence information from a mitochondrial port thefirst evidence ofa vestimentiferan tubeworm as- gene verified the identity of the SCB specimen. A 639- sociated with a whale fall (Fig. 1). The tubeworm, Es- bp portion of the COI gene from the SCB vestimentif- carpia spicata. was identified by morphological criteria eran was identical to that ofan Escarpia spicata individ- and DNA sequence datafrom a portion ofthe mitochon- ual sampled from cold seeps (Transform Fault) in the drial cytochrome oxidase C subunit I (COI) gene. Addi- Gulf of California (Table I). The COI sequence of the tionally, the bacterialendosymbiont in the tubewormpos- SCB specimen differed by 0.32% from those ofEscarpia sesseda 16S rRNA gene that was similarto that ofendo- spicata from hydrothermal vents (Guaymas Basin) in symbiontsfrom vestimentiferans in sedimented cold-seep the Gulf of California (Table I). COI sequence differ- environments. ences less than 0.4% are consistent with intraspecific A chemosynthetic community associated with whale levels ofdivergence in vestimentiferans (7). Intergeneric bones in the Santa Catahna Basin (SCB) off southern COI sequence divergence in the Vestimentifera ranges CaHfomia (depth 1240 m, 33°12'N, 118°30'W) is the site from 8.5%-21.3% (7). ofongoingecological studies (1,2). Werevisited the SCB The bacterial endosymbiont from the SCB E. spicata site in May 1995 with the U.S. Navy's advanced tethered was phylogenetically compared to other endosymbionts vehicle (ATV dive #118) and found a persistent commu- as part ofa separate study (8). Sequence information from nity composed of filamentous sulfur bacteria (Beggiatoa a 1361-bp region ofthe 16S rRNA gene (Genbank acces- sion number U77842) revealed that the endosymbiont is a memberofthe gamma subdivision ofthe Proteobacteria. Received 6 January 1997; accepted 16 January 1998. The SCB endosymbiont was closely related (1.45% se- San*CDuirergeon.tCadAdr9es2s1:21D.iversaCorporation, 10665 SorrentoValleyRoad. quence divergence) to endosymbionts found in vestimen- tTowhomcorrespondence should be addressed. E-mail: rfeldman@ tiferans that live in sedimented seep localities; its relation- diversa.com ship to endosymbionts from vestimentiferans found in 116 DEEP-SEA TUBEWORM ON A DEAD WHALE 117 Figure 1. Skull bonesofan IS-ni baleanopteridskeleton, andassociatedchemoautotrophiccommunity, (a) A broad view ofthe skull region with the first recorded adult vestimentiferan (arrow) at a whale fall (forscale, the squamo.salbone in the right foreground is 80-cm long).The vestimentiferan plume (left) and posteriorendofthetube(nghl) are visible inthe magnifiedimage(b). This individual wasmorphologically and genetically identified as Escarpia spicata. With the exception ofLamellibrachia cotumna. supplied by C. Cary (Univ. ofDelaware), the specimens examined in this study were collected during oceanic cruises by one or more of the authors. DNA was extracted using the hexadecyl-trimethyl-ammonium bromide (CTAB) technique,modified from Doyle and Dickson (12). Forthe amplificationofthe mitochondrial COI gene; LCOI490: 5'-GGTCAACAAATCATAAAGATATTGG-3' and HC02I98: .V-ACTAAAAAACCA- GTGGGACTTCAAAT-3'primerswereusedinstandardconditions(13).Toamplifysymbiontandsequence the 16S rDNA, we used the universal bactenal pnmers 27Fand I492R (14); 27F: 5'-AGAGTTTGATCM- TGGCTCAG-3', and I492R: 5'-TACGGYTACCTTGTTACGACTT-3' and a suite ofinternal pnmers (8). PCR products were purified and quantified on a video imaging .system (Fotodyne Inc.), 100 ng of pure DNA was used as template for ABI Prism DNA sequencing reactions. The sequenced fragments were electrophoretically separated and detected using a Perkin ElmerABI 373A DNA sequencer. 118 R. A. FELDMAN ET AL. Table I Percentgenetic distances between the Escarpia spicata individualfrom llie Santa Catalina Basin whale fall cnid related yestimemiferansfara 639-bpportion ofthe mitochondrial COIgene Collectiiin Site' Genetic Distance Matiix" Substrate, Depth Species Location Lat Long Community Type (m) Ref 1 4 E. spicata DEEP-SEA TUBEWORM ON A DEAD WHALE 119 5. Jclmert, A., and O.D. Oppen-Berntsen. 1996. Whaling and 1 1 Black, M.B., R.A. Lutz, and R.C. Vrijenhoek. 1994. Gene deep-sea biodiversity. Consen'. Biol. 10: 653-654. flow among vestimentiferan tube worm (RifliafKuhypiHa) popula- 6. Jones,M.L. 1985. On the Vestimentifera.new phylum: Six new tions from hydrothermal vents of the eastern Pacific. Mar. Biol. species, and other taxa, from hydrothermal vents and elsewhere. 120: 33-.39. Bull. Biol. Sac. Wa.sh. 6: 117-158. 12, Doyle,J.J.,and E.Dickson. 1987. Preservationofplantsamples 7 Black,M. B., K.Halanych,P. Maas,W.R.Hoeh,J.Hashimoto, for DNA restriction endonuclease analysis. 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