Marine Biology (1997) 130: 151±161 Ó Springer-Verlag 1997 A. S. Peek á R. G. Gustafson á R. A. Lutz R. C. Vrijenhoek Evolutionary relationships of deep-sea hydrothermal vent and cold-water seep clams (Bivalvia: Vesicomyidae): results from the mitochondrial cytochrome oxidase subunit I Received: 24 July 1997 / Accepted: 22 August 1997 Abstract Phylogenetic relationships among vesicomyid (e.g. whale-falls). The intense interest in deep-sea vent clams (Bivalvia: Vesicomyidae) and their placement and seep fauna has led to descriptions of many new within the order Heterodonta were examined using mi- species since the family was last revised by Boss (1970). tochondrial encoded cytochrome oxidase subunit I Boss and Turner (1980) noted that ``... systematics of the (COI) DNA sequences. The presently analyzed ve- family Vesicomyidae is beset with diculties because sicomyids represent a recent monophyletic radiation there is at present no satisfactory diagnosis that would that probably occurred within the Cenozoic. Nucleotide exclude the constituent taxa from other heterodonts phylogenetic analyses resolved discrete clades that were based on shared derived characters.''. Attempts to rec- consistent with currently recognized species: Ca- oncile vesicomyid relationships with molecular tech- lyptogena magni®ca, C. ponderosa, Ectenagena extenta, niques also have proved frustrating. An allozyme C. phaseoliformis, Vesicomya cordata, Calyptogena n. sp. analysis of taxa from the eastern Paci®c and Gulf of (Gulf of Mexico), C. kaikoi, C. nautilei, C. solidissima Mexico revealed many morphologically cryptic species; and C. soyoae (Type-A). However, specimens variously however, the data could not resolve phylogenetic identi®ed as: V. gigas, C. kilmeri, C. paci®ca, and V. lepta (or taxonomic) relationships above the level of species comprised two ``species complexes'', each composed of (Vrijenhoek et al. 1994). An analysis of mitochondrial multiple evolutionary lineages. Most taxa are limited to DNA from western Paci®c taxa also identi®ed divergent hydrothermal-vent or cold-seep habitats, but the ``vent'' sequences from morphologically similar individuals (i.e. versus ``seep'' clams do not constitute separate mono- cryptic taxa) and, to complicate matters, found simi- phyletic groups. Current applications of the generic lar sequences in morphologically distinct individuals names Calyptogena, Ectenagena, and Vesicomya are not (Kojima et al. 1995). This combination of cryptic consistent with phylogenetic inferences. morphospecies and phenotypic plasticity within species creates a vexing problem for biologists studying evolu- tionary history and biogeography of vent and seep or- Introduction ganisms. A number of characteristics unite the family Ve- The Vesicomyidae, a family of heterodont clams erected sicomyidae. All extant members are restricted to habi- by Dall and Simpson (1901), includes about 50 described tats that furnish reduced sul®des for chemoautotrophic species that are associated with sul®de-rich habitats in endosymbiotic bacteria, which in turn provide nutriment the vicinity of hydrothermal vents, cold-water sul®de/ for these clams (Cavanaugh 1983; Childress and Fisher hydrocarbon seeps, and other reducing environments 1992). Such environments occur at hydrothermal vents along the mid-ocean ridge system and back-arc basins (German et al. 1995) and at cold-water sul®de/hydro- carbon seeps (Paull et al. 1984; Brooks et al. 1990). Communicated by N.H. Marcus, Tallahassee Additionally, vesicomyids were found in reducing sedi- A.S. Peek (&) á R.G. Gustafson1 á R.A. Lutz á R.C. Vrijenhoek ments associated with a decomposing whale carcass Center for Theoretical and Applied Genetics, and (Bennett et al. 1994). Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, The observation that typical vent species are absent New Jersey 08903-0231, USA at seeps, and vice versa, led to speculation about fun- Present address: damental dierences between vent and seep ecosystems 1 NOAA/NMFS/CZES 2725 Montlake Boulevard East, (Tunniclie 1991; Craddock et al. 1995). Eastern Paci®c Seattle, Washington 98112, USA hydrothermal vent ®elds tend to be ephemeral, lasting 152 perhaps a few decades (Fustec et al. 1987), whereas cold- pli®cation primers: VesLCO (5¢-TTAATAggAACTgCTTTTAg-3¢), water seeps may be very stable, lasting for many thou- and VesHCO (5¢-TCACCCAAACCAgCAggATC-3¢). Sequencing templates were puri®ed by column gel-®ltration (Qiagen, Chats- sands of years (Hecker 1985). Many vent communities worth, California) or ethanol precipitation. Sequencing reaction live on hard basaltic substrates, although sedimented mixtures included either 5¢-P33 end-labeled primers (Perkin-Elmer, sites are known. Seeps typically are covered with soft Foster City, California), or dye-terminated dideoxy labeling (ABI, sediments, although some have hard carbonate out- Foster City, California) under standard cycle-sequencing condi- croppings. Dierences in sul®de concentration, ¯ux, and tions. accessibility also exist between these habitats (Lowell et al. 1995; Von Damm 1995). What are the evolutionary Phylogenetic analysis relationships among taxa found in these ecologically dierent habitats? Do vent and seep vesicomyids com- To facilitate alignment of DNA sequences, we inferred amino acid (a.a.) sequences with the Drosophila yakuba amino acid code (Clary prise separate evolutionary lineages? How old are the and Wolstenholme 1985). We used the program ClustalW vesicomyids as a group? Evolutionary questions such as (Thompson et al. 1994) to provide a preliminary alignment of a.a. these can only be addressed with robust phylogenetic sequences, and the GDE 2.2a (Smith) editor to optimize alignments models. In this study, we use DNA sequence informa- by eye. The a.a. analysis was based on 172 positions. We used the protdist program from PHYLIP 3.57 (see Felsenstein 1989) to as- tion from a 516 base pair (bp) portion of the mito- sess Kimura protein distances (Nei 1987) among the representative chondrial cytochrome c oxidase subunit I (COI) gene to heterodont bivalves. Assuming a JTT (Jones et al. 1992) model of infer phylogenetic relationships of vesicomyids from the a.a. substitution, we applied the protml program from the MO- Paci®c Ocean and Gulf of Mexico. LPHY 2.2 package (Adachi and Hasegawa 1992) to estimate phylogenetic relationships by the maximum-likelihood method. Nucleotide sequences were used to examine evolutionary rela- tionships within the family Vesicomyidae. Nucleotide composition Materials and methods of these sequences was estimated with MEGA 1.0 (Kumar et al. 1993). Using the observed nucleotide frequencies and transi- tion:transversion ratio, we applied the fastDNAml 1.0.6 program Specimens (Olsen et al. 1994) or the dnaml program from PHYLIP 3.57. Parsimony analysis was performed with PAUP 3.1.1 (Swoord Vesicomyid clams were obtained from a variety of locations in the 1993). Alternate phylogenetic hypotheses were constructed with Paci®c Ocean and Gulf of Mexico (Table 1). Specimens were col- MacClade 3.06 (Maddison and Maddison 1992), and tested with lected with the deep submergence vehicles ``Alvin'', ``Nautile'', likelihood by the Kishino±Hasegawa method (Kishino and Has- ``Shinkai 6500'', ``Johnson Sea Link'' and ``Turtle''; the unmanned egawa 1989). To test hypotheses based on parsimony methods, we vehicles ``HYSUB'' (renamed ``ROPOS'') and ``Ventana'', and by used the Wilcoxon rank-order test to determine whether alterna- ocean surface-dredges. Preliminary identi®cations of morphospe- tive trees diered signi®cantly in the number of steps (Templeton cies (Table 1) were performed by the authors (R.G.G., R.A.L., 1983). R.C.V.), R.D. Turner, or the providers of specimens: J. Barry, C. Cavanaugh, J.J. Childress, J. Hashimoto, or A. Fiala-Me dioni. Specimens that were of questionable identi®cation were labeled with location designation. (See Vrijenhoek et al. 1994 for an ac- Results count of sample preparations). In most cases, large specimens were dissected soon after retrieval, while small specimens were frozen whole at )80 °C. Shells were cleaned and dried, and coded for We obtained new COI nucleotide sequences spanning permanent storage. Pending formal taxonomic descriptions of new 516 bp for 52 vesicomyid and two other heterodont species, we will deposit representative voucher shell specimens in an specimens. These 54 original sequences were deposited appropriate museum collection. with GenBank (accession numbers in Table 1 and Fig. 1 legend). For comparative purposes, we examined pub- DNA extraction, PCR, and sequencing lished sequences from ®ve additional heterodont bi- valves (Baldwin et al. 1996). Subsequent phylogenetic Mantle and adductor muscle tissues were dissected from fresh or comparisons of these sequences and published COI se- frozen specimens. Nucleic acids were extracted using CTAB (hex- adecyltrimethyl ammonium bromide) (Doyle and Dickson 1987) or quences from eight western Paci®c species of ve- proteinase K (Mullenbach et al. 1989) protocols followed by phe- sicomyids (Kojima et al. 1995) used only the 278 nol extraction and ethanol precipitation (Sambrook et al. 1989). positions common to both data sets. Puri®ed nucleic acids were hydrated in TE buer (10 mM Tris- HCl, pH 7.5; 1 mM EDTA) and stored at )20 °C. A 710 base pair (bp) portion of the mitochondrial cytochrome oxidase subunit I (COI) gene was ampli®ed by the polymerase chain-reaction Amino acid (a.a.) sequences (PCR) with universal primers designed by