Eighteen strains of flagellated protists representing nine species were isolated and cultured from four deep-sea hydrothermal vents: Juan de Fuca Ridge (2,200 m), Guaymas Basin (2,000 m), 21 degrees N (2,550 m) and 9 degrees N (2,000 m). Light and electron microscopy were used to identify flagellates to genus and, when possible, species. The small subunit ribosomal RNA genes of each vent species and related strains from shallow-waters and the American Type Culture Collection were sequenced then used for comparative analysis with database sequences to place taxa in an rDNA tree. The hydrothermal vent flagellates belonged to six different taxonomic orders: the Ancyromonadida, Bicosoecida, Cercomonadida, Choanoflagellida, Chrysomonadida, and Kinetoplastida. Comparative analysis of vent isolate and database sequences resolved systematic placement of some well-known species with previously uncertain taxonomic affinities, such as Ancyromonas sigmoides, Caecitellus parvulus, and Massisteria marina. Many of these vent isolates are ubiquitous members of marine, freshwater, and terrestrial ecosystems worldwide, suggesting a global distribution of these flagellate species.

The chemical stress factors for microbial life at deep-sea hydrothermal vents include high concentrations of heavy metals and sulfide. Three hyperthermophilic vent archaea, the sulfur-reducing heterotrophs Thermococcus fumicolans and Pyrococcus strain GB-D and the chemolithoautotrophic methanogen Methanocaldococcus jannaschii, were tested for survival tolerance to heavy metals (Zn, Co, and Cu) and sulfide. The sulfide addition consistently ameliorated the high toxicity of free metal cations by the formation of dissolved metal-sulfide complexes as well as solid precipitates. Thus, chemical speciation of heavy metals with sulfide allows hydrothermal vent archaea to tolerate otherwise toxic metal concentrations in their natural environment.Hyperthermophilic archaea grow in the steep thermal and chemical gradients in hydrothermal vent chimney rock; in this habitat, they are exposed to metal-and sulfide-rich vent fluid, which is transported through the porous chimney matrix with concomitant precipitation of metal-sulfides and -sulfates and in some chimney silica (15) upon mixing with seawater. Although metal and sulfide concentrations in the rock matrix have not been measured in situ, end-member concentrations represent upper-limit approximations. Sulfide concentrations are typically in the millimolar range and can be higher than 12 mM. Metal concentrations are typically in the range of 10 to 40 M for Cu, 20 to 220 nM for Co, and 40 to 780 M for Zn (8, 30). Site-specific peak concentrations can reach 1 to 2 M for Co (22) and 1,000 to 3,000 M for Zn (30). The chemical speciation of metals and sulfide, in particular metal-sulfide complex formation, might play a critical role in shaping the environmental niches and survival strategies of hydrothermal vent microorganisms. Metal-sulfide complexes play an important role in biological contexts (23), for example, by relieving cadmium toxicity to amphipods in marine sediments (6) and by influencing the distribution of hydrothermal vent invertebrates such as Riftia pachyptila and Alvinella pompeiana (19).For hydrothermal vent archaea, efforts to characterize their growth conditions and survival capabilities have focused on extremes of temperature and pH, oxygen sensitivity, and electron acceptor and donor range (13, 27). Genomic information on metal tolerance and metabolism is limited to tentatively identified metal transport proteins (primarily Co, Cu, and Fe transporters) in the genomes of Pyrococcus furiosus, P. abyssi, P. horikoshii (http://www.ncbi.nlm.nih.gov), and Methanocaldococcus jannaschii (4, 26). As a consequence, the tolerances of vent archaea to the high concentrations of metals in their native habitat, their response to the chemical speciation of those metals, and their defense mechanisms against these environmental stress factors have remained largely obscure. Silver (26) has pointed out that a surprisingly small number of detoxicification genes appear to be present in the genome of M. jannaschii given its high-metal habitat in hydrothermal vent chimneys.In or...

The ability of metabolically diverse hyperthermophilic archaea to withstand high temperatures, low pHs, high sulfide concentrations, and the absence of carbon and energy sources was investigated. Close relatives of our study organisms, Methanocaldococcus jannaschii, Archaeoglobus profundus, Thermococcus fumicolans, and Pyrococcus sp. strain GB-D, are commonly found in hydrothermal vent chimney walls and hot sediments and possibly deeper in the subsurface, where highly dynamic hydrothermal flow patterns and steep chemical and temperature gradients provide an ever-changing mosaic of microhabitats. These organisms (with the possible exception of Pyrococcus strain GB-D) tolerated greater extremes of low pH, high sulfide concentration, and high temperature when actively growing and metabolizing than when starved of carbon sources and electron donors/acceptors. Therefore these organisms must be actively metabolizing in the hydrothermal vent chimneys, sediments, and subsurface in order to withstand at least 24 h of exposure to extremes of pH, sulfide, and temperature that occur in these environments.

Six isolates of deep-sea flagellated protozoa \yere grown in culture at 1 to 300 atm to measure their growth response to increasing hydrostatic pressure. Three kinetoplastid flagellates and 1 choanoflagellate were isolated from deep-sea hydrothermal vent samples and 2 chrysomonads were isolated from deep continental shelf sediments. The growth rates of 2 species isolated from the vent, CaeciteUus parvulus and Rhynchomonas nasuta, were compared to the growth rates of shallow-water strains of the same species. Deep-sea isolates of C. parvulus and R. nasuta had a higher rate of growth at higher pressures than did their shallow-water counterparts. Thls feature could result from adaptation to higher pressure upon sinking to depth or with time on the ocean bottom. Four of the 6 deep-sea isolates-C. parvulus, R. nasuta and the 2 chrysomonads-were capable of growth at pressures corresponding to their respective depths of collect~on, indicating that these species could be metabolically active at these depths. C. parvulus and R. nasuta encysted at pressures greater than their depth of collection. The choanoflagellate isolate was observed to encyst at pressures greater than 50 atm. These findings suggest a potential ecological role for encystment In deep-sea/water-column coupling. Cosmopolitan or epipelag~c species, such as C. parvulus and R nasuta, may be transported on sedimenting particles into the deep sea, where encystment at high pressure could serve as a mechanism for pelagic renewal by advection via hydrothermal plume entrainment and thermohaline circulation.

Molecular and morphological evidence points to the ancyromonad Ancyromonas as a plausible candidate for the closest relative to the common ancestor of metazoans, fungi, and choanoflagellates (the Opisthokonta). Using 18S rDNA sequences from most of the major eukaryotic lineages, maximum-likelihood, minimum-evolution, and maximum-parsimony analyses yielded congruent phylogenies supporting this hypothesis. Combined with ultrastructural similarities between Ancyromonas and opisthokonts, the evidence presented here suggests that Ancyromonas may form an independent lineage, the Ancyromonadida Cavalier-Smith 1997, closer in its relationship to the opisthokonts than is its nearest protist relatives, the Apusomonadida. However, the very low bootstrap support for deep nodes and hypothesis testing indicate that the resolving power of 18S rDNA sequences is limited for examining this aspect of eukaryotic phylogeny. Alternate branching positions for the Ancyromonas lineage cannot be robustly rejected, revealing the importance of ultrastructure when examining the origins of multicellularity. The future use of a multigene approach may additionally be needed to resolve this aspect of eukaryotic phylogeny.