Autochthonous eukaryotic diversity in hydrothermal sediment and experimental microcolonizers at the Mid-Atlantic Ridge Purificacio´ nLo´ pez-Garcı´a†‡, Herve´ Philippe§¶, Franc¸oise Gail†, and David Moreira‡§ʈ †Biologie Marine, Unite´Mixte de Recherche, Centre National de la Recherche Scientifique 7622, Universite´Pierre et Marie Curie, 7 Quai St. Bernard, 75005 Paris, France; and §Phyloge´nie, Bioinformatique et Ge´nome, Unite´Mixte de Recherche, Centre National de la Recherche Scientifique 7622, Universite´Pierre et Marie Curie, 9 Quai St. Bernard, 75005 Paris, France Edited by Rita R. Colwell, National Science Foundation, Arlington, VA, and approved November 27, 2002 (received for review September 24, 2002) The diversity and mode of life of microbial eukaryotes in hydro- having an insight on the in situ microbial colonization process. thermal systems is very poorly known. We carried out a molecular For this, we analyzed the eukaryotic diversity in experimental survey based on 18S ribosomal RNA genes of eukaryotes present microcolonizers containing different substrates that were col- in different hydrothermal niches at the Mid-Atlantic Ridge. These lected after a 15-day exposure at a fluid emission source. These included metal-rich and rare-earth-element-rich hydrothermal sed- data also should provide information about the potential spec- iments of the Rainbow site, fluid–seawater mixing regions, and ificity of microbes for a particular substrate. colonization devices (microcolonizers) containing organic, iron- rich, and porous mineral substrates that were exposed for 15 days Methods to a fluid source. We identified considerable phylogenetic diver- Sampling. Samples were taken with the aid of the remotely sity, both at kingdom level and within kinetoplastids and alveo- operated vehicle (ROV) Victor during the French cruise ATOS lates. None of our sequences affiliates to photosynthesizing 2001 to the Mid-Atlantic Ridge hydrothermal area. A sediment lineages, suggesting that we are targeting only autochthonous core was obtained from Rainbow hydrothermal sediment deep-sea communities. Although sediment harbored most phylo- (36°6ЈN, 33°11ЈW, depth 2,264 m). After removal of the Ϸ1- to genetic diversity, microcolonizers predominantly contained 2-mm uppermost layer in a laminar flux chamber, a fraction of bodonids and ciliates, indicating that these protists pioneer the the sediment corresponding to the Ϸ1-cm upper part was frozen colonization process. Given the large variety of divergent lineages in liquid nitrogen until used. Fluid–seawater mixtures were detected within the alveolates in deep-sea plankton, hydrothermal collected from vents by using 0.75-liter titanium bottles at Lucky sediments, and vents, alveolates seem to dominate the deep ocean Strike (37°17ЈN, 32°16ЈW, depth 1,695 m) and Rainbow chim- in terms of diversity. Compared with data from the Pacific Guaymas neys; they were then filtered sequentially through 5- and 0.2- basin, some protist lineages seem ubiquitous in hydrothermal ␮m-diameter Millipore filters. Filters then were frozen in lysis areas, whereas others, notably kinetoplastid lineages, very abun- buffer (40 mM EDTA͞50 mM Tris⅐HCl͞0.75 M sucrose) and dant and diverse in our samples, so far have been detected only in stored in liquid nitrogen. Sterile microcolonizers were deployed Atlantic systems. Unexpectedly, although alvinellid polychaetes adjacent to a fluid emission at the Tour Eiffel chimney (Lucky are considered endemic of Pacific vents, we detected alvinellid- Strike site) for 15 days (Fig. 2) and collected in a closed sterile related sequences at the fluid–seawater interface and in micro- container by the ROV Victor. Microcolonizers consisted of colonizers. This finding can boost further studies on deep-sea vent different substrates placed into extensively perforated 50-ml animal biology and biogeography. Corning tubes. Two of them consisted of an inert plastic mesh containing, respectively, a meat-based substrate and iron frag- ompared with prokaryotes, microbial eukaryotes thriving in ments, and a third one was made of basalt and pumice fragments. MICROBIOLOGY Cextreme environments have rarely been studied. This fact is The container was opened in a laminar flux chamber on board, partly because of the difficulties imposed by classical cultivation and the microcolonizers were stored at 4°C in 75% ethanol͞2% approaches. Recent eukaryotic diversity surveys based on 18S NaCl. Because of the turbulent mixture of cold deep-sea water rRNA are revealing an unexpected variety of often divergent and hot hydrothermal fluid at the sampling sites, reliable tem- lineages in different biotopes, including some extreme environ- perature measurements are unavailable. ments (1–6). The only available molecular survey of microbial eukaryotes from deep-sea vents was recently carried out in Electron Microscopy and Sediment Chemical Analysis. Samples were hydrothermal sediments from the Pacific Guaymas basin and dehydrated in increasing ethanol concentrations (50%, 70%, revealed an important diversity of hitherto unknown lineages 90%, and 100%), critical-point-dried, and gold-coated. Obser- (5). Surprisingly, many of these sequences affiliated to typical vation was carried out with a JEOL (JSM-840A) scanning photosynthesizing groups (such as green algae or diatoms), leading to the conclusion that autochthonous eukaryotes cannot be distinguished from those deposited from the water column This paper was submitted directly (Track II) to the PNAS office. (5). There were two objectives in this study. First, we aimed at Abbreviations: MP, maximum parsimony; ML, maximum likelihood. characterizing the diversity of autochthonous microbial eu- Data deposition: The sequences reported in this paper have been deposited in the GenBank karyotes from Mid-Atlantic Ridge hydrothermal systems. Thus, database (accession nos. AF530516–AF530552). we have carried out a molecular survey of hydrothermal sedi- ‡Present address: Unite´d’Ecologie, Syste´matique et Evolution, Unite´Mixte de Recherche ment and seawater–fluid interface. These results should consti- Centre National de la Recherche Scientifique 8079, Universite´ Paris-Sud, Baˆtiment 360, tute a first base for comparison with data from the Pacific 91405 Orsay Cedex, France. systems. To date, whereas prokaryotes seem ubiquitous in ¶Present address: De´partement de Biochimie, Universite´de Montre´al, C.P. 6128 Succursale Centre-Ville, Montreal, QC, Canada H3C 3J7. different oceanic regions (7), possibly including vent areas, ʈTo whom correspondence should be addressed at: Unite´ d’Ecologie, Syste´matique et metazoans are subject to a defined biogeographical distribution Evolution, Unite´Mixte de Recherche Centre National de la Recherche Scientifique 8079, (8). At present, molecular data on the oceanic distribution of Universite´ Paris-Sud, Baˆtiment 360, 91405 Orsay Cedex, France. E-mail: david.moreira@ microbial eukaryotes are still negligible. Second, we aimed at ese.u-psud.fr. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0235779100 PNAS ͉ January 21, 2003 ͉ vol. 100 ͉ no. 2 ͉ 697–702 Downloaded by guest on October 1, 2021 identification of identical or nearly identical sequences and the selection of clones for complete sequencing. Representative clones (n ϭ 37) were completely sequenced, and their sequences were automatically aligned, by using the program BABA (H.P., unpublished work), with 4,575 eukaryotic 18S rDNA sequences retrieved from GenBank (http://ncbi.nlm.nih.gov/). The multiple alignment was then manually edited by using the program ED from the MUST package (11). NJ trees were constructed for the different eukaryotic taxa to choose a representative subset of sequences, avoiding partial, redundant, and fast-evolving ones, for further phylogenetic analyses. Three different subsets of 18S rDNA sequences were selected: one included alveolate se- quences (55 sequences), another included representatives of most major eukaryotic groups (55 sequences), and a third included metazoan sequences (46 sequences). Gaps and ambig- uously aligned positions were excluded from our analyses, re- sulting in alignments of 883, 877, and 1,256 positions, respec- tively. The smaller number of positions used for the first two Fig. 1. Scanning electron microscopy photographs of Rainbow sediment analyses was because of the inclusion of environmental partial showing coccoliths and foraminifer shells. [Bars ϭ 1 ␮m(A and B), 10 ␮m(C), and 100 ␮m(D).] sequences from a molecular survey of eukaryotic diversity in the Guaymas basin (5), which were Ϸ550 nucleotides shorter than our sequences (the average length was Ϸ1,200 nucleotides vs. Ϸ electron microscope operated at 17 kV at the Service de 1,750 nucleotides, respectively). The three data sets were Microscopie Electronique de l’Institut Federatif de Recherche subjected to maximum parsimony (MP) and maximum likeli- de Biologie Inte´grative (Paris). Major elements in sediment were hood (ML) analysis by using PAUP* 4b8 (12). By using an MP analyzed by inductively coupled plasma (ICP)–atomic emission tree, the parameter values for a general time-reversible model of spectrometry; trace elements and rare-earth composition were nucleotide substitution were estimated, with a six-category dis- crete approximation of a ⌫ distribution plus invariable sites analyzed by ICP–mass spectrometry by the Service d’Analyze ϩ⌫ϩ des Roches et Mine´raux du Centre National de la Recherche (GTR I model). The ML trees were constructed by using Scientifique (Nancy, France). 20 random additions