Eukaryotic Diversity Associated with Carbonates and Fluid–Seawater

Home , Haplosporidium nelsoni, Reticulamoeba

Environmental Microbiology (2007) 9(2), 546–554 doi:10.1111/j.1462-2920.2006.01158.x

Brief report

Eukaryotic diversity associated with carbonates and ﬂuid–seawater interface in Lost City hydrothermal ﬁeld

Purificación López-García,1 Alexander Vereshchaka2 Introduction and David Moreira1* Since their discovery in the late 1970s, hydrothermal 1Unité d’Ecologie, Systématique et Evolution, UMR vents associated with mid-oceanic ridges have fascinated CNRS 8079, Université Paris-Sud, bâtiment 360, 91405 scientists of various disciplines. In these ecosystems, Orsay Cedex, France. chemolithoautotrophic prokaryotes are responsible for an 2Institute of Oceanology of the Russian Academy of important primary production that sustains dense micro- Sciences, 117997 Moscow, Russia. bial communities and, often, crowded colonies of endemic fauna. However, whereas the prokaryotic and animal Summary components of known deep-sea hydrothermal systems have been extensively described, very few studies have Lost City is a unique off-axis hydrothermal vent field been devoted to the microbial eukaryotes associated with characterized by highly alkaline and relatively low- deep-sea vents. The presence of ciliates based on temperature fluids that harbours huge carbonate microscopy observations was reported early at East chimneys. We have carried out a molecular survey Pacific Rise vents (Small and Gross, 1985). Also, a few based on 18S rDNA sequences of the eukaryotic com- classical protistology studies based on isolation and cul- munities associated with fluid–seawater interfaces tivation led to the description of a limited number of ther- and with carbonates from venting areas and the mophilic ciliates (Baumgartner et al., 2002) and a variety chimney wall. Our study reveals a variety of lineages of psychrophilic or mesophilic protists (Atkins et al., 2000). belonging to eight major taxa: Metazoa, Fungi, Heter- Recently, two molecular surveys based on the amplifica- okonta (Stramenopiles), Alveolata, Radiolaria, Cerco- tion, cloning and sequencing of 18S rDNAs were carried zoa, Heterolobosea and Euglenozoa. We detected out in deep-sea hydrothermal sites of the East Pacific one fungal lineage that appears to be widespread Rise and the Mid-Atlantic Ridge, revealing an astonishing in hydrothermal systems both submarine and diversity of eukaryotic lineages (Edgcomb et al., 2002; continental. Alveolates were the most abundant and López-García et al., 2003). The first of these studies, diverse group in Lost City samples, although their carried out in the sulfide- and hydrocarbon-rich hydrother- distribution was very different in carbonate, where mal sediment of the Guaymas basin (2000 m deep) in the ciliates dominated, and in fluid–seawater libraries, Gulf of California, revealed the presence of putative indig- where dinoflagellates, Group I and Group II (Syndini- enous lineages but also of contaminating organisms ales) marine alveolates were profuse. Similarly, coming from euphotic water layers, as a large diversity of Euglenozoa also displayed a differential distribution, typical photosynthetic lines was observed (Edgcomb kinetoplastids being present on carbonates and a et al., 2002). The second study analysed the 18S rDNA novel group of diplonemids so far exclusively eukaryotic diversity associated with Rainbow hydrother- observed in the deep sea being dominant in fluid– mal sediments (2200 m deep) and with artificial sub- seawater libraries. Protist lineages identified in this strates having stayed for 15 days at a fluid source of the ecosystem likely correspond to grazers, decompos- Eiffel Tour chimney in the Lucky Strike site (1600 m deep) ers and parasites, playing key roles in the food web of (López-García et al., 2003). Both sites are located at the Lost City ecosystem. similar depth and relatively close in the Mid-Atlantic Ridge, but whereas Rainbow fluids are very hot (~365°C), acidic (pH ~2.8) and extremely enriched in metals and rare-earth elements (Douville et al., 2002), Lucky Strike Received 31 August, 2006; accepted 31 August, 2006. *For correspondence. E-mail [email protected]; Tel. fluids are slightly less hot (generally 200–212°C, up to (+33) 1 69 15 76 08; Fax (+33)169154697. 333°C in black smokers) and hydrogen sulfide-rich

(Langmuir et al., 1997). By contrast to the Guaymas study, field were selected: venting fluids as they mixed with the none of the Mid-Atlantic Ridge lineages detected surrounding seawater (samples LC22 and LC23) and belonged to typical photosynthetic groups, suggesting carbonates (samples LC103 and LC104) (Fig. 1, and that they were actually autochthonous to the deep ocean Table S1 in Supplementary material). The temperature of (López-García et al., 2003). In addition to these hydro- the fluids sampled fluctuated, ranging from 40°C to 75°C, thermal vent systems located in the deep sea, a third and their pH varied between 9 and 9.8. Fluids were 2+ 2– recent survey was carried out in the anoxic environment enriched in Mg and SO4 compared with other hydro- around the fumaroles of a shallow (200 m deep) subma- thermal fluids (Lein et al., 2004). After preliminary micros- rine caldera (‘Tagiri’ sites) at the Kagoshima bay in Japan copy observations, we applied molecular methods to (Takishita et al., 2005). study the eukaryotic diversity associated to these Lost City, a spectacular hydrothermal field located samples. Once DNA was extracted, we amplified near approximately 15 km off the Mid-Atlantic Ridge axis at full-length 18S rDNA by PCR, cloned the amplified prod- 30°N and at a depth of 750–900 m, was discovered in ucts and sequenced partially inserts of expected size (see 2000 (Kelley et al., 2001). The site is unique because, Supplementary material). A total of 266 good-quality being off-axis and located in the Mid-Atlantic Ridge slow- partial sequences (~800 pb) were analysed initially (71 spreading system, its fluids have a very particular com- sequences from LC22; 85 sequences from LC23; 65 position derived from serpentinization reactions that is sequences from LC103; 45 sequences from LC104), from characterized by abundant methane, relatively little metal which a total of 47 clones (at least one from each of the content, high pH (9–11) and cooler temperatures clusters of sequences with > 98% identity detected) were (40–90°C) compared with mid-axis fluids. The alkaline selected as representatives to be completely sequenced. conditions promote carbonate precipitation and the forma- Complete sequences were incorporated to an alignment tion of large chimneys that host-specific prokaryotic com- of ~3000 eukaryotic 18S rDNA together with their closest munities where both anaerobic methane oxidation and homologues identified by BLAST in GenBank (Table S2 in methanogenesis appear to be important (Schrenk et al., Supplementary material). Subsequently, we constructed 2004; Kelley et al., 2005). These microbial communities three data sets that included Lost City sequences and a associated with carbonates in venting areas can be choice of representative sequences covering the taxo- dense, containing 107-108 cells per gram of wet weight. nomic diversity of the groups identified in order to carry However, although different animal species have been out detailed phylogenetic analysis by maximum likelihood identified and the presence of foraminifera has been and Bayesian analyses. They corresponded to animal and acknowledged based on microscopy observation (Kelley fungal sequences within the opisthokonts (Fig. 2), a et al., 2005), detailed data about protist diversity associ- variety of alveolates and one heterokont sequence, and ated with Lost City vents are still missing. diverse protist groups spanning the rest of the eukaryotic In this work, we report the first 18S rDNA survey of diversity found (Fig. 3). eukaryotic diversity from Lost City carbonates and the fluids percolating through them. Our analysis reveals a Opisthokont sequences relatively wide diversity of lineages, none of which is typically exclusively photosynthetic, including members of We identified a number of phylotypes belonging to the eight different taxonomic groups: Metazoa, Fungi, Heter- Metazoa and the Fungi in Lost City samples. Metazoan okonta (Stramenopiles), Alveolata, Radiolaria, Cercozoa, sequences were often abundant in our libraries (Table S2 Heterolobosea and Euglenozoa. Comparison with the in Supplementary material), but they tend to be over- eukaryotic diversity in deep-sea plankton, and the represented in 18S rDNA libraries due to a higher contri- Guaymas and mid-axis Atlantic deep-sea vents suggests bution of template DNA by pluricellular organisms. Within that most of the lineages associated to these deep-sea the Metazoa, copepods, which were found only in the vents are those thriving in deep-sea plankton, in the case fluid–seawater samples, and abundantly observed in the of fluid–seawater interfaces, and lineages typically asso- water column above the vent, were the most diverse ciated to anoxic settings. (Fig. 2). Copepods appear to be abundant in other deep- sea hydrothermal areas, suggesting that they may be attracted by the rich primary production in these ecosys- Eukaryotic diversity in Lost City tems (Humes, 1987; Heptner and Ivanenko, 2002). The samples that we used in our study were collected with Sequences of members of the Porifera, Nematoda and the deep-sea submersibles ‘MIR’ during the 50th cruise of Polychaeta were exclusively detected in carbonates, R/V ‘Akademik Mstislav Keldysh’ (see Experimental pro- which would be in accordance with their benthic lifestyle. cedures in Appendix S1 in Supplementary material). Two Indeed, these animals were observed and sampled types of samples from the highest pylon in Lost City vent for independent taxonomic analyses (A. Vereshchaka,

Sample LC103

Sample LC104

Fig. 1. Lost City sampling sites. The map indicates the position of Lost City relative to other Mid-Atlantic Ridge deep-sea vent sites. The schematic drawing on the left (down) shows the relative position of the sites from which samples were taken. Panels on the right show the MIR arm coring the top of the main pylon to collect sample LC103 and the lateral carbonate wall (some hydroids and a crab are visible on it) from which sample LC104 was collected. unpublished). Sequences belonging to the Cnidaria were noes in Bohemia, Czech Republic (J. Berthon, D. Moreira detected in both carbonates and seawater (Fig. 2). We and P. López-García, unpublished). The other clone in this only identified two fungal phylotypes, one within the Asco- group, BAQA52 was obtained in a study of brackish sedi- mycota, and the other within the Basidiomycota. Interest- ment in Berkeley Aquatic Park (Dawson and Pace, 2002). ingly, the basidiomycete phylotype (LC23-5EP-14), which Therefore, this group may represent a group of anaerobic was relatively abundant in one fluid–seawater sample fungi that is ubiquitous in hydrothermal environments. (Table S2 in Supplementary material), formed a cluster with other environmental sequences relatively far from Heterokont and alveolate diversity cultivated species that were retrieved from other hydrothermal settings, either deep-sea vents (A1-E024 from We identified only one heterokont (stramenopile) Guaymas basin and AT9-6 from fluid–seawater at Lucky sequence, which was closely related to the group of envi- Strike) or cool terrestrial geothermal systems. Thus, PAT6- ronmental sequences MAST-9, represented by the clone EK5-11 was retrieved from near-neutral pH, saline mud BL010625-32 (Fig. 3). This group appears to occur mostly volcanoes in the Paterno area close to the Etna in Sicily, in hydrothermal vents and has been proposed to be Italy, and BOH3 clones from carbonate-rich mud volca- adapted to anoxic or microoxic habitats (Massana et al.,

Saccharomyces cerevisiae Fig. 2. Bayesian phylogenetic tree of 18S .99 Lost City LC23 4EP 18 [DQ504331] Ascomycota Fungi rDNA metazoan and fungal sequences 1 Aureobasidium pullulans 1 Aspergillus zonatus retrieved from Lost City hydrothermal ﬁeld. 1 Eupenicillium javanicum Phylotypes shown in bold have been retrieved 1 Clone AT2-4 [AF530541] from hydrothermal sites. Bayesian posterior 1 Trichosporon pullulans 1 Cryptococcus aquaticus Basidiomycota probabilities are indicated at nodes. Maximum Sistotrema eximum likelihood bootstrap values higher than 50% 1 Trametes versicolor are shown with empty and ﬁlled circles to 1 1 Boletus satanas Exobasidium vaccinii indicate values between 50% and 75%, and Sympodiomycopsis paphiopedili between 75% and 100% respectively. 1 Malassezia furfur 1 Clone BOH3 EK2 20 [DQ504358] Hydrothermal 1 Clone AT9-6 [AF530542] .97 Clone Guaymas A1 E024 [AY046691] and/or 1 Clone PAT6 EK5 11 [DQ504360] anaerobic Lost City LC23 5EP 14 [DQ504335] fungal Clone BOH3 EK4 7 [DQ504359] Basidiomycete BAQA52 [AF372708] group Axinella polypoides Porifera 1 Geodia neptuni Metazoa [DQ504344] 1 Lost City LC104 3EP 39 .58 Lost City LC104 3EP 2 [DQ504342] .86 Cinachyrella sp. Leucosolenia sp. Lost City LC104 3EP 44 [DQ504345] 1 Corynactis californica Cnidaria 1 Clausophyes ovata .67 1 Lost City LC23 4EP 17 [DQ504348] .68 Lost City LC23 4EP 2 [DQ504347] Neocalanus cristatus 1 [DQ504357] Copepoda .95 Lost City LC22 5EP 47 1 Clone AT2-18 [AF530545] [DQ504311] .97 Lost City LC22 4EP 1 [DQ504333] 1 Lost City LC23 5EP 3 1 Clone SCM28C58 [AY665128] 1 Clone AT4-36 [AF530550] 1 Lost City LC104 3EP 33 [DQ504343] Nematoda 1 Achromadora cf. terricola .70 Lost City LC104 3EP 47 [DQ504346] 1 Caulleriella parva Polychaeta 1 Unclassified metazoan [AF025935] [AF530549] .85 Clone AT4-37 [AF530546] 0.1 1 Clone AT6-18 1 Clone AT7-8 [AF530547] 1 Clone AT7-21 [AF530548]

Carbonate Fluid-seawater mix

2004). It is distantly related to the osmotrophic labyrin- correspond indeed to the Syndiniales, a group of small thulids and thraustochytrids and is probably formed by parasites traditionally classed within the dinoflagellates heterotrophic organisms. that appears to be highly diverse and ubiquitous in the By contrast to this limited diversity of heterokonts, we water column and around hydrothermal vents (López- retrieved a wide variety of alveolates (Fig. 3). Ciliates García et al., 2001; Moon-van der Staay et al., 2001; were the most abundant in our clone libraries, especially Moreira and López-García, 2003). Lost City sequences in the carbonates (Table S2 in Supplementary material branched within two distinct clusters in the Syndiniales and Fig. 4). Lost City sequences were affiliated to three (Fig. 3). One of them corresponded to the Syndiniaceae, ciliate classes, Hypotrichea (LC103-4EP-1), Spirotrichea grouping Hematodinium and Syndinium species, which (LC23-5EP-24, LC22-5EP-7) and Oligohymenophorea generally infect invertebrates, including copepods, (LC22-5EP-48, LC22-5EP-20, LC103-5EP-27), and likely whereas the other group, the Amoebophryaceae, includ- correspond to grazing organisms. One possible exception ing Amoebophrya species, are generally endoparasites could be LC103-5EP-27, closely related to members of of other protists, such as dinoflagellates, ciliates or the genus Cohnilembus, which contains also commensal radiolaria. Alveolate Group I was described on the basis of species in the gut of various invertebrates (Lynn and environmental sequences exclusively. It encompasses a Strüder-Kypke, 2005). Different dinoflagellate sequences large genetic diversity and appears also ubiquitous in the were also detected, but in this case, all of them were oceanic water column. Unfortunately, to date, there is no identified in the seawater above the carbonate chimneys, information about their morphology or lifestyle. in accordance with their planktonic way of life. Finally and, as in the case of dinoflagellates only in the seawater–fluid Other protist lineages mix, various phylotypes related to the marine alveolates Groups I and II and an alveolate sequence of unclear In addition to metazoa, fungi, heterokonts and alveolates, affinity (LC22-4EP-19) were obtained. Group II alveolates we retrieved sequences belonging to four protist phyla,

Wobblia lunata Clone HA2 [AF382824] 1 Blastocystis hominis Heterokonta 1 Collozoum serpentinum Radiolaria .70 Caecitellus parvulus 1 Clone AT8-54 [AF530524] Developayella elegans Clone DH147-EKD17 [AF290072] 1 1 1 Sargassum sp. 1 Hexaconus serratus .85 Nannochloropsis oculata .54 Amphiacon denticulatus .55 Labyrinthula sp. AN1565 .65 Lost City LC22 5EP 23 [DQ504355] [DQ504337] .84Sticholonche zanclea Lost City LC23 5EP 40 .99 Clone KW16 [AF382825] .56 1 Clone BL010625 32 [AY381217] [AF530525] .90 Clone OLI11150 [AJ402355] .76 Clone AT4-94 Clone BL000921 26 [AY381196] .90 Dictyocoryne profunda .96 1 Clone DH148-EKD30 Clone AT4-38 [AF530528] 1 strain Reunion [DQ504338] Gromia oviformis Lost City LC103 4EP 1 Ciliophora .90 1 Gromia oviformis strain Guam 1 Aspidisca steini Reticulamoeba sp. JJP2003 Cercozoa Oxytricha granulifera Haplosporidian cf. .77 Pandalus platyceros 1 Oxytricha nova Lost City LC104 3EP 36 [DQ504354] [AF530527] .58 1 Clone AT6-4 Urosporidium crescens Lost City LC23 5EP 24 [DQ504336] .69 1 Urosporidium cf. Stictodora lari .95 1 Tintinnopsis fimbriata 1 Haplosporidium nelsoni 1 Codonellopsis americana .74 1 Haplosporidium costale 1 1 Lost City LC22 5EP 7 [DQ504317] 1 Bonamia sp. [DQ504353] 1 Strombidium sp. SNB99 2 Lost City LC104 3EP 8 .51 Clone UEPAC Jp1 [AY129030] 1 Lotharella globosa Pseudomicrothorax dubius 1 Gymnochlora stellata [AF530529] 1 Clone AT1-2 .57 Chlorarachnion reptans [AF530531] .57 Bigelowiella natans 1 Clone AT7-37 1 Chlorarachniophyte sp. BC52 1 Clone AT7-23 [AF530530] 1 Euglypha acanthophora Cryptocaryon irritans 1 Lost City LC22 5EP 48 [DQ504328] Tracheleuglypha dentata [DQ504324] .71 Metopion fluens 1 .60 Lost City LC22 5EP 20 Clone 9-23 [AY620313] Frontonia lynni .63 Lost City LC103 5EP 6 [DQ504340] 1 Cyclidium glaucoma 1 Massisteria marina Schizocaryum dogieli Allas sp. JJP2003 .99 Lost City LC103 5EP 27 [DQ504341] 1 Cryothecomonas aestivalis 1 Cohnilembus verminus Gymnophrys cometa .78 Philasterides dicentrarchi .60 Hedriocystis reticulata .98 Anophyroides haemophila .68 Phagomyxa bellerocheae .92 Toxoplasma gondii 1 Spongospora subterranea Plasmodium falciparum Apicomplexa 1 Plasmodiophora brassicae 1 Theileria sp. Percolomonas cosmopolitus 1 Cryptosporidium parvum Lost City LC103 5EP 3 [DQ504339] Soil amoeba AND9 [AY965861] .59 Monocystis agilis 1 .84 .62 Clone AT4-16 [AF530523] 1 Vahlkampfia damariscottae Clone AT4-98 [AF530536] .59 Naegleria gruberi [AF530535] 1 Vahlkampfia lobospinosa 1 Clone AT2-6 Perkinsozoa 1 Heterolobosea 1 Perkinsus marinus 0.33 Didasculus thorntoni .65 Hematodinium sp. MF2000 Heteramoeba clara sp. ex .82 Paravahlkampfia ustiana 1 Syndinium Corycaeus Syndiniales [DQ504356] .71 Psalteriomonas lanterna .53 Lost City LC22 5EP 37 1 .98 Lost City LC22 5EP 44 [DQ504327] (marine alveolate Monopylocystis visvesvarai 1 Clone DH148-EKD14 [AF290079] Eutreptiella gymnastica [AF530532] Group II) 1 Trachelomonas echinata Euglenida 1 Clone AT4-21 1 Euglena gracilis 1 Amoebophrya cf.Karlodinium micrum [DQ504321] 1 Lost City LC22 5EP 17 Deep-sea 1 Clone UEPAC 46p4 [AY12905] [DQ504349] 1 Lost City LC22 5EP 32 Diplonemida Lost City LC22 5EP 11 [DQ504320] Lost City LC22 5EP 19 [DQ504323] diplonemid .89 Clone UEPAC 15p4 [AY12904] 1 Lost City LC22 5EP 10 [DQ504319] group .90 Clone OLI11012 [AJ402330] .99 1 Lost City LC22 5EP 8 [DQ504318] Clone DH147-EKD6 [AF290068] 1 1 .98 Clone DH148-EKB1 [AF290080] Clone DH147-EKD3 [AF290069] .69 Lost City LC22 5EP 18 [DQ504322] 1 Clone W159G6 [AY429070] Diplonema papillatum 1 Lost City LC22 5EP 36 [DQ504326] .86Diplonema sp. ATCC 50225 Other Clone CCW85 [AY180037] .62 Gymnodinium mikimotoi .58 .54 .83 Lessardia elongata 0.36 Diplonema ambulator Lost City LC23 4EP 25 [DQ504332] dinoflagellates Diplonema sp. .84 Gymnodinium sp. .88 .84 Rhynchopus sp. ATCC 50226 Pentapharsodinium sp. CCMP771 1 Lost City LC23 5EP 5 [DQ504350] Pentapharsodinium tyrrhenicum .99 Rhynchopus sp. SH 2004 IVQ Lost City LC22 5EP 3 [DQ504315] Clone AT4-96 [AF530521] 1 Clone AT4-103 [AF530522] Kinetoplastida Pyrodinium bahamense .91 Clone L8 9 [AY753947] Prorocentrum panamensis Clone AT4-56 [AF530520] .72 Lost City LC22 4EP 12 [DQ504312] -like sp AFSM3 [DQ504325] 1 1 Perkinsiella .87 Lost City LC22 5EP 33 Lost City LC103 5EP 19 [DQ504351] .53 Gymnodinium sp. MUCC284 sp. AAN2003 [DQ504314] .82 Ichthyobodo Lost City LC22 4EP 19 Unclassified alveolate 1 Ichthyobodo sp. .95 Euglenozoa Lost City LC22 5EP 6 [DQ504316] .95 Ichthyobodo necator Clone Guaymas C2 E003 [AY046794] Clone DH144-EKD3 [AF290064] Parabodo caudatus 1 Procryptobia sorokini 1 Clone Guaymas C2 E006 [AY046797] 1 1 Clone AT1-3 [AF530519] [AF530537] 1 Clone AT4-42 .74 Bodo saltans 1 Clone DH148-EKD22 [AF290078] Rhynchobodo sp. ATCC 50359 [AY046624] .94 1 Clone Guaymas C1 E023 Marine .79 Rhynchomonas nasuta 1 Clone AT8-27 [AF530538] .94 Dimastigella trypaniformis .81 Lost City LC23 4EP 1 [DQ504329] alveolate .80 Clone AT5-25 [AF530518] .85 [DQ504330] [AF530517] Lost City LC23 4EP 9 .68 Clone AT5-48 Lost City LC22 4EP 17 [DQ504313] Cruzella marina Group I .95 strain Dune 2 Clone DH147-EKD18 [AF290073] 0.1 .97 Neobodo designis 1 Clone OLI11033 [AJ402353] 1 Lost City LC103 5EP 36 [DQ504352] 1 Lost City LC23 5EP 9 [DQ504334] .59 Neobodo saliens 0.1 .92 Clone BL010320.28 [DQ186536] .96 Neobodo designis .88 [AF530516] .98 Clone Guaymas C3 E034 [AY046862] Clone AT5-9 1 Clone AT4-47 [AF530539]

Carbonate Fluid-seawater mix

Fig. 3. Bayesian phylogenetic trees of 18S rDNA heterokont and alveolate sequences (left) and of Radiolaria, Cercozoa, Heterolobosea and Euglenozoa (right) retrieved from Lost City hydrothermal ﬁeld. Phylotypes shown in bold have been retrieved from hydrothermal sites. Bayesian posterior probabilities are indicated at nodes. Maximum likelihood bootstrap values higher than 50% are shown with empty and ﬁlled circles to indicate values between 50% and 75%, and between 75% and 100% respectively.

Radiolaria, Cercozoa, Heterolobosea and Euglenozoa the most diverse and species-rich groups of ﬂagellated (Fig. 3). The clone LC22-5EP-23 from the ﬂuid–seawater protists (Bass and Cavalier-Smith, 2004). LC104-3EP-36 mix branched among the Radiolaria, but its affiliation emerged as a very short branch with moderate support at within this phylum remains uncertain. Three Lost City the base of a group including the described species of carbonate sequences belonged to the Cercozoa, one of Haplosporidia, a long-branching group composed exclu-

100

Deep-sea plankton Un Apu Guaymas sediment Heterolo c Cerc ertain R s Kinetopla ad ozoa Rainbow sediment Diplo ozoa Una io Ciliate Lucky Strike colonisers laria bo Alv ne Alveolate ss s Lost City carbonate ea Dino eolate i mid Per s g s Apicomplex ned alveol tid Lost City fluid-SW Non s kin fla s Photo s Greena g -phto s s Group II - Fun ozoa ellat Group I

s g ynthetic e ate i s s lg ynt a ae s hetic S yn Heter hete d iniale

okont r okont s

s s

Fig. 4. Relative abundance of protist clones retrieved from Lost City carbonate and hydrothermal fluid-bearing seawater (fluid-SW) libraries compared with that found in deep-sea plankton and other oceanic deep-sea hydrothermal systems. Deep-sea plankton data correspond to the average of clones obtained at the Antarctic Polar Front at 500, 2000 and 3000 m depth (López-García et al., 2001). sively by parasites of invertebrates (Perkins, 1990). It may Massisteria marina appears therefore to be ubiquitous be well a basal haplosporidian, as it is close to another and to show some affinity for hydrothermal vents, likely sequence also basal to the described haplosporidians because of their high primary productivity. that apparently corresponds to the haplosporidian para- We also identified one sequence (LC103-5EP-3) dis- site of the shrimp Pandalus platyceros (Reece et al., tantly related to members of the Heterolobosea, a phylum 2004) (Fig. 3). If this is the case, this phylotype could of widely diverse flagellated free-living amoeba. Some correspond to a parasite as well. The clone LC104-3EP-8 heterolobosean amoeba are thermotolerant, such as occupies an interesting phylogenetic position, as it Naegleria spp., and have been isolated and detected in branched at the base of the Chlorarachniophyta, thus hot springs and effluents from power plants (Sheehan breaking the relatively long branch leading to this group. et al., 2003). The third cercozoan clone (LC103-5EP-6) was very After the alveolates, and similarly to the fungi, the most closely related to Massisteria marina, a heterotrophic represented phylum in our Lost City libraries was that of flagellate that was isolated from four hydrothermal vents the Euglenozoa (Table S2 in Supplementary material and along the East Pacific Rise (Juan de Fuca Ridge, Fig. 4). Members of two classes of this phylum, Diplone- Guaymas basin, 21°N and 9°N) (Atkins et al., 2000). mida and Kinetoplastida, were detected in Lost City

© 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 546–554 552 P. López-García, A. Vereshchaka and D. Moreira samples. Kinetoplastid sequences were detected only in and Group II (Syndiniales) phylotypes were found in the carbonate samples, whereas diplonemid sequences were fluid–seawater interface. Similarly, in the case of Eugle- exclusively detected in the fluid–seawater interface nozoa, diplonemids were only detected in fluid–seawater (Fig. 3). One kinetoplastid phylotype (LC103-5EP-36) libraries, whereas kinetoplastids were exclusively identi- was very close to Neobodo species, among the free-living fied in carbonate samples. Fungi were also exclusively bodonid flagellates, whereas the other kinetoplastid detected in fluid–seawater libraries. A differential distri- sequence (LC103-5EP-19) was related to a group of the bution of lineages was also observed for the less parasitic Ichthyobodo species. Within the diplonemids, represented phyla. Thus, heterokont and radiolarian one phylotype (LC23-5EP-5) was closely related to the sequences were retrieved only from fluid–seawater heterotrophic genus Rhynchopus. Interestingly, all the samples, whereas cercozoan and heterolobosean other phylotypes detected formed a well-supported sequences were found only in carbonates (Fig. 4). It is cluster with the environmental sequence DH148-EKB1 also worth mentioning that we did not detect any lineage coming from 3000-m-deep plankton (López-García et al., of uncertain phylogenetic affiliation in Lost City, in con- 2001). This cluster appears to form a new group within the trast with observations from hydrothermal sediments at diplonemids, which has been detected so far only in the Guaymas and Rainbow that revealed the existence of deep ocean. On the one hand, the detection of this new potential new high-rank taxonomic groups (Edgcomb group of phylotypes spans the described genetic diversity et al., 2002; López-García et al., 2003). Some lineages within the Diplonemida, a class still poorly known and little in those sediments can indeed represent novel eukary- observed in natural samples. On the other hand, given the otic phyla as deduced from careful analyses that ruled phylogenetic position of this group as sister to known out the possibility of methodological artefacts linked to diplonemids, it would be particularly interesting to isolate the presence of chimeric or fast-evolving sequences, members of this cluster in order to characterize their mito- and to the lack of adequate taxonomic representation in chondrial DNA. Diplonemids are the sister group to the 18S rDNA phylogenies (Berney et al., 2004; Cavalier- kinetoplastids, which are characterized by complex net- Smith, 2004). These observations suggest that potential works of massed mitochondrial DNAs (Lukes et al., 2005). novel divergent lineages are generally confined to Very recently, the mitochondrial genome structure of anoxic sediments, whose study is more difficult and has Diplonema has been shown to be not only different from been often disregarded. that of kinetoplastids but unique among eukaryotes as a When comparing protist diversity among different deep- whole (Marande et al., 2005). Therefore, having cultured sea samples, including both plankton and a variety of representatives of related organisms could help to under- deep-sea hydrothermal samples (sediments, artificial sub- stand the evolutionary origin of the complex mitochondrial strates, natural substrates – carbonates – and fluid– genomes and their regulation, perhaps leading to the seawater mix), the most remarkable trend is the discovery of new mitochondrial genome organization vari- dominance of alveolate lineages in all 18S rDNA libraries ants in these euglenozoans. from deep-sea samples (Fig. 4). Nevertheless, the pro- portion of the different alveolate subgroups varies depending on the type of deep-sea environment, probably Comparison of protist diversity in Lost City with reflecting a preference for particular sets of physicochemi- that of deep-sea plankton and other deep-sea vents cal and/or ecological conditions. For instance, Perkinso- and some functional predictions zoa, typical parasites of bivalve molluscs, are found in Although protist diversity data are still quite limited for the Mid-Atlantic Ridge samples where extensive colonies of deep sea and deep-sea hydrothermal vents, it is possible endemic hydrothermal bivalves have been described to carry out preliminary comparisons between types of (López-García et al., 2003). The atypical case of samples (plankton and benthos) and between sites to see Guaymas sediment, where the autochthonous microbial whether general trends in the distribution of particular taxa community appeared to be contaminated by organisms occur. sedimenting from the whole water column (Edgcomb Important differences in the distribution of lineages et al., 2002), becomes apparent in this comparison. In between carbonate and fluid–seawater were apparent in addition to the abundance of different alveolate phylo- Lost City although, in both types of samples, regardless types in Guaymas sediment, there is not only a notable of metazoan sequences, the most abundant and diverse occurrence of photosynthetic eukaryotes (green algae phylum was by far that of the Alveolata, followed by and photosynthetic heterokonts) but also a high propor- Euglenozoa and Fungi (Fig. 4). However, although tion of heterotrophic heterokonts (Fig. 4). The latter alveolates dominated both types of samples, only cili- appear to be particularly abundant in the plankton of the ates were detected in carbonates, whereas more or less photic zone (Massana et al., 2004) rather than the deep equivalent proportions of dinoflagellate, ciliate, Group I sea. As a matter of fact, a comparable abundance of

© 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 546–554 Eukaryotes in Lost City hydrothermal system 553 heterokonts is detected in the shallow (200 m deep) Tagiri References hydrothermal site (Takishita et al., 2005) (Fig. S1 in Atkins, M.S., Teske, A.P., and Anderson, O.R. (2000) A Supplementary material), confirming the association of survey of flagellate diversity at four deep-sea hydrothermal large heterotrophic heterokont diversity with surface vents in the Eastern Pacific Ocean using structural and oceanic waters. molecular approaches. J Eukaryot Microbiol 47: 400–411. Although less apparent that the dominance of alveo- Bass, D., and Cavalier-Smith, T. (2004) Phylum-specific envi- lates in the deep sea, another observation in this com- ronmental DNA analysis reveals remarkably high global parison is the preference of some taxonomic groups for biodiversity of Cercozoa (Protozoa). Int J Syst Evol Micro- biol 54: 2393–2404. solid substrates. Despite the fact that ciliates are present Baumgartner, M., Stetter, K.O., and Foissner, W. (2002) Mor- in all deep-sea samples and are quite diverse, they phological, small subunit rRNA, and physiological charac- appear particularly abundant in 18S rDNA libraries from terization of Trimyema minutum (Kahl, 1931), an anaerobic artificial colonization substrates and carbonates. Kineto- ciliate from submarine hydrothermal vents growing from 28 plastids, and more particularly bodonids, seem to exhibit a degrees C to 52 degrees C. J Eukaryot Microbiol 49: 227– similar trend, as they have not been detected in deep-sea 238. plankton or fluid–seawater mix up to date. This trend may Berney, C., Fahrni, J., and Pawlowski, J. (2004) How many novel eukaryotic ‘kingdoms’? Pitfalls and limitations of be related to the grazing lifestyle of free-living bodonids environmental DNA surveys. BMC Biol 2: 13. and many ciliates, which may feed easily on the microbial Cavalier-Smith, T. (2004) Only six kingdoms of life. Proc R mats that colonize substrates around vents. Soc Lond B Biol Sci 271: 1251–1262. The wide eukaryotic diversity identified in Lost City con- Dawson, S.C., and Pace, N.R. (2002) Novel kingdom-level firms that protists are present in deep-sea hydrothermal eukaryotic diversity in anoxic environments. Proc Natl Acad systems and possibly play a variety of ecological func- Sci USA 99: 8324–8329. tions in the trophic chain. Lost City microbial eukaryotes Douville, E., Charlou, J.L., Oelkers, E.H., Bienvenu, P., Jove Colon, C.F., Donval, J.P., et al. (2002) The rainbow vent do not only include typical predators or grazers that likely fluids (36°14′N, MAR): the influence of ultramafic rocks and exert a control on bacterial populations, such as the phase separation on trace metal content in Mid-Atlantic bodonids, many ciliates, radiolaria and possibly the Ridge hydrothermal fluids. Chem Geol 184: 37–48. dinoflagellates and some cercozoa, but also decompos- Edgcomb, V.P., Kysela, D.T., Teske, A., De Vera Gomez, A., ers and parasites. Fungi are most likely decomposers, and Sogin, M.L. (2002) Benthic eukaryotic diversity in the and it is possible that many Lost City fungi are anaerobic Guaymas Basin hydrothermal vent environment. Proc Natl or microaerophilic, as at least one fungal phylotype Acad Sci USA 99: 7658–7662. Heptner, M.V., and Ivanenko, V.N. (2002) Copepoda branches within a group of fungi formed by environmental (Crustacea) of hydrothermal ecosystems of the World sequences exclusively retrieved so far from deep-sea Ocean. Arthropoda Selecta 12: 117–134. vents, continental mud volcanoes and brackish sediments Humes, A.G. (1987) Copepoda from deep-sea hydrothermal (Fig. 2). Parasitic lineages appear to be also present in vents. Bull Mar Sci 41: 645–788. Lost City, such as the Syndiniales, the haplosporidian Kelley, D.S., Karson, J.A., Blackman, D.K., Fruh-Green, G.L., cercozoa and, perhaps, some ciliates and basal kineto- Butterfield, D.A., Lilley, M.D., et al. (2001) An off-axis plastids related to Ichthyobodo. This is in agreement with hydrothermal vent field near the Mid-Atlantic Ridge at 30 degrees N. Nature 412: 145–149. previous observations of diverse parasitic protists in asso- Kelley, D.S., Karson, J.A., Fruh-Green, G.L., Yoerger, D.R., ciation with deep-sea vent environments (Moreira and Shank, T.M., Butterfield, D.A., et al. (2005) A serpentinite- López-García, 2003). In the case of divergent lineages, hosted ecosystem: the Lost City hydrothermal field. such as the new deep-sea diplonemid cluster, it is not Science 307: 1428–1434. possible to ascertain their mode of life for the moment. Langmuir, C., Humphris, S., Fornari, D., Van Dover, C., Von Now that their presence has been detected, it should be Damm, K., Tivey, M.K., et al. (1997) Hydrothermal vents possible to design specific probes to identify and quantify near a mantle hot spot: the Lucky Strike vent field at 37°N on the Mid-Atlantic Ridge. Earth Planet Sci Lett 148: them by fluorescent in situ hybridization as well as to 69–91. attempt directed isolation and cultivation to gain insights Lein, A.Y., Bogdanov, Y.A., Sagalevich, A.M., Ul’yanov, A.A., in their physiology and lifestyle. Chernyshev, I.V., Dubinina, E.O., and Ivanov, M.V. (2004) A new type of vent fields at the Mid-Atlantic Ridge (vent Acknowledgements field Lost City, 30 S). Doklady Akademii nauk (Proc Acad Rus Sci) 394: 380–383. We are grateful to the team involved in the 50th cruise of R/V López-García, P., Rodríguez-Valera, F., Pedrós-Alió, C., and ‘Akademik Mstislav Keldysh’ and to the Russian Academy of Moreira, D. (2001) Unexpected diversity of small eukary- Sciences. This work was supported by an ATIP Plus grant by otes in deep-sea Antarctic plankton. Nature 409: 603–607. the French Centre National de la Recherche Scientifique, López-García, P., Philippe, H., Gaill, F., and Moreira, D. section ‘Dynamique de la biodiversité’, to P.L.-G. (2003) Autochthonous eukaryotic diversity in hydrother-

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