Erwan G Roussel
email: rousseleg@cardiff.ac.uk

Cécile Konn

Jean-Luc Charlou

Jean-Pierre Donval

Yves Fouquet

Joël Querellou

Daniel Prieur

Marie-Anne Cambon Bonavita

Comparison of microbial communities associated with three Atlantic ultramafic hydrothermal systems

Keywords: Archaea, hydrothermal vent, Mid-Atlantic Ridge, 16S rRNA gene, Sediment, ultramafic

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Introduction

Deep-sea hydrothermal environments are characterized by intense physico-chemical gradients providing a large range of habitats for chemolithoautotrophic microorganisms [START_REF] Kelley | Volcanoes, fluids, and life at mid-ocean ridge spreading centers[END_REF]. Most of the studies of microbial diversity associated with deep-sea hydrothermal environments have mainly investigated basaltic-hosted hydrothermal systems [START_REF] Kelley | Volcanoes, fluids, and life at mid-ocean ridge spreading centers[END_REF]. However, a few studies showed that ultramafic-hosted hydrothermal systems contained specific microbial communities [START_REF] Brazelton | Methane-and sulfur-metabolizing microbial communities dominate the Lost City hydrothermal field ecosystem[END_REF][START_REF] Perner | The influence of ultramafic rocks on microbial communities at the Logatchev hydrothermal field, located 15 degrees N on the Mid-Atlantic Ridge[END_REF][START_REF] Voordeckers | Culture dependent and independent analyses of 16S rRNA and ATP citrate lyase genes: a comparison of microbial communities from different black smoker chimneys on the Mid-Atlantic Ridge[END_REF][START_REF] Flores | Microbial community structure of hydrothermal deposits from geochemically different vent fields along the Mid-Atlantic Ridge[END_REF]. To date, only three ultramafic sites were fully described on the Mid-Atlantic Ridge (Rainbow, Lost City and Logatchev), and were characterized by high concentrations of methane and hydrogen, in contrast with basaltichosted hydrothermal systems [START_REF] Kelley | An off-axis hydrothermal vent field near the Mid-Atlantic Ridge at 30[deg][END_REF][START_REF] Charlou | Geochemistry of high H 2 and CH 4 vent fluids issuing from ultramafic rocks at the Rainbow hydrothermal field (36 degrees 14 ' N, MAR)[END_REF][START_REF] Schmidt | Geochemistry of hydrothermal fluids from the ultramafic-hosted Logatchev hydrothermal field, 15 degrees N on the Mid-Atlantic Ridge: Temporal and spatial investigation[END_REF]. Moreover, Ashadze, a novel hydrothermal site, was recently reported on the Mid-Atlantic Ridge (MAR) [START_REF] Bel'tenev | New hydrothermal sites at 13ºN, Mid-Atlantic Ridge[END_REF][START_REF] Charlou | High hydrogen and abiotic hydrocarbons from new ultramafic hydrothermal sites between 12°N and 15°N on the Mid Atlantic Ridge-Results of the Serpentine cruise[END_REF][START_REF] Fouquet | Diversity of ultramafic hosted hydrothermal deposits on the Mid Atlantic Ridge: first submersible studies on Ashadze, Logatchev 2 and Krasnov vent fields during the Serpentine cruise[END_REF][START_REF] Mozgova | Mineralogy of massive sulfides from the Ashadze hydrothermal field, 13°N, Mid-Atlantic Ridge[END_REF][START_REF] Bassez | Organic analysis of peridotite rocks from the Ashadze and Logatchev hydrothermal sites[END_REF][START_REF] Charlou | High production and fluxes of H 2 and CH 4 and evidence of abiotic hydrocarbon synthesis by serpentinization in ultramafic-hosted hydrothermal systems on the Mid-Atlantic Ridge[END_REF]. This part of the MAR is characterized by rock compositions indicating that anomalously enriched mantle domains are involved in the melting region [START_REF] Dosso | Geochemical morphology of the North Mid-Atlantic Ridge, 10-Degrees-24-Degrees-N -Trace Element-Isotope Complementarity[END_REF], and also by numerous outcrops of serpentinized mantlederived rocks [START_REF] Bougault | Fast and slow-spreading ridges -Structure and hydrothermal activity, ultramafic topographic highs, and CH 4 output[END_REF][START_REF] Cannat | Ultramafic and gabbroic exposures at the Mid-Atlantic Ridge: geological mapping in the 15 degrees N region[END_REF]. However, these ultramafic systems expelling fluids characterized by moderate to high temperatures are also probably linked to magmatic heating processes [START_REF] Allen | Serpentinization and heat generation: Constraints from Lost City and Rainbow hydrothermal systems[END_REF]. Ultramafic hydrothermal fluids are highly enriched in abiogenic methane and hydrogen as a result of serpentinization reactions between the ultramafic rocks and seawater [START_REF] Holm | Initial indications of abiotic formation of hydrocarbons in Rainbow ultramafic hydrothermal system, Mid-Atlantic Ridge[END_REF][START_REF] Charlou | Geochemistry of high H 2 and CH 4 vent fluids issuing from ultramafic rocks at the Rainbow hydrothermal field (36 degrees 14 ' N, MAR)[END_REF][START_REF] Allen | Serpentinization and heat generation: Constraints from Lost City and Rainbow hydrothermal systems[END_REF], and could therefore supply twice as much chemical energy as basaltic-hosted hydrothermal systems [START_REF] Mccollom | Geochemical constraints on sources of metabolic energy for chemolithoautotrophy in ultramafic-hosted deep-sea hydrothermal systems[END_REF]. Hence, most of the prokaryotes found at these sites seemed to be related to methane and hydrogen cycling [START_REF] Boetius | Lost City Life[END_REF][START_REF] Perner | The influence of ultramafic rocks on microbial communities at the Logatchev hydrothermal field, located 15 degrees N on the Mid-Atlantic Ridge[END_REF][START_REF] Voordeckers | Culture dependent and independent analyses of 16S rRNA and ATP citrate lyase genes: a comparison of microbial communities from different black smoker chimneys on the Mid-Atlantic Ridge[END_REF][START_REF] Flores | Microbial community structure of hydrothermal deposits from geochemically different vent fields along the Mid-Atlantic Ridge[END_REF].

A large number of microbial communities from hydrothermal environments could be fuelled by inorganic compounds [START_REF] Amend | Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and Bacteria[END_REF]. Although these microbial communities occupy both aerobic and anaerobic habitats, anaerobic hyper/thermophilic Archaea are reported to be usually associated with the hottest parts of these environments [START_REF] Kelley | Volcanoes, fluids, and life at mid-ocean ridge spreading centers[END_REF][START_REF] Schrenk | Incidence and diversity of microorganisms within the walls of an active deep-sea sulfide chimney[END_REF]Takai et al., 2004a), some of which could be entrained by hydrothermal fluids from subsurface ecosystems [START_REF] Deming | Deep-sea smokers: windows to a subsurface biosphere?[END_REF][START_REF] Holden | Thermophilic and hyperthermophilic microorganisms in 3-30 degrees C hydrothermal fluids following a deep-sea volcanic eruption[END_REF]Summit & Baross, 1998). Moreover, it was suggested that Archaea could encompass up to 33-50% of the total microbial community in deep-sea hydrothermal environments [START_REF] Harmsen | Distribution of microorganisms in deep-sea hydrothermal vent chimneys investigated by whole-cell hybridization and enrichment culture of thermophilic subpopulations[END_REF][START_REF] Nercessian | Archaeal diversity associated with in situ samplers deployed on hydrothermal vents on the East Pacific Rise (13 °N)[END_REF].

Although an increasing number of thermophilic prokaryotes are cultivated from hydrothermal environments [START_REF] Huber | A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont[END_REF][START_REF] Miroshnichenko | Recent developments in the thermophilic microbiology of deepsea hydrothermal vents[END_REF][START_REF] Reysenbach | A ubiquitous thermoacidophilic archaeon from deep-sea hydrothermal vents[END_REF][START_REF] Wagner | Diversity of thermophilic anaerobes[END_REF][START_REF] Slobodkina | Geoglobus acetivorans sp. nov., an iron(III)-reducing archaeon from a deep-sea hydrothermal vent[END_REF], molecular phylogenetic approaches have revealed several new uncultivated lineages [START_REF] Takai | Genetic diversity of Archaea in deep-sea hydrothermal vent environments[END_REF][START_REF] Nercessian | Archaeal diversity associated with in situ samplers deployed on hydrothermal vents on the East Pacific Rise (13 °N)[END_REF][START_REF] Kormas | Bacterial and archaeal phylotypes associated with distinct mineralogical layers of a white smoker spire from a deep-sea hydrothermal vent site (9 degrees N, East Pacific Rise)[END_REF]Moussard et al., 2006a).

Metagenomic approaches and functional gene analyses have also contributed to the characterization of metabolic and physiological properties of these communities [START_REF] Nercessian | Diversity of functional genes of methanogens, methanotrophs and sulfate reducers in deep-sea hydrothermal environments[END_REF]Moussard et al., 2006b;Moussard et al., 2006c). However, to our knowledge, rRNA-based molecular approaches have seldom been used to compare the microbial diversity from multiple different hydrothermal sites (López-García et al., 2003a;López-García et al., 2003b;[START_REF] Voordeckers | Culture dependent and independent analyses of 16S rRNA and ATP citrate lyase genes: a comparison of microbial communities from different black smoker chimneys on the Mid-Atlantic Ridge[END_REF][START_REF] Flores | Microbial community structure of hydrothermal deposits from geochemically different vent fields along the Mid-Atlantic Ridge[END_REF].

In the present study, we characterized the molecular genetic diversity, using 16S rRNA gene and functional genes of methanogens, methanotrophs and sulfate-reducers, associated with three ultramafic-hosted hydrothermal sites: Rainbow, Lost City and Ashadze. As these hydrothermal fluids are highly enriched in methane and hydrogen, these environments could harbour specific prokaryotic communities possibly associated with potential subsurface chemolithoautotrophic ecosystems. Hence, the aim of this study was to compare the microbial communities of these ultramafic-hosted hydrothermal sites using molecular genetic methods, in order to correlate their phylogeny with ecological niches.

Materials and methods

Site location and sampling techniques

Fluid, chimney and sediment samples were collected during the scientific cruises EXOMAR (2005), SERPENTINE (2007) and MoMARDREAM-Naut (2007) conducted with the R. V. "L'Atalante" and "Pourquoi pas ?" and using the ROV "Victor 6000" and DSV "Nautile". The three hydrothermal fields explored, Rainbow (36°13'N; 33°54'W; ~ 2300 m depth), Lost City (30°07'N; 42°07'W; ~ 750 m depth) and Ashadze 1 (12°58'N; 44°51'W; ~ 4090 m depth) were all located along the Mid-Atlantic Ridge (MAR), although Lost City and Ashadze were further from the axis (Fig. 1a).

Fluid samples from Rainbow, Lost City and Ashadze were collected respectively from the "thermitière" chimney (36°13'76''N; 33°54'16''W; 2294 m depth, Fig. 1e), from a flange near the EXOMAR 11 Marker (30°07'43'' N; 42°07'16''W; 748 m depth, Fig. 1c), and from two chimneys in the SE2 area at Ashadze 1 site (12°58'33''N; 44°51'78''W; 4097 m depth, Fig. 1f). Chimney samples were also retrieved from the two chimneys in the SE2 area of Ashadze 1 site where the fluids were previously collected. The sediment samples were retrieved from the Rainbow site close to the active hydrothermal area (36°13'76''N; 33°54'04''W; 2287 m depth, Fig. 1d) and from the immediate periphery of the Lost City site (30°07'57'' N; 42°07'05''W; 752 m depth, Fig. 1b).

In order to describe the microbial communities from the surrounding seawater, the water column from the Rainbow site (36°13'76''N; 33°54'06''W; 2291 m depth) was also sampled.

All fluid samples were collected using titanium syringes and analyzed as described elsewhere [START_REF] Charlou | Geochemistry of high H 2 and CH 4 vent fluids issuing from ultramafic rocks at the Rainbow hydrothermal field (36 degrees 14 ' N, MAR)[END_REF]. On board, the fluid samples were immediately removed aseptically from the titanium syringes and stored at -80°C for molecular genetic analyses.

On board the sediment cores (~ 20 cm in length, 5 cm diameter) collected from the Rainbow site, using a push-core device operated by the arm of the DSV "Nautile", were sectioned in three equal samples and were designated as top, middle and bottom. The sediment surface sample from the Lost City site was collected using PSDE system (Fig. 1b, Kato C., unpublished). The chimney fragments were collected in biobox and sediment samples were stored aseptically at -80°C for molecular genetic analyses.

DNA extractions and PCR amplification

To avoid contaminations, all manipulations were carried out in a PCR cabinet (Biocap™ RNA/DNA, erlab ® ), using Biopur ® 1.5 mL Safe-Lock micro test tubes (Eppendorf™), Rnase/Dnase Free Water (MP Biomedicals™) and UV-treated (>60 min) plasticware and pipettes.

DNA extractions from fluids were performed from 50 mL of fluid left to thaw on ice prior centrifugation (15000 g for 60 min). Supernatant was carefully discarded and DNA was extracted from the pellet, following a modified FastDNA ® Spin Kit for Soil (Bio101 Systems, MP Biomedicals™) protocol [START_REF] Webster | Assessment of bacterial community structure in the deep sub-seafloor biosphere by 16S rDNA-based techniques: a cautionary tale[END_REF]Roussel et al., 2009a). The DNA extractions from sediments and chimney fragments were also performed using the modified FastDNA ® Spin Kit for Soil as described elsewhere (Roussel et al., 2009a).

All amplifications were performed using a "GeneAmp PCR system" 9700 ® (Applied Biosystems™). All PCR mixtures (50 µL) contained 5 µL of DNA template, 1X Taq DNA polymerase buffer (MP Biomedicals™), 1 µL of dNTP (10 mM of each dATP, dCTP, dGTP and dTTP), 10 µM of each primer and 0.5 µL of Taq DNA polymerase (MP Biomedicals™).

Negative controls were also carried out with DNA extractions performed without any sample.

For all controls, no PCR products were detected. Inhibition of PCR amplification by soluble contaminants in the DNA extracts was also tested as described elsewhere [START_REF] Juniper | Extraction and purification of DNA from organic rich subsurface sediments (ODP Leg 169S)[END_REF].

Archaeal 16S rRNA gene amplification was conducted by nested PCR with combination of primers A8f (5'-CGG TTG ATC CTG CCG GA-3') and A1492r (5'-GGC TAC CTT GTT ACG ACT T-3') in the first round [START_REF] Teske | Microbial diversity of hydrothermal sediments in the Guaymas Basin: evidence for anaerobic methanotrophic communities[END_REF][START_REF] Lepage | Molecular diversity of new Thermococcales isolates from a single area of hydrothermal deepsea vents as revealed by randomly amplified polymorphic DNA fingerprinting and 16S rRNA gene sequence analysis[END_REF], and with A344f (5'-AYG GGG YGC ASC AGG SG-3') and A915r (5'-GTG CTC CCC CGC CAA TTC CT-3') in the second round (Stahl & Amann, 1991;Sørensen et al., 2004). PCR cycles for the first round (A8f/A1492r), and for the second round (A344f/A915r) were as previously described (Roussel et al., 2009a). To minimize PCR bias, five independent PCR products from the first round were pooled and purified (QIAquick PCR purification Kit; Qiagen™) and used as template for the second round. This nested PCR was necessary to obtain visible PCR products on a 0.8% (w/v) agarose gel stained with ethidium bromide.

A portion of the mcrA gene was amplified using the ME primers [START_REF] Hales | Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis[END_REF] with the following reaction conditions as described elsewhere (Roussel et al., 2009a). A fragment of the pmoA gene was amplified using the pmoA189-mb661 primer couple [START_REF] Holmes | Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily Related[END_REF][START_REF] Costello | Molecular characterization of functional and phylogenetic genes from natural populations of methanotrophs in lake sediments[END_REF] with the following reaction conditions: 1 cycle of 4 min at 92°C, 35 cycles of 1 min at 92°C, 1.5 min at 55°C and 1 min at 72°C, and 1 cycle of 9 min at 72°C. A portion of the dsrA gene was amplified using the DSR1F+ and DSR-R primers [START_REF] Kondo | Detection and enumeration of sulphatereducing bacteria in estuarine sediments by competitive PCR[END_REF] with the following reaction conditions: 1 cycle of 5 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 54°C and 2.5 min at 72°C, and 1 cycle of 8 min at 72°C. For all functional genes, two rounds with the previous reaction conditions were required to obtain visible amplification products. An aliquot (5 µL) of three pooled PCR products of the primary amplification was used as template for the second amplification round.

CM-DGGE analysis

In order to obtain the general archaeal 16S rRNA gene diversity associated with the hydrothermal environment and to compare it with the seawater diversity, a preliminary CM-DGGE analysis was performed as described elsewhere (Roussel et al., 2009b).

After amplification of the nested PCR products, using two different fluorescent reverse labelled (Cy3 or Cy5) primers from total DNA from either a hydrothermal sample or seawater, these were pooled and loaded into the same lane. Archaeal 16S rRNA gene amplification was performed with primers Saf-PARCH 519r, labelled with either Cy3 (hydrothermal samples) or Cy5 (seawater), following touchdown PCR protocol as previously described [START_REF] Nicol | The impact of grassland management on archaeal community structure in upland pasture rhizosphere soil[END_REF]. All manipulations were performed in the dark. The PCR products were analyzed by DGGE using a DCode Universal Mutation Detection System ® (BioRad™) on a 1 mm thick (16 × 16 cm) 8% (w/v) polyacrylamide gel (acrylamide/bisacrylamide, 40%, 37.5:1, BioRad™) with a denaturant gradient between 30 and 70% prepared with 1 × TAE buffer (pH 8, 40 mM Tris Base, 20 mM acetic acid, 1 mM EDTA, MP Biomedicals™) and poured with a "Gradient maker" (Hoefer SG30 ® ). Electrophoresis was carried in 1 × TAE buffer at 60°C for 330 min at 200 V (initially at 80 V for 10 min). The gel was scanned using a Phospho fluorimager Typhoon 9400 ® (Amersham Biosciences™).

Cloning and sequencing

Fourteen 16S rRNA gene, one dsrA gene, four mcrA gene, and eight pmoA gene clone libraries were constructed. To minimize PCR bias [START_REF] Polz | Bias in template-to-product ratios in multitemplate PCR[END_REF], five independent PCR products were pooled, purified (QIAquick PCR purification Kit; Qiagen™), and cloned into Escherichia coli (XL10-Gold; Stratagene™) using the pGEM-T Easy vector system I (Promega™) following the manufacturer's instructions. Positive transformants were screened by PCR amplification of the insert using the vector-specific M13 primers. Plasmid extraction, purification and sequencing of the insert were carried out by the sequencing Ouest-Genepole platform ® of Roscoff Marine laboratory (France).

Phylogenetic analysis and statistical analyses

Chimeras [START_REF] Cole | The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy[END_REF] were excluded from the clone libraries and a total of 759 sequences (including those from the 16S rRNA gene and functional genes) were used for further phylogenetical analysis. The phylogenetic placement was carried out using NCBI BLAST search program within GenBank (http://www.ncbi.nlm.nih.gov/blast) [START_REF] Altschul | Basic local alignment search tool[END_REF]. The 16S rRNA gene sequences (~553 bases) were then edited in the BioEdit 7.0.5.3 program [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF] and aligned using CLUSTALW [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF]. The phylogenetic trees were constructed by the PHYLO_WIN program (http:// pbil.univ-lyon1.fr/) [START_REF] Galtier | SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny[END_REF] with Neighbour-Joining method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF] and Jukes and Cantor correction. The nonchimeric mcrA (~0.76 kb), pmoA (~0.51 kb) and dsrA (~0.22 kb) sequences were translated into amino acids using BioEdit and then aligned using CLUSTALW, and the PHYLO_WIN program with Neighbour-Joining algorithm, and PAM distance [START_REF] Dayhoff | A model of evolutionary change in proteins[END_REF] was then used for phylogenetic tree construction. For the entire phylogenetic reconstruction, the robustness of inferred topology was tested by bootstrap resampling (1000), values over 50% are shown on the trees. The richness from the clone libraries was estimated, with the rarefaction curves at 99%, 97% and 95% sequence identity levels, using the DOTUR program [START_REF] Schloss | Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness[END_REF]. Operational taxonomic units (OTUs), using a 95% or 97% sequence similarity, were generated with the SON program [START_REF] Schloss | Introducing SONS, a tool for operational taxonomic unitbased comparisons of microbial community memberships and structures[END_REF], and the percentage of coverage (Cx) of the clone libraries was calculated by Good's method [START_REF] Good | The Population frequencies of species and the estimation of population parameters[END_REF] as described by Singleton and colleagues [START_REF] Singleton | Quantitative comparisons of 16S rRNA gene sequence libraries from environmental samples[END_REF]. Statistical estimators, the significance of population differentiation among clone libraries (F ST ) [START_REF] Martin | Phylogenetic approaches for describing and comparing the diversity of microbial communities[END_REF], and the exact tests of population genetic differentiation [START_REF] Raymond | An exact test for population differentiation[END_REF], were calculated using Arlequin 3.11 [START_REF] Excoffier | Arlequin ver. 3.0: An integrated software package for population genetics data analysis[END_REF].

Nucleotide sequence accession numbers

The sequences are available from GenBank database under the following accession numbers and names: 16S rRNA gene (FN650174 to FN650288), mcrA gene (FN650315 to FN650322), dsrA (FN650289 to FN650291) and pmoA (FN650292 to FN650314).

Results

Site description

A total of 15 samples encompassing fluids, chimney fragments and sediments, were retrieved from three Atlantic ultramafic-hosted hydrothermal sites: Rainbow, Lost City and Ashadze (Fig. 1). The dilution of the hydrothermal fluid sample was estimated according to pH measurements. Overall, the three sites had much higher hydrogen (<16 mM) and abiogenic methane (<2.5 mM) concentrations than the MAR basaltic-hosted hydrothermal sites [START_REF] Charlou | High production and fluxes of H 2 and CH 4 and evidence of abiotic hydrocarbon synthesis by serpentinization in ultramafic-hosted hydrothermal systems on the Mid-Atlantic Ridge[END_REF].

All the hydrothermal fluid samples from the Rainbow site were retrieved from the "thermitière" chimney group (Fig. 1e), except the "PP27 swarm" sample which was obtained in close proximity to a shrimp swarm on the side of the PP27 chimney. The "thermitière" chimney group was composed of both diffuse and black smoker venting. The Rainbow sediment samples were retrieved nearby the hydrothermal chimneys and were predominantly made of pelagic sediment (98% calcite) with a small amount of hematite, indicating a small hydrothermal contribution (Fig. 1d). For this study, the maximum temperature measured at Rainbow was 324°C, and the less diluted hydrothermal fluid analyzed had a pH of 3.40 (Fig. 2b), and high concentrations of hydrogen (>10 mM), carbon dioxide (17 mM), iron (>17 mM) and methane (>1mM) [START_REF] Charlou | High production and fluxes of H 2 and CH 4 and evidence of abiotic hydrocarbon synthesis by serpentinization in ultramafic-hosted hydrothermal systems on the Mid-Atlantic Ridge[END_REF].

The Lost City fluid samples were obtained from one of the hottest venting areas of this site, which was located above a flange (Fig. 1c). To date, Lost City is a unique off-axis hydrothermal site expulsing fluids with a high pH (~ 11), as opposed to the other known ultramafic environments that are acidic (Rainbow, Ashadze pH = ~ 3). The maximum temperature recorded at Lost City (93°C) was lower than for Rainbow and Ashadze. The less diluted hydrothermal fluid analyzed had a pH of 11.75 (Fig. 2a), and high concentrations of hydrogen (>7 mM) and methane (0.9 mM).

Ashadze, a hydrothermal field that was recently explored for the first time during the French-Russian Serpentine cruise [START_REF] Fouquet | Serpentine cruise -ultramafic hosted hydrothermal deposits on the Mid-Atlantic Ridge: First submersible studies on Ashadze 1 and 2, Logatchev 2 and Krasnov vent fields[END_REF], is one of the deepest active black smoker fields discovered so far (4100 m depth). Ashadze is characterized by an ultramafic rock environment [START_REF] Charlou | High hydrogen and abiotic hydrocarbons from new ultramafic hydrothermal sites between 12°N and 15°N on the Mid Atlantic Ridge-Results of the Serpentine cruise[END_REF][START_REF] Fouquet | Diversity of ultramafic hosted hydrothermal deposits on the Mid Atlantic Ridge: first submersible studies on Ashadze, Logatchev 2 and Krasnov vent fields during the Serpentine cruise[END_REF][START_REF] Fouquet | Serpentine cruise -ultramafic hosted hydrothermal deposits on the Mid-Atlantic Ridge: First submersible studies on Ashadze 1 and 2, Logatchev 2 and Krasnov vent fields[END_REF]. Several groups of active one to two meter high chimneys were observed at Ashadze 1 site. The fluid and chimney fragments were obtained from two different active chimneys in a unique group near the SE-2 marker (Fig. 1f). For this study, the maximum temperature measured at Ashadze was 353°C. The less diluted hydrothermal fluid analyzed had a pH of 4.02 (Fig. 2b), and high concentrations of hydrogen (>10 mM), carbon dioxide (>2.5 mM), iron (7.3 mM) and methane (>0.80 mM).

Archaeal 16S rRNA gene analyses

CM-DGGE. All the 16S rRNA gene PCR products from all the samples were screened by Co-Migration DGGE (CM-DGGE) prior cloning, in order to estimate the archaeal phylogenetic diversity of each hydrothermal sample and to compare it directly with the seawater diversity (Fig. 2a). Band pattern intensities from all Lost City samples, and from the less diluted hydrothermal fluids, were weaker than for all the other samples, suggesting a lower biomass and/or high concentration of PCR inhibitors (Fig. 2a). The archaeal seawater CM-DGGE band pattern was different from all the hydrothermal fluid and chimney band patterns (Fig. 2a), suggesting low levels of seawater contamination. The band patterns from hydrothermal samples were mostly composed of DGGE fragments with higher melting points, a probable consequence of higher GC content of the 16S rRNA gene. The high-GC content of these 16S rRNA gene sequences indicates that the Archaea could be hyper/thermophiles [START_REF] Kimura | Selective phylogenetic analysis targeted at 16S rRNA genes of thermophiles and hyperthermophiles in deep-subsurface geothermal Environments[END_REF], as also suggested by the several putative hyper/thermophilic lineages detected in the clone libraries from hydrothermal fluids and chimneys (Archaeoglobales, Methanococcales, Thermococcales, Methanopyrales, Desulfurococcales, Nanoarchaeota, DHVE; Fig. 2b).

Clone libraries. After technical optimization and removal of soluble PCR inhibitors and in order to amplify sufficient PCR product for cloning, archaeal amplifiable DNA from all samples was retrieved by nested PCR. However, no sufficient amplified PCR product was obtained for cloning from the less diluted fluid samples (pH 11.75) and from the chimney samples from Lost City. F ourteen different 16S rRNA gene clone libraries were constructed, representing a total of 610 sequences. The coverage values for the 16S rRNA gene clone libraries ranged from 68 to 97%, based on a 97% sequence similarity level (Fig. 2b). On the whole, rarefaction curves were asymptotic for all clone libraries, based on a 95% sequence similarity level, confirming sufficient sampling effort (Fig. S1).

The overall archaeal diversity analyzed was similar to previous studies (e.g. [START_REF] Brazelton | Methane-and sulfur-metabolizing microbial communities dominate the Lost City hydrothermal field ecosystem[END_REF][START_REF] Flores | Microbial community structure of hydrothermal deposits from geochemically different vent fields along the Mid-Atlantic Ridge[END_REF] and very heterogeneously distributed between the sites (Lost City, Rainbow, and Ashadze) and between types of samples (seawater, hydrothermal fluid, chimney and sediment). The number of OTUs per clone library ranged from five to nineteen, based on a 95% genus level of phylotype differentiation [START_REF] Schloss | Status of the microbial census[END_REF], and the Shannon-Wiener index of diversity ranged between 0.63 and 3.04 (Fig. 2b). The archaeal diversity indices of all the samples were in the same range, except for the fluid associated with Lost City which, as previously described [START_REF] Schrenk | Low archaeal diversity linked to subseafloor geochemical processes at the Lost City Hydrothermal Field, Mid-Atlantic Ridge[END_REF], displayed the lowest detectable diversity (Fig. 2a and2b). On average, the hydrothermal samples contained six different lineages, except for Lost City (Fig. 2a and2b), which is also in agreement with most published studies on hydrothermal environments (e.g. [START_REF] Takai | Distribution of Archaea in a black smoker chimney structure[END_REF][START_REF] Nercessian | Archaeal diversity associated with in situ samplers deployed on hydrothermal vents on the East Pacific Rise (13 °N)[END_REF][START_REF] Schrenk | Incidence and diversity of microorganisms within the walls of an active deep-sea sulfide chimney[END_REF][START_REF] Schrenk | Low archaeal diversity linked to subseafloor geochemical processes at the Lost City Hydrothermal Field, Mid-Atlantic Ridge[END_REF]Takai et al., 2004b;[START_REF] Kormas | Bacterial and archaeal phylotypes associated with distinct mineralogical layers of a white smoker spire from a deep-sea hydrothermal vent site (9 degrees N, East Pacific Rise)[END_REF][START_REF] Page | Temporal and spatial archaeal colonization of hydrothermal vent deposits[END_REF][START_REF] Nunoura | Archaeal diversity and distribution along thermal and geochemical gradients in hydrothermal sediments at the Yonaguni Knoll IV hydrothermal field in the southern Okinawa Trough[END_REF]. All the 16S rRNA gene sequences obtained from the clone libraries were assigned to 95 OTUs, based on a 95% sequence similarity level, forming a total of 21 different phylogenetic lineages (Fig. 2b, 3a and3b). On the whole, 16S rRNA gene sequences were related to Euryarchaeota (51%), Crenarchaeota (48%) and Nanoarchaeota (1%). The 16S rRNA gene clone libraries obtained from hydrothermal samples (fluid and chimney) were dominated by sequences related to Euryarchaeota (69%), whereas sequences related to Crenarchaeota were a majority in the sediment (92%) and seawater samples (70%) (Fig. 2b). Seven of the fifteen Euryarchaeota lineages detected had at least one known cultured representative, and six of these seven had known thermophilic Archaea (Halobacteriales, DHVE2, Archaeoglobales, Methanococcales, Thermococcales, Methanopyrales). Although three of the five Crenarchaeota lineages detected had at least one cultured representative, only one was known to be thermopilic (Desulfurococcales).

Moreover, Marine Group I (MG-I) Archaea had the highest intra-lineage diversity representing 25 OTUs based on a 95% genus level of phylotype differentiation.

Functional gene clone libraries

Diversity of mcrA gene. The operon coding for the MCR-I, which includes McrA subunit, is found in all known methanogens [START_REF] Reeve | Methanogenesis: genes, genomes, and who's on first?[END_REF]. Four mcrA clone libraries were obtained from sediment, fluid and chimney samples from Ashadze and Rainbow sites.

Although detected by previous studies [START_REF] Kelley | A serpentinite-hosted ecosystem: the Lost City hydrothermal field[END_REF], no mcrA gene sequences were detected from Lost City samples. The diversity of the four mcrA libraries was limited to sequences related to the H 2 /CO 2 methanogens Methanopyrales and Methanococcales orders (Fig. 4a), congruently with the 16S rRNA gene clone libraries (Fig. 3a). mcrA gene sequences affiliated to Methanopyrales were only detected at Rainbow. Moreover, the mcrA gene sequences from Rainbow and Ashadze matched the two groups of uncultured methanogenic Archaea previously retrieved from Rainbow [START_REF] Nercessian | Diversity of functional genes of methanogens, methanotrophs and sulfate reducers in deep-sea hydrothermal environments[END_REF].

Diversity of dsrA gene. Sequences coding for the dsrA gene were only retrieved from Ashadze chimney 1 (Fig. 4b). dsrA gene sequences were previously detected in chimney samples from Lost City [START_REF] Gerasimchuk | The search for sulfate-reducing bacteria in mat samples from the lost city hydrothermal field by molecular cloning[END_REF], and in sediments from Rainbow [START_REF] Nercessian | Diversity of functional genes of methanogens, methanotrophs and sulfate reducers in deep-sea hydrothermal environments[END_REF]. All dsrA gene sequences detected from Ashadze site were all related to sequences from marine sediments and East-Pacific Rise hydrothermal vents, as a probable consequence of a lack of dsrA gene sequences from the MAR in the databases. dsrA gene sequences were mainly affiliated to sequences from the Desulfobulbaceae family (Fig. 4b).

Diversity of pmoA gene. The pmoA gene was the most widespread functional gene detected, as a PCR amplification was obtained on eight out of the fifteen samples tested (Fig. 2b and4c). The phylogeny of the pmoA gene is usually poorly resolved, the bacterial pmoA gene being distantly related to the ammonia monooxygenase subunit A (amoA) [START_REF] Holmes | Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily Related[END_REF][START_REF] Nicol | Ammonia-oxidising Crenarchaeota: important players in the nitrogen cycle?[END_REF], as revealed by incongruence between tree topologies performed with different phylogenetic methods. However, two groups of pmoA sequences from Rainbow fluids and Ashadze chimney samples clustered (cluster pmoA 1 and cluster P-A) with sequences related to thermophilic methylotrophs [START_REF] Inagaki | Distribution and phylogenetic diversity of the subsurface microbial community in a Japanese epithermal gold mine[END_REF][START_REF] Hirayama | Culture-dependent and -independent characterization of microbial communities associated with a shallow submarine hydrothermal system occurring within a coral reef off Taketomi Island, Japan[END_REF](Fig. 4c). Moreover, pmoA gene sequences from sediments from Rainbow grouped into two major clusters (cluster pmoA 2 and pmoA 3). Sequences from cluster pmoA 2 did not have any closely related sequences (Fig. 4c).

Community structures and distribution analyses

Although the seawater and the Rainbow sediment CM-DGGE band patterns were quite similar (Fig. 2a), all the sediment clone library community structures were indistinguishable from the combined communities and significantly different (P <0.001) from the seawater (Fig. 2b). Insignificant F ST and P tests (P < 0.001), based on an analysis at a 97% sequence similarity level, suggested that community structures from all the Rainbow hydrothermal fluids and Ashadze chimney 1 clone libraries were similar and indistinguishable from the combined communities (Fig. 2b). However, although the archaeal community structures from all the Rainbow hydrothermal fluids were also from similar lineage distributions, all the Ashadze chimney and fluid samples had significantly different population structures (P <0.001; Fig. 2a and2b). The archaeal diversity of all the other clone libraries was also significantly different from the seawater clone library (P < 0.001), showing that the hydrothermal vent archaeal communities are probably adapted to their environment.

According to pH measurements, the archaeal diversity in the hydrothermal fluids was always the most reduced in the less diluted fluids (Fig. 2b). Moreover, a correlation was also observed between Methanococcales (P < 0.001) and Thermococcales (P < 0.05) lineages and the Rainbow fluids. Correlations were also shown between MG-I lineage and the hydrothermal sediments (P < 0.01), and between the unaffiliated Methanosarcinales cluster and the Lost City fluids (P < 0.0001).

Discussion

High diversity of putative chemolithoautotrophs

Overall, the analysis of the phylogenetic data showed a specific distribution of different putative metabolic processes over the different MAR ultramafic-hosted hydrothermal environments that were mainly dominated by putative chemolithoautotrophs.

Putative ammonia-oxidizing Crenarchaeota. Marine Group I (MG-I) was the most ubiquitous lineage found in the MAR ultramafic-hosted hydrothermal environments, as sequences related to the MG-I Archaea were detected in the majority of clone libraries (93%).

Interestingly, the archaeal community structure of the seawater clone library was dominated by sequences related to MG-I (41%) and Marine Group II (48%), but was significantly different from all the other clone libraries (P < 0.001). Congruently, Takai and colleagues showed that the highest proportion of MG-I members in a hydrothermal environment from the Central Indian Ridge, was found in the seawater adjacent to the hydrothermal emissions (Takai et al., 2004c). MG-I sequences also dominated the sediment 16S rRNA gene clone libraries (≥ 86%), as commonly found in marine surface sediments [START_REF] Inagaki | Archaeology of Archaea: geomicrobiological record of Pleistocene thermal events concealed in a deep-sea subseafloor environment[END_REF][START_REF] Teske | Uncultured Archaea in deep marine subsurface sediments: have we caught them all?[END_REF]Roussel et al., 2009b). The highest diversity indices were also observed in these sediment samples, as a consequence of the very high intra-phylum diversity observed within the MG-I. However, all the MG-I 16S rRNA gene sequences from the sediment clustered in phylogenetic groups different from the seawater MG-I, suggesting that specific MG-I communities could be associated with sedimentary environmental conditions (Roussel et al., 2009b). Moreover, the G + C content of all the MG-I sequences ranged between 48% to 52%, in opposition to the high-GC content of the 16S rRNA gene sequences from hydrothermal fluids or chimneys, thus supporting the hypothesis that these MG-I Archaea are probably adapted to the cooler ecological niches of the hydrothermal environments [START_REF] Kimura | Selective phylogenetic analysis targeted at 16S rRNA genes of thermophiles and hyperthermophiles in deep-subsurface geothermal Environments[END_REF][START_REF] Ehrhardt | Evidence for hydrothermal Archaea within the basaltic flanks of the East Pacific Rise[END_REF]. Several studies also showed that specific phylogenetic groups of MG-I Archaea appear to be endemic to basaltic crust environments [START_REF] Ehrhardt | Evidence for hydrothermal Archaea within the basaltic flanks of the East Pacific Rise[END_REF][START_REF] Mason | The phylogeny of endolithic microbes associated with marine basalts[END_REF][START_REF] Mason | Prokaryotic diversity, distribution, and insights into their role in biogeochemical cycling in marine basalts[END_REF]. Some of these specific subclades of MG-I Archaea could therefore be adapted to various environments as they were also detected in aerobic and anaerobic basalt enrichment cultures and sediment slurries [START_REF] Mason | The phylogeny of endolithic microbes associated with marine basalts[END_REF][START_REF] Webster | Prokaryotic functional diversity in different biogeochemical depth zones in tidal sediments of the Severn Estuary, UK, revealed by stable-isotope probing[END_REF]. Hence, as the MG-I Archaea were widespread in our hydrothermal fluid and chimney clone libraries, their presence could be the result of the mixing of ambient seawater with cool niche water in the rocks of the hydrothermal system.

MG-1 Archaea, regularly described as aerobic autotrophic ammonia oxidizers [START_REF] Francis | Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean[END_REF][START_REF] Konneke | Isolation of an autotrophic ammonia-oxidizing marine archaeon[END_REF][START_REF] Hallam | Pathways of carbon assimilation and ammonia oxidation suggested by environmental genomic analyses of marine Crenarchaeota[END_REF], are commonly found in seawater and marine sediments, forming several phylogenetic clusters with several cultured relatives (e.g. [START_REF] Preston | A psychrophilic crenarchaeon inhabits a marine sponge: Cenarchaeum symbiosum gen nov, sp, nov[END_REF][START_REF] Konneke | Isolation of an autotrophic ammonia-oxidizing marine archaeon[END_REF]. Moreover, based on the analysis of the first sequenced genome of a cultured relative (Cenarchaeum symbiosum), the MG-1 were proposed as a novel archaeal phylum named Thaumarchaeota [START_REF] Brochier-Armanet | Mesophilic crenarchaeota: proposal for a third archaeal phylum, the Thaumarchaeota[END_REF]. Interestingly, moderate thermophilic ammonia-oxidizing crenarchaeotes were recently isolated from hot springs (de la [START_REF] De La Torre | Cultivation of a thermophilic ammonia oxidizing archaeon synthesizing crenarchaeol[END_REF][START_REF] Hatzenpichler | A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring[END_REF] and may also play a major role in the nitrogen cycle in these environments [START_REF] Zhang | Global occurrence of archaeal amoA genes in terrestrial hot springs[END_REF][START_REF] Wang | Diversity and abundance of ammonia-oxidizing Archaea in hydrothermal vent chimneys of the Juan de Fuca Ridge[END_REF]. High ammonium concentration and removal rates were previously measured from a Pacific hydrothermal system [START_REF] Lam | Microbial ammonia oxidation and enhanced nitrogen cycling in the Endeavour hydrothermal plume[END_REF], and a thermophilic origin for anaerobic ammonium oxidation was also suggested [START_REF] Canfield | Early anaerobic metabolisms[END_REF]. Hence, according to their widespread dissemination in hydrothermal systems, as shown in several studies, and due to the high mixing processes occurring in these dynamic systems (e.g. Takai et al., 2004c), MG-I may also play a role in ammonium oxidation in hydrothermal systems as previously suggested for marine basalts [START_REF] Mason | The phylogeny of endolithic microbes associated with marine basalts[END_REF][START_REF] Mason | Prokaryotic diversity, distribution, and insights into their role in biogeochemical cycling in marine basalts[END_REF].

Putative hydrogen-oxidizing chemolithoautotrophs. All the ultramafic hydrothermal fluids from Rainbow, Ashadze and Lost city were highly enriched in abiogenic methane and hydrogen as a result of serpentinization reactions between ultramafic rocks and seawater [START_REF] Holm | Initial indications of abiotic formation of hydrocarbons in Rainbow ultramafic hydrothermal system, Mid-Atlantic Ridge[END_REF][START_REF] Charlou | Geochemistry of high H 2 and CH 4 vent fluids issuing from ultramafic rocks at the Rainbow hydrothermal field (36 degrees 14 ' N, MAR)[END_REF][START_REF] Allen | Serpentinization and heat generation: Constraints from Lost City and Rainbow hydrothermal systems[END_REF]. Moreover, McCollom showed that ultramafic-hosted hydrothermal systems could theoretically provide twice as much chemical energy as comparable basaltic-hosted systems [START_REF] Mccollom | Geochemical constraints on sources of metabolic energy for chemolithoautotrophy in ultramafic-hosted deep-sea hydrothermal systems[END_REF]. More than half of the archaeal lineages detected from Rainbow, Ashadze and Lost City were related to known cultured species, two thirds of which were involved in hydrogen or methane cycling processes, supporting the theory that these ecosystems could be mainly fuelled by hydrothermal fluids highly enriched in hydrogen and methane.

Methanogenesis was the most common putative hydrogen-oxidizing metabolism detected among ultramafic hydrothermal fluids and chimney samples. Indeed, among all archaeal lineages in the hydrothermal samples, Methanococcales was the most widespread, as it was detected in a majority of the clone libraries (78%) obtained from hydrothermal fluids or chimney samples. Interestingly, all the sequences related to Methanococcales from Rainbow and Ashadze grouped with sequences previously detected at Rainbow [START_REF] Nercessian | Diversity of functional genes of methanogens, methanotrophs and sulfate reducers in deep-sea hydrothermal environments[END_REF], suggesting that these could be long-term stabilized population in these chemically slow evolving environments [START_REF] Charlou | Geochemistry of high H 2 and CH 4 vent fluids issuing from ultramafic rocks at the Rainbow hydrothermal field (36 degrees 14 ' N, MAR)[END_REF]. Methanococcales Archaea are strictly anaerobic autotrophic methanogens, using hydrogen and carbon dioxide or formate as energy sources [START_REF] Whitman | Bergey's Manual of Systematic Bacteriology. The Archaea and The Deeply Branching and Phototrophic Bacteria[END_REF]. Strains affiliated to Methanocaldococcus infernus were successfully cultured from hydrothermal chimney samples from Rainbow and Ashadze (Jeanthon C. and L'Haridon S. personal communication, respectively). Moreover, the methanogenic potential of Methanococcales at Rainbow and Ashadze was also confirmed by the detection of mcrA genes related to Methanothermococcus thermolithotrophicus (> 97% similarity) and Methanocaldococcus infernus (> 97%). Although hyperthermophilic or thermophilic members of the Methanococcales are commonly cultured and detected with molecular tools from marine hydrothermal vent systems (e.g. [START_REF] Kelley | Volcanoes, fluids, and life at mid-ocean ridge spreading centers[END_REF][START_REF] Nercessian | Archaeal diversity associated with in situ samplers deployed on hydrothermal vents on the East Pacific Rise (13 °N)[END_REF][START_REF] Schrenk | Incidence and diversity of microorganisms within the walls of an active deep-sea sulfide chimney[END_REF]Takai et al., 2004a;[START_REF] Perner | The influence of ultramafic rocks on microbial communities at the Logatchev hydrothermal field, located 15 degrees N on the Mid-Atlantic Ridge[END_REF][START_REF] Page | Temporal and spatial archaeal colonization of hydrothermal vent deposits[END_REF], Schrenk and colleagues reported that Methanococcales encompassed a low proportion (<5%) of the hydrothermal prokaryotic communities associated with the walls of a sulphide chimney, whereas on different hydrothermal sites Takai and colleagues reported proportions up to 76.5% [START_REF] Schrenk | Incidence and diversity of microorganisms within the walls of an active deep-sea sulfide chimney[END_REF]Takai et al., 2004a). These differences could be linked to environmental factors such as the high hydrogen production from these ultramafic systems, which fuel these communities.

Moreover, putative hyperthermopilic methanogens were also represented by Methanopyrales. Methanopyrales were rarely detected on the MAR by molecular methods [START_REF] Flores | Microbial community structure of hydrothermal deposits from geochemically different vent fields along the Mid-Atlantic Ridge[END_REF], probably as a consequence of technical biases or of the restricted number of microbial studies of the MAR, though the first isolated member originates from a hydrothermal system north of Iceland [START_REF] Kurr | Methanopyrus kandleri, Gen and sp-nov represents a novel group of hyperthermophilic methanogens, Growing at 110°C[END_REF]. Sequences related to Methanopyrales were rarely detected elsewhere [START_REF] Nercessian | Archaeal diversity associated with in situ samplers deployed on hydrothermal vents on the East Pacific Rise (13 °N)[END_REF]Takai et al., 2004a;[START_REF] Ehrhardt | Evidence for hydrothermal Archaea within the basaltic flanks of the East Pacific Rise[END_REF][START_REF] Page | Temporal and spatial archaeal colonization of hydrothermal vent deposits[END_REF]. However, in this study, eighteen sequences related to Methanopyrus kandleri (>96% similarity) were detected from Rainbow and Ashadze fluids.

Interestingly, Takai and colleagues also reported recently an isolate related to Methanopyrus kandleri capable of methanogenesis with H 2 /CO 2 under elevated hydrostatic pressures and at 122°C [START_REF] Takai | Cell proliferation at 122°C and isotopically heavy CH 4 production by a hyperthermophilic methanogen under high-pressure cultivation[END_REF]. As mcrA gene sequences related to Methanopyrus kandleri (88% similarity) were also detected at Rainbow and as strains affiliated to Methanopyrales were successfully cultured from Rainbow and Ashadze (Jeanthon and L'Haridon personal communication, respectively), the Methanopyrales detected were probably capable of methanogenesis, thus supporting the hypothesis that these sites may harbour large methanogenic communities.

Archaeoglobales, another putative hydrogen-oxidizing archaeal lineage, was also found to be widespread among hydrothermal samples from Rainbow and Ashadze. 16S rRNA gene sequences closely related (> 96% similarity) to members of genus Archaeoglobus, Geoglobus and Ferroglobus, were retrieved from Rainbow and Ashadze. Interestingly, a new dissimilatory Fe(III)-reducing Archaeoglobaceae was also isolated from Ashadze and reported as growing autotrophically on hydrogen [START_REF] Slobodkina | Geoglobus acetivorans sp. nov., an iron(III)-reducing archaeon from a deep-sea hydrothermal vent[END_REF]. As several Archaeoglobales are also iron-cycling Archaea (e.g. [START_REF] Kashefi | Geoglobus ahangari gen. nov., sp. nov., a novel hyperthermophilic archaeon capable of oxidizing organic acids and growing autotrophically on hydrogen with Fe(III) serving as the sole electron acceptor[END_REF], the high concentrations of iron (> 3 mM) released by acidic ultramafic-hosted hydrothermal environments could possibly fuel specific members of these Archaeoglobaceae communities.

Putative sulphur-cycling and methane-oxidizing communities. Members of the

Archaeoglobales lineage also belong to the hyperthermophilic sulfate-reducing Archaea [START_REF] Miroshnichenko | Recent developments in the thermophilic microbiology of deepsea hydrothermal vents[END_REF]. Interestingly, putative sulphur-cycling Archaea related to Thermococcales and Archaeoglobales lineages were detected in more than half of the clone libraries (56%) obtained from hydrothermal samples (fluid or chimney) and were always detected together, suggesting they could require similar environmental conditions. However, contrary to all Rainbow hydrothermal fluid samples, Thermococcales and Archaeoglobales at Ashadze were only detected from the Ashadze chimney 1 samples, suggesting that all hydrothermal vents from Ashadze site did not share optimal conditions for putative sulphur-cycling microorganisms. Interestingly, the first obligate piezophilic hyperthermophilic microorganism, Pyrococcus CH1, was also recently isolated from the Ashadze chimney 1 [START_REF] Zeng | Pyrococcus CH1, an obligate piezophilic hyperthermophile: extending the upper pressure-temperature limits for life[END_REF]. Members of the Thermococcales order are mainly characterized as thermophilic to hyperthermophilic anaerobic heterotrophs that ferment peptides and sugars, and their growth can also be stimulated by sulphur reduction [START_REF] Miroshnichenko | Recent developments in the thermophilic microbiology of deepsea hydrothermal vents[END_REF][START_REF] Zeng | Pyrococcus CH1, an obligate piezophilic hyperthermophile: extending the upper pressure-temperature limits for life[END_REF]. However, some members of the Thermococcales were also able to grow on acetate-utilising Fe(III) [START_REF] Summit | A novel microbial habitat in the mid-ocean ridge subseafloor[END_REF] or capable of lithotrophic growth on carbon monoxide coupled with hydrogen production [START_REF] Sokolova | The first evidence of anaerobic CO oxidation coupled with H 2 production by a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent[END_REF], thus matching the environmental conditions of ultramafic-hosted hydrothermal systems. It has also been suggested that Thermococcales and hyper/thermophilic members of the Methanococcales order could inhabit sub-seafloor ecosystems (Summit & Baross, 1998;[START_REF] Summit | A novel microbial habitat in the mid-ocean ridge subseafloor[END_REF][START_REF] Kelley | Volcanoes, fluids, and life at mid-ocean ridge spreading centers[END_REF]Takai et al., 2004a), and could be part of a hydrogen-driven subsurface lithoautotrophic microbial ecosystem [START_REF] Nealson | Hydrogen-driven subsurface lithoautotrophic microbial ecosystems (SLiMEs): do they exist and why should we care?[END_REF].

Within methane cycling communities associated with Rainbow, putative methanotrophic ANME-2 sequences were detected, suggesting occurrence of anaerobic methane oxidation communities associated with anoxic habitats below 90°C [START_REF] Kallmeyer | Effects of temperature and pressure on sulfate reduction and anaerobic oxidation of methane in hydrothermal sediments of Guaymas Basin[END_REF].

Interestingly, dsrA gene sequences detected at Ashadze clustered with sequences previously detected in methane-rich hydrothermal systems and related to the Desulfobulbaceae family [START_REF] Teske | Microbial diversity of hydrothermal sediments in the Guaymas Basin: evidence for anaerobic methanotrophic communities[END_REF][START_REF] Nercessian | Diversity of functional genes of methanogens, methanotrophs and sulfate reducers in deep-sea hydrothermal environments[END_REF], indicating that these putative sulfate-reducing bacteria could be linked to these specific environmental conditions.

Besides, some members of Desulfobulbaceae can live syntrophically with ANME-3 members [START_REF] Niemann | Novel microbial communities of the Haakon Mosby mud volcano and their role as a methane sink[END_REF]. However, although 16S rRNA gene sequences related to ANME-2 Archaea were detected, no ANME-3 Archaea were found. Nevertheless, the most widespread functional gene (pmoA) detected in ultramafic-hosted hydrothermal environments remained related to methanotrophic communities as a probable consequence of the high methane concentration prevailing in these ultramafic-hosted hydrothermal systems. Methanotrophic bacteria were also previously detected in Bathymodiolus species and among the gill chamber of Rimicaris exoculata at the Rainbow hydrothermal field [START_REF] Duperron | A dual symbiosis shared by two mussel species, Bathymodiolus azoricus and Bathymodiolus puteoserpentis (Bivalvia: Mytilidae), from hydrothermal vents along the northern Mid-Atlantic Ridge[END_REF][START_REF] Zbinden | New insigths on the metabolic diversity among the epibiotic microbial communitiy of the hydrothermal shrimp Rimicaris exoculata[END_REF], suggesting that these symbionts could also be present in seawater. However, no known sequences related to symbionts were detected in the seawater, as a possible consequence of low cell concentrations or of a technical bias.

Moreover, the phylogenetic distribution of the pmoA gene was related to the habitat, suggesting that different methanotrophic communities were specifically adapted to different ecological niches (e.g. sediments and fluid/seawater mixing zones).

Specific distribution and ecological niches

The different environmental conditions (temperature, pH, hydrostatic pressure, metabolic substrates) at the different MAR ultramafic-hosted hydrothermal sites generate diverse microbial ecological niches (hydrothermal fluid, chimney, sediment, and seawater) that seem to strongly select for specific communities.

Site specific phylotypes. Although molecular techniques (PCR and cloning) used to build clone libraries are known to be inherently biased [START_REF] Suzuki | Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR[END_REF][START_REF] Von Wintzingerode | Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis[END_REF][START_REF] Polz | Bias in template-to-product ratios in multitemplate PCR[END_REF][START_REF] Nocker | Genotypic microbial community profiling: A critical technical review[END_REF], we assumed that the biases were equal for all samples as they were analyzed under the same strict conditions (storage, DNA extraction, PCR amplification, cloning, sequencing) [START_REF] Von Wintzingerode | Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis[END_REF]. However, comparisons of population structures from other studies using different experimental conditions remain unreliable. For example, archaeal diversity from Rainbow chimneys as described by Flores and colleagues (2011) was much higher than that described by Voordeckers and colleagues (2008), suggesting either spatial and temporal heterogeneity or a technical bias. Interestingly, archaeal diversity observed from the Rainbow fluids in the present study was similar to that from the chimneys analysed using pyrosequencing reported by Flores and colleagues (2011). The present study also shows that some communities seemed to be site-specific and specifically adapted to different ecological niches (e.g. sediments and fluid/seawater mixing zones).

Sequences related to Nanoarchaeota, for example, were only detected in the Rainbow hydrothermal system, showing that the nanoarchaeal habitat extends to at least one of the deep hot marine hydrothermal systems of the MAR. The recently discovered novel Nanoarchaeota phylum has shown a wide distribution in high temperature ecosystems [START_REF] Hohn | Detection of 16S rDNA sequences representing the novel phylum "Nanoarchaeota": Indication for a wide distribution in high temperature biotopes[END_REF][START_REF] Huber | A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont[END_REF], and may represent pioneering communities in deep-sea hydrothermal vents [START_REF] Mccliment | Colonization of nascent, deep-sea hydrothermal vents by a novel Archaeal and Nanoarchaeal assemblage[END_REF]. Nanoarchaeota could also represent a fastevolving euryarchaeal lineage related to Thermococcales [START_REF] Brochier | Nanoarchaea: representatives of a novel archaeal phylum or a fast-evolving euryarchaeal lineage related to Thermococcales?[END_REF]. Moreover, the nano-sized Nanoarchaeota were previously described to have a symbiotic relationship with Ignicoccus hospitalis, a member of the Desulfurococcales order isolated from the Kolbeinsey Ridge, north of Iceland [START_REF] Paper | Ignicoccus hospitalis sp nov, the host of 'Nanoarchaeum equitans[END_REF]. Interestingly, 16S rRNA gene sequences with 94% similarity to the hyperthermophilic chemolithoautotrophic suphur and hydrogen-utilizing Ignicoccus hospitalis, were also retrieved exclusively from the same Rainbow hydrothermal fluids, suggesting that a similar symbiotic relationship could also occur between the Nanoarchaeota and specific Desulfurococcales from Rainbow.

Differences between the composition of archaeal communities associated with the two hydrothermal chimneys from Ashadze could be probably linked to environmental factors as the Ashadze chimney 3 has a higher copper concentration than chimney 1 (Charlou JL., Donval JP. and Konn C., unpublished). Moreover, 16S rRNA gene sequences related to Halobacteriales were only detected from Ashadze, which is to date the deepest known hydrothermal site. The highest similarity with a cultured relative was Natronomonas pharaonis (98%), an extremely halo-alkaliphilic archaeon. The occurrence of halotolerant prokaryotes in hydrothermal environments, growing at higher NaCl concentrations than most marine microorganisms, was previously reported [START_REF] Takai | Distribution of Archaea in a black smoker chimney structure[END_REF]. Due to phase separation it is admitted that venting of a condensed vapor phase with low salinity will generate a high salinity phase at depth. This phase may be venting later or be trapped in the subsurface environments. In addition some authors have suggested a double diffusive hydrothermal system where brines are trapped in the deepest part of the system and exchange only heat with the upper convective system [START_REF] Bischoff | Salinity variations in submarine hydrothermal systems by layered double-diffusive convection[END_REF][START_REF] Fouquet | Metallogenesis in Back-Arc Environments -the Lau Basin Example[END_REF]. If partially cooled, this deep high salinity reservoir may constitute an extensive location for halotolerant prokaryotes. Hence, it was also suggested that these communities could be associated with a sub-vent ecosystem, as well as with hydrothermal chimneys [START_REF] Kaye | High incidence of halotolerant bacteria in Pacific hydrothermalvent and pelagic environments[END_REF][START_REF] Takai | Distribution of Archaea in a black smoker chimney structure[END_REF].

As previously described, the off-axis Lost City hydrothermal system is remarkable by its geological, geochemical and biological settings [START_REF] Kelley | A serpentinite-hosted ecosystem: the Lost City hydrothermal field[END_REF]. The archaeal diversity associated with hot and very alkaline Lost City hydrothermal fluid was limited to unaffiliated Methanosarcinales and to MG-I sequences. The detected unaffiliated Methanosarcinales sequences matched the Lost City Methanosarcinales cluster (99% similarity) described by Schrenk and colleagues [START_REF] Schrenk | Low archaeal diversity linked to subseafloor geochemical processes at the Lost City Hydrothermal Field, Mid-Atlantic Ridge[END_REF], suggesting that these Archaea were involved in methane cycling processes [START_REF] Schrenk | Low archaeal diversity linked to subseafloor geochemical processes at the Lost City Hydrothermal Field, Mid-Atlantic Ridge[END_REF][START_REF] Boetius | Lost City Life[END_REF]. However, no mcrA gene sequences were detected at Lost City, as a likely consequence of low cell densities in the Lost City fluids [START_REF] Brazelton | Methane-and sulfur-metabolizing microbial communities dominate the Lost City hydrothermal field ecosystem[END_REF]. Members of the Lost City uncultured Methanosarcinales cluster are probably endemic communities associated to cooler (<95°C) and very alkaline habitats as they were not detected from any other hydrothermal sites. The occurrence of molecular genetic evidences in hot and very alkaline fluids also suggests that the Lost City Methanosarcinales have physiological potentials beyond the capacities of any known cultured isolates [START_REF] Mesbah | Life at extreme limits: the anaerobic halophilic alkalithermophiles[END_REF]. Specific ecological niches. To summarize, besides some specific Archaea that seemed endemic to some hydrothermal sites, the distribution of archaeal phylotypes and putative metabolic processes was linked to different microbial niches (seawater, sediments, macrofaunal communities, hydrothermal chimneys and fluids, Fig. 5). The cold and oxygenated seawater (< 10°C) overlaying the hydrothermal systems probably represented one of the largest microbial niches, and was characterized only by marine group lineages, some of which could be aerobic ammonia oxidizers. These psychrophilic seawater communities surrounding hydrothermal vents are most likely to benefit from high ammonium inputs from the chemolithotrophic primary producer associated with the hydrothermal structures. Another large ecological niche is probably the cold (< 10°C) and porous sediments surrounding the hydrothermal systems, which may represent a stable environment and suitable substrate for selection of specific seawater phylotypes (MG-I) and for colonization by specific psychrophilic unaffiliated Euryarchaeota and methylotrophic bacteria.

The specific sedimentary microbial communities could be fuelled by the products from organic matter degradation, but also by methane seepage from these ultramafic systems. In contrast, the warm (~15°C) and relatively unstable mixing zones colonized by macrofaunal communities were probably the most metabolically active microbial niche benefiting from oxidized seawater compounds and from reduced compounds from the hydrothermal system.

Mixing zones between the adjacent ecological niches also occur as a result of steep physicochemical gradients characterizing these dynamic hydrothermal environments, therefore resulting in exchanging microbial communities. Mesophilic to thermophilic methane-oxidizing bacteria could dominate the moderate oxic habitats in the mixing environment as revealed by the pmoA gene analyses. The detection of ANME-2 members suggests that moderate thermophilic (<90°C) anaerobic methanotrophs could occur in probably restricted anoxic habitats, as a consequence of the very steep oxygen and temperature gradients.

Methanotrophic archaeal communities fuelled by hydrogen and carbon dioxide could probably dominate the more chemically reduced zones of this niche, which is closer to the hydrothermal chimney. The thermophilic communities composed of Methanococcales, Methanopyrales, Thermococcales, Archaeoglobales, and Desulfurococcales were in all likelihood harboured by the hydrothermal chimneys and could mainly be composed of hydrogen-oxidizing members. Although hydrothermal fluids from ultramafic systems such as Rainbow do not have significant levels of hydrocarbons from biogenic origin, methanogenesis could still be the dominant archaeal metabolic process, as the high abiogenic methane concentration may mask the biogenic methane. Moreover, the hyper/thermophilic methanogen Methanococcales order and the Thermoccocales order could be typical members of the hot anaerobic microbial ecosystem that could extend below the Rainbow hydrothermal system seafloor.

Captions

Fig. 1 .

 1 Fig. 1. (a) Location map of Atlantic ultramafic-hosted hydrothermal sites mentioned in this

Fig. 2 .

 2 Fig. 2. (a) Co-migration denaturant gradient gel electrophoresis (CM-DGGE) analysis of

Fig. 3 .

 3 Fig. 3. (a) Phylogenetic tree representing the Euryarchaeota 16S rRNA gene sequences.

Fig. 4 .

 4 Fig. 4. Phylogenetic trees based on translated partial amino acid sequences of functional

Fig. 5 .

 5 Fig. 5. Hypothetical model (not to scale) of microbial ecological niches in acidic Atlantic

Fig. S1 .

 S1 Fig. S1. Rarefaction curves for the 16S rRNA gene clone libraries from the Fairway and New Caledonia Basin sites[START_REF] Schloss | Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness[END_REF]. The sequence identity levels are represented in brackets.

Acknowledgements

We thank Anne Godfroy, Françoise Gaill, Yves Fouquet and Georgy Cherkashov chief scientists of Exomar, MoMARDREAM-Naut and SERPENTINE cruises. All crew members and the Scientific Party were crucial in this effort, especially the ROV "Victor 6000" and DSV "Nautile" crews for the sampling efforts. This work was supported by the Université de Bretagne Occidentale, the Institut français de recherche pour l'exploitation de la mer, the Centre National de la Recherche Scientifique, the Région Bretagne, and MoMARnet (Monitoring deep-sea floor hydrothermal environments on the MAR: a Marie Curie Research Training Network). We are also grateful to Ouest-Génopole for the use of their facilities.

Erwan Roussel was supported by a grant from the Ministère de la Recherche.

Supplementary material

Caledonia Basin sites [START_REF] Schloss | Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness[END_REF]. The sequence identity levels are represented in brackets.


V Ardizzone

Y Chassagneux

F Vialla

G Delport

C Delcamp

N Belabas

E Deleporte

Ph Roussignol

I Robert-Philip

C Voisin

J S Lauret

Strong reduction of exciton-phonon coupling in high crystalline quality single-wall carbon nanotubes: a new insight into broadening mechanisms and exciton localization

Strong reduction of exciton-phonon coupling in high crystalline quality single-wall carbon nanotubes: a new insight into broadening mechanisms and exciton localization

Carbon nanotubes are quantum sources whose emission can be tuned at telecommunication wavelengths by choosing the diameter appropriately. Most applications require the smallest possible linewidth. Therefore, the study of the underlying dephasing mechanisms is of utmost interest. Here, we report on the low-temperature photoluminescence of high crystalline quality individual single-wall carbon nanotubes synthesized by laser ablation (L-SWNTs) and emitting at telecommunication wavelengths. A thorough statistical analysis of their emission spectra reveals a typical linewidth one order of magnitude narrower than that of most samples reported in the literature. The narrowing of the PL line of L-SWNTs is due to a weaker effective exciton-phonon coupling subsequent to a weaker localization of the exciton. These results suggest that exciton localization in SWNTs not only arises from interfacial effects, but that the intrinsic crystalline quality of the SWNT plays an important role.

Photoluminescence (PL) emission in semiconducting carbon nanotubes arises from exciton recombination [1][2][3] and has been extensively studied in view of possible applications in opto-electronics, bio-imaging or photovoltaics [4][5][6][7]. Observation of photon antibunching in the near infrared [8,9] suggests that SWNTs are also promising single-photon sources for the implementation of quantum information protocols. Interestingly, the PL emission energy (i.e. the excitonic recombination energy) strongly depends on the tube diameter and can be easily tuned in the telecommunication bands at 0.83 eV (1.5µm) by choosing SWNTs with a diameter of about 1-1.2 nm [10]. SWNTs could therefore make up a very versatile light source for quantum optics. Several studies suggested that the optical properties of SWNTs at low temperature are best described in terms of localized excitons (zero-dimensional confinement), leading to a quantum dot like behavior [11,12]. Nevertheless, the nature of the traps responsible for this exciton localization is not elucidated yet. In order to address the issue of exciton localization, we studied carbon nanotubes produced by high-temperature synthesis methods such as electric arc or laser ablation methods, which are known for their higher crystalline quality, with a lower density of defects [13][14][15][16][17].

In addition to the large Coulomb interaction responsible for the huge exciton binding energy in SWNTs, many other effects -both intrinsic (exciton -phonon coupling [11,18,19], exciton -exciton interaction [20], structural defects [21,22],. . . ) and extrinsic (residual doping [23], interfacial inhomogeneities [24], fluctuating environment [9,25],. . . ) -are known to significantly alter the emission properties of SWNTs and act as dephasing sources for the exciton. The corresponding figure of merit is the full width at half maximum (FWHM) of the PL line that contains the optical signatures of exciton dephasing processes. Importantly, this line-width has to be measured at the single emitter scale to avoid inhomogeneous broadening [26], but only the statistical analysis of the linewidth distribution is relevant to benchmark the quality of a sample and thus of a growth/post-processing method.

In this work, we study the low-temperature (10 K) PL emission from surfactant dispersed laser ablation carbon nanotubes (L-SWNTs) [27,28]. These nanotubes result from a high-temperature growth process and are wellknown for having a higher crystalline quality. With a mean diameter of the order of 1.1 nm, they typically emit in the telecommunication band at about 1.5 µm [10]. Our sample is obtained by spinning the solutionprocessed SWNTs on the flat surface of a solid immersion lens coated with poly-lysine, following the previously reported procedure [8]. All nanotube suspensions are obtained by dispersing raw powders in water with 2% in weight of sodium cholate under vigorous sonication for one hour. The microphotoluminescence setup consists of a home-built confocal microscope, with a 0.4 NA microscope objective. The sample is excited by a laser diode emitting at 830 nm and the signal is then analyzed by a 50cm spectrometer equipped with a 600 l/mm grating blazed at 1.6 µm. A nitrogen cooled InGaAs array is used to record the spectra. By performing a statistical study on more than 40 SWNTs, we find a FWHM distribution centered around 500 µeV with values as low as 250 µeV (resolution limited). We stress that, despite the lack of control of the microscopic dielectric environment in our sample, the observed FWHM distribution is centered at values that are smaller than most of the values presented in the literature [20,29,30], or comparable to the values reported in studies focusing on the control of the environment-induced dephasing (air-bridging or polymer wrapped SWNTs) [25,31]. As a reference for CVD-grown SWNTs, we compare typical spectra of L-SWNTs with those of similarly processed CoMoCat SWNTs. We show that the reduction of the effective exciton-phonon coupling due to a weaker exciton localization in L-SWNTs is the key factor for the narrowing of the PL lines. The emission profiles are fitted using the extended exciton-phonon coupling model [11,19]. We deduce that the exciton localization length σ [START_REF]σ is the extension of the exciton center-of-mass gaussian envelope wave function[END_REF] is in the range 10-16 nm, which is up to ∼6 times larger than the value reported for CVD-grown SWNTs [11,19]. This suggests that the exciton localization in SWNTs is not enterly due to interfacial effects, but that the intrinsic crystalline quality of the SWNT matters. Figure 1 (a,c) shows a sequence of 60 spectra (acquisition time of 1 second for each spectrum) for two typical individual SWNTs found in our sample, with emission wavelengths of 1530 nm and 1507 nm respectively. These temporal traces show the usual features of PL from individual surfactant dispersed SWNT, that are spectral diffusion (SD) and blinking. In figure 1 (b,d) two spectra extracted from the sequence are represented. They show a FWHM of 360 and 250 µeV respectively. In presence of SD, the measured FWHM can be considered as an upper limit to the intrinsic FWHM. In the following, we study this upper limit by setting the acquisition time to 1 second. This value ensures a good signal to noise ratio for a great number of SWNTs in our sample. Moreover, no significant change in FWHM is observed in the pump power range used in this work, and the PL intensity shows a linear dependence on the incident power (See Supplemental Material for further details [39]). This suggests that exciton-exciton interactions [20] give a negligible contribution to the measured FWHMs.

In order to check if L-SWNTs show a statistically narrower FWHM than previously reported samples, we measured the FWHM distribution. We analyzed 43 SWNTs, resulting in the histogram shown in figure 2. The FWHM distribution is centered around 500 µeV, with a lower (resolution limited) FWHM of 250 µeV. The asymmetry of this distribution, due to instrumental constraints, strongly suggests that even narrower PL lines could be observed in this sample. This FWHM distribution shows that L-SWNTs have a narrower PL emission than most solution processed SWNTs which show typical FWHMs of several meV [12,24,29,31]. Strikingly, these FWHM values seem comparable to and even narrower (at least in term of distribution) than those observed for most airbridging CVD SWNTs [31] and for most neutral polymerembedded SWNTs [9,25,32], i.e. in samples in which the fluctuations of the microscopic dielectric environment are reduced.

The FWHM distribution of figure 2 results from a complex interplay of different broadening phenomena. In the case of carbon nanotubes, FWHM measurements essentially encompass two different mechanisms, namely the so-called Ohmic broadening and the pure dephasing broadening. The former corresponds to the non-perturbative coupling of a localized exciton to a 1D phonon bath, that is known to give rise to a broad and asymmetric PL lineshape [11,19]. The latter gives an additional contribution to the global FWHM and andd) were acquired with an integration time of 60s and show small phonon wings on either sides of the ZPL. In c) a broader profile with an asymmetric shape (Integration time 20 s). In red, fits to the experimental data using the non perturbative exciton phonon coupling model (see main the text and SI). Insets: magnified view on the phonon sidebands.

becomes the dominant dephasing mechanism when the Ohmic broadening is suppressed. The intermediate case known as extended exciton-phonon coupling model accounts for all the distinct PL profiles observed at low temperature, consisting of a sharp zero-phonon line (ZPL) with phonon side-bands of variable magnitudes [19]. In the following, we show that the reduction of the effective exciton-phonon coupling subsequent to a larger exciton delocalization plays a key role in the interpretation of our experimental findings.

Figure 3 displays high signal to noise ratio spectra recorded in L-SWNTs. Note that despite the 60-fold increase of the integration time used to acquire these highresolution spectra, the extra broadening is on the order of 100-200 µeV at most, which remains small as compared to the extension of the typical spectral features (several meV), showing that spectral diffusion is not the dominant broadening mechanism. The observation of all the representative PL profiles predicted by the extended exciton-phonon coupling model shows that the diversity of line-profiles reported in the literature [19] is not specific to the case of CVD nanotubes but is also encountered in L-SWNTs. To highlight this point, we compare in Figure 4 some examples of PL spectra of L-SWNTs with those of CoMoCat nanotubes showing an identical line-shape. The profile similarity is striking except that the line-width is consistently lower for L-SWNTs.

This means that the nature of the broadening mechanisms is similar in all types of nanotubes but that their magnitude is reduced in L-SWNTs. In this respect, it is worth noting that the typical SD magnitude (as measured by the standard deviation of the peak energy in a series of 60 spectra acquired with a 1 s integration time) is of the same order for CoMoCat and L-SWNTs (e.g. ∼ 100µeV for the tubes of figure 4-a), ruling out that SD is the source of the difference between the two types of sample.

Spectra a), b)and d) of figure 3 show the presence of a pedestal formed by small asymmetric phonon wings on either sides of the ZPL. In c) the ZPL is merged in the phonon wings, forming an overall broader and asymmetric PL line-shape. The red lines in figure 3 are the fits obtained with the extended exciton-phonon coupling model [11,19](See Supplemental Material for the details). Here, we briefly recall that for a localized exciton coupled to a truly 1D phonon bath, the exciton-phonon coupling diverges for the low-energy phonon modes leading to a typical broad and asymmetric lineshape. In contrast, when the local density of states of acoustic phonons at the exciton location is reduced (e.g. by a local mechani-cal coupling to the environment), a sharp ZPL is restored and emerges from the phonon wings. Most importantly, in both cases the extension of the phonon sidebands directly mirrors the size of the (Gaussian) exciton envelope σ used to describe the localization of the exciton center of mass. In fact, a finite value of σ corresponds to a finite extension of the exciton wave-function in the k-space leading to a cut-off energy of the exciton-phonon coupling at ∆E σ = 2 v/σ [START_REF]9 km/s is the sound velocity for the effective phonon mode obtained by merging low energies stretching and twisting phonon branches[END_REF], due to the momentum conservation in the exciton-phonon interaction. Therefore, the spectral width of the phonon sidebands is a probe of the exciton localization. The values of σ extracted from the fits (red curves in figure 3) are reported on the graphs. Interestingly, all the tubes studied here show values of σ in the range 10 -16 nm. These values are larger than the values found for CoMoCat tubes with the same fitting model (∼3nm) [11,19]. Large values of σ are hallmarks of weaker exciton localization in L-SWNTs, which we interpret as a consequence of the higher crystalline quality of this material. This shows that several degrees of exciton localization are achievable in SWNTs. Therefore, identification of high crystalline quality nanotubes is a key step towards the control of exciton localization, going continuously from 0D to 1D confinement.

Previous studies reported a diameter dependence of the magnitude of the electron-phonon coupling [33][34][35]. In fact, the increase of the tube linear mass density, which is proportional to the tube diameter, also gives rise to a reduction of the exciton-phonon coupling [36]. In order to see if such diameter related effects are visible in our data, we checked for a possible correlation between the FWHM and the energy of the PL emission. In the inset of figure 2, the FWHM of the PL spectra are plotted as a function of the emission energy. Three groups of points are visible and are tentatively assigned to (11,7), (14,3), (12,4) chiralities [37]. Due to the scattering of the experimental points and due to the poor statistics for the smaller diameters, it is hard to identify any clear trend. Moreover, we note that previous studies with CVD nanotubes in the same range of diameters led to much larger linewidths ruling out the diameter dependence of the electron-phonon coupling as a main source of narrowing of the PL spectra for L-SWNTs [30]. Finally, we discuss the possible origin of the residual dephasing in SWNTs with prominent ZPL (non Ohmic exciton-phonon coupling). This broadening can be due to residual SD on a time-scale smaller than the integration time. Qualitatively, we do observe a correlation between the FWHM and the SD measured at the 1 s time-scale (See Supplemental Material). With a contribution of the order of 100 µeV as compared to an average FWHM of 500 µeV, SD plays a non-negligible role on the ZPL linewidth. This is exemplified in the spectra of Figure 4-a, where the L-SWNT and the CoMocat spectra show comparable ZPL widths, which is consistent with the comparable magnitude of SD measured in each tube.

In conclusion, we studied the low-temperature PL emission from laser ablation solution-processed SWNTs emitting in the telecommunication band around 1.5 µm. A statistical analysis shows a distribution of line-widths centered around 500 µeV, which is significantly narrower than the previously reported values for solution-processed nanotubes. Moreover, L-SWNTs show a diversity of PL profiles which is very similar to that of CVD nanotubes, showing that their emission spectra is also driven by the exciton-phonon coupling. However, the consistently weaker and narrower phonon wings point to an up to 6-fold larger localization length of the excitons in L-SWNTs, as deduced from a fitting of the line profiles to the extended exciton-phonon coupling model. This finding shows that the exciton localization at low-temperature is not governed by the sole interfacial coupling to the environment but also to a large extend by the intrinsic crystalline quality (i.e. the defect density) of the nanotube. Finally, it is worth noting that this study reveals that several degrees of exciton localization are achievable in SWNTs at low temperature, ranging from nearly 0D quantum dot like excitons in CVD-nanotubes to a pronounced 1D flavor in L-SWNTs with localization lengths exceeding 10 times the exciton Bohr radius. Identification of high crystalline quality SWNTs is therefore a key issue since it could provide a model system for the study of confinement effects in one-dimensional excitons going continuously from near one-dimensional excitons to truly zero-dimensional fully confined excitons. Finally, it may open the way to the control of the exciton localization, an important step towards the realization of deterministic single photon source at telecommunication wavelengths based on carbon nanotubes. This work is supported by a public grant overseen by the French National Research Agency (ANR) as a part of the "Investissements d'Avenir" program (reference: ANR-10-LABX-0035, Labex NanoSaclay), by the GDR-I GNT, the ANR grant TRANCHANT. C.V. is a member of Institut Universitaire de France. Laser ablation SWNTs were provided by O. Jost (now at Fraunhofer IWS).

FFIG. 1 :

 1 FIG. 1: a) and c) Temporal traces of the PL emission of two different SWNTs consisting of 60 consecutive spectra (1 second integration time for each spectrum). b) and d) Example of a spectrum extracted from the trace in a) and c) (indicated by the white arrows). The power density used here is 60 kW/cm 2 .

FIG. 2 :

 2 FIG. 2: FWHM distribution measured for 43 individual SWNTs. The data reported on the histogram are obtained by picking the minimum FWHM in the temporal traces such as those showed in figure 1. The most probable values are around 500 µeV. The low-values tail is cut off by the optical resolution of the setup at 250 µeV (gray dashed line). Inset : dependence of FWHM on emission energy.

FIG. 3 :

 3 FIG.3: PL spectra of L-SWNTs : a), b) are the tubes of figure1. a), b) and d) were acquired with an integration time of 60s and show small phonon wings on either sides of the ZPL. In c) a broader profile with an asymmetric shape (Integration time 20 s). In red, fits to the experimental data using the non perturbative exciton phonon coupling model (see main the text and SI). Insets: magnified view on the phonon sidebands.

FIG. 4 :

 4 FIG.4: In black two typical PL spectra of L-SWNT. In red PL spectra of CoMoCAT SWNT with the same type of line profile. a) Exemple of sharp ZPL emerging from the phonon wings. In the inset : zoom on the phonon wings. b) Example of triangular asymmetric shape. In the inset : zoom on the L-SWNT PL spectra to highlight the asymmetric shape. The energy axes are shifted in order to superimpose the PL peaks.