This article is protected by copyright. All rights reserved. 1

Received Date: 26-Nov-2013 1

Accepted Date: 05-Jun-2014 2

Spatial distribution of microbial communities in the shallow submarine alkaline 3

hydrothermal field of the Prony Bay, New Caledonia1 4

5

Marianne Quéméneur1*

, Méline Bes1, Anne Postec

1, Nan Mei

1, Jérôme Hamelin

2, Christophe 6

Monnin3

,

Valérie Chavagnac3,

Claude Payri4, Bernard Pelletier

4, Linda Guentas-7

Dombrowsky4,5

, Martine Gérard6, Céline Pisapia

6, Emmanuelle Gérard

6, Bénédicte Ménez

6, 8

Bernard Ollivier1, Gaël Erauso

1. 9

10

1 Aix Marseille Université, CNRS, Université de Toulon, IRD, MIO UM 110, 13288, 11

Marseille, France 12

2 INRA, UR0050, Laboratoire de Biotechnologie de l’Environnement, 11100 Narbonne, 13

France 14

3 GET UMR5563 (CNRS/UPS/IRD/CNES), Observatoire Midi-Pyrénées, Université de 15

Toulouse, CNRS, IRD, 14 Avenue Edouard Belin, 31400 Toulouse, France 16

4 Institut pour la Recherche et le Développement (IRD) Centre de Nouméa, MIO UM 110, 17

promenade Laroque, 98848 Nouméa, Nouvelle Calédonie 18

5 Université de Toulon, Laboratoire des Matériaux Polymères Interfaces Environnement 19

Marin (MAPIEM), ISITV- Avenue Georges Pompidou BP56 - 83162 La Valette-du-Var 20

Cedex, France 21

6 Institut de Physique du Globe de Paris, Sorbonne Paris Cité, Université de Paris Diderot, 22

UMR CNRS 7154, 75005 Paris, France 23

24

* Corresponding author: Marianne Quéméneur 25

Address: Aix Marseille Université, CNRS/INSU, IRD, Mediterranean Institute of 26

Oceanography (MIO), UM 110, 13288 Marseille, France 27

E-mail: marianne.quemeneur@ird.fr; Phone: +0033(0)491828577; Fax: +0033(0)491828570 28

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process, which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

10.1111/1758-2229.12184

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29

Running title: Microbial communities of Prony hydrothermal field 30

Keywords: Microbial diversity, serpentinization, hydrothermal system, alkaliphilic, Prony bay 31

32

Abstract 33

34

The shallow submarine hydrothermal field of the Prony Bay (New Caledonia) discharges 35

hydrogen- and methane-rich fluids with low salinity, temperature (<40°C) and high pH (11) 36

produced by the serpentinization reactions of the ultramafic basement into the lagoon 37

seawater. They are responsible for the formation of carbonate chimneys at the lagoon 38

seafloor. CE-SSCP fingerprinting, quantitative PCR and sequence analysis of 16S rRNA 39

genes revealed changes in microbial community structure, abundance and diversity depending 40

on the location, water depth and structure of the carbonate chimneys. The low archaeal 41

diversity was dominated by few uncultured Methanosarcinales similar to those found in other 42

serpentinization-driven submarine and subterrestrial ecosystems (e.g. Lost City, The Cedars). 43

The most abundant and diverse bacterial communities were mainly composed of Chloroflexi, 44

Deinococcus-Thermus, Firmicutes, and Proteobacteria. Functional gene analysis revealed 45

similar abundance and diversity of both Methanosarcinales methanoarchaea and 46

Desulfovibrionales- and Desulfobacterales sulfate-reducers in the studied sites. Molecular 47

studies suggest that redox reactions involving hydrogen, methane and sulfur compounds (e.g. 48

sulfate) are the energy driving forces of the microbial communities inhabiting the Prony 49

hydrothermal system. 50

51

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Introduction 52

53

A wide variety of submarine hydrothermal systems has been investigated worldwide since 54

their first discovery in the late 1970s (Corliss et al., 1979). Depending on the basement rock 55

hosting the hydrothermal system, different energy sources are available and thus 56

preferentially used as electron donors by the indigenous microorganisms. In ultramafic-hosted 57

oceanic hydrothermal systems, hydrogen (H2) and abiotic organic compounds dissolved in the 58

hydrothermal fluids are the dominant energy sources generated by serpentinization processes 59

(i.e. the metamorphic hydration of ultramafic rocks/minerals into serpentine) (Charlou et al., 60

2002; Kelley et al., 2005; Ludwig et al., 2006; Proskurowski et al., 2008). 61

The Lost City Hydrothermal Field (LCHF) (30°07′N, 42°07′W) is a well-known serpentinite-62

hosted submarine hydrothermal system (at water depth of ~800 meters below sea level, mbsl) 63

(Kelley et al., 2005). LCHF hosts carbonate chimneys up to 60 meters high above the 64

seafloor, and generate fluids with moderate temperatures (40-80°C at chimney vents, ca. 90-65

110°C in the subsurface), high pH (9-10.8), high dissolved H2 (1-15 mM), methane (1-2 mM), 66

and sulfate (5.9-12.9 mM) concentrations (Lang et al., 2010; Ludwig et al., 2006; 67

Proskurowski et al., 2008). Molecular surveys of the LCHF microbial diversity provided 68

evidence for a limited number of dominant phylogenetic prokaryotic lineages (Brazelton et 69

al., 2013; Kelley et al., 2005; Schrenk et al., 2004). The LCHF archaeal communities 70

consisted mainly of Lost City Methanosarcinales (LCMS), and ANaerobic MEthanotrophic 71

archaea (ANME-1 group), while bacterial communities included sulfide/methane-oxidizing 72

Gammaproteobacteria, H2/sulfide-oxidizing Epsilonproteobacteria, and sulfate-reducing and 73

H2-producing Firmicutes (Schrenck et al., 2004; Brazelton et al., 2006; Brazelton et al., 74

2013). In this respect, H2, CH4 and sulfur compounds are considered as the main components 75

sustaining the growth of LCHF microbial communities (Schrenk et al., 2013). 76 Acc

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Shallow submarine vents support complex biological communities, characterized by the 77

coexistence of both chemosynthetic and photosynthetic organisms (Canet and Prol-Ledesma, 78

2007; Maugeri et al., 2009). To our knowledge, the Prony Hydrothermal Field (PHF) is the 79

only example of a shallow ultramafic-hosted submarine hydrothermal systems so far (at water 80

depth <50 mbsl). The PHF is located in the Prony bay on the New Caledonia ophiolitic nappe 81

(southwest Pacific Ocean) (Launay and Fontes, 1985; Quesnel et al., 2013). PHF comprises 82

large carbonate chimneys where H2- and CH4-rich and high pH (up to pH 11.3) fluids 83

discharge at low temperature (<40°C) into the lagoon (Launay and Fontes, 1985; Monnin et 84

al., submitted). In contrast to LCHF, but similarly to subterrestrial serpentinite-hosted 85

hydrothermal systems, such as those found in ophiolite massifs (e.g., Oman, Liguria) (Boulart 86

et al., 2013; Chavagnac et al., 2013; Monnin et al., 2011), the highly alkaline PHF fluids have 87

a low salinity inherited from their meteoric origin (Launay and Fontes, 1985; Monnin et al., 88

submitted), which may variably impact the PHF-associated microbial communities, 89

depending on the complexity of hydrothermal circulation from land to sea and thus the 90

location of the vents. Easy accessibility of PHF for sampling, as compared to deep-sea 91

hydrothermal systems (e.g. Lost City), makes PHF an ideal natural observatory to study 92

biogeochemical processes linked to serpentinization in submarine hydrothermal systems. 93

This study aimed to determine the abundance, the structure and the diversity of both archaeal 94

and bacterial communities using both functional- and 16S rRNA gene-based PCR methods in 95

several shallow and active carbonate chimney samples from four distinct sites located at 96

different water depths in the Prony bay. The microbial community of the shallow submarine 97

PHF and their potential in H2, CH4 or sulfur cycling was then compared with those of other 98

serpentinization-driven sites studied so far. 99

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Results and discussion 101

102

Sampling sites in PHF and geochemistry 103

Samples of carbonate concretions and fluids were collected by scuba divers in four active 104

springs at different water depths in the Prony Bay (Fig. 1A): (i) at the Bain des Japonais 105

(BdJ), a spring in shallow water uncovered at low tide in the Carenage Bay, (ii) at the Needle 106

of Prony (ST07) at 16 mbsl in the North Bay, (iii) at Site ST08 at 47 mbsl in the Central Bay, 107

and (iv) at Site ST12 at 38 mbsl in the West Bay (Supplementary Experimental Procedures). 108

The PHF carbonate structures have different characteristics, depending on their size, age and 109

the associated level of discharged fluids (Fig. 1B): (i) at the BdJ, numerous fluid outlets build 110

a flat carbonate plateau on top of which grow small needle-like brittle structures with thin 111

walls (a few tens of cm high and a few centimeters in diameter) (Fig. S1), (ii) the concretion 112

sampled at ST07 (up to 30 cm in diameter) was part of a large-sized and competent 113

carbonated edifice (Fig. S2). The presence of encrusted marine shells inside the highly-114

consolidated chimney along with coral coating likely attest for a marine fingerprint in the 115

edification along with a long lasting existence and a reduced permeability, (iii) the ST08 116

hydrothermal vent (10 to 15 cm in diameter) was fairly active, with white tips at its top 117

demonstrating an active fluid discharge (Fig. S3), and (iv) ST12 massive chimney (~25 cm in 118

diameter) had several fluid diffusion centers and black spots of bitumen that correspond to 119

degraded organic matter (Fig. S4). Submarine chimney surfaces were colonized by various 120

organisms (e.g. ascidia, sponges, corals), which probably further contribute to reduce 121

permeability with the surrounding seawater of the site. PHF carbonate chimneys were mainly 122

composed of aragonite (CaCO3), brucite [Mg(OH)2], calcite (CaCO3), and magnesium 123

carbonates (MgCO3) (Pisapia et al., 2013), as observed at LCHF (Ludwig et al., 2006). 124 Acc

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PHF fluids had high pH values ranging from 10.1 (ST07) to 11.1 (BdJ) (Table S1). The 125

highest concentrations of Cl- (163.9 mM), SO4

2- (7.5 mM), and major cations (e.g. 97.3 mM 126

Na+), measured in ST07, were 3 to 4 times lower than those of Prony ambient seawater. 127

Although these values are probably overestimated due to the likely contamination of the 128

hydrothermal fluid by the surrounding seawater, they indicated the low salinity level of PHF 129

fluids. The temperature of the Prony alkaline fluids ranged from 30°C to 37°C (BdJ). Gas 130

bubbles escaping at BdJ were composed of N2 (66.7%vol), CH4 (17.8%vol) and H2 131

(15.0%vol) (CH4/H2~1-2; Monnin et al., submitted) which is similar to those reported for 132

gases emitted at The Cedars, California, hyperalkaline springs (pH 11.5) (Morrill et al., 2013; 133

Suzuki et al., 2013). The CH4 concentration (8.0 mM) was 4 times higher than that of Lost 134

City fluids (1-2 mM), whereas the H2 concentration (6.4 mM) was two times lower than the 135

highest LCHF values (Schrenk et al., 2013). The variable CH4/H2 ratios were interpreted as 136

reflecting the different tectono-metamorphic evolution of the ultrabasic substratum (Boulart et 137

al., 2013). CO2 and O2 levels in gas bubbles were below the detection limit, as found in other 138

serpentinization-derived fluids, such as alkaline (pH 11.4) groundwater from Cabeço de Vide 139

Aquifer (CVA) in Portugal (Tiago and Verissimo, 2013; Morill et al., 2013). 140

141

Microbial community abundance and structure in PHF 142

The 16S rRNA gene was used as target gene for qPCR experiments to assess the archaeal and 143

bacterial abundance in the four studied chimney samples, while the dsrB and mcrA genes 144

were used to quantify the sulfate-reducers and methanoarchaea (methanogens and/or 145

anaerobic methanotrophs), respectively. The abundance of bacterial 16S rRNA genes was 146

more than one order of magnitude higher than that of archaeal 16S rRNA genes in all samples 147

(Table S2). According to the average 16S rRNA copy number in archaeal and bacterial 148

genomes (1.62 and 3.82, respectively) (Sun et al., 2013), Archaea accounted for 15.8% 149 Acc

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(ST08) to 24.9% (ST07) of the total prokaryotic cells in PHF. Similar total cell numbers 150

(ranging from 0.02 to 8.4 x 108 cells per g of chimneys) and Archaea percentages were 151

obtained from LCHF samples using fluorescence in situ hybridization (Kelley et al., 2005). 152

Assuming that archaeal and bacterial cells contain, respectively, only one copy of mcrA and 153

dsrB genes, methanoarchaea accounted for 18.2% (BdJ) to 77.9% (ST12) of the archaeal 154

community, whereas the percentage of sulfate-reducers only represented from 2.2% (ST12) to 155

8.1% (ST07) of the bacterial community. 156

The CE-SSCP profiles of both bacterial and archaeal 16S rRNA genes allowed comparing the 157

PHF microbial community structures (Fig. S5), and were used to construct the dendrograms 158

shown in Fig. 2. The cluster analysis revealed two groups of archaeal CE-SSCP patterns: 159

BdJ/ST07 and ST08/ST12. Compared to BdJ and ST07, which shared a large dominant peak, 160

the two other encrusted chimneys (ST08 and ST12) collected at the greater depths showed a 161

more diverse archaeal community. The bacterial CE-SSCP profiles displayed two times the 162

number of peaks than the archaeal CE-SSCP profiles on average (Fig. S5). Cluster analysis 163

demonstrated a higher similarity among the three submarine samples ST07, ST08 and ST12, 164

with a slight difference for BdJ, the only site uncovered at low tide. 165

The dominant members of the microbial community of the four Prony chimney samples were 166

identified by analyzing clone libraries of bacterial and archaeal 16S rRNA gene sequences, as 167

well as, functional dsrB and mcrA sequences. Table S2 lists the number of clones, OTUs, 168

species richness, coverage and diversity indices calculated for each clone libraries. These 169

coverage values, supported by the Shannon index of diversity (H’) and the Chao1 estimator of 170

total species richness, demonstrated that PHF bacterial communities were more diverse than 171

archaeal communities, as already reported in other serpentinization-related ecosystems 172

(Brazelton et al., 2010; Brazelton et al., 2006; Suzuki et al., 2013; Tiago and Verissimo, 173

2013). Pyrosequencing 16S rRNA gene data obtained from ST07 sample was comparable 174 Acc

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with that derived from clones (Fig. S6), and confirmed the low archaeal diversity and 175

abundance (20-25% of prokaryotic community). The extreme conditions and energy 176

limitations inside the porous carbonate chimneys may contribute to the establishment in it of 177

low and specific archaeal diversity (Brazelton et al., 2011; Valentine, 2007; Schrenk et al., 178

2004). Low diverse Archaea could live in direct contact with hyperalkaline and reduced fluids 179

and should be considered as primary producers using CH4 and possibly H2 in these alkaline 180

ecosystem. The more diverse bacteria could colonize different niches within the chimney both 181

in direct and indirect contacts with hydrothermal fluids. 182

183

Phylogenetic composition of microbial communities in different PHF sites 184

All sites combined, the PHF microbial communities consisted mainly of: Alpha- (16%), Beta- 185

(8%), Delta- (10%), Gammaproteobacteria (4%), Chloroflexi (28%), Firmicutes (12%) and 186

Deinococcus-Thermus (6%) within the Bacteria domain, and of Euryarchaeota (88%) and 187

Thaumarchaeota (12%) within the Archaea domain (Fig. 2). Both PHF archaeal and bacterial 188

community compositions changed depending on chimney sites and structure. 189

Sample BdJ and ST07 displayed the highest occurrence and diversity of Proteobacteria (Fig. 190

2). Alphaproteobacteria were mainly represented in BdJ by phototrophic anaerobic sulfur-191

oxidizing Rhodobacterales (Fig. 7). Betaproteobacteria, detected in BdJ (24%) and ST07 192

(7%), were related to H2-oxidizing chemolithoautotrophic Hydrogenophaga members 193

(Willems et al., 1989) (Fig. 3A). ST07 displayed the highest occurrence of 194

Deltaproteobacteria sequences (17%), which were divided in two groups in both 16S rRNA- 195

and dsrB-based phylogenetic trees (Fig. 4). The first and most abundant deltaproteobacterial 196

group was closely affiliated with the alkaliphilic Desulfonatronum cooperativum (97-98% 197

identity) (Zhilina et al., 2005) (Fig. 4). The second deltaproteobacterial group, only retrieved 198

in ST07 and ST12, was related to the alkaliphilic chemolithoautotrophic Desulfurivibrio 199 Acc

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alkaliphilus (91% identity) (Sorokin et al., 2008). Both H2-oxidizing Desulfonatronum and 200

Desulfurivibrio genera, isolated from soda lakes, can use sulfate, thiosulfate, elemental sulfur 201

and/or polysulfide as terminal electron acceptors (Pikuta et al., 1998; Sorokin et al., 2008; 202

Zhilina et al., 2005). Deinococcus-Thermus sequences constituted 24% of the ST07 library 203

and closely clustered with the CVA sequences (Fig. S8A). Deinococci are known to be 204

chemoorganotrophic and resistant to extreme conditions (i.e., alkaline, moderately saline, 205

high temperature, ionizing radiations) (Albuquerque et al., 2005). The majority of BdJ and 206

ST07 archaeal sequences (94-96%) were closely affiliated with the uncultured Lost City 207

Methanosarcinales (LCMS) (Schrenk et al., 2004) (96-97% identity) (Fig. 5), as related to 208

ANME-3 and more distantly related to the Methanomethylovorans hollandica (91% identity), 209

which can use methylamines for methanogenesis (Lomans et al., 1999). 210

ST08 and ST12 samples were mainly represented by Chloroflexi sequences (>47%) belonging 211

to Dehalococcoidia class (87-96% identity) (Hug et al., 2012) (Fig. 3B), the function of which 212

is still unknown in the natural environment (Wasmund et al., 2013). The majority of 213

Firmicutes sequences detected in ST08 sample (23%) clustered with Clostridiales sequences 214

from CVA, The Cedars, and the hydrothermal alkaline groundwater of a deep South African 215

gold mine (pH 9.2; depth of 1290m) (96-97% identity) (Lin et al., 2006; Suzuki et al., 2013; 216

Tiago and Verissimo, 2013) (Fig. S9). PHFST08_B19 OTU was related to the alkaliphilic H2-217

oxidizing Dethiobacter alkaliphilus isolated from Mongolian soda lakes (Sorokin et al., 218

2008). PHFST12_B13 was affiliated with subsurface Clostridiales from LCHF carbonate 219

chimneys (91% identity) (Brazelton et al., 2006). Contrary to samples BdJ and ST07, the 220

ST08 and ST12 Euryarchaeota sequences were mainly (31-40%) related to The Cedars 221

Methanosarcinales sequences (98-99% identity) (Suzuki et al., 2013) (Fig. 5). South African 222

Gold Mine Euryarchaeotic Group (SAGMEG) sequences were exclusively found in samples 223

ST08 and ST12 (15% and 29%, respectively). The metabolic role of SAGMEG, in both 224 Acc

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marine and terrestrial subsurface environments, still remains unknown. Thaumarchaeota 225

sequences were also exclusively found in samples ST08 (13%) and ST12 (38%) (Fig. S10), 226

and were affiliated with uncultured symbionts of marine sponges, which covered the surface 227

of these chimneys. PHFST08_A6 OTU clustered with Nitrosopumilus maritimus, the first 228

cultivated ammonia-oxidizing archaeon isolated from the rocky bottoms of a marine aquarium 229

(Konneke et al., 2005). 230

231

Comparison of PHF microbial community with other serpentinizing systems 232

PHF microbial diversity, as well as chemistry and mineralogy, are comparable to other 233

continental and submarine serpentinized sites (Schrenk et al., 2013; Suzuki et al., 2013). 234

Regarding Bacteria, the members of the H2-oxidizing Hydrogenophaga detected in BdJ and 235

ST07 chimneys were already found in many continental hyperalkaline springs (Brazelton et 236

al., 2013; Suzuki et al., 2013; Tiago and Verissimo, 2013). Such Hydrogenophaga sequences 237

were detected neither in the LCHF, nor in both encrusted deepest ST08 and ST12 PHF 238

chimneys. In the Tablelands ultrabasic springs (pH 10.5-12.6), these Hydrogenophaga 239

members were specifically associated with oxic-anoxic transition zones where H2-rich 240

subsurface fluids mix with oxygenated surface water (Brazelton et al., 2013). Both alpha- and 241

gammaproteobacterial sequences related to Methylosinus and Methylomicrobium genera were 242

detected at BdJ, suggesting potential aerobic methane oxidation in porous BdJ structures. In 243

contrast, Firmicutes and Chloroflexi were mainly recovered from submarine PHF chimneys, 244

which could be considered as more anoxic. Similar patterns have recently been observed in 245

The Cedars serpentinizing sites, where Chloroflexi and Firmicutes dominated the spring fed 246

solely by deep groundwater, while Beta- and Gammaproteobacteria dominated the shallow 247

groundwater-fed springs (Suzuki et al., 2013). These findings suggest the key role of 248

Chloroflexi members in anoxic zones of the PHF carbonate chimneys, while Hydrogenophaga 249 Acc

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members seem to be important in oxic/anoxic transition zones. Both 16S rRNA- and dsrB 250

gene library analyses showed that the alkaliphilic H2-oxidizing and sulfate-reducing 251

Desulfonatronum members were common in PHF (Fig. 4), but they were retrieved neither 252

from LCHF, nor from the continental serpentinizing sites. Desulfurivibrio members were 253

detected only in the PHF submarine chimneys and in The Cedars (Suzuki et al., 2013), but not 254

in the LCHF. In contrast to Lost City (Brazelton and Baross, 2010), sulfur compounds could 255

be oxidized at PHF by gammaproteobacterial sulfur/sulfide-oxidizing 256

Thioalkalivibrio/Thiohalophilus genera (vs. Thiomicrospira genus at LCHF) (Fig. S8B). 257

Regarding Archaea, the most abundant Euryarchaeota phylotypes of both 16S rRNA gene 258

and mcrA clone libraries were associated with the Methanosarcinales (Fig. 5). In the PHF 259

system, Methanosarcinales similar to those found at The Cedars surprisingly dominated the 260

concretion samples collected at the greatest depths (40% at ST08), while the LCMS were 261

predominant in the shallower ones (96% at BdJ) (Fig. 2). Methanosarcinales members have 262

been isolated from diverse environments and are known to produce methane from different 263

substrates (e.g. H2, acetate, and methylated compounds) in a wide range of temperature and 264

pH conditions (Kendall and Boone, 2006). Moreover, some Methanosarcinales sequences had 265

those of LCMS, which are able to produce and oxidize methane (Brazelton et al., 2011), as its 266

closest phylogenetic relatives. Interestingly, the former co-occurred with those of sulfate-267

reducing bacteria belonging to Desulfobacterales (e.g. Desulfobulbus spp.) in some 268

submarine chimneys (e.g. ST08, ST12). Thus, a potential Anaerobic Methane Oxidation 269

(AMO) could take place in the inner parts of the PHF carbonate chimney in direct contact 270

with the H2 and CH4-rich fluids (anoxic zones), as previously demonstrated in LCHF 271

(Brazelton et al., 2011; Schrenk et al., 2004). Thaumarchaeota were detected only in 272

submarine PHF chimneys and have also been detected at LCHF, but it is unclear whether they 273 Acc

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are autotrophic nitrifiers or representatives of a hydrothermal ecotype with a different 274

physiology (Lincoln et al., 2013). 275

276

Conclusion 277

PHF is an alkaline hydrothermal system hosted in an ultrabasic formation and is composed of 278

several springs (emerging at low tide or completely under water) discharging into the lagoon 279

seawater anoxic, H2- and CH4-rich, high pH fluids. Its shallow submarine location (at less 280

than 50 mbsl) and the low salinity of hydrothermal fluids originating from meteoric waters are 281

unique features for serpentinization-associated environments studied so far. Microbial 282

diversity and structure changed in the four PHF carbonate concretions collected at different 283

water depths in the Prony Bay. Factors that could affect PHF microbial diversity include age, 284

porosity, and colonization by various organisms of the chimney which may have impact on its 285

permeability and availability of nutrients. It is possible that distance to the recharge area and 286

presence of a fault close to the deepest sites with involvement in deep fluids circulation, might 287

also influence the PHF microbial structure. A large-scale study integrating numerous 288

chimneys and fluid samples from other geographically distant sites in the Prony Bay should 289

help in demonstrating the impact of various physico-chemical environmental factors (Eh, 290

organic carbon content, dissolved H2, CH4 and O2 levels, nutrient concentrations, age of the 291

edifice and permeability) on the existing microbial communities. In view of their abundance, 292

diversity and broad distribution in PHF carbonate chimneys, prokaryotes are likely to play a 293

key role in the carbon and sulphur budgets in this shallow submarine serpentinite-hosted 294

environment, similarly to what is observed in deep submarine hyperalkaline settings. 295

296

Acknowledgments 297 Acc

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This project was financially supported by IRD and by the French national program EC2CO-298

Biohefect/Ecodyn/Dril/MicrobiEn (MicroProny) CNRS/INSU. We are grateful to the divers’ 299

team of IRD Nouméa (Eric Folcher, John Butcher, and Bertrand Bourgeois) for collecting 300

samples, and to Captain Jean-François Barazer and his crew on board the R/V Alis. 301

302

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1001-1006. 431

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Figure legends 433

434

Figure 1. Sampling sites in the Prony bay. Simplified geological map of the Prony bay 435

(Maurizot and Vendé-Leclerc, 2009) displaying the location of the four sampling sites: Bain 436

des Japonais (BdJ), the Needle of Prony (ST07), ST08 and ST12 (A). Photographs of the 437

chimneys (B) collected at BdJ, ST07, ST08 and ST12. Chimney and fluid samples were 438

collected in 2011 by scuba divers, who appear in picture ST07. The top of chimneys 439

(approximately 30 cm high) were collected at ST07, ST08 and ST12 and the inner parts were 440

sampled for DNA extraction. BdJ chimney samples corresponded to the crushed small 441

chimney shown on picture BdJ. 442

443

Figure 2. Structures of archaeal (A) and bacterial (B) communities at the phylum and 444

subphylum level in the four chimney samples collected in the Prony bay. The clustering 445

analyses were based on euclidean distance and Ward's linkage of CE-SSCP profiles of 446

archaeal and bacterial 16S rRNA gene fragments retrieved from chimneys of the four sites: 447

BdJ, ST07, ST08 and ST12. The diagram of relative phylogenetic abundance was based on 448

frequencies of 16S rRNA gene sequences affiliated with major phylogenetic groups in the 449

clone libraries from the four chimneys of the Prony Bay. MGII and SAGMEG mean Marine 450

Group II and South Africa Gold Mine Euryarchaeotic Group, respectively. 451

452

Figure 3. Neighbor-joining phylogenetic tree of the Betaproteobacteria (A) and the 453

Chloroflexi (B) 16S rRNA gene sequences from the PHF chimneys. Each OTU is 454

represented by one sequence with ≥97% similarity grouping. Numbers between brackets 455

indicate the number of clones obtained for each sequence type. PHF sequences are marked in 456

bold and their origin is indicated by different font colors and by the following markers: BdJ 457 Acc

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This article is protected by copyright. All rights reserved. 21

(, green font), ST07 (, pink font), ST08 (, blue font), and ST12 (, red font). Bootstrap 458

values <50% are not shown. 459

460

Figure 4. Neighbor-joining phylogenetic trees showing the relationship between 461

Deltaproteobacteria 16S rRNA gene sequences (A) and DsrB protein sequences from four 462

PHF chimneys. Each OTU is represented by one sequence with ≥97% similarity grouping. 463

Numbers between brackets indicate the number of clones obtained for each sequence type. 464

PHF sequences are marked in bold and their origin is indicated by different font colors and by 465

the following markers: BdJ (, green font), ST07 (, pink font), ST08 (, blue font), and 466

ST12 (, red font). Bootstrap values <50% are not shown. Genus name have been 467

abbreviated in the DsrB tree as follows: Dsv., Desulfovibrio; Dsnv., Desulfonatronovibrio; 468

Dsn., Desulfonatronum; Dsrv., Desulfurivibrio; Dsb., Desulfobulbus; Dfm., 469

Desufotomaculum. 470

471

Figure 5. Neighbor-joining phylogenetic trees showing the relationship between 472

Methanosarcinales 16S rRNA gene sequences (A) and McrA protein sequences from 473

four PHF chimneys. Each OTU is represented by one sequence with ≥97% similarity 474

grouping. Numbers between brackets indicate the number of clones obtained for each 475

sequence type. The PHF Methanosarcinales sequences are marked in bold and their origin is 476

indicated by different font colors and by following markers: BdJ (, green font), ST07 (, 477

pink font), ST08 (, blue font), and ST12 (, red font). Bootstrap values <50% are not 478

shown. Genus name have been abbreviated in the McrA tree as follows: Msr., 479

Methanosarcina; Mlo., Methanolobus; Mmt., Methanomethylovorans; Msl., Methanosalsum; 480

Mcc., Methanococcoides. Environmental (uncultured) Methanosarcinales clusters have been 481

abbreviated as follows: LCMS, Lost City Methanosarcinales; ANME-3, Anaerobic 482

Methanotrophs of group 3; TCMS, The Cedars Methanosarcinales. 483

Acc

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Figure 1

ST12

BdJ

ST08

BdJ ST07

A B

Acc

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100 100

Unclassified Bacteria

OD1

OP11

OP3

Cyanobacteria

Nitrospirae

BdJ

ST

07

ST

08

ST

12

0

10

20

Arc

haeal C

E-S

SC

P

sim

ilarity

(%

)

3.6

BdJ

ST

07

ST

08

ST

12

Bacte

rial C

E-S

SC

P

sim

ilarity

(%

)

A - Archaea B - Bacteria

Figure 2

0

10

20

30

40

50

60

70

80

90

100

BJ ST12

Arc

haeal clo

ne f

requencie

s (

%)

MG II

Thaumarchaeota

SAGMEG

The Cedars

Methanosarcinales

Lost City

Methanosarcinales

0

10

20

30

40

50

60

70

80

90

100

Bacte

rial clo

ne f

requencie

s (

%)

Deinococcus-Thermus

Chloroflexi

Actinobacteria

Firmicutes

Bacteroidetes

Gammaproteobacteria

Deltaproteobacteria

Betaproteobacteria

AlphaproteobacteriaST07 ST08 BJ ST12 ST07 ST08

Acc

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Burkholderiales

100

94

93

75

75

73

61

57

100

91

0.05

PHFBdJ_B4 (2) (KF886116)Ralstonia pickettii (NR 102967)

Hydrogenophaga intermedia (FJ009392) Clone KSB18 (JX047095)

Hydrogenophaga atypica (AJ585992)PHFBdJ_B6 (18) (KF886128)

The Cedars clone Ced_B12 (KC574882)Malikia spinosa (AB681972)

The Cedars clone Ced_B11 (KC574880)CVA clone CVCloAm3Ph105 (AM778002)

PHFST07_B1 (6) (KF886164)

Clone LC1524B-50 (DQ270636)

A - Betaproteobacteria

Figure 3

PHFST08_B10 (2) (KF886088)PHFST12_B5 (3) (KF886100)

Abyssal marine sediment clone SPG12 461 471 B7 (FJ746255)Subseafloor sediment clone 1H3M40 (JN230084)

PHFST12_B10 (4) (KF886095)Dehalogenimonas sp. SBP1 (JQ994267)

Dehalococcoides ethenogenes 195 (NR 074116)Dehalococcoides sp. GT strain G (NR 074288)

Holocene sediments clone KM35B-155 (AB300104)Siliciclastic sediment clone CK 2C4 105 (EU488394)

PHFST08_B7 (8) (KF886076)PHFST12_B1 (17) (KF886112)

Mud volcano clone AMSMV-5-B75 (HQ588482)Hypersaline microbial mat clone SBZO 4493 (JN533673)

The Cedars clone Ced B01 (KC574890)PHFST07_B6 (7) (KF886162)PHFST08_B3 (33) (KF886074)PHFST12_B3 (16) (KF886101)

Dehalococcoidia

99

86

100

100

100

61

100

99

100

9971

67

10098

87

85

0.05

B - Chloroflexi

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22

62

92

80

96

90

62

87

90

84

67

0.05

PHFST07_D10 (5) (KF886061)PHFST07_D2 (2) (KF886060)

PHFST12_D13 (1) (KF886064)

PHFST12_D1 (46) (KF886063)

PHFST08_D2 (22) (KF886067)

PHFBdJ_D2 (26) (KF886072)

PHFBdJ_D1 (5) (KF886071)

Mono Lake strain ML-1 (ADW23606)

Dsn. thioautotrophicum (AFI13797)

Dsn. lacustre (AAL57452)

Dsnv. hydrogenovorans (AAL57468)

Dsv. vulgaris (AAS94886)

Desulfovibrionales

PHFST12_B4 (10) (KF886103)

PHFST07_B3 (11) (KF886171)

PHFBdJ_B8 (9) (KF886124)

PHFST08_B5 (1) (KF886075)

Desulfonatronum cooperativum (NR043143)

Clone SYNH02 C3-03B-070 (JQ245528)

Desulfobulbus elongus (NR029305)

0.05

A - 16S rDNA Deltaproteobacteria B - DsrB

74

100

75

52

Figure 4

95

98

100

88

100

79

72

22

28

77

96

89

100

59

Clone NTd-III07 (BAE96488)

Clone ng7d1037 (AAX21319) PHFST08_D1 (1) (KF886066)

Dfm. alkaliphilum (AAL57464)

Dfm. ruminis (AAC24104)

Dfm. hydrothermale (CCO08396)

Dsb. propionicus (ADW19074)

Dsrv. alkaliphilus AHT2 (ADH85008)

Mono Lake strain MLMS-1 (EAT04804)

PHFST08_D54 (10) (KF886070)

PHFST12_D61 (3) (KF886065)

PHFST07_D1 (17) (KF886062)

Clone DSR-L (AAQ56688)PHFST08_D3 (15) (KF886068)

PHFST08_D34 (3) (KF886069)

PHFST07_D10 (5) (KF886061)

PHFST12_B20 (1) (KF886111)

PHFST07_B11 (3) (KF886157)

Desulfobulbus elongus (NR029305)

Desulfurivibrio alkaliphilus (CP001940)

The Cedars clone Ced_B15 (KC574885)

Desulfobacterales

Nitrospirales

99

83

66

100

Acc

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PHFBdJ_A1 (50) (KF886028)

PHFST07_A1 (43) (KF886031)

PHFST12_A2 (1) (KF886041)

PHFST08_A3 (15) (KF886034)

PHFBdJ_A3 (4) (KF886030)

PHFST07_A3 (1) (KF886034)

LCHF LC1149a-56 (AY299515 )

LCHF SGXU457 (FJ791612 )

Methanosarcina barkeri (AJ002476)

Methanomethylovorans hollandica (CP003362)

clone AMOS4A 4113 H07 (AY323224)

100

70

99

93

100

96

100

100

clone Eel-36a2H11 (AF354136) Clone Kuro-mcrA-5.04 (BAD16558)Clone HMMVBeg-ME94 (CAL58676)

Mcc. methylutens (AAC43410)Mcc. burtonii (AAC43406)Msl. zhilinae (AAC43429)

Mmt. hollandica (AAP20897)Mlo. tindarius (AAC43425)

Mlo. taylorii (AAC43424)Msr. barkeri (YP_304447

Msr. lacustris (AAP20898)

99

69

96

100

90

66

LCMS

ANME-3

A - 16S rDNA Methanosarcinales B - McrA Methanosarcinales

Figure 5

clone HMMVBeg-34 (AJ579327) clone HydBeg92 (AJ578119)

Methanosaeta pelagica (AB679167)

Methanosaeta thermophila PT (NR 028157)

Clone SYNH02 C3-03A-040 (JQ245658)

Clone MD-178-10-3291 KP12N-P S150 01A06 (JQ817506) PHFST08_A4 (19) (KF886037)

The Cedars clone Ced_A01 (KC574884)

PHFST07_A2 (2) (KF886032)

PHFBdJ_A4 (1) (KF886029)

PHFST12_A5 (13) (KF886044)

100

85

96

95

70

clone Eel-36a2H11 (AF354136)

PHFST07_M24 (16) (KF886052)PHFST07_M1 (7) (KF886053)

PHFBdJ_M21 (1) (KF886050)PHFBdJ_M1 (38) (KF886048)

Clone C9001C_mcrA_13_4_C05 (BAL42891)LCHF LCM1228-3 (AAW31861)

LCHF clone LCM1443-49 (AAW31859)LCHF clone LCM1404-22 (AAW31858)

PHFBdJ_M37 (1) (KF886051) PHFST08_M44 (26) (KF886054)PHFST12_M1 (20) (KF886057)PHFST12_M29 (1) (KF886059)

PHFST12_M11 (14) (KF886058)PHFBdJ_M5 (1) (KF886049)

PHFST08_M6 (3) (KF886055) PHFST08_M26 (2) (KF886056)

Clone K8MV-C22mcrA-19 (BAF96815)Clone Kuro-mcrA-5.04 (BAD16558)

95

93

57

90

56

51

99

58

61

90

73

0.05

Methanococcoides burtonii (NR 074242)

ANME-3

0.05

TCMS

Acc

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