Dominique Boeuf

Matthew T Cottrell

David L Kirchman

Philippe Lebaron

Christian Jeanthon
email: jeanthon@sb-roscoff.fr

Summer community structure of aerobic anoxygenic phototrophic bacteria in the western Arctic Ocean

Keywords: photoheterotroph, aerobic anoxygenic phototrophic bacteria, pufM gene, bacteriochlorophyll, Arctic

Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophs that require oxygen for their growth and for bacteriochlorophyll a (Bchl a) synthesis. They inhabit a wide variety of illuminated habitats in diverse terrestrial, freshwater, and marine environments [START_REF] Béjà | Unsuspected diversity among marine aerobic anoxygenic phototrophs[END_REF][START_REF] Csotonyi | A new environment for aerobic anoxygenic phototrophic bacteria: biological soil crusts[END_REF][START_REF] Atamna-Ismaeel | 4: 209-216. and PSI-BLAST: a new generation of protein database search programs[END_REF]. First discovered in coastal marine waters [START_REF] Shiba | Distribution of aerobic bacteria which contain bacteriochlorophyll a[END_REF], AAP bacteria have been intensively studied in the 30 marine environment [START_REF] Cottrell | Aerobic anoxygenic phototrophic bacteria in the Mid-Atlantic Bight and the North Pacific Gyre[END_REF][START_REF] Mašín | Seasonal changes and diversity of aerobic anoxygenic phototrophs in the Baltic Sea[END_REF][START_REF] Lehours | Summer distribution and diversity of aerobic anoxygenic phototrophic bacteria in the Mediterranean Sea in relation to environmental variables[END_REF]. Their abundance and distribution vary greatly among oceanic regimes, suggesting that there is a broad range of potential ecological niches for these microbes. AAP bacteria seem to be more abundant in shelf and coastal areas than in the open ocean [START_REF] Schwalbach | Wide-ranging abundances of aerobic anoxygenic phototrophic bacteria in the world ocean revealed by epifluorescence microscopy and quantitative PCR[END_REF][START_REF] Sieracki | Distribution of planktonic aerobic anoxygenic photoheterotrophic bacteria in the northwest Atlantic[END_REF]. Although their abundance can be high in some oligotrophic regions 35 [START_REF] Lami | High abundances of aerobic anoxygenic photosynthetic bacteria in the South Pacific Ocean[END_REF], AAP bacteria typically constitute a small percentage (2 to 4%) of total prokaryotes in oceanic environments [START_REF] Cottrell | Aerobic anoxygenic phototrophic bacteria in the Mid-Atlantic Bight and the North Pacific Gyre[END_REF][START_REF] Jiao | Distinct distribution pattern of abundance and diversity of aerobic anoxygenic phototrophic bacteria in the global ocean[END_REF]. However, their proportions can exceed 10% in eutrophic estuaries [START_REF] Waidner | Aerobic anoxygenic phototrophic bacteria attached to particles in turbid waters of the Delaware and Chesapeake estuaries[END_REF]. Despite the lower abundances in most oligotrophic pelagic marine environments, AAP bacteria constitute a very dynamic part of the bacterial community and potentially contribute 40 significantly to the cycling of organic carbon in the upper ocean [START_REF] Koblížek | Rapid growth rates of aerobic anoxygenic phototrophs in the ocean[END_REF].

Culture-dependent and -independent studies have shown AAP bacteria to be genetically diverse with members of the Alpha-, Beta-, and Gammaproteobacteria [START_REF] Béjà | Unsuspected diversity among marine aerobic anoxygenic phototrophs[END_REF][START_REF] Yutin | Assessing diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of the Atlantic and 750 Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF][START_REF] Jeanthon | Diversity of cultivated and metabolically active aerobic anoxygenic phototrophic bacteria along an 620 oligotrophic gradient in the Mediterranean Sea[END_REF]. The environmental factors controlling the population structure and diversity of these photoheterotrophic bacteria remain poorly properties. In the Beaufort Sea, the Mackenzie River delivers considerable freshwater, nutrients and organic matter which exert strong controls on the productivity, salinity and 50 water circulation [START_REF] Aagaard | The Role of Sea Ice and Other Fresh Water in the Arctic Circulation[END_REF][START_REF] Opsahl | Major flux of terrigenous dissolved organic matter through 675 the Arctic Ocean[END_REF]. Despite a short growing season and extensive sea ice coverage, the most river-influenced of the world's oceans supports a productive aquatic ecosystem. Recent studies over the Mackenzie Shelf in the Beaufort Sea have revealed diverse and active communities of Bacteria and Archaea that vary across salinity gradients [START_REF] Galand | Remarkably diverse and contrasting archaeal communities in a large arctic river and the coastal Arctic Ocean[END_REF][START_REF] Garneau | Prokaryotic community 595 structure and heterotrophic production in a river-influenced coastal arctic ecosystem[END_REF][START_REF] Galand | Microbial community diversity and 590 heterotrophic production in a coastal Arctic ecosystem: A stamukhi lake and its source waters[END_REF]. 55 Our current knowledge of the abundance, distribution, and activity of AAP bacteria in perennially cold marine waters is still limited. In the only study in the Arctic, AAP bacteria contributed 5 to 8% of the total prokaryotic community in spatially close coastal sites of the western Beaufort and eastern Chukchi Seas [START_REF] Cottrell | Photoheterotrophic microbes in the Arctic Ocean in summer and 575 winter[END_REF]. Seasonal shifts in the abundance of AAP bacteria between summer and winter paralleled changes in the 60 abundance of the total prokaryotic community. There were no substantial shifts in the community structure of AAP bacteria between the two seasons. Although pufM genes in Arctic surface waters could be assigned to taxonomic groups previously identified in metagenomic shotgun sequences from the GOS expedition [START_REF] Yutin | Assessing diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of the Atlantic and 750 Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF], their sequences were distinct from those retrieved in lower-latitude waters (Cottrell & Kirchman, 65 2009).

In this study, we collected samples across a several-thousand kilometers transect above the 60°N latitude that yielded an unprecedented resolution of the Beaufort Sea during summer and explored transition zones between offshore and large Arctic rivers that have a major influence on coastal ocean biogeochemistry. To examine the AAP bacterial community 70 structure and factors that govern it, we combined the use of infrared epifluorescence microscopy and culture-dependent and independent approaches. The Beaufort Shelf surface circulation is strongly influenced by ice, winds and freshwater input resulting from river discharge and sea-ice melt. We hypothesized that AAP bacterial abundance and composition would change substantially along this transect and that riverine inputs would have a 75 substantial influence on the AAP bacterial populations.

Materials and methods

Study area, sampling and oceanographic parameters 80

The MALINA cruise took place onboard the Canadian research icebreaker CCGS Amundsen during summer 2009 from Victoria (BC, Canada) to the Beaufort Sea (Leg 1b) and then throughout the Beaufort Sea (Leg 2b) (Fig. 1). Most of the stations sampled on the west-east transect in the Beaufort Sea (leg2) were ice-free. However, surface waters of eastward offshore waters were still ice-covered. Surface seawater samples were collected with an acid 85 cleaned bucket during Leg 1b and in the Mackenzie plume (stations 395, 398, 694, and 697) during Leg 2b. In the Beaufort Sea, seawater was collected from six depths using Niskin bottles mounted on a CTD (conductivity temperature depth probe) rosette. Ancillary data of temperature, salinity, pH, dissolved oxygen, colored dissolved organic matter, inorganic and organic nutrients, and chlorophyll a are given in Table S1. 50 mM Tris-HCl, pH 8), immediately frozen in liquid nitrogen, and stored at -80°C until analysis. To limit the degradation of mRNA, small volumes of seawater (~ 1 liter) were filtered as rapidly as possible, immediately upon retrieval of the CTD. RNA filters were transferred in collection tubes containing 0.8 ml of RLT buffer (Qiagen, Hamburg, Germany) 100 with 1 % of β-mercaptoethanol, frozen in liquid nitrogen, and stored at -80°C until analysis.

The time from the start of filtration to storage was 15-20 min.

Microscopic enumeration of AAP bacteria and total prokaryotes

Prokaryotes were enumerated by epifluorescence microscopy of paraformaldehyde-fixed 105 samples that were filtered onto 0.2-µm pore-size black polycarbonate filters [START_REF] Cottrell | Aerobic anoxygenic phototrophic bacteria in the Mid-Atlantic Bight and the North Pacific Gyre[END_REF]. Filters were stored at -80°C for up to 4 months prior to analysis. Total prokaryotes were enumerated after staining with 4',6-diamidino-2-phenylindole (DAPI), 1 μg.ml -1 final concentration in 1× phosphate-buffered saline (PBS) for 10 min. The AAP bacteria were enumerated using an Intensified Retiga charge-couple device camera (Qimaging, Surry, BC 110 Canada) and image analysis routines to enumerate cells that fluoresce blue when stained with DAPI and have infrared fluorescence (BChl a signal), but not red or orange fluorescence (Chl a and phycoerythrin signals, respectively) [START_REF] Cottrell | Aerobic anoxygenic phototrophic bacteria in the Mid-Atlantic Bight and the North Pacific Gyre[END_REF]. AAP bacterial abundance data were imported into Ocean Data View version 4 [START_REF] Schlitzer | Ocean Data View[END_REF] and interpolated using the DIVA gridding tool.

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Isolation and identification of AAP bacterial strains

Seawater samples (50-100 µl) collected at the surface and at the deep chlorophyll maximum (DCM) were spread onto low nutrient heterotrophic medium [START_REF] Cho | Cultivation and growth characteristics of a diverse group of oligotrophic marine Gammaproteobacteria[END_REF] Dimethylsulfoniopropionate (DMSP) was added to the medium at a final concentration of 100 nM. Culture plates were incubated for up to 2 months at 4°C into thermostatic cabinets illumined by luminescent tubes at irradiance levels of 120 µmol photons m -2 s -1 (for surface samples) and 25 µmol photons m -2 s -1 (for DCM samples). BChl a-containing colonies were 125 detected as previously described [START_REF] Jeanthon | Diversity of cultivated and metabolically active aerobic anoxygenic phototrophic bacteria along an 620 oligotrophic gradient in the Mediterranean Sea[END_REF].

Bacterial DNA was extracted using the QuickExtract™ Bacterial DNA Extraction Kit (Epicentre Biotechnologies, Madison, WI, USA). Small-subunit (16S) rRNA genes were amplified by polymerase chain reaction (PCR) using universal reverse primer 1492R and Bacteria-specific forward primer 27F (Table 1), whose 5' ends were labeled with 6-130 hexachlorofluorescein (6-HEX) and with 6-carboxyfluorescein (6-FAM), respectively. Reaction mixtures (25 μL) contained 5 X buffer (5 μl), 2mM MgCl 2 , 10 pmoles of each deoxyribonucleotide triphosphate (dATP, dCTP, dGTP, dTTP; Eurogentec, Seraing, Belgium), 10 pmoles of each oligonucleotide primer, 1.25 U of GoTaq Flexi DNA polymerase (Promega, Madison, WI, USA) and 1 μl of cell lysate. The initial denaturation step consisted of heating 135 the reaction mixture at 95 °C for 5 min, and the thermal profile then consisted of 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1.5 min.

A final extension step was carried out at 72 °C for 10 min. The PCR products were digested with the restriction endonucleases HinfI and MspI (New England BioLabs, Ipswich, MA, USA) for 4 h at 37°C. After enzymatic inactivation at 80°C for 20 min, T-RFLP digests were diluted 16S rRNA gene sequences of at least two randomly chosen strains from each T-RFLP group were amplified by PCR using 8F-1492R primers (Table S2) following the same 145 conditions as described above. PCR products were purified by ExoSAP-IT PCR purification kit (GE Healthcare) and sequenced using the latter primers with an ABI 3130 POP7 sequencer (Applied Biosystems).

16S rRNA gene sequences were compared to sequences in public databases with BLASTn (Altschul et al 1997). The new sequences were aligned using the Q-INS-I algorithm of 150 the MAFFT sequence alignment program version 6.5 [START_REF] Katoh | Multiple alignment of DNA sequences with MAFFT[END_REF]. A set of 16S rRNA sequences with high similarity to those determined in this study were retrieved and added to the alignment. Phylogenetic trees were computed using neighbor-joining, maximum parsimony and maximum likelihood inference approaches using MEGA 5 [START_REF] Tamura | MEGA5: Molecular 715 evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]. Bayesian analyses (MrBayes; [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF] were also performed 155 to provide additional statistical support for the nodes.

The presence of pufM was determined by using PCR amplification with specific primers [START_REF] Lehours | Summer distribution and diversity of aerobic anoxygenic phototrophic bacteria in the Mediterranean Sea in relation to environmental variables[END_REF]. When necessary, bands of the targeted size were isolated from the gel using a DNA gel extraction kit (Millipore, Billerica, MA, USA). Gel-purified products were concentrated with a Microcon YM50 centrifugal filter devices (Millipore) and 160 sequenced using pufMF and pufM-WAW primers (Table S2).

Diversity analysis of pufM gene

A total of 42 DNA samples were extracted as previously described [START_REF] Marie | Eukaryotic picoplankton communities of the Mediterranean Sea in summer assessed by molecular approaches (DGGE, TTGE, QPCR)[END_REF]. To select samples for pufM diversity analysis, amplification and fingerprinting of pufM 165 communities using temporal temperature gradient electrophoresis (TTGE) were performed as described by [START_REF] Lehours | Summer distribution and diversity of aerobic anoxygenic phototrophic bacteria in the Mediterranean Sea in relation to environmental variables[END_REF]. For the construction of partial pufM DNA clone libraries, fresh PCR products were cloned directly or after gel purification using the TOPO-TA cloning kit (Invitrogen) according to the manufacturer's instructions. The sequences were trimmed to remove any vector and primer sequences. DNA sequences were translated into 170 amino acid sequences and aligned using the ClustalW program in MEGA 5 [START_REF] Tamura | MEGA5: Molecular 715 evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]. The resulting protein alignment was back-translated to nucleotide acid sequences that were realigned manually. A conservative value of 94% nucleic acid sequence similarity was chosen for clustering sequences into Operational Taxonomic Units (OTUs) using MOTHUR [START_REF] Schloss | Introducing mothur: open-source, platformindependent, community-supported software for describing and comparing microbial communities[END_REF]. Representative sequences (defined as the sequence with the 175 minimum distance to all other sequences in the OTU) were obtained using MOTHUR. The coverage value [START_REF] Mullins | Genetic comparisons reveal the same 670 unknown bacterial lineages in Atlantic and Pacific bacterioplankton communities[END_REF], the Shannon index, H Shannon [START_REF] Shannon | The mathematical theory of communication[END_REF] and the bias-corrected richness-estimator, S Chao1 [START_REF] Chao | A new statistical approach for assessing similarity of species composition with incidence and abundance data[END_REF] were calculated for each library.

A pufM database containing 35 aligned sequences (720 total nucleotide positions) of 180 cultured species and environmental clones retrieved from GenBank and the GOS scaffold nucleotide sequences was constructed using MEGA 5 [START_REF] Tamura | MEGA5: Molecular 715 evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF] according to [START_REF] Yutin | Assessing diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of the Atlantic and 750 Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF]. A phylogenetic tree was constructed by Bayesian inference using MrBayes with substitution rates varying according to codon positions. Four parallel MCMC chains of 3.5 million generations were run; trees were sampled every 100 generations. A 185 consensus tree was constructed after the exclusion of 10.000 'burnin' trees. The pufM database and the consensus tree were imported into ARB software [START_REF] Ludwig | ARB: a software environment for sequence data[END_REF].

Representative sequences of each OTU (245 pb) and short pufM environmental reference sequences were aligned as above and added to the consensus Bayesian tree using the ADD_BY_PARSIMONY algorithm implemented in ARB. Non-informative taxa were removed 190 from the final tree. For cluster analysis, the dataset was randomly resampled before analysis. A distance matrix was computed from the presence of OTUs in clone libraries at a 94% identity threshold using Dice coefficient [START_REF] Dice | 26: 297-302. suspended particles in the Mackenzie River plume (Canadian Arctic Ocean) and implications for ocean colour remote sensing[END_REF] and a dendrogram was inferred with the unweighted pair-group average algorithm (UPGMA) as implemented in XLSTAT (Addinsoft,195 New York, USA).

In situ abundance of OTU 6 members

OTU 6, the most abundant group retrieved in the pufM libraries, was targeted by qPCR.

Primers targeting specifically pufM genes from OTU 6 (Table S2) were designed using ARB.

200

Primer specificity was confirmed by constructing a clone library as described above and sequencing 31 clones. All sequences were identical to those of OTU 6 members. QPCR conditions were as in Table S2. Two plasmid clones from our pufM and 16S rRNA libraries were used as positive controls and for standard curves (10-fold dilution series) in the qPCR assays. Plasmid DNA was linearized with HindIII and quantified using the Picogreen assay 205 (Invitrogen).

QPCR was performed in triplicate, with 1 µl of environmental sample DNA normalized to 100 pg.µl -1 or plasmid DNA solution in a total reaction volume of 10 µl using the SYBR Green/ROX qPCR Master Mix kit (Applied Biosystems). QPCR assay were performed using a Chromo4 Real-Time PCR Detection System and the Opticon Monitor software package (Bio-210 Rad) with the following thermal cycling conditions: 95°C for 15 min, 45 cycles of denaturation at 95°C for 45 s, primer annealing at the primer-specific annealing temperature (Table S2) for 45 s with fluorescence measurements after this step, and polymerase extension at 72°C for 15 s. Melting curves were generated after each assay to check the specificity of the amplification by heating from 65 to 95°C at a rate of 0.1°C.s -1 and taking No inhibition was detected when a known quantity of standard was added to DNA 220 extracts from surface waters of stations 697, 680, 620 and 398 (data not shown). The estimates of OTU 6-pufM abundance were normalized to 16S rRNA gene abundance in order to estimate the fraction of the bacterial community bearing OTU 6-pufM type [START_REF] Campbell | Abundant proteorhodopsin genes in the North Atlantic Ocean[END_REF].

225

Activity of OTU 6 members

Reverse-transcription PCR was used to produce cDNA from mRNA transcripts to determine whether pufM was being transcribed by OTU 6 members at the time of sampling. RNA samples were extracted using the RNeasy Mini kit (Qiagen) following the manufacturer's instructions with several modifications. Glass beads (50 µl) were added to the samples after 230 thawing on ice and the tubes were mixed vigorously for 10 min. Genomic DNA was removed using a Turbo DNA-free kit (Ambion, Life Technologies, Paisley, UK). DNA removal in RNA samples was confirmed by control PCR amplifications without the reverse transcription step.

No amplification was detected in these controls. ThermoScript RT-PCR system (Invitrogen, Carlsbad, USA) was used for the reverse transcription of pufM mRNA from total RNA 235 samples. All reverse transcription reactions were performed in a total reaction volume of 10 μl. cDNA synthesis was performed at 55 °C using the reverse primer as gene-specific primer.

PCR products obtained as above were cloned directly or after gel extraction and sequenced.

Results

Oceanographic context

250 Using salinity and temperature profiles, several water masses were delineated in the Beaufort Sea/Mackenzie system (Fig. S1; Table S1). Surface waters in the Mackenzie plume stations and under the influence of the Firth river (station 680) displayed the highest temperatures and lowest salinities. The influence of the Mackenzie plume decreased eastward in the shelf where colder and denser surface waters were recorded, which was 255 likely due to the addition of ice melt from offshore waters. Below 40-50m depth, cold, high salinity deep ocean waters consistent with Pacific Ocean intrusion were observed. Nitrate concentrations in surface waters were low. Surface-water phosphate increased with salinity, whereas silicate and nitrate decreased, as expected in this river-influenced region [START_REF] Carmack | Phytoplankton productivity on the Canadian Shelf of the Beaufort Sea[END_REF]. Similar to hydrographic features, total Chl a levels decreased greatly from the 260 river plume region towards the more oligotrophic marine shelf and offshore waters.

Abundance of total prokaryotes and AAP bacteria

Total prokaryotic abundances ranged from 9.1 X 10 4 cells ml -1 to 3.4 X 10 5 cells ml -1 in surface waters sampled from the North Pacific Ocean to the Chukchi Sea. Along this transect, 265 AAP bacterial abundances as measured by infrared epifluorescence microscopy were low (about 0.1% to 0.5%) except in the Chukchi Sea (ARC1) where their contribution reached 14.8% of the total prokaryotic community. In the Beaufort Sea, abundances of total prokaryotes decreased slightly from the river plume to offshore surface waters (Table S1; Fig. 2a to 2d). Similar to total Chl a, AAP abundances dropped 10-fold between the plume 270 and the shelf surface waters and by another 10-fold between the shelf and offshore surface waters (Fig. 2e; Table S1). AAP bacterial abundances were highest in surface waters of the Mackenzie plume (mean abundance of 6.3 %), reaching 14% of total prokaryotes at station 398 (Fig. 2f and2g). High contributions (up to 10%) were also found in the bottom nepheloid layer (BNL) of transects 200 and 300 (Fig. 2g and2h).

275

For the overall dataset, total prokaryotes and AAP bacterial abundances were positively correlated with temperature and negatively with salinity (Table S3). AAP proportions correlated significantly with ammonium, silicate, total Chl a and the different forms of organic carbon, nitrogen, and phosphorus. In contrast to relative AAP bacterial abundance, total prokaryote abundance did not covary significantly with most of the 280 parameters. High AAP bacterial abundances in the BNL were related to dissolved inorganic nitrogen and phosphorus, suggesting the presence of a diffusive flux of nutrients from the organic-rich sediments, the BNL, or both [START_REF] Sundby | The phosphorus cycle in coastal marine sediments[END_REF]. S4) while the coverage index was low. However, 290 coverage estimates (> 83%) and rarefaction curves (data not shown) indicated that most of the diversity was detected in the libraries from marine stations. The diversity of AAP bacteria was generally lower in surface than in deeper waters. Cluster analysis of the AAP bacterial community composition separated the Beaufort Sea samples in three clusters according to water masses (Fig. S2). The two main clusters separated surface samples (upper polar mixed 295 layer) and waters below 50m (Pacific summer water). North Pacific Ocean and Bering Sea samples grouped together.

Diversity and distribution of environmental pufM sequences

A phylogenetic tree was constructed for all 43 OTUs from this study and from previous studies of pufM sequences (Fig. 3). The phylogenetic analysis revealed that 41 OTUs were tentatively assigned to Alpha-(22 OTUs, ~ 49 % of the sequences) or 300 Betaproteobacteria (17 OTUs; ~ 48% of the sequences). Two OTUs belonged to Gammaproteobacteria. Two singletons could not be confidently assigned.

The betaproteobacterial OTU 6 was the most frequent OTU (38 % of the sequences) and had pufM sequences identical (153 bp out of 245 bp) to that of environmental clones from the Monterey Bay [START_REF] Béjà | Unsuspected diversity among marine aerobic anoxygenic phototrophs[END_REF]. OTU 6 belonged to a large cluster of 13 OTUs that [START_REF] Waidner | Aerobic anoxygenic photosynthesis genes and operons in uncultured bacteria in the Delaware River[END_REF] 310 and Rhodoferax fermentans [START_REF] Page | Representative freshwater bacterioplankton isolated from Crater Lake, Oregon[END_REF]. Betaproteobacterial sequences dominated in clone libraries from river-influenced waters (Fig. 4). OTU 6, the most abundant OTU, was recovered in all Beaufort Sea stations and in the Bering Sea but was absent in the North Pacific Ocean and Chukchi Sea.

Alphaproteobacterial OTU 1 and OTU 5, the second and third most abundant OTU 315 (18.6% and 6.6% of the sequences, respectively) together with OTU 4, -12 and -29 were closely related to environmental clones recovered from the Chukchi and Beaufort Seas [START_REF] Cottrell | Photoheterotrophic microbes in the Arctic Ocean in summer and 575 winter[END_REF]) (Fig. 3). These sequences dominated libraries from offshore waters of the Beaufort and Chukchi Seas. Among them, OTUs 4 and 5 were only detected in the Beaufort Sea (Fig. 4). In contrast, OTU 1 was retrieved from the North Pacific Ocean to 320 the Chukchi Sea with relative proportions increasing northwardly. It also occurred in most Beaufort Sea samples.

Other Alphaproteobacteria grouped mainly with cultivated members of the Bradyrhizobiaceae and of genera Citromicrobium, Methylobacterium and Sphingomonas (Fig. 3). Bradyrhizobiaceae-and Citrobacterium-like sequences showed similar distribution 325 patterns. They co-occurred in surface waters of the North Pacific Ocean and in shelf and offshore DCM layers of the Beaufort Sea. Sequences related to Sphingomonas and Methylobacterium were only found at the surface in the Mackenzie mouth and at the DCM level along the plume (Fig. 4). Gammaproteobacterial OTUs belonged to the phylogroup K defined by [START_REF] Yutin | Assessing diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of the Atlantic and 750 Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF]. They prevailed in surface water libraries of the North Pacific 330 Ocean but were not detected in higher latitude samples.

Diversity of AAP isolates

To gain more insights into the origin of the pufM genes obtained in this study, AAP bacteria 335 were isolated that grew on low nutrient heterotrophic medium. BChl a-containing colonies were detected only from shelf and offshore surface waters of the Beaufort Sea. Based on T-RFLP analyses of 16S rRNA gene, the 92 pufM-positive strains were distributed in four groups (Table 1). In accordance with their position in the pufM phylogenetic tree (Fig. 3), representative strains of these groups had 16S rRNA gene sequences closely related to 340 Sulfitobacter and Loktanella (Fig. 5). Interestingly, the pufM partial sequences of most isolates were identical or nearly identical to environmental sequences obtained in this study.

About half of the isolates were affiliated with S. marinus (Fig. 5) and had pufM sequences that matched with OTU 5, the third most abundant OTU in the pufM libraries (Fig. 3). The 16S rRNA gene sequences of both Sulfitobacter representatives RCC 2406 and RCC 2401 345 were most closely related with that of isolates from Antarctic Sea ice and Arctic marine sediments, respectively (Table 1). The two other T-RFLP groups were affiliated to the genus Loktanella. Both Loktanella groups had pufM sequences almost identical to that of OTU 29.

On the basis of their 16S rRNA gene sequences, both groups clustered with strains isolated from Antarctic lakes [START_REF] Van Trappen | Loktanella salsilacus gen. nov., sp. nov., Loktanella fryxellensis sp. nov. and Loktanella vestfoldensis sp. nov., new members of the Rhodobacter group, isolated from microbial mats in Antarctic lakes[END_REF], the major one having a sequence similar to 350 that of Loktanella sp. WM1 isolated from the Baltic Sea [START_REF] Salka | Abundance, depth distribution, and composition of aerobic bacteriochlorophyll a-producing bacteria in four basins of 690 the central Baltic Sea[END_REF]. No gamma-or betaproteobacterial AAP strains were isolated.

Abundance and activity of OTU 6 members

Since pufM in the OTU 6 clade clustered with betaproteobacterial sequences, we 355 hypothesized that OTU 6 members would have a freshwater origin. The relative abundance of OTU 6 was investigated using quantitative PCR along the two salinity gradients of the Mackenzie River (transects 300 and 600). Proportions of OTU 6-like pufM genes decreased gradually from higher-temperature and lower-salinity to lower-temperature and highersalinity waters in both transects (Fig. 6). However, we noticed a high proportion of OTU 6 at 360 station 680 associated with increasing temperature and decreasing salinity. This contribution was higher than the expected based on simple dilution. It could be related to a local input of OTU 6 members released from the Firth River. A subsequent PCR survey of our samples using specific primers detected OTU 6 members in all Beaufort Sea surface samples except at the three northern offshore stations (110, 220, and 320).

365

To investigate if members of this clade actively express photosynthetic genes in cold marine waters, we extracted mRNA from surface samples of transect 600 and amplified OTU 6-specific genes. We confirmed by sequencing that all pufM cDNA clones were OTU 6 members. Active OTU 6 members were detected in all samples including the most offshore station 620, although their contribution to total abundance was low according to qPCR 370 assays (Fig. 6).

Discussion

Little is known about AAP bacteria in polar environments. The main objective of this study was to explore their abundance, diversity, and distribution patterns in a significant and 375 typical arctic region and to evaluate their ecology and biogeographical trends. Our results indicate that the distribution patterns generally observed in oligotrophic open ocean and eutrophic estuarine environments of temperate and tropical regions [START_REF] Jiao | Distinct distribution pattern of abundance and diversity of aerobic anoxygenic phototrophic bacteria in the global ocean[END_REF][START_REF] Lamy | Ecology of aerobic anoxygenic phototrophic bacteria along an oligotrophic gradient in the Mediterranean Sea[END_REF]Waidner & Kirchman, 2008) also occur in the Beaufort Sea.

Links between AAP population abundance and environmental variables

Our results indicate that particulate and dissolved organic matter and Chl a concentrations positively influenced the relative abundance of AAP bacteria but did not contribute to the 385 success of the prokaryote community as a whole. This suggests that AAP bacteria may respond to organic supply differently and have higher mineralization capacities than the bacterial community. In addition to AAP bacteria associated with the thick surface turbidity layer, proportions of AAP bacteria were high in the thinner but more turbid bottom nepheloid layer. Interestingly, AAP bacteria in surface and bottom layers showed contrasting 390 relationships to several environmental variables, suggesting that the two particle-rich environments represent substantially different habitats. The two habitats could differ because of different types of particles in the water column, phytoplankton, and mineral-rich fine particles in the surface layer and coarse aggregates of suspended sediments in the bottom layer (Doxaran et al., 2012). These particle-rich waters might be important not only 395 in terms of sediment transport but also as sites of microbially mediated organic transformations. The latter possibility is consistent with observations of increased microbial production and activity in particle-rich surface and bottom waters of the Mackenzie plume [START_REF] Vallières | Bacterial production and microbial food web structure in a large arctic river and the coastal Arctic Ocean[END_REF][START_REF] Garneau | Importance of particle-associated bacterial heterotrophy in a coastal Arctic ecosystem[END_REF]. Although we did not quantify the proportion of particle-associated AAP bacteria, earlier studies in other nutrient-rich environments 400 [START_REF] Waidner | Aerobic anoxygenic phototrophic bacteria attached to particles in turbid waters of the Delaware and Chesapeake estuaries[END_REF] suggest that they may represent a significant part of the AAP bacterial community in the Mackenzie plume. Being associated with particles seems to enhance the synthesis of BChl a in estuaries [START_REF] Cottrell | Bacteriochlorophyll and community structure of aerobic anoxygenic phototrophic bacteria in a particle-rich estuary[END_REF]. It is likely that the energy gained by AAP bacteria via phototrophy in these environments is suitable for cost-intensive

AAP bacterial diversity and distribution

Most OTUs recovered were closely related (>94% similarity) to existing Arctic sequences or belonged to novel OTUs. Only 8 OTUs matched sequences from elsewhere. The arctic pufM 410 genes were also different from those in Antarctic sea-ice and coastal seawater [START_REF] Koh | Aerobic anoxygenic phototrophic bacteria in Antarctic sea ice and seawater[END_REF]. Consistent with previous findings [START_REF] Cottrell | Photoheterotrophic microbes in the Arctic Ocean in summer and 575 winter[END_REF], this suggests that most OTUs found in this study might be restricted to the Beaufort Sea, which seems to constitute a microbial province favoring endemism (Lovejoy et al., 2007).

The most striking observation from our data set is the widespread distribution of a 415 AAP betaproteobacterial clade (OTU 6) in the entire shelf. Betaproteobacteria are usually low in abundance in the open ocean and the few betaproteobacterial sequences that have been retrieved from the marine environment are from coastal environments [START_REF] Rappé | Phylogenetic comparisons of a coastal bacterioplankton community with its counterparts in open ocean and freshwater systems[END_REF][START_REF] Riemann | The native bacterioplankton community in the central Baltic Sea is influenced by freshwater bacterial 685 species[END_REF]. In contrast, they represent a consistently large fraction of the bacterioplankton in freshwater lakes and diverse river types [START_REF] Glöckner | Comparative 16S rRNA analysis of lake bacterioplankton reveals globally distributed phylogenetic clusters including an abundant group of actinobacteria[END_REF]Zwart et 420 al., 2002) including the Mackenzie River where they are abundant [START_REF] Garneau | Prokaryotic community 595 structure and heterotrophic production in a river-influenced coastal arctic ecosystem[END_REF][START_REF] Galand | Microbial community diversity and 590 heterotrophic production in a coastal Arctic ecosystem: A stamukhi lake and its source waters[END_REF]. Since the Arctic Ocean receives about 10% of global riverine discharge [START_REF] Aagaard | The Role of Sea Ice and Other Fresh Water in the Arctic Circulation[END_REF], the stratified surface waters of the Arctic Ocean share many characteristics of an estuary where Betaproteobacteria form a minor fraction of the total prokaryotic community [START_REF] Galand | Hydrography shapes bacterial biogeography of the deep Arctic Ocean[END_REF][START_REF] Kirchman | The structure of bacterial communities in the western Arctic Ocean as revealed by pyrosequencing of 16S rRNA genes[END_REF]. Most 425 betaproteobacterial OTUs recovered in this study were new (< 94% similarity to existing sequences) and do not group with the Rhodoferax, Roseateles, and Rubrivivax pufM clusters often identified in other estuarine and freshwater systems (Waidner & Kirchman, 2008; [START_REF] Salka | Rhodoferax-related pufM gene cluster dominates the aerobic anoxygenic phototrophic communities in German freshwater lakes[END_REF]. They formed several clusters divergent from the phylogroup I [START_REF] Yutin | Assessing diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of the Atlantic and 750 Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF] that include arctic sequences [START_REF] Cottrell | Photoheterotrophic microbes in the Arctic Ocean in summer and 575 winter[END_REF] and single amplified genome 430 (SAG) sequences from temperate freshwater lakes [START_REF] Martinez-Garcia | High-throughput single-cell sequencing identifies photoheterotrophs and 660 chemoautotrophs in freshwater bacterioplankton[END_REF].

Interestingly, the 16S rRNA sequences from the latter pufM-containing SAGs were primarily related to the betaproteobacterial Polynucleobacter cluster that are common in the Mackenzie river [START_REF] Galand | Microbial community diversity and 590 heterotrophic production in a coastal Arctic ecosystem: A stamukhi lake and its source waters[END_REF]. Since horizontal gene transfer of the photosynthetic gene cluster is possible [START_REF] Igarashi | Horizontal transfer of the photosynthesis gene cluster and operon rearrangement in purple bacteria[END_REF], phylogenetic data need to be interpreted with 435 caution especially in groups with only a few species with known pufM and 16S sequences.

Our data clearly indicate that betaproteobacterial AAP bacteria exhibited a strong river to ocean gradient, suggesting that these bacteria grew in the Mackenzie River and then were mixed with Beaufort coastal waters. The distribution of OTU 6 in the Beaufort Sea was consistent with satellite observations of the offshore extension of Mackenzie turbid waters 440 over the continental shelf (Doxaran et al., 2012). However, the presence and activity of OTU 6 in Beaufort coastal waters contrasted with that of other betaproteobacterial OTUs found only near the mouth of the Mackenzie River. Since mortality of freshwater bacteria can be an important process in estuaries [START_REF] Painchaud | Assessment of salinity-related mortality of freshwater bacteria in the Saint Lawrence estuary[END_REF], one explanation is that OTU 6 members have a broader salinity tolerance than other AAP betaproteobacteria. The 445 presence of partial pufM sequences identical to that of OTU 6 in marine waters of Monterey Bay [START_REF] Béjà | Unsuspected diversity among marine aerobic anoxygenic phototrophs[END_REF] supports this hypothesis. Further studies are required to estimate their contribution in other marine Arctic regions and to identify parameters that control their distribution.

Previous studies showed that some Betaproteobacteria are still active in marine 450 waters [START_REF] Cottrell | Single-cell analysis of bacterial growth, cell size, and community structure in the Delaware estuary[END_REF], including in the Arctic [START_REF] Alonso-Sáez | Winter-to-summer changes in 545 the composition and single-cell activity of near-surface Arctic prokaryotes[END_REF]. Here, we showed that the OTU 6 pufM gene was actively transcribed in saline waters during the [START_REF] Yurkov | Impact of light/dark regimen on growth rate, biomass formation and bacteriochlorophyll synthesis in Erythromicrobium hydrolyticum[END_REF][START_REF] Tomasch | Transcriptional response of the photoheterotrophic marine bacterium Dinoroseobacter shibae to changing light regimes[END_REF]. Our data suggest that BChl a synthesis in OTU 6 members is not fully repressed by 455 light, like in Dinoroseobacter shibae and Roseateles depolymerans [START_REF] Suyama | 710 Photosynthetic apparatus in Roseateles depolymerans 61A is transcriptionally induced by carbon limitation[END_REF][START_REF] Tomasch | Transcriptional response of the photoheterotrophic marine bacterium Dinoroseobacter shibae to changing light regimes[END_REF]. In most AAP bacteria isolated so far, photoheterotrophy generates additional metabolic energy enhancing AAP bacterial growth under light [START_REF] Koblížek | Isolation and characterization of Erythrobacter sp. strains from the upper ocean[END_REF][START_REF] Cooney | Influence of photoperiod on pigmentation and 570 metabolic efficiency of the marine aerobic anoxygenic photosynthetic bacterium Erythrobacter longus Strain NJ 3 Y[END_REF]. Considering the variety of sharp environmental gradients encountered by OTU 6 members in the transition zone between freshwater and marine 460 waters, it is likely that, when light is sufficient, photoheterotrophy may have different physiological roles in warm, organic matter-rich freshwater and in cold, saline oligotrophic waters. The nature of their physiological adaptation and metabolic versatility needs to be elucidated to understand the selective advantage that photoheterotrophy provides them.

To our knowledge, AAP strains have not previously been isolated from the Arctic 465 Ocean. In this study, the isolates provided a valuable set of data that allowed the taxonomic identification of two predominant pufM phylotypes. We successfully isolated pufMcontaining bacteria, but only from surface waters, consistent with the phototrophic character of AAP bacteria. Our arctic isolates were restricted to different species of the genera Sulfitobacter and Loktanella. Although these species were cultivated from temperate 470 to polar regions [START_REF] Labrenz | Staleya guttiformis gen. nov., sp. nov. and Sulfitobacter brevis sp. nov., alpha-3-Proteobacteria from hypersaline, heliothermal and meromictic antarctic Ekho Lake[END_REF][START_REF] Van Trappen | Loktanella salsilacus gen. nov., sp. nov., Loktanella fryxellensis sp. nov. and Loktanella vestfoldensis sp. nov., new members of the Rhodobacter group, isolated from microbial mats in Antarctic lakes[END_REF][START_REF] Salka | Abundance, depth distribution, and composition of aerobic bacteriochlorophyll a-producing bacteria in four basins of 690 the central Baltic Sea[END_REF], the strains isolated in this study were most similar to microorganisms residing in polar waters. Among them, the Sulfitobacter strains clustering into OTU 5 were absent outside the Beaufort Sea, suggesting that they potentially represent typical arctic members. Sulfitobacter and Loktanella species are often found in surface waters and numerous interactions with 475 phytoplankton have been reported [START_REF] Moran | Ecological genomics of marine Roseobacters[END_REF]. These metabolically versatile bacteria can satisfy a significant part of their carbon and sulfur demands by assimilating DMSP released during the decay of phytoplankton blooms [START_REF] González | Transformation of sulfur compounds by an abundant 605 lineage of marine bacteria in the α-subclass of the class Proteobacteria[END_REF][START_REF] Mou | Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations[END_REF]. The Beaufort Sea waters sampled during this study exhibited post-bloom characteristics, with low levels of Chl a in the surface layer. Since surface waters of the 480 Beaufort Sea are oligotrophic in summer, the capacity to derive energy from light and reduced sulfur compounds would give a physiological advantage for competing in this extreme environment. The influence of light and starvation on BChl a and biomass formation has been studied in AAP bacteria such as Sulfitobacter and Loktanella that contain low amounts of BChl a [START_REF] Biebl | Growth and bacteriochlorophyll a formation in taxonomically diverse aerobic anoxygenic phototrophic bacteria in chemostat culture: influence of light regimen 560 and starvation[END_REF]. Since this type of AAP bacteria seems to 485 use phototrophy genes only under simultaneous illumination and extreme shortage of organic nutrients, their distribution in arctic waters may be linked to their photoheterotrophic mode of metabolism.

AAP diversity was highest at the shelf and offshore DCM layers of the Beaufort Sea where Pacific Summer Water mixes with the BNL. Methylobacterium-and Sphingomonas-490 like bacteria, mostly absent in surface waters, were common at these depths along the Mackenzie plume. Although a few species of both genera have been isolated from seawater [START_REF] Vancanneyt | Sphingomonas alaskensis sp. nov., a dominant bacterium from a marine oligotrophic environment[END_REF][START_REF] Wang | Methylobacterium salsuginis sp. nov., isolated from seawater. 735[END_REF], these bacteria have a widespread distribution in diverse terrestrial habitats including soil, freshwater, and lake sediments [START_REF] White | The genus Sphingomonas: physiology and ecology[END_REF][START_REF] Green | Methylobacterium. The Prokaryotes: A Handbook on the Biology of Bacteria[END_REF]. Our data suggest that Methylobacterium-and Sphingomonas-like 495 bacteria share the same habitat, more likely associated with soluble humic material in river water [START_REF] Kirk | Light and Photosynthesis in Aquatic Ecosystems[END_REF]. In line with this hypothesis, these bacteria were found to actively participate in humic matter degradation [START_REF] Balkwill | Sphingomonas and related genera. The Prokaryotes: A Handbook on the Biology of Bacteria[END_REF][START_REF] Hutalle-Schmelzer | Enrichment and cultivation of pelagic bacteria from a humic lake using phenol and humic matter additions[END_REF]. Two groups of Bradyrhizobiaceae-related sequences were detected mostly in DCM samples of the Beaufort Sea shelf. One group was loosely associated to the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris that is widely distributed in nature including coastal marine sediments [START_REF] Oda | Biogeography of the purple nonsulfur bacterium Rhodopseudomonas palustris[END_REF]. The other grouped with strictly aerobic anoxygenic phototrophic Bradyrhizobium strains that form nitrogen-fixing nodules on stems and/or roots of many legumes [START_REF] Giraud | Nitrogen-fixing symbiosis between photosynthetic bacteria and 600 legumes[END_REF]. Like Methylobacterium and Sphingomonas clusters, these clades were mostly recovered in the 505 deep layers of the Mackenzie plume, suggesting that they were also entrained by the river sediment load. However, their distribution was similar to that of Citromicrobium-like clades that are typical marine bacteria [START_REF] Yurkov | Citromicrobium bathyomarinum, a novel aerobic bacterium isolated from deep-sea hydrothermal vent plume waters that contains 745 photosynthetic pigment-protein complexes[END_REF][START_REF] Jeanthon | Diversity of cultivated and metabolically active aerobic anoxygenic phototrophic bacteria along an 620 oligotrophic gradient in the Mediterranean Sea[END_REF]. Furthermore, we also retrieved them from North Pacific Ocean surface waters, supporting earlier reports of Bradyrhizobiaceae in the North Pacific Ocean gyre and the Bering Sea where terrestrial 510 inputs are unlikely [START_REF] Hu | Abundant presence of the γ-like Proteobacterial pufM gene in oxic seawater[END_REF]. It is therefore tempting to speculate that arctic Bradyrhizobiaceae-related pufM sequences are marine photosynthetic bacteria possibly transported in Pacific waters.

Conclusion 515

Our transect analysis in the coastal arctic ecosystem of the Mackenzie River and Beaufort Sea shelf revealed strong spatial gradients in environmental properties and in the abundance and composition of AAP bacterial communities. We showed that AAP bacteria represent a significant fraction of the total prokaryotic community in the arctic coastal environments impacted by riverine inputs. Our results highlight that OTU 6 members are an 520 important and widely distributed but largely unknown component of AAP bacterial communities. However, we do not know the impact of phototrophic light harvesting on energy requirements and carbon metabolism of these bacteria. Future studies are needed to explore their role in biogeochemical cycling in coastal ecosystems. 2.56 10 5 (8.7 10 4 ; 1.2 10 6 ) 2.85 10 5 (6.3 10 4 ; 1.0 10 6 ) 2.78 10 5 (1.4 10 5 ; 6.5 10 5 ) 1.99 10 5 (3.7 10 4 ; 9.7 10 5 ) AAP bacteria (cells.ml -1 ) 1.60 10 4 (1.2 10 4 ; 2.3 10 4 ) 1.02 10 3 (0 ; 8.6 10 3 ) 1.51 10 2 (0 ; 2.2 10 3 ) 2.33 10 2 (0 ; 3.6 10 4 ) AAP bacteria (%) 6.31 (1.96 ; 14.23) 0.23 (0 ; 1.53) 0.07 (0 ; 0.52) 0.16 (0 ; 9.71) a Ancillary data of temperature, salinity, pH, dissolved oxygen, colored dissolved organic matter, inorganic and organic nutrients, and chlorophyll a were provide by D. Doxaran, P. Raimbault, N. Garcia and J. Ras.

b The detection limit is 3 nM for nitrate and nitrite and 5 nM for ammonium.

F o r P e e r R e v i e w

  marine ecosystems are characterized by strong seasonality in incident light, ice cover, temperature, and river discharge which affect salinity and other biogeochemical

90

  Bacterioplankton biomass for DNA and total RNA extraction were collected onboard.Seawater samples were prefiltered through 47 mm diameter and 3 µm pore size polycarbonate filters (Cyclopore, Whatman, USA) before the final collection of bacterioplankton cells onto 0.22 μm-pore-size Sterivex cartridges (Millipore, USA) for DNA extraction or onto 27 mm diameter and 0.22 µm-pore-size Durapore filters (Millipore, USA) 95 for RNA extraction. Sterivex cartridges were filled with 1.6 ml of lysis buffer (0.75 M sucrose,

  collected at station 860 at the surface and the DCM, respectively.

  140 in Hi-Di™ formamide (Applied Biosystems, Carlsbad CA, USA) and terminal-restriction fragments (T-RFs) were separated in a 3130 xl Genetic Analyzer (Applied Biosystems). Data were analyzed using the PeakScanner™ Software (Applied Biosystems).

  215

  0.5°C. Only single peaks were observed in the dissociation curves for both the standards and samples, indicating specific amplification with each set of primers. Average amplification efficiencies were as follows: 16S rDNA = 99.2% and OTU 6-pufM= 97%.

  using cDNA from station 620 were cloned and sequenced as described above. Of 31 clones that were analyzed, all contained the expected pufM gene sequences.240Nucleotide sequence accession numbersUnique environmental pufM gene sequences obtained in this study are available in GenBank database under the accession numbers JN248465 to JN248539. The 16S rRNA and pufM gene sequences of the isolates are available in GenBank database under the accession 245 numbers JN248461 to JN248464 and JN378828 to JN378831, respectively.

  TTGE band patterns and their location, 13 samples were selected for further cloning and sequencing. Of 365 clones analyzed, 43 distinct OTUs were identified after grouping the sequences at 94% nucleic acid sequence similarity. The diversity in the riverinfluenced stations was high as indicated by the S Chao1 values (estimated numbers of expected OTUs) and diversity indices (Table

  305also contained two sequences obtained by single-cell sequencing[START_REF] Martinez-Garcia | High-throughput single-cell sequencing identifies photoheterotrophs and 660 chemoautotrophs in freshwater bacterioplankton[END_REF] and a mixture of arctic sequences[START_REF] Cottrell | Photoheterotrophic microbes in the Arctic Ocean in summer and 575 winter[END_REF], but it did not have any close relationship to known AAP bacteria. Two singleton OTUs belonged to betaproteobacterial phylogroup I defined by[START_REF] Yutin | Assessing diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of the Atlantic and 750 Pacific Oceans using the Global Ocean Sampling expedition metagenomes[END_REF] that formed a sister lineage fosmid clones from the Delaware river

  380

  as degradation of humic-rich DOM from riverine and terrestrial 405 run-off.

  which is surprising because BChl a synthesis is partly or totally inhibited by light in AAP bacteria currently in culture

Fig. 1 .

 1 Fig. 1. Locations of stations sampled during legs 1b and 2b of MALINA cruise. Leg 1b stations

Fig. 2 .

 2 Fig. 2. Spatial distribution of total prokaryotes (cells ml -1 ) and AAP bacteria (%) in surface

Fig. 3 .Fig. 4 .

 34 Fig. 3. Phylogenetic tree showing the relationships between pufM gene sequences from

Fig. 5 .

 5 Fig. 5. Phylogenetic analysis based on the 16S rRNA sequences indicating the position of

Fig. 6 .

 6 Fig. 6. Surface relative abundance of OTU 6 members (qPCR) and AAP bacteria (IR

Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .Fig. 6 .

 123456 Fig. 1. Locations of stations sampled during legs 1b and 2b of MALINA cruise. Leg 1b stations (black squares) were sampled only at the surface; leg 2b stations were sampled at several depths (black dots) or only at the surface (black triangles). Circles indicate stations where pufM clone libraries were constructed. Grey shades correspond to bottom depths. 178x209mm (150 x 150 DPI)

  Fig. S1. Temperature-salinity diagram of the water masses observed in the Beaufort Sea during the MALINA cruise. Black dots correspond to water samples; numbers indicate the station and depth sampled. The inset gives an enlarged view of the diagram below. 178x195mm (150 x 150 DPI)

Table 1 .

 1 Main features of T-RFLP groups determined among the 92 AAP isolates and closest relatives based on the 16S rRNA and pufM gene sequences.

	T-RF size (bp) FAM a HEX b	Nb of strains	Representative strains	16S rRNA gene closest relative Accession % Name number similarity	pufM gene closest relative Name Accession number similarity %
	300	121; 123	47	RCC 2406 (RCC 2405; RCC 2400)	Sulfitobacter sp. ZS5-11	FJ196046	99	Unc.bacterium MALINA OTU5	JN248469	100
	298	123; 125	2	RCC 2401	Sulfitobacter sp. ZS2020	FJ889550	99.6	Unc.bacterium MALINA OTU4	JN248468	92
	299	123; 125	41	RCC 2403 (RCC 2402; RCC 2404)	Loktanella sp. WM1	EF421433	100	Unc.bacterium MALINA OTU29	JN248513	99
					Loktanella			Unc.bacterium
	298	123; 124;125	2	RCC 2642	salsilacus	AJ582229	99.5	MALINA OTU29	JN248513	99
					LMG 22002
	a FAM, carboxyfluorescein; a HEX, hexachlorofluorescein; T-RF, terminal restriction fragment;

Table S1 .

 S1 Summary of environmental parameters of water masses delineated during the MALINA cruise. For each, the median and range are reported.

	Parameters a	River plume surface	Water samples categories Shelf surface Offshore surface	Other depths
	n =	5	29	17	60
	Max depth sampled (m)	surface	50	50	151
	Temperature (°C)	8.8 (7.99 ; 10.32)	1.03 (-0.87 ; 5.02)	-0.58 (-1.04 ; 1.13)	-1.21 (-1.43 ; -0.93)
	Salinity (psu)	14.77 (0.15 ; 19.17)	30.17 (21.98 ; 32.2)	28.02 (22.09 ; 30.2)	31.92 (30.3 ; 33.65)
	O 2 (mg.l -1 )	8.07 (7.34 ; 10.48)	8.74 (6.94 ; 10.21)	8.89 (7.98 ; 9.22)	7.68 (6.18 ; 9.35)
	pH	8.04 (8.04 ; 8.32)	8.1 (7.91 ; 8.34)	8.11 (8.03 ; 8.15)	8.02 (7.84 ; 8.17)
	SiOH 4 (µM)	30.27 (3.52 ; 64.53)	3.92 (3.12 ; 27.9)	3.27 (2.89 ; 7.82)	17.95 (3.38 ; 37.19)
	cDOM (V.m -1 )	0.12 (0.09 ; 0.15)	0.09 (0.08 ; 0.14)	0.08 (0.07 ; 0.13)	0.1 (0.08 ; 0.12)
	NO 3 (µM) b	0.7 (0 ; 3.62)	0.02 (0 ; 6.49)	0.01 (0 ; 0.02)	6.15 (0 ; 15.65)
	NO 2 (µM) b	0.01 (0 ; 0.07)	0.01 (0 ; 0.12)	0 (0 ; 0.03)	0.05 (0 ; 0.15)
	NH 4 (µM) b	0.2 (0.07 ; 0.64)	0.02 (0 ; 0.43)	0 (0 ; 0.08)	0.02 (0 ; 0.43)
	PO 4 (µM)	0.06 (0 ; 0.21)	0.6 (0.31 ; 1.36)	0.58 (0.33 ; 0.72)	1.24 (0.62 ; 2.97)
	TOC (µM)	313.7 (199 ; 462.5)	79.01 (66.15 ; 135)	65.45 (60.62 ; 112)	64.09 (52.55 ; 101)
	POC (µM)	16.15 (12.49 ; 52)	7.91 (0 ; 41.16)	2.18 (0.92 ; 5.61)	3.15 (0.39 ; 43.2)
	DOC (µM)	295.8 (183 ; 454.6)	68.65 (58.34 ; 107)	62.34 (57.8 ; 108.4)	60 (43.19 ; 83.2)
	TON (µM)	17.36 (12.65 ; 29.7)	6.75 (5.11 ; 13.87)	5.85 (4.52 ; 16.27)	7.28 (3.21 ; 15.02)
	PON (µM)	3.42 (1.71 ; 15.84)	0.83 (0 ; 3.14)	0.28 (0.17 ; 0.77)	0.36 (0.14 ; 2.65)
	DON (µM)	13.69 (10.94 ; 26.7)	5.91 (4.43 ; 11.22)	5.57 (4.34 ; 15.83)	6.76 (3.02 ; 14.68)
	TOP (µM)	0.88 (0.23 ; 2.82)	0.24 (0.11 ; 0.66)	0.21 (0.12 ; 0.59)	0.2 (0.02 ; 0.78)
	POP (µM)	0.56 (0.13 ; 4.96)	0.06 (0 ; 0.24)	0.02 (0.01 ; 0.06)	0.02 (0.01 ; 0.24)
	DOP (µM)	0.22 (0.1 ; 1.99)	0.17 (0.09 ; 0.53)	0.19 (0.11 ; 0.56)	0.17 (0 ; 0.76)
	Total Chl a (µg.l -1 )	2.3 (0.18 ; 4.53)	0.16 (0.02 ; 3.23)	0.05 (0.01 ; 0.1)	0.17 (0.01 ; 6.43)
	Prokaryotes (cells.ml -1 )

Table S2 .

 S2 Summary of primers used in this study

	Page 43 of 47			FEMS Microbiology Ecology
	Experiment t-RFLP PCR PCR / TTGE qPCR qPCR	Gene 16S rDNA 16S rDNA pufM 16S rDNA pufM	Group Bacteria Bacteria AAPB Bacteria AAPB-OTU6	Primer name Primer sequence (5'-3') 27F AGAGTTTGATCMTGGCTCAG 1492R CGGTTACCTTGTTACGACTT 8F AGAGTTTGATCCTGGCTCAG 1492R CGGTTACCTTGTTACGACTT PufMF TACGGSAACCTGTWCTAC Puf-WAW AYNGCRAACCACCANGCCCA BACT1369F CGGTGAATACGTTCYCGG PROK1492R GGWTACCTTGTTACGACTT OTU6-f23 CCACGCACTATCCATTGT OTU6-r34 GGGCGGCGCGCTCGGAAG F o r P e e r R e v i e w Annealing temp (°C) length (bp) Amplicon 55 1465 55 1465 58 245 56 142 60 146	Reference (Lane et al., 1991) (Lane et al., 1991) (Lane et al., 1991) (Lane et al., 1991) (Beja et al., 2002) (Yutin et al., 2005) (Suzuki et al., 2000) (Suzuki et al., 2000) This study This study

Table S3 .

 S3 Correlation of total and AAP bacterial abundances (p < 0.05) over the entire data set and along Mackenzie-influenced transects.

				FEMS Microbiology Ecology	Page 44 of 47
	Variables	All samples (n=110) Prokaryotic cells AAP proportions	All depths of transects 600 & 300 (n=40) Prokaryotic cells AAP proportions
	Temperature	0.24	0.46**	ns a	0.59**
	Salinity O 2 cDOM PAR TIC HCO 3 NO 3 NO 2 NH 4 PO 4 SiOH 4 POC PON POP DOC DON DOP TOC	-0.23 ns ns 0.2 -0.24 -0.24 -0.20 ns ns -0.23 ns 0.24 ns ns ns ns ns ns	-0.45** -0.20 0.28* ns ns ns ns ns 0.48** ns 0.35** 0.42** 0.74** 0.77** 0.65** 0.59** 0.62** 0.66** F o r P e e r R e v i e w ns -0.55** ns ns ns 0.32 ns ns -0.32 ns ns ns -0.31 ns ns ns ns 0.47* -0.35 ns -0.34 0.47* ns 0.57** ns 0.80** ns 0.82** ns 0.74** ns 0.74** ns 0.68** ns 0.73**
	TON	0.22	0.63**	ns	0.74**
	TOP	ns	0.70**	ns	0.76**
	Total Chla	ns	0.43**	ns	0.78**
	a ns, not significant ; *, p < 0.005 ; **, p < 0.001

Table S4 .

 S4 Clone library comparison of seawater samples collected during the MALINA cruise in summer 2009. LCI and UCI are the lower and upper bound of 95% confidence interval of the Chao1 and Shannon indexes, respectively.

	Location	Station-depth	Nb. of clones Nb. of OTUs a	Coverage (%)	S Chao1 (LCI-UCI) b	H Shannon (LCI-UCI)
		PAC2 0m	18	7	83	8 (7-18)	1.67 (1.29-2.05)
	leg1b	BER 0m	18	4	89	5 (4-17)	0.98 (0.6-1.35)
		ARC1 0m	32	2	100	2 (2-2)	0.48 (0.28-0.69)
		170 3m	26	4	92	4.5 (4-12)	0.59 (0.19-0.98)
		360 3m	28	7	89	8 (7-18)	1.26 (0.82-1.69)
	leg2b	360 60m	27	8	96	8	1.94 (1.71-2.17)
	Beaufort	620 3m	25	3	100	3 (3-3)	1.05 (0.91-1.2)
	Sea	620 80m	35	10	83	25 (13-78)	1.84 (1.53-2.14)
		680 3m	27	3	100	3 (3-3)	0.81 (0.56-1.07)
		680 45m	35	11	86	13.5 (11-28)	1.85 (1.48-2.23)
	leg2b	694 0m	23	6	78	16 (8-58)	0.87 (0.37-1.38)
	Mackenzie	697 0m	41	20	63	72.5 (34-213)	2.59 (2.27-2.91)
	plume	398 0m	30	5	90	6.5 (5-20)	0.67 (0.27-1.08)

a Operational taxonomic units (OTUs) were defined with Mothur assuming a 94% sequence similarity level. b

Doxaran D, Ehn J, Bélanger S, Matsuoka A, Hooker S & Babin M (2012) Optical characterisation of

Acknowledgements

This study was conducted as part of the Malina Scientific Program funded by ANR (Agence Nationale de la Recherche), INSU-CNRS (Institut National des Sciences de l'Univers -Centre National de la Recherche Scientifique), CNES (Centre National d'Etudes Spatiales) and ESA 530 (European Space Agency). We thank all participants to the MALINA cruises for their help, especially M. Babin who coordinated the project, K. Lévesque for the logistics, and all CCGS Amundsen crew members. We warmly thank D. Doxaran, P. Raimbault, N. Garcia, and J. Ras for providing ancillary data. We are grateful to M. Koblížek for help with detection of AAP bacterial colonies using the infrared system available in his laboratory, M. Perennou and G. 535 Tanguy (Biogenouest Sequencing Platform-FR2424-Station Biologique) for help with sequencing. This work was supported by the European Union programs MicroB3 (UEcontract-287589) and MaCuMBA (FP7-KBBE-2012-6-311975). D. Boeuf was supported by a grant from the French Ministry of Higher Education and Research. D. L. Kirchman and M. T. Cottrell were supported by NSF grant OCE 1030306.

540

Supporting information

The supporting information is composed of 6 files and contains 4 tables (Tables S1 to S4) and 2 figures (Fig. S1 andS2).

Below are the legends of the supplementary tables and figures: Table S1. Summary of environmental parameters of water masses delineated during the MALINA cruise. For each, the median and range are reported.

Table S2. Summary of primers used in this study.

Table S3. Correlation of total and AAP bacterial abundances (p < 0.05) over the entire data set and along Mackenzie-influenced transects.