Gene Sequencing Revealed via High-Throughput 16S rRNA

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Phylogenetic Differences in Attached and Free-Living Bacterial Communities in a Temperate Coastal Lagoon during Summer, Revealed via High-Throughput 16S rRNA Gene Sequencing Vani Mohit, Philippe Archambault, Nicolas Toupoint and Connie Lovejoy Appl. Environ. Microbiol. 2014, 80(7):2071. DOI: 10.1128/AEM.02916-13. Published Ahead of Print 24 January 2014.

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Phylogenetic Differences in Attached and Free-Living Bacterial Communities in a Temperate Coastal Lagoon during Summer, Revealed via High-Throughput 16S rRNA Gene Sequencing Vani Mohit,a Philippe Archambault,b Nicolas Toupoint,b Connie Lovejoya Département de Biologie, Québec-Océan and Institut de Biologie Intégrative et des Systèmes, Université Laval, Québec, Québec, Canadaa; Institut des Sciences de la Mer (ISMER), Université du Québec à Rimouski (UQAR), Rimouski, Québec, Canadab

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n marine systems, bacteria are the main agents of carbon cycling and nutrient regeneration, converting dissolved organic matter to biomass, which fuels microbial food webs and transfers energy and carbon to higher trophic levels (1). Bacterioplankton are frequently categorized as either free living or attached to particles (2, 3). Attached bacteria may have very high local concentrations compared to free-living bacteria (4) and also provide nutrition for macroscopic filter feeders (5). However, free-living bacteria are often more abundant than particle-attached bacteria in diverse marine (6) as well as freshwater ecosystems (7). Free-living and attached communities can differ both morphologically and physiologically, for example, attached bacteria are often larger (8) and are reported to have lower growth efficiency than free-living bacteria, with comparatively less bacterial biomass produced per quantity of organic substrate taken up (9). Some studies report higher per-cell metabolic activity for particle-attached communities compared to free-living communities (10, 11), while other studies report the opposite (12, 13). Interestingly, Ghiglione et al. (6) reported diel changes in bacterial activity, with the free-living fraction being more active during the day and the attached fraction more active at night, consistent with different functional capacities in the two communities, which may be reflected in taxonomy. Such observations suggest that the two communities are favored under different conditions, and understanding the dynamics and diversity of bacterial communities is an important step in characterizing an ecosystem as well as developing indicators to study ecosystem health and function. There have been many studies on freshwater, estuarine, open ocean, and coastal ocean free-living and attached bacteria (2, 8, 14). Recently, high-throughput sequencing has been used to test

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whether attached and free-living communities are taxonomically distinct, and results have tended to indicate that along estuarine salinity gradients, the two communities differ when salinities are lower but are similar at higher oceanic salinities (15, 16). Highthroughput 16S rRNA gene surveys have also tended to support the notion that bacteria are strongly influenced by estuarine salinity gradients (17, 18), and the question arises as to whether there are true marine bacteria that form attached communities or whether they are fundamentally pelagic bacteria that are temporarily associated with particles; if this were the case, all or the majority of attached bacteria would be represented in the pelagic community in systems where such salinity gradients did not exist (19). Alternatively, attached communities that formed in coastal versus open oceans could be fundamentally different. One approach to test these scenarios is to investigate enclosed coastal marine systems, such as coastal lagoons that are not influenced or are little influenced by freshwater but maintain oceanic salinities. The microbial ecology and biodiversity of coastal lagoons are important for recreational and ecosystem services but are often se-

Received 28 August 2013 Accepted 10 January 2014 Published ahead of print 24 January 2014 Editor: K. E. Wommack Address correspondence to Connie Lovejoy, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.02916-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.02916-13

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Most of what is known about coastal free-living and attached bacterial diversity is based on open coasts, with high particulate and nutrient riverine supply, terrestrial runoffs, and anthropogenic activities. The Magdalen Islands in the Gulf of St. Lawrence (Canada) are dominated by shallow lagoons with small, relatively pristine catchments and no freshwater input apart from rain. Such conditions provided an opportunity to investigate coastal free-living and attached marine bacterial diversity in the absence of confounding effects of steep freshwater gradients. We found significant differences between the two communities and marked temporal patterns in both. Taxonomic richness and diversity were greater in the attached than in the free-living community, increasing over summer, especially within the least abundant bacterial phyla. The highest number of reads fell within the SAR 11 clade (Pelagibacter, Alphaproteobacteria), which dominated free-living communities. The attached communities had deeper phylum-level diversity than the free-living fraction. Distance-based redundancy analysis indicated that the particulate organic matter (POM) concentration was the main variable separating early and late summer samples with salinity and temperature changes also significantly correlated to bacterial community structure. Our approach using high-throughput sequencing detected differences in free-living versus attached bacteria in the absence of riverine input, in keeping with the concept that marine attached communities are distinct from cooccurring free-living taxa. This diversity likely reflects the diverse microhabitats of available particles, implying that the total bacterial diversity in coastal systems is linked to particle supply and variability, with implications for understanding microbial biodiversity in marine systems.

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verely impacted by anthropogenic stressors (20), and deriving the natural state of bacterial communities in such coastal systems is problematic. Although there are reports on the community composition of attached and free-living bacteria in coastal oceans (21– 23), coastal lagoons, which are defined as “shallow water bodies separated from the ocean by a barrier, connected at least intermittently to the ocean by one or more restricted inlets. . .” (24), are little investigated (25). The few such studies were in lagoons severely impacted by anthropogenic activities and freshwater inputs from rivers or streams (25). To address this lack of data, we investigated the attached and free-living communities in a lagoon in the Magdalen Island Archipelago, which is in the southern Gulf of St. Lawrence (Canada). The Magdalen Islands are a narrow archipelago linked by shallow lagoons with limited interchange with the Gulf of St. Lawrence (26). The lagoons are relatively pristine, with no industrial development in the surrounding catchment and little agricultural activity. The Havre-aux-Maisons (HAM) lagoon, with only minor mussel farming activity (⬍5% of the lagoon surface area) (27), restricted inflow from the sea, and little freshwater influence except from rainfall (28), is an ideal site to investigate differences between attached and free-living communities in a shallow, exclusively marine environment. We used a high-throughput amplicon approach targeting the V6-V8 region of the 16S rRNA gene (23) to facilitate comparisons to other marine studies (23, 29, 30). Our goals were to (i) identify the attached and free-living communities in the water column, including the less common taxa; (ii) investigate temporal community changes during summer, given that coastal marine bacterial communities vary over time (6); and (iii) to identify likely drivers of any temporal pattern in the distribution of these two types of bacterial communities. For this, we examined the influence of meteorological events, as well as temperature, salinity, nutrients, particulates, and other environmental variables, on the communities by way of constrained ordination analysis. We hypothesized that bacterial communities would be influenced relative to the magnitude of environmental change over time. In the Magdalen islands, persistent winds mean that the water column is well mixed, providing no physical structure and a uniform environment for free-living bacteria. In contrast, particulate material ar-

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rives from many diverse sources, and particles could represent many distinct habitats for attached bacteria. We would predict increased diversity with increasing particle concentrations if the two communities were not the same. If the two communities were the same, they would be predicted to change in concert over the summer. MATERIALS AND METHODS Site description. Havre-aux-Maisons lagoon (Fig. 1) is a shallow lagoon with a maximum depth of 6 m, surface area of 30 km2, and catchment area of ca. 63 km2 (Centre d‘Expertise Hydrique du Québec). HAM is classified as a restricted coastal lagoon (26) with only two connections for exchange. The first is a restricted tidal inlet, where water enters from the Gulf of St. Lawrence along the southeast corner via a narrow channel. The second, in the northeast corner, acts mostly as an outlet and is a narrow channel connected to Grande-Entrée lagoon (GEL). Because of the lower tidal amplitude in HAM (26), it has a higher residual water level than the adjacent GEL, and water mostly flows out from HAM into the GEL. Frequent high winds, ⬎15 m s⫺1 (28), are a defining characteristic of the Magdalen Islands, and water column mixing is mainly due to the winddriven currents, as tides alone are not sufficient for complete water renewal in the lagoon (31). Water residence time decreases from ca. 45 days, when only tidal action is considered, to 25 days, when prevailing winds essentially push water out toward the GEL (31). Nutrient concentrations and phytoplankton biomass are characteristically low throughout the summer (28, 32). Sample collection. Because of the low spatial heterogeneity within the well-mixed lagoon (B. Pequin and C. Lovejoy, personal communication) and logistic restraints, samples along with ancillary data were collected from a single sampling site (47°25= 730⬙N, 61°48=832⬙W). Sampling was approximately every 2 weeks during the summer, from 16 June to 8 September 2009. The 5-m-deep site was also part of a concurrent study examining growth of juvenile mussels with 24 pearl nets, each containing 50 mixed 0⫹ and 1⫹ age class mussels suspended 2 m from the superficial sediment. The water was collected via a submersible electric pump within the well-mixed water column. To avoid inadvertent contamination by mussel fecal pellets from the pearl nets, the pump was kept above the nets at 2.5 m. Oxygen saturation was measured every meter down the water column using a YSI 550A oxygen profiler, and salinity and temperature were taken with a YSI 30/25 FT profiler (both from Yellow Springs Instruments Inc., OH, USA). Rainfall and wind data for the 4 days prior to and on the day of sampling were from the airport weather station located ca. 4

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FIG 1 Study site in Havre-aux-Maisons (HAM) lagoon, Magdalen Islands, Gulf of St. Lawrence. The Grande-Entrée lagoon (GEL) is also indicated on the

Attached and Free-Living Bacteria in a Coastal Lagoon

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a 1/4 plate of the 454 GS FLX system (Roche Applied Science, Indianapolis, IN) at the Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval Plate-forme d’Analyses Génomiques, Québec, Canada. The target amplification product was 400 to 500 nucleotides (nt) long. Data analysis. The resulting reads were analyzed using MOTHUR (44) and BioEdit v7.0.9 (Tom Hall, Ibis Biosciences, Carlsbad, CA). MOTHUR was run by the Calcul Canada’s CLUMEQ high-performance computing facility, Université Laval. Preliminary denoising was performed in MOTHUR. Reads less than 300 nt and those containing homopolymers of ⬎7 indels were removed at this point. A preliminary classification was done on the remaining reads using a modified GreenGenes97 database (greengenes.lbl.gov/); briefly, we removed sequences with little taxonomic information (those unclassified at the phylum level) and added genus information based on a consensus between the original GreenGenes classification and the results of the Classifier tool from the RDP database (45) using the 95% bootstrap cutoff value (46). Reads classed as chloroplasts were removed at this step. Reads were then compared against the bacterial SILVA database (47) using the ksize ⫽ 9 parameter. Chimeras were identified using the UCHIME (48) command in MOTHUR and removed. The remaining read alignment was verified using BioEdit (49), and badly aligned reads (i.e., reads causing large gaps or many gaps, suggesting sequencing errors) were deleted. The remaining reads were grouped into operational taxonomic units (OTUs) based on 97% similarity using the furthest neighbor clustering method in MOTHUR. Singletons, defined as OTUs containing only 1 read with a single occurrence in the combined data from all samples, were removed. Finally, to provide statistical robustness when comparing diversity measures among samples, the reads were randomly resampled so that all samples had the same number of reads (50). The final 3,894 reads per sample were then classified based on the modified GreenGenes97 database. Chao1 diversity and rarefaction estimates were carried out in MOTHUR. Beta diversity measures were performed using QIIME v1.7.0 (51) in which phylogenetic information was integrated to compare microbial communities. FastTree (http://www.microbesonline.org/fasttree/) was used for the construction of test phylogenetic trees, which were further used in QIIME. The unweighted-pair group method using arithmetic means (UPGMA) clustering was performed on both weighted and unweighted UniFrac distance matrices to build a UPGMA tree. Unweighted UniFrac analysis gives greater importance to the rare taxa than weighted analysis. To determine the robustness of the UPGMA clustering, jackknife beta diversity and clustering analyses were carried out using 1,000 permutations by resampling 2,920 reads per sample, which represented 75% of the total number of sequences per sample (52). Linear regression analysis was used to test for significant trends in the diversity indices over the summer, and a residual plot was constructed to verify normality and homoscedasticity (53). PRIMER software (v6) was used for a similarity percentage (SIMPER) (54) test to determine which taxa contributed the most to the average Bray-Curtis dissimilarity between the attached and free-living communities. SIMPER calculates the overall percent contribution that each taxon makes to the average dissimilarity between two groups and lists the taxa in decreasing order with respect to their importance in discriminating two sets of samples (54). Both abundance and presence/absence data were used for the SIMPER analysis. To further assess the beta diversity between attached and free-living bacteria, a phylogenetic accumulation curve was constructed for which the number of OTUs was plotted against sequence similarity thresholds (80% to 100%) used to define the OTUs. The range of similarity cutoffs within the V6 to V8 regions, 80%, 90%, 97%, and 99%, nominally estimates phylum, class, genus, and species within Bacteria (55, 56). The phylogenetic diversity accumulation analysis is a way of examining the degree of phylogenetic relatedness among taxa within each sample or community (30). One-way analysis of similarity (ANOSIM) (PRIMER v6) was performed on square root-transformed data to test the null hypothesis that there was no difference between the phylogenetic diversity

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km from the study site and were available from Environment Canada (http://www.climate.weatheroffice.gc.ca). Water level measurements above the chart datum (water level) were from Fisheries and Oceans Canada (http://www.meds-sdmm.dfo-mpo.gc.ca). Day length data were estimated using the National Research Council Canada sunrise/sunset calculator (http://www.nrc-cnrc.gc.ca/eng/services/sunrise/index.html). Total chlorophyll (Chl a) samples were collected by filtering 500 ml of water onto GF/F filters (0.7-␮m pore size; Whatman, USA). For the small fraction, water was first prefiltered through a 3.0-␮m-pore-size polycarbonate (PC) filter (Millipore, USA), and the filtrate was collected on GF/F filters. Chl a concentrations were determined by spectrofluorimetry (Cary Eclipse, USA) before and after acidification (33). Nutrients were analyzed with a Seal Autoanalyzer 3 (Seal Analytical, Germany) specifically for nitrite plus nitrate (referred to as nitrate) (34), soluble reactive phosphate (SRP) (35), and silicate (36). Samples for flow cytometry (FCM) were first fixed in the dark with gluteraldehyde (final concentration, 1% [vol/vol]) and stored at ⫺80°C until analysis. High-nucleic-acid (HNA) and lownucleic-acid (LNA) Bacteria and Archaea (referred to as bacteria) were detected following nucleic acid staining with SYBR green (Invitrogen, USA). Chlorophyll autofluorescence was used to detect picophytoeukaryotes (0.2 to 2 ␮m) and nanophytoeukaryotes (2 to 20 ␮m), and phycoerythrin was used to detect pico- and nanocyanobacteria by FCM as in Belzile et al. (37). Particulate organic matter (POM) was determined by collecting material from 2 liters of water filtered through prewashed and preweighed GF/F filters, which were dried at 45°C for 48 h and combusted at 450°C for 4 h (38). Duplicate samples for Chl a, nutrients, and FCM and triplicate samples for POM analysis were run. Environmental microbial DNA was collected by filtering water through a 50-␮m nylon mesh to remove any fecal pellets and zooplankton with associated microbiomes (C. Lovejoy, unpublished data). Material then was sequentially filtered onto a 3-␮m-pore-size PC filter and a 0.2␮m-pore-size Sterivex unit (Millipore, USA) with a peristaltic pumping system at a flow rate of ca. 0.75 to 1 liter h⫺1. When the filtration rate dropped below this level, the volume filtered (1.5 to 2 liter) was noted and the filtrations were stopped, since generally this indicates that the first filter (3 ␮m) is becoming obstructed. Throughout this study, we consider the 3- to 50-␮m fraction as attached and the ⬍3-␮m fraction as free living (2, 15, 23, 25). The filters were stored at ⫺80°C in a buffer solution (40 mmol liter⫺1 EDTA, 50 mmol liter⫺1 Tris, 0.75 mol liter⫺1 sucrose, pH 8.3) until processing. DNA extraction and pyrosequencing. Samples were treated with lysozyme and proteinase K (39). DNA from the 3.0-␮m filters and 0.2-␮m Sterivex units was then extracted using a saline extraction protocol (40) with added lysozyme (1 mg/ml, final concentration), proteinase K (0.2 mg/ml), and sodium dodecyl sulfate (0.01%). Following extraction, DNA was eluted in 1⫻ TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and stored at ⫺80°C. The V6 to V8 region of the 16S rRNA gene (41–43) was amplified. PCR amplification was performed with the forward primer B969F (5=-ACGCGHNRAACCTTACC-3=), which included Roche’s multiplex identifiers (MIDs), and the reverse primer BA1406R (5=-ACGGGC RGTGWGTRCAA-3=) (42). PCRs were run in triplicate in a 50-␮l reaction mixture that consisted of 3 different concentrations of DNA template per sample, varying from ca. 2.3 ⫻ 10⫺3 to 2.2 ng ␮l⫺1, in 1⫻ Phusion HF buffer (New England BioLabs, Inc.), 200 ␮M each deoxynucleoside triphosphate (dNTP), 0.5 ␮M each forward and reverse primer, and 0.02 U/␮l Phusion DNA polymerase. Amplification of the 16S rRNA gene was performed on a Bio-Rad C1000 thermal cycler with the following amplification program: initial denaturation at 98°C for 30 s, 30 cycles of 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and a final extension of 4.30 min at 72°C. PCRs were run and purified as in Comeau et al. (42). The resulting amplicon length was verified by electrophoresis on 1% agarose gels, and the absence of primer dimers (⬍100-bp products) was verified. Purified products were quantified spectrophotometrically on a Nanodrop ND 1000 (Nanodrop). Equal concentrations of PCR products from each of the MID-tagged amplicons were pooled and run on

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TABLE 1 Environmental dataa Date

Temp (°C)

O2 (%)

Sal

SRP (␮M)

Nitrate (␮M)

Silicate (␮M)

WLb (m)

DL (h)

16 June 02 July 16 July 28 July 11 Aug 25 Aug 03 Sept 08 Sept

13.3 15.4 18.3 19.1 19.7 21.0 16.2 16.6

101.0 103.3 102.2 95.0 88.2 89.2 99.6 93.3

30.4 30.6 30.7 30.9 30.9 30.8 30.7 30.6

0.16 0.03 0.20 0.27 0.45 0.11 0.03 0.01

0.98 0.85 0.92 0.85 0.94 0.93 1.02 0.95

0.42 0.82 1.21 0.65 1.32 1.34 0.71 1.07

0.69 0.30 0.76 0.75 1.13 0.88 0.96 1.17

17.29 17.20 16.81 16.31 15.59 14.81 14.29 14.00

a Data were taken at the 2.5-m sampling depth and include water level (WL) and day length (DL). Nitrate indicates nitrate plus nitrite. Sal, salinity; SRP, soluble reactive phosphorus. b Average water level above chart datum measured between 9 and 11 a.m.

TABLE 2 Biological data Bacteria (⫻103 cells ml⫺1)

Cyanobacteria Phytoplankton Chlc (⫻103 cells (⫻103 cells a (␮g liter⫺1) ml⫺1) ml⫺1)

Date

Total

HNAa

Pico

Nano

Pico

Nano

Total

Smallb

16 June 02 July 16 July 28 July 11 August 25 August 03 September 08 September

1,440 2,803 2,303 2,491 3,316 5,265 5,181 4,422

573 (40) 1,208 (43) 1,147 (50) 1,007 (40) 1,233 (37) 3,012 (57) 3,429 (66) 2,373 (54)

0.3 2.1 4.6 12.0 12.7 26.8 56.1 14

0.0 0.1 0.0 0.1 0.2 0.1 0.3 0.1

6.6 43.4 77.8 35.2 7.3 62.7 73.1 96.4

3.2 8.2 7.8 9.0 8.8 6.8 14.2 7.8

1.7 1.2 1.1 2.0 1.9 2.0 2.1 1.6

0.4 (24) 0.9 (73) 1.4 (127) 0.9 (47) 1.1 (56) 1.8 (91) 2.0 (95) 1.2 (70)

a b c

Data in parentheses are percentages of bacteria classified as HNA. Data in parentheses indicate the percentage of the total. Chl a in total (ⱖ0.7 ␮m) and small (0.7 to 3 ␮m) fractions.

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FIG 2 Chao1 richness of the attached and free-living bacterial communities and POM concentrations plotted on the secondary axis; the trend line (broken line) shows the significant increase through summer for POM (P ⫽ 0.02). The significant increasing trend is shown for the attached bacteria (P ⫽ 0.018) versus a nonsignificant trend (P ⫽ 0.239) for free-living bacteria. Abbreviations: June, JUN; July, JUL; August, AUG; September, SEP.

(in PAST) was also used for some of the pairwise comparisons. A 0.01 significance level was chosen over a 0.05 level to reduce type I error (61). All normality tests were performed with PAST via the Shapiro-Wilk test. SRA accession number. The raw reads have been deposited in the NCBI Sequence Read Archive (SRA; http://www.ncbi.nlm.nih.gov/sra) under the study accession number SRP027405.

RESULTS

Environmental setting. The water column of the HAM lagoon was well mixed on all days sampled, with no indication of stratification by either temperature or oxygen saturation values (not shown), and values from a depth of 2.5 m are given (Table 1). The lagoon was well oxygenated throughout summer 2009, and the warmest temperatures were in August. Nutrient concentrations were low, with nitrate at ⱕ1 ␮mol liter⫺1, SRP below detection (⬍0.0l ␮mol liter⫺1) to 0.45 ␮mol liter⫺1, and silicate from 0.42 to 1.34 ␮mol liter⫺1 (Table 1). Values for POM ranged from 0.7 to 1.33 mg liter⫺1 and were greatest in late summer (Fig. 2). Over the summer, total Chl a values ranged from 1.1 ␮g liter⫺1 on 16 July to 2.1 ␮g liter⫺1 on 3 September. Over the summer, the ⬍3-␮m Chl a fraction contributed from 24 to 95% of the total (Table 2), with the highest proportions on 25 August (91%) and 3 September (95%). For the 16 July sample, the level for the ⬍3-␮m Chl a fraction was slightly greater than the total Chl a concentration, which was likely because of sample variability at low chlorophyll concentrations. Phytoplankton cell concentrations from FCM showed that picophytoeukaryotes were always in excess of nanophytoeukaryotes, except on 11 August. Similarly, concentrations of picocyanobacteria were always greater than those for the nanocyanobacteria (Table 2). For the bacteria, the proportion of HNA cells relative to the total bacteria increased over summer, with the lowest proportion on 16 June (39.6%) and the highest proportion on 3 September (66.1%). Total bacterial concentrations also increased over the summer, with a maximum of 5.26 ⫻ 106 cells ml⫺1 on 25 August. Spearman rank correlation and Pearson cor-

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patterns of attached compared to free-living bacterial communities. If the R statistic (57) is closer to 1, then samples within the same category are more similar to each other than to those of other categories. Distance-based redundancy analysis (dbRDA) (58), which is a constrained ordination analysis, was used to determine the influence of environmental and meteorological parameters on sample distribution. Tested explanatory variables included temperature, oxygen, salinity, SRP, nitrate, silicate, water level, and day length (Table 1), as well as pico- and nanocyanobacteria, pico- and nanophytoplankton, total (⬎0.7 ␮m) and small-fraction Chl a (0.7 to 3 ␮m) (Table 2), and rain and wind data for the actual sampling day and the 4 preceding days (see Fig. S1 in the supplemental material). dbRDA uses sample scores from the principle coordinate analysis (PCoA) as the species data for the redundancy analysis (RDA). Bray-Curtis distance was calculated for the square root-transformed data, which were then used for the PCoA analysis. Forward selection and Monte Carlo estimation were used to select the environmental variables that best explained the sample distribution and to test the significance of those variables, respectively. Explanatory variables having significant conditional effects or partial contribution to the variation in OTU distribution (P ⬍ 0.15) were retained. At this significance level, i.e., 0.15, elimination of variables that have a biological contribution in the model is avoided (59). A final RDA was run that included only the best explanatory environmental variables. The constrained ordination analysis was performed using CANOCO v4.5 (60). The cumulative average values of rain and wind data for the actual sampling day and 4 preceding days (see Fig. S1 in the supplemental material) were calculated for inclusion in the dbRDA analysis. Spearman’s rho correlation with permutations was calculated using PAST software (http://folk.uio.no/ohammer/past/) to test for significant relationships (P ⬍ 0.01) between environmental variables and/or bacterial taxa. For normally distributed data, Pearson correlation

Attached and Free-Living Bacteria in a Coastal Lagoon

each sample is less than 1%. Note that out of a total of 24 phyla, 22 occur in the attached and 10 in the free-living communities. (B) Percent contribution of separate classes within the Proteobacteria.

relation were used to explore potential relationships among biological and environmental variables with significance detected for the following. Salinity and water temperature were strongly positively correlated (Spearman’s rho ⫽ 0.93; P ⬍ 0.01). Concentrations of nanocyanobacteria were correlated with POM (Spearman’s rho ⫽ 0.83; P ⬍ 0.01) and LNA bacteria (Spearman’s rho ⫽ 0.77; P ⬍ 0.01). Concentrations of picocyanobacteria were correlated with LNA, HNA, and POM (Spearman’s rho ⫽ 0.73 to 0.80; P ⬍ 0.01). POM was also significantly correlated with HNA cell abundance (Pearson’s r ⫽ 0.89; P ⬍ 0.01). Rainy days were infrequent during summer, with high rain events (up to 60 mm) only occurring twice: 4 days before 16 June and 4 days before 3 September (see Fig. S1 in the supplemental material). The total monthly rainfall recorded was 111.4 mm in June, 72.6 mm in July, 128.4 mm in August, and 51.2 mm in September. However, there was no rain on most days over the summer. Wind was mostly from the northeast (not shown), and the highest wind speeds were during a storm 4 days before the 3 September sampling date, reaching gusts of over 90 km h⫺1. High winds were also recorded 2 days before the 25 August sample (see Fig. S1). Diversity and richness indices. We identified 3,455 OTUs from the attached communities and 1,690 OTUs in the free-living communities (see Table S1 in the supplemental material). Rarefaction analysis (not shown) suggested that both the total attached and total free-living diversity approached that of an asymptote (at the 97% similarity level). Plotting Chao1 richness (Fig. 2) over the summer indicated that OTU richness increased

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significantly in the attached community (by linear regression, r2 ⫽ 0.71; P ⫽ 0.018) but not in the free-living community (Fig. 2). Attached bacterial richness was always greater than that of the free-living bacterial communities. Taxonomic identification and betadiversity. The attached bacterial community was dominated by Proteobacteria and Bacteroidetes (Fig. 3A), which together accounted for ⬎60% of the reads. The Proteobacteria in the attached fraction were mostly Alphaproteobacteria and Gammaproteobacteria (Fig. 3B); within the Gammaproteobacteria were Oceanospirillales, Chromatiales, and Enterobacteriales (not shown). The free-living proteobacterial community was dominated by Alphaproteobacteria (Fig. 3B). On 25 August, 50% of the free-living bacterial reads were Cyanobacteria (Fig. 3A), with little change in the proportions of different Proteobacteria classes (Fig. 3B). Other frequent phyla in all of the samples were Verrucomicrobia and Actinobacteria. Less common bacterial phyla, here defined as phyla having ⬍1% of the total reads of a sample, were much more prevalent in the attached bacterial fraction (Fig. 3A). The phylogenetic diversity accumulation curves (Fig. 4) highlight the significantly greater differences (by ANOSIM, R ⫽ 0.96; P ⫽ 0.001; 999 permutations) between attached and free-living communities compared to differences within the two categories. Over the wide range of similarity thresholds, the phylogenetic diversity in the attached fraction was always greater than that of the free-living bacterial fraction, as also suggested by the Chao1 results (Fig. 2). The wider distance within attached community curves compared to the free-living curves also indicate greater

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FIG 3 (A) Phylum-level percent contribution to the total number of reads. The category “Less common” includes the phyla where the percent contribution to

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differences between the individual attached compared to free-living communities. In addition, both unweighted (Fig. 5) and weighted UniFrac clustering (not shown) revealed a clear separation between the attached and free-living bacterial communities but also showed that early summer samples (June and July) differed from those of late summer (August and September). The unweighted UniFrac analysis is shown, providing information on the influence of less common taxa compared to a weighted UniFrac. Less common taxa were more prevalent in the attached bacterial community, strongly contributing to the difference between

FIG 5 UPGMA tree indicating the unweighted-UniFrac clustering of the attached and free-living communities based on UniFrac phylogenetic distance. Bootstrap support values according to the Jackknife analysis are at the nodes. The bubble chart shows the top 12 genera contributing to the dissimilarity between attached and free-living bacterial communities (SIMPER analysis); bubble size indicates the proportion of each taxon to the total in each sample. The class is in parentheses. Attached bacteria are shown as black circles; free-living bacteria are shown as gray circles.

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FIG 4 Phylogenetic diversity accumulation curve for the attached versus freeliving bacterial communities. The proportion of OTUs (log scale) was plotted against the sequence similarity threshold (%) used to define the OTUs for attached (ATT) or free-living (FREE) results.

the attached and free-living communities. The unweighted UniFrac showed the strongest temporal clustering. At the phylum level, the greatest contribution to the dissimilarity between the two fractions was by the less common phyla (SIMPER, unweighted analysis) that were more frequent in the attached community. Planctomycetes and Chlorobi had the largest contribution (each 8.38%) to the dissimilarity between attached and free-living bacterial communities (see Table S2 in the supplemental material). Twelve genera were identified (SIMPER, weighted analysis) as contributing the most to the dissimilarity between the attached and free-living bacterial communities based on relative abundance. These were plotted against community similarity determined by the UniFrac analysis based on presence/ absence data (Fig. 5). These 12 genera contributed 18.5% of the dissimilarity between the two categories, with differences in the proportion of Pelagibacter (Alphaproteobacteria) contributing the most to the dissimilarity (4.2%). Pelagibacter was the most frequently recovered genus in the samples, contributing up to a maximum of 40% of reads in the attached and 74% of reads in the free-living fractions. An approximate maximum likelihood tree was built using FastTree just with the Pelagibacter reads (not shown). Although there were a number of clades, none were distinguished as exclusively or predominantly attached versus freeliving Pelagibacter. In addition to Pelagibacter, Synechococcus, Ulvibacter, Winogradskyella, Sulfitobacter, and Haliscomenobacter were among these top 12 genera and occurred in all samples. Most of these, including Pelagibacter and Sulfitobacter, had more variable relative abundances in the attached compared to the freeliving fraction. Maximum Synechococcus reads were detected in the free-living fraction in the 25 August sample, and the largest proportion of Sulfitobacter occurred in the 16 July samples for both communities. This maximum in Synechococcus 16S rRNA

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gene reads did not correspond to picocyanobacterial abundance from the FCM data on 3 September. However, Synechococcus 16S rRNA gene reads were relatively high in both the free-living (12.2%) and attached fraction (13.5%). A separate MAFFT alignment and phylogeny of the Synechococcus reads failed to detect any consistent differences between the attached and free-living Synechococcus (not shown). A relatively large proportion of unclassified Gomphosphaeriaceae (62) was detected in the 3 September attached sample. These were correlated with attached Synechococcus (Spearman’s rho, r2 ⫽ 0.88; P ⬍ 0.01). Escherichia and unclassified Sinobacteraceae were only found in the attached community, with the highest proportions of Escherichia reads on 2 July and 11 August (Fig. 5). The Escherichia reads were subjected to a separate BLASTn search in the NCBI database (http://www.ncbi .nlm.nih.gov/), and the closest matches were to E. coli. The other taxa which contributed significantly to the dissimilarity between the attached and free-living bacterial communities were Verrucomicrobium, Congregibacter genera, and unidentified genus-level taxa in Flammeovirgaceae and Sinobacteraceae families (Fig. 5). All of these were more frequent in the attached fraction. dbRDA analysis of summer samples. Out of the 25 environmental variables tested, POM and salinity were the only two that were significantly correlated to the distribution of the attached bacterial OTUs (P ⬍ 0.05) Fig. 6A). POM and salinity had the highest partial contributions, 24% and 18%, respectively, to the variation in OTU abundance of the attached community. Both also contributed significantly to the separation between early and late summer samples (Fig. 6A). Since Pelagibacter, which is usually described as free-living, was dominant in both attached and freeliving bacterial communities, a dbRDA analysis was run with Pelagibacter removed, and the output was the same: POM and salinity were still significantly correlated with the variability in the OTU distribution (not shown). For the free-living fraction, POM and temperature (Fig. 6B) were significantly (P ⬍ 0.05) correlated with the variation in OTU distribution; they had the highest partial contributions of 28% and 19%, respectively, to the variation. As with the attached fraction, early summer (16 June, 02 July, and 16 July) and late summer samples (28 July, 11 August, 25 August, 03 September, and 08 September) were separated in the ordination plot, with the greatest correlation attributed to POM.

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DISCUSSION

Attached versus free living. Our pyrosequencing approach identified differences between the attached and free-living communities in the HAM lagoon and a temporal clustering in both communities with a separation of early and late summer samples. The phylogenetic differences and seasonal patterns were evident irrespective of the clustering method: the UniFrac weighted and unweighted analyses indicated clear differences in phylogenetic diversity between the two fractions. OTU abundance data and the phylogenetic accumulation analysis also showed differences between the two fractions regardless of the OTU similarity cutoffs used. The SIMPER analysis detected differences in relative abundance of different genera as well as differences relative to presence/ absence of bacterial phyla. The weighted SIMPER analysis pointed to the most common taxa contributing to the separation of the attached and free-living bacterial communities, and the unweighted SIMPER analysis showed the importance of the less common taxa. The most common reads in both fractions were classified as Pelagibacter from the SAR 11 clade, but these were proportionally fewer in the attached fraction, in which Gammaproteobacteria were also common. To our knowledge, this is the first report of the attached and free-living bacterial communities in a closed temperate coastal lagoon. By comparison, open coastal regions and estuaries are relatively well studied, for example, offshore from the Columbia River (2), the Mediterranean sea (23), and the southwestern Bay of Fundy (22). Differences between attached and free-living communities were reported in all of these studies. Tropical and subtropical lagoons are closed systems more often than temperate coastal areas, but as of yet there are no studies using high-throughput sequencing published to our knowledge. By way of comparison, clone library studies, which target the most abundant taxa, have shown that the degree of similarity between the attached and free-living communities in tropical and subtropical lagoons is linked to seasonal patterns, with greater differences during the dry season in tropical regions (63) and greater differences during the rainy season in the subtropical lagoons (25). The dominance of Pelagibacter in the water column was not surprising, since SAR 11 is the most abundant bacterial clade in the world’s ocean and is usually considered free living (64, 65). There are few cultured representatives and a single described spe-

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FIG 6 Distance-based RDA ordination plot representing the environmental variables that have a significant influence (arrows) on the distribution of summer attached (A) and free-living (B) bacterial communities (samples are black circles) based on OTU abundance.

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methodological differences between those studies and our study make comparisons difficult, since the earlier studies’ cutoff values for the different size fractions were ⱖ1 in reference 77 and ⱖ0.8 ␮m in reference 6 and may have included a higher proportion of free-living bacteria in the attached fraction. In addition, denaturing gradient gel electrophoresis (6) and capillary electrophoresissingle-strand conformation polymorphisms (77) do not capture rarer taxa (78). Overall, our use of high-throughput sequencing highlighted the contributions of those rare taxa to the attached bacterial communities. In HAM, the two fractions always clustered apart, with the attached bacterial community more diverse at all taxonomic levels, including phyla. Finally, we note that groups such as Flavobacteria (Bacteroidetes), especially Ulvibacter and Winogradskyella, were predominantly in the attached fraction, consistent with these genera being commonly attached to algal cells (79, 80). Overall, while likely pelagic bacteria were found in the attached fraction, the converse was not true, and within the attached fraction were groups that are exclusively associated with particles. Other dominant genera in the summer bacterial communities. Reads matching Sulfitobacter were detected in both attached and free-living bacterial communities. Rooney-Varga et al. (22) and Blažina et al. (73) also recorded Sulfitobacter in both communities. Despite the well-oxygenated water column, the HAM sediments are mostly anoxic, with detectable sulfide concentrations (81, 82, and V. Mohit, personal observation). Sulfitobacter may persist in low-oxygen microzones within particles and oxidize sulfites (83) released into the water column by mixing or bioturbation of the sediment (84). Sulfitobacter has also been reported associated with zooplankton fecal pellets (85), consistent with active particle degradation. Representatives of the Verrucomicrobia class were among the most common taxa in our attached community. Verrucomicrobia are difficult to cultivate and were considered rare prior to the application of metagenomics. This class is now frequently reported, and Verrucomicrobia seem to be widely distributed in the marine environment (86). Another unusual find in the summer attached bacteria was Escherichia coli, which may be alarming given that attached bacteria can be assimilated by mussels (5). However, the serotype or toxicity of the strain was not resolved here. Other attached bacterial taxa included the bacteriochlorophyll-containing gammaproteobacterium genus Congregibacter (87) and the cyanobacterium family Gomphosphaeriaceae. Gomphosphaeriaceae are likely colonial (62) and are larger than 3 ␮m. In this study, we estimated the diversity of bacteria in two size fractions and compared them. Although bacteria living on ⬎50-␮m particles may have contributed to the overall diversity in the system, others have found that most attached bacterial diversity is in the ⬍40-␮m fraction. Almeida and Alcantara (88) observed a sharp drop in the contribution of bacteria attached to ⬎40-␮m particles to 6.7% of the total attached bacterial abundance in a tidal lagoon, Ria de Alveiro, Portugal. Particles in the size ranges of ⬎3 to 10 and ⬎10 to 40 ␮m had 41.3 and 44.8% of the total attached bacteria, respectively, compared to 7.2% for the ⬎1- to 3-␮m particles (88). In addition, because larger particles sediment more quickly, their persistence in the water column would be more ephemeral and they would be poorly sampled in the 2-liter volumes. Since they were not sampled, it could be that a proportion of bacterial diversity in the lagoon was not detected.

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cies, Pelagibacter ubique (65), which is thought to scavenge nutrients at very low concentrations (66), such as those in the lagoon. Here and elsewhere, attached bacteria are defined as those that are retained on a filter of a given pore size and free-living bacteria as those that pass though the filter (2, 67). The presence of Pelagibacter in the attached fraction may be a technical artifact where, for example, the filters clog and retain smaller particles (6). The opposite may also occur, and attached bacteria can be released into the free-living fraction during sampling manipulation (2). Biological explanations for Pelagibacter in both fractions would include it forming close physical associations with other cells (68) or forming colonies under certain conditions (69), in which case the bacteria are attached to each other and not necessarily to particles. Steindler et al. (70) observed clumping of Pelagibacter cells through pilus production in carbon-limiting and dark conditions in cultures, suggesting that Pelagibacter cells are able to form close associations with each other via upregulation of the pilin gene (pilA); whether this occurs under natural conditions is unknown. Members of the SAR 11 clade, to which Pelagibacter belongs, have been recovered elsewhere in the attached fraction using 3-␮mpore-size filters (23) and on larger filters of up to 30-␮m pore size (67); however, determining whether this is a filtration artifact is not possible in the absence of microscopy observations. To address this, Fuchsman et al. (67) took into account the relative abundance of taxa between the two fractions and defined freeliving bacteria as those with at least 4-fold greater abundance than the same taxon in the attached fraction and defined SAR 11 in the Black Sea as free living. Given the high abundance of Pelagibacter cells in HAM, this taxa would have the highest probability of being retained on clogged filters, and as it was 5-fold more abundant in the free-living fraction, it likely belongs in that category. Synechococcus was also persistently recovered in both size fractions, although it is also considered free living (71). However, there was only one date (25 August) on which the difference between the 3to 50-␮m and 0.2- to 3-␮m Synechococcus samples was sufficiently high to categorize it as free living using the criteria of Fuchsman et al. (67). Interestingly, there are reports of Synechococcus attached to particles (72), and Malfatti and Azam (68) observed Synechococcus cells attached to one or more heterotrophic bacterial cells using atomic force microscopy (AFM), suggesting that at least some Synechococcus cells are legitimate members of an attached community. Attached Synechococcus in the HAM lagoon could benefit from the dissolved organic carbon released by their bacterial partners and nutrients made available from the extracellular enzymatic activities of the associated bacteria (73). Some freeliving bacteria can even spend most of their time in nutrient agglomerations in the surroundings of or within particles (74). This could be the case for the other free-living cells found in the attached fraction. Some of the taxonomic overlap may also have been due to exchange between attached and free-living fractions, with release of cells from the aggregates that are free until they encounter another particle and rejoin the attached bacterial fraction (75). Grossart (74) described bacteria as either truly free living or as those that spend most but not all of their lifestyle attached to particles. Particles are, in fact, risky environments for attached bacteria, given that they are more liable to be preyed upon. One strategy to counteract this is to attach to particles part of the time (76). Hollibaugh et al. (77) and Ghiglione et al. (6) concluded that most attached bacteria were also in the free-living fraction, suggesting little difference between the two communities. However,

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linity is a strong selector of bacterial taxa (104–106). It has an impact on cellular osmotic stress, and different phylogenetic groups may react differently to this physiological stress (104). The gradual increase in salinity over the summer and periodic input of rain could also directly influence the character of particulate material. Slight changes in salinity were linked to the makeup of the attached bacterial communities and may have been an indirect or direct consequence of periodic rains either by increased flocculation, runoff from land, or increased winds and resuspension of particles that accompanied rain. However, we found no significant correlation of POM with rain and wind events going back 5 days prior to sampling (not shown), consistent with a gradual accumulation of POM in the lagoon over summer (107). There are the caveats that some of the increasing diversity could be due to the accumulation of nonliving DNA over the summer, since DNA from dead cells can persist in the environment (108), and that the residual water level is higher in HAM than in the adjacent GEL (26), contributing to the longer residence time of cells in the system. Arguing against this as the primary source of diversity was the fact that the increasing diversity was attributable to a higher diversity of less common taxa (unweighted SIMPER analysis) entering the pool of species, which is more consistent with increasing numbers of niches for particular groups. Less common taxa may act as a type of seed bank of taxa that become abundant when conditions are favorable (109) or may always be rare if they are functional specialists (110). Attached bacteria have a metabolic advantage in that they can minimize time of starvation, increase the efficiency for resource exploitation in terms of carbon and energy, and have a higher evolutionary success rate (74). Therefore, it is more advantageous for a bacterial phylotype to spend part of its lifestyle as attached cells, and this is probably why we find a higher diversity of attached bacteria. The concentration of POM was also a significant factor which appears to influence the variation in free-living communities over the summer. The free-living community could profit from the activity of the attached bacteria releasing dissolved organic matter into the water via exoenzymatic activities, providing needed substrate for the free-living microbial community (73). In addition to POM, temperature also appeared to influence the free-living fraction of the HAM. Salinity and temperature are among the main environmental drivers of marine bacterial diversity (111), and even small changes may have had an effect. Temperature and POM quality have also been reported to directly affect free-living bacterial abundance and production (21) but may also influence community composition. Cyanobacteria, for example, thrive in warmer waters (112), and Synechococcus populations decline with temperature, especially when coupled to high rain events (113). In HAM, the sudden increase in Synechococcus reads coincided with the highest temperature recorded, on August 25. Abundance of HNA bacterial cells. The increase in the proportion of HNA bacteria from 25 August followed temperature, Chl a, and POM maxima and was also positively correlated to picocyanobacterial cell abundance and POM. A positive link between the picocyanobacteria and HNA bacteria was previously reported in a study of the Iberian Peninsula (114). Several studies have inferred that HNA content is indicative of more active bacterial cells (115, 116), but we were not able to address this question using the data available. The positive correlation of HNA bacteria and POM could be because HNA are associated with the attached

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Environmental variables explaining the summer bacterial community structure. Bacterial diversity may follow seasonal patterns in coastal waters, e.g., in the Western English Channel (50, 89) and in the coastal lagoon in Arroyo Burro, Santa Barbara (25). These patterns have been linked over shorter time scales to environmental variability, including temperature, phosphate and silicate levels (50), and rainfall (25); on longer time scales, day length seemed to explain seasonal variation in a 6-year study (86). The phylogenetic diversity of bacterioplankton has also been linked to the diversity of available substrate (90). Among all environmental variables tested, we found that POM concentration in the water column was the main factor influencing both fractions. This was especially marked for the attached bacterial community, which showed a significant increase in diversity over the summer, with POM following the same trend. Attached bacterial diversity was also higher in the Arroyo Burro coastal lagoon (25) and was correlated with external sources of particles during the wet season or resuspension of sediment. Increased POM concentrations over the summer is common in other coastal open waters, for example, in the northwest Mediterranean (91), the Bay of Biscay (21), and the Ionian Sea (92). These increases are thought to be due to the accumulation of phytoplankton following blooms, particulate material from runoffs following rainfall, and resuspension of sediment from high winds. POM is not chemically or structurally uniform, as it is generated from many sources and with large spatiotemporal heterogeneity (3). Its properties can also change over time due to bacterial activity (93). Attached bacteria produce exoenzymes that target specific complex molecules, and the availability and stoichiometry of particulate organic matter likely selects for bacteria with specific degradation systems (94). Similar to other coastal lagoons (20), the eelgrass Zostera marina occurs in the shallow regions of the Magdalen Islands lagoons (95–97). Zostera marina increases in biomass over the summer growing season, consistent with a seasonal contribution to POM; maximum foliar biomass is in August, and stem density increases from July to September (98). Other sources of particulate material may include input from aquatic bird colonies mostly located along the southern end of HAM lagoon on the Bird Rock Sanctuary Islet, material derived from mussel farming activity, such as mussel drop offs (27), and fecal material that can be rapidly colonized by bacteria (99). Phytoplankton can also contribute to formation of transparent exopolymer particles (TEP), which is an important agent for aggregation of particles (100). Over the summer, winds are constant, mixing the shallow water column of the HAM lagoon (101) and contributing to persistent particle resuspension from the sediment. In addition, residual mean flows and water level are relevant for the transport of dissolved and suspended organic matter. Circulation within the HAM lagoon is restricted (26), especially near our sampling site at the center of the lagoon, where water residence time can be as long as 45 days or more when only tide action is considered (31). These factors would result in an accumulation of particles in multiple states of degradation in the water column, overall increasing the diversity of substrates available for attached bacteria over time. In addition, salinity, which has a direct link with suspended matter in the flocculation of particles, was the second most significant variable contributing to the variation in attached OTU abundance. Increasing salinity decreases the electrostatic repulsion between particles, causing them to approach close to each other, which then leads to flocculation (102) and facilitates attachment of bacterial cells to particles (103). Sa-

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ACKNOWLEDGMENTS We thank Claude Belzile from ISMER (UQAR, Rimouski) and Lisandre Solomon for providing the nutrient and FCM data and Bérangère Pequin for laboratory assistance and valuable discussions. Comments from anonymous reviewers on earlier versions of the manuscript were greatly appreciated. We acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) for a strategic grant led by G. Fussmann (McGill) and coinvestigators P.A., C.L., and others. Additional funds were provided by Fonds de Recherche du Québec (FQRNT) to Québec Océan and Ressources Aquatiques Québec (RAQ). We acknowledge the contribution from the Centre d’Innovation de l’Aquaculture et des Pêches du Québec (Merinov) for laboratory space and access to the experimental site.

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fraction and have higher activity than LNA bacteria. Consistent with this was the increased diversity of attached bacteria in late summer (3 and 8 September) along with more HNA. Concluding remarks. Our approach via pyrosequencing captured sufficient bacterial diversity to clearly separate attached and free-living communities and highlight the temporal pattern in the bacterial community structure over the summer in a coastal lagoon. The retention of POM was the best explanation for the increasing attached bacterial diversity and temporal pattern observed in the bacterial community. Significant differences were found between the attached and free-living bacterial communities and overall diversity in the absence of estuarine circulation and large freshwater inputs in the Havre-aux-Maisons lagoon. This suggests that multiple specialist attached bacterial taxa contribute to overall marine bacterial biodiversity, and that particles contribute to overall coastal bacterial diversity in marine natural systems that are not under the influence of rivers.

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49. 50.

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