Biogeosciences, 13, 2319–2337, 2016 www.biogeosciences.net/13/2319/2016/ doi:10.5194/bg-13-2319-2016 © Author(s) 2016. CC Attribution 3.0 License.

Succession within the prokaryotic communities during the VAHINE mesocosms experiment in the New Caledonia lagoon

Ulrike Pfreundt1, France Van Wambeke2, Mathieu Caffin2, Sophie Bonnet2,3, and Wolfgang R. Hess1 1Faculty of Biology, University of Freiburg, Schaenzlestr. 1, 79104 Freiburg, Germany 2Aix Marseille Université, CNRS/INSU, Université de Toulon, IRD, Mediterranean Institute of Oceanography (MIO) UM110, 13288 Marseille, France 3Institute de Recherche pour la Développement (IRD) Nouméa, 101 Promenade R. Laroque, BPA5, 98848, Nouméa CEDEX, New Caledonia

Correspondence to: Wolfgang R. Hess ([email protected])

Received: 4 December 2015 – Published in Biogeosciences Discuss.: 18 December 2015 Revised: 31 March 2016 – Accepted: 11 April 2016 – Published: 21 April 2016

Abstract. N2 fixation fuels ∼ 50 % of new primary produc- teria (Cyanothece). Growth of UCYN-C led to among the tion in the oligotrophic South Pacific Ocean. The VAHINE highest N2-fixation rates ever measured in this region and experiment has been designed to track the fate of diazotroph- enhanced growth of nearly all abundant heterotrophic groups derived nitrogen (DDN) and carbon within a coastal lagoon in M1. We further show that different Rhodobacteraceae ecosystem in a comprehensive way. For this, large-volume were the most efficient heterotrophs in the investigated sys- (∼ 50 m3) mesocosms were deployed in the New Caledo- tem and we observed niche partitioning within the SAR86 nian lagoon and were intentionally fertilized with dissolved clade. Whereas the location in- or outside the mesocosm had inorganic phosphorus (DIP) to stimulate N2 fixation. This a significant effect on community composition, the tempo- study examined the temporal dynamics of the prokaryotic ral effect was significantly stronger and similar in both loca- community together with the evolution of biogeochemical tions, suggesting that overarching abiotic factors were more parameters for 23 consecutive days in one of these meso- influential than the enclosure. While temporal community cosms (M1) and in the Nouméa lagoon using MiSeq 16S changes were evident, prokaryotic diversity (Shannon index) rRNA gene sequencing and flow cytometry. Combining these only declined slightly from ∼ 6.5 to 5.7 or 6.05 in the lagoon methods allowed for inference of absolute cell numbers from and M1, respectively, throughout the experiment, highlight- 16S data. We observed clear successions within M1, some ing the importance of multiple and varying sources of organic of which were not mirrored in the lagoon. The dominat- matter maintaining competition. ing classes in M1 were Alpha- and Gammaproteobacteria, Cyanobacteria, eukaryotic microalgae, Marine Group II Eur- yarchaeota, Flavobacteriia, and Acidimicrobia. Enclosure led to significant changes in the M1 microbial community, prob- 1 Introduction ably initiated by the early decay of Synechococcus and di- atoms. However, we did not detect a pronounced bottle ef- The Southwest Pacific Ocean is recognized as an area with fect with a copiotroph-dominated community. The fertiliza- one of the highest dinitrogen (N2)-fixation rates in the global tion with ∼ 0.8 µM DIP on day 4 did not have directly ob- ocean (Garcia et al., 2007; Luo et al., 2012). In this re- servable effects on the overall community within M1, as the gion, plankton taxa capable of N2 fixation (N2-fixing or di- data samples obtained from before and 4 days after fertil- azotrophic organisms) are very diverse (Moisander et al., ization clustered together, but likely influenced the develop- 2010) and fuel up to 60 % of primary production (Bon- ment of individual populations later on, like Defluviicoccus- net et al., 2015a), yet their interactions with surrounding related bacteria and UCYN-C-type diazotrophic cyanobac- planktonic communities are rarely studied. Within this vast oceanic region, most of the studies investigating coupling

Published by Copernicus Publications on behalf of the European Geosciences Union. 2320 U. Pfreundt et al.: Succession within the prokaryotic communities between phytoplankton and heterotrophic bacteria have tar- net et al., 2015b). Unicellular N2-fixing cyanobacteria of the geted the Nouméa lagoon in New Caledonia. Phytoplank- UCYN-C type (Cyanothece-like) dominated the diazotroph ton and heterotrophic bacterial production (BP) shows sea- community in the mesocosms during the second period, but sonal patterns, with maxima in December and January and they did not appear in Nouméa lagoon waters (Turk-Kubo et an annual BP representing 21 to 34 % of primary production al., 2015). N2-fixation rates increased during days 15–23 and −1 −1 (Torréton et al., 2010). High N2-fixation rates are a recur- reached > 60 nmol N L d , which is among the highest rent feature in the Nouméa lagoon (Biegala and Raimbault, rates measured in marine waters (Bonnet et al., 2015b; Luo 2008; Garcia et al., 2007), and diazotroph-derived N (DDN) et al., 2012). These two periods corresponded to character- was shown to be significantly channeled through the het- istic successions in phytoplankton taxa, chlorophyll stocks, erotrophic bacterial compartment in this environment (Berth- primary production and BP, discussed in detail in compan- elot et al., 2015; Bonnet et al., 2015b). However, no informa- ion studies in this issue (Berthelot et al., 2015; Leblanc et al., tion is available on the potential effects of this N2 fixation on 2016; Van Wambeke et al., 2015). bacterial successions. In this research paper, we focus on prokaryotic commu- The production and quality of varying sources of organic nity dynamics and possible parallels between or successions matter is expected to vary as blooms of diazotrophic organ- among different groups of phytoplankton, heterotrophic bac- isms are transient events. Such variation is expected to in- teria, and N2-fixing organisms. fluence not only biogeochemical fluxes but also the succes- sion of different heterotrophic bacteria, according to their metabolic capabilities. Indeed, the importance of quality 2 Material and methods and quantity of dissolved organic matter (DOM) in struc- turing bacterioplankton communities has been well estab- 2.1 Mesocosm description, sampling strategy, and lished (Alonso-Sáez and Gasol, 2007; Beier and de Al- stocks/fluxes analyses buquerque, 2015), by either comparing the continuum of trophic conditions in situ (West et al., 2008) or following Three large mesocosms (∼ 50 m3) were deployed in the phytoplankton blooms under natural (Teeling et al., 2012) nutrient-poor waters of the Nouméa lagoon close to the or experimental (Lebaron et al., 2001) conditions. Since Boulari passage (22◦29.073 S–166◦26.205 E), 28 km off the the 1970s, heterotrophic bacterial succession during phyto- coast from 13 January to 4 February 2013. Details of the lo- plankton blooms has been observed with culture-dependent cation, deployment, and sampling strategy are described in techniques (Fukami et al., 1981a) and linked to the possi- Bonnet et al. (2016). The three triplicate mesocosms were ble origin of DOM sources (release, lysis, sloppy feeding, fertilized with ∼ 0.8 µM KH2PO4 between day 4 and day 5 degradation of phytoplankton detritus), DOM size (low- or of the experiment to promote a diazotroph bloom. Samples high-molecular weight molecules), or quality (Biddanda and were collected every morning at 07:00 over a period of 23 Pomeroy, 1988; Murray et al., 2007; Nagata, 2000; Riemann days from three selected depths (1, 6, 12 m) in each meso- et al., 2000). cosm (M1, M2, and M3) and in surrounding waters (hereafter The VAHINE experiment (Bonnet et al., 2016) provided referred to as Nouméa lagoon waters). For sequencing of the a unique opportunity to study the fate of DDN in the ma- microbial community and metatranscriptomics, only one of rine planktonic food web and the interactions between phy- the mesocosms, together with the Nouméa lagoon as a con- toplankton and heterotrophic bacteria by simultaneously ad- trol, was sampled. Further details regarding the sampling are dressing biogeochemical parameters, stocks, and fluxes, as given separately (Pfreundt et al., 2016; Van Wambeke et al., well as biodiversity based on 16S rRNA gene sequencing and 2015). flow cytometry cell counts. Taking into account different 16S Subsampling and analysis procedures for stocks and fluxes copy numbers, we used the absolute cytometry cell counts for are not given in detail here as they were mainly used for Synechococcus and Prochlorococcus to calculate a sample- statistical analyses (see Sect. 2.3), but they are described in specific cells count to 16S ratio and infer cells mL−1 for all full in companion papers from this issue: inorganic nutri- operational taxonomic units (OTUs) obtained from MiSeq ents and chlorophyll a (Chl) concentrations, DIP turnover sequencing. time, N2-fixation rates, and primary production (PP) were After fertilization with dissolved inorganic phosphorus analyzed according to procedures detailed in Berthelot et (DIP: ∼ 0.8 µmol L−1) on the evening of day 4, to allevi- al. (2015). Synechococcus and Prochlorococcus cell num- ate potential limitation often observed in the region (Moutin bers were counted by flow cytometry (Leblanc et al., 2016). et al., 2008), two periods of about 10 days each (P1 and Heterotrophic bacterioplankton abundances were also deter- P2, see description in Sect. 2.1) were identified in terms mined by flow cytometry and corresponded to the sum of of diazotroph succession. During the first period, diatom- cells with high and low nucleic acid content, discarding au- diazotroph associations were dominating (Turk-Kubo et al., totrophic cell counts (Van Wambeke et al., 2015). BP was 3 2015) the diazotrophic community with N2-fixation rates de- determined by the H leucine technique, and alkaline phos- creasing from 17.9 ± 2.5 to 10.1 ± 1.3 nmol N L−1 d−1 (Bon- phatase activity (APA) was determined on the total frac-

Biogeosciences, 13, 2319–2337, 2016 www.biogeosciences.net/13/2319/2016/ U. Pfreundt et al.: Succession within the prokaryotic communities 2321 tion (i.e., unfiltered samples) using MUF-P substrate (Van mation on the weight of each unique sequence, sorting of Wambeke et al., 2015). Three periods of the experiment were unique sequences by decreasing weight and discarding of defined. P0 was defined as days 2–4, before DIP fertiliza- singletons (weight = 1), and clustering into OTUs with the tion of the mesocosms. P1 (days 5–14) was characterized -cluster_otus command using a maximum dissimilarity of by DIP availability and correspondingly lower APA inside 2 %. The last steps includes chimera filtering (Edgar, 2013). the mesocosms (Van Wambeke et al., 2015). P2 (days 15– Finally, the merged reads from the different samples were 23) was characterized by increasing Chl a concentrations, mapped back onto the generated OTUs using VSEARCH N2-fixation rates, PP, and BP inside the mesocosms as well v1.1.3 (Rognes et al., 2015) with at least 98 % identity to cre- as in the Nouméa lagoon, albeit to a lower extent. Further, ate the final OTU table. For taxonomic classification of the APA increased steeply, corresponding to DIP becoming lim- OTUs, they were submitted to https://www.arb-silva.de/ngs/ iting in the mesocosms during P2 (Berthelot et al., 2015; Van and classified using the SILVA SSU taxonomy and database Wambeke et al., 2015). release SSU 119.1 (Quast et al., 2013). Cyanobacterial OTUs that SILVA had only classified to family level were manually 2.2 16S rRNA gene amplicon sequencing and OTU curated by BLASTN searches of these OTUs against NCBI’s clustering nucleotide collection. If a hit with > 95 % sequence identity was found, the genus of this classification was added to the DNA was extracted from seawater samples collected every OTU table. For all further use, relative OTU abundances in 2 days at 1 and 12 m depth in M1 and in Nouméa lagoon each sample were calculated. waters. A volume of 10 L was pre-filtered through a 1 mm mesh to keep out large eukaryotes and filtered on 0.45 µm 2.2.1 Inference of absolute cell numbers from 16S data pore size, 47 mm diameter polyethersulfone filters (Pall Su- and flow cytometry por), immersed in RNA resuspension buffer (10 mM NaAc pH 5.2, 200 mM D(+)-sucrose, 100 mM NaCl, 5 mM EDTA) Absolute cell numbers were inferred from these relative OTU abundances in three steps. First, raw read counts per OTU and snap frozen in liquid N2. Tubes with filters were vor- texed, then agitated in a Precellys bead beater (Peqlab, Er- were normalized to equal 16S copy numbers (“16S-norm langen, Germany) 2 × (2 × 15 s) at 6500 rpm after adding reads”) using a manually curated table of 16S copies per 0.25 mL glass beads (0.10–0.25 mm, Retsch, Frimley, UK) genome for all OTUs (Supplement). For OTUs with no avail- and 1 mL PGTX (39.6 g phenol, 6.9 mL glycerol, 0.1 g 8- able genome, an average copy number of all genomes from hydroxyquinoline, 0.58 g EDTA, 0.8 g NaAc, 9.5 g guanidine marine representatives of the next higher phylogenetic clade was used (for the detailed procedure see Supplement). Sec- thiocyanate, 4.6 g guanidine hydrochloride, H2O to 100 mL; Pinto et al., 2009). RNA and DNA were extracted simulta- ond, the cumulated raw reads for Synechococcus (Syn) and neously by adding 0.7 mL chloroform, shaking vigorously, Prochlorococcus (Pro) were divided by the respective sum of incubating at 24 ◦C for 10 min, and subsequent phase sepa- Synechococcus and Prochlorococcus cytometric cell counts rating by centrifugation. RNA and DNA were retained in the for each sample, producing a sample-specific ratio (16S- −1 aqueous phase, precipitated together, and stored at −80 ◦C norm reads / cells mL ). We used Syn and Pro, because for further use for metatranscriptomics analysis (Pfreundt et flow cytometry is well established for them and we could ex- al., 2016) and 16S tag sequencing. For the latter, ∼ 100 ng of pect them to be reasonably precise. This ratio was then used −1 RNase-treated total DNA was sent to a commercial provider to calculate cells mL for all OTUs by dividing their 16S- (LGC Genomics, Berlin, Germany) for amplicon sequenc- norm reads in each sample by the sample-specific ratio. We ing with primer pair S-D-Bact-0341-b-S-17/S-D-Bact-0785- are aware that missing genomic information may lead to er- a-A-21 (Klindworth et al., 2013), which targets bacteria and rors in this calculation. OTUs for which no 16S copy number archaea. Briefly, 16S hypervariable regions 3–4 were ampli- could be estimated were set to 1 copy/genome. fied and the amplicons sequenced on a MiSeq (Illumina) se- 2.2.2 Calculation of diversity measures. quencer, generating between 31 466 and 749 629 paired-end × reads (2 300) per sample. The Shannon and Simpson indices as measures of diversity All bioinformatic steps were done using the USEARCH were calculated separately for M1 and Nouméa lagoon wa- package and following the UPARSE pipeline (Edgar, 2013) ters using the diversity function in the R package vegan (Ok- unless mentioned otherwise. Briefly, lowest quality tails were sanen et al., 2015). truncated from all reads and the paired reads merged us- ing the -fastq_mergepairs command and yielding between 2.3 Multivariate statistics of 16S-abundance 25 221 and 604 457 reads of average length > 400 nt per sam- community data, samples, and environmental data ple. Merged reads were then quality filtered, reads shorter than 350 nt discarded, and converted to FASTA. All FASTA Statistical tools were all used with relative 16S abundance files were concatenated for OTU clustering, with the follow- data. A canonical correspondence analysis (CCA; XLSTATS ing steps: dereplication at 100 % identity and keeping infor- Excel software) was used to test for significant effects of bio- www.biogeosciences.net/13/2319/2016/ Biogeosciences, 13, 2319–2337, 2016 2322 U. Pfreundt et al.: Succession within the prokaryotic communities geochemical parameters on variations in the bacterial 16S- nificance of the derived correlation coefficients. A coefficient derived community structure. These parameters were PP, BP, was defined as significant if p ≤ 0.05. Chl, APA, DIP, N2-fixation rates, particulate organic car- bon concentrations (POC), and temperature (T ). CCA gen- erates ordination axes which are linear combinations of the 3 Results environmental variables that best explain microbial diversity composition data (Braak, 1986). The variables were the log- 3.1 Biogeochemical context transformed biogeochemical stocks and fluxes, the objects Here, we present data on the prokaryotic community in M1 were the 34 dominating OTUs in terms of abundance (ex- and the Nouméa lagoon from depths of 1 and 12 m, which pressed in relative OTU abundance), and the sites were sam- was close to the bottom of the mesocosm. As a foundation pling time and origin of the samples. for understanding and discussing these data, selected biogeo- Non-metric multidimensional scaling (NMDS) was used chemical parameters are shown in Fig. 1 for the correspond- to find the best display of differences in 16S-derived commu- ing sampling locations. On the evening of day 4, M1 was nity structures. The full OTU table containing relative abun- fertilized with ∼ 0.8 µM DIP, leading to lower APA than in dances of each OTU per sample, with samples as rows and the lagoon until day 19, when APA in M1 increased steeply species as columns, was used for unconstrained ordination (Fig. 1c, d) and DIP turnover times dropped below initial lev- with the R package vegan (Oksanen et al., 2015). Addition- els (Van Wambeke et al., 2015). Of the three mesocosms, the ally, the same procedure was run with the subset tables con- here investigated M1 was the one with the lowest increase of taining only “heterotroph” OTUs (i.e., all that are not classi- Chl a during P2, still reaching higher levels than in Nouméa fied as Cyanobacteria or chloroplasts) or only “autotroph” lagoon waters, whereas PP in M1 did not exceed the values OTUs (i.e., all classified as chloroplasts or Cyanobacteria measured in the lagoon (Fig. 1a, b). BP exhibited two promi- without Melainabacteria). First, the abundances were stan- nent peaks in M1 with a 4-fold and 2-fold increase, on day 4 dardized using Wisconsin double standardization, which di- and on day 21, and was otherwise close to values measured vides abundances by the species maximum and then stan- for Nouméa lagoon waters, only slightly exceeding them on dardizes samples to equal totals. This converts abundances most days after day 11 (Fig. 1e). per species into relative values and therefore reduces the weight of dominant species and increases the weight of 3.2 Bacterial community based on 16S tag MiSeq rare species. Then, Bray–Curtis distance matrices were com- sequencing puted and an NMDS ordination calculated using metaMDS. MetaMDS did not use any further transformations or stan- 3.2.1 An overview dardizations. Assuming that samples of days 2–8, days 10– 16, and days 18–21 cluster together, ellipses were drawn us- We identified a total of 3600 OTUs belonging to bacteria, ing the standard error for each cluster in the ordination plot archaea, and chloroplasts using 16S rRNA gene sequencing with a confidence limit of 0.95. Like this, we could consider data from all samples combined (17 samples from M1, 20 clusters with non-overlapping ellipses as significantly differ- samples from the Nouméa lagoon; see Supplement). On this ent from each other at a significance level α = 0.05. Permuta- basis, Shannon and Simpson indices as measures for over- tional multivariate analysis of variance (PERMANOVA) was all diversity were calculated. With values between 5.6 and conducted using the adonis function in the R package vegan 7 (Shannon) at both investigated depths over the 23-day ex- on the Bray–Curtis distance matrices described above. We periment, diversity was relatively high. Values above 5 have tested the dependence of sample distances on the factors lo- been reported for the edge of the South Pacific gyre (Yin et cation (M1 or lagoon waters), depth (1 or 12 m), and time al., 2013), but they are based on clone libraries, which might (separation of samples into three phases: days 2–8, 10–16, underestimate diversity. Over the course of the experiment and 18–21). we observed a slight decrease in diversity both in M1 and in To test significance of higher Cyanothece (UCYN-C) 16S the lagoon (Supplement Fig. S1). abundances in M1 during P2, we defined two groups, days 2– Throughout results and discussion, we will use absolute 14 (P0/P1) and days 16–21 (P2), both in M1 and the lagoon, abundances inferred from 16S sequencing data and flow cy- leading to four groups. We applied ANOVA and Tukey’s tometry counts for Synechococcus and Prochlorococcus (see honestly significant difference (HSD) test to see whether the methods Sect. 2.2 and Supplement). The temporal evolu- means of these groups were significantly different. We found tion of relative (16S) and absolute (flow cytometry) Syne- that P2 in M1 was significantly different from the other three chococcus abundances in M1 diverged substantially after groups (Tukey’s HSD p values 0.00002, 0.00009, 0.00003) day 4 (compare Figs. S4f and 2a). As Synechococcus and and that P2 in the lagoon was not significantly different from Prochlorococcus together constituted up to 30 % of all 16S P0/P1. reads, changes in absolute cell counts for these groups can Pearson and Spearman rank correlations were performed highly influence the temporal evolution of relative 16S data. in R using the rcorr function, which also calculates the sig- To elude possible ambiguous interpretation of these data,

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(a) (d) 2.5 20

) 18 ) -1

2.0 -1 16 d h -1

-1 14 1.5 12 10 1.0 8 A (nmol L

P 6 PP (µmol C L 0.5 A 4 2 0.0 0 5 10 15 20 25 5 10 15 20 25 (b) DIP fertilization (e) DIP fertilization 0.8 900

) 800

0.7 -1 h ) 700 -1

-1 0.6 0.5 600 500 0.4

l a (µg L 400

0.3 P (ng C L B Ch 300 0.2 200 0.1 100 0.0 0 5 10 15 20 25 5 10 15 20 25 (c) (f) 0.9 )

-1 80 0.8 M1 1 m 0.7 M1 12 m 70 0.6 Nouméa lagoon 1 m 60 M)

µ 0.5 Nouméa lagoon 12 m 50 (

P 0.4 40 DI 0.3 30 0.2 20 fixation rates (nM d

2 10

0.1 N 0.0 0 5 10 15 20 25 5 10 15 20 25 Days after the deployment of the mesocosms Days after the deployment of the mesocosms

Figure 1. Evolution of selected parameters in mesocosm M1 and the Nouméa lagoon at depths of 1 and 12 m. (a) Primary production (PP), (b) chlorophyll a (Chl a) concentration, (c) dissolved inorganic phosphorous (DIP), (d) alkaline phosphatase activity (APA), (e) heterotrophic 15 bacterial production (BP, measured by 3H-leucine assimilation), and (f) N2-fixation rates as measured by N incorporation in M1 and the Nouméa lagoon. we used the combined Synechococcus and Prochlorococ- Euryarchaeota, and Flavobacteriia showed similar trends cus cytometric counts to calculate a “16S / cells mL−1” ra- (Figs. 3, 4, correlations in Fig. S2a). Amongst the classes tio for each sample (see Sect. 2.2). This ratio was then used contributing less than 5 % to the total 16S community, dy- to calculate absolute cell numbers from relative 16S abun- namics were generally different between M1 and the lagoon dance in M1 for all other groups investigated here (Figs. 3, (Fig. S2b). Inside M1, Deferribacteres (almost entirely the 4). Different 16S copy numbers per genome for different SAR406 clade) and Deltaproteobacteria abundances halved OTUs/phylogenetic groups were taken into account (Supple- after the DIP fertilization and increased again when DIP con- ment). We hope that this combinatorial approach using 16S centrations fell below 0.1 nM on day 12 (Fig. 3c). All other sequencing, flow cytometry, and 16S copies/genome may groups increased at similar times, except for Actinobacte- serve as a blueprint for future 16S-based studies. In the fol- ria, which responded later (Fig. 3b). Except for the increase lowing, the term “16S community” will be used for the total in Flavobacteriia and Cyanobacteria (predominantly Syne- inferred cell count per sample. chococcus), none of these late-phase increases were observed The dominating classes in M1 were Alpha- and in the lagoon (Fig. 4), although several of the less abun- Gammaproteobacteria, Cyanobacteria, chloroplasts, Marine dant classes (Cytophaga, Betaproteobacteria, Actinobacte- Group II (MGII) Euryarchaeota (Thermoplasmata), Acidimi- ria) more than doubled from day 16 to 18 in the lagoon but crobia, and Flavobacteriia (Fig. 3a). Of these, Alpha- and decreased just as quickly after that (Fig. 4b). Gammaproteobacteria exhibited clearly different dynam- ics between M1 and the lagoon with completely different peaking times, whereas cyanobacteria, chloroplasts, MGII

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(a) Mesocosm M1 (c) Nouméa lagoon 140 000 140 000 1 m Synechococcus 1 m Prochlorococcus

120 000 12 m Synechococcus 12 m Prochlorococcus 120 000 100 000 100 000 -1 80 000 80 000 60 000 60 000

Cells mL 40 000 40 000 20 000 20 000 0 0 5 10 15 20 25 (d) 5 10 15 20 25 (b) DIP fertilization 1 200 000 1 200 000 1 m tot al heterotrophs 1 000 000 12 m total heterotrophs 1 000 000 -1 800 000 800 000

600 000 600 000 Cells mL 400 000 400 000

200 000 200 000

0 0 5 10 15 20 25 5 10 15 20 25 Days after the deployment of the mesocosms Days after the deployment of the mesocosms

Figure 2. Evolution of absolute abundances as counted by flow cytometry at depths of 1 and 12 m of Synechococcus and Prochlorococcus in M1 (a) and the lagoon (c); evolution of non-photosynthetic bacteria (total heterotrophs) in M1 (b) and the lagoon (d).

3.2.2 Cyanobacteria quences as descendants of the Calothrix node. This is dis- cussed in Sect. 4. Candidatus Atelocyanobacterium thalassa (UCYN-A) constituted the third most abundant diazotroph Synechococcus sp. (OTU-9) was the dominant OTU through- until day 14. UCYN-A may be seen as a heterotrophic di- out the experiment in M1 and the lagoon, being almost an azotroph, as it misses photosystem II, cannot fix CO2 into order of magnitude more abundant than the second ranked biomass (Tripp et al., 2010), and lives in symbiosis with uni- OTU at some time points. The steep drop of Synechococcus cellular algae (Thompson et al., 2012). Between days 14 and abundance on day 4 in M1, 2 days after the mesocosm clo- 16, Cyanothece (UCYN-C) 16S tags increased by an order of sure, was equally seen in absolute counts by flow cytometry magnitude, exhibiting significantly higher levels in M1 dur- ∼ ∼ −1 (from 78 000 to 44 000 cells mL ; Fig. 2a) and in the ing P2 than during P0/P1 and in the lagoon (ANOVA fol- relative 16S data (Fig. S4f). Please note that the inferred ab- lowed by Tukey’s HSD test, all p values < 0.0001). UCYN- solute abundances shown in Figs. 3f and 4f are not the same C was the most abundant diazotroph on day 16 in M1 with as the flow cytometry counts for Syn and Pro. We used the ∼ 400 cells mL−1 (Fig. 3f). Heterotrophic diazotrophs like sum of Syn and Pro to infer the sample-specific ratios for the Bradyrhizobium and Mesorhizobium were, with a maximum calculation to reduce putative counting biases in one of the of 0.00013 % of the community for the former, not present two groups (see discussion). Inferred cell counts for Syne- −1 in numbers anywhere close to N2-fixing cyanobacteria (Sup- chococcus were only 61 000 and 25 000 cells mL for these plement). In the lagoon samples, the two dominant dia- 2 days. At the same time in M1, Prochlorococcus inferred −1 zotrophs (Trichodesmium and Calothrix/Richelia) were the abundance reached a maximum of 37 000 cells mL on day same but with different dynamics (Fig. 4f). Notably, Cyan- ∼ −1 6, corresponding to 16 000 cells mL counted with flow othece (UCYN-C) OTUs did not increase in abundance in cytometry (Figs. 2a and 3f). Variations in Synechococcus the lagoon. abundance correlated with changes in Chl concentrations, ex- cept for days 2–5 with high Synechococcus cell counts but 3.2.3 Heterotrophic bacteria low Chl concentrations (Figs. 1b, 2a). N2-fixing cyanobacte- ria were generally 1 to 2 orders of magnitude less abundant As alpha- and gammaproteobacterial 16S abundances than Prochlorococcus and Synechococcus and were in M1 showed close to zero correlation between M1 and the la- most prevalent on days 2–4, owing mainly to Trichodesmium goon (Fig. S2a), we examined the dynamics within these with ∼ 5000 cells mL−1 (Fig. 3f). After the DIP fertiliza- classes at family level. We observed clear successions within tion, Trichodesmium 16S tags dropped quickly by a factor M1 that were not mirrored in the lagoon (Figs. 3c, d of 10, leaving Calothrix as the most abundant diazotroph un- and 4c, d). On day 4 (2 days after the mesocosms had til day 12. Important to mention, the SILVA database used been closed around the water column), Rhodobacteraceae for 16S classification lists all Richelia intracellularis 16S se- increased from < 10 000 to almost 35 000 cells mL−1 in M1,

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(a) Classes with > 5 % maximum abundance (b) Classes with 0.5–5 % maximum abundance

● 200 000 ● DIP ● DIP 15 000 ● -1 150 000 ● ● ● ● ● 10 000 ● ● ● ● ● ● 100 000 ● ● ● ● ● ● ● ● ● ● ● ● ● ● Inferred cells mL ● ● ● ● ● ● ● 5000 ● 50 000 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0 0 ● ● ● ● ● ● ● ●

2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20 ● Cyanobacteria ● Flavobacteriia ● Deferribacteres (SAR406) ● Sphingobacteriia ● Alphaproteobacteria Thermoplasmata ● Deltaproteobacteria ● Actinobacteria

Chloroplasts ● Acidimicrobiia ● Phycisphaerae ● Opitutae ● Gammaproteobacteria ● Cytophagia (c) (d) Alphaproteobacteria >5000 cells ml-1 Gammaproteobacteria >5000 cells ml-1

80 000 ● ●

● DIP 100 000 DIP ●

-1 60 000 ● 80 000

● ● ● ● 60 000 ● ● 40 000 ● ● ● ● ● ● ● 40 000 ●

Inferred cells mL ● ● ● ● ● ● ● ● 20 000 ● 20 000 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0 0 2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20

● Rickettsiales/ SAR116 clade ● Rhodospirillales/Rhodospirillaceae ● Oceanospirillales/SAR86 clade ● SAR11 clade/Surface 1 ● Rhodobacterales/Rh odobacteraceae ● Alteromonadales/Alteromonadaceae SAR11 clade/other Rickettsiales/other Oceanospirillales/other (e) (f) Flavobacteriia >5000 cells ml-1 Cyanobacteria >250 cells ml-1 140 000 40 000 120 000

100 000 DIP DIP ● ● 80 000 30 000 -1 ● ● 60 000 ● ● 40 000 ● ● ● 20 000 ● ● ● ● ● ● ● ● ● ● ● 20 000 ● ● ● Inferred cells mL ● 10 000 10 000 ● ● 5000 ● ● ● ● ● ● ● ● 1000 ● ● ● ● ● ● 500 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0 ● ● ● ● ● ● ● ●

2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20

● Flavobacteriales/Flavobacteriaceae ● Synechococcus ● Trichodesmium ● Flavobacteriales/NS9 marine group ● Prochlorococcus ● Atelocyanobacterium thalassa (UCYN-A) ● Calothrix Cyanothece (UCYN-C)

Days after the deployment of the mesocosms

Figure 3. Evolution of the 16S community with time for surface samples (1 m depth) inside mesocosm M1. Absolute abundances in cells mL−1 were inferred in three calculation steps. Initially, raw read counts per OTU were normalized to equal 16S copy numbers (“16S-norm reads”). These reads were summed for all Prochlorococcus (Pro) and Synechococcus (Syn) OTUs in each sample and divided by the sum of flow cytometry cell counts for Syn and Pro in that sample, creating a ratio. For each OTU, the 16S-norm read count was then divided by this ratio to produce the inferred absolute abundance of that OTU. This procedure accounts for library size in each sample. For better visualization, the classes with (a) a maximum abundance above 5 % and (b) a maximum abundance between 0.5 and 5 % were plotted separately. (c–e) Families with > 5000 cells mL−1 (at their respective maximum) amongst the dominant non-photosynthetic classes Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia. (f) All genera with > 250 cells mL−1 (at their respective maximum) within photosynthetic Cyanobacteria. Dashed lines were used to connect the data points for visualization purposes only. The DIP fertilization event in the evening of day 4 is indicated by a black vertical line. www.biogeosciences.net/13/2319/2016/ Biogeosciences, 13, 2319–2337, 2016 2326 U. Pfreundt et al.: Succession within the prokaryotic communities

(a) Classes with > 5 % maximum abundance (b) Classes with 0.5–5 % maximum abundance ●

350 000 ● 15 000 ●

-1 150 000 ● ● ● ● ● ● ● ● ● 10 000 ● ● ● ● 100 000 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Inferred cells mL ● ● ● ● 5000 ● ● ● ● ● ● 50 000 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0 0 ● ● ● ● ●

2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20 ● Cyanobacteria ● Flavobacteriia ● Deferribacteres ● Betaproteobacteria ● Alphaproteobacteria Thermoplasmata ● Deltaproteobacteria ● Sphingobacteriia Chloroplasts ● Acidimicrobiia ● Phycisphaerae ● Actinobacteria

● Gammaproteobacteria ● Cytophagia ● Opitutae

(c) Alphaproteobacteria >5000 cells ml-1 (d) Gammaproteobacteria >5000 cells ml-1 150 000

100 000 ●

-1 100 000 80 000 ● ● ● ● ● ● ● ● 60 000 40 000 ● ● ● ● ● ● ● 40 000

Inferred cells mL ● ● ● ● ● ● ● 20 000 ● ● ● ● ● ● ● ● ● ● ● ● 20 000 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0 ● ● ● ● 0

2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20 ● Rickettsiales/SAR116 clade ● SAR11 clade/Surface 1 ● Oceanospirillales/SAR86 clade ● Rhodospirillales/Rhodospirillaceae Rickettsiales/other ● Alteromonadales/Alteromonadaceae SAR11 clade/other ● Caulobacterales/Hyphomonadaceae ● Rhodobacterales/Rhodobacteraceae (e) (f) Flavobacteriia >5000 cells ml-1 Cyanobacteria >250 cells ml-1 140 000 40 000 ● ● 120 000 100 000 ● ● 80 000 ● 30 000 ● ● ● ● -1 ● 60 000 ● ● ● ● ● ● ● 40 000 ● 20 000 ● 20 000 ●

Inferred cells mL ● ● ● ● ● 10 000 ● 10 000 ● ● ● ● ● 5000 ● ● ● ● ● 1000 ● ● ● ● ● 500 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0 ● ● ●

2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20

● Flavobacteriales/Flavobacteriaceae ● Synechococcus ● Trichodesmium ● Flavobacteriales/ NS9 marine group ● Prochlorococcus● Calothrix

Days after the deployment of the mesocosms

Figure 4. Evolution of the 16S community with time for surface samples in the Nouméa lagoon waters. All abundances are given as inferred absolute abundances in cells mL−1 as described for Fig. 3. Data were plotted in the same way as for Fig. 3. and dropped (Fig. 3c). One particular OTU (297), classi- cumulation from Rhodobacteraceae seen in the metatran- fied as Thalassobium sp., contributed 79 % of Rhodobac- scriptome analysis (Pfreundt et al., 2016) and a 4-fold in- teraceae sequences on that day, but only 2–20 % in other crease in bacterial production on that day (Fig. 1e). This samples. This peak corresponded to increased transcript ac- situation occurred the morning before the DIP fertilization

Biogeosciences, 13, 2319–2337, 2016 www.biogeosciences.net/13/2319/2016/ U. Pfreundt et al.: Succession within the prokaryotic communities 2327 and was accompanied by a 30–50 % increase in SAR86, 23 (Fig. 1f). Preceding and overlapping this reaction of het- SAR11, and Rhodospirillaceae. Notably, a similar increase erotrophs was a constant increase in Synechococcus from of SAR11 was observed in the lagoon (Fig. 4c), speaking its minimum of ∼ 20 000 cells mL−1 on day 9 to maximally against a M1-specific trigger for this group. In addition, a 104 000 cells mL−1 on days 19–20 (Fig. 2a). M1-specific severe drop of Synechococcus from > 60 000 to 25 000 cells mL−1 was observed (Fig. 3f). While Rhodobac- 3.2.4 Temporal dynamics of the 16S community teraceae dropped to ∼ 5000 cells mL−1 on day 6 – similar to lagoon values – and stayed fairly stable until day 14, SAR11, Using all OTUs as a basis, we did unconstrained ordination dominated by the Surface 1 group, increased from 30000 to of all samples on Bray–Curtis dissimilarities calculated from > 100 000 cells mL−1 on day 8 and outcompeted all other het- standardized relative abundances. NMDS showed that inside erotrophic bacteria in M1 (Fig. 3c). SAR11 was replaced by M1 as well as in lagoon waters, the microbial communities SAR116 2 days later, the most numerous alphaproteobac- defined three distinct clusters (Fig. 5a). Days 2–8, days 10– teria for most of the experiment, and surpassed in numbers 16, and days 18–21 (the last day 16S rRNA gene sequencing only by SAR86 at the end of the experiment (Fig. 3d). After was done for) were grouped together and the clusters sep- day 14, Rhodobacteraceae other than Thalassobium became arated along the first MDS axis both in lagoon waters and more numerous again both in M1 and the lagoon (Figs. 3c, in M1, whereas the locations lagoon waters and M1 (color- 4c). coded in blue and orange, respectively) were also clearly sep- Within Alphaproteobacteria, SAR11 abundance dynam- arated along the second MDS axis. These clusters in time will ics were completely uncorrelated between M1 and the la- be called early, mid-time, and late from here on and were goon, whereas other groups were slightly positively corre- time delayed from the periods P0, P1, and P2 defined by bio- lated (Fig. S2c). Within Gammaproteobacteria, Alteromon- geochemical parameters, production rates, and N2 fixation adaceae and SAR86 together constituted between 75 and (see Sect. 2.1). Thus, communities from M1 and the lagoon almost 90 % (Fig. 3d) and whereas the former showed were clearly different from each other but followed a simi- very similar dynamics between M1 and the lagoon waters, lar trajectory with time. When we separated the autotrophic SAR86 did not (Fig. S2d). A second abundance peak of community (547 OTUs) from the heterotrophic community SAR86 occurred only in M1 on days 16 and 18 and co- (2981 OTUs, all OTUs other than cyanobacteria and chloro- incided with similar peaks of less abundant heterotrophic plasts) for ordination, it became evident that the heterotrophs classes (Fig. 3b, d). While in M1, SAR86 never de- showed a pattern very similar to the full community (Fig. 5b). creased below 30 000 cells mL−1 with peaks of 80 000 and The autotrophic community also clearly separated early, mid- 110 000 cells mL−1, these bacteria were mostly around or be- time, and late phases from each other in M1 and the lagoon, low 30 000 cells mL−1 in the lagoon after day 4. but not as far, and separation by location (M1 vs. lagoon wa- Within Flavobacteriia, the dominant families exhibited ters) only really became evident in the late phase (Fig. 5c). highly correlated temporal dynamics in M1 and the la- PERMANOVAshowed that indeed the community structures goon and also between the different families (Fig. S2e). The of the full OTU set, as well as both separate OTU sets, were Flavobacteriaceae family dominated, increasing from 4000 significantly different between the three phases (predictor to 32 500 cells mL−1 between days 10 and 18 in M1 (Fig. 3e), time) and were also different according to their origin, M1, or in the period of DIP consumption, coinciding with higher Chl lagoon waters (predictor location; p values < 0.001, for au- concentrations from day 12 onward (Fig. 1b, c). A maximum totrophs p = 0.003). Using the interaction of the predictor abundance of 38 000 cells mL−1 was reached in the lagoon location with time additionally showed that time still had a also on day 18 (Fig. 4e). The NS9 marine group, the sec- significant effect after taking out the location effect but not ond most abundant flavobacterial group, had very low abun- the other way around. This shows that the effect of time was dances until day 16 and increased towards the end of the ex- stronger and included the effect of location. Depth had no periment in both locations. significant effect. Generally, a steep increase (> 2-fold) of total non- photosynthetic cells (abundant classes except Cyanobacteria 3.2.5 Effect of hydrological and biogeochemical and chloroplasts) from day 12 to day 16 was seen in M1 parameters on the community structure of (Fig. 3a, b), which was not seen in flow cytometry counts dominant OTUs of total heterotrophic bacteria (Fig. 2b) but was accompa- nied by a moderate increase (50 %) in BP (Fig. 1e), sup- The 34 dominant OTUs (in terms of summed relative abun- porting the former. Spearman rank correlation between BP dances of all samples) were subjected to constrained ordina- and inferred cell counts for non-photosynthetic bacteria was tion within the frame of eight environmental variables (BP, 0.56, p = 0.09 (0.72, p = 0.03 when omitting day 21 with PP, APA, Chl, POC, N2 fixation, DIP, and temperature T ), its prominent BP peak). N2-fixation rates were also substan- taking all samples from M1 and the lagoon from 1 and 12 m tially higher throughout this period (days 12 to 16) than be- depth into account (Fig. 6). The sum of all unconstrained fore and stayed high until the end of the experiment on day eigenvalues was 0.304 and the sum of canonical eigenvalues www.biogeosciences.net/13/2319/2016/ Biogeosciences, 13, 2319–2337, 2016 2328 U. Pfreundt et al.: Succession within the prokaryotic communities

(a) All OTUs (3600) (b) Heterotroph OTUs only (2981) (c) Autotroph OTUs only (547)

T19 T10 T19 T10 T8 T10 T10 T21 T8 0.2 T16 T6 T14 T21 T21 T14 T18 T6 0.2 T21

T18 T16 0.2 T6 T18 T2 T6 T19 T18 T18 T16 T21 T2 T4 T12 T4 T18 T14 T12 T14T14T12 T21 T14 T10T10T14T12

0.0 T10 T14 T2 T2 T10 T10T12T16 T21 0.0 T12 0.0 T19

MDS2 T14 T4 T8T12 T4 T10 T2T2T2T8 T19 T21 T2 T21 T8 T6T8 T18 T6 T10T14T16 T19 T21 T12T14 T4 T6 T19 T2 T12 T6 T6 T14 T19 T18 T6T2T4 T10 T2 T6 T21 T18 T16 T19 T8 −0.2 T2 T18 −0.2 T8 −0.2 T6 T18 T21 T8 T18 Stress 0.162 Stress 0.169 Stress 0.155

−0.2 0.0 0.2 0.4 −0.2 0.0 0.2 0.4 −0.2 0.0 0.2 0.4 0.6 MDS1 MDS1 MDS1

Figure 5. NMDS ordination of all samples using Bray–Curtis distances based on the relative abundances of (a) all OTUs, (b) only non- cyanobacterial OTUs (“heterotrophic bacteria”), or (c) only OTUs classified as photosynthetic cyanobacteria. The samples are color-coded as follows: orange for the M1 surface, red for M1 12 m, light blue for the Nouméa lagoon surface, and blue for Nouméa lagoon 12 m. Ellipses are color-coded in a similar way to distinguish M1 sample clusters and Nouméa lagoon sample clusters. Ellipses denote a confidence interval of 0.95 around the weighted average of each cluster. Thus, clusters can be considered significantly different with α = 0.05, if ellipses do not overlap. was 0.130. Thus, the amount of total variation in the species with the very early samples from days 2 to 4 in M1 and days data that could be explained by the employed environmen- 2 to 8 in the lagoon. One MGII and one SAR116 OTU (47 tal variables was 43 % (permutation test; p < 0.001 after 999 and 19) were also separated from the rest, possibly profit- permutations). The canonical axes F1 and F2 accounted for ing from higher temperatures. More tightly linked with in- 50.5 and 22.3 % of this total explainable variance and 21.6 creasing temperatures and APA was the dominant flavobac- and 9.5 % of the total variance, respectively. Both axes were terial OTU (Sufflavibacter OTU 67) in lagoon samples from significant and displayed strong species–environment corre- days 16 to 21. Noteably, there was an opposition between lations (p < 0.001 for both axes). Samples (location and time; the two dominant autotroph OTUs, Synechococcus (9) and Fig. 6b) were mainly separated along F1 with days 6 to 14 Prochlorococcus (4), where the former was closely linked to towards negative values and days 16 to 21 towards posi- the increase of PP and Chl at the end of the experiment. Rho- tive values. Interestingly, samples from M1 were separated dospirillaceae of the Aegean-169 group were positioned op- further apart from each other than those from the lagoon. posite to APA, T , and PP and close to Prochlorococcus and The main parameters separating samples and OTUs (Fig. 6a) two chloroplast OTUs (3 and 1677), suggesting they might along the F1 axis were DIP, which was clearly higher in M1 profit from these autotrophs specifically. samples following the DIP fertilization (days 6–10), and BP, The quadrants resulting from this CCA nicely correspond Chl, and PP in opposition to DIP. Highly associated with BP to the clusters in time found through NMDS (Sect. 3.2.4), and Chl were the late-phase samples from M1 and lagoon, with few overlaps between quadrants: the bottom left corre- together with Rhodobacteraceae OTUs 1 and 37 and the sponds to the early phase, the top left to the mid-time phase, flavobacterial NS4 marine group OTU 44, suggesting these and both right quadrants together to the late phase (compare directly profited from primary producers at the end of the Figs. 6 and 5). experiment. Interestingly, another Rhodobacteraceae OTU (23) was tightly associated with N2 fixation and with the OM60(NOR5) OTUs 2 and 18 from the Halieaceae family of 4 Discussion Gammaproteobacteria. Other gammaproteobacterial OTUs, Alteromonas OTU 13 and SAR86 OTUs 7 and 26, were po- 4.1 Inference of absolute abundances from relative 16S sitioned oppositely, closer to high DIP values, and together data with all three SAR11 OTUs (43, 60, 2922), three of four SAR116 OTUs (16, 20, 34), the Rickettsiales group S25(593) Our results rely on the calculation of absolute cell counts OTU 27, and Candidatus Actinomarina OTU 6 (Actinobac- from relative 16S abundance data obtained by MiSeq se- teria). Most tightly associated with high DIP concentrations quencing. This is possible by supplementing these data with was Defluviicoccus OTU 40 (Rhodospirillaceae). Also in the flow cytometry data and 16S copy numbers per genome. same quadrant, but forming a separate cluster, were MGII Whereas major trends could likewise be observed in the rel- Euryarchaeota. Two more SAR86 OTUs (98, 1229) were ative data (Figs. S4, S5), the inferred absolute data give a separated from the rest and linked with Prochlorococcus and much more precise picture of the bacterial community and have the power to reveal absolute abundance changes of spe-

Biogeosciences, 13, 2319–2337, 2016 www.biogeosciences.net/13/2319/2016/ U. Pfreundt et al.: Succession within the prokaryotic communities 2329

(a) 0.6

T°C 0.5 MGII-123 APA 0.4 MGII-141

0.3 MGII-53 Sufflavibacter-67 SAR86-26 MGII-47 0.2 SAR11-43 SAR116-19 SAR11-2922 S25(593)-27

%) 0.1 Alteromonas-13 Actinomarina-6 P P DIP SAR116-20 Synechococcus-9

SAR11-60 28 SAR86-7 Defluvicoccus-40 SAR116-16 Rhodob-1 0 SAR116-34 (22. Chloroplast-3 2 Chloroplast-1677 Marinimicrobia-17 NS4margroup-44 Ch l F - 0.1 Objects Rhodob-37 Aegean169-35 Aegean16 9-1177 Gammproteobacteria POC B P OM60(NOR5)-18 -0 .2 Alphaproteobacteria Aegean169-39 Rhodob-23 Flavobacteriia SAR86-1229 -0 .3 Euryarchaea SAR86-98 photoautotrophs N2 fixation OM60(NOR5)-2 -0 .4 Prochlorococcus-4 Environmental -0 .5 variables

(b) 0.6

0.5 T°C APA 0.4 10 M1 s 19 NL s 0.3 12 NL s 18 NL s 0.2 10 M1 d 14 NL d 14 M1 s 14 NL s 10 NL s 19 NL d

%) P P 0.1 DI P 8 M1 s 16 NL s 12 NL d 12 M1 s 21 N L d 28 16 M1 s 18 M1 s 0 19 M 1 d 21 M1 s

(22. 21 NL s 8 NL s 18 M1 d

2 6 M1 s 10 NL d 14 M1 d Ch l F - 0.1 2 M1 s 8 NL d 4 NL s POC 21 M1 d B P 6 M1 d 6 NL s -0 .2 18 NL d 4 M1 s 2 NL s 6 NL d -0 .3 Sites 2 NL d Nouméa lagoon - NL N2 fixation 2 M1 d -0 .4 Mesocosm 1 - M1

-0 .5

0. 8 - 0. 7 - 0. 6 - 0. 5 - 0. 4 - 0. 3 - 0. 2 - 0. 1 0 0. 1 0. 2 0. 3 0. 4 0. 5 0. 6 0. 7 0. 8 0.9 1 F1 (50,49 %)

Figure 6. Canonical correspondence analysis (CCA) biplot of bacterial 16S-community structure. Correlations between environmental vari- ables and the first two CCA axes are represented by the lengths and angles of the arrows. The position of OTUs or samples relative to arrows indicates the extent to which the distribution of that OTU or the community composition of that sample is influenced by the environmental parameter represented by that arrow. (a) Ordination of OTUs constrained by the given environmental variables (black lines). Color-coded pen squares mark the 34 most abundant OTUs in the two-dimensional space of the plot. These were one Synechococcus, one Prochlorococcus, and two chloroplast OTUs (photoautotrophs, red); three SAR11, four SAR116, three Aegean169 marine group, three not further classified Rhodobacteraceae (Rhodob), one Rickettsiales S25(593), and one Rhodospirillaceae (Defluviicoccus) OTU (Alphaproteobacteria, blue); four SAR86, two OM60(NOR5) clade (Alteromonadaceae), and one Alteromonas OTU (Gammaproteobacteria, green); one Sufflavibacter and one NS4 marine group OTU (Flavobacteriia, orange); four Marine Group II archaea (MGII, Euryarchaeota, pink); one Candidatus Actinomarina OTU (Acidimicrobiales, black) and one unclassified Marinimicrobia OTU (black). (b) Ordination of sampling sites in the same two-dimensional space. Sites are coded “M1” and “NL” for their origin from mesocosm M1 and the Nouméa lagoon, then “s” or “d” according to the depth sampled (surface 1 m and deep 12 m, respectively). These codes are prefixed by a number giving day of sampling (from days 2 to 21). cific groups within. However, there are major potential error flow cytometry might miss some Syn or Pro, if cells are part sources that need to be mentioned. First of all, the curated of aggregates. This would matter if the fraction of Syn or Pro table of 16S copy numbers per genome is a conscientious es- in aggregates changed between samples. To test for biases, timate and some estimates might well be corrected as more we calculated the ratio of Syn to Pro for 16S-copy-number- genomes of marine bacteria are being sequenced. Second, normalized 16S read counts (16S-norm reads) and for flow www.biogeosciences.net/13/2319/2016/ Biogeosciences, 13, 2319–2337, 2016 2330 U. Pfreundt et al.: Succession within the prokaryotic communities cytometry counts. In an error-free scenario, both should give 2015), which is consistent with the results of our metatran- the same ratios. With a systematic error source, e.g., constant scriptomic analysis (Pfreundt et al., 2016). Measurements of percentage of Syn in aggregates, the ratios would differ by a BP and PP revealed that the organic matter produced by non- similar value in all samples. However, we observed varying diazotrophic phytoplankton was the major carbon source fu- ratio differences (Supplement Table S1). In M1, Pro was ei- eling heterotrophic prokaryotes (Van Wambeke et al., 2015). ther underestimated in flow cytometry or Syn underestimated Our community analysis identified among these primary pro- in the 16S data, leading to higher Syn/Pro ratios in flow cy- ducers Synechococcus as most numerous contributor. tometry data than in 16S data for most samples. In the la- goon, this was the case until day 16, the samples after that 4.3 Effects of enclosure and DIP fertilization on the showed the opposite. Apart from differential aggregation be- microbial community tween samples, a reason for this might be that the assumed 16S copy number of two for Syn and one for Pro did not re- Apart from investigating the effect of changing diazotroph flect the real populations, but that the initial Syn population communities on general community structure, we judged to had only one copy (or the Pro population two copies), leading which extent the enclosure and the DIP fertilization in the to Syn underestimation in 16S data. These errors are reduced deployed large mesocosms changed community composition by using the sum of Syn and Pro for the calculation of the and development compared to the surrounding lagoon wa- 16S-norm / cells mL−1 ratio. However, the reader should be ters. Roughly, the 16S community partitioned the M1 sam- aware that the inferred absolute counts are mere estimates. ples into three periods, an early period of DIP availability (in- cluding samples before the spike) until day 8, a mid-time pe- 4.2 Synechococcus as the most numerous primary riod of DIP consumption, and a late period of Chl accumula- producer tion/P depletion starting with day 18. Remarkably, although we enriched the mesocosms with ∼ 0.8 µM DIP and noticed a In the world’s oceans, the unicellular cyanobacteria significant difference between the communities in M1 and in Prochlorococcus and Synechococcus constitute the most lagoon waters (Fig. 5a, separation along the second ordina- abundant marine picophytoplankton, amounting together up tion axis, p < 0.001), the evolution of both communities with to 106 cells mL−1, and are responsible for up to 50 % of the time was mostly parallel, shown by their parallel separation total CO2 fixation in some regions (Liu et al., 1997; Li, along the first ordination axis. This indicates an overarching 1994; Veldhuis et al., 1997). Previous studies (Zwirglmaier influence of abiotic factors acting on both locations equally, et al., 2008) showed that Synechococcus abundance is low in like temperature, which increased during the course of the the oceanic oligotrophic gyres, reaching only 4 × 103, but is study. Individual OTUs, like Sufflavibacter, were shown to up to 105 cells mL−1 in nutrient-rich upwelling regions (see be positively linked with increasing temperatures. These re- also the data compilation in Flombaum et al. (2013). There- lations may be linked to significantly enhanced chemotactic fore, the cell counts reported here for Synechococcus in the abilities at higher temperatures, as was recently shown for New Caledonian lagoon were, with up to 105 cells mL−1, a coral pathogen (Garren et al., 2015). Despite the parallel among the highest reported thus far for marine waters. In evolution of the full community (rare and abundant OTUs contrast, the maximum values for Prochlorococcus were rel- had similar weights in NMDS), community composition and atively low. Several studies indicated that Prochlorococcus is dynamics of single abundant groups in M1 were different to adapted to ultra-oligotrophic conditions and is outcompeted those in the lagoon from the beginning. These differences by Synechococcus in waters of higher nutrient content, such first became evident with the drop in Synechococcus on days as upwelling and coastal regions (Partensky et al., 1999a, b), 2 to 4 (Figs. 2a, 3a). In this process, cyanophages might have which is consistent with the findings in this study (Leblanc played a role as suggested by the high gene expression from et al., 2016). The Synechococcus group can be further delin- Myoviridae detected in M1 compared to the lagoon (Pfreundt eated into several major clades with different ecophysiolo- et al., 2016). Based on the known host association of these gies (Scanlan et al., 2009). Due to the insufficient phyloge- T4-like phages (Frank et al., 2013; Ma et al., 2014; Sabehi netic resolution, this was not possible based on 16S rRNA et al., 2012), these were likely targeting Synechococcus. This gene sequencing. However, in the accompanying metatran- event and its putative consequences on the availability of or- scriptome analysis (Pfreundt et al., 2016), we found that the ganic material coincided with diatoms sinking out of the wa- Synechococcus population was dominated by representatives ter column in the mesocosms, roughly halving in numbers of clade II and to a lower extent, clade IX, within the pi- (Leblanc et al., 2016), and overlapped with DIP fertilization cophytoplankton subcluster 5.1A. Recent analyses have sug- on day 4. Thus, it cannot be conclusively determined which gested that the different frequencies of co-occurring Syne- had a higher influence on the community differences we ob- chococcus ecotypes partition the ocean into four distinct served in the early phase of the experiment, decaying phy- regimes that can be distinguished by temperature, macronu- toplankton or increased DIP concentrations. Van Wambeke trients, and iron availability, suggesting clade II ecotypes are et al. (Van Wambeke et al., 2015) showed that BP was not competitive in warmer, oligotrophic conditions (Sohm et al., P limited on day 4, suggesting that phytoplankton decay had

Biogeosciences, 13, 2319–2337, 2016 www.biogeosciences.net/13/2319/2016/ U. Pfreundt et al.: Succession within the prokaryotic communities 2331 the greater effect. Indeed, POC concentrations in M1, but not Strikingly, for both SAR11 and SAR86, 16S abundance in the lagoon, increased from ∼ 10 on day 2 to 15 µmol L−1 was decoupled from the respective total transcript accumula- on day 4 (Berthelot et al., 2015). tion presented in the companion paper, whereas the transcript We correlated the relative abundances in M1 and the abundances of both clades were highly correlated (Pfreundt Nouméa lagoon for all investigated phylogenetic groups et al., 2016), and specific abundant SAR11 and SAR86 OTUs (Fig. S2). This approach revealed Alpha- and Gammapro- shared a similar environmental niche (Fig. 6). Dupont et al. teobacteria as the least correlated classes between M1 and (Dupont et al., 2012) hypothesized that, due to their differ- the lagoon. Investigating this aspect at higher taxonomic res- ent resource specializations, SAR11 and SAR86 compete olution showed that SAR11 and SAR86 temporal dynam- only slightly for dissolved organic carbon (DOC), which ics differed the most, suggesting that these groups were the would allow for their co-existence, albeit without explain- most sensitive to changing nutrient sources. SAR11 was ing their tight transcriptional correlation. Dominant SAR86 present at comparatively low numbers compared to other OTUs exhibited niche partitioning in the constrained ordina- reports (Morris et al., 2002). We used filters with 0.45 µm tion (Fig. 6). The temporal variation of two OTUs was close pore size, so it cannot be excluded that SAR11 (average size to SAR11 and Alteromonas and of another two OTUs close to 0.2 µm × 0.4 µm) and the even smaller Candidatus Actino- Prochlorococcus, suggesting different sources of DOC were marina were lost at the beginning of filtering and retained utilized by these SAR86 subtypes. On 16S sequence level, when more other cells were already on the filter. Thus, we pairwise alignments of these four SAR86 OTUs showed se- have possibly missed a substantial fraction of these groups quence identities between 86.3 % (OTUs 26 and 1229) and in this study. Nevertheless, we can expect the loss rate to 94.9 % (OTUs 7 and 98). Thus, those SAR86 OTUs sharing be very similar across all samples, due to the following rea- a niche were not more similar on 16S level than in between sons: 16S and metatranscriptomics analyses were done from niches. DNA and RNA isolated together from the same filter. Tran- An aspect of special interest was to recognize community script abundances were tightly correlated (Pearson correla- changes which could be attributed to the DIP fertilization in tion 0.88 in M1, and 0.96 in the lagoon) between SAR86 M1. CCA suggested that the taxon profiting most from DIP and SAR11 (Pfreundt et al., 2016) across all samples. With fertilization was Defluviicoccus (Rhodospirillaceae). Apart different loss rates of SAR11 in different samples this cor- from the maximum on day 6 in M1, this genus had sim- relation should not be possible. SAR86, like SAR11, shows ilar abundances also in the Nouméa lagoon (Supplement). metabolic streamlining, but while SAR11 genomes contain Defluviicoccus-related bacteria were initially described from putative genes for synthesis of most amino acids (Grote et enhanced biological phosphorus removal systems (Maszenan al., 2012), a substantial fraction of SAR86 is probably aux- et al., 2005; McIlroy and Seviour, 2009; Mielczarek et al., otroph for Met, His, and Arg (Dupont et al., 2012). Op- 2013). In such systems, used to treat wastewater, Defluviic- positely, SAR86 genomes encode a disproportionally high occus are known as glycogen-accumulating bacteria, com- number of TonB-dependent outer membrane receptors (TB- peting with polyphosphate-accumulating organisms (Burow DRs) that allow transport of > 600 Da molecules, including et al., 2007; McIlroy and Seviour, 2009; Wong et al., 2004; carbohydrates and lipids, combined with lipase genes and Wong and Liu, 2007). There are only few studies reporting an expanded sugar utilization metabolism, whereas SAR11 Defluviicoccus-related bacteria in marine samples, and, if so, genomes are not known to contain any TBDR genes and these were associated with marine invertebrates (Enomoto et have strongly reduced sugar utilization capabilities (Dupont al., 2012; Fan et al., 2013). Our data show a minor population et al., 2012). In our accompanying metatranscriptome study, of Defluviicoccus-related bacteria in the Nouméa lagoon that transcripts for TBDRs and phospholipase (days 14–16) from could benefit from the addition of fresh phosphorus in M1. SAR86 were highly abundant, suggesting that membrane Note, however, that the dominant OTU in this genus was only lipids were used as an energy source (Pfreundt et al., 83 % identical to Defluviicoccus vanus 16S but 99 % identi- 2016). Cultivation experiments of SAR11 representatives cal to environmental sequences that, according to SILVA tax- have shown their ability to utilize a wide variety of small onomy for SSU release 119 and 123, are leaves of the same organic molecules, including peptides, to generate energy node as D. vanus and were thus classified as Defluviicoccus (Tripp, 2013). The SAR11 subgroup Ia (surface 1) represen- (name of the node). Phylogenetic placement of the here de- tative Pelagibacter ubique was able to substitute one of its scribed Defluviicoccus OTUs showed their association with glycine by glycolate (Carini et al., 2013), which is one of marine environmental Defluviicoccus 16S (Fig. S3). the main excretion byproducts of phytoplankton and is as- sumed to be abundant in phytoplankton release. Finally, both 4.4 Effects of DIP depletion at the end of the SAR11 and SAR86 genomes contain proteorhodopsin genes, experiment potentially providing an additional energy source, but the corresponding transcript levels were much higher in SAR11 Some aspects of the taxonomic succession after day 13, when than in SAR86 during the VAHINE experiment (Pfreundt et DIP was mainly depleted, can be related to the strategies al., 2016). used for adaptation to P limitation. Among autotrophs, the www.biogeosciences.net/13/2319/2016/ Biogeosciences, 13, 2319–2337, 2016 2332 U. Pfreundt et al.: Succession within the prokaryotic communities main trend observed in M1 and the lagoon waters was the op- matic activities generally associated with the end of blooms position of Prochlorococcus and Synechococcus with Syne- (Chróst, 1992; Riemann et al., 2000). The quality of organic chococcus reaching higher abundances in M1 at the end of matter issued from specific phytoplankton detritus influences the experiment compared to the beginning, when DIP was bacterial community succession, and while the potential for comparatively low. Both expressed AP mainly 2 days after polymer degradation appears to be widespread among differ- their respective 16S abundance peak (Pfreundt et al., 2016), ent phyla it is restricted to some specialized species (Landa suggesting that the increase in cell numbers led to a higher et al., 2014; Murray et al., 2007). Our data indicate that both phosphorous demand that was subsequently adapted to. In- in M1 and the lagoon, available organic components favored corporation of sulfolipids instead of phospholipids into mem- growth of diverse flavobacteria, probably enhanced by in- brane lipids is an ecological strategy developed by cyanobac- creasing temperatures during P2. teria for an advantage in P deficient environments (Van Mooy Our data further indicate that Rhodobacteraceae, when et al., 2006, 2009). The ability to synthesize Sulfoquinovosyl abundant, assimilate much more organic matter than other diacylglycerols in place of phosphatidylglycerol in P-limited heterotrophs seen in this study, as dynamics in BP were cultures can save 5 to 43 % of total cellular phosphorus de- closely linked to this group. This was not only shown by mand and is highly variable according to the strain tested CCA (Fig. 6) but is also evident from directly comparing (Van Mooy et al., 2009). SqdB, a gene encoding a sulfolipid BP (Fig. 1e) to Rhodobacteraceae abundance. Both striking synthesis protein, was mainly transcribed by Synechococcus BP peaks on day 4 and day 21 in M1 correspond to con- and less by Prochlorococcus (Pfreundt et al., 2016) and tran- comitant Rhodobacteraceae abundance peaks, while no other script abundance changes corresponded to changes in 16S heterotrophic groups showed these peaks. Rhodobacteraceae abundance. In the North Atlantic Ocean, Synechococcus are are considered ecological generalists, with large gene inven- also good competitors with Prochlorococcus for DIP and tories, but they cannot be easily condensed in an ecologi- ATP uptake (Michelou et al., 2011). A variety of heterotrophs cally differentiated cluster (Newton et al., 2010). Roseobac- expressed AP mainly after day 10, possibly to the benefit ter, a common marine member of the Rhodobacteraceae, of other populations. Indeed, metagenomic analyses showed have a variety of metabolic pathways that confer advantages that 59 % of marine bacterial APs are extracellular, periplas- among other bacteria and facilitate interaction with, and at- mic, or located at the outer membrane (Luo et al., 2009). tachment to, phytoplankton detritus. They can produce quo- Possibly, DIP release through extracellular APA may have rum sensing molecules, vitamins, and antimicrobial com- provided P to neighboring cells that cannot synthesize this pounds (Buchan et al., 2014). CCA showed two dominat- enzyme, and such effects could possibly be emphasized in ing Rhodobacteraceae OTUs linked with Chl concentrations macro-aggregate gels seen during P2 in the VAHINE experi- (Fig. 5), which argues for their ability to access phytoplank- ment (Berman-Frank et al., 2016). ton detritus, while one OTU was closely linked with N2 fixa- tion. Notably, none of these three OTUs represented the Tha- 4.5 Possible functional roles of bacterial lassobius sp. OTU that was responsible for the Rhodobacter- subcommunities in the degradation of organic aceae peak on day 4. matter 4.6 The diazotroph community and its impact on the The concerted increase of Cytophaga and Flavobacteriia to- heterotroph-community structure wards the end of the experiment is a recurrent feature seen in older studies based on plateable strains (Fukami et al., 1981b; Diazotroph-community structure (Figs. 3f, 4f) probably Painting et al., 1989) and in molecular studies allowing ac- changed in M1 due to DIP fertilization and reduced turbu- cess to the uncultivable community (Teeling et al., 2012). lence, with Cyanothece-like cells profiting and forming large Both Cytophaga and Flavobacteriia profit from phytoplank- aggregates during P2 (Bonnet et al., 2015b). Our 16S-based ton blooms and have often been found in marine snow (De- diazotroph abundances compare reasonably well with those Long et al., 1993; Fontanez et al., 2015; Teeling et al., 2012). found by quantitative PCR of the nifH gene (Turk-Kubo et Members of the Flavobacteriia are known to degrade organic al., 2015), if we assume that those 16S reads classified as matter, some even specializing on algal-derived organic mat- Calothrix are indeed mostly Richelia (Het-1). The SILVA ter (Bauer et al., 2006; Bowman, 2006). In the lagoon, mem- database positions Richelia below the Calothrix node, and bers of the Flavobacteriaceae reached even higher abun- the metatranscriptome data published in this issue show a dances than in M1, arguing against M1-specific effects be- substantial Richelia read fraction and only little Calothrix ing the reason for their increase. The occurrence of different (Pfreundt et al., 2016), supporting this assumption. Further, Flavobacteriaceae phylotypes seems to depend on the na- UCYN-C (Cyanothece) nifH gene abundance was at max- ture of the dominant phytoplankton species (Pinhassi et al., imum 100 copies mL−1 in M1 (Turk-Kubo et al., 2015), 2004). Information retrieved from analysis of cultured repre- which matches well the maximal 400 cells mL−1 that we in- sentatives showed a high capacity to degrade high molecular ferred from relative 16S values for Cyanothece (Fig. 3f). weight molecules, which is in line with the high ectoenzy- Note that there are also non-diazotrophic Cyanothece repre-

Biogeosciences, 13, 2319–2337, 2016 www.biogeosciences.net/13/2319/2016/ U. Pfreundt et al.: Succession within the prokaryotic communities 2333 sentatives, and thus the second number can be expected to be to UCYN-C after day 14, together with very high N2-fixation larger. This is remarkable given the different methods used rates (Bonnet et al., 2015b), led to an increase in abundance to produce these numbers. of almost all heterotrophic groups investigated here, consis- The higher Trichodesmium abundance inside M1 after its tent with N limitation of BP (Van Wambeke et al., 2015). deployment might be attributed to entrapment of filaments that were not present in the lagoon anymore when the sam- pling started, likely due to high tide currents. The reduction in the number of 16S tags belonging to Trichodesmium fol- 5 Conclusions lowing the DIP fertilization on the evening of day 4 was un- expected. To the contrary, the combined effects of reduced We show that despite their large size (> 50 m3), the VAHINE turbulence and P limitation was thought to trigger a Tri- mesocosms led to significant changes in the microbial com- chodesmium bloom. However, a Trichodesmium bloom was munity structure, probably initiated by decay of Synechococ- observed at the end of the VAHINE experiment in the lagoon cus and diatoms (Leblanc et al., 2016), albeit not exhibit- (Spungin et al., 2016), demonstrating that either the small ing a typical bottle effect with a copiotroph-dominated com- numbers of existing filaments were competent to build up a munity. The accompanying metatranscriptome study for M1 bloom rapidly or that most Trichodesmium resided in deeper showed gene expression from cyanophages, which might waters before the bloom. While we did see cyanophage gene have contributed to Synechococcus decay. Fertilization with expression in M1 in the accompanying metatranscriptome 0.8 µM DIP on day 4 did not have directly observable effects study (Pfreundt et al., 2016), the known host ranges of those on the overall community, as samples from before and 4 days phages suggest that they rather would act on Synechococcus, after fertilization clustered together, but likely influenced excluding viral lysis as the reason for the Trichodesmium development of individual populations, like Defluviicoccus- decline. Thus, there were likely other factors involved that related bacteria or UCYN-C (Cyanothece) diazotrophs. The deserve further investigation. Trichodesmium has compara- latter increased after day 10 only in the mesocosms and were tively low growth rates (Turk-Kubo et al., 2015), so enhanced probably limited by temperature before that (Turk-Kubo et competition after DIP fertilization might be such a factor. al., 2015). The shift of the dominant diazotroph from Riche- Average N -fixation rates of 27.3 ± 1.0 nmol N L−1 d−1 2 lia and Trichodesmium to UCYN-C after day 14 (Turk-Kubo were measured inside the mesocosms during P2 (days 15– et al., 2015), together with extremely high N -fixation rates 23), and with 69.7 nmol N L−1 d−1 very high maximum rates 2 (Bonnet et al., 2015b), led to an increase in abundance of were reached in M1 between days 18 and 21. A short- almost all heterotrophic groups investigated here, consistent term experiment performed by Bonnet et al. (Bonnet et with N-limited BP (Van Wambeke et al., 2015). al., 2015b) indicated that DDN was efficiently transferred Linked with the observation of tight correlation between to non-diazotrophic microbial communities during P2: af- SAR11 and SAR86 transcription during a diel cycle (Ayl- ter 24 h, 16 ± 6 % of the fixed N2 was released in the dis- ward et al., 2015) and over 3 weeks (Pfreundt et al., 2016), solved nitrogen pool and 18 ± 4 % was transferred to non- we show that the temporal dynamics of individual OTUs of diazotrophic picoplankton. We show that almost all het- these clades over 3 weeks can be explained by very simi- erotrophic classes analyzed here (> 0.5 % relative abundance) lar combinations of environmental variables. With the results increased during this period (Fig. 3). Another publication of this work we present for the first time in this ecosystem an in this issue showed that BP was stimulated after short- in-depth analysis of the prokaryotic diversity and community term ammonium plus nitrate enrichments on days 4 and 20 structure and of their changes over time with a focus on di- (Van Wambeke et al., 2015). This speaks for a positive ef- azotrophic organisms. We correlate the prokaryotic commu- fect of released DDN on heterotrophic growth during P2, but nity structure with effects of DIP fertilization, of enclosure effects through phytoplankton growth and decay probably vs. open lagoon waters, and with the fate of DDN and DOC. also played a role. Indeed, nitrogen inputs from N fixation 2 The determined microbial diversity was very high, and we alone were not sufficient to sustain BP (Van Wambeke et al., provide evidence for previously unknown niche partitioning, 2015). Constrained ordination showed that generally, tem- e.g., among dominant SAR86 OTUs. These results suggest poral variation of dominant Rhodobacteraceae, Halieaceae a plethora of still unknown metabolic and regulatory inter- (OM60(NOR5), in SILVA release 119 within the Alteromon- actions to occur within these marine microbial communities adaceae), and Flavobacteriia could be partially be explained that are worth being explored further. with changes in N2 fixation, whereas variation in other al- phaproteobacteria (mainly SAR11, SAR116, and Rhodospir- illaceae), SAR86, Euryarchaeota, and Alteromonas abun- Data availability dances were not explainable by N2 fixation (Fig. 6), indicat- ing the involvement of additional factors. However, it appears All raw sequencing data can be downloaded from NCBI’s very clear that the shift within the diazotroph community BioProject database under the accession number BioProject from the dominant diazotrophs Richelia and Trichodesmium PRJNA304389 (Pfreundt et al., 2015). www.biogeosciences.net/13/2319/2016/ Biogeosciences, 13, 2319–2337, 2016 2334 U. Pfreundt et al.: Succession within the prokaryotic communities

The Supplement related to this article is available online transcriptional networks are conserved in three domains at at doi:10.5194/bg-13-2319-2016-supplement. ocean basin scales, P. Natl. Acad. Sci. USA, 112, 5443–5448, doi:10.1073/pnas.1502883112, 2015. Bauer, M., Kube, M., Teeling, H., Richter, M., Lombardot, T., Allers, E., Würdemann, C. A., Quast, C., Kuhl, H., Knaust, F., Woebken, D., Bischof, K., Mussmann, M., Choudhuri, J. V., Author contributions. Sophie Bonnet is the chief scientist responsi- Meyer, F., Reinhardt, R., Amann, R. I., and Glöckner, F. O.: ble for the VAHINE program; she designed and executed the meso- Whole genome analysis of the marine Bacteroidetes “Gramella cosm experiment and measured N2 fixation. France Van Wambeke forsetii” reveals adaptations to degradation of polymeric organic sampled for and prepared Figs. 1 and 6, Mathieu Caffin performed matter, Environ. Microbiol., 8, 2201–2213, doi:10.1111/j.1462- flow cytometry, Wolfgang R. Hess and Ulrike Pfreundt took sam- 2920.2006.01152.x, 2006. ples for DNA extraction, Ulrike Pfreundt prepared and analyzed Beier, P. and de Albuquerque, F. S.: Environmental diversity as a 16S rRNA gene amplicons and prepared all other figures, Ul- surrogate for species representation, Conserv. Biol., 29, 1401– rike Pfreundt, France Van Wambeke, and Wolfgang R. Hess drafted 1410, doi:10.1111/cobi.12495, 2015. the manuscript, and all authors reviewed the manuscript. Berman-Frank, I., Spungin, D., Rahav, E., Van Wambeke, F., Turk- Kubo, K., and Moutin, T.: Dynamics of transparent exopoly- mer particles (TEP) during the VAHINE mesocosm experi- ment in the New Caledonia lagoon, Biogeosciences Discuss., Acknowledgements. Funding for this research was provided by the doi:10.5194/bg-2015-612, in review, 2016. Agence Nationale de la Recherche (ANR starting grant VAHINE Berthelot, H., Moutin, T., L’Helguen, S., Leblanc, K., Hélias, S., ANR-13-JS06-0002), INSU-LEFE-CYBER program, GOPS, IRD, Grosso, O., Leblond, N., Charrière, B., and Bonnet, S.: Dinitro- and MIO. The participation of UP and WRH was supported by gen fixation and dissolved organic nitrogen fueled primary pro- the German–Israeli Research Foundation (GIF), project number duction and particulate export during the VAHINE mesocosm 1133-13.8/2011, and the MiSeq-based microbial community experiment (New Caledonia lagoon), Biogeosciences, 12, 4099– analysis by the EU project MaCuMBA (Marine Microorganisms: 4112, doi:10.5194/bg-12-4099-2015, 2015. Cultivation Methods for Improving their Biotechnological Ap- Biddanda, B. and Pomeroy, L.: Microbial aggregation and plications; grant agreement no. 311975) to Wolfgang R. Hess. degradation of phytoplankton-derived detritus in seawater. The authors thank the captain and crew of the R/V Alis. We I. Microbial succession, Mar. Ecol.-Prog. Ser., 42, 79–88, acknowledge the SEOH divers’ service from the IRD research doi:10.3354/meps042079, 1988. center of Nouméa (E. Folcher, B. Bourgeois, and A. Renaud) and Biegala, I. C. and Raimbault, P.: High abundance of diazotrophic from the Observatoire Océanologique de Villefranche-sur-Mer picocyanobacteria (< 3 µm) in a Southwest Pacific coral lagoon, (OOV, J. M. Grisoni) as well as the technical service of the IRD Aquat. Microb. Ecol., 51, 45–53, doi:10.3354/ame01185, 2008. research center of Nouméa for their helpful technical support. Bonnet, S., Rodier, M., Turk-Kubo, K. A., Germineaud, C., C. Guieu, F. Louis, and J. M. Grisoni (Laboratoire d’Océanographie Menkes, C., Ganachaud, A., Cravatte, S., Raimbault, P., Camp- de Villefranche and Observatoire céalonologique de Villefranche- bell, E., Quéroué, F., Sarthou, G., Desnues, A., Maes, C., and sur-Mer) are warmly thanked for the mesocosms design and their Eldin, G.: Contrasted geographical distribution of N2 fixation useful advice for deployment. The authors thank Anne Desnues rates and nifH phylotypes in the Coral and Solomon Seas and Dina Spungin for help in sampling, T. Moutin, M. Rodier, (South-Western Pacific) during austral winter conditions, Global and H. Berthelot for providing their data, Danny Ionescu (Leibniz Biogeochem. Cy., 29, 1874–1892, doi:10.1002/2015GB005117, Institute for Freshwater Ecology and Inland Fisheries, Berlin) for 2015a. introduction to and initial help with the SILVA pipeline, and Craig Bonnet, S., Berthelot, H., Turk-Kubo, K., Fawcett, S., Rahav, E., Nelson (University of Hawai’i at Manoa) for advice on NMDS l’Helguen, S., and Berman-Frank, I.: Dynamics of N2 fixation analysis. We are grateful to the Regional Flow Cytometry Platform and fate of diazotroph-derived nitrogen in a low nutrient low for Microbiology (PRECYM) of the Mediterranean Institute of chlorophyll ecosystem: results from the VAHINE mesocosm ex- Oceanography for the flow cytometry analyses. We thank the periment (New Caledonia), Biogeosciences Discuss., 12, 19579– reviewers of this manuscript for their valuable comments. 19626, doi:10.5194/bgd-12-19579-2015, 2015b. Bonnet, S., Moutin, T., Rodier, M., Grisoni, J. M., Louis, F., Folcher, Edited by: E. Marañón E., Bourgeois, B., Boré, J. 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