N2 fixation in the Mediterranean Sea related to the composition of the diazotrophic community and impact of dust under present and future environmental conditions

. N 2 ﬁxation rates were measured in the 0–1000 m layer at 13 stations located in the open western and central Mediterranean Sea (MS) during the PEACETIME cruise (late spring 2017). While the spatial variability in N 2 ﬁxation was not related to Fe, P nor N stocks, the surface composition of the diazotrophic community indicated a strong longitudinal gradient increasing eastward for the relative abundance of non-cyanobacterial diazotrophs (NCDs) (mainly γ -Proteobacteria) and conversely decreasing eastward for photo-heterotrophic group A (UCYN-A) (mainly UCYN-A1 and UCYN-A3), as did N 2 ﬁxation rates. UCYN-A4 and UCYN-A3 were identiﬁed for the ﬁrst time in the MS. The westernmost station inﬂuenced by Atlantic waters and characterized by highest stocks of N and P displayed a patchy distribution of diazotrophic activity with an exceptionally high rate in the euphotic layer of 72.1 nmolNL − 1 d − 1 , which could support up to 19 % of primary production. At this station at 1 %PAR (photosynthetically available radiation) depth, UCYN-A4 represented up to 94 % of the diazotrophic community. These in situ observations of greater relative abundance of UCYN-A at stations with higher nutrient concentrations and dominance of NCDs at more oligotrophic stations suggest that nutrient conditions – even in the nanomolar range – may determine the composition of diazotrophic communities and in turn N 2 ﬁxation rates. The impact of Saharan dust deposition on N 2 ﬁxation and diazotrophic communities was also investigated, under present and future projected conditions of temperature and pH during short-term d) experiments at three stations. New nutrients from simulated dust deposition triggered a signiﬁcant stimulation of

Abstract. N 2 fixation rates were measured in the 0-1000 m layer at 13 stations located in the open western and central Mediterranean Sea (MS) during the PEACETIME cruise (late spring 2017). While the spatial variability in N 2 fixation was not related to Fe, P nor N stocks, the surface composition of the diazotrophic community indicated a strong longitudinal gradient increasing eastward for the relative abundance of non-cyanobacterial diazotrophs (NCDs) (mainly γ -Proteobacteria) and conversely decreasing eastward for photo-heterotrophic group A (UCYN-A) (mainly UCYN-A1 and UCYN-A3), as did N 2 fixation rates. UCYN-A4 and UCYN-A3 were identified for the first time in the MS. The westernmost station influenced by Atlantic waters and characterized by highest stocks of N and P displayed a patchy distribution of diazotrophic activity with an exceptionally high rate in the euphotic layer of 72.1 nmol N L −1 d −1 , which could support up to 19 % of primary production. At this station at 1 % PAR (photosynthetically available radiation) depth, UCYN-A4 represented up to 94 % of the diazotrophic community. These in situ observations of greater relative abundance of UCYN-A at stations with higher nutrient concentrations and dominance of NCDs at more oligotrophic stations suggest that nutrient conditions -even in the nanomolar range -may determine the composition of diazotrophic communities and in turn N 2 fixation rates. The impact of Saharan dust deposition on N 2 fixation and diazotrophic communities was also investigated, under present and future projected conditions of temperature and pH during short-term (3-4 d) experiments at three stations. New nutrients from simulated dust deposition triggered a significant stimulation of N 2 fixation (from 41 % to 565 %). The strongest increase in N 2 fixation was observed at the stations dominated by NCDs and did not lead on this short timescale to changes in the diazotrophic community composition. Under projected future conditions, N 2 fixation was either increased or unchanged; in that later case this was probably due to a too-low nutrient bioavailability or an increased grazing pressure. The future warming and acidification likely benefited NCDs (Pseudomonas) and
The MS is strongly impacted by periodic dust events, originating from the Sahara, which have been recognized as a significant source of macro-and micronutrients to the nutrientdepleted SML during stratified periods (Guieu and Ridame, 2020a and references therein;Mas et al., 2020). Results from Saharan dust seeding experiments during open-sea microcosms and coastal mesocosms in the MS showed stimulation of both PP (Herut et al., 2005;Ternon et al., 2011;Ridame et al., 2014;Herut et al., 2016) and heterotrophic bacterial production (BP) (Pulido-Villena et al., 2008Lekunberri et al., 2010;Herut et al., 2016). Experimental Saharan dust seeding was also shown to enhance N 2 fixation in the western and eastern MS Ternon et al., 2011;Ridame et al., 2013;Rahav et al., 2016a) and to alter the composition of the diazotrophic community (Rahav et al., 2016a), as also shown in the tropical North Atlantic .
The MS has been identified as one of the primary hotspots for climate change (Giorgi, 2006). Future sea surface warming and associated increase in stratification (Somot et al., 2008) might reinforce the importance of atmospheric inputs as a source of new nutrients for biological activities during that season, including diazotrophic microorganisms. This fertilizing effect could also be enhanced by the expected decline in pH (Mermex Group, 2011), which could increase the nutrient dust solubility in seawater. Under nutrient-repleted conditions, predicted elevated temperature and CO 2 concentration favor the growth and N 2 fixation of the filamentous cyanobacteria Trichodesmium and of the photo-autotrophic UCYN-B and UCYN-C (Webb et al., 2008;Hutchins et al., 2013;Fu et al., 2008Fu et al., , 2014Eichner et al., 2014;Jiang et al., 2018), whereas effects on UCYN-A and non-cyanobacterial diazotrophs (NCDs) are uncertain.
In this context, the first objective of this study is to investigate during the season characterized by strong stratification and low productivity the spatial variability in N 2 fixation rates in relation to nutrient availability and diazotrophic community composition. The second objective was to study, for the first time, the impact of a realistic Saharan deposition event in the open MS on N 2 fixation rates and diazotrophic community composition under present and realistic projected conditions of temperature and pH for 2100.

Oceanographic cruise
All data were acquired during the PEACETIME cruise (Pro-cEss studies at the Air-sEa Interface after dust deposition in the MEditerranean sea) in the western and central MS on board the R/V Pourquoi Pas? from 10 May to 11 June 2017 (http://peacetime-project.org/, last access: 17 January 2022) (see the detailed description in Guieu et al., 2020a). The cruise track, including 10 short stations (ST1 to ST10) and 3 long stations (TYR, ION and FAST), is shown in Fig. 1 (coordinates in Table S1 in the Supplement). Stations 1 and 2 were located in the Liguro-Provencal Basin; Stations 5, 6 and TYR in the Tyrrhenian Sea; Stations 7, 8 and ION in the Ionian Sea; and Stations 3, 4, 9, 10 and FAST in the Algerian Basin.

Dust seeding experiments
Experimental dust seedings into six large tanks were conducted at each of the three long stations (TYR, ION and FAST) under present and future conditions of temperature and pH. Based on previous studies, the location of these stations was chosen based on several criteria and because they represent three main bioregions of the MS (Guieu et al., 2020a, their Fig. S1). They are located along the longitudinal gradient in biological activity, including the activity of diazotrophs decreasing eastward Rahav et al., 2013). The experimental setup is fully described in a companion paper (Gazeau et al., 2021a). Briefly, six climate reactors (volume of about 300 L) made in high-density polyethylene were placed in a temperature-controlled container and covered with a lid equipped with LEDs to reproduce the natural light cycle. The tanks were filled with unfiltered surface seawater collected at ∼ 5 m with a peristaltic pump at the end of the day (T − 12 h) before the start of the experiments the next morning (T0). Two replicate tanks were amended with mineral Saharan dust (dust treatments D1 and D2) simulating a high but realistic atmospheric dust deposition of 10 g m −2 (Guieu et al., 2010b). Two other tanks were also amended with Saharan dust (same dust flux as in the dust treatment) under warmer (∼ +3 • C) and more acidic water conditions (∼ −0.3 pH unit) (greenhouse treatments G1 and G2). This corresponds to the Intergovernmental Panel on Climate Change (IPCC) projections for 2100 under RCP8.5 (IPCC, 2019). Seawater in G1 and G2 was warmed overnight to reach +3 • C and acidified through the addition of CO 2saturated 0.2 µm filtered seawater (∼ 1.5 L in 300 L). The difference in temperature between G (greenhouse) tanks and other tanks (C, controls, and D, dust) was +3, +3.2 and +3.6 • C at TYR, ION and FAST, respectively, and the decrease in pH was −0.31, −0.29 and −0.33 at TYR, ION and FAST, respectively (Gazeau et al., 2021a). Two tanks were filled with untreated water (Controls C1 and C2). The experiment at TYR and ION lasted 3 d, while the experiment at FAST lasted 4 d. The sampling session took place every morning at the same time over the duration of the experiments.
The fine fraction (< 20 µm) of a Saharan soil collected in southern Tunisia used in this study has been previously used for the seeding of mesocosms in the framework of the DUNE project (a DUst experiment in a low-Nutrient, lowchlorophyll Ecosystem). Briefly, the dust was previously subjected to physico-chemical transformations mimicking the mixing between dust and pollution air masses during atmospheric transport (see details in Desboeufs et al., 2001;Guieu et al., 2010b). This dust contained 0.055 ± 0.003 % of P, 1.36 ± 0.09 % of N and 2.26 ± 0.03 % of Fe in weight (Desboeufs et al., 2014). Right before the artificial seeding, the Table 1. Integrated N 2 fixation over the surface mixed layer (SML; from surface to the mixed layer depth), from the surface to the base of the euphotic layer (1 % PAR depth), over the aphotic layer (1 % PAR depth to 1000 m) and from the surface to 1000 m at all the sampled stations. Contribution (in percent) of SML-integrated N 2 fixation to euphotic-layer-integrated N 2 fixation and contribution of euphotic-layerintegrated N 2 fixation to total (0-1000 m) integrated N 2 fixation. N 2 Fix SML N 2 Fix euphotic N 2 Fix aphotic N 2 Fix 0-1000 m N 2 Fix SML / N 2 Fix euphotic / N 2 Fix euphotic N 2 Fix 0-1000 m µmol N m

N 2 fixation and primary production
All materials were acid-washed (HCl Suprapur 32 %) following trace metal clean procedures. Before sampling, bottles were rinsed three times with the sampled seawater. For the in situ measurements, seawater was sampled using a trace metal clean (TMC) rosette equipped with 24 GO-FLO bottles (Guieu et al., 2020a). At each station, seven to nine depths were sampled between the surface and 1000 m for N 2 fixation measurements and five depths between surface and ∼ 100 m for primary production measurements (one sample per depth). During the seeding experiments, the six tanks were sampled for simultaneous determination of net N 2 and CO 2 fixation rates before dust seeding (initial time T0) and 1 d (T1), 2 d (T2) and 3 d (T3) after dust addition at TYR and ION stations. At FAST, the last sampling took place 4 d (T4) after dust addition. After collection, 2.3 L of seawater was immediately filtered onto pre-combusted GF/F (glass microfiber) filters to determine natural concentrations and isotopic signatures of particulate organic carbon (POC) and particulate nitrogen (PN). Net N 2 fixation rates were determined using the 15 N 2 gas tracer addition method (Montoya et al., 1996) and net primary production using the 13 C tracer addition method (Hama et al., 1983). Immediately after sampling, 1 mL of NaH 13 CO 3 (99 %, Eurisotop) and 2.5 mL of 99 % 15 N 2 (Eurisotop) was introduced to 2.3 L polycarbonate bottles through a butyl septum for simultaneous determination of N 2 and CO 2 fixation. 15 N 2 and 13 C tracers were added to obtain a ∼ 10 % final enrichment. Then, each bottle was vigorously shaken before incubation for 24 h. The in situ samples from the euphotic zone were incubated in on-deck containers with circulating seawater, equipped with blue filters with different sets of blue neutral density filters (LEE Filters) (percentages of attenuation: 70, 52, 38, 25, 14, 7, 4, 2 and 1 %) to simulate an irradiance level (% PAR) as close as possible to the one corresponding to their depth of origin. Samples for N 2 fixation determination in the aphotic layer were incubated in the dark in thermostated incubators set at in situ temperature. In situ 13 C PP will not be discussed in this paper as 14 C PP rates are presented in Maranon et al. (2021) (see details in Fig. S1 in the Supplement). The in situ 13 C PP and molar C : N ratio in the organic particulate matter, measured simultaneously in our samples (see below for details), were used to estimate the contribution of N 2 fixation to PP.
Samples from the dust addition experiments were incubated in two tanks dedicated to incubation: one tank at the same temperature and irradiance as the C and D tanks and another one at the same temperature and irradiance as the G tanks. It should be noted that 14 C PP was also measured during the seeding experiments (Gazeau et al., 2021b;Fig. S1 in the Supplement).
After 24 h incubation, 2.3 L was filtered onto precombusted 25 mm GF/F filters, and filters were stored at −25 • C. Filters were then dried at 40 • C for 48 h before analysis. POC and PN as well as 15 N and 13 C isotopic ratios were quantified using an online continuous flow elemental analyzer (Flash 2000 HT) coupled with an isotopic ratio mass spectrometer (Delta V Advantage via a ConFlow IV interface from Thermo Fisher Scientific). For each sample, POC (in the 0-100 m layer) and PN (0-1000 m) were higher than the analytically determined detection limit of 0.15 µmol for C and 0.11 µmol for N. Standard deviations were 0.0007 at. % and 0.0005 at. % for 13 C and 15 N enrichment, respectively. The atomic percent excess of the dissolved inorganic carbon (DIC) was calculated by using measured DIC concentrations at the LOCEAN laboratory (SNAPO-CO 2 ). N 2 fixation rates were calculated by isotope mass balance equations as described by Montoya et al. (1996). For each sample, the 13 C and 15 N uptake rates were considered to be significant when excess enrichment of POC and PN was greater than 3 times the standard deviation obtained on natural samples. According to our experimental conditions, the minimum detectable 13 C and 15 N uptake rates in our samples were 5 nmol C L −1 d −1 and 0.04 nmol N L −1 d −1 , respectively. CO 2 uptake rates were above the detection limit in the upper 0-100 m, while N 2 fixation was not quantifiable below 300 m depth except at Stations 1 and 10 with rates of ∼ 0.05 nmol N L −1 d −1 at 500 m depth. From these measurements, the molar C : N ratio in the organic particulate matter was calculated and used to estimate the contribution of N 2 fixation to primary production. As a rough estimate of the potential impact of bioavailable N input from N 2 fixation on BP, we used the BP rates presented in companion papers (Gazeau et al., 2021b;Van Wambeke et al., 2021) and converted them to N demand using the molar ratio C : N of 6.8 (Fukuda et al., 1998). Trapezoidal method was used to calculate integrated rates over the SML, the euphotic layer (from surface to 1 % photosynthetically available radiation (PAR) depth) and the 0-1000 m water column.
It must be noted that N 2 fixation rates measured by the 15 N 2 tracer gas addition method may have been underestimated due to incomplete 15 N 2 gas bubble equilibration (Mohr et al., 2010). However, this potential underestimation is strongly lowered during long incubation (24 h).
The relative changes (RC, in percent) in N 2 fixation in the dust experiments were calculated as follows: with N 2 FIXATION Tx the rate in D1, D2, G1 or G2 at Tx; N 2 FIXATION Control the mean of the duplicated controls (C1 and C2) at Tx; and Tx the time of the sampling.

Composition of the diazotrophic community
Samples for characterization of the diazotrophic communities were collected during the dust seeding experiments in the six tanks at initial time before seeding (T0) and final time (T3 at TYR and ION and T4 at FAST); 3 L of water was collected in acid-washed containers from each tank, filtered onto 0.2 µm PES (polyethersulfone) filters (Sterivex) and stored at −80 • C until DNA extraction. The composition of the diazotrophic community was also determined at four depths (10, 61, 88 and 200 m) at Station 10. Here, 2 L seawater was collected from the TMC rosette. Immediately after collection, seawater was filtered under low vacuum pressure through a 0.2 µm nuclepore membrane and stored at −80 • C in cryovials. Nucleic acids were obtained from both filter types using phenol-chloroform extraction followed by purification (NucleoSpin ® PlantII kit; Macherey-Nagel). DNA extracts were used as templates for PCR (polymerase chain reaction) amplification of the nifH gene by nested PCR protocol as fully described in protocol.io). Following polymerase chain reactions, DNA amplicons were purified and quantified using NanoQuant Plate™ and Tecan Spark ® (Tecan Trading AG, Switzerland). Each PCR product was normalized to 30 ng µL −1 in the final 50 µL and sent to Genotoul (https://www.genotoul.fr/, last access: 17 January 2022; Toulouse, France) for high-throughput sequencing using paired-end 2x250bp Illumina MiSeq. All reads were processed using the Quantitative Insight Into Microbial Ecology 2 pipeline (QIIME2 v2020.2; Bolyen et al., 2019). Reads were truncated to 350 bp based on sequencing quality, denoised, merged and chimera-checked using DADA2 (Callahan et al., 2016). A total of 1 029 778 reads were assigned to 635 amplicon sequence variants (ASVs). The table was rarefied by filtering at 1 % relative abundance per sample cut-off that reduced the dataset to 97 ASVs, accounting for 98.27 % of all reads. Filtering for homologous genes was done using the NifMAP pipeline (Angel et al., 2018) and translation into amino acids using FrameBot (Wang et al., 2013). This yielded 235 ASVs, accounting for 1 022 184 reads (99 %). These remaining ASVs were classified with DIAMOND blastp (Buchfink et al., 2015) (Farnelid et al., 2016;Turk-Kubo et al., 2017). All sequences associated with this study have been deposited under the BioProject ID PRJNA693966. Alpha and beta diversity indices for community composition were estimated after randomized subsampling. Analyses were run in QIIME 2 and in the Primer v.6 software package (Clarke and Warwick, 2001). Table 2. Initial physico-chemical and biological properties of surface seawater before the perturbation in the dust seeding experiments at TYR, ION and FAST (average at T0 in C and D treatments (n = 4) or value at T − 12 h (n = 1) in the pumped surface waters). The relative abundances of diazotrophic cyanobacteria and NCDs (non-cyanobacterial diazotrophs) are given as proportion of total nifH sequence reads. DIP: dissolved inorganic phosphorus; DFe: dissolved iron. The C : N ratio corresponds to the ratio in the organic particulate matter from IRMS (isotopic ratio mass spectrometer) measurements (> 0.7 µm). Means that did not differ significantly between the experiments (p > 0.05) are labeled with the same letter (in parentheses

Complementary data from PEACETIME companions papers
Bacterial production. Heterotrophic bacterial production (BP; sensus stricto referring to prokaryotic heterotrophic production) was determined on board using the microcentrifuge method with the 3 H-leucine ( 3 H-Leu) incorporation technique to measure protein production (Smith and Azam, 1992). The detailed protocol and the rates of BP are presented in Van Wambeke et al. (2021) for measurements in the water column and in Gazeau et al. (2021b) for measurements over the course of the dust seeding experiments. Dissolved Fe. Dissolved iron (DFe) concentrations (< 0.2 µm) were measured by flow injection analysis with online preconcentration and chemiluminescence detection (FIA-CL). The detection limit was 15 pM . DFe concentrations in the water column along the whole transect are presented in Bressac et al. (2021) and for the dust seeding experiments in Roy- Barman et al. (2021).
Dissolved inorganic phosphorus and nitrate. Concentrations of DIP and nitrate (NO − 3 ) were analyzed immediately after collection on 0.2 µm filtered seawater using a segmented flow analyzer (AAIII HR Seal Analytical) according to Aminot and Kérouel (2007) with respective detection limits of 0.02 and 0.05 µmol L −1 . Samples with concentrations below the limit of detection with standard analysis were analyzed by spectrophotometry using a 2.5 m long waveguide capillary cell (LWCC) for DIP  and a 1 m LWCC for NO − 3 (Louis et al., 2015); the limit of detection was 1 nM for DIP and 6 nM for NO

Statistical analysis
Pearson's correlation coefficient was used to test the statistical linear relationship (p < 0.05) between N 2 fixation and other variables (BP, PP, DFe, DIP, NO − 3 ); it should be noted that the DIN stocks estimated at Stations 1 to 4 (Table S1) were excluded from statistical analysis. In the dust seeding experiments, means at initial time (T0) before dust amendment (average at T0 in C and D treatments, n = 4; see Table 2) were compared using a one-way ANOVA followed by a Tukey means comparison test (α = 0.05). When assumptions for ANOVA were not respected, means were compared using a Kruskal-Wallis test and a post hoc Dunn test. To test significant differences (p < 0.05) between the slopes of N 2 fixation as a function of time in the C, D and G treatments (n = 8), an ANCOVA was performed on data presenting a significant linear relationship with time (Pearson's correlation coefficient, p < 0.05). Statistical tests were done using XLSTAT and R (version 4.1.1 with the stats, tidyverse and FactoMineR packages). Over the cruise, the water column was well stratified with a shallow SML varying from 7 to 21 m depth (Table S1). Detectable N 2 fixation rates in the 0-1000 m layer ranged from 0.04 to an exceptionally high rate of 72.1 nmol N L −1 d −1 at Station 10 (Fig. 2). Vertical N 2 fixation profiles exhibited a similar shape at all stations, with maximum values within the euphotic layer and undetectable values be-low 300 m depth (except at Stations 1 and 10, with rates of ∼ 0.05 nmol N L −1 d −1 at 500 m depth). Within the euphotic layer, all the rates were well above the detection limit (DL = 0.04 nmol N L −1 d −1 , minimum in situ N 2 fixation = 0.22 nmol N L −1 d −1 ). The highest rates were generally found below the SML and the lowest at the base of the euphotic layer or within the SML (Fig. 2). The lowest N 2 fixation rates integrated over the euphotic and aphotic (defined as 1 % PAR depth to 1000 m) layers were found at Station 8 and the highest at Station 10 (Table 1). On average, 59 ± 16 % of N 2 fixation (min 42 % at TYR and ION, max 97 % at Station 10) took place within the euphotic layer (Table 1). The contribution of the SML-integrated N 2 fixation to Figure 3. Volumetric surface (∼ 5 m) (a) and integrated N 2 fixation from surface to euphotic layer depth (b) along the longitudinal PEACETIME transect (Station 10 was excluded). Integrated N 2 fixation rate from Station 10 was excluded from statistical analysis.

N 2 fixation and composition of diazotrophs at Station 10
The westernmost station, Station 10, was in sharp contrast to all other stations, with an euphotic-layer-integrated N 2 fixation on average 44 times higher (Table 1) due to high rates of 2.9 at 37 m and 72.1 nmol N L −1 d −1 at 61 m (i.e., at the deep chlorophyll a maximum, DCM) (Fig. 2). That rate at 61 m was associated with a maximum in PP but not with a maximum in BP. From the surface to 200 m depth, the nifH community composition was largely dominated by ASVs related to different UCYN-A groups (Fig. 4)

N 2 fixation versus primary production, heterotrophic bacterial production, nutrients
For statistical analysis, due to the high integrated N 2 fixation rate from Station 10, this rate was not included in order to not bias the analysis. N 2 fixation rate integrated over the euphotic layer correlated strongly with PP (r = 0.71, p < 0.05) and BP (r = 0.76, p < 0.05) (Fig. 5). Integrated N 2 fixation over Figure 5. N 2 fixation rate integrated over the euphotic layer versus 13 C primary production (a) and bacterial production (b); data at Station 10 were removed.
the euphotic layer (and over the SML) was not correlated with the associated DFe, DIP and NO − 3 stocks (p > 0.05). It should be noted that DIP and NO − 3 stocks correlated positively with PP and BP (p < 0.05) over the euphotic layer (no correlation between DFe stock, PP and BP).
3.2 Response of N 2 fixation and composition of the diazotrophic communities to dust seeding 3.2.1 Initial characteristics of the tested seawater N 2 fixation and BP were the highest at FAST, while PP was the highest at FAST and ION ( Table 2). The N 2 fixation rates were similar at ION and TYR and significantly higher (factor ∼ 2.6) at FAST. At TYR and ION, the diazotroph community was largely dominated by NCDs (on average 94.5 % of the total diazotrophic community), whereas at FAST, diazotrophic cyanobacteria, mainly UCYN-A, represented on average 91.4 % of the total diazotrophic community. NO − 3 concentration was the highest at FAST (59 nM), while DIP concentration was the highest at TYR (17 nM) and the lowest at ION (7 nM). The molar NO − 3 : DIP ratio was strongly lower than the Redfield ratio (16 : 1), indicating a potential N limitation of the phytoplanktonic activity in all experiments. DFe concentrations were all higher than 1.5 nM.
3.2.2 Changes in N 2 fixation in response to dust seeding and relationship with changes in primary and heterotrophic bacterial production All the dust seedings led to a significant stimulation of N 2 fixation relative to the controls under present and future climate conditions (D and G treatments) (Figs. 6 and S3 in the Supplement). The reproducibility between the replicated treatments was good at all stations (mean coefficient of variation (CV, in percent) < 14 %). The maximum N 2 fixation relative change (RC) was the highest at TYR (+434-503 % in D1 and D2, +478-565 % in G1 and G2), then at ION (+256-173 % in D1 and D2 and +261-217 % in G1 and G2) and finally at FAST (+41-49 % in D1 and D2 and +97-120 % in G1 and G2) (Fig. 7). At TYR and FAST, dust addition stimulated N 2 fixation more in the G treatment than in D, whereas at ION the response was similar between the treatments (Fig. S3). N 2 fixation measured during the dust seeding experiments correlated strongly with PP at FAST (r = 0.90, p < 0.05) and with BP at TYR and ION (r > 0.76, p < 0.05) (Fig. S4 in the Supplement).

Changes in the diazotrophic composition in response to dust seeding
At TYR and ION, the diazotrophic communities before seeding were largely dominated by NCDs (∼ 94.5 % of total ASVs; Fig. 8, Table 2). These were mainly γ -Proteobacteria related to Pseudomonas. Some of these ASVs had low overall relative abundance and therefore did not appear in the top 20 ASVs (Fig. 8, Tables S2 and S3 in the Supplement) but could nevertheless account for up to 16 % in a specific sample. Filamentous cyanobacteria (Katagnymene) were also observed at both stations (∼ 4.7 % of the total diazotrophs). The community at FAST was initially dominated by UCYN-A phylotypes, mostly represented by UCYN-A1 and UCYN-A3 (relative abundance of UCYN-A1 and UCYN-A3 in C and D treatments at T0; n = 4: 34 ± 6 % and 45 ± 2 % of the total diazotrophic composition, respectively; Fig. 8). At TYR and ION, the variability at T0 between replicates was higher than at FAST (Fig. S5 in the Supplement; C1T0 at TYR was removed due to poor sequencing quality). Also, the diversity (Shannon H ) was generally higher at TYR and ION at the start of incubations compared to FAST (T0; Fig. S6 in the Supplement). For ION and FAST experiments, Pseudomonas-related ASVs were more abundant in G treatments at T0 relative to control and dust treatments (T0). At  the end of the TYR and ION experiments, the community from all treatments appeared to converge (Fig. S5) due to the increase of a few γ -Proteobacteria (mainly Pseudomonas) that strongly increased in all treatments (Fig. 8). At FAST, no difference in the relative abundances of diazotrophs was recorded between D treatment and the controls at T4. However, when comparing G treatment relative to D at T4, the relative contribution of NCDs was higher (82 % in G vs. 63 % in D), and the relative abundance of UCYN-A was lower (13 % in G vs. 31 % in D).

Discussion
Late spring, at the time of sampling, all the stations were well stratified and characterized by oligotrophic conditions increasing eastward (Maranon et al., 2021; Fig. 8 in Guieu et al., 2020a). NO − 3 and DIP concentrations were low in the SML, from 9 to 135 nM for NO − 3 (Van Wambeke et al., 2021) and from 4 to 17 nM for DIP ; the highest stocks were measured at the westernmost station (Station 10) (Table S1).

General features in N 2 fixation and diazotroph community composition
N 2 fixation rates in the aphotic layer were in the range of those previously measured in the western open MS (Benavides et al., 2016) and accounted, on average, for 41 % of N 2 fixation in the 0-1000 m layer, suggesting that a large part of the total diazotrophic activity was related to heterotrophic NCDs in the aphotic layer. N 2 fixation rates in the euphotic layer were of the same order of magnitude (data from Station 10 excluded) as those previously measured in the open MS in spring and summer Rahav et al., 2013). At the tested stations, the surface diazotrophic cyanobacteria were largely dominated by UCYN-A (∼ 93 % of the total diazotrophic cyanobacteria, mostly UCYN-A1 and UCYN-A3) and the NCD community by γ -Proteobacteria (∼ 95 % of the total NCDs). This is the first time that UCYN-A3 and UCYN-A4 are detected in the MS. The photo-autotrophic N 2 fixation was negligible as no UCYN-B and UCYN-C were detected, and very low abundance of filamentous cyanobacteria was observed.

Longitudinal gradient of N 2 fixation related to the composition of the diazotrophic communities
At Station 10 and FAST, the surface diazotrophic communities were largely dominated by UCYN-A (> 91 %), whereas at TYR and ION they were dominated by NCDs (> 94 %), which highlights the predominance of photo-heterotrophic diazotrophy in the western waters of the Algerian Basin and of NCD-supported diazotrophy in the Tyrrhenian and Io- nian basins. Surface N 2 fixation exhibited a longitudinal gradient decreasing eastward, as previously reported , Rahav et al., 2013. Strong longitudinal gradients decreasing eastward for the relative abundance of UCYN-A (r = −0.93, p < 0.05) and inversely increasing eastward for NCDs were observed (r = 0.89, p < 0.05) (Fig. 9). Despite no quantitative abundances of distinct diazotrophs for the studied area (in this and previously published studies), the intensity of the bulk N 2 fixation rate was likely related to the overall composition of the diazotrophic communities (here relative abundance of UCYN-A versus NCDs). Indeed, surface N 2 fixation rates correlated positively with the relative abundance of UCYN-A (mainly A1 and A3) (r = 0.98, p < 0.05) and negatively with the relative abundance of NCDs (r = −0.99, p < 0.05) (Fig. S7 in the Supplement). This could be related, in part, to the variability in the cell-specific N 2 fixation rates that were shown to be higher for UCYN-A relative to NCDs Bentzon-Tilia et al., 2015;Martinez-Perez et al., 2016;Pearl et al., 2018;Mills et al., 2020). Besides, in Atlantic and Pacific Ocean areas when the diazotrophic community is dominated by unicellular organisms, high N 2 fixation rates are mostly associated with a predominance of UCYN-A and low rates with a predominance of NCDs , Martinez-Perez et al., 2016Moreiro-Coello et al., 2017Tang et al., 2019).

Intriguing Station 10
The patchy distribution of the diazotrophic activity at Station 10 was related to an exceptionally high rate at the DCM (72.1 nmol N L −1 d −1 ). High N 2 fixation rates have previously been observed locally: 2.4 nmol N L −1 d −1 at the Strait of Gibraltar (Rahav et al., 2013), ∼ 5 nmol N L −1 d −1 in the Bay of Calvi (Rees et al., 2017), 17 nmol N L −1 d −1 in the northwestern MS (Garcia et al., 2006) and 129 nmol N L −1 d −1 in the eastern MS (Rees et al., 2006). Station 10 was also hydrodynamically "contrasted" com-pared to the other stations: it was located almost at the center of an anticyclonic eddy (Guieu et al., 2020a), with the core waters (0-200 m) of Atlantic origin (colder, fresher). In such anticyclonic structures, enhanced exchange with nutrientrich waters from below take place, which, combined with lateral mixing, could explain higher stocks of NO − 3 and DIP in the euphotic layer (Table S1). Nevertheless, the anomaly of N 2 fixation at the DCM was associated with anomalies of neither PP, BP nor NO − 3 and DIP concentrations. It only coincided with a minimum in DFe concentration (0.47 nM compared to 0.7 to 1.4 nM at the nearby depths; Bressac et al., 2021). Based on a range of Fe : C (from 7 to 177 µmol : mol) and associated C : N ratios for diazotrophs (Trichodesmium, UCYN) from the literature (Tuit et al., 2004;Berman-Frank et al., 2007;Jiang et al., 2018), we found that 0.004 to 0.08 nM of DFe is required to sustain this N 2 fixation rate. Consequently, the minimum in DFe concentration at 61 m could not be explained solely by the diazotroph uptake.
Despite no correlation between N 2 fixation and the relative abundance of specific diazotrophs (p > 0.05) along the profile, the huge heterogeneity in N 2 fixation rate was likely related to the patchy distribution of diazotroph taxa. Indeed, patchiness seems to be a common feature of unicellular diazotrophs (Robidart et al., 2014;Moreira-Coello et al., 2019). The exceptionally high N 2 fixation rates coincided with the highest relative contributions of UCYN-A and more precisely UCYN-A4. Exceptional N 2 fixation rates at Station 10, impacted by northeast Atlantic surface waters of subtropical origin, could thus be related to incoming waters. Indeed, Fonseca-Batista et al. (2019) reported high N 2 fixation rates (45 and 65 nmol N L −1 d −1 , with euphotic N 2 fixation rates up to 1533 µmol N m −2 d −1 ) associated with a predominance of UCYN-A in subtropical Atlantic surface water mass along the Iberian Margin (∼ 40 • N, 11 • E). It should be noted that UCYN-A4 was only detected at Station 10, and its relatively high contribution to the whole diazotrophic community in the euphotic layer coincided with the highest stocks of P (and N) (Table S1). This could reflect higher nutrient requirement(s) of the UCYN-A4 and/or of its eukaryotic partner relative to other sublineages. Another intriguing feature was the high contribution (∼ 86 %) of UCYN-A in the mesopelagic zone (200 m). As UCYN-A lives in obligate symbiosis with haptophytes, from which it receives fixed carbon from photosynthesis (Thompson et al., 2012(Thompson et al., , 2014, this suggests that this contribution was probably derived from sinking senescing prymnesiophyte UCYN-A cells and that the weak N 2 fixation rate at 200 m depth is likely only driven by γ -Proteobacteria (Pseudomonas).

Supply of bioavailable N from diazotrophic activity
for fueling primary and heterotrophic bacterial production -relationship with potential controlling factors of N 2 fixation The relationship established between N 2 fixation and PP and BP (Fig. 5) illustrated that in the studied area, N 2 fixation is promoted by UCYN and NCDs and/or could indicate that all processes have the same (co)-limitation. Overall, N 2 fixation was a poor contributor to PP (1.0 ± 0.3 %; Fig. S8 in the Supplement), as previously shown in the MS Yogev et al., 2011;Rahav et al., 2013) and BP (7 ± 1 %; Fig. S8), except at Station 10, where N 2 fixation could support up to 19 % of PP (Fig. S8) and supply the entire bioavailable N requirements for heterotrophic prokaryotes (199 % of BP). As expected, our results suggest no control of N 2 fixation by DFe and NO − 3 , as previously shown through nutrient additions in microcosms (Rees et al., 2006;Ridame et al., 2011, Rahav et al., 2016b. No correlation was observed between N 2 fixation and DIP, which may highlight the spatial variability in the controlling factor of diazotrophs as DIP was shown to control N 2 fixation in the western basin but not in the Ionian Basin . Moreover, DIP concentration does not reflect the rapid turnover of P in the open MS and thus could be a poor indicator of DIP availability .

Diazotrophic activity and composition in response to dust addition under present climate conditions
General features. In all experiments, simulated wet dust deposition under present climate conditions triggered a significant (41 to 503 %) and rapid (24-48 h) stimulation of N 2 fixation relative to the controls. Despite this strong increase, N 2 fixation rates remained low (< 0.7 nmol N L −1 d −1 ) as well as their contribution to PP (< 7 %) and BP (< 5 %), as observed in situ (Sect. 4.4). All of these results are consistent with those found after dust seeding in mesocosms at a coastal site in the northwestern MS (Ridame et al., 2013) and in the open Cretan Sea (Rahav et al., 2016a). Temporal changes in the composition of the diazotrophic community. Dust addition under present climate conditions did not impact the diazotrophic communities' composition. At TYR and ION, the large increase in N 2 fixation recorded after dust addition might be attributed to NCDs (mainly γ -Proteobacteria), as suggested by the positive correlation between N 2 fixation and BP (Fig. S4). At FAST, the community shifted from a large dominance of UCYN-A towards a dominance of NCDs in both the dust treatments and unamended controls due to the increase of a few fast growing γ -Proteobacteria (mainly Pseudomonas). This shift could be attributed to a bottle effect imposed by the tanks, which can favor fast-growing heterotrophic bacteria (Sherr et al., 1999;Calvo-Diaz et al., 2011). Nevertheless, the increased N 2 fixation after dust seeding at FAST cannot be explained by the shift in composition of the diazotrophic communities because the rates remained quite stable in the controls all along the experiment. Rather, the abundances of diazotrophs have likely increased due to dust input, and UCYN-A in association with prymnesiophytes could still be responsible for the majority of the enhanced N 2 fixation as N 2 fixation correlated strongly with PP (Fig. S4).
Variability in the N 2 fixation response among stations. The highest stimulation of N 2 fixation to dust addition was observed at TYR (mean RC D = 321 %), then at ION (mean RC D = 161 %) and finally at FAST (mean RC D = 21 %) (Fig. 7). The differences in the intensity of the diazotrophic response were not related to differences in the initial nutrient stocks (Table S1) and in the nutrient input from dust, which was quite similar between experiments (Gazeau et al., 2021a). Briefly, dust input led to a strong increase of 11.2 ± 0.2 µM NO − 3 a few hours after seeding in the three experiments, and the maximum DIP release was slightly higher at FAST (31 nM) than at TYR and ION (23 ± 2 nM) (Gazeau et al., 2021a). As DFe concentration before seeding was high (≥ 1.5 nM; Table 2), the bioavailability of Fe did not appear to drive the response of N 2 fixation (Ridame et al., 2013). Also, we evidenced in this experiment that NO − 3 release from dust did not inhibit N 2 fixation rate driven by UCYN-A and NCDs. This was expected for UCYN-A as it lacks NO − 3 assimilation pathways (Tripp et al., 2010;Bombar et al., 2014). N 2 fixation was initially more limited at TYR and ION (as evidenced by the lowest initial rates) compared to FAST, thereby explaining the highest stimulation of N 2 fixation by dust seeding at these stations. Interestingly, the stimulation of N 2 fixation was higher at TYR than at ION (Fig. 7), while these stations presented the same initial rate supported by NCDs. One major difference is that PP was not enhanced by dust seeding at TYR, while BP increased in both experiments (Gazeau et al., 2021b), suggesting that NCD-supported N 2 fixation was not limited by organic carbon at this station. As N 2 fixation and BP correlated strongly after the dust seeding (Fig. S4), it means that dust-derived DIP could relieve the ambient limitation of both heterotrophic prokaryotes (BP was co-limited by NP; Van Wambeke et al., 2021) and NCDs at TYR. This could explain why DIP concentration in the D treatments became again similar to the controls at the end of this experiment (Gazeau et al., 2021a). At ION, characterized by the lowest initial DIP concentration, N 2 fixation and PP were likely DIP (co-)limited as shown for BP . Consequently, diazotrophs as well as non-diazotrophs (heterotrophic prokaryotes and photoautotrophs) could all take up the dust-derived DIP, reducing then potentially the amount of DIP available for each cell that could explain the lower stimulation of N 2 fixation relative to TYR.
At FAST, initially dominated by UCYN-A, N 2 fixation and PP correlated strongly after the dust seeding (Fig. S4c). This indicated that dust could relieve either directly the ambient nutrient limitation of both N 2 fixation and PP (Fig. S9 in the Supplement) or indirectly through first the relief of the PP limitation of the UCYN-A photoautotroph hosts, inducing an increase in the production of organic carbon, which could be used by UCYN-A to increase its N 2 -fixing activity. Nutrients from dust could also first enhance the UCYN-A-supported N 2 fixation, which in turn could relieve the N limitation of the UCYN-A photoautotrophic host as the initial NO − 3 : DIP ratio indicates a potential N limitation of the PP (Table 2).

Response to dust addition under future relative to present climate conditions
General features. At TYR and FAST, N 2 fixation was more stimulated by dust input under future than present climate conditions (mean RC G-TYR = 478 % and mean RC G-FAST = 54 %), whereas at ION the response was similar (Figs. 7 and S3). These differences between future and present climate conditions were not related to the nutrients supplied from dust (Gazeau et al., 2021a). The purpose of our study was to study the combined effect of warming and acidification, but we can expect on the short timescale of our experiments (< 3-4 d) that NCDs and UCYN-A would not be directly affected by the changes in the CO 2 concentration as they do not fix CO 2 (Zehr et al., 2008). Indeed, no impact of acidification (or increase in the partial pressure of CO 2 ) on N 2 fixation was detected when the diazotrophic communities were dominated by UCYN-A in the North and South Pacific (Law et al., 2012;Böttjer et al., 2014). Nevertheless, the decrease in pH may indirectly impact UCYN-A through changes affecting its autotrophic host.
TYR and ION. Under future climate conditions, the composition of the diazotrophic communities did not change after dust input at TYR and ION relative to present conditions. At TYR, the highest N 2 fixation stimulation might be linked to the increase in the NCD abundances and/or in their cell-specific N 2 fixation rates under future climate conditions. Unfortunately, the impact of increased temperature and decreased pH on the cell-specific N 2 fixation rates of NCDs is currently unknown. However, some studies suggest a positive relationship between temperature and abundances of NCDs: diazotrophic γ -Proteobacteria (γ -24774A11) gene copies correlated positively with temperature (from ∼ 20 to 30 • C) in surface waters of the western South Pacific Ocean (Moisander et al., 2014), and Messer et al. (2015) suggested a temperature optimum for these γ -Proteobacteria around 25-26 • C in the Australian tropical waters. At ION, the similar stimulation of N 2 fixation by dust under future climate conditions compared to present climate conditions could be explained by a greater mortality of diazotrophs due to a higher grazing pressure and/or a higher viral activity. Indeed, higher bacterial mortality in the G treatment that could be related to a higher grazing pressure has been observed . Another explanation is that in spite of the DIP supply from the dust, the DIP bioavailability, initially the lowest at ION, was not sufficient to allow an additional N 2 fixation stimulation.
FAST. Some differences in the composition of the diazotrophic communities were observed between present and future climate conditions at FAST after dust input: the contribution of NCDs (mainly Pseudomonas) increased, and that of UCYN-A decreased. It must be noted that the duration of the experiment was longer at FAST (4 d) relative to TYR and ION (3 d), which could explain at least partly differences between stations. A direct response of increased temperature and/or decreased pH can be considered on a very short timescale (12 h) by comparing the results in the G treatment at T0 (+3 • C, −0.3 pH unit) with those in C and D treatments. The increased contribution of Pseudomonas in the G treatment at T0 (before dust addition) reveals a likely positive effect of temperature on the growth of this NCD as an increase in the top-down control on the bacterioplankton was observed after dust seeding under future climate conditions . Interestingly, despite the decrease in the relative contribution of UCYN-A to the total diazotroph community after dust addition, we observed contrasted responses within the UCYN-A group relative to present climate conditions: the relative abundance of UCYN-A3 strongly decreased (4.6 % in G vs. 25.4 % in D), whereas the relative abundance of UCYN-A2 was twice as high (7 % in G vs. 3.4 % in D). Notably, the relative contribution of UCYN-A1 did not appear to be impacted during the dust addition experiment. These respective changes could be explained by the difference in the temperature tolerance between UCYN-A2 and UCYN-A3. Temperature is one of the key drivers explaining the distribution of UCYN-A, which appeared to dominate in most of the temperate regions, with temperature optima around ∼ 20-24 • C (Langlois et al., 2008;Moisander et al., 2010). However, the temperature optima for the different UCYN-A sublineages, in particular for UCYN-A2 and UCYN-A3, are poorly known. Interestingly, Henke et al. (2018) observed that the absolute UCYN-A2 abundance was positively affected by increasing temperature, within a range of temperature from about 21 to 28 • C, which is in agreement with our results, although only relative abundances were measured in our study. Based on the strong positive correlation between N 2 fixation and PP after dust addition (and no correlation between N 2 fixation and BP; Fig. S4) and despite the decrease in the relative abun-dance of UCYN-A3, the increased stimulation of N 2 fixation under future climate conditions could likely be sustained by the increase in the relative abundance of UCYN-A2, which is bigger than UCYN-A3 (Cornejo-Castillo et al., 2019) and could consequently have a higher cell-specific N 2 fixation rate.

Conclusions
In the MS, N 2 fixation is a minor pathway to supply new bioavailable N for sustaining both PP and BP but can locally support up to 20 % of PP and provide all the N requirements for bacterial activity. UCYN-A might be supporting extremely high rates of N 2 fixation (72 nmol L −1 d −1 ) in the core of an eddy in the Algerian Basin influenced by Atlantic waters. The eastward-decreasing longitudinal trend of N 2 fixation in the surface waters is likely related to the spatial variability in the composition of the diazotrophic communities, as shown by the eastward increase in the relative abundance of NCDs towards more oligotrophic waters, while we observed a westward increase in the relative abundance of UCYN-A. This could reflect lower nutrient requirements for NCDs relative to UCYN-A. Through the release of new nutrients, simulated wet dust deposition under present and future climate conditions significantly stimulated N 2 fixation. The degree of stimulation depended on the metabolic activity of the diazotrophs (degree of limitation) related to the composition of diazotrophic communities and on the ambient potential nutrient limitations of diazotrophs, including that of the UCYN-A prymnesiophyte host. The strongest increase in N 2 fixation, not accompanied with a change in the composition of the diazotrophic communities, was observed at the stations dominated by NCDs (TYR, ION), where the nutrient limitation was the strongest. Under projected future levels of temperature and pH, the dust effect is either exacerbated or unchanged. Knowing that NCDs and UCYN-A do not fix CO 2 , we suggest that, on the timescale of our experiments (3-4 d), the exacerbated response of N 2 fixation is likely the result of the warming (from about 21 to 24 • C), which may increase the growth of NCDs when nutrient availability allows it and may alter the composition of UCYN-A community. However, to date, the effect of acidification and temperature optima of the different UCYN-A sublineages is poorly known (or unknown) as these UCYN-A remain uncultivated.
Future changes in climate, desertification and land use practices could induce an increase in dust deposition to the oceans (Tegen et al., 2004;Moulin and Chiapello, 2006;Klingmüller et al., 2016). The predicted future increase in surface temperature and the resulting stronger stratification are expected to expand the surface of low-nutrient, lowchlorophyll (LNLC) areas, consequently reinforcing the role of new nutrient supply from eolian dust on the N 2 fixation and probably on the structure of the diazotrophic communities.
Data availability. The PEACETIME dataset will be available at https://doi.org/10.17882/75747 (Guieu et al., 2020b) upon publication of all papers in the special issue (January 2022).
Author contributions. FG and CG designed the dust seedings experiments. CR, JD, EB, MB, FVW, FG, VT, ATS and CG participated in the sampling and analysis. CR and EB performed DNA extraction; EB performed library preparation. CR, JD and SH analyzed the data; CR wrote the manuscript with contributions from all authors.
Competing interests. The contact author has declared that neither they nor their co-authors have any competing interests.
Disclaimer. Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Special issue statement. This article is part of the special issue "Atmospheric deposition in the low-nutrient-low-chlorophyll (LNLC) ocean: effects on marine life today and in the future (ACP/BG interjournal SI)". It is not associated with a conference.
Acknowledgement. The authors thank the captain and the crew of the RV Pourquoi Pas? for their professionalism and their work at sea. We warmly acknowledge our second chief scientist Karine Desboeufs. We gratefully thank Eric Thiebaut and Pierre Kostyrka for their precious advice with statistical tests. We also thank Kahina Djaoudi and Thibaut Wagener for their assistance in sampling the tanks and TMC rosette and Magloire Mandeng-Yogo and Fethiye Cetin for IRMS measurements at the Alyses platform (SU, IRD). The DIC data used in this study were analyzed at the SNAPO-CO 2 service facility at the LOCEAN laboratory and supported by the CNRS-INSU and OSU Ecce-Terra. We greatly appreciate the interest of the reviewers and Christine Klaas and thank them for their relevant comments and time spent reviewing our manuscript.
Financial support. This study is a contribution to the PEACETIME project (http://peacetime-project.org, last access: 17 January 2022), a joint initiative of the MERMEX and ChArMEx components supported by the CNRS-INSU, IFREMER, CEA and Météo-France as part of the program MISTRALS coordinated by the INSU. PEACE-TIME was endorsed as a process study by GEOTRACES. Julie Dinasquet was funded by a Marie Curie Actions International Outgoing Fellowship (PIOF-GA-2013-629378). Søren Hallstrøm and Lasse Riemann were funded by grant 6108-00013 from the Danish Council for Independent Research.

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C. Ridame et al.: N 2 fixation in the Mediterranean Sea Review statement. This paper was edited by Carolin Löscher and reviewed by two anonymous referees.