Salinity as a key control on the diazotrophic community composition in the Southern Baltic Sea

. Over the next decade, the Baltic Sea is predicted to undergo severe changes including decreased salinity due to altered precipitation related to climate changes. This will likely impact the distribution and community composition of Baltic Sea dinitrogen (N 2 ) fixing microbes, of which especially heterocystous cyanobacteria are adapted to low salinities and may expand to waters with currently higher salinity, including the Danish Strait and Kattegat, while other high-salinity adapted 10 N 2 fixers might decrease in abundance. In order to explore the impact of salinity on the distribution and activity of different diazotrophic clades, we followed the natural salinity gradient from the Eastern Gotland and Bornholm Basins through the Arkona Basin to the Kiel Bight and combined N 2 fixation rate measurements with a molecular analysis of the diazotrophic community using the key functional marker gene for N 2 fixation nifH , as well as the key functional marker genes anf and vnf, encoding for the two alternative 15 nitrogenases. We detected N 2 fixation rates between 0.7 and 6 nmol N L -1 d -1 , and the diazotrophic community was dominated by the cyanobacterium related to Nodularia spumigena and the small unicellular, cosmopolitan cyanobacterium UCYN-A. Nodularia was present in gene abundances between 8.07 x 10 5 and 1.6 x 10 7 copies L -1 in waters with salinities of 10 and below, while UCYN-A reached gene abundances of up to 4.5 x 10 7 copies L -1 in waters with salinity above 10. Besides those 20 two cyanobacterial diazotrophs, we found several clades of proteobacterial N 2 fixers and alternative nitrogenase genes associated with Rhodopseudomonas palustris , a purple non-sulfur bacterium. Based on principal component analysis (PCA), salinity was identified as the primary parameter describing the diazotrophic distribution, while pH and temperature did not have a significant influence on the diazotrophic distribution. While this statistical analysis will need to be explored in direct experiments, it gives an indication for a future development of diazotrophy in a freshening Baltic Sea with UCYN-A retracting to more saline North Sea waters and heterocystous cyanobacteria expanding as salinity decreases.


Introduction
The Baltic Sea (Fig. 1) is a marginal, brackish sea characterized by a natural salinity gradient increasing from the  to the South-West. The Baltic Sea covers an area of 415000 km 2 with a permanent halocline in the Baltic Sea proper, preventing vertical mixing, oxygen (O2)-depleted waters in the deeper basins and coastal systems, accompanied with the occasional accumulation of hydrogen sulfide (H2S) and ammonium (NH4 + ) below the chemocline (Conley et al., 2002;Hietanen et al., 2012;Lennartz et al., 2014). It is further challenged by a high land-derived influx of phosphorus leading to a substantial internal surface water and sedimentary phosphorus load (Gustafsson et al., 2017;Stigebrandt and Andersson, 35 2020). Prior studies have shown that the Baltic Sea experienced a 10-fold increase in O2-depleted areas over the last 115 years, covering an area of 12000 -70000 km 2 making it one of the ocean areas most severely affected by deoxygenation (Diaz and Rosenberg, 2008;Reusch et al., 2018). Between 1871 and 2013, the Baltic Sea showed an increase in temperature of 0.1 o C decade -1 exceeding the average global trend of about 0.06 o C decade -1 (Reusch et al., 2018;Rutgersson et al., 2014) and decreased salinity (1-2 kg -1 ) (Liblik and Lips, 2019). Nitrogen (N) deposition rates are now among the highest in marine 40 areas (Reusch et al., 2018). The resulting eutrophication severely affects sensitive coastal areas resulting in high pelagic production, frequent events of anoxia, and decreased biodiversity (Breitburg et al., 2018;Carstensen et al., 2014;Maar et al., 2016;Reusch et al., 2018;Rutgersson et al., 2014). In this context, the Baltic Sea has been described as a "time machine" for how future oceans will respond to climate change (Reusch et al., 2018), making it an ideal environment to investigate biological fixation of dinitrogen gas (N2 fixation) in response to such changes. 45 N2 fixation is the primary external source of new N to life in the ocean and plays a crucial role for primary production, and thus carbon dioxide (CO2) uptake from the atmosphere (Capone and Carpenter, 1982;Gruber, 2005). Specialized organisms, called diazotrophs, carry out this highly energy-demanding process. The diversity of diazotrophs is not easy to describe, as those organisms spread across the microbial tree of life, and are found in both the bacterial and archaeal domains (Zehr et al., 1998). N2 fixation is catalyzed by an enzyme complex, the nitrogenase, which exists in three different subtypes: 50 the molybdenum (Mo)-iron (Fe) type referred to as Nif, the vanadium (V)-Fe type called Vnf, and a Fe-Fe type called Anf (Chisnell et al., 1988;Robson et al., 1986). The different metal cofactors are essential in the context of the redox sensitivity of the three nitrogenases, respectively. Mo is preferentially available in the presences of at least traces of O2, contrary to V and Fe, which are more soluble and thus available under anoxic conditions (Bennett and Canfield, 2020;Bertine and Turekian, 1973;Crusius et al., 1996;Dixon and Kahn, 2004;Morford and Emerson, 1999). The differential availability of 55 those trace metals along redox gradients might have played a role in the evolutionary development of those nitrogenase types (Anbar, 2008;Mus et al., 2019) and their distribution might change in a future ocean impacted by acidification, deoxygenation (Keeling et al., 2010;Löscher et al., 2014;Schmidtko et al., 2017;Stramma et al., 2008) and desalination (Olofsson et al., 2020). To date, most studies focused mainly on Nif-type nitrogenases in the marine environment, while Vnf and Anf nitrogenases were often overlooked. Molecular detection of the nifH gene does not fully recover the diversity of 60 vnfH and anfH, possibly resulting in an underestimation of alternative nitrogenases in the environment (Affourtit et al., 2001; available studies focusing on alternative nitrogenases identified them to be abundant and diverse and suggested to play a role for N2 fixation in various ecosystems, including the upper water column of the ocean, O2-depleted waters, and Baltic Sea sediments (Bellenger et al., 2011(Bellenger et al., , 2014Betancourt et al., 2008;Christiansen and Löscher, 2019;Farnelid et al., 2009;65 Loveless et al., 1999;McRose et al., 2017;Tan et al., 2009;Zehr et al., 2003;Zhang et al., 2016).
In the Baltic Sea, N2 fixation is considered to take place in sunlit surface waters mainly and carried out by three genera of heterocystous cyanobacteria, Aphanizomenon sp, N. spumigena and Anabaena spp. (now referred to as Dolichosperum spp. (Klawonn et al., 2016;Wacklin et al., 2009)), all of them containing the classic Nif-nitrogenase (Janson et al., 1994;Leppanen et al., 1988), and contributed massively to N2 fixation with rates of up to 115 -295 nmol N L -1 d -1 (Larsson et al., 70 2001). At the same time, cyanobacteria consume only a fraction of the N they fix with estimates in the range of 33.33 -85.72 nmol N L -1 d -1 (Janson et al., 1994;Larsson et al., 2001;Wasmund, 1997). Using an EA-IRMS-based approach, Klawonn et al., 2016 measured N2 fixation rates up to 300 ± 216 nmol N L -1 d -1 and based on cell-specific measurement of Aphanizomenon spp. and N. spumigena, revealed that they release up to 35% of fixed N, translating into a substantial fraction of fixed N leaking out, available for other primary producers (Ploug et al., 2010(Ploug et al., , 2011. In addition, low rates of N2 75 fixation (0.44 nmol N L -1 d -1 ) have been described in anoxic waters of the Baltic Sea (Farnelid et al., 2013). These rates were accompanied by a highly diverse Nif-containing heterotrophic diazotroph community at and below the chemocline in anoxic NH4 + -rich waters (Farnelid et al., 2013). Heterotrophic diazotrophs were closely related to those in other O2-depleted marine systems regions including the eastern tropical South (ETSP) and North Pacific (ETNP), the Arabian Sea (AS), and an anoxic basin in the Californian Bight (Fernandez et al., 2011;Hamersley et al., 2011;Jayakumar et al., 2012Jayakumar et al., , 2017Löscher et al., 80 2014). Non-cyanobacterial diazotrophs have been found to fix N2 actively. However, it is still inconclusive what regulates and controls their N2 fixation activity.
Over the last three decades, the Baltic Sea has experienced a trend of freshening in the range of 1-2 kg -1 in surface waters (Liblik and Lips, 2019) or between 0.4 to 1.2 (Saraiva et al., 2019). Earlier studies suggested that salinity impacts the diazotrophic community composition and the activity of nitrogenases in pure cultures of Azotobacter sp. marine microbial 85 mats and estuaries (Dicker and Smith, 1981;Marino et al., 2006;Severin et al., 2012). Surface salinity in the Baltic Sea ranges between 0 and 10 ( Fig. 1) making it one of the major drivers for microbial composition and distribution in the Baltic Sea (Dupont et al., 2014;Olofsson et al., 2020;Wulff et al., 2018). Typically, Aphanizomenon sp. dominates less saline waters (e.g., Bothnian Sea) and grow best with salinities around 5 (Lehtimäki et al., 1997). Aphanizomen sp., however, has also been frequently reported to grow at salinities reaching up to 5-8 such as the northern Baltic Proper (Olofsson et al., 90 2020). N. spumigena prefers the higher saline waters in southern part (e.g. the Southern Baltic Proper) with an optimum growth in salinities of 8-10 (Lehtimäki et al., 1997;Rakko and Seppälä, 2014). Additionally, the small, unicellular cyanobacterial symbiont UCYN-A has been detected in the Baltic Sea (Bentzon-Tilia et al., 2015). This cosmopolitan diazotroph has previously been shown to be abundant throughout most marine systems (Tang et al., 2019;Zehr et al., 2016), and to substantially contribute to N2 fixation rates (Martínez-Pérez, C., Mohr, W., Löscher, 2016;Mills et al., 2020). 95 However, the factors controlling the biogeography of UCYN-A is poorly understood. Though, latest study suggested salinity having a positive correlation to UCYN-A biogeography (Li et al., 2021). These observations indicate potential changes in the cyanobacterial composition in future freshening events (Olofsson et al., 2020;Wasmund et al., 2011). To now explore the impact and importance of salinity for diazotrophy in the Baltic Sea and in order to complement existing datasets on N2 fixation from high-productivity seasons, we carried out a survey including chemical profiling, and rate measurements in the 100 low productive fall season. The focus of this study is a molecular genetic assessment of the diazotrophic community diversity and genetic activity including heterocyst and unicellular cyanobacteria, heterotroph diazotrophs and diazotrophs carrying alternative nitrogenase genes. In addition, we compared our data to available and to be able to predict the future distribution of Baltic Sea diazotrophs and of N2 fixation rates.

Seawater sampling
Samples were collected during the cruise AL528 using the German research vessel RV Alkor from 17.09.2019 to 28.09.2019 along a transect through the major basins of the Baltic Sea (see Fig. 1 for a cruise plan and table S2 for overview of all stations). Specifically, samples were collected at a station in the Kiel fjord (KB06), one in the Arkona basin (H21), four stations in the Bornholm Basin (BB08, BB15, BB23, BB31) and the Eastern Gotland Basin (GB84, GB90a, GB107, 110 GB108), respectively, at water depths between 3 to 115 meters (m) using a 10 L Niskin bottle rosette equipped with a conductivity-temperature-depth (CTD) sensor. Water for dissolved inorganic nitrogen (NOx (Nitrate (NO3 -)+ Nitrite (NO2 -)) was collected from the Niskin bottles, filtered through an acid-washed 0.8 µm cellulose acetate filter (Sartorius™), using a syringe and stored in 20 ml scintillation vials at -20°C until analysis. Samples for PO4 3analysis were filtered through a 200 µm mesh to avoid contamination with large zooplankton. Samples were filled in 20 ml scintillation vials and stored at -80°C 115 until analysis. NOx and PO4 3samples were analyzed on a SKALAR SAN plus analyzer (Thermo Fischer, Waltham, US) according to Grasshoff (1999) with a detection limit of 0.05 µmol N L -1 for NOx and 0.03 µmol P L -1 for PO4 3-. Nutrient samples were taken in triplicates, handled with nitrile gloves and all equipment was rinsed with MilliQ water in between stations to avoid contamination. Samples for dissolved inorganic carbon (DIC) were collected by filling triplicates of 12 mL exetainers bubble-free at stations KB06, H31, H21 and BB15. Samples were fixed with 20µl of a saturated mercury chloride 120 solution and stored at room temperature until analysis. DIC measurements were carried out using a 2 mM NaHCO3 solution as standard and as described previously (Hall and Aller, 1992). From all stations and depths 0.5 to 1 L of seawater were collected from Niskin bottles and were immediately filtered onto a 0.22 µm pore size membrane filters (Millipore, Bilaterica, USA; exact seawater volumes were constantly recorded), and filters were stored at -80˚ C until DNA extraction and further analysis. 125

15 N2 and 13 C seawater incubations
N2 and C fixation rates were determined using stable isotope labelling at KB06, H21, BB15, BB08 and GB107, at water depths between 3 and 41 m. We used the bubble addition method (Montoya et al., 1996) to ensure comparability to previous studies from the Baltic Sea. Triplicate water samples were filled from Niskin bottles into 2.4 L glass bottles until it was topfilled (Schott-Duran, Wertheim, Germany). To each incubation bottle, 1 mL 15 N2 gas ( 15 N2 98%+ Lot#I-16727, Cambridge 130 Isotope Laboratories, Inc., USA) and 1 mL H 13 CO3 (1g 50 mL -1 , Sigma-Aldrich, Saint Louis Missouri US) was added through an air-tight septum and bottles were inverted to ensure mixing with final calculated concentrations of 0.8 atom % 15 N2 and 10 µg mL -1 H 13 CO3 (approximately 3.8 atom %). After an incubation time of 24 h, samples were filtered onto precombusted GF/F filters (GE Healthcare Life Sciences, Whatman, USA). A parallel set of triplicate samples was collected from each sampling depth for determining the natural abundance of N and C isotopes in the particulate organic material. 135 Filters were stored at -20 o C until further analysis. Filters were then acidified and dried in an oven at 65 o C. Dried filters were analysed on an Elemental Analyzer Flash EA 1112 series (Thermo Fisher), coupled to an isotope ratio mass spectrometer (Finnigan Delta Plus XP, Thermo Fisher).

Molecular methods
For nucleic acid extraction, 0.22 µm pore sized Millipore membrane filters (Millipore, Bilaterica, USA) were flash-frozen in 140 liquid N and subsequently crushed. DNA was purified with the MasterPure Complete DNA and RNA purification Kit (Lucigen, Wisconsin, US) according to the manufacturer's protocol, with the minor modification of using 1 mL of lysis buffer to completely cover the filter pieces. RNA was purified using Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany). The remaining DNA was removed with the gDNA wipeout mix, and a cDNA library was constructed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) with the supplied RT primer set. The nucleic acid 145 concentration and quality were checked spectrophotometrically on a MySpec spectrofluorometer (VWR, Darmstadt, Germany).
A total of 15 DNA samples were amplified for nifH using nested PCR with primers and PCR conditions as described in Zani et al. (2000). anf/vnfD were amplified using a nested PCR with primers and conditions as described in Bellenger et al. (2014) and McRose et al. (2017). Amplicons were TOPO TA-cloned (Topo TA cloning Kit for sequencing, Thermo Fisher 150 Scientific, Waltham, US) and Sanger-sequenced. Sequencing was carried out at the Institute of Clinical Molecular Biology in Kiel, Germany. Samples for nifH and anf/vnfD amplification were the same as the ones where N2 rate measurements were carried out (KB06, H21, BB15, BB08 and GB107 between 3 and 41 meters, see table S1).
Sequences were quality checked and trimmed with BioEdit (Hall, 1999). nifH sequences were trimmed to 321 bp and vnf/anfD to 591 bp. This resulted in 182 nifH and 69 vnf/anfD amplicon sequences. Sequences were BLASTX-searched 155 against the NCBI database. A reference library was created, and sequences with lower than 80% identity were discarded.
Quantitative real-time PCRs were carried out targeting nifH clade-specifically for Nodularia, UCYN-A and gammaproteobacterial diazotrophs (Gamma AO, Gamma PO) as described previously (e.g., Boström et al., 2007;Langlois et al., 2008;Loescher et al., 2014). As standards, serial dilutions of plasmids (10 7 to 10 1 copies) containing the target nifH genes were used. Samples, standards, and non-template controls were run in duplicates on a Biorad qPCR machine (Biorad,170 Hercules, USA), and reactions were considered uncontaminated if no amplification could be detected in non-template controls after 40 qPCR cycles. To further ensure that no DNA contamination was present in our cDNA, we ran additional qPCR reactions of RNA samples. No amplification was observed. Sequences were submitted to GenBank with accession numbers MZ063808-MZ063873 and MZ063874-MZ064057 and 16S rDNA with accession number SAMN20309768-SAMN20309783. 175

Statistical Methods
Principle component analyses (PCA) on qPCR and environmental data were performed in R using the vegan package (Oksanen et al., 2020;R Foundation for Statistical Computing, 2017). The simper function (from vegan package) was used to identify parameters with the highest impact on diazotroph abundance. A principal component analysis (PCA) was subsequently performed using prcomp and visualized with the factoextra package (Kassambara and Mundt, 2020). To 180 maximize the amount of explained variance of the dataset, the figure included both component one versus two (Fig. 8A), and one versus three (Fig. 8B).

Hydrochemistry
During the cruise, surface waters above 40 m water depth exhibited the typical Baltic Sea salinity gradient with salinities 185 decreasing from South-West to North-East (10 -24 in the Kiel and Arkona Basin and 7-9 in the Bornholm and Eastern Gotland Basins), accompanied by lower temperatures of 13-14 o C in the Bornholm and Eastern Gotland Basin compared to slightly higher temperatures of 15-16 o C in the Kiel Fjord and Arkona Basin (Fig. 2). A thermocline was observed at 45 m in the Bornholm Basin and 65 m in the Eastern Gotland Basin. The coastal station we sampled in the Arkona basin showed a similar stratification pattern (Fig. 2) with a salinity of 10 on the surface and 17 below 30 m water depth. This stratification 190 pattern is typical for the region and time of the year and could also be observed from annual data from the International Council for the Exploration of the Sea (ICES Copenhagen, 2020) (Fig. 1, B). Surface waters were well-oxygenated with O2 concentrations above 156.31 µmol L -1 in the top 40 m of the water column. Low O2 water masses with concentrations below 15 µmol L -1 were only observed in the Bornholm Basin in waters deeper than 65 m (Fig. 2). Our dataset indicates an N:P ratio [µmol L -1 :µmol L -1 ] well below the Redfield ratio of 16:1 in surface waters along the cruise track, with excess P classically supposed to promote N2 fixation. However, NOx was still available to the 210 phytoplankton and microbial community as supported by a positive intercept of the trendline, which might be considered unfavourable for N2 fixation (Fig. 3). Remaining NOx concentrations in the surface waters above 40 m thus speak for a limitation of primary production by either micronutrients, temperature, or light availability and less for a limitation by N or PO4 3- (Arrigo, 2005;Saito et al., 2008). Nitrogen repletion is supported by POC: PON ratios of 3.6 to 4.94 in the Kiel Fjord and Arkona Basin and 2.92 to 6.15 in the Bornholm and Eastern Gotland Basins (Fig. 3), which are close to the Redfield 215 ratio (Redfield, 1934). Altogether, nutrient concentrations do not strongly support a niche for N2 fixation.

Nitrogen fixation
While no clear niche for N2 fixation could be identified, we still detected N2 fixation rates in our samples from water depths between 3 and 41 m (Fig. 4). N2 fixation rates were between 0 to 6 ± 4.73 nmol N L -1 d -1 in the Arkona Basin, 0.7 ± 0.97 to 27.3 ± 38.21 nmol N L -1 d -1 in Bornholm Basin and 0.67 ± 0.51 to 2 ± 0.4 nmol N L -1 d -1 in the Eastern Gotland Basin (Fig.  220   4). In Kiel Fjord, N2 fixation rates were below the detection limit (0.03 nmol N L -1 d -1 ) of our method. One outlier was observed in the Bornholm Basin at a water depth of 5 m, with an N2 fixation rate of 27.3 nmol N L -1 d -1 . This high rate was only detected in one out of our three replicates and was not included in our further analysis. Without this outlier, a conservative estimate of N2 fixation in the Bornholm Basin would be between 0.7 ± 0.97 and 5.27 ± 1.37 nmol N L -1 d -1 .
Though these rates are at the lower end of previous observations from the Baltic Sea (Table S3), they are comparable to a 225 previous study from the same region, but not the same season, with N2 fixation rates of 7.6 ± 1.76 nmol N L -1 d -1 from July and August (Farnelid et al., 2013). N2 fixation activity is supported by the δ 15 N -PON in our control samples which was in the range of control samples was found to be 0.3 to 2 ‰ in samples from surface waters above 40 m from the Eastern Gotland and Bornholm Basins (Fig. 4). Values between -2 to +2 ‰ are considered indicative for N2 fixation (Dähnke and Thamdrup, 2013;Delwiche and Steyn, 1970). 230 Seasonal variations might explain the observed lower N2 fixation rates if compared to previous studies. N2 fixation rates are generally described sensitive to low temperatures (Brauer et al., 2013;Church et al., 2009;Englund and Meyerson, 1974;Moisander et al., 2010;Stal, 2009)  N2 fixation rates were accompanied by low C fixation rates compared to rates in the micromolar range as typically detected in the Baltic Sea (e.g., (Klawonn et al., 2016))( Fig. 4), which were, however, increasing over depth, with an average C fixation of 29.16 ± 10.89 nmol C L -1 d -1 in Kiel Fjord, 42.62 ± 7.86 nmol C L -1 d -1 the Arkona Basin, 40.52 ± 13.16 nmol C 240 L -1 d -1 in Bornholm Basin and 58.52 ± 15.42 nmol C L -1 d -1 in Eastern Gotland Basin. Assuming Redfield stoichiometry, N2 fixation sustained between 8.8 and 108% with an average of 43% of C fixation during our cruise. These rates are comparable with the contribution of N2 fixation to C fixation in other oceanic regions, including the Atlantic (20-25%) and Pacific Ocean (1-4%), the Mediterranean Sea (8%), and the Bay of Bengal (<1%) (Church et al., 2009;Dore et al., 2002;Fonseca-Batista et al., 2019;Rahav et al., 2013;Saxena et al., 2020;Tang et al., 2019) (Table 1). The high contribution to primary production 245 indicates that N2 fixation may promote or extend primary production into the fall season if only at low rates. Other sources, however, might include land or riverine input.

Diazotrophic community composition
We assessed the diazotrophic diversity based on nifH and vnf/anfD amplicons (Fig. 5 and 6). Sequences belonging to N. spumigena (19%) and UCYN-A (26%) were dominant in the sequence pool. While Nodularia clades are classically 250 abundant in the Baltic Sea waters (Boström et al., 2007;Farnelid et al., 2013;Klawonn et al., 2016;Larsson et al., 2001), only one previous report of UCYN-A from the Baltic Sea exists, where it had been detected in waters of the Danish Strait and the Great Belt (Bentzon-Tilia et al., 2015). Moreover, we detected Pseudanabaena-like sequences representing 8 % of the sequence pool consistent with previous studies (e.g. Acinas et al., 2009;Farnelid et al., 2013;Klawonn et al., 2016;Stal et al., 2003). Only 1.5 % of the sequences belonged to Aphanizomenon sp., a diazotroph known to dominate N2 fixation early 255 in the season with salinity at 5-6 (Klawonn et al., 2016;Svedén et al., 2015). The low number found in this study might be seasonally related.
Notably, based on our nifH sequence analysis, we did not identify, Dolichospermum spp., one of the common N2 fixing cyanobacteria for the Baltic Sea (Bentzon-Tilia et al., 2015;Boström et al., 2007;Farnelid et al., 2013). However, sequences related to A. lemmermannii were recovered from our 16S rDNA amplicon sequencing (Fig. S1). This discrepancy might 260 speak for a bias against their specific nifH genes by our approach, or the clades we found in the 16S rDNA dataset might lack a nifH gene.
Nodularia-specific nifH gene and transcript abundances were in the same range as previously described (10 6 gene copies L -1 and 10 5 transcripts L -1 (Farnelid et al., 2013)). UCYN-A was identified based on both nifH and 16S rDNA sequences and 270 quantified using cluster-specific qPCR. UCYN-A was present in abundances of 3.73 x 10 3 -9.97 x 10 7 copies L -1 , similar to the one available previous study where the abundance of nifH specific for UCYN-A peaked in September ( approximately 10 6 copies L -1 ) (Bentzon-Tilia et al., 2015) (Fig. 7). The same study showed nifH expression (roughly 10 3 transcript L -1 ) of UCYN-A in the Baltic Sea surface waters (1 m). These transcript abundances are comparable with those obtained from our study with transcript abundances between 2.98 x 10 2 and 3.73 x 10 2 transcripts L -1 at 5 m water depth and up to 3.71 x 10 5 275 transcripts L -1 at a water depth of 20 m (Fig. 7). Notably, the above-mentioned study took place in the Danish Strait, and thus in more saline waters (salinity of 13-17).
Contrary to Nodularia, UCYN-A reached highest abundances (up to 4.52 x 10 7 copies L -1 ) in higher saline waters impacted by the North Sea (the Kiel Fjord and Arkona Basin) and the lowest abundance (up to 6.29 x 10 5 copies L -1 ) in low saline waters (Fig. 7, Supplementary Figure S1). UCYN-A is increasingly found throughout the oceans, often playing an 280 important role in N2 fixation (e.g. Martínez-Pérez, C., Mohr, W., Löscher, 2016;Tang and Cassar, 2019). Over the recent years, UCYN-A has been shown to have a crucial part in N2 fixation and has been identified all over the globe, from polar to tropical regions and tolerating between 4 o C and 30 o C (Gradoville et al., 2020;Harding et al., 2018;Martínez-Pérez, C., Mohr, W., Löscher, 2016;Mills et al., 2020;Short and Zehr, 2007;Tang and Cassar, 2019). Despite UCYN-A exposing highest abundances in Baltic Sea waters with higher salinity, we found UCYN-A present and active across the salinity 285 gradient.
Besides those cyanobacterial diazotrophs, we detected several clades of proteobacteria related to Rhodopseudomonas palustris (4%), Pelobacter sp (9%) and gamma proteobacteria of the Gamma-AO clade (3%, Langlois et al., 2008). We quantified gamma-proteobacterial nifH in abundances between 1.78 x 10 4 -1.82 x 10 5 copies L -1 (Gamma AO) and their transcripts in abundances of 2.89 x 10 3 -3.91 x 10 3 transcripts L -1 . Despite lack of clones clustering with Gamma PO, the 290 group could be detected in abundances between 8.21 x 10 2 -1.37 x 10 5 copies L -1 and their transcripts in abundances between 3.04 x 10 3 -8.05 x 10 3 transcripts L -1 , indicating a potential contribution to N2 fixation of those clades (Fig. 7). Diazotrophic proteobacteria are common in the ocean, however, their role for N2 fixation is not fully understood (Benavides et al., 2018;Chen et al., 2019;Turk-Kubo et al., 2014). Diazotrophs related to Cluster III were dominated by Desulfovibrio-like (1.5%), Opitutaceae-like (6%) and Clostridium-like sequences (1.5%). However, both nifH gene and transcript abundances were 295 below the detection limit of our qPCR. Altogether, the gene and transcript abundances of cyanobacterial diazotrophs were three to four orders of magnitude higher than those of non-cyanobacterial clusters suggesting that heterotrophic microbes may have only played a minor role for N2 fixation in Baltic Sea surface waters during our cruise.
In the pool of alternative nitrogenase sequences of the anfD type, we identified the purple non-sulfur bacterium Rhodopseudomonas palustris. We could not recover any vnfD sequences, which might be an adaptation to low vanadium 300 concentrations in the Baltic Sea (Bauer et al., 2017). The role of alternative nitrogenases in the environment is poorly understood, partly due to a lack of data and detection systems. Our data shows however the presence of alternative nitrogenases in the Baltic Sea surface water, which may sustain N2 fixation under certain conditions, for example in anaerobic micro-niches (Bertagnolli and Stewart, 2018;Farnelid et al., 2019;Paerl and Prufert, 1987;Pelve et al., 2017), including sinking particles (Chakraborty et al., 2021;Pedersen et al., 2018). 305

Factors impacting the diazotrophic distribution
To identify parameters impacting on the distribution of diazotrophs during our cruise, we carried out a simper analysis.
Interestingly, pH and temperature were less important for describing the diazotrophic distribution. The PCA suggested that depth and salinity explained the distribution of both the diazotrophic community and N2 fixation best (Fig. 8). Moreover, it revealed a correlation of N2 fixation and the Nodularia abundance and less of a correlation between N2 fixation and UCYN-310 A or other diazotroph clusters (Fig. 8). A decreasing salinity would thus not only promote the distribution of Nodularia-like diazotrophs, and decrease UCYN-A abundances, but also promote N2 fixation rates in such a future scenario. Intuitively, it is of little surprise that our data (Fig. 8) and previous studies indicate salinity to be a key control for cyanobacterial distribution and N2 fixation in the Baltic Sea (Dupont et al., 2014;Olofsson et al., 2020;Rakko and Seppälä, 2014). A recent study also suggest a positive correlation between UCYN-A and salinity (Li et al., 2021). While a study based on a compiled dataset 315  indicated that salinity does not affect the biovolumes of the filamentous N. spumigena but rather speciesinteractions (Karlberg and Wulff, 2013;Olofsson et al., 2020). Our study cannot evaluate such interactions and their impact of N2 fixation; however, our data do point towards Nodularia expanding into low salinity waters in the future thus complementing UCYN-A and possibly increasing N2 fixation rates in those waters.
Our analysis did not show temperature and pH to be major descriptors of N2 fixation during this cruise, consistent with 320 previous findings with elevated CO2 concentrations not causing any response in N2 fixation in the Baltic Sea Paul et al., 2016;Wulff et al., 2018). Moreover, a very recent study showed that ocean acidification has an impact on the diazotroph community composition and ecology and can decrease bulk N2 fixation rates in the subtropical Atlantic Ocean (Singh et al., 2021). Most likely, the contradicting results are due to other factors obscuring the stimulation by N2 fixation (Karlberg and Wulff, 2013;Wannicke et al., 2018). Besides pH, temperature has also been suggested as a control on 325 diazotrophy, either directly or indirectly, by impacting on O2 solubility (Stal, 2009).
In case of a potential future freshening of the upper water column (Liblik and Lips, 2019) in combination with increased PO4 3availability both through land-derived influx and through phosphorus mobilization via bottom-water anoxia (Gustafsson et al., 2017;Ingall and Jahnke, 1997;Stigebrandt and Andersson, 2020;Vahtera et al., 2007), N2 fixation by e.g.
Nodularia will likely increase in the region covered by our cruise. This could lead to a scenario with increased bloom 335 formations and expansion of such large heterocystous cyanobacteria across the Baltic Sea, as previously observed (Kahru et al., 1994;Kahru and Elmgren, 2014). An increased organic matter load would possibly lead to enhanced respiration by heterotrophic microbes promoting the further expansion of O2 depleted waters. This could lead to increased denitrification resulting in N-loss and emission of N2O, fuelling global warming, an increase in euxinic events as already observed in coastal Baltic waters (Breitburg et al., 2018;Carstensen et al., 2014;Lennartz et al., 2014). Further, blooms of Nodularia 340 could lead to an increased load of Baltic Sea waters with the toxin Nodularin (Sivonen et al., 1989), which can harm Baltic Sea biota and poison animals (Main et al., 1977;Nehring, 1993) and ultimately humans by impacting e.g. fishery industry (Karjalainen et al., 2007). Latest studies, however, shows that projection of increased sea level rise counteracts freshening events in the Baltic Sea making it uncertain whether salinity increases or decreases in the future (Meier et al., 2021(Meier et al., , 2022.
Regardless, potential changes in salinity might impact the diazotrophic community as described above. The uncertainty in 345 changing salinity makes it unclear in which extent diazotrophs might be impacted.

Conclusion
During our cruise, we explored the diazotrophic community composition and N2 fixation rates along the natural salinity gradient in the South Baltic Sea in the fall season. N2 fixation rates were detectable but low for the Baltic Sea and sustained cyanobacterium UCYN-A. The two different types of cyanobacteria occupied two different niches defined by salinity ranges, respectively, with Nodularia dominating in lower saline waters (8-9) and UCYN-A in high-salinity waters (>10).
Statistical analysis revealed that N2 fixation is quantitatively mainly driven by Nodularia clades and that both, Nodularia abundances and N2 fixation rates, are best explained by salinity. In the context of a predicted freshening of the Baltic Sea, the habitat of Nodularia-like heterocystous cyanobacteria would extend towards the South-Western part of the Baltic Sea, 355 possibly replacing the community of UCYN-A and increasing N2 fixation in those waters. Enhanced N2 fixation might facilitate primary productivity, and organic matter export to waters below the euphotic zone and could thus have severe impacts on the Baltic Sea biogeochemistry including increased respiration, O2 and N loss.

Author contribution
C. Reeder and C. Löscher designed the experiments and C. Reeder carried out the experimental work. J. Javidpour and I. 360 Stoltenberg designed the sampling strategy during the expedition and contributed essential datasets. C. Reeder and C.
Löscher prepared the manuscript with contributions from all the co-authors.

Competing interest
The authors declare that they have no conflict of interest.