Microbial associates of an endemic Mediterranean seagrass enhance the access of the host and the surrounding seawater to inorganic nitrogen under ocean acidification

Seagrasses are important primary producers in oceans worldwide. They live in shallow coastal waters that are experiencing carbon dioxide enrichment and ocean acidification. Posidonia oceanica, an endemic seagrass species that dominates the Mediterranean Sea, achieves high abundances in seawater with relatively low concentrations of dissolved inorganic nitrogen. Here we tested whether microbial metabolisms associated with P. oceanica and surrounding seawater enhance seagrass access to nitrogen. Using stable isotope enrichments of intact seagrass with amino acids, we showed that ammonification by free-living and seagrass-associated microbes produce ammonium that is likely used by seagrass and surrounding particulate organic matter. Metagenomic analysis of the epiphytic biofilm on the blades and rhizomes support the ubiquity of microbial ammonification genes in this system. Further, we leveraged the presence of natural carbon dioxide vents and show that the presence of P. oceanica enhanced the uptake of nitrogen by water column particulate organic matter, increasing carbon fixation by a factor of 8.6–17.4 with the greatest effect at CO2 vent sites. However, microbial ammonification was reduced at lower pH, suggesting that future ocean climate change will compromise this microbial process. Thus, the seagrass holobiont enhances water column productivity, even in the context of ocean acidification.


Results
The movement of 15

N amino acids in P. oceanica and seawater
There were differences in the fate of the 15 N amino acids that were incubated with P. oceanica versus seawater only (Fig. 1), with enrichment of seawater ammonium greatest at the control site and greatest when in association with P. oceanica and at night (Fig. 2a).The 15 N in ammonium in seawater (δ 15 N of NH 4 ) increased during the first 7 daylight hours ('day') and increased again overnight ('night').Initial conditions for the incubation bottles are in Table S1; statistics and all measurements of P. oceanica are in Supplementary Tables 2 and 3, respectively.
The ammonification rate in nmol per hour across the two sites (Eq. 1, Methods), revealed greater ammonification rates at the control site with ambient pH compared with the vent site, both in seawater and when P. oceanica was present (Fig. 2b, p = 0.002).Across all treatments, ammonification rates were more than 4 times greater at control sites.There was a trend for greater ammonification with P. oceanica compared with seawater (p = 0.045), and ammonification with seagrass was 2.5× greater when averaged across both sites.Rates did not differ between night and day (p = 0.512).Comparing the ammonification rates where P. oceanica was in the incubation bottles, there was no diel pattern, while incubations with seawater only were associated with greater ammonification by particulate organic matter (POM) during the day (p = 0.002).
The very newest Posidonia tissue growth in the meristem showed almost 3× greater uptake of 15 N-enriched ammonium during the day compared to night (p = 0.002, Fig. 2c), but the increase seen at the vent during the day switched at night to greater uptake in control sites (interaction between time of the experiment and site, www.nature.com/scientificreports/p = 0.020, Fig. 2c).Rates of ammonium uptake by Posidonia reached up to 6-8 μmol N per g dry mass per hour based on estimates of 15 N enrichment and using Eq. ( 2) (Fig. 2c, Table S3).P. oceanica plants also showed similar patterns of 15 N enrichment in the midblade and on the blade under epiphytes (Table S3).
The nitrogen content in Posidonia tissue did not differ between the vent and control site, either in the meristem (t = 0.993, p = 0.360) or midblade (t = 0.517, p = 0.623), and averaged 2.1% in the meristem and 1.5% in the midblade region overall (Fig. S1).
The daylight carbon fixation rates per unit tissue mass of P. oceanica did not differ whether Posidonia was in proximity to CO 2 vents or in control areas (p = 0.646, Fig. 3a, Table S2).At night, respiration rates too were indistinguishable (Fig. 3a).The lack of difference in carbon fixation by Posidonia and the nitrogen content of the blades were consistent with a statistically indistinguishable C:N ratio of P. oceanica tissue among sites, whether we considered the meristem tissue or the midblade underneath epiphytes (Fig. S1a,b).

Dissolved and inorganic nutrient dynamics
The dynamics of dissolved organic nutrients was affected by P. oceanica, proximity to CO 2 vents, and time of day.Dissolved organic carbon (DOC) was relatively unaffected by P. oceanica, with a nearly significant interaction between time of day (day or night), likely reflecting the increase DOC at the vents at night (p = 0.017) (Fig. S2a).DON release in association with P. oceanica showed no difference between control and vent site as a main effect (p = 0.220) but there was an interaction over the diel cycle, with DON increased during the day when P. oceanica was fixing the most carbon (p = 0.003, Fig. S2b).The pattern of dissolved organic matter release in seawater only showed that DOC was greater at the vent site (p = 0.005) and greater at night (p = 0.041).DON release was also greater at the vent site, though the interaction between site and time of day (p < 0.001) indicated that DON release was greater during the day at the vent site and greater at night at the control site (p < 0.001).DOC and DON tended to have net production during the day and net uptake at night.In contrast, the use of inorganic nutrients was greater during daylight hours and is illustrated by decreased concentrations of ammonium, nitrite, nitrate and silica (Fig. S2).

The effect of P. oceanica on surrounding POM
The presence of P. oceanica was associated with increased uptake of 15 N in POM during nighttime hours, an effect that was greatest at the control site, based on the interaction between site and treatment (p = 0.001, Fig. 2d).Combining the day and night interval, POM 15 N uptake was greater with P. oceanica (Table S2), resulting in 1.6 times more nitrogen uptake in chambers with P. oceanica compared with seawater at the control site, and 2.5× higher at the vent site.
Carbon uptake by POM was also enhanced with P. oceanica though only during the day; POM carbon uptake decreased with P. oceanica at night (Fig. 3b).The mean C:N ratio of POM was 5.09 at the control site and 5.97 at the vent site (Table S3).If the carbon:nitrogen uptake by POM was relatively constant, water column carbon

Metagenome analyses
After quality control and filtering, we obtained an average of 33.7 million sequence reads per sample (range 28.6-43.8 million).Metagenomic short reads were assembled into contigs of at least 1000 nucleotides in length, resulting in 26,460-123,697 contigs per sample with a mean of 68,852 across all 6 samples (Table S4).Across all the metagenomes in our study, the taxonomy of Posidonia blades had the greatest alpha diversity, with rhizomes at the vent having the fewest (Fig. 4, Table S4), though due to lack of replication we are precluded from statistical tests of diversity.
The metagenomic reads allowed us to construct metagenome-assembled genomes (MAGs), ranging from only a single MAG on rhizomes at the vent site to 21 MAGs on a blade metagenome at the control site.We used  15 N amino acids added to incubation bottles with and without P. oceanica.In a, δ 15 N ammonium (‰) in seawater in incubation bottles with P. oceanica or seawater only.A comparison of the values during the day and night, showed a significant increase in the bottles at the end of the night, with increased values at control site and with P. oceanica.There was also an interaction between time and treatment; the increase at night was highest with P. oceanica.In b.The ammonification rate in nmol per hour across the two sites and attributed to P. oceanica and seawater versus seawater only based on Eq. (1).The control site had greater ammonification compared with the vent site (p = 0.002) and P. oceanica was nearly associated with greater ammonification rates than seawater (p = 0.060), while rates did not differ overall between night and day (p = 0.732).Examining P. oceanica separate from seawater during day versus night, P.oceanica has no diel pattern, while the POM in seawater is associated with greater ammonification during the day.In c, ammonium uptake rate in umol N per g dry mass per hour in the Posidonia meristem during both day and night periods using Eq.(2).Uptake was greater during the day (p = 0.002), with a significant interaction (p = 0.020), indicating a relatively greater uptake of 15 N ammonium at night in the control site.In d., the uptake of ammonium by POM was greater when P. oceanica was absent from the incubation bottles during the day, and this effect was greatest at the control site, based on the interaction between site and treatment (p = 0.033).At night, the situation is reversed and the incubation bottles with P. oceanica resulted in a greater incorporation of 15 N into the POM in the seawater compared with bottles that had no P. oceanica (p = 0.013).All values were log transformed prior to statistical analyses shown in Table S2.
Vol Bowers et al. 36 criteria to define a MAG as high quality if it had a competition score of > 90% and a redundancy (or contamination) of < 10% (Table S5), yielding 22 high quality MAGs.A remaining 42 were medium quality MAGs with completion scores between 42 and 90% and redundancy scores between 0 and 11% (Table S5).All MAGs were bacterial and represented 5 phyla (Fig. 4a).A notable difference among blades versus rhizomes was the presence of the Cyanobacteria and Bacteroidota phyla on blades but not on rhizomes.In three cases, we were unable to taxonomically resolve a metagenome-assembled genome.Taxonomic diversity at the class and family level was higher on blades compared with rhizomes (Fig. 4b,c).The metabolism of these metagenomes was diverse, with some functional differences (Fig. 5).Photosynthetic and anoxygenic photosystem II were present only on blades, an expected result given the presence of Cyanobacteria only on blades.The KEGG module for nitrogen fixation was partially annotated within three MAGs, and especially the two MAGs in the genus Thiodiazotropha.The third MAG was an unidentified Gammaproteobacteria and was only 33% complete for nitrogen fixation genes (Table S7).Further, nitrogen fixation was indicated Carbon uptake rates by a. P. oceanica and b. seawater.In a, carbon fixation or respiration, in mg C per hour per g P. oceanica dry mass in incubation bottles during daylight and nighttime hours.P. oceanica carbon fixation was based on oxygen change, following subtraction of water column rates.P. oceanica carbon fixation was greater during the day, but did not differ between control and vent sites.Two-way ANOVA (site effect 0.646, diel effect p < 0.001, interaction p = 0.728).In b, carbon uptake by unfiltered seawater (in mg C per L per hour) is shown for both sites and in incubation bottles with and without P. oceanica.POM carbon uptake differed between day and night (p < 0.001) and with P. oceanica (p = 0.002); there were significant interactions with time of day and site (p = 0.004) and time of day and treatment (p < 0.001).Boxplots show mean values and delimit 25th and 75th percentiles; all points are shown, statistical tests in Table S2.S4 and S5.only on rhizome tissue and at the control site based on these three MAGs (Fig. 5, Table S7).Ammonification was a prevalent metabolism across all bacterial MAGs on P. oceanica.Of the 67 MAGs, all had enzymes classified as EC:1.4.* or EC:3.5.*, while 61 MAGs had enzyme function classified as EC:4.3.1*(Table S6).Nitrate reduction capability was present in the rhizome of seagrass from control areas (MAG 1 and MAG4), including denitrification in control rhizomes (MAG4).
Other microbial metabolisms that could be beneficial to P. oceanica included the presence of B vitamin metabolism on both tissue types at both vent and control areas.Vitamin B6 (pyridoxal) biosynthesis was only present in control rhizomes, while thiamin biosynthesis, vitamin B1, was present in all samples except vent rhizomes and vitamin B7 (biotin) biosynthesis was in multiple MAGS in both tissue types and sites.Vitamin B12 (cobalamin) synthesis was suggested in multiple MAGs at both sites, with aerobic biosynthesis only on the blades and anaerobic biosynthesis suggested in MAGs from all samples.Multiple MAGs on blades and rhizomes at both sites had sulfur oxidizing capability.

Discussion
Proximity to CO 2 vents changed microbial processes in association with the seagrass P. oceanica, reducing rates of ammonification.Carbon and nitrogen uptake by POM was also increased at the vent sites.Thus, water column processes differed between the control and vent area, possibly due to microbial nitrogen processing in association with seagrass.Despite differences in plant morphology 29,32 and grazing pressure 30 at vent sites, we measured similar rates of carbon fixation by P. oceanica.
Benthic-pelagic coupling and enhancement of pelagic productivity through the host-associated microbes on P. oceanica have implications due to global declines in seagrass cover 37 and the prevalence of seagrass wasting diseases 38 .Marine macrophytes have increasingly been shown to host a species rich microbiome 39 , with diverse functions that include not only ammonification, but also nitrogen fixation 8,35,40 , nitrification 41 and nitrate reduction 14,42 .Seagrasses are important to carbon sequestration due to carbon fixation 43 and the carbon storage in the root phyllosphere 44 and sediment.Seagrasses also ameliorate pH decreases 45 .Here we show that P. oceanica enhanced carbon fixation in the water column by over eightfold at the control site and over 17-fold at the vent site, suggesting that the contribution of seagrass to the carbon cycle is likely greater than previously recognized due to water column effects.The presence of seagrass also enhanced water column nitrogen uptake to 2.5 times at the vent site and 1.6 times at the ambient site when we integrated over both day and night intervals (Fig. 2d).A possible reason for enhanced POM carbon and nitrogen uptake is the increased dissolved organics available to heterotrophic bacteria when P. oceanica is present (Fig. S2).Nitrogen cycling in association with P. oceanica has been shown previously to be elevated near vents 35 .P. oceanica mirrors the findings for other foundational species, such as corals and sponges 4,46,47 where there are indirect effects through a food web due to the nutrient provisioning by particular species.Our findings with P. oceanica suggest that its role as a host for an active microbiome amplifies its role in these shallow Mediterranean systems.

Photosynthesis
Nitrogen Metabolism Nitrogen Metabolism Vitamin B7 B1 Photosynthesis Nitrogen Metabolism The metabolisms in 67 microbial metagenome assembled genomes (MAGs) assembled on P. oceanica at Ischia, Italy across 5 Phyla and on four different tissue types.The completion (black = complete, white = absent) in each microbial MAG for metabolic modules that showed differences across many of the MAGs.A full list of MAG metabolisms is in Table S7.Every MAG had some ammonification genes (Table S6).The figure was generated from anvi' o 76  www.nature.com/scientificreports/Ammonification occurred during day and night and with or without P. oceanica, suggesting that there is an abundance of microbes that metabolize amino acids and enhance carbon fixation and primary productivity in seagrass ecosystems.Further, every genome that we assembled had the functional capacity for deamination of amino acids to ammonium (Table S6).The use of unfiltered, in situ seawater allowed us to quantify ammonification in the water column, both with and without P. oceanica.Bacteria in the water column was likely a significant contributor to ammonification. 15N uptake to water column POM was detected in all incubation bottles, though an unknown amount of this uptake could be due to bacteria or eukaryotic phototrophs.
Regardless of whether the 15 N went into bacteria first, then into water column phytoplankton, the presence of Posidonia enhanced the uptake of 15 N into POM, suggesting that seagrass-associated bacteria, or even fungi, enhance water column productivity both at vent and control sites.The comparison of light versus dark uptake of 15 N into POM demonstrated that at least some of this uptake can be attributed to bacteria given the continued incorporation of 15 N into POM at night (Fig. 2d).
One enigmatic aspect of our results is ammonification rates in units of nmol per hour, while 15 NH 4 uptake rates could be in umol per hour.We can think of several explanations for this, including that amino acid concentrations that were greater than the 1uM that we assumed would have overestimated enrichment (R source ) and underestimated ammonification in Eq. (1).A second reason is that these processes could be spatially restricted, where microbial production in a biofilm could be immediately followed by host use, causing us to underestimate ammonification in the water column.Thirdly, if amino acid metabolisms are rapid and amino acid concentrations have high flux, we may have underestimated the processing and recycling of nitrogen during incubation.Ammonium concentrations are often not measured to the extent that nitrate concentrations are measured, despite preferential use of the former by macrophytes 48,49 .Yet, our estimates of ammonium uptake by P. oceanica during the day from the addition of 15 N enriched amino acids ranged from 1.17 to 15.33 umol per g dry mass per hour, comparable to the 2.77 umol rate estimated in 50 for P. oceanica in the Mediterranean and suggesting ammonification may contribute to ammonium fluxes and uptake rates here and in other areas.
Estimates of ammonium use and turnover in nature can be high 50 , though bacterial ammonification has been measured only rarely 51 .Also measured rarely is the concentration of DON, including any component parts such as amino acids.Amino acid concentrations have been estimated at 05-1.9 uM in other areas in the Mediterranean 52,53 , but a single flux estimate in the Caribbean suggests the flux could be high 51 .DON could be a persistent source of ammonium, and DON metabolisms warrant increased understanding of their contribution to nitrogen concentrations and fluxes.
Our demonstration of increased δ 15 N in seagrass tissue does not conclusively prove that these plants are taking up 15 NH 4 from microbial processes.Only pulse-chase experiments with more precise imaging, for example using NanoSIMS 15,16 , could show that microbial metabolism was the intermediate between 15 N-amino acids and the higher δ 15 N of macrophyte tissue.However, ammonification genes were ubiquitous across MAGs here (Table S4) and in other studies 14,54 , and suggests that microbes may mediate the availability of ammonium.Several previous studies that suggest seagrasses directly take up DON did not control or account for microbial activity 18 , and thus direct DON uptake by marine angiosperms remains unknown.
Amino acid concentrations in the ocean are rarely reported, though there are several studies in the Mediterranean in proximity to P. oceanica beds that report 0.5 to 1.9 uM dissolved free amino acids (DFAA) concentrations 52,53 , several orders of magnitude greater than those reported in open ocean settings 55,56 .DFAA can be a reduced source of nitrogen for both phytoplankton and bacteria 20 , and may be rapidly renewed via the release from animals 57,58 , phytoplankton 59 , and possibly the host themselves, such as P. oceanica here 52 .Concentrations can be greater in the sediment surrounding P. oceanica when compared to the water column 52 .Although estimates are that DFAA is only from 1 to 10% of DON in coastal areas 60 , the estimates are few and DFAA and DON may have rapid turnover rates which make their contribution to overall nitrogen use difficult to estimate.
Posidonia oceanica has other means of acquiring dissolved inorganic nitrogen from microbial associates.Nitrogen fixation genes were found in MAGs from the rhizome at the control site, which is consistent with other studies that have identified nitrogen fixation 61 , nifH genes 62 , or nitrogen-fixing taxa in association with P. oceanica 15 in the low-oxygen environment of the rhizomes.Mohr et al. 15 assembled a MAG for a novel taxon that fixes nitrogen within the cells of P. oceanica roots.We did not find the 'Candidatus Celerinatantimonas neptuna' they described in any of the MAGs we assembled, nor in the family Celerinatantimonadaceae, perhaps due to either incomplete DNA sequencing or to our use of surface swabs rather than grinding the root tissue of P. oceanica, as they did.Our analysis of P. oceanica blades did not reveal nifH genes.Dissimilatory nitrate reduction to ammonium is an additional bacterial metabolism that may help the seagrass host access nitrogen.We found genes for this function in rhizomes in control areas, consistent with previous studies of denitrification in association with seagrass 63 .
Ambient nitrate values were similar between the two sites at the single timepoint when we estimated them; previously published values also cite similar low nitrate concentrations regardless of venting that are < 1 uM 64,65 .Both vent and control sites had microbes that could increase nitrogen to enhance P. oceanica primary production.In addition to the abundance of enzymes with deaminating function (Table S4), nitrate reduction genes were indicated, as was nitrogen fixation.Even though estimates of ammonification were greater in control areas, P. oceanica carbon fixation did not differ between the control area and the CO 2 vent area.Carbon fixation rates were similar to the rates estimated at the same location previously 8 for blades without epiphytes.The NPP values reported by Berlinghof et al. 8 in μmol O 2 convert to ~ 0.7 to 0.9 mg C per g dry mass per hour, assuming a photosynthetic quotient of 1.0 (a 1:1 molar ratio of oxygen release to carbon uptake), a range nearly identical to what we estimated, though they show increased net primary production in proximity to vents.Here, our estimates of primary production included the rhizome tissue which may have obscured carbon fixation differences due to respiration.www.nature.com/scientificreports/There were microbial metabolisms that were unique to P. oceanica tissue types, such as cyanobacteria and anoxygenic photosynthesis only on seagrass blades.B vitamin synthesis was also prevalent, but the rhizomes in vent sites lacked Vitamin B1 and B6 synthesis.Vitamin B12, suggested to be auxotrophic for marine eukaryotic hosts 66,67 was in some MAGs in both tissue types and at both control and vent sites.Whether it plays the critical role in host fitness that has been demonstrated for other marine macrophytes (e.g. 1 ) remains to be investigated.In general, alpha diversity and the number of high quality MAGs was low in vent samples.Whether this reflects lower bacterial abundance in these areas is unclear, though certain functions were absent.
Our finding that P. oceanica and its microbial associates stimulate water column carbon fixation adds an important dimension to the role of seagrass beds in the carbon cycle.Seagrasses are suggested to alter the dissolved organic nitrogen 51 and seaweeds can influence water column microbes 68 .As ocean acidification continues, our results suggest that microbial ammonification rates in association with seagrass will decrease.However, the demonstrated role that P. oceanica plays in enhancing carbon fixation by surrounding POM, particularly in low pH seawater, suggests an underappreciated role for resilient foundational species in a changing ocean.

Study system
Underwater CO 2 vent systems are a powerful tool for exploring the effects of low pH on the features of species and biological systems.In Ischia, Italy, CO 2 vent-influenced P. oceanica meadows (Castello South, "vent", 40.73068047, 13.96303709) are separated by less than a km from areas unaffected by CO 2 vents and ("control", Sant' Anna, 40.73053186, 13.96102293).The vent sites at Castello South have a mean pH near 7.7, while the control pH site at Sant' Anna has ambient pH ~ 7.95 28 .Seawater temperature and other environmental variables (e.g.depth, light, salinity) are similar in CO 2 vent and in control pH sites 28 .While carbon dioxide is the main gas from the vents, other gasses include N 2 (3.2-6.6%),O 2 (0.6-0.8%), Ar (0.08-0.1%), and CH 4 (0.2-0.8%) but not sulfur gas 32 .Trace metal concentrations can be elevated near vents, although detrimental effects on P. oceanica have not been recorded 69 .
15 N amino acid incubation with P. oceanica P. oceanica was collected in situ at the control pH site at 1000 h and the CO 2 vent site at 1040 h on 14 Sept 2021.At each site, two blades attached to a single rhizome unit were selected via SCUBA to have both a new blade developing as well as a corresponding older blade that was generally free of epiphytes.The rhizome was removed so that the blades (from 4 to 6) remained within a sheath.P. oceanica wet mass was quantified with a Pesola and ranged from 2.0 to 3.5 g (mean = 2.75 g).At each site, 8 P. oceanica shoots were collected.While we collected plants that were approximately a half meter distant from each other, we do not know if individuals were genetically distinct.
Simultaneously, gas-tight 500 ml polycarbonate bottles (actual volume is 660 ml) were filled at 1-2 m depth at each site, holding the bottle underwater above the P. oceanica beds, filling to eliminate all bubbles, and capping underwater.For each site, P. oceanica was immediately added to 8 of the bottles, while 4 held seawater only to quantify water column processes.Oxygen and temperature were recorded immediately with 3 mm optical fiber probe (#OXROB3, Pyroscience, Germany) and temperature sensor (Firesting FS02-4, Pyroscience, Germany) assuming 38 ppt Salinity.
We amended each of the 16 bottles with P. oceanica and the 8 with seawater only with amino acids that were enriched in 15 N (98%, Cambridge Isotope, NLM-2151, Lot#PR-24163).We assumed a concentration of dissolved free amino acids (DFAA) of 1 uM based on previous work in Mediterranean Posidonia beds that ranged from 0.5 to 1.9 uM 52,53 .We added 50 uL of a 0.05 M solution to an assumed existing concentration of 1 uM to achieve an enrichment of ~ 0.76 at%.Each bottle was gently inverted 5 times to distribute the isotope, then attached to a line with dive weights at either end and deployed at 2 m depth at noon.All 32 bottles were deployed across 4 lines in the same embayment (Cartaromana Bay), approximately 0.5 km shoreward from where both sets of seagrasses originated, though in waters that were unaffected by the vent activity.The bottles rested on the benthos and they received natural light and some gentle water surge throughout the experiment.
To quantify nitrogen metabolic processes during the daylight versus darkness, we analyzed half of the P. oceanica and seawater contained within the bottles after 6 h at 1800 h; the remaining 16 experimental units were removed from the water immediately after sunrise the following day on 15 Sept 2021 (0720 h).
For each of the end of day and end of night censuses, the oxygen concentration in each bottle was measured and seawater was preserved for dissolved organic matter concentration, inorganic nutrient concentrations, including the concentration of 15 N-ammonium.We also filtered 360-420 ml of seawater through a 0.7 uM gf/f filter (25 mm, Whatman) to quantify the 15 N of particulate organic matter (POM) in the seawater and dried at 50 °C for 48 h.Dissolved inorganic nutrient samples were filtered through a 0.2 um PE filter and frozen at − 20 °C.Seawater samples for dissolved organic C and N analysis were filtered through pre-combusted GF/F filters into acid-washed HDPE vials, immediately fixed with 160 uL of 18.5% HCl and stored at 4 °C until analysis on a total organic carbon analyzer (TOC-L with TNM-L Unit, Shimadzu Corporation, Japan).DON was obtained from TDN subtracting total dissolved inorganic N (nitrate, nitrite and ammonium), determined on seawater samples preserved frozen and analyzed on a Continuous Flow Analyzer (Flowsys, SYSTEA SpA., Italy).
We analyzed the changes to both organic nutrient (DOC and DON) and inorganic nutrients (nitrate, nitrite, ammonium, phosphorus, silica) when incubation bottles had seawater only or P. oceanica, using a univariate three-way ANOVA, followed by two-way ANOVAs with site and treatment and their interaction for both daytime and nighttime.

Figure 1 .
Figure 1.Illustration of the possible paths of δ 15 N amino acids in with the seagrass Posidonia oceanica or seawater only.Enlarged arrows on the left indicate processes that were greater at the control site than the CO 2 venting site.All processes were also greater with Posidonia present, even if only during either the day or the night, indicated with either a sun or moon icon.All processes correspond to Fig. 2a-d and the statistical tests can be found in TableS2.

Figure 2 .
Figure 2. The fate of15 N amino acids added to incubation bottles with and without P. oceanica.In a, δ15 N ammonium (‰) in seawater in incubation bottles with P. oceanica or seawater only.A comparison of the values during the day and night, showed a significant increase in the bottles at the end of the night, with increased values at control site and with P. oceanica.There was also an interaction between time and treatment; the increase at night was highest with P. oceanica.In b.The ammonification rate in nmol per hour across the two sites and attributed to P. oceanica and seawater versus seawater only based on Eq. (1).The control site had greater ammonification compared with the vent site (p = 0.002) and P. oceanica was nearly associated with greater ammonification rates than seawater (p = 0.060), while rates did not differ overall between night and day (p = 0.732).Examining P. oceanica separate from seawater during day versus night, P.oceanica has no diel pattern, while the POM in seawater is associated with greater ammonification during the day.In c, ammonium uptake rate in umol N per g dry mass per hour in the Posidonia meristem during both day and night periods using Eq.(2).Uptake was greater during the day (p = 0.002), with a significant interaction (p = 0.020), indicating a relatively greater uptake of15 N ammonium at night in the control site.In d., the uptake of ammonium by POM was greater when P. oceanica was absent from the incubation bottles during the day, and this effect was greatest at the control site, based on the interaction between site and treatment (p = 0.033).At night, the situation is reversed and the incubation bottles with P. oceanica resulted in a greater incorporation of15 N into the POM in the seawater compared with bottles that had no P. oceanica (p = 0.013).All values were log transformed prior to statistical analyses shown in TableS2.

Figure 3 .
Figure 3.Carbon uptake rates by a. P. oceanica and b. seawater.In a, carbon fixation or respiration, in mg C per hour per g P. oceanica dry mass in incubation bottles during daylight and nighttime hours.P. oceanica carbon fixation was based on oxygen change, following subtraction of water column rates.P. oceanica carbon fixation was greater during the day, but did not differ between control and vent sites.Two-way ANOVA (site effect 0.646, diel effect p < 0.001, interaction p = 0.728).In b, carbon uptake by unfiltered seawater (in mg C per L per hour) is shown for both sites and in incubation bottles with and without P. oceanica.POM carbon uptake differed between day and night (p < 0.001) and with P. oceanica (p = 0.002); there were significant interactions with time of day and site (p = 0.004) and time of day and treatment (p < 0.001).Boxplots show mean values and delimit 25th and 75th percentiles; all points are shown, statistical tests in TableS2.

Figure 4 .
Figure 4.The taxonomic assignments of 67 metagenome-assembled genomes (MAGs) in the 6 samples from the blade or rhizome surface of Posidonia as (a).Phylum, (b).Class and (c).Family.There were 22 high quality MAGs and 42 medium quality MAGs, while taxonomy was not found for 3.In (c), alpha diversity in each sample is shown and based on 'anvi-estimate-scg-taxonomy' .See Supplemental Table S4 and S5.

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