Effect of marine heat waves on carbon metabolism, optical characterization, and bioavailability of dissolved organic carbon in coastal vegetated communities

Dissolved organic carbon (DOC) plays an essential role in the global marine carbon cycle, with coastal vegetated communities as important DOC producers. However, the ultimate fate of this DOC remains still largely unknown due to the lack of knowledge about its chemical composition and lability. Furthermore, global change could alter both DOC ﬂ uxes and its bioavailability, affecting the carbon sequestration capacity of coastal vegetated communities. This study explores, in two contrasting seasons (winter and summer), the effects of an in situ simulated marine heatwave on carbon metabolism and DOC ﬂ uxes produced by seagrass ( Cymodocea nodosa ) and macroalgae ( Caulerpa prolifera ) communities. In addition, the ﬂ uorescent

. In recent years, the effect of sudden and temporary temperature increases, such as marine heatwaves, on these communities has been assessed.However, most previous papers are either laboratory studies (Deguette et al. 2022) or studies that reported widespread mortality or reduced abundance of individuals following marine heatwaves (Wernberg et al. 2013;Arias-Ortiz et al. 2018).To date, little attention has been paid to the effects of in situ experimental marine heatwaves, where unexpected responses may be recorded as a consequence of integrating the entire community (i.e., sediment, fauna, macroalgae, epiphytes, plankton, etc.) into the experimental design (Macreadie and Hardy 2018;Egea et al. 2019a).
Seagrasses have been recognized for their carbon storage potential, leading us to consider these communities as a key element for carbon sequestration in marine areas (i.e., blue carbon) (Nellemann et al. 2009;Kennedy et al. 2010).Like seagrasses, benthic macroalgal communities have also been recently suggested as an important contributor to carbon sequestration in marine sediments (Krause-Jensen and Duarte 2016).The ability of natural ecosystems to act as carbon sinks has usually been related to the huge deposits of organic carbon buried in sediments, which are mainly formed by belowground refractory biomass (Kennedy et al. 2010).However, recent studies have highlighted the importance of the recalcitrant fraction of the dissolved organic carbon (DOC) as an important carbon sink with potential climate regulating capacity (Jiao et al. 2010).DOC is one of the largest exchangeable organic carbon pools in the marine environment, being a central factor in the global carbon cycle (Hansell 2013).Recently, the relevance of DOC generated by macrophyte communities has been highlighted, as it is a significant fraction of their net community production (NCP) (e.g., represents up to 46% of the global seagrass NCP; Barr on et al. 2014) and is a critical component of the carbon exchange between communities (Krause-Jensen and Duarte 2016; Egea et al. 2019b).
Changes in environmental conditions can alter DOC fluxes, but most of our knowledge is limited to studies in freshwater habitats (Larsen et al. 2011;Godin et al. 2017), although recently some studies have shown that DOC release in seagrass populations is highly dependent on environmental factors, such as hydrodynamics (Egea et al. 2018a), nutrient load (Egea et al. 2020;Liu et al. 2020), or increasing temperature (Egea et al. 2019a).However, the ultimate fate of DOC released from vegetated coastal communities (i.e., consumed or sequestered in the long term) may depend not only on the amount, but also on its turnover time (Jiao et al. 2010;Hansell 2013).That is, a significant fraction of DOC (i.e., the labile fraction), consisting of bioavailable material such as carbohydrates, amino acids, and proteins, is rapidly consumed by bacteria (average lifetime $ 0.001 years; Hansell 2013) transferring carbon through the food web and fully involved in carbon exchange between communities (Navarro et al. 2004;Egea et al. 2019b).However, another fraction of DOC (i.e., recalcitrant fraction such as humic-like substances) is resistant to rapid microbial degradation (average lifetime from $ 1.5 years (semi-labile) to $ 40,000 years (ultra-refractory); Hansell 2013).This fraction may be sequestered in continental shelf sediments or in the deep sea, and thus may contribute to long-term carbon sequestration (Krause-Jensen and Duarte 2016;Jiménez-Ramos et al. 2022).Therefore, changes driven by natural and anthropogenic factors in the labile/recalcitrant ratio of DOC produced by benthic communities may have a large and significant effect on its ultimate fate, but this is still a research gap that needs to be addressed.
The optical properties (i.e., absorbance and fluorescence) of dissolved organic matter (DOM) have been used extensively to assess both the sources and behavior of different fractions of DOM in marine systems (Coble 1996;Coble et al. 1998).The fraction of DOM that absorbs light in the ultraviolet (UV) and visible wavelengths is referred to as colored DOM (CDOM) (Coble et al. 1998).The subfraction of CDOM that emits induced fluorescent light is termed fluorescent DOM (FDOM).Two main groups of fluorophores have been differentiated (Coble et al. 1998): protein-like substances, which are considered bioavailable and can contribute substantially to bacterial demand for carbon and nitrogen in marine systems, and humic-like substances, which have traditionally been considered photodegradable but resistant to bacterial degradation.Thus, when humic-like FDOM is not exposed to natural UV radiation, it can accumulate in the ocean on centennial to millennial time scales within the global conveyor belt (Yamashita and Tanoue 2008).Therefore, the protein-and humic-like fluorescence could be used as a proxy to estimate the labile/recalcitrant ratio of the DOC pool produced in macrophyte benthic communities.
The optical characterization and bioavailability of DOC produced by vegetated coastal communities are not yet fully understood.Previous observations in some seagrass systems suggested that DOC released by seagrasses would contribute little to the refractory fraction of DOC (Ferguson et al. 2017;Akhand et al. 2021).In contrast, other studies observed how seagrass meadows directly produce recalcitrant DOM, which is retained in the water column (Watanabe and Kuwae 2015).Similarly, the fate of DOC exuded by macroalgae is still poorly studied.Opportunistic macroalgal species often release highly labile and biodegradable DOC (Zhang and Wang 2017;Chen et al. 2020), while other species such as the kelp Ecklonia cava appear to release more recalcitrant DOC (Wada et al. 2008;Zhang et al. 2017).The optical characterization and bioavailability of DOC released by Caulerpa sp. is still unknown.
In this study, an in situ manipulative experiment was conducted in which a sudden, short-duration temperature increase (as a proxy for a simulated marine heatwave) was designed and replicated in two contrasting seasons (winter and summer) to address the response of two vegetated coastal communities (one dominated by the seagrass Cymodocea nodosa and the other dominated by the macroalga Caulerpa prolifera) to a simulated marine heatwave.In addition, the labile/recalcitrant ratio and chemical composition of DOC produced by both communities were also evaluated for the first time under all experimental conditions.Therefore, we have presented a pioneering study that aims to understand how vegetated coastal communities cope with the effects of marine heatwaves and the contributions of DOC released by these ecosystems as an overlooked tool in Blue Carbon strategies.

Study area
The study was conducted in a shallow macrotidal and sheltered embayment (3545 ha) in the Santib añez salt marsh, in the inner part of C adiz Bay, southern Spain (36.47 N;6.25 W).The benthic community consisted predominantly of dense, monospecific stands of the seagrass Cymodocea nodosa Ucria (Ascherson) and the green rhizophyte Caulerpa prolifera (Forsskål) J. V. Lamouroux.These populations inhabit shallow areas, at a depth of approximately 1 m below the lowest astronomical tide.Climatically, it fits into a semi-warm subtropical thermal regime whose normal seawater temperature range varies between 9 C and 28 C, with an average annual rainfall of 593 mm.The freshwater input into the system is negligible, so the average salinity ranges between 34.1 and 35.6 PSU.In the water column, nutrient peaks usually occur in winter, with values up to 1.4 μmol 4 , and 1.5 μmol L À1 PO 3À 4 (Tovar et al. 2000).For detailed information of the study area, see previous descriptions in Jiménez-Ramos et al. (2021) andPeralta et al. (2021).

In situ experimental set-up of a simulated marine heatwave
To test the effects of a short-term (110 h) marine heatwave on carbon metabolism and DOC fluxes in two seasons (i.e., winter and summer) and in two different communities (i.e., C. nodosa and C. prolifera), an in situ temperature-rise experiment was set up.The experiments were conducted in February 2019 and September 2019, hereafter referred to as winter and summer trials, as they correspond to the period of maximum and minimum macrophyte growth and production in the area (Egea et al. 2019b;Peralta et al. 2021).To better compare the two study periods, sampling days were chosen at each season with a similar tidal range as well as weather forecast (e.g., presence of clouds, no rain, wind, etc.) to reduce environmental variability.Although each community was dominated by macrophytes, it is actually an assemblage of several biological components, such as plankton, epiphytes, other macroalgae, fauna, and sediment microbes.Therefore, the results integrate the entire community as a way to recreate a more realistic approach.In each season, for each community, three replicated benthic chambers (hereafter called incubators) were randomly placed for each treatment (control and high temperature) by scuba diving.The minimum distance between replicates was 6 m and the locations of both treatments were mixed in each benthic communities, avoiding any bias due to the location of the treatments (distance to coast, meadow density, etc.) (Fig. 1).
Incubators were similar to those used in previous studies analyzing carbon metabolism and DOC fluxes in situ (Egea et al. 2019b) and manufactured by ©Aquatic-Biotechnology (Spain).Incubators consisted of two parts: a rigid polyvinyl chloride cylinder (diameter = 20 cm; height = 17 cm) and a transparent polyethylene plastic bag (height ≈ 37 cm; width ≈ 33 cm) attached to a polyvinyl chloride ring (width = 4 cm).Both parts were joined by a silicone gasket and tightly held by four rubber bands (Fig. 1).The cylinders were inserted into the sediment (15 cm) 2 h before fitting the plastic bags to reduce the effect of sediment disturbance.In addition, all six hightemperature incubators had underwater heaters (Easyheater 100 W; height ≈ 15.5 cm and width ≈ 4.5 cm) attached to the rigid polyvinyl chloride ring, separated from it by 2.5 cm and about 5 cm from the seafloor to heat the water to about 3 C (above water temperature) during the experimental period (110 h).HOBO data loggers (UA-002-64) were placed within each incubator and on the bare sediment (n = 3) near the experimental incubators to record temperature ( C) and light (lumens m À2 ) every 10 min throughout the experimental period.To transform lumens m À2 into μmol photons m À2 s À1 , the most common conversion factor given in the literature for sunlight was used (1 lumen m À2 = 51.2μmol photons m À2 s À1 ; Carruthers et al. 2001).The daily light dose was calculated using the average daily hours of light (photoperiod) in each season (10.75 and 12.38 h in winter and summer trials, respectively).
During the simulated marine heatwave experiment, two types of polyethylene plastic bags were used alternatively: (i) a gas-permeable bag, and (ii) an air-tight bag.First, the gaspermeable bags were used for the first 90 h of the in situ experimental heating, which helped to minimize the risk of oxygen over-accumulation during the light hours and anoxic conditions during the night.After the first 90 h of the in situ experimental heating, the gas-permeable bags were replaced with air-tight bags to achieve a complete isolation of the community and thus allow the study of changes in dissolved oxygen and DOC fluxes under simulated marine heatwave conditions.To minimize heat loss from the water in the high-temperature treatments during bag replacement, we changed the bags rapidly (< 1 min) just before sunset, when the water temperature naturally decreased.Each air-tight bag was provided with a sampling port located in the upper half of the bag (≈ 20 cm) to draw water samples.The walls of both bags (wall thickness ≈ 0.07 mm) were flexible enough to allow movement with hydrodynamics, preventing water stagnation.Light penetration measured by the HOBOs inside the incubators was approximately 99.15% AE 0.01% of incident light outside the bag.Oxygen diffusion controls were performed on the two types of plastic bags, showing oxygen permeability and oxygen nonpermeability of the plastic bags, respectively.

Simulated marine heatwave characteristics
Mean seawater temperature in control temperature treatments ranged from 15.6 AE 0.07 C in winter to 24.2 AE 0.08 C in summer.The heaters produced a steady temperature rise from ambient water temperature, and so the high-temperature treatments exhibited a temperature oscillation between day and night (Fig. 2).Seawater temperature in high-temperature treatments was statistically higher in both sampling events (about 3 C; p < 0.001) compared to the control treatments, with a mean of 18.7 AE 0.05 C and 27.5 AE 0.07 C in winter and summer, respectively.To assess the similarity of the temperature reached in the high-temperature treatments to the natural marine heatwaves in the area, the sea surface temperature (SST) and the occurrence of oceanic marine heatwaves close to C adiz Bay were evaluated.The daily temperature dataset, displayed on the Marine Heatwaves Tracker app (Schlegel 2020), contains SST, climatology, threshold data, and the records of marine heatwave events from 1982 to present.A pixel near C adiz Bay (Lon = 6.375 W, Lat = 36.375N) was selected, and a time series of ocean SST was plotted from 08 June 2017 to 08 June 2022 (Supporting Information Fig. S1).The hightemperature treatments reached mean temperatures higher than the high SST threshold for the last 5 years in February (16.3 AE 0.03 C) and September (24.4 AE 0.03 C).In turn, 17 marine heatwaves occurred in this period, lasting between 5 and 44 d and with an increase in SST between 1.21 C and 2.63 C.However, in shallow waters the temperature rise can be even higher than the average SST in marine areas (Deguette et al. 2022).Therefore, the increase of about 3 C reached in this experiment during 110 h was relevant to simulate a marine heatwave in the shallow water conditions of the Bay of C adiz.

Sample procedure
Samples for community carbon metabolism (i.e., dissolved oxygen) and DOC fluxes were collected using acid-washed 50-mL syringes at three times during the last hours of the experimental period (on the 4 th day): (i) just before sunset (S1), (ii) right after sunrise (S2), and (iii) 6 h after sunrise (S3).In this way, community carbon metabolism (through changes in dissolved oxygen concentration) and DOC fluxes in dark and light periods can be distinguished (Egea et al. 2019b).To calculate the exact water volume in each incubator, 20 mL of a 0.1 M uranine solution (sodium fluorescein, C 20 H 10 Na 2 O 5 ) were injected into each incubator bag at the end of the simulated marine heatwave experiment, allowing 15 min for mixing, and shaking the bag manually to facilitate rapid mixing of the uranine.The water samples were then collected and kept frozen until spectrophotometric determination according to Egea et al. (2019b).The mean volume of water enclosed in the incubator bags at the end of experimental period was 11.4 AE 0.2 L (n = 24).
Finally, upon disassembly of the experimental set-up, the macrophyte biomass within the incubators was harvested, rinsed and dried at 60 C to estimate the photosynthetic biomass (i.e., aboveground), subterranean biomass (i.e., belowground) and opportunistic macroalgal biomass (dry weight; g DW m À2 ) of both communities.In this study, aboveground biomass of C. prolifera refers to fronds whereas belowground biomass refers to the subterranean network of cylindrical stolons with a series of rhizoid clusters, which is consistent with previous studies with this species (e.g., Vergara et al. 2012).For C. nodosa, aboveground biomass is shoots, while belowground biomass is rhizomes and roots (Brun et al. 2002).A fraction of the biomass was used to measure nonstructural carbohydrates (NSCs; i.e., sucrose and starch in aboveground and belowground tissues) following Brun et al. (2002).

Bioavailability assay of DOC produced by vegetated communities
At the end of the in situ simulated marine heatwave experiment and before removing the experimental set-up, 300 mL of seawater were taken from each incubator through the sampling port using acid-washed 50-mL syringes.The seawater collected was filtered and added to hermetic closure glass bottles at a ratio of 9 : 1 according to Jiménez-Ramos et al. (2022).For this purpose, 270 mL of water from the in situ incubators was filtered through a 0.2-μm polycarbonate filter into glass bottles.Bacterial cultures were then inoculated with the natural bacteria community collected in 30 mL of water from each in situ incubator and were filtered through a 0.8-μm polycarbonate filter to remove bacterial predators such as small flagellates.Incubation bottles (n = 12) were stored in darkness at 4 C until transported to the laboratory.Once in the laboratory, the DOC bioavailability assay was carried out for 7 d in a temperature-controlled room set at 18 C and under dark conditions, in accordance with previous works (Jiménez-Ramos et al. 2022).In this study, the term bioavailable/labile fraction of DOC refers to the fraction of DOC that is utilized by heterotrophic bacteria during the incubation time (i.e., 7 d).Likewise, we use the term recalcitrant fraction of DOC to refers to the remaining fraction of DOC.Ammonium (NH 4 Cl) and phosphate (NaH 2 PO 4 ) were supplied at the beginning to a final concentration of 10 and 2 μM, respectively, to avoid growth limitation by nitrogen or phosphorus availability.At time zero and every other days, samples were collected from each incubation bottle to measure DOC.

Carbon community metabolism analysis
Water samples for dissolved oxygen concentration from the simulated marine heatwave in situ experiment were fixed immediately after collection in the supporting vessel, kept in darkness and refrigerated, and determined using a spectrophotometric modification of the Winkler titration method (Pai et al. 1993;Roland et al. 1999).Hourly rates of community respiration (CR h ) and NCP (NCP h ) were calculated using the following formulas: where DO S1-S3 are the dissolved oxygen concentrations at sampling times S1-S3, ΔT is the elapsed time between sampling events, and "Vol" and "Area" are the volume and area of the incubators, respectively.Finally, daily rates of community gross primary production (GPP d ), community respiration (CR d ), and NCP (NCP d ) were estimated following the calculations: where photoperiod corresponds to the number of hours of sunlight measured on each sampling day.Metabolic rates in units of dissolved oxygen were converted to carbon units assuming photosynthetic (PQ = moles O 2 : moles CO 2 ) and respiratory quotients (RQ) of 1, a widely used value for benthic macrophyte communities (Tuya et al. 2014;Egea et al. 2019b).

DOC analysis
DOC samples from the in situ simulated marine heatwave experiment and the the produced DOC bioavailability assay were filtered through pre-combusted (450 C for 4 h) Whatman GF/F filters (0.7 μm) and kept with 0.08 mL of H 3 PO 4 (diluted 30%) at 4 C in acid-washed material (encapsulated glass vials with silicone-PTFE caps) until analyzed.Concentrations of DOC were obtained by catalytic oxidation at high temperature (720 C) and chemiluminescence using a Shimadzu TOC-VCPH analyzer.Certified reference material for DOC (low and deep), provided by D. A. Hansell and W. Chen (University of Miami), were used to assess the accuracy of the estimates.
Hourly rates of DOC production during the night and light periods in the in situ simulated marine heatwave experiment were calculated using the following formulas: where DOC S1-S3 , are the DOC concentrations at sampling times S1-S3, ΔT is the elapsed time between sampling events, and "Vol" and "Area" are the volume and the area of the incubator, respectively.Finally, daily rate of net DOC flux was estimated following the calculation: where photoperiod and dark-period correspond to the number of hours of sunlight and darkness measured on each sampling day.Thus, when net DOC flux was positive, the community was considered to act as a net DOC producer (i.e., source).However, when net DOC flux was negative, the community was considered to act as a net DOC consumer (i.e., sink).
In the bioavailability assay of the DOC produced, the labile (DOC L ) and recalcitrant (DOC R ) fractions of the community DOC fluxes were calculated using the following formulas: where DOC initial and DOC final are the DOC concentrations at the initial and final periods of the bacterial incubation assay.
Finally, the ratio between recalcitrant and labile DOC concentrations (DOC R : DOC L ) at each sampling event was calculated as the recalcitrant DOC concentration (i.e., the DOC concentration at the end of the incubations with bacteria) divided by the labile DOC concentration (i.e., the difference between the initial and the final DOC concentrations during the incubations with bacteria) using the following formula: FDOM analysis FDOM samples from the in situ simulated marine heatwave experiments were filtered through pre-combusted (450 C for 4 h) Whatman GF/F filters (0.7 μm) and kept at 4 C in acidwashed material (glass vials encapsulated with silicone-PTFE caps) until analyzed.For FDOM analyses, excitation emission matrices (EEMs) were performed with a LS 55 PerkinElmer Luminescence spectrometer equipped with a xenon discharge lamp, equivalent to 20 kW for 8 ms duration.The detector was a red-sensitive R928 photomultiplier.Measurements were performed at a constant room temperature of 20 C in a 1-cm quartz fluorescence cell.The excitation wavelength ranged from 240 to 440 nm with 10 nm increments and the emissions wavelength from 300 to 560 nm at 0.5 nm increments, with excitation and emission bandwidths of 5 nm, and the integration time was 0.1 s (scan speed, 250 nm min À1 ).The EEMs of the samples were corrected for Raman and Rayleigh scattering (Murphy et al. 2010) and normalized to the Raman area of the milli-Q water blanks.Raman area and its baseline correction were calculated from the emission scan of milli-Q water excited at 350 nm (Lawaetz and Stedmon 2009).Samples with an absorption coefficient at 254 nm greater than 10 m À1 were diluted with MQ water to avoid inner-filter correction (Stedmon and Bro 2008).The drEEM 0.2.0 toolbox was used to standardize the EEMs (Murphy et al. 2013).
The changes in intensities of each peak were calculated during the dark period (i.e., the differences between S2 and S1) and during the light period (i.e., after extrapolating the differences between S3 and S2 for all daylight hours).Thus, the intensity changes at each peak during the whole day (24 h) were calculated by summing the hourly peak in daylight multiplied by the photoperiod and the hourly peak at night multiplied by the night hours using the following formula: where Peak S1 , Peak S2 , and Peak S3 are the intensity of peaks at different sampling times respectively, ΔT is the elapsed time between sampling events, PP (h) is the photoperiod and NP (h) is the night period.

Data and statistical analysis
The effects of treatment, community, and season on each response variable were tested using generalized linear models (GLMs).For each response variable, we selected a particular family error structure and link function to achieve the assumptions of linearity, homogeneity of variances and the absence of overdispersion, which were checked by visual inspection of residuals and Q-Q plots (Harrison et al. 2018) after modeling.Aboveground biomass, belowground biomass, community biomass, opportunistic macroalgal biomass, GPP, CR, NSC content, and DOC bioavailability were modeled using Gamma distribution with inverse link, whereas NCP and DOC flux were modeled using Gaussian distribution with identity link.Pairwise comparisons were tested using estimated marginal means with a Bonferroni correction ("emmeans" R package; Lenth et al. 2019).A linear model was used to test the relationships between the main fluorescent peaks and temperature for each community and the relationships between humic-like peak-M and protein-like peak-T with GPP.Assumptions of normality and homoscedasticity were evaluated by examining the residuals of all linear models.Statistical analyses were computed with R statistical software 4.0.2(R Development Core Team 2020).

In situ marine heatwave simulated experiment
The simulated marine heatwave in Cymodocea nodosa produced a tendency to increase both CR and, especially, GPP (approximately 2.1 and 1.5 times higher in winter and summer, respectively), although differences were not statistically significant.As a result, the NCP increased significantly (i.e., approximately 1.9 and 1.7 times more than the control treatments in winter and summer, respectively) (Fig. 3).In contrast, the simulated marine heatwave in Caulerpa prolifera caused the opposite effect, showing a slight decrease in NCP as a consequence of a marked tendency to increase its CR (approximately 1.7 and 2.4 times higher in winter and summer, respectively), although differences were not statistically significant for this community (Fig. 3; Supporting Information Table S1).
DOC fluxes during daylight hours were higher than during night hours in C. nodosa (p < 0.05).Regarding C. prolifera, higher DOC release was found during night hours in the winter trial, whereas higher DOC release was recorded during daylight hours in the summer trial in the control treatments (p < 0.05).The simulated temperature increase shifted C. prolifera from DOC-consuming to DOC-producing during daylight hours in winter, whereas it shifted from DOC-producing to DOC-consuming during daylight hours in summer (Fig. 4a).In terms of daily DOC rates, both communities were DOC producers in the control treatments, especially C. nodosa in the winter trial.The simulated temperature increase raised the release of DOC in C. nodosa community (approximately 1.3 and 1.9 times more than the control treatments in the winter and summer trial, respectively) although no statistically significant differences were found.As for C. prolifera, the simulated temperature increase significantly reduced DOC fluxes in the summer trial to negative values, whereas a tendency to raise the daily DOC flux was found in winter (Â2.7-fold), but without significant statistical difference (Fig. 4c; Supporting Information Table S2).
Aboveground biomass was significantly higher in summer than in winter in C. nodosa (average 111 AE 19 and 27 AE 5 g DW m À2 respectively).In contrast, aboveground biomass was similar in C. prolifera in both seasons (average 32 AE 4 g DW m À2 ) (Supporting Information Fig. S2a).Aboveground biomass, belowground biomass, and community biomass (i.e., the sum of seagrasses, epiphytes and other opportunistic macroalgae) were similar between treatments in both communities and seasons (Supporting Information Fig. S2).A high proportion of opportunistic macroalgae (especially Ulva sp.) was found in C. nodosa in winter (average 71 AE 19 g DW m À2 , Â3.4 higher than the average found in this community at summer and in the community dominated by C. prolifera) (Supporting Information Fig. S2d).As for NSCs content, they varied between macrophytes and seasons, but were not affected by increasing temperature in both communities (Supporting Information Fig. S3).Sucrose was the main NSC in C. nodosa, especially in belowground (rhizomes and roots) tissues, whereas starch was the main NSC in C. prolifera.C. nodosa increased its sucrose content in aboveground and belowground tissues in summer, while its belowground starch content decreased.No pattern was found between seasons in NSC content in C. prolifera.

Bioavailability assay of DOC produced by the vegetated communities
The DOC pool from the C. nodosa community experienced a sharp decline during the 1 st 4-6 d after bacterial inoculation in the bioavailability assay (Supporting Information Fig. S4).The same was recorded for DOC from the C. prolifera community, but the decrease was milder.This initial phase of sharp decrease in the DOC concentration was followed by a final phase of stagnation in all the cultures.The lowest fraction of recalcitrant DOC flux (DOC R ) was found in C. nodosa in the winter control treatment (0.42), whereas the highest DOC R was found in C. prolifera in the winter high-temperature treatment (0.92).All treatments showed DOC R : DOC L ratios higher than 1, except C. nodosa in the winter control treatment.The highest DOC R : DOC L ratios were found in the high-temperature treatments, especially for C. prolifera (Fig. 5; Supporting Information Table S3).

Optical characterization of the DOM produced
The main humic-like peaks observed in the EEMs of both communities were peak-A, peak-C, and peak-M (Fig. 6, Coble 1996).Peak-A was found at < 240/400-450 nm in all in situ incubators in summer, but shifted to Ex/Em $ 262/455 nm in winter.Peak-C (usually categorized as terrestrial humiclike substances) and peak-M (usually described as marine humiclike substances) were found at Ex/Em $ 350/450 nm and Ex/Em $ 320/420 nm, respectively (Coble 1996).Since peak-A, peak-C, and peak-M presented a significant relationship between them (Pearson correlations r ≥ 0.6; p < 0.05), only peak-C and peak-M data are shown in this study.As for the protein-like peaks, the peak-T maximum was observed at Ex/Em $ 280/375 nm.
In winter, the control treatments of both communities showed a peak-C maximum around $ 360/450 that shifted to shorter wavelengths (peak-M, $ 325/420) with the simulated marine heatwave conditions.In summer, the main peak observed in the visible region was the peak-M for all treatments and communities.Also, a shift of the humic-like maxima in peak-C to peak-M was observed under higher temperature conditions (i.e., high-temperature treatments in both seasons).Changes in the intensities of each fluorescent peak throughout the day (24 h) showed the pattern of FDOM consumption or production for each community and treatment (Supporting Information Table S4).Significantly higher FDOM intensity values were found in summer than in winter for both communities.The marine heatwave condition increased FDOM production in C. nodosa in summer.Meanwhile, increasing temperature appeared to elevate humic-like peaks in the winter trial in C. prolifera, from being a consumer of humic-like peaks to a producer of humic-like peaks.The peak-T/peak-C ratio was less than 1 or negative in all treatments as a consequence of the peak-T consumption, except in C. prolifera in winter (Supporting Information Table S4).
A significant and positive linear regression (p < 0.05) was found between humic-like peaks with temperature (both natural, i.e., derived from seasonal temperature changes, and simulated, i.e., derived from the marine heatwave simulated in the experiment) for both communities (Fig. 7a,b).The linear regression between protein-like peak-T with temperature was significantly positive in the C. nodosa-dominated community but no significant regression was found in the C. proliferadominated community (Fig. 7c).A significant and positive linear regression (p < 0.05) was found between humic-like peak-M and protein-like peak-T with GPP in the C. nodosadominated community (Supporting Information Fig. S5).In contrast, no relationship has been found between FDOM and GPP in the C. prolifera-dominated community (Supporting Information Fig. S5).

Effect of marine heat waves on vegetated community production
The simulated marine heatwave condition resulted in a significant increase in NCP in both seasons for the Cymodocea nodosa-dominated community, which is likely due to the enhanced photosynthetic rate.The optimum temperature for C. nodosa is higher than for other macrophyte species (up to 29.5 C; Savva et al. 2018) and temperature seems to favor enzymatic action in photosynthesis (Terrados and Ros 1995).In addition, the maximum quantum yield of photosystem II is also favored (Deguette et al. 2022) when the temperature increase is maintained within the thermal tolerance limits for this species (12.9-36.9C; Savva et al. 2018;Egea et al. 2018b).This increase in photosynthetic rates and production with temperature has been demonstrated in other seagrass species such as Halophila ovalis (Collier et al. 2011) and Zostera marina (Marsh et al. 1986).High productivity in seagrasses may result in a high potential for substantial carbon burial rates (Duarte et al. 2013).Therefore, our results could suggest that a higher frequency of marine heatwaves in the coming decades could lead to an increase in net production of C. nodosa, which would add further arguments for the protection of this seagrass species, as they could sequester even more carbon under predicted conditions of climate change (i.e., higher temperatures and frequency of marine heatwaves).However, it should be noted that our results were obtained in a healthy community where C. nodosa reaches a high density and biomass, and inhabits sandy/muddy sediments with a medium organic matter content (2%) (Peralta et al. 2021).In other seagrass meadows where the canopy density was lower or the organic matter content in the sediment was higher, the effect of temperature on respiratory processes would probably gain more relevance (Malinverno and Martinez 2015), decreasing this potential carbon uptake.In addition, other seagrass communities growing near their thermal tolerance limit or under the effect of other limiting factors such as light or nutrients may show a different response than those recorded in this study (Arias-Ortiz et al. 2018).Therefore, the cumulative effects of several marine heatwaves on the growth and production of the seagrass community should be evaluated in future research.Unlike the C. nodosa community, the Caulerpa.prolifera community did not show significant differences in both seasons, although a trend of reduced NCP was observed, probably as a consequence of the higher rates of respiration than photosynthesis recorded in this species under higher temperatures (Terrados and Ros 1992).This trend is in line with previous studies that showed a decrease in the production of this macroalga in the summer period (Egea et al. 2019b) as a consequence of both high irradiance (Vergara et al. 2012) and elevated temperature (Vaquer-Sunyer et al. 2012).
Production values (i.e., GPP, CR, and NCP) in both communities in the control treatments were similar to those reported in the same area by Egea et al. (2019b), and similar to those reported by Duarte et al. (2010) for the seagrass and by Tuya et al. (2014) for the macroalga.The methodology used in this study has been widely utilized (Egea et al. 2019b) allowing for true independent replication and modification of a single factor (i.e., temperature), but it is not without methodological limitations.Because it is based on long-term incubations, pH and dissolved oxygen may differ from nearby natural meadows, where turbulent mixing prevents oversaturation during daylight hours and hypoxia during darkness (Champenois and Borges 2012).To address this, we used flexible gas-permeable bags for the 1 st 90 h, which were replaced by air-tight bags on the last day to completely isolate the community.In our experimental set-up, the NCP was estimated for 6 h after sunrise, which may underestimate the NCP up to 25% (Olivé et al. 2016).As the DO concentrations measured in the S2 period were higher than the accepted threshold of 2 mg O 2 L À1 for hypoxia (Vaquer-Sunyer and Duarte 2008), CR was probably not underestimated.Overall, it is possible that the NCP estimated in this study has some degree of underestimation as a result of the isolation of the community inside the incubator bag, indicating that these communities may be even more autotrophic than our results suggested.

Effect of marine heat waves on DOC fluxes
A substantial amount of autochthonous DOC was generated by both coastal vegetated communities derived from their high productivity, which is in agreement with previous studies (Watanabe and Kuwae 2015;Egea et al. 2019b).The values reported in the control treatments were within the range of values previously described for these species throughout the year in the area (Egea et al. 2019b) and similar to those reported by Barr on et al. (2014) (average of 12.4 AE 2.9 and 23.2 AE 12.6 mmol C m À2 d À1 for seagrass and macroalgae, respectively).The only exception were the slightly higher values found in winter in C. nodosa, which can be attributed to the high biomass values of opportunistic macroalgae present in the community, which probably elevated DOC production.Our results also showed that the C. nodosa meadow displayed higher net DOC release during sunlight hours than in darkness, when the community may even act as DOC consumer in the summer trial (Fig. 4).This is in agreement with previous studies in seagrasses (Barr on et al. 2014;Egea et al. 2019b), and suggests that a significant fraction of the DOC released by this community is mostly done by photosynthesis activity.In contrast, C. prolifera acted as a DOC consumer during sunlight hours, which may suggest that the contribution of photosynthesis activity to DOC released by this community may not be as important.This is consistent with previous studies on other macroalgae such as kelps (Weigel and Pfister 2021) or rhodophytes like Pyropia haitanensis (Xu et al. 2022).Given that the higher organic matter content and smaller sediment grain size (i.e., muddy sediments) are typical in bottoms where this species thrives (Vergara et al. 2012), DOC release from POC remineralization in the sediment could also be more relevant in this community (Loginova et al. 2020).
Marine heatwave conditions significantly reduced the daily DOC flux in C. prolifera in summer, which became negative, whereas a tendency was found to increase the daily DOC flux in winter (Â2.7-fold but no significant statistical difference).These contradictory changes in DOC fluxes between the two trials could be explained by the role of other community components such as plankton, as the latter is an important DOC consumer from macroalgae, especially in summer (Egea et al. 2019b).As for C. nodosa, marine heatwave conditions did not produce significant differences in both seasons, although there was a tendency to increase its daily DOC flux, especially in summer, when the DOC flux in high-temperature treatments doubled that of the control treatments, although differences were not statistically significant.

Bioavailability of DOC produced by vegetated communities
Overall, both communities showed a DOC R : DOC L ratio > 1 in all seasons and treatments, indicating that both communities tend to release a higher proportion of recalcitrant DOC than labile fraction.The only exception was C. nodosa in the winter control treatment, which is likely related to the large biomass of opportunistic macroalgae (Ulva sp.) present in the community, which tend to release mainly labile and biodegradable DOC (Zhang and Wang 2017).The C. nodosadominated community released a similar proportion of recalcitrant (DOC R ; 42% in winter and 56% in summer) and labile fraction (DOC L ) in control treatments.The high DOC L fraction found here explains the close coupling between DOC production and bacterioplankton productivity recorded in some seagrass communities (Ziegler and Benner 1999;Ziegler et al. 2004) and with the high concentrations of easily degradable proteinaceous components found recently in a seagrass system (Akhand et al. 2021).Similarly, the high fraction of DOC R found in our experiment may also explain the recalcitrant character of DOC released in other seagrass communities (Watanabe and Kuwae 2015;Jiménez-Ramos et al. 2022).On the other hand, while opportunistic macroalgal species usually release highly labile and biodegradable DOC (Zhang and Wang 2017;Chen et al. 2020), other species like the kelp Ecklonia cava show a more recalcitrant character in released DOC (Wada et al. 2008;Zhang et al. 2017).Our results indicated that C. prolifera appears to release a high proportion of recalcitrant DOC in control treatments (87% in winter and 64% in summer).These results are in agreement with those obtained by optical characterization of the DOM produced.Our results suggest that humic-like fluorescence may dominate in vegetated coastal communities as the peak-T/peak-C ratio (Supporting Information Table S4), a measure of the ratio between protein-like and humic-like components, was less than 1 or negative in most treatments.
Our results on the recalcitrant DOC fraction have important implications for conservation and management, as they support the idea that vegetated coastal communities may not only contribute to ocean carbon sequestration as recalcitrant carbon buried in their sediments (Nellemann et al. 2009;Duarte et al. 2013), but also as recalcitrant carbon sequestered in dissolved form in the water column, similar to the recalcitrant DOC fraction of plankton in the open ocean (Jiao et al. 2010(Jiao et al. , 2014)).However, the scaling-up of our DOC bioavailability assay results should be viewed with caution.There are multiple reasons why results under controlled laboratory conditions may not fully reflect the natural long-term fate of DOC released by these macrophytes.Recalcitrance may vary between different bacterial species, and different environments (Jiao et al. 2014;Watanabe et al. 2020).Although previous work on DOC bioavailability reached the stationary phase in DOC concentration within the incubation time used here (7 d; Jiménez-Ramos et al. 2022), some previous studies indicated that short-term degradation experiments may underestimate the long-term persistence of organic carbon (Trevathan-Tackett et al. 2020).Finally, there are DOC degradation processes that were not measured in this study (photochemical degradation) that could also be important in driving DOC degradation (Wada et al., 2015).Regardless, our study shows how seagrasses and macroalgae can directly release a significant fraction of nonlabile DOC, with a high potential to contribute to the recalcitrant fraction of oceanic DOC.This DOC can help counteract climate change through the fraction exported to deep waters, where it remains "trapped" long enough to qualify as sequestration, even if fully respired to CO 2 (Krause-Jensen and Duarte 2016).Furthermore, it should be noted that although marine prokaryotes consume labile DOC, they can also produce recalcitrant DOC as a metabolic byproduct (Jiao et al. 2010).Therefore, long-term studies on the bioavailability of DOC released by macrophytes that include molecular fingerprints and carbon fluxes through the microbial loop are needed to better understand the role of DOC released by these important communities in climate regulation capacity.
One of the most remarkable results of this study was the increase in the fraction of recalcitrant DOC in both communities under marine heatwave conditions.Increase temperature also changed the quality (i.e., chemical structure) of DOM produced by both communities, as indicated by the shift of Ex/Em humic-like maxima from longer (peak-C) to shorter wavelengths (peak-M).Moreover, a positive and significant correlation between humic-like substances and temperature for both communities were found (Fig. 7).This is probably due to both the increased production of recalcitrant DOC directly by the macrophytes themselves and the microbial transformation of labile to recalcitrant DOM by the marine prokaryotes.The production of recalcitrant DOC by macrophytes may be related to the production of secondary metabolites, as it is in summer (i.e., higher temperatures) that macrophytes increase secondary metabolism characterized by humic compounds (Rotini et al. 2013).Also, higher temperatures increase bacteria abundance, production and respiration (Joint and Smale 2017), which may drive the consumption of labile DOM by these marine prokaryotes, which may also produce recalcitrant DOM as a metabolic by-product (Jiao et al. 2010;Koch et al. 2013).These are hypotheses that need to be tested in future research.Increased release of recalcitrant DOC by macrophyte communities with increasing temperature has important conservation and management implications, especially for C. nodosa, as this study shows how increasing seawater temperature could not only enhance its productivity, but also increase the recalcitrant fraction of DOC produced.

Conclusions
The results of this study indicated that marine heatwaves can increase the productivity of seagrass-dominated communities such as Cymodocea nodosa and shift DOC fluxes from Caulerpa prolifera-dominated community to negative in summer.Our results also showed that both communities are highly DOC-producing, especially during sunlight hours for C. nodosa and dark hours for C. prolifera.C. nodosa produced a substantial amount of labile (58% and 44% in winter and summer, respectively) and recalcitrant (42% and 56% in winter and summer, respectively) DOC, whereas C. prolifera released mainly recalcitrant DOC (87% and 64% in winter and summer, respectively).This research also revealed that temperature is an important factor determining the chemical structure and bioavailability of DOC produced by these communities, as both vegetated coastal communities tend to produce more humic-like and less bioavailable DOC with increasing temperature.Finally, our results follow in line with previous studies highlighting the role of vegetated coastal communities in sequestering recalcitrant carbon in dissolved form in the ocean, adding further arguments to the growing need for their protection and conservation as valuable ecosystems to combat climate change now and under future global change scenarios.

Fig. 1 .
Fig. 1.(a) Simplified diagram of the incubators and experimental design.See detailed description in the text.The images show a representative in situ incubator in (b) Cymodocea nodosa and in (c) Caulerpa prolifera communities.

Fig. 2 .
Fig. 2. Temperature-time series during the in situ experiment of the simulated marine heatwave.Lines are the mean of the control treatments (n = 6) (black) and high-temperature treatments (n = 6) (red), respectively.

Fig. 3 .
Fig. 3. Effect of an in situ simulated marine heatwave on (a) community GPP, (b) CR, and (c) NCP in communities dominated by Cymodocea nodosa and Caulerpa prolifera in winter and summer.Different letters indicate significant differences among treatments, communities and seasons.Bars represent mean (n = 3) and dots represent the data.

Fig. 4 .
Fig. 4. Effect of an in situ simulated marine heatwave on (a) DOC fluxes in daylight hours, (b) DOC fluxes at night hours, and (c) daily DOC fluxes in communities dominated by Cymodocea nodosa and Caulerpa prolifera in winter and summer.Note the scale differences on the Y-axis.Different letters indicate significant differences among treatments, communities, and seasons.Bars represent mean (n = 3) and dots represent the data.

Fig. 5 .
Fig. 5. (a) Labile DOC fraction, (b) recalcitrant DOC fraction, and (c) the recalcitrant DOC: labile DOC ratio in the different treatments, communities and seasons.Bars represent mean (n = 3) and dots represent the data.

Fig. 6 .
Fig. 6.FDOM EEMs of in situ incubators at the end of the marine heatwave experiment.Fluorescent intensity is reported in Raman units (R.U.).The main Coble's peaks are indicated in the 1 st plot.Humic-like peaks are A, C, and M, while peak-T is classified as protein-like.

Fig. 7 .
Fig. 7. Relationship between temperature with (a) fluorescent humic-like peak-C, (b) fluorescent humic-like peak-M and (c) fluorescent protein-like peak-T for each community.Solid and dotted lines represent significant linear regression for Cymodocea nodosa and Caulerpa prolifera, respectively.