F L Figueroa

J Bonomi-Barufi

P S M Celis-Plá

U Nitschke

F Arenas

S Connan

M H Abreu

E-J 10 Malta

R Conde-Álvarez

F Chow

Figueroa

Celis - Plá

Chow

Stengel Robledo D 14

Short-term effects of increased CO2, nitrate and temperature on photosynthetic activity in Ulva rigida (Chlorophyta) estimated by different pulse amplitude modulated fluorometers and oxygen evolution

Keywords: Acidification, climate change, electron transport rates, in vivo chlorophyll a fluorescence, nitrate, photosystem I and II, temperature

published or not. The documents may come

INTRODUCTION

Ocean acidification will increase by 0.3-0.4 pH unit by 2100 due to the anticipated rise in atmospheric CO 2 levels to 800-1000 ppm by the end of this century, according to the "business-as-usual" CO 2 emission scenario [START_REF] Caldeira | Anthropogenic carbon and ocean pH[END_REF]. This acidification process may significantly change the water carbonate chemistry [START_REF] Hall-Spencer | Volcanic carbon dioxide vents show ecosystem effects of ocean acidification[END_REF][START_REF] Pelejero | Paleo-perspectives on ocean acidification[END_REF] and then affect not only calcifying organisms [START_REF] Riebesell | Reduced calcification of marine plankton in response to increased atmospheric CO 2[END_REF] but also non-calcifying photoautotrophs [START_REF] Roleda | Ocean acidification and seaweed reproduction: increased CO 2 ameliorates the negative effect of lowered pH on meiospore germination in the giant kelp Macrocystis pyrifera (Laminariales, Phaeophyceae)[END_REF]. So far, the cumulative effects of climatically realistic CO 2 -driven pH change on non-calcifying seaweeds remain poorly understood [START_REF] Kroeker | Meta-analysis reveals negative yet variable effects of ocean acidification on marine organisms[END_REF]. Studies on the ecological and physiological impacts of elevated CO 2 concentrations on macroalgae were initiated in the early 1990s: growth of Porphyra yezoensis juveniles was significantly enhanced in cultivation enriched with CO 2 up to 1000 ppmv [START_REF] Gao | Enhanced growth of the red alga Porphyra yezoensis Ueda in high CO 2 concentrations[END_REF]. Macroalgae are a highly diverse group with complex functional morphologies and varied ecological roles. Morphological plasticity, in addition to physiological mechanisms, may further determine the algal capacity to acclimate to global change scenarios [START_REF] Falkenberg | Contrasting resource limitations of marine primary producers: implications for competitive interactions under enriched CO 2 and nutrient regimes[END_REF][START_REF] Gao | Ocean acidification and nutrient limitation synergistically reduce growth and photosynthetic performances of a green tide alga Ulva linza[END_REF][START_REF] Gao | Diverse responses of sporophytic photochemical efficiency and gametophytic growth for two edible kelps, Saccharina japonica and Undaria pinnatifida, to ocean acidification and warming[END_REF]. In macroalgae, doubling CO 2 level caused an increase in photosynthetic activity of 52-130% depending on the species [START_REF] Riebesell | Enhanced biological carbon consumption in a high CO 2 ocean[END_REF][START_REF] Longphuirt | Seasonal and species-specific response of five brown macroalgae to high atmospheric CO 2[END_REF][START_REF] Celis-Plá | Macroalga responses to ocean acidification depend on nutrient and light levels[END_REF], but the magnitude of the effects of a CO 2 increase can be related to the presence or absence of a carbon concentration mechanism (CCM). Since Ulva spp. species possess carbon concentration mechanismsor incorporate bicarbonate directly through an anion exchanger (AE) system [START_REF] Axelsson | Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca[END_REF], the effects of a CO 2 increase on their photosynthetic capacity can be expected to be moderate. Moreover, studies on the effect of climate change on aquatic organisms have mostly been conducted applying only one or two variables, and interactions among multiple factors have been studied only scarcely [START_REF] Gordillo | Non-photosynthetic enhancement of growth by high CO 2 level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta)[END_REF][START_REF] Gao | Ocean acidification and nutrient limitation synergistically reduce growth and photosynthetic performances of a green tide alga Ulva linza[END_REF]. The sensitivity of algae to acidification is also expected to be complex, due to interactive effects of pH and CO 2 on photosynthesis. Although elevated seawater CO 2 concentrations may enhance photosynthetic and growth rates (particularly in species without carbon concentrating mechanisms), such increase may be limited by nutrient availability [START_REF] Raven | Algae lacking carbon concentrating mechanisms[END_REF][START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF][START_REF] Li | Photoperiod mediates the effects of elevated CO 2 on the growth and physiological performance in the green tide alga Ulva prolifera[END_REF]. Photosynthesis, nutrient uptake, growth, and other metabolic processes are affected by temperature, light and nutrient availability, and seasonal changes in these parameters are further likely to interact with CO 2 effects on the algal metabolism (Tyrrell et al., 2008;[START_REF] Martin | Response of Mediterranean coralline algae to ocean acidification and elevated temperature[END_REF][START_REF] Mercado | Inorganic carbon acquisition in algal communities: are the laboratory data relevant to the natural ecosystems?[END_REF][START_REF] Brodie | The future of the northeast Atlantic benthic flora in a high CO 2 world[END_REF].

Therefore, outdoor mesocosm studies (in situ measurements) are particularly useful for Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraa473/5926739 by guest on 20 October 2020 A c c e p t e d M a n u s c r i p t monitoring CO 2 impacts over time capturing natural temperature, nutrient and light fluctuations (Celis-Plá et al., 2017a, b).

The mechanisms for algal acclimatization to climate change have been evaluated through the effect on photoinhibition, photoprotection, nutrient uptake systems and patterns of growth, reproduction and morphogenesis of different developmental stages of macroalgae [START_REF] Villafañe | Photosynthesis in the aquatic environment as affected by UVR[END_REF][START_REF] Celis-Plá | Seasonal changes in photoprotectors and antioxidant capacity of the fucoid macroalga Cystoseira tamariscifolia[END_REF][START_REF] Gao | Diverse responses of sporophytic photochemical efficiency and gametophytic growth for two edible kelps, Saccharina japonica and Undaria pinnatifida, to ocean acidification and warming[END_REF]. experiments have been performed and reveal mixed responses, depending on algal species tested and culture conditions applied [START_REF] Porzio | Effects of ocean acidification on macroalgal communities[END_REF][START_REF] Cornwall | Carbon-use strategies in macroalgae: differential responses to lowered pH and implications for ocean acidification[END_REF]Figueroa et al., 2014a, b, c). Recently, long term studies on the impact of acidification were also conducted in mesocosms in which the target pCO 2 was controlled via direct analysis of pCO 2 in seawater [START_REF] Sordo | A direct CO 2 control system for ocean acidification experiments: testing effects on the coralline red algae Phymatolithon luisitanicum[END_REF].

The aim of this study was to evaluate the short-term (six days) impact of increasing pCO 2 on photosynthetic responses of the marine macroalga Ulva rigida (Chlorophyta) which is locally dominant on Mediterranean coasts. This study simulated a range of a global change scenarios under different nitrogen levels provided through nitrate pulses (high nitrate level vs. low nitrate level), comparable to elevated nitrogen supply from urban wastewaters [START_REF] Bermejo | Application of the CARLIT index along a biogeographical gradient in the Alboran Sea (European Coast)[END_REF]. The high capacity of nutrient biofiltration of Ulva species can provide ecological services both in the coastal areas and in aquaculture systems. Therefore, it is critical to evaluate the likely effects of climate change on the physiology and biomass productivity of this species through the simulation of anticipated future conditions. The Mediterranean Sea is recognised as a priority area for climate studies due to the previously reported increases in average seawater temperature and the occurrence of heat extremes by 200 to 500% [START_REF] Diffenbaugh | Heat stress intensification in the Mediterranean climate change hotspot[END_REF][START_REF] Mieszkowska | Intertidal Species in Marine Climate Change Impacts[END_REF][START_REF] Louanchi | Decadal changes in surface carbon dioxide and related variables in the Mediterranean Sea as inferred from a coupled data diagnostic model approach[END_REF].

This study was conducted in the frame of the Group of Aquatic Primary production (GAP) at the workshop "Influence of the pulsed-supply of nitrogen on primary productivity in phytoplankton and marine macrophytes: an experimental approach". In situ photosynthesis under solar radiation measured using Diving PAM [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF] and a Monitoring-PAM (Figueroa et al., 2014a) under artificial light in laboratory [START_REF] Longstaff | An in situ study of photosynthetic oxygen exchange and electron transport rate in the marine macroalga Ulva lactuca (Chlorophyta)[END_REF][START_REF] Jerez | Chlorella fusca (Chlorophyta) grown in thin-layer cascades: estimation of biomass productivity by in-vivo chlorophyll a fluorescence monitoring[END_REF]. Thus, a comparison of in situ versus ex situ measurements is key to identifying an appropriate and reliable protocol to estimate algal photosynthetic activity. The value of applying different approaches in the determination of photosynthesis in Ulva rigida under different CO 2 , nitrate and temperature conditions is also discussed.

MATERIAL AND METHODS

Algae and experimental design

Ulva rigida C. Agardh (Chlorophyta) was collected at La Araña Beach (Malaga, Southern Iberian Peninsula, Spain) (Fig. 1A-D). U. rigida is a two-cell layer laminar macroalga, characterized by a rapid growth and nitrophilic pattern.

The algae were transported to the laboratory in an icebox, cleaned from epibionts and acclimated during four days to low Carbon (C) and low Nitrate (N) conditions prior to experimental exposure. The experimental design is described in detail in [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF].

Briefly, the algal species was cultivated in a mesocosm system at two CO 2 levels, i.e. high (HC, 700 ppm) and low CO 2 (LC, 380 ppm), and two nitrate levels, i.e. high (HN, 50 M) and low nitrate (LN, 5 M). Nitrate was added as KNO 3 . Phosphate as K 3 PO 4 (1 M) was added daily in both LN and HN treatments to avoid any phosphate limitation. The experiment was conducted at the Grice-Hutchinson experimental centre of Malaga University (Fig. 1E).

The experimental system was composed of 3 open vessels (0.094 m 2 surface area, 14 L volume) per treatment, each vessel being considered as a replicate unit (Fig. 1F-G). Each vessel was connected independently to a 60-L tank (header tank), the water flow rate between both being 0.84 ±0.05 L min -1 , representing a turnover rate of 26±1% h -1 . The open vessels were located within 1000-L tanks with circulating fresh water permanently cooled by two cooling units Titan-500 (Aqua Medic GmbH, Bissendorf, Germany) in order to maintain the temperature, as reported by [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF].

The chosen low CO 2 level (380 ppm) falls within the range of surface pCO 2 in the Alborán Sea (367-394 ppm of pCO 2 ), according to [START_REF] Mcelhany | Appropriate pCO 2 treatments in ocean acidification[END_REF]. This region is characterized as oligotrophic, with maximum nitrate level reaching 5 M at certain periods of the year [START_REF] Ramírez | Seasonal dynamics of inorganic nutrients and phytoplankton biomass in the NW Alboran Sea[END_REF]. Thus, the LCLN treatment was considered a control treatment, being closest to natural conditions. In addition to the different C and N treatments, after a 6-days, the algae were subjected to a 4°C temperature increase (AT+4 o C) relative to ambient temperature (AT) for a further three days. Effeltrich, Germany). In the case of the Mini-PAM, data were assessed at three different periods throughout the day: morning (M; 7:00-8:00 GMT), noon (N; 11:30-12:30 GMT) and evening (E; 17:00-18:00 GMT). Data were collected at two different days under each temperature condition, representing a total of six measurements per condition (n=6). The samples were transported under dark conditions from the out-door vessels to the laboratory (25 m distance), where the samples were kept for 15 min in darkness. Fluorescence parameters were then determined under ex-situ conditions in the laboratory. In the case of the Dual-PAM, data were measured only at the morning (M; 7:00-8:00 GMT, three times) and evening (E; 17:00-18:00 GMT, two times) periods. Data were collected at two different days only after temperature increase (AT+4°C). To allow statistical analyses, as a different number of measurements were conducted at each daytime, all data were pooled by period, and time was not considered as a factor, for a total of ten measurements per treatment (n=10).

The data produced by the Mini-PAM and Dual-PAM are compared to previous reported in situ data (under solar radiation) using a Diving-PAM [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF] and a Monitoring-PAM (Figueroa et al., 2014a).

In vivo chlorophyll a fluorescence of PSII using a Mini-PAM

Maximum quantum yield (F v /F m ) was determined according to [START_REF] Schreiber | Continuous recording of photochemical and nonphotochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer[END_REF] using the fluorometer Mini-PAM (Walz GmbH, Germany). After 15 min of dark acclimation, rapid light curves (RLC) were conducted. Following an initial quasi-dark measurement (~1.5 µmol photons m -2 s -1 ) to provide estimates of minimum fluorescence (F o ), a saturating flash of 0.8 s with intensity of > 4000 mol photons m -2 s -1 was applied to obtain the maximum fluorescence level from fully reduced PSII reaction centres (F m ) and

F v /F m was obtained, with F v =F m -F o .
Effective quantum yield of PSII (Y(II)) of algae under different actinic lights were calculated by formula (1) according to [START_REF] Genty | The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence[END_REF], that is equivalent to F/F m " or  II [START_REF] Kromkamp | The use of variable fluorescence measurements in aquatic ecosystems: differences between multiple and single turnover measuring protocols and suggested terminology[END_REF]:

Y(II) = (F m "-F) / F m "
(1)

ETR(II) [µmol electrons m -2 s -1 ] = Y(II) x E PAR x A x F II (2)
Where E PAR is the incident irradiance of PAR (Photosynthetically Active Radiation, =400-700 nm), A is the absorptance, and F II is the fraction of cellular chlorophyll a associated with LHCII (Light Harvesting Complex associated to PSII); 0.5 being the value for green algae [START_REF] Grzymski | The significance of intracellular self-shading on the biooptical properties of brown, red and green macroalgae[END_REF]. Although variation in F II due to light conditions has been demonstrated, such changes were lower in green macroalgae than in other algal groups [START_REF] Grzymski | The significance of intracellular self-shading on the biooptical properties of brown, red and green macroalgae[END_REF][START_REF] Johnsen | Biooptical characteristics of PSII and PSI in 33 species (13 different groups) of marine phytoplankton and the relevance for pulse-amplitudemodulated and fast-repetition-rate fluorometry[END_REF]; in our study, the value of F II was considered as a constant. Absorptance was determined as A = 1 -T as reported by [START_REF] Figueroa | Effects of nutrient supply on photosynthesis and pigmentation in Ulva lactuca (Chlrophyta): responses to short-term stress[END_REF]. T is the transmittance estimated as shown in equation ( 3):

T = E t / E o (3)
Where E o is the incident irradiance of a high pressure lamp determined by a cosine corrected sensor Li-189 (LI-COR Ltd, Nebraska, USA) connected to a radiometer Li-250 (LI-COR Ltd, Nebraska, USA), and E t is the transmitted irradiance, measured by placing a small thallus piece above the PAR sensor [START_REF] Figueroa | Effects of nutrient supply on photosynthesis and pigmentation in Ulva lactuca (Chlrophyta): responses to short-term stress[END_REF]. Reflectance was not determined in this study since according to other reports, the reflectance of Ulva sp. measured by using integrating sphere, opal glass or other techniques, was very low, i.e. 0.04-0.06 [START_REF] Mercado | Comparison methods for measuring light absorption by algae and their application to the estimation of the package effect[END_REF]Figueroa et al., 2003a). ETR values in U. rigida were then overestimated by only 4-6%.

Rapid light curves (RLC, ETR vs. irradiance curves) were conducted using 20-s exposure to eight incremental irradiances (53,157,300,473,686,971,1241 and 1640 µmol photons m -2 s -1 ) of actinic red light. Light curves were fitted according to different models [START_REF] Jassby | Mathematical formulation of relationship between photosynthesis and light for phytoplankton[END_REF][START_REF] Platt | Modeling primary production[END_REF]Eilers and Peters, 1988) to obtain the maximum ETR (ETR max ), the initial slope of the curves ( ETR ) and the irradiance of ETR saturation (E k ). The proportions of absorbed irradiance at ETR max used as electron transport (E YII ) and energy dissipation (E YNPQ , E YNO ) were calculated according to [START_REF] Hendrickson | A simple alternative approach to assessing the fate of absorbed light energy using chlorophyll fluorescence[END_REF].

Y(NPQ) = (F t /F m ") -Y(NO) (5)
Considering that the sum of the three yield components (Y(II), Y(NO) and Y(NPQ)) adds up to 1, i.e. the total absorbed energy by the algal tissues, it was possible to determine the amount of energy used in photochemistry and the dissipated energy according to [START_REF] Hendrickson | A simple alternative approach to assessing the fate of absorbed light energy using chlorophyll fluorescence[END_REF]. The values of Y(II), Y(NPQ) and Y(NO) were obtained from the rapid light curves, considering the samples incubated at each actinic light for 20 s. These values were used to calculate the energy destined to each route by multiplying them by the absorbed PAR. This allowed the calculation of E Y(II) , E Y(NO) and E Y(NPQ) . These values were calculated at each absorbed PAR and are presented to illustrate the rates of energy destination.

Non-photochemical quenching (NPQ), as the expression of total energy dissipation, was calculated according to the ratio of both yield losses, which is similar to Stern-Volmer NPQ parameter [START_REF] Bilger | Determination of the quantum efficiency of photosystem II and non-photochemical quenching of chlorophyll fluorescence[END_REF] as follows (equation 6):

NPQ = Y(NPQ)/Y(NO)= (F m -F m ")/F m (6)
NPQ was calculated after each saturating pulse during the RLCs, and NPQ vs. irradiance relationships were determined. These curves were then fitted to different models [START_REF] Jassby | Mathematical formulation of relationship between photosynthesis and light for phytoplankton[END_REF][START_REF] Platt | Modeling primary production[END_REF]Eilers and Peters, 1988), allowing the calculation of In vivo chlorophyll a fluorescence of PSI and PSII using a Dual PAM All measurements were carried out using a Dual PAM-100 instrument (Walz, Effeltrich, Germany). Weak modulated measuring light from a 620 nm light emitting diode (LED) and blue actinic light of 250 μmol photons m -2 s -1 from 460 nm LED arrays were delivered to the upper algal surface by a DUAL-DR measuring head. Saturating light pulses of 300 ms duration and 10000 μmol photons m -2 s -1 intensity from 620 nm LED arrays were given simultaneously to the upper and lower algal side by the DUAL-DR and a DUAL-E emitter unit, respectively. The oxidation/reduction status of PSI reaction centres was followed by changes in the absorbance at 830 nm [START_REF] Schreiber | Measuring P700 absorbance changes around 830 nm with a new type of pulse modulation system[END_REF]. For this purpose, 830 nm sample and 875 nm reference beams, both emitted by the DUAL-E unit, were passed through the alga and received by a photodiode located in the DUAL-DR measuring head. Both the DUAL-DR and the DUAL-E units were placed into a leaf holder (DUAL-B, Walz) in which Perspex rods guided the light between the alga and measuring units.

Experiments were carried out using the automated routine provided by the Dual-PAM software, with repetitive application of saturation pulses for assessment of fluorescence and P700 parameters, from which the quantum yields of PSI and PSII were derived by the software [START_REF] Klughammer | An improved method, using saturating light pulses, for the deter-mination of photosystem I quantum yield via P700+-absorbance changes at 830 nm[END_REF]. Firstly, using low-light acclimated algae, the basal fluorescence was established and subsequently the F m fluorescence was determined by the saturation pulse method. Secondly, the maximal P700 signal was determined by application of a saturation pulse after far-red pre-illumination. It is assumed that under donorside limited conditions (produced by far-red exposure), a saturation pulse transiently induces full P700 oxidation. Briefly after the saturation pulse the minimum P700 signal is measured when P700 is fully reduced. The difference in signal between the fully reduced and oxidized states is denoted by P m . Actinic illumination was started, and then saturation pulses were applied every 20 s, with the same pulses used for both fluorescence and P700 analyses.

Twelve incremental irradiances were applied: 28,40,53,100,163,211,342,522,796,1209,1844,2745 mol photons m -2 s -1 . Each saturation pulse was followed by a 1-s dark-period to determine the minimal P700 signal level P o (P700 fully reduced). Values for F and F m ' were recorded immediately before and during each saturation pulse, according to [START_REF] Oxborough | Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical componentscalculation of qP and Fv/Fm without measuring Fo[END_REF]. P700 was measured in the dual-wavelength mode (photodetector set to A c c e p t e d M a n u s c r i p t measure 875 nm and 830 nm pulse modulated light) [START_REF] Schreiber | New accessory for the Dual PAM 100. The P515/535 module and examples of its application[END_REF]. The P700 signal (P) was recorded just before a saturation pulse; then, briefly after onset of a saturation pulse (P m ) when maximum P700 oxidation under the effective experimental conditions is observed; and, finally, at the end of the 1-s dark interval following each saturation pulse (P o determination). The P and P m signals are referenced against P o . In this way, unavoidable signal drifts, e.g. due to changes in the water status, do not disturb the actual measurements of P700 parameters.

The photosynthetic electron transport rates of PSI (ETR(I)) were calculated as follows (equation 7):

ETR(I) [µmol electrons m -2 s -1 ] = Y(I) x E PAR x A x F I (7)
where Y(I) is the effective quantum yield of PSI, E PAR the incident irradiance of PAR, A the absorptance in the PAR region and F I the fraction of chlorophyll a related to PSI. F I was calculated as F I = 1 -F II according to the F II values of 0.5.

Photosynthetic parameters related to PSII were also obtained using the Dual PAM, and calculations of Y(II) DP and ETR(II) DP followed the same method as explained above for data obtained with the Mini-PAM.

ETR(I) and ETR(II) vs. irradiance curves were fitted according to different models [START_REF] Jassby | Mathematical formulation of relationship between photosynthesis and light for phytoplankton[END_REF]Eilers and Peters, 1988) to obtain the maximum ETR (ETR(I) max and ETR(II) max ), the initial slope of ETR vs. irradiance curves ( ETR(I) and  ETR(II) ) and the saturation irradiance of ETR (E k(ETR(I)) and E k(ETR(II)) ).

The technical characteristics of the PAM fluorometers used in this study and those used in previous reports of the same experiment [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF]Figueroa et al., 2014a) are presented in Table S1 (Supplementary data section). In addition, the Y(II) data of an intercalibration exercise among all fluorometers is presented. No significant difference (p<0.01)

was found among all PAM fluorometers used (Table S1).

Oxygen measurements

The oxygen consumption and production was determined under solar radiation in closed UV transparent chambers using OPTODES Fibox 3, (Presens, Nürnberg, Germany). The temperature was maintained at the same level as in the experiment, i.e. AT and AT +4 o C using refrigerated circulating water. Each measuring chamber contained 0.019 L of seawater and 20 mg fresh weight of algal biomass. Three replicates were conducted for each nitrogen and carbon treatment on two consecutive days (n=6 per treatment) under each of the two temperature conditions. Net photosynthesis (NP) was determined under solar natural radiation at noon, followed by measurements of respiration (R) in darkness (for 30 min).

Gross photosynthesis (GP) was determined as NP+R. Oxygen evolution was expressed in terms of algal area as mol O 2 m -2 s -1 .

Statistical analyses

Interactive effects on physiological parameters obtained from the experimental procedures were analysed using Analyses of Variance (ANOVA) (according to [START_REF] Underwood | Experiments in Ecology: Their Logical Design and Interpretation, using Analysis of Variance[END_REF]. After checking for normality, data were tested for homogeneity of variances by the Cochran"s test. In the case of the Mini-PAM data (F v /F m , ETR max ,  ETR and E k ), a Multifactorial ANOVA was performed including Temperature (Ambient Temperature, AT, and Ambient Temperature +4°C, AT+4°C), Nitrogen (N; Low and High Nitrogen), CO 2 (C; Low and High Carbon) and Time of sampling (M, Morning; N, Noon; E, Evening), all considered fixed crossed factors, and Vessel as a random factor nested in the interaction N x C and crossed with the other two factors. The factor "Vessel" or their interactions were always not significant (p > 0.05) and this factor was thus not included in the final models. In the case of PAR-energy destination data, they were pooled and a linear regression was performed on the averaged data. The data set of the different variables corresponded to two measurement days (n=6). In the case of the Dual PAM data, a one-way ANOVA was conducted, considering the factor treatment with four levels (LCLN, LCHN, HCLN and HCHN) (n=10). When significant differences were detected, post hoc tests were performed using Student-Newman-Keuls tests [START_REF] Underwood | Experiments in Ecology: Their Logical Design and Interpretation, using Analysis of Variance[END_REF]. The significance level was set at 0.05. Pearson correlation coefficients were calculated and tested between physiological variables as; ETR max, NPQ max,

F v /F m, ⍺ ETR , E Y(NO) and E Y(NPQ) (Sigma plot, IBM).
Principal Coordinates Analyses (PCA) were performed to detect patterns among parameters obtained with the Mini-PAM fluorometer, based on Euclidean distance using PERMANOVA+ with PRIMER6 package. PCA analyses were conducted for F v /F m , ETR max , and NPQ max considering treatment (4 levels), time (3 levels) and temperature (2 levels). This procedure corresponding to equivalent ordination of a PCA calculates the percentage of variation explained by each axis in the multidimensional scale; the overlay of the vectors on the PCA was performed using Spearman correlation [START_REF] Anderson | PERMANOVA+ for Primer: Guide to Software and Statistical Methods[END_REF].

RESULTS

Fluorescence parameters of PSII using a Mini-PAM fluorometer

Photosynthetic parameters measured at the beginning of the experimental period (i.e., after acclimation time and before the application of any treatment) by the Mini-PAM are presented in Table 1, with values of 0.69 ± 0.03 for F v /F m and 1.26 ± 0.62 for NPQ max . E k was 674 ± 103.77 mol photons m -2 s -1 .

Values for F v /F m determined after exposure to different levels of CO 2 and nitrate were significantly impacted (p<0.05) by the interaction of CO 2 enrichment and time (Table 2), with significant differences through the day: morning, noon or evening (Fig. 2). F v /F m significantly decreased at noon due to photoinhibition; and values were higher for thalli under high carbon (HC) than low carbon (LC) condition, in the morning or evening (Fig. 2). F v /F m values recorded in the morning and evening were similar, and also increase from initial values (Table 1, Fig. 2).

Maximal electron transport rate (ETR max ) as an indicator of photosynthetic capacity was influenced by a significant interaction (p<0.05) between C, time and temperature (Table 2, Fig. 3A). At the beginning of the experiment, U. rigida displayed the highest ETR max values (37.39 ± 8.76 µmol e -m -2 s -1 ; Table 1) of the experimental period (Fig. 3A). At ambient temperature, ETR max increased at noon and was higher for samples under LC than under HC (Fig. 3A). At AT+4°C, a similar trend with higher ETR max values at noon was observed (Fig. 3A), although no significant difference was found between LC and HC. At noon, ETR max under LC was also higher at ambient temperature than at AT+4 o C (Fig. 3A). After pooling the data (Fig. 3B), ETR max was greater at noon, with the exception of ETR max in HCLN samples. In the evenings, ETR max was significantly lower at HCLN than at LCLN (Fig 3B).

The photosynthetic efficiency ( ETR ) was significantly affected by the interaction among C, N and temperature (Table 2). No significant difference was found between C and N treatments at either ambient temperature or AT+4°C (Fig. 3C), except for thalli under HCLN at AT+4°C which reached higher values compared to the other C and N treatments. When data were pooled,  ETR significantly decreased at noon under HCLN treatment and increased in the evening (Fig. 3D).

Photosynthetically saturating irradiance (E k ) of PSII was impacted by different factors such as time, temperature and the interactions between C and N, and between C and time (Table 2). When the data were pooled (Fig. 4), E k significantly increased at noon A c c e p t e d M a n u s c r i p t independently of C supply, with higher E k under HC than LC, whereas the opposite trend was observed in the evening (Fig. 4).

Non-photochemical quenching (NPQ max ), as indicator of the energy dissipation, revealed a significant effect (p<0.05) of the interaction between CO 2 , time and temperature (Table 2, Fig. 5). NPQ max presented a daily pattern that depended on C level, the interaction with N, and on an interactive effect of C, time and temperature (Table 2, Fig. 5). Taking into account this triple interaction, the highest values were detected at noon under LC at both temperatures (Fig. 5). At noon, the increase in C (HC) resulted in a decrease in NPQ max under both temperature treatments. In the evening, HC samples showed a reduction in NPQ max only under increased temperature (Fig. 5). Overall, NPQ max values were generally higher than initial values at the beginning of the experimental period (Table 1).

The Pearson correlations between the different fluorescence parameters measured are shown in indicate that less than 2% of the energy was used effectively for photosynthesis (Fig. 6E-F), regardless of temperature.

The slopes of the curves representing the light energy destined for photochemical and non-photochemical yields the different C and N treatments, were also estimated (Fig. 6).

Significant effects of an interaction were found between the availability of N and temperature and NPQ max were also correlated positively (Table 4).

Electron transport rates and yields of PSI and PSII using the

Comparison of ETR max values obtained with different fluorometers

ETR max values at noon under solar radiation were calculated from previously reported data [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF]Figueroa et al., 2014a). For Diving-PAM data (based on Stengel et al., 2014), ETR was calculated using reported effective quantum yield, average irradiance at noon in the experimental vessels, the measured absorptance (Table 5) and 0.5 as F II value.

For the Monitoring-PAM data (based on Figueroa et al., 2014a), rETR values were used to calculate ETR by multiplying the absorptance data (Table 5). The data of maximal ETR determined by the Mini-PAM and Dual-PAM are presented in the Tables 1 -5 and Fig. 3).

Regardless of the type of fluorometer used, the highest ETR max measuring through in vivo chlorophyll a fluorescence related to PSII was consistenly measured under HCHN, and 5). The ETR max determined from Y(II) measurements in situ under solar radiation (Diving-PAM) was higher than ETR max determined in the laboratory from the rapid light curves using red light as actinic light (Mini-PAM). ETR max determined from Y(II) measurement using the Monitoring-PAM was lower than that obtained using the Diving-PAM, although both measurements were conducted under solar radiation (Table 5).

Under LC and ambient temperature, ETR max values determined using the Mini-PAM were not significantly different than those determined by the Monitoring-PAM, whereas for the other treatments, ETR max values were higher when using the Monitoring-PAM than the Mini-PAM. In general, ETR max was higher under AT+4 o C, except when the measurements were conducted in the laboratory using the Mini-PAM.

Oxygen measurements

The highest gross photosynthesis determined under solar radiation was reached under HCHN, and the lowest under HCLN at both temperatures (Table 5). Gross photosynthesis reached intermediate values under LCLN and LCHN (Table 5). No significant difference was found between N treatments under LC (Table 5). Respiration rate was similar across the different C, N and temperature treatments, except under HCLN at ambient temperature, where a significant decrease occurred compared to the other treatments (Table 5). Under solar radiation (where in vivo chlorophyll a fluorescence was determined using the Diving-PAM), the lowest ETR max / GP ratio, i.e. transport electrons per oxygen produced, was observed under HCHN; the highest ratio was determined for HCLN at both temperatures. This ratio reached intermediate values under LCHN and LCLN (Table 5).

Principal components analyses

Based on Mini-PAM measurements, Principle Component Analysis (PCA) was undertaken for photosynthetic parameters across all treatments (Fig. 9). The first axis of PCA represented 59.1% of the total variation, and the second axis 19.4% of the total variation. The first two dimensions of the PCA based on different photosynthetic responses represented 79.1% of total the variation (Fig. 9). The first axis could be related to a combination of C and N treatments characterized by the highest productivity (ETR max ) and photoprotection (NPQ max ) indicators. By contrast, the photoinhibition or F v /F m responses appears to represent the reverse part of X-axis. The combination of these variables allowed a separation of the treatments revealing that intertidal Ulva rigida exhibited responses linked to the C and N treatments. were tested on Ulva rigida, an abundant eulittoral Mediterranean green macroalga.

Interactive effects of C, N and temperature

Most studies on the effects of global change factors on aquatic organisms have been conducted using only one or two environmental variables, and studies on the interaction of multiple stressors remain scarce [START_REF] Cornwall | Carbon-use strategies in macroalgae: differential responses to lowered pH and implications for ocean acidification[END_REF][START_REF] Koch | Climate change and ocean acidification effects on seagrasses and marine macroalgae[END_REF][START_REF] Yildiz | Ultraviolet radiation modulates the physiological responses of the calcified rhodophyte Corallina officinalis to elevated CO 2[END_REF]Celis-Plá et al., 2017a, b;[START_REF] Gao | Ocean acidification and nutrient limitation synergistically reduce growth and photosynthetic performances of a green tide alga Ulva linza[END_REF]2019). In this study, we focused on the interactive effects of four variables, i.e., temperature, carbon and nitrogen availability, and time. Little is known about how seaweeds living under nutrient-limited conditions will respond to ocean acidification. In addition, the effect of acidification (as linked to high-CO 2 levels) and nutrient limitation on Ulva species is of interest [START_REF] Rautenberger | Saturating light and not increased carbon dioxide under ocean acidification drives photosynthesis and growth in Ulva rigida (Chlorophyta)[END_REF][START_REF] Gao | An Ocean Acidification Acclimatised Green Tide Alga Is Robust to Changes of Seawater Carbon Chemistry but Vulnerable to Light Stress[END_REF][START_REF] Li | Photoperiod mediates the effects of elevated CO 2 on the growth and physiological performance in the green tide alga Ulva prolifera[END_REF] due to their ecological importance, their capability to form green tides [START_REF] Smetacek | Green and golden seaweed tides on the rise[END_REF], their delivery of crucial ecosystem services such as wastewater bioremediation [START_REF] Alam | Bioremediation potential, growth and biomass yield of the green seaweed, Ulva lactuca in an integrated marine aquaculture system at the Red Sea coast of Saudi Arabia at different stocking densities and effluent flow rates[END_REF], and their uses as animal feed [START_REF] Rico | Influence of the dietary inclusion of Gracilaria cornea and Ulva rigida on the biodiversity of the intestinal microbiota of Sparus aurata juveniles[END_REF], human food [START_REF] Yaich | Chemical composition and functional properties of dietary fibre extracted by Englyst and Prosky methods from the alga Ulva lactuca collected in Tunisia[END_REF], biofuel (Neveusx et al., 2014).

Although young and adult thalli of Ulva spp. may display differential responses to ocean acidification and nutrient limitation [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF]Figueroa et al., 2014a, c;[START_REF] Gao | PSI-driven cyclic electron flow allows intertidal macroalgae Ulva sp. (Chlorophyta) to survey under desiccated conditions[END_REF][START_REF] Gao | Ocean acidification and nutrient limitation synergistically reduce growth and photosynthetic performances of a green tide alga Ulva linza[END_REF], the ability to respond to CO 2 enrichment is generally dependent on ambient N availability [START_REF] Gordillo | Non-photosynthetic enhancement of growth by high CO 2 level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta)[END_REF][START_REF] Zou | Short-and long-term of elevated CO 2 on photosynthesis and respiration in the marine macroalgae Hizikia fusiforme (Sargassaceae, Phaeophyta) grown at low and high nitrogen supply[END_REF]. In coastal areas occupied by Ulva spp. including U. rigida, ammonia and nitrate concentrations are typically higher due to by urban waste effluents, commonly resulting in the formation of green tides. [START_REF] Corneliesen | Patterns in the δ 13 C and δ 15 N signature of Ulva pertusa: Interaction between physical gradients and nutrient source pools[END_REF] showed interactions between physical gradients and nutrient source pools on the N signature of U. pertusa, affecting both carbon and nitrogen assimilation. Data from this present study clearly indicate that a decrease in ETR max under elevated CO 2 (HC) occurred only under nitrate limitation (LN). In fact, higher nitrate availability (HN) resulted in a large increase in ETR max not only under HC, but also LC treatments. Under HNLC, the observed high ETR max values in the experimental time were probably transient since the stoichiometry C:N could be affected and ETR max could be reduced by increasing the culture time.

Previously, in Ulva rotundata, an increase in nitrate supply reduced photoinhibition at noon during daily cycles (Henley et al., 1991b). [START_REF] Gao | Ocean acidification and nutrient limitation synergistically reduce growth and photosynthetic performances of a green tide alga Ulva linza[END_REF]). An increase in CO 2 can enhance the uptake and assimilation of nitrogen in U. rigida [START_REF] Gordillo | Non-photosynthetic enhancement of growth by high CO 2 level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta)[END_REF] and U. lactuca [START_REF] Zou | Effects of elevated CO 2 concentration on photosynthesis and nutrients uptake of Ulva lactuca[END_REF]. This would allow a decrease in the energy required for nitrate assimilation due to a release of nitrogen, and any saved available energy can thus be channelled into other processes, such as repair or photoprotection [START_REF] Zou | Photosynthetic utilization of inorganic carbon in the economic brown alga, Hizikia fusiforme (Sargassaceae) from the South China Sea[END_REF]. In U. rigida an increase in photosynthetic rates under HC supply is linked to a stimulation of nitrate reductase activity under high N supply [START_REF] Gordillo | Non-photosynthetic enhancement of growth by high CO 2 level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta)[END_REF]2003) but acidification and nitrate depletion (HCLN) reduced soluble protein level (Figueroa et al. 2014c). In the present study, a decrease in pH and elevated CO 2 levels in the HC treatments lowered the photosynthetic activity and photoprotective capacity in U. rigida, probably due to a negative effect on C acquisition, concentration mechanism and energy dissipation processes. Ulva rigida uses preferentially bicarbonate when Ci saturated and growth is mainly controlled by light, rather than CO 2 [START_REF] Rautenberger | Saturating light and not increased carbon dioxide under ocean acidification drives photosynthesis and growth in Ulva rigida (Chlorophyta)[END_REF]. These authors concluded that future increased CO 2 (aq) levels alone cannot stimulate Ulva growth and resultant blooms, and ocean acidification, when combined with increased irradiance, might even suppress green tide blooms. Locally, ocean acidification and nutrient limitation may thus synergistically reduce growth of Ulva species and limit the occurrence of green tides in a future ocean environment. Specifically in the coastal area of Malaga (Southern Iberian Peninsula), nitrate concentration are reduced as a consequence of the weakening of wind-induced upwelling; as a result, oligotrophication is becoming more recurrent here [START_REF] Mercado | Decadal weakening of the wind-induced upwelling reduces the impact of nutrient pollution in the Bay of Málaga (western Mediterranean Sea)[END_REF], and ocean acidification may reduce productivity of Ulva species.

The decrease in photosynthetic production (ETR max ) under increased temperature observed in this study (when using a Mini-PAM) could be related to a decrease in chlorophyll a level previously described by [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF]. A reduced chlorophyll a content of U.

lactuca under elevated (700 ppm) compared to preindustrial (280 ppm) CO 2 levels was also previously reported by [START_REF] Olischläger | Effects of ocean acidification on growth and physiology of Ulva lactuca (Chlorophyla) in a rockpool-scenario[END_REF]. Increased temperature also reduced maximum gross photosynthesis and dark respiration in Ulva pertusa, and the interaction of elevated carbon and elevated temperature had negative impacts on photosynthesis and biochemical composition [START_REF] Kang | Effects of future climate conditions on photosynthesis and biochemical component of Ulva pertusa (Chlorophyta)[END_REF]. Similar results are also reported in this (pigments and proteins) previously described (Figueroa et al., 2014c). [START_REF] Xu | Future CO 2 -induced ocean acidification mediates the physiological performance of a Green Tide Algal[END_REF] reported that growth and ETR of U. prolifera was enhanced by elevated CO 2 concentration. However, a reduction in the maximal photosynthetic oxygen evolution rate, the light use efficiency and the efficiency to dissipate excessive light energy was described as [START_REF] Liu | CO 2 -driven seawater acidification increases photochemical stress in a green alga[END_REF] for the same algal species when grown under the same high CO 2 level over a longer period (80 d). Some Ulva species exhibit a very high affinity for dissolved inorganic carbon, dominating tidal pools with pH higher than 10.5 [START_REF] Axelsson | Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca[END_REF]. In the present experimental set-up, under LC, pH reached maximal values of pH 9.5-10 [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF], corresponding to a CO 2 concentration lower than 1  as conditions previously measured in tidal pools [START_REF] Björk | Why is Ulva intestinalis the only macroalga inhabiting isolated rockpools along the Swedish Atlantic coast?[END_REF]. The conductance for CO 2 together with the uptake of nitrate is high in U. rigida (250 x10 -6 m s -1 ) [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF], and it is further thought that U. rigida has a high capacity to use bicarbonate and retains high carbonic anhydrase activity compared to other macroalgae [START_REF] Mercado | External carbonic anhydrase and affinity to inorganic carbon in intertidal macroalgae[END_REF]. Ulva lactuca could thus modify the seawater carbonate system via its photosynthetic activity:

under a CO 2 pre-industrial treatment (280 ppm), HCO 3 -is incorporated via an anionexchanger (AE-state) which has the highest affinity for HCO 3 - [START_REF] Axelsson | Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca[END_REF].

Uunder a future scenario of increased CO 2 in rockpools, Ulva biomass may increase [START_REF] Olischläger | Effects of ocean acidification on growth and physiology of Ulva lactuca (Chlorophyla) in a rockpool-scenario[END_REF]. Using inhibitors of photosynthetic inorganic carbon utilization, [START_REF] Gao | An Ocean Acidification Acclimatised Green Tide Alga Is Robust to Changes of Seawater Carbon Chemistry but Vulnerable to Light Stress[END_REF] demonstrated that both extracellular carbonic anhydrase (CA) and intracellular CA were active in Ulva linza grown under LC whereas under HC, extracellular

CA was completely inhibited. The down-regulated CCMs (Carbon Concentration Mechanisms) in U. linza was not affected by changes of seawater carbon chemistry but a decrease in net photosynthetic rate was observed when thalli were exposed to increased light intensity [START_REF] Gao | An Ocean Acidification Acclimatised Green Tide Alga Is Robust to Changes of Seawater Carbon Chemistry but Vulnerable to Light Stress[END_REF].

Fluorescence parameters of PSII and comparison between in-situ vs. ex-situ measurements

In U. rigida, the maximal quantum yield (F v /F m ) presented a decrease at noon, indicating the presence of photoinhibition. Photoinhibition was higher under LC than under HC treatments, whereas the non-photochemical quenching increased at noon under LC at both temperature treatments, indicating a high capacity for energy dissipation and photoprotection.

A decrease in NPQ when more carbon is available has also been demonstrated in microalgae A c c e p t e d M a n u s c r i p t [START_REF] Berteotti | Increased biomass productivity in green algae by tuning non-photochemical quenching[END_REF]. In this study, the 4 o C increase showed no effect on NPQ max . However, other species, such as the brown macroalga Fucus serratus, showed a decrease in NPQ max with increasing temperatures [START_REF] Figueroa | Yield losses and electron transport rate as indicators of thermal stress in Fucus serratus (Ochrophyta)[END_REF]. In contrast to Ulva sp., Fucus serratus is suffering a decrease in biomass due to climate change (Duarte and Viejo, 2018). A positive temperature dependence of NPQ has been previously reported in vascular plants but when applying a higher temperature increase (+10 o C) than in this study (+4 o C) [START_REF] Hendrickson | A simple alternative approach to assessing the fate of absorbed light energy using chlorophyll fluorescence[END_REF]. The high NPQ max values recorded at noon, principally under LC treatment, indicates an effective acclimation of U. rigida to high irradiance levels since a high dissipation of energy is related to photoprotection mechanism, which was also observed by Figueroa et al. (2014b) and [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF] in the same algal species after increased UV-B radiation. A large decrease in F v /F m due to an increase of PAR and/or UVR, but also a high capacity of recovery in the evening, has been reported previously for the same species (Figueroa et al. 2003b).

In contrast to F v /F m, ETR max and E k increased at noon. Diurnal variations in E k and ETR have previously been observed in the red macroalga Kappaphycus alvarezii [START_REF] Schubert | Circadian rhythm of photosynthesis in Kappaphycus alvarezii (Rhodophyta): independence of cell cycle and possible clock[END_REF]. The negative relationship between  ETR and ETR max (R=-0.351, p<0.05) and between  ETR and E k (R=-0.735, p<0.05) indicated photoacclimation to high light, as respresented by an increase in ETR max and Ek and decrease in α ETR . Under most conditions, light-saturated photosynthesis (P max ) appears to be limited by downstream processes [START_REF] Behrenfeld | Compensatory changes in photosystem II electron turnover rates protects photosynthesis form photoinhibition[END_REF] as carbon or nitrogen assimilation. Nitrogen limitation forces photosynthetic organisms to reallocate available nitrogen to essential functions as protein accumulation, enzymatic process and growth. At the same time, it increases the probability of photodamage by limiting the rate of energy-demanding metabolic processes, downstream of the photosynthetic apparatus [START_REF] Salomon E Bar-Eyal | Balancing photosynthetic electron flow is critical for cyanobacterial acclimation to nitrogen limitation[END_REF]. Thus, the maximal quantum yield (F v /F m ) but not the maximal photosynthetic capacity (ETR max ) was negatively affected by noon irradiance. As defined by [START_REF] Franklin | UV-B and algal photosynthesis[END_REF], photoinhibition is a generic outcome of the failure of photoprotection to mitigate photo-inactivation. Photoinhibition has been suggested as a photoprotective mechanism in Ulva spp. (Henley et al., 1991a;Figueroa et al., 2003b), and a decrease in F v /F m as a consequence of different ambient stress conditions has previously been reported in Ulva (Figueroa et al., 2003b;Figueroa et al., 2014b;[START_REF] Li | Photoperiod mediates the effects of elevated CO 2 on the growth and physiological performance in the green tide alga Ulva prolifera[END_REF] and other macroalgal species [START_REF] Celis-Plá | Seasonal changes in photoprotectors and antioxidant capacity of the fucoid macroalga Cystoseira tamariscifolia[END_REF].

ETR max in U. rigida was affected by temperature but its pattern depended on the measuring system. When measuring Y(II) under solar radiation using a Diving-PAM or a Monitoring-PAM, ETR max increased under AT+4 o C [START_REF] Stengel | CO 2 , temperature, and nutrient impacts on morphologically different macroalgae under solar radiation: a mesocosm study[END_REF]Figueroa et al., 2014a). By contrast, when ETR max was calculated from rapid light curves conducted in this study by a Mini-PAM in the laboratory, it decreased with at the higher temperature. Thus, show that the photosynthetic conditions are more favourable under HCHN than HCLN for U.

rigida.

Similar to ETR max, rates of gross photosynthesis quantified by oxygen evolution were maximal under HCHN, at both temperatures; by contrast, lowest rates were reached under HCLN, which was again similar to observations on ETR max . A good relationship between ETR max (determined under solar radiation using the Diving-PAM) and GP was thus found.

However, the ratio of ETR max /GP was far from the expected theoretical values, i.e. 4-6 including only PSII [START_REF] Flameling | Light dependence of quantum yields for PSII charge separation and oxygen evolution in eukaryotic algae[END_REF]Figueroa et al., 2003a). This ratio indicates the number of moles of electron transported in the electron transport chain related to the number of moles of oxygen produced. In this study, the ETR max /GP ratio ranged from about 31 to 77, indicating that the algae were not under optimal conditions. The lowest values, related to less stressful physiological conditions, were observed under HCHN, both at AT and AT+4 o C (ETR max /GP values: 33.9 and 31.62, respectively), whereas the least assimilation expressed as mole of electron per mole of C ranged from 5 to 60 and were determined in situ for phytoplankton [START_REF] Lawrenz | Predicting the electron requirement for carbon fixation in seas and oceans[END_REF][START_REF] Carrillo | Interactive effect of UVR and phosphorus on the coastal phytoplankton community of the western Mediterranean. Sea: Unravelling ecophysiological mechanisms[END_REF] and under laboratory conditions for macroalgae (Figueroa et al., 2003a). ETR max /GP values that are higher than 4-6 are reached under unfavourable irradiance, nutrients and temperature conditions and indicate the activation of the alternative electron flow as Mehler reaction, the cyclic electron flow around PSII and/ or PSI, and photorespiration, mechanisms whose activation is probably taxa-specific [START_REF] Flameling | Light dependence of quantum yields for PSII charge separation and oxygen evolution in eukaryotic algae[END_REF][START_REF] Suggett | Comparing electron transport with gas exchange: parameterising exchange rates between alternative photosynthetic currencies for eukaryotic phytoplankton[END_REF][START_REF] Schuback | Diurnal variation in the coupling of photosynthetic electron transport and carbon fixation in iron-limited phytoplankton in the NE subarctic Pacific[END_REF].

In general, the ETR max values in U. rigida were higher when Y(II) was determined under solar radiation than under artificial actinic light, as previously reported for Ulva lactuca [START_REF] Longstaff | An in situ study of photosynthetic oxygen exchange and electron transport rate in the marine macroalga Ulva lactuca (Chlorophyta)[END_REF] and the green microalga Chlorella fusca [START_REF] Jerez | Chlorella fusca (Chlorophyta) grown in thin-layer cascades: estimation of biomass productivity by in-vivo chlorophyll a fluorescence monitoring[END_REF]. A possible explanation is that, under solar radiation, not only chlorophyll but accessory pigments are excited and transfer photons to chlorophyll to a greater extent than that under red actinic artificial light. On the other hand, the ETR max values determined using a Diving-PAM were higher than those determined using a Monitoring-PAM. This result could be explained by the fact that, when using the Monitoring-PAM, the algal thalli were fixed in a clip at 180 o angle whereas, when using the Diving-PAM, the algae were free-floating, thus receiving solar radiation from different angles but were also subject to self-shading due to the high algal density in the vessels (27 g FW L -1 ). Consequently, measured Y(II) was higher when using the Diving-PAM than the Monitoring-PAM system, because in this latter system samples were exposed to higher solar irradiance. The irradiance used to calculate ETR when using the Monitoring-PAM was measured directly next to the algae fixed within the clip, whereas for the Diving-PAM measurements, average irradiances determined at three depths within the vessels were used (surface, 5-cm and 10-cm depth) since algae moved within the water column due to air injection. In the experimental set-up that Diving-PAM measurements were based on, algae were thus subjected to greater light fluctuations than in the Monitoring-PAM system. Similar to our conclusions derived here for U. rigida, [START_REF] Longstaff | An in situ study of photosynthetic oxygen exchange and electron transport rate in the marine macroalga Ulva lactuca (Chlorophyta)[END_REF] previously highlighted that in situ Y(II) data more realistically related to natural physiological responses than those obtained under the laboratory conditions using artificial actinic light.

We thus suggest that ETR measurements in situ are more closely related to productivity than ETR measurements ex situ. The importance of in situ fluorescence measurement on Ulva spp. A c c e p t e d M a n u s c r i p t either in the field or in outdoor tanks, rather than under laboratory conditions, has been previously reported in order to achieve most accurate estimation of algal photosynthesis and productivity [START_REF] Häder | Pulse amplitude modulated fluorescence in the green macrophytes Enteromorpha muscoides, Ulva rigida, Ulva gigantea and Codium adherens from the Atlantic coast of Southern Spain[END_REF]Altamirano et al, 2000a,b;[START_REF] Cabello-Pasini | Photoinhibition of photosynthesis in Macrocystis pyrifera (Phaeophyte), Chondrus crispus (Rhodophyta) and Ulva lactuca (Chlorophyta) in out-door culture systems[END_REF][START_REF] Figueroa | Effects of nutrient supply on photosynthesis and pigmentation in Ulva lactuca (Chlrophyta): responses to short-term stress[END_REF][START_REF] Cabello-Pasini | Effect of nitrate concentration on UVR on photosynthesis, respiration, nitrate reductase activity and phenolic compounds in Ulva rigida (Chlorophyta)[END_REF]. Importantly, the response to experimental variables was consistent across all PAM systems and also when measuring oxygen evoluation, i.e. the highest photosynthetic activity observed was under HCHN, and lowest under HCLN.

In vivo chlorophyll fluorescence of PSII compared to that of PSI

Another distinct feature among algal species and treatments is the relative contribution of PSI and PSII to photosynthesis. Here, the ETR(I):ETR(II) ratio was about 2.01, and this value was not affected by increased CO 2 or N supply. As a higher activity of PSI has been related

to stress conditions (Bukhov and Carpentier, 2004;[START_REF] Gao | PSI-driven cyclic electron flow allows intertidal macroalgae Ulva sp. (Chlorophyta) to survey under desiccated conditions[END_REF], in U. rigida, PSI could have an important role in the stress acclimation. For example, [START_REF] Gao | PSI-driven cyclic electron flow allows intertidal macroalgae Ulva sp. (Chlorophyta) to survey under desiccated conditions[END_REF] reported that the ratio ETR(I):ETR(II) in fully hydrated Ulva sp. was 1.35, similar to the lowest values found in U. rigida in this study. Under desiccated conditions, this ratio decreased because PSII activity was much more strongly affected by desiccation than that of PSI. In addition, upon rehydration, PSI activity recovered faster than PSII activity [START_REF] Gao | PSI-driven cyclic electron flow allows intertidal macroalgae Ulva sp. (Chlorophyta) to survey under desiccated conditions[END_REF]. In our study, changes in the PSII/PSI activity were also related to CO 2 and N supply: under LC conditions, the increase in N enhanced the ETR(I):ETR(II) ratio indicating a higher PSII activity under high N levels. By contrast, under HC conditions, the increase in N resulted in a slight increase in PSI activity. The average value of ETR(II):ETR (I) ratio was 0.54, and thus similar to the ratio reported by [START_REF] Gao | The enhancement of cyclic electron flow around photosystem I improves the recovery of severely desiccated Porphyra yezoensis (Bangiales, Rhodophyta)[END_REF] for Pyropia yezoensis under hydrated conditions (0.53). The cyclic electron flow around PSI in the thylakoid membranes has been shown to occur in vascular plants, eukaryotic algae, and cyanobacteria [START_REF] Ravenel | The cyclic electron pathways around photosystem I in Chlamydomonas reinhardtii as determined in vivo by photoacoustic measurements of energy storage[END_REF][START_REF] Mi | Thylakoid membrane bound pyridine nucleotide dehydrogenase complex mediates cyclic electron transport in the cyanobacteria Synechocystis PCC6803[END_REF]. During steady-state photosynthesis, cyclic electron flow also contributes to ATP synthesis and adjusts the ATP:NADPH ratio [START_REF] Backhausen | Electron acceptors in isolated intact spinach chloroplasts act hierarchally to prevent over-reduction and competition for electrons[END_REF]Kramer et al., 2004b), which is essential for preventing stroma over-reduction [START_REF] Munekage | Cyclic electron flow around photosystem I is essential for photosynthesis[END_REF].

CONCLUSIONS

This study clearly demonstrates that the photosynthetic activity of U. rigida decreased under increased CO 2 levels (acidification) but only under N-limitation. Under N-replete conditions, ETR max was higher at elevated CO 2 levels. Following an increase temperature pattern of ETR max observed under different treatments depended on the measuring system and conditions: under solar radiation (in situ), ETR max increased with temperature, but in the laboratory under artificial actinic light, no difference or decrease in ETR max with increased temperature was observed. In addition, ETR max was higher when calculated using Y(II)

measurements obtained under natural solar radiation than those obtained under artificial light.

The activity of PSI was higher than that of PSII under all treatments, and both PSI and PSII activities were reduced under HCLN. This study highlights the importance of 1) research on interactive effects of environmental factors and 2) the measuring approach used (in-situ vs.

ex-situ conditions) to evaluate the impact of global change on marine macroalgae. during the day: morning (M; 7:00-8:00 GMT), noon (N; 11:30-12:30 GMT) and evening (E; 17:00-18:00 GMT). Data represent the pooled results (n=24) in accordance with significant effects obtained by an ANOVA (Table 2). The different letters indicate significant differences among treatments (p < 0.05, SNK post hoc test).

  photosynthetic capacity, showing higher in situ fluorescence values under solar radiation than

  Photosynthetic performance estimated by in vivo chlorophyll a fluorescence was determined using two different fluorometers, a Mini-PAM and a Dual-PAM (Walz,

Fig. 6 .

 6 Fig. 6. The effective quantum yield represented the smallest fraction of light use under any of the experimental conditions, and the regulated non-photochemical quenching (E Y(NPQ) ) was the main destination of the energy absorbed in samples under LC treatment (Fig. 6A-D).Moreover, samples under LCLN and increased temperature displayed the lowest capacity of light absorption (close to the maximal amount of 200 mol photons m -2 s -1 ). When the thalli under HCHN were kept at increased temperature, approximately twice as much light was absorbed, reaching ~400 mol photons m -2 s -1 (Fig.6H). However, based on a comparison of slopes of the three route of energy dissipation, more than 95% of this energy acquired was lost through non-photochemical quenching (Y(NO) and Y(NPQ)). When samples were kept under HCLN, Y(NO) was the main component for energy allocation, and the slopes of E Y(II)

  Dual-PAM fluorometerValues of Y(I) vs. Y(II) for all treatments exhibited a direct linear relationship, and Y(I) was always higher than Y(II), under all treatments (Fig.7A-D). Specific slopes of Y(I) vs. Y(II) based on the regression linear model were 0.7421 for LCLN (Fig 7A), 0.8389 for LCHN (Fig. 7B), 0.8032 for HCLN (Fig. 7C) and 0.7893 for HCHN (Fig. 7D), respectively. The NPQ calculated from the rapid light curve decreased significantly under HCHN (Fig. 8A). By contrast, the rapid light curves of ETR(I) and ETR(II) vs. irradiance indicate that the photosynthetic parameters ETR(I) (Fig. 8B) and ETR(II) (Fig. 8C) decreased significantly under this (HCLN) treatment. Generally, significant effects of treatments were observed for ETR(I) max (F=5.763, p<0.05), ETR(II) max(DP) (F=13.1728, p<0.05), as well as PSI and PSII photosynthetic efficiency ( ETR(I) , F=3.0739, p<0.05;  ETR(II)(DP) , F=5.024, p<0.05). The lowest ETR(I) max and ETR(II) max were reached under HCLN, and the highest under HCHN. The photosynthetic efficiency of PSI decreased under HCLN. E k values did not present significant differences among the different treatments (p>0.05).

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  under in situ measurements (natural solar radiation), a different pattern related to growth temperature on ETR max was found. The saturation light pulses of the different fluorometers represent different light qualities, i.e. produced by halogen lamp (Diving-PAM), or blue (Monitoring-PAM) or red (Mini-PAM and Dual-PAM) LEDs, but the effect of the light quality on the measuring pattern remains obscure; the observed results may be explained by differences in saturation pulse spectra that could contribute differently affect the electron flow from accessory pigments (carotenoids) to chlorophyll a[START_REF] Celis-Plá | Seasonal changes in photoprotectors and antioxidant capacity of the fucoid macroalga Cystoseira tamariscifolia[END_REF].The energy dissipation pattern was affected by C treatment. Under LC, the main energy loss was produced under regulated photoprotective mechanisms, i.e. Y(NPQ), whereas under HC, the main energy loss was passively dissipated as heat, i.e. Y(NO). Again, the more favourable treatment for the use of energy for electron transport was HCHN since approximately twice as much light was absorbed, i.e.~400 mol photons m -2 s -1 , than under the LC treatment. Despite the fact that more than 95% of this energy acquired was lost by non-photochemical quenching (Y(NO) and Y(NPQ)), when comparing the slopes of the three energy destinations, Y(NO) was the main component of energy fate under HCLN; by contrast, Y(NPQ), related to photoregulated energy dissipation mechanisms, was higher under HCHN than under HCLN. The observed differences in energy losses, interestingly,

  FIGURE CAPTIONS

Figure 2 .

 2 Figure 2. Maximum quantum yield (F v /F m ; mean ± SD) in Ulva rigida, measured using a Mini-PAM fluorometer, exposed for six days to High Carbon (HC, 700 ppm) or Low Carbon (LC, 380 ppm) level, combined with High Nitrate (HN, 50 M) or Low Nitrate (LN, 5 M) concentration during the daytime: morning (M; 7:00-8:00 GMT), noon(N;11:30-12:30 GMT) and evening (E; 17:00-18:00 GMT). Data represent the pooled results (n=24) in accordance with significant effects obtained by an ANOVA (Table2).The different letters indicate significant differences among treatments (p < 0.05, SNK post hoc test).

Figure 3 .

 3 Figure 3. A) and B) Maximum electron transport rates (ETR max ; mean ± SD, n=12), and C) and D) Photosynthetic efficiency ( ETR ; mean ± SD; C, n=18 and D, n=12) in Ulva rigida, measured using a Mini-PAM fluorometer, exposed for six days to High Carbon (HC, 700 ppm) or Low Carbon (LC, 380 ppm) level, combined with High Nitrate (HN, 50 M) or Low Nitrate (LN, 5 M) concentration at Ambient Temperature (AT) vs. Ambient Temperature +4°C (AT+4°C) during the daytime: morning (M; 7:00-8:00 GMT), noon (N;11:30-12:30 GMT) and evening (E; 17:00-18:00 GMT). Data represent the pooled results in accordance with significant effects obtained by an ANOVA (Table2). The different letters indicate significant differences among

Figure 5 .

 5 Figure 5. Maximum non-photochemical quenching (NPQ max ; mean ± SD; n = 12) in Ulva rigida, measured using a Mini-PAM fluorometer, exposed for six days to High Carbon (HC, 700 ppm) or Low Carbon (LC, 380 ppm) level, combined with High Nitrate (HN, 50 M) or Low Nitrate (LN, 5 M) concentration at Ambient Temperature (AT) vs. Ambient Temperature +4°C (AT+4°C) during the daytime: morning (M; 7:00-8:00 GMT), noon (N; 11:30-12:30 GMT) and evening (E; 17:00-18:00 GMT). Data represent the pooled results in accordance with significant effects obtained by an ANOVA (Table2). The different letters indicate significant differences among treatments (p < 0.05, SNK post hoc test).

Figure 6 .

 6 Figure 6. Absorbed PAR energy destined to different photochemical routes in Ulva rigida (mean of n=6), using a Mini-PAM fluorometer: i.e. PAR-energy to effective quantum yield (E Y(II) ), PAR-energy to non-regulated non-photochemical quenching (E Y(NO) ), and PAR-energy to regulated non-photochemical quenching (E Y(NPQ) ), under High Carbon (HC, 700 ppm) or Low Carbon (LC, 380 ppm) level, High Nitrate (HN, 50 M) or Low Nitrate (LN, 5 M) concentration in combination with Ambient Temperature (AT) vs. Ambient Temperature +4°C (AT+4°C). A) LCLN under AT, B) LCLN under AT+4°C, C) LCHN under AT, D) LCHN under AT+4°C, E) HCLN under AT, F) HCLN under AT+4°C, G) HCHN under AT, and H) HCHN under AT+4°C. Regression linear equations are included.

Figure 7 .

 7 Figure 7. Correlation between Y(I) and Y(II) in Ulva rigida, according to four treatments; A) LCLN (Low Carbon, 380 ppm and Low Nitrogen, 5 M), B) LCHN

Figure 8 A

 8 Figure 8

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The energy dissipated as non-photochemical quenching

	Yield of losses, Y(NO) and Y(NPQ), were determined according to Kramer et al.
	(2004a) and Hendrickson et al. (2004). Y(NO) is the fraction of energy passively dissipated
	as heat and fluorescence, mainly due to closed PSII reaction centres. High values indicate an
	inability of the macroalga to protect itself against photodamage by an excess of radiation.
	Y(NO) was calculated as in equation (4):
	A c c e p t e d Y(NO) = F t /F m M Y(NPQ) is the fraction of energy dissipated as heat via regulated photoprotective (4) mechanisms. High values are indicative of photoprotection capacity, and this parameter was a n u s c r i p t calculated according to equation (5):	Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraa473/5926739 by guest on 20 October 2020

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Table 3 .

 3 F v /F m was negatively correlated to ETR max , E k and NPQ max but positively correlated to  ETR . ETR max was positively correlated to both E k and NPQ max .  ETR was negatively correlated to both E k and NPQ max and, finally, E k and NPQ max were also positively

correlated. All Pearson correlation coefficients exhibited a significant p-value lower than 0.05. The energy utilized by U. rigida during different quenching responses is presented in the

  Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraa473/5926739 by guest on 20 October 2020 (Table4; F=7.6697; p<0.05) on the slopes of energy for the effective quantum yield (Slope E Y(II) ). The slopes of non-photochemical quenching parameters (E YNO and E YNPQ ) were not affected by any interaction among the evaluated factors.The effective quantum yield was positively correlated to both energy losses ETR max and NPQ max (Table4); energy conducted to Y(NO) was positively related to the energy associated with other losses Y(NPQ) and ETR max , but negatively correlated to NPQ max . The energy loss corresponding to Y(NPQ) was positively correlated to ETR max and NPQ max . Finally, ETR max

	M a n u s c r i p t
	A c c e p t e d

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	M a n u s c r i p t
	A c c e p t e d

the lowest under HCLN (Table

  [START_REF] Baydend | Synergistic effects of climate change and local stressors: CO 2 and nutrient driven change in subtidal rocky habitats[END_REF] demonstrated that both ocean acidification and elevated nutrient concentration reduced the growth of coralline crusts, and the combination of these factors led to a further decrease in growth. Elevated pCO 2 reduced net photosynthetic rate under N, P and N-P limitations, but increased nitrate reductase activity and soluble protein content, under

	P-replete conditions; by contrast, N or P limitation reduced nitrate reductase activity and
	soluble protein content in U. linza
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  study for U. rigida relating to ETR max , taking into account the biochemical composition

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Table 1 .

 1 Photosynthetic parameters (mean ± SD; n = 3) of Ulva rigida, using a Mini-PAM fluorometer, under low nitrate (5 M) and Ambient Temperature (23-26 o C), i.e. at the beginning of the experiment. F v /F m = Maximal quantum yield, ETR max = Maximal electron transport rate, α ETR = Photosynthetic efficiency, E k = Saturating irradiance of the function ETR vs. irradiance and NPQ max = Maximal non-photochemical quenching (relative units).

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Table 3 .

 3 Values of Pearson correlations among photosynthetic parameters of Ulva rigida (N = 144) using a Mini-PAM fluorometer. The significant differences (p < 0.05) are shown in bold. Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraa473/5926739 by guest on 20 October 2020

		ETR max	 ETR	E k	NPQ max
	F v /F m	-0.2678 p = 0.001	0.2104 p = 0.011	-0.3993 p = 0.000	-0.519 p = 0.000
	ETR max		-0.247 p = 0.003	0.8004 p = 0.000	0.4757 p = 0.000
	 ETR			-0.583 p = 0.000	-0.2546 p = 0.002
	E k				0.4075 p = 0.000

Table 4 .

 4 Values of Pearson correlation among the energy used in photochemistry (Y(II)) as E Y(II) , the dissipation or yield losses (Y(NO) and Y(NPQ)) as E Y(NO) and E Y(NPQ) , respectively, the maximal electron transport rate (ETR max ) and the maximal non photochemical quenching (NPQ max ) of Ulva rigida for the entire experiment measured using a Mini-PAM fluorometer.

	The significant differences (p < 0.05) are shown in bold.
	E Y(II) E Y(NO) E Y(NPQ) ETR max	E Y(NO) 0.4803 p=0.001	E Y(NPQ) 0.6505 p=0.000 0.7988 p=0.000	ETR max 0.9789 p=0.000 0.5757 p=0.000 0.7183 p=0.000	NPQ max 0.392 p=0.006 -0.2638 p=0.070 0.2773 p=0.056 0.3357 p=0.020	Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraa473/5926739 by guest on 20 October 2020

Table 5 .

 5 Maximal electron transport rate (ETR max ) expressed as mol electrons m -2 s -1 calculated from Y(II) measurements with different Pulse Amplitude Modulated (PAM) fluorometers; Diving-PAM, Monitoring-PAM, Mini-PAM and oxygen evolution in Ulva rigida exposed to High Carbon (HC, 700 ppm) or Low Carbon; (LC, 380 ppm), combined with High Nitrate (HN, 50 M) or Low Nitrate (LN, 5 M) at Ambient Temperature (AT) and Ambient Temperature +4°C (AT +4°C). Gross photosynthesis and respiration are expressed as mol O 2 m -2 s -1 as gross photosynthesis and dark respiration. The ratio ETR max /GP under solar radiation (Diving-PAM measurements) and the absorptance measurements are also indicated. The light quality of the actinic light (AL) and the saturation light pulses (SLP) of the different fluorometers are indicated. The different letters indicate significant differences (p < 0.05, SNK post hoc test). 5±0.04 a 0.41±0.03 a 0.33±0.028 b 0.45±0.035 a 0.53±0.06 a 0.47±0.03 a 0.42±0.05 a 0.51±0.06 a This study

			Ambient temperature				Ambient temperature +4ºC
	Fluorometer	LCLN	LCHN	HCLN	HCHN	LCLN	LCHN	HCLN		HCHN	AL	SLP	Reference
	Diving PAM	66.4±6.8 a	60.1±7.3 a 45.6±6.3 b 72.2±7.8 c 83.2±9.2 c 82.7±9.8 c 67.3±7.2 a 93.3±10.1 c	Sun	Halogen	Stengel et al. (2014)
	Monitoring PAM	32.9±3.5 d	29.6±4.2 d 16.2±3.1 e 42.2±5.2 b 60.6±8.2 a 59.1±6.1 a 27.3±3.1 d	61.3±7.5 a	Sun	Blue	Figueroa et al. (2014)
	Mini PAM	30.8±3.27 d 31.7±3.2 d 8.8±1.66 f 33.3±6.2 d 20.3±3.2 d 16.4±2.8 g	8.9±2.2 f	25.0±1.3 d	Red	Red	This study
		Ambient temperature				Ambient temperature +4ºC
	Oxygen measurement	LCLN	LCHN	HCLN	HCHN		LCLN	LCHN	HCLN	HCHN	AL	Reference
	Gross Photosynthesis	1.59±0.3 a	1.46±0.2 a	0.78±0.21 b	2.13±0.25 c	1.80±0.3 a	1.70±0.6 a	1.20±0.3 b	2.95±0.30 c	Sun	This study
	Respiration	0.49±0.08 A 0.42±0.1 A 0.24±0.05 B	0.39±0.07 A 0.49±0.1 A 0.34±0.07 A	0.31±0.07 A	0.48±0.08 A	Dark	This study
	ETRmax/GP	LCLN	LCHN	HCLN	HCHN		LCLN	LCHN	HCLN	HCHN	Reference
	Diving PAM	41.7	41.2	58.43	33.9		45.9		39.5	77.7	31.62	This study
			Ambient temperature					Ambient temperature +4ºC
	Treatments	LCLN	LCHN	HCLN	HCHN		LCLN	LCHN	HCLN	HCHN	Reference
	Absorptance	0.

  Table 2). The different letters indicate significant differences among treatments (p < 0.05, SNK post hoc test). from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraa473/5926739 by guest on 20 October 2020Figure 4. Photosynthetic saturating irradiance (E k ; mean ± SD) in Ulva rigida, measured using a Mini-PAM fluorometer, exposed for six days to High Carbon (HC, 700 ppm) or Low Carbon;(LC, 380 ppm) level, combined with High Nitrate (HN, 50 M) or Low Nitrate (LN, 5 M) concentration at a temperature increased by +4°C

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ACKNOWLEDGEMENTS

The financial contributions to the GAP 9 workshop "Influence of the pulsed-supply of nitrogen on primary productivity in phytoplankton and marine macrophytes: an experimental approach" by: Walz GmbH (making available several PAM fluorometers for comparative measurements); Redox; University of Malaga; Ministry of Economy and Competitivity of Spanish Government (Acción Complementaria CTM2011-15659-E); the Project Interacid (RNM5750) of Junta de Andalucía and Spanish Institute of Oceanography are extensively gratefully acknowledged. Paula S.M. Celis-Plá acknowledges partial support from FONDECYT through grant Project FONDECYT N° 11180197, ANID, Chile. Udo Nitschke received financial support from the Marine Institute and the Marine Research Sub-program of the National (Irish) Development Plan 2007-2013. Erik Jan Malta acknowledges funding by the project "The Salty Gold" SIA Foundation, The Netherlands. Francisco Arenas was funded by FCT-Portugal, project Physiography (PTDC/MAR/105147/2008) co-funded by FEDER through the program Compete-Qren. Fungyi Chow thanks the project funding of FAPESP (São Paulo Research Foundation, Brazil). The help of GAP workshop participants with measurements is acknowledged. The measurements of oxygen evolution by Paulina Flores are are thankfully acknowledged. Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraa473/5926739 by guest on 20 October 2020

A c c e p t e d M a n u s c r i p t Table 2. Multifactorial ANOVA effects on photosynthetic parameters of Ulva rigida, using a Mini-PAM fluorometer under different Carbon (C; 700-ppm vs 380 ppm), Nitrate (N; 50 M vs 5 µM), Time (Time; morning, noon and evening) and Temperature (Ambient Temperature vs. Ambient Temperature +4ºC) conditions. df: degree of freedom; MS: mean square; F: F-statistic. The significant differences (p < 0.05) are shown in bold.