Short-term ecophysiological and biochemical responses of Cystoseira tamariscifolia and Ellisolandia elongata to environmental changes

Short-term ecophysiological and biochemical responses of Cystoseira tamariscifolia and Ellisolandia elongata to changes in solar irradiance and nutrient levels were analyzed in situ in oligotrophic coastal waters by transferring macroalgae collected at 0.5 and 2.0 m depth and exposing them to 2 irradiance levels (100 and 70% of surface irradiance) and nutrient conditions (nutrient-enriched and non-enriched). Both species were affected by changes in irradiance and nutrient levels. Few interactive effects between these 2 physical stressors were found, suggesting major additive effects on both species. C. tamariscifolia collected at 0.5 m and exposed to 70% irradiance had the highest maximal electron transport rate (ETRmax), saturated irradiance (EkETR) and chl a content and the lowest antioxidant activity. Under the same conditions, E. elongata had increased EkETR, antheraxanthin and β-carotene content. At 100% irradiance, C. tamariscifolia collected at 2.0 m had higher maximal quantum yield (Fv/Fm), photosynthetic efficiency (αETR), ETRmax, maximal non-photochemical quenching (NPQmax), saturation irradiance for NPQ (EkNPQ), and antheraxanthin and polyphenol content increased, whereas in E. elongata only αETR increased. In nutrient-enriched conditions, phenolic compounds, several carotenoids and N content increased in C. tamariscifolia at both depths. E. elongata from 2.0 m depth at 100% irradiance and nutrient-enriched conditions showed increased N content and total mycosporine-like amino acids (MAAs). Our results show rapid photophysiological responses of C. tamariscifolia to variations in in situ irradiance and nutrient conditions, suggesting efficient photoacclimation to environmental changes. In E. elongata, Fv/Fm and ETRmax did not change in the transplant experiment; in contrast, N content, pigment and MAAs (biochemical variables) changed. The responses of these macroalgae to nutrient enrichment indicate oligotrophic conditions at the study site and environmental stress.


INTRODUCTION
Environmental stressors can interact and have synergistic or antagonistic effects on physiological re sponses (Bischof et al. 2006).When multiple stressors act synergistically, there can be unpredict able effects on organisms (Xenopoulos et al. 2002).In contrast, when stressors operate in an additive way, species' responses are easier to predict (Martínez et al. 2012).It is important to understand the mechanisms of combined environmental stressors in order to predict an organism's responses to future climate scenarios.Experimental transplants can provide a better understanding of such effects (Marzinelli et al. 2009(Marzinelli et al. , 2011)).
Benthic intertidal organisms are subjected to major changes during the tidal cycle (Davison & Pearson 1996).The responses of intertidal and benthic organisms to stressors can be very rapid, and involve ad justments in their photosynthetic and respiratory activities (Southward et al. 1995, Hoegh-Guldberg & Bruno 2010, Sorte et al. 2010).Temperate intertidal rocky communities can be dominated by habitat-forming macroalgae that drive the biodiversity and functioning of these ecosystems.The algae provide food and shelter, and also reduce environmental stress (Davison & Pearson 1996, Jones 1997, Helmuth et al. 2002, 2006).However, the in creasing environmental stresses associated with climatic changes and anthropogenic impacts (e.g.coastal eutrophication, increase in UV light) can af fect macroalgal communities at the biochemical, ecophysiological, morphological and population levels (Figueroa & Gómez 2001, Bischof et al. 2006).
Light availability is a key factor affecting marine environments (Huovinen & Gómez 2011).Light promotes photosynthetic activity, but can inhibit many biological processes if radiation becomes excessive (Hanelt & Figueroa 2012).Macroalgae have several photoprotective mechanisms such as energy dissipation by specific pigments (e.g.ca ro tenoids) through the xanthophyll cycle (Goss & Jakob 2010); dynamic photo inhibition, i.e. reversible changes in photosynthetic efficiency and capacity, accumulation of ultraviolet screen compounds and increase of antioxidant activity (Gómez et al. 2011).For instance, brown algae accumulate UV screen compounds (polyphenols) with a strong antioxidant activity under high photosynthetically active radiation (PAR) and UVR (Pavia et al. 1997, Connan et al. 2004, Cruces et al. 2012), whereas the tolerance of most red algae to excessive light, including UV, is driven by the accumulation of myco -sporine-like amino acids (MAAs) (de la Coba et al. 2009).
In this study, the physiological and biochemical responses of C. tamariscifolia and E. elongata, collected from 2 different depths, were investigated in relation to the independent and/or interactive effects of ambient radiation and nutrient availability.Based on previous research on the additive effects of physical stressors on fucoid algae (Martínez et al. 2012), we hypothesized that changes in light and nitrogen will have an additive effect on C. tamariscifolia and E. elongata.Algae collected from 0.5 m depth and under nutrient enriched conditions were expected to be less vulnerable under the transplant conditions.

Studied species
Cystoseira tamariscifolia is a habitat-forming species that dominates intertidal and shallow-subtidal Mediterranean communities in pristine sites and oligo trophic waters.Although this is a perennial species, receptacles are most developed in spring and summer (Gómez-Garreta et al. 2001).Ellisolandia elongata is an articulated calcareous species that dominates benthic intertidal communities replaced by ulvacean algae at intermediate levels of nutrient enrichment (Arévalo et al. 2007).Resembling a small bush and up to 20 cm in height (Braga et al. 2009), it is a perennial species and can occupy both well-lit and shaded habitats (Algarra & Niell 1987, Häder et al. 1997, Figueroa & Gómez 2001).It has been recorded to be in the fertile tetrasporophyte phase throughout the year (Rodríguez & Polo 1986).

Experimental design
The experiment was performed from September 19 to 21, 2012.C. tamariscifolia and E. elongata were randomly collected from 2 different depths (0.5 and 2.0 m) (Fig. 1a) at the 'Cabo de Gata-Níjar' Natural Park (36°51' 0" N; 2°6' 0" W; southwestern Mediterranean Sea, Spain).Immediately after collection, macroalgal samples (5 g fresh weight [FW]) were placed into mesh cylinders (15 cm long × 5 cm in diameter) and suspended in the water column (at a depth of 0.2 m) by a floating longline system anchored to the bottom and parallel to the coast (Fig. 1b).This system comprised 4 lines of 12 m length.Each line contained 12 cylinders (separated by 1 m).Two lines were placed at one site for the enriched nitrogen treatment and the other 2 lines were placed at another site for the non-enrichment treatment (Fig. 1b).Both sites were separated by 50 m with a small artificial breakwater between them.Each cylinder contained specimens of one uni que species and collection depth (in triplicate) was fixed along each line (Fig. 1c).Two light levels were assigned within each treatment, i.e. 70 and 100% of surface irradiance defined as PAB irradiance (PAR + UVR) under nutrient-enriched and non-en riched conditions (Fig. 1c).With regard to the irradiance treatment, a neutral screen was used which attenuates 30% of the incident light.Half of the cylinders (containing algae from both depths) were covered with mesh (1 mm 2 ) to attain 70% incoming irradiance (simulating conditions at a depth of 2.0 m, thereafter 70% PAB ), and the remaining cylinders were without the screen to attain 100% incoming irradiance (simulating a depth of 0.5 m, thereafter 100% PAB ).Thereby, algae collected at 0.5 m depth (shallow waters) were exposed to 70% PAB (as a transplant treatment) and 100% PAB (as a control of natural conditions at 0.5 m depth).On the other hand, those algae collected at 2.0 m depth were exposed to 100% PAB (as a transplant treatment) and 70% PAB (as a control of natural conditions at 2.0 m depth) (Fig. 1b).For the nutrient-enriched treatments, mesh bags containing 100 g of a slow-release resin-coated fertilizer (Multicote ® , Haifa Chemicals) (modified from Martínez et al. 2012) and fixed below each cylinder was used to simulate nutrient enrichment.Fertilizer composition was 17% N (NH 4 + and NO 3 − ), 17% P (P 2 O 5 ) and 17% K.For non-enriched treatments, a neutral bag with 100 g of sand was used as a control of the effect of the fertilizer bag and the modifying buoyancy (Fig. 1b).
Three replicate cylinders were used for each combination of treatment level, species and depth (2 species × 2 depths × 2 irradiance levels × 2 nutrient levels), resulting in a total of 48 cylinders with macroalgal samples (Fig. 1b).Several physiological variables were obtained from the algae within each cylinder after the in situ experiment.These variables were also measured in C. tamariscifolia and E. elongata from natural populations (at 0.5 and 2.0 m depth) in order to know the initial values.Additionally, water nutrient concentrations, irradiance (PAR and UVR) and underwater temperature were measured during the experiment.
Irradiance of solar radiation was continuously measured in the air at 3 wavelength bands (UVB = 280−315 nm, UVA = 315−400 nm and PAR = 400−700 nm) using 2 hyperspectral irradiance sensors for UV and PAR (Ramses, TrioS).Attenuation coefficients in water (Kd PAR and Kd UVA ) were measured using PAR (QSO-SUN 2.5V) and UV-R (USB-SU 100, Onset Computer) sensors sealed within a waterproof poly-carbonate box (OtterBox3000).Kd UVB was not measured due to the high absorption of the polycarbonate box in the UVB spectral band (Quintano et al. 2013).
Underwater temperature was continuously measured using a HOBO U22 Water Temp Pro v2 logger (Onset Computer).

Physiological and biochemical variables
Carbon and nitrogen contents on a dry weight (DW) basis were determined using an element analyzer CNHS-932 (LECO).
In vivo chlorophyll a (chl a) fluorescence associated with Photosystem II (PSII) was determined by using a portable pulse amplitude modulated fluorometer Diving-PAM (Walz).Algal pieces were collected from natural populations (initial time) and after 60 h of incubation (for each cylinder) and were placed in 10 ml incubation chambers in order to conduct rapid light curves, one for each cylinder.F o and F m were determined after 15 min in darkness to obtain the maximum quantum yield (F v /F m ), where F v = F m − F o , F o is the basal fluorescence of dark-adapted thalli after 15 min and F m is the maximal fluorescence after a saturation light pulse of > 4000 µmol m −2 s −1 (Schreiber et al. 1995, Figueroa et al. 2009).The electron transport rate (ETR, µmol electrons m −2 s −1 ) as rapid light curves (RLC) was determined after a 20 s exposure period in 8 increasing irradiances (E1 = 9.3, E2 = 33.8,E3 = 76, E4 = 145, E5 = 217, E6 = 301, E7 = 452, E8 = 629, E9 = 947 µmol m −2 s −1 ) of white light (halogen lamp provided by the Diving-PAM).ETR was calculated according to Schreiber et al. (1995) as follows: where ΔF/F m ' is the effective quantum yield, ΔF = F m ' − F t (F t is the intrinsic fluorescence of alga incubated in light and F m ' is the maximal fluorescence reached after a saturation pulse of algae incubated in light), E is the incident PAR irradiance ex pressed in µmol photons m −2 s −1 , A is the thallus absorptance as the fraction of incident irradiance that is absorbed by the algae (see Figueroa et al. 2003) and F II is the fraction of chlorophyll related to PSII (400-700 nm), being 0.8 in brown and 0.15 in red macroalgae (Grzymski et al. 1997, Figueroa et al. 2003).Maximum ETR (ETR max ) and the initial slope of ETR versus irradiance function (α ETR ), as an estimator of photosynthetic efficiency, were obtained from the tangential function reported by Eilers & Peeters (1988).Finally, the saturation irradiance for ETR (Ek ETR ) was calculated from the intercept between ETR max and α ETR .
Non-photochemical quenching (NPQ) was calculated according to Schreiber et al. (1995) (2) Maximal NPQ (NPQ max ) and the initial slope of NPQ versus irradiance function (α NPQ ) were obtained from the tangential function of NPQ versus irradiance function according to Eilers & Peeters (1988).Finally, the saturation irradiance for NPQ (Ek NPQ ) was calculated from the intercept between NPQ max and α NPQ .
Chl a and carotenoid pigments were determined in both species, whereas chlorophyll c (chl c) only in C. tamariscifolia and phycobiliproteins only in E. elongata.
Chl a was determined spectrophotometrically, whilst chl c was identified and quantified using HPLC.Both chlorophyll analyses were made by extracting pigments from thalli (25 mg FW) using 1 ml of N,N-dimethylformamide (DMF) and maintained in darkness at 4°C for 12 h.After centrifugation at 5000 × g for 10 min (Labofuge 400R, Heraeus, Kendro Laboratory Products), each supernatant was used to measure chlorophyll spectrophotometrically.In the case of chl c, the extracts were filtered (0.2 µM) before analyzing with HPLC.The chlorophyll concentrations were calculated using equations by Wellburn (1994).Carotenoid composition was determined by HPLC according to García-Sánchez et al. (2012), using commercial standards (DHI LAB Products).
Total phenolic compounds (polyphenols) were determined only in C. tamariscifolia using 0.25 g FW.Samples were pulverized in a mortar and pestle with sea sand using 2.5 ml of 80% methanol.After keeping the samples overnight, the mixture was centrifuged at 2253 × g for 30 min at 4°C, and then the super natant was collected.Total phenolic compounds were determined colorimetrically using Folin-Ciocalteu reagent (Folin & Ciocalteu 1927) and phloroglucinol (1, 3, 5-trihydroxybenzene, Sigma P-3502) as standard.Finally, the absorbance was determined at 760 nm using a Shimadzu UVMini-1240 spectro photometer.Phenolic concentration was expressed as mg g −1 DW after determining the fresh to dry weight ratio in the tissue (4.3 and 1.5 for C. tamariscifolia and E. elongata, respectively).The results are expressed as mean ± SE from 3 replicates of each treatment.
Antioxidant activity, determined by the 2, 2diphenyl-1-picrylhydrazyil (DPPH) method, was measured on the polyphenol compound extracts according to Blois (1958).Each extract had 150 µl of DPPH, prepared in 90% methanol, added.The reaction was complete after 30 min in darkness at ambient temperature (~20°), and the absorbance was read at 517 nm in a spectrophotometer UVmini-1240 (Shimadzu).The calibration curve made from DPPH was used to calculate the remaining concentration of DPPH in the reaction mixture after incubation.Values of DPPH concentration (mM) were plotted against plant extract concentration (mg DW ml −1 ) in order to obtain the EC 50 value (oxidation index), which represents the concentration of the extract (mg ml −1 ) required to scavenge 50% of the DPPH in the reaction mixture.Ascorbic acid was used as a positive control (Connan et al. 2006).
Total MAA content was determined only in E. elongata using HPLC (Waters 600) as described by Korbee-Peinado et al. (2004).Results were expressed as mg g −1 DW after determining the fresh to dry weight ratio in the tissue (1.5 for E. elongata).

Statistical analysis
The effects of the in situ treatments on the ecophysiological response variables of C. tamariscifolia and E. elongata were assessed using ANOVA (Under wood 1997).For that purpose, 2 factors were considered: Nutrient (fixed with 2 levels) and Irradiance (fixed with 2 levels).This design allows the testing of interactive and additive effects of the variables on the ecophysiological responses.Data used in the analyses were those obtained at the end of the experimental period (after 60 h of photo acclimation).Student-Newman-Keuls tests (SNK) were performed after significant ANOVA interactions (Underwood 1997).Homogeneity of variance was tested using Cochran tests and by visual inspection of the residuals.Analyses were performed by using SPSS v.21 (IBM).

Physiological response variables
Internal N content was higher in Cystoseira tamaris cifolia than in Ellisolandia elongata (Table 1, Fig. 2).ANOVA results showed that both species from 0.5 m depth presented significantly higher N content and a lower C:N ratio under the nutrient-enriched treatment (Table 1, Figs. 2 & 3).However, the N content from 2.0 m depth samples was different for both species (Table 1, Fig. 2).C. tamariscifolia specimens collected from 2.0 m showed similar N content to those from 0.5 m and the C:N ratio increased under the non-enriched treatments (Figs. 2a & 3a).In contrast, E. elongata showed a significant interaction be tween nutrients and irradiance (Table 1).N content in the nutrient-enriched treatment was lower under the 100% PAB treatments and the C:N ratio was higher under the same conditions (Figs. 2b & 3b).
F v /F m in C. tamariscifolia showed a significant interaction with nutrients and irradiance in algae collected at 2.0 m depth (Table 2).Specimens of C. tamariscifolia transplanted to 100% PAB presented higher F v /F m under non-enriched treatments (Table 3).Neither of the species collected at 0.5 m   2).In contrast, ETR max of C. tamariscifolia showed significant differences among irradiance treatments (70% PAB and 100% PAB ) at 0.5 m depth (Table 2).This value was higher when they were transplanted to 70% PAB (Table 3).Conversely, specimens of both species collected at 2.0 m depth did not show any significant differences for either depth.α ETR in C. tamariscifolia showed a significant inter action with nutrients and irradiances at both depths (Table 2).This value was lower at 70% PAB (transplant treatment) and non-nutrient enriched conditions.In both cases, α ETR equaled initial observations from its natural habitat after incubation in the cylinders.(Table 3).To compare, E. elongata α ETR values showed 2 different significant results depending on the depth.α ETR in algae collected from 0.5 m depth showed a significant increase at the nutrient-enriched site and in the 70% PAB treatment (Tables 2 & 3).In contrast, algae collected from 2.0 m had higher α ETR values under the nonenriched treatment (Tables 2 & 3).
In C. tamariscifolia collected from 0.5 m depth, Ek ETR showed a significant interaction with nutrients and irradiance.In algae collected at 0.5 m depth under 70% PAB in the non-enriched treatment, Ek ETR was higher than in the other 3 combinations of treatments (Table 3).However, in algae collected from 2.0 m depth, Ek ETR did not show any significant differences (Table 2).On the other hand, in E. elongata, Ek ETR at both depths showed significant differences with the nutrients (Table 2).Ek ETR values for algae collected from 0.5 m depth were higher in nonenriched treatments, whereas in algae from 2.0 m depth, the values were higher in nutrient-enriched treatments (Table 2).
NPQ max in C. tamariscifolia showed significant differences due to nutrient treatments in algae collected from 0.5 m depth, and a significant interaction was observed with nutrients and irradiance in algae collected from 2.0 m depth (Table 2).In algae from both depths, NPQ max was higher in non-enriched treatments, whereas the NPQ max increased under 100% PAB conditions in algae collected from 2.0 m depth (Table 3).NPQ max did not show any significant differences among treatments in E. elongata (Table 2), in contrast to C. tamariscifolia which showed significant differences due to nutrients at both depths (Table 2).Ek NPQ values in algae collected from 0.5 m were higher in enriched treatments, whereas values were higher under non-enriched treatments in algae from 2.0 m (Table 3).Finally, Ek NPQ showed no significant differences among treatments in E. elongata (Table 2).

Pigment content
Chl a in C. tamariscifolia increased significantly when algae from 0.5 m depth were exposed to lower irradiance levels (70% PAB treatment).Similar results were found for chl c in algae collected from 2.0 m (Tables 4 & 5).Chl c content in C. tamariscifolia collected from 0.5 m was significantly higher in the nutrient-enriched treatment than in the non-enriched one (Tables 4 & 5).Chl a and c contents were initially higher in algae collected from 0.5 m (Table 5).Chl a in E. elongata did not present any significant differences among treatments (Tables 4 & 5).
PC content was significantly higher in the nutrientenriched treatment in E. elongata collected from 0.5 m depth.In contrast, PE content did not show any differences after the experiment (Tables 4 & 5).
The carotenoids fucoxanthin and violaxanthin in C. tamariscifolia showed a significant increase under nutrient-enriched treatment in algae from 0.5 m depth  4 & 5).In contrast, carotenoid content in algae collected from 2.0 m depth was significantly higher the under 70% PAB treatment (Tables 4 & 5).Additionally, antheraxanthin and β-carotene in C. tamariscifolia collected at the same depth had a significant interaction between nutrients and irradiance.Both com pounds increased significantly at 70% PAB in the non-enriched treatment site (Tables 4 & 5).In E. elongata, fucoxanthin, antheraxanthin and β-carotene con tents in algae collected from 0.5 m depth showed a significant increase in the 70% PAB irradiance treatment (Tables 4 & 5).Additionally, fucoxanthin content increased significantly in algae cultured under nutrient-enrichment conditions (Tables 4 & 5).Zeaxanthin content did not show any differences after the in situ experiment (Tables 4 & 5) for either species.
Total phenolic compounds.Total phenolic compounds in C. tamariscifolia were significantly different among nutrient treatments in algae from both 0.5 and 2.0 m depths (Table 6).Additionally, algae collected from 2.0 m showed significant differences in both irradiance treatments (Table 6).In algae collected from 0.5 m depth, the total phenolic compounds were higher in the nutrient-enriched treatment (Fig. 4a).In C. tamariscifolia from 2.0 m depth, the increase of phenolic compounds was higher under 100% PAB than under 70% PAB , whereas this increase was higher under non-enrichment than that under the enrichment treatment (Fig. 4a).
Antioxidant activity (EC 50 ).EC 50 in C. tama riscifolia collected at 0.5 m depth showed a significant interaction between nutrients and irradiance  6).In the non-enriched treatment, EC 50 was higher (lower antioxidant activity) than in the other treatment combinations (Fig. 4b).In algae collected at 2.0 m depth, significant differences were only found in nutrient-enriched treatments (Table 6), i.e.EC 50 was higher (lower antioxidant activity) in the nutrient-enriched treatment (Fig. 4b) than in the non-enriched treatment.
Total MAA content.Total MAA content in E. elongata was higher in algae collected at 0.5 m depth than in those collected at 2.0 m (Fig. 5a).MAA content in algae from 0.5 m depth showed a significant increase under 100% PAB in nutrienten riched treatments (Table 7, Fig. 5a).In contrast, total MAA content in algae collected from 2.0 m depth was significantly higher at 100% PAB for both enriched and non-enriched nutrient treatments (Table 7, Fig. 5a).The most abundant MAAs de tected in this species were shinorine (50 to 60%) and palythine (approx.40%), other MAAs such as asterina-330 were present in trace amounts.After the in situ experiment, algae collected from 2.0 m depth showed significantly higher palythine content under nutrient-enriched treatments, and shinorine in creased in nonenriched treatments (Table 7, Fig. 5b,c).In contrast, algae collected from 0.5 m did not show any differences (Table 7).

DISCUSSION
We found high photoacclimation in Cystoseira tamariscifolia and Ellisolandia elongata, with photo synthetic parameters and biochemical composition changing in response to the short-term irradiance and nutrient treatments (60 h).The algae collected from 0.5 m depth had a higher production (ETR) and efficiency (α ETR ) than those from 2.0 m depth.These differences can be explained by the high transparency in the coastal waters of Cabo de Gata-Níjar Natural Park, allowing high penetration of both PAR and UVR, which can produce negative biological effects such as photoinhibition or DNA damage.In our study, the attenuation coefficients for PAR (Kd PAR ) and UVA (Kd UVA ) were 0.076 m −1 and 0.137 m −1 , respectively.Figueroa & Gómez (2001) de scribed these coefficients with similar results for PAR (Kd PAR ) and UVA (Kd UVA ), 0.070 m −1 and 0.100 m −1 , respectively, and a Kd UVB value of 0.22 m −1 in the same coastal area.The C:N ratio was more favorable physiologically (< 23) in C. tamariscifolia from 0.5 m than in algae from 2.0 m (> 30).On the other hand, the elevated NPQ max indicated high photo protection capacity.The suntype photosynthetic pattern of the species analyzed is shown by the high Ek ETR values (200 to 220 µmol photons m −2 s −1 ) in algae collected at both 0.5 and 2.0 m (initial conditions).These values were lower than those reported by Celis-Plá (2011) andFigueroa et al. (2014, this Theme Section) in C. tamariscifolia growing in a nearby coastal area of the Mediterranean Sea but subjected to emersion conditions, in contrast to the subtidal species of Cabo de Gata-Níjar, i.e. higher nutrient and irradiance levels than those found in this study.
According to the physiological status, algae grown at 0.5 m will be less vulnerable to higher irradiance conditions (100% PAB ) than algae grown at 2.0 m.At the initial natural conditions, the phenolic compounds (photoprotectors) in C. tamariscifolia are expected to be higher in algae grown at 0.5 m than at 2.0 m.However, in algae collected at 0.5 m depth, the phenolic compounds were lower than algae collected at 2.0 m, during the initial period.This can be explained as a consequence of the high irradiance found at 0.5 m, since phenolic compounds could be re leased under high solar irradiance, preventing the photodamage as a photoprotection strategy (Abdala-Díaz et al. 2006).Photoacclimation responses were also af fected by nitrate supply in general; nitrate enrichment increased the photosynthetic rate and the accumulation of photoprotectors.This indicates that the algae are nutrient-limited in this oligotrophic system (Figueroa & Gómez 2001).
C. tamariscifolia collected from 0.5 m depth maintained ETR values 60 h after transferring to 100% PAB in both nutrient conditions, but phenolic compounds and internal N content increased only in nutrientenriched conditions.The transplantation to 70% PAB provoked an increase in ETR max , indicating that algae at 0.5 m depth were photoinhibited under initial conditions.The increase of ETR max at 70% PAB is related to a decrease in NPQ max , indicating less energy dissipation as a consequence of decreased    6. ANOVA results after in situ experiment testing for the effect of irradiance and nutrients on the phenolic compounds and antioxidant activity (EC 50 ) of Cystoseira tamariscifolia collected at 2 different depths.We used a significance level of α = 0.05, shown in bold PAR and UVR can cause photoinhibition, which can be defined as the light-dependent decline in photosynthetic capacity and maximal photosynthetic efficiency as a consequence of the dominance of photo damage versus photorepair processes (Osmond 1994, Gómez et al. 2004).It is also thought that photo inhibition is a down-regulation mechanism to quench excessive solar energy (Demmig-Adams et al. 2008).However, in C. tamariscifolia, no photoinhibition was observed.Intertidal macroalgae from southern Spain have low photoinhibition at noon and high recovery capacity during daily cycles due to high energy dissipation (Figueroa et al. 1997, Häder et al. 1997, 1998).
Photosynthetic efficiency α ETR , ETR max and MAAs in E. elongata collected from 0.5 m depth de creased after transfer to 100% PAB under both nutrient conditions, but internal N contents increased only under nutrient-enriched conditions.The transplant to 70% PAB provoked an increase of α ETR and ETR max only under nutrient-enriched conditions; however, internal N content and MAAs decreased in both nutrient treatments, indicating that algae grown at 0.5 m depth can be photoinhibited under initial conditions.The level of ETR max , α ETR and MAAs in algae collected from 2.0 m depth increased when they were transplanted to 100% PAB under both nutrient treatments; however, the internal N content de creased in both nutrient treatments.The transplantation of algae collected from 2.0 m depth to 70% PAB caused a higher α ETR and ETR max in both nutrient conditions; however, internal N content and MAAs increased in nutrient-enriched conditions.
In general, in both species collected from 0.5 m depth, the addition of nutrients in creased their photosynthetic efficiency.The photosynthetic response was also af fected by irradiance levels.Although the initial values of NPQ max in C. tama ris ci folia were similar, NPQ max decayed at both depths under nutrient enrichment and Ek NPQ only increased in the enriched treatment.Furthermore, in C. tama riscifolia collected at 2.0 m depth, an interaction between light and nutrients was ob served, where transplanted algae (to 100% PAB ) under nonenriched treatment showed an increase in NPQ max and Ek NPQ in all treatments.At high nutrient availability, it seems that algae collected from 0.5 m depth had higher levels of photoprotective compounds (phenols) or increased size of antenna (higher content of chl c and fucoxanthin were ob served).This could be due to high antioxidant activity and less requirement for the dissipation of energy in the form of heat (low NPQ max ) or due to less UV radiation that could be reaching the photosynthetic apparatus.However, in C. tamariscifolia collected at 2 m depth after the transplant conditions (70% PAB ), high levels of accessory pigments were found.These differences were independent of the nutrient treatment.The phenolic compounds and antioxidant activity were affected by irradiance and nutrients as single factors in the first case, and by the interaction of both factors in the second case.For the other caro te noids, similar results were found in C. tamariscifolia collected from 0.5 m depth.
Carotenoid contents were less influenced by irradiance or nutrients with the exception of violaxanthin that had higher content after nutrient enrichment.On the other hand, in C. tamariscifolia collected from 2.0 m depth, violaxanthin content was higher in the simulated deeper irradiance (70% PAB ), as was found in other accessory pigments.However, antheraxanthin and β-carotene were significantly affected by the interaction of irradiance and nutrients.In E. elongata collected from 0.5 m depth, an effect of irradiance was found.The responses found in this study for both species are similar to those described by Demmig-Adams & Adams (1996) flect a regulatory and photoprotective response that down-regulates the delivery of excitation energy into the electron-transport chain to match the rates at which products of electron transport can be consumed in these leaves.Goss & Jakob (2010) indicate that the xanthophyll cycle represents an important photoprotection mechanism in plant cells.This suggests a relationship between higher photosynthetic rates and a higher activity of the xanthophyll cycle.However, the presence of a functional xanthophyll cycle in red algae is uncertain (Andersson et al. 2006, Schubert et al. 2006).In fact, the predominant presence of red algae in intertidal zones and coral reefs suggests a highly efficient capacity to withstand elevated irradiance levels and large diurnal light fluctuations due to tides and aerial exposure (Schubert et al. 2011).E. elongata possesses high reflectance under high solar radiation, allowing it to live in areas of high radiation and sun exposure due to a skeleton composition of calcium carbonate (Häder et al. 1997).These authors de scribed a high reflectance under high solar radiation exposure in E. elongata, which can be advantageous under elevated solar irradiance reducing photo inhibition in this species.Connan et al. (2004) found higher levels of phenols in summer in several brown macroalgae off Brittany related to higher solar irradiance.Similarly, Abdala-Díaz et al. (2006) found higher phenol content in summer than in winter in C. tamariscifolia collected in southern Spain in the morning.However, at noon the levels were similar in both seasons due to the high release of polyphenols in summer.In our study, the phenolic content in C. tamariscifolia increased with nutrient enrichment in algae collected at 0.5 m depth in the non-enriched treatment and in transplanted specimens (to 100% PAB ) under non-enrichment treatments in those collected from 2.0 m depth.In brown algae, UV screen compounds (polyphenols) accumulate under high PAR and UVR and these compounds have strong antioxidant activity (Pavia et al. 1997, Connan et al. 2004, Cruces et al. 2012).This may suggest that this is probably more related to the nitrate availability than to solar irradiance conditions.Pavia & Toth (2000) indicate that the N content can enhance the accumulation of phenolic compounds in some brown algae.In fact, concentrations of phenolic compounds show phenotypic plasticity in response to changes in environmental parameters, such as salinity, nutrients, light quality and availability, and intensity of herbivores (Peckol et al. 1996, Pavia et al. 1997, Pavia & Toth 2000, Honkanen et al. 2002, Swanson & Druehl 2002, Amsler & Fairhead 2006).Moreover, C. tama ris cifolia had higher antioxidant activity at 0.5 m depth in transplanted conditions (70% PAB ) without nutrient enrichment, and also in algae collected from 2.0 m depth in transplant conditions (100% PAB ) with nutrient enrichment.
As has been mentioned, the response of E. elongata collected from 0.5 m depth was de pendent mostly on irradiance.However, the content of MAAs (UV-screening substance) of algae collected at 0.5 m depth depended on the interaction between irradiance and nutrients, as reported by Korbee-Peinado et al. (2004).Karsten et al. (1998) and Franklin et al. (2001) have shown that accumulation of MAAs depend on both quality and quantity of radiation, with higher accumulation of MAAs with high daily PAR doses and UV exposure.Korbee-Peinado et al. (2004) found that high ammonium concentrations significantly in creased the content of MAAs in Pyropia columbina (as Porphyra columbina).In their study, an interaction between irradiance and nutrients was found.Similar results were found for other Porphyra species, Grateloupia lanceola and Gracilaria spp.(Korbee et al. 2005a, Huovinen et al. 2006, Barufi et al. 2011, Figueroa et al. 2012).In our study, the MAA total content decreased in algae transplanted from 100% PAB to 70% PAB and after nutrient enrichment, whereas no effect of nutrient was observed in algae collected from 2.0 m depth waters.It seems that the short-term effect of the nutrient addition is not enough to produce an increase of total MAA content under nitrogen-enriched conditions as has been reported in other algae (Barufi et al. 2011, Figueroa et al. 2012).However, the effect of nutrients was reflected by a preferential accumulation of some types of MAAs, but only in E. elongata collected from 2.0 m depth.The relative content of palythine in creased in nutrient-enriched algae, which has been associated with higher antioxidant activities compared to shinorine (de la Coba et al. 2009).
In conclusion, C. tamariscifolia and E. elongata showed different physiological responses under different nutrient and irradiance conditions.Few interactive effects between these 2 physical stressors were found, suggesting major additive effects on the responses of both species.In fact, environmental variables acting in additive forms can act as more powerful stress factors (Martínez et al. 2012) leading to changes in the physiology of these macroalgae.Therefore, understanding the physiological consequences of the potential additive effects of these physical stressors on these dominant species is needed to predict future environmental fluctuations related to climate change.
Acknowledgements.We thank the office of the 'Cabo de Gata-Níjar' Natural Park of the Junta de Andalucía for the use of their facilities.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 (including the use of several PAM fluorometers), Redox, the University of Málaga General Foundation, the Ministry of Economy and Competitivity of Spain Government (Acción Complementaria CTM2011-15659-E) and the Spanish Institute of Oceano graphy are extremely appreciated.P.S.M.C.-P.gratefully acknowledges financial support from 'Becas-Chile' (CONICYT) of the Chilean Ministry of Education.We thank the reviewers for their helpful and constructive comments which significantly improved the manuscript.We also thank Dr. Jason Hall-Spencer for English corrections.

Fig. 2 .
Fig. 2. Total internal N content (mean ± SE, n = 3) of (a) Cystoseira tamariscifolia and (b) Ellisolandia elongata from 0.5 and 2.0 m depth under irradiance and nutrient treatments.Black bars indicate 100% PAB , and grey bars indicate 70% PAB .N+ and N− indicate nutrient-enriched and nonenriched treatments, respectively.Upper values in each box indicate initial values (I S : 0.5 m depth; I D : 2.0 m depth).Lowercase letters denote significant differences after SNK test for 0.5 m and capital letters for 2.0 m algae

Fig. 3
Fig. 3. C:N ratio (mean ± SE, n = 3) of (a) Cystoseira tamariscifolia and (b) Ellisolandia elongata from 0.5 and 2.0 m depth under irradiance and nutrient treatments.Black bars indicate 100% PAB , and grey bars indicate 70% PAB .N+ and N− indicate nutrient-enriched and nonenriched treatments, respectively.Upper values in each box indicate initial values (I S : 0.5 m depth; I D : 2.0 m depth)

DW
Cystoseira tamariscifolia andEllisolandia elongata collected at 2 different depths (0.5 m and 2.0 m) in relation to irradiance (70% PAB and 100% PAB ) and nutrient (Nutrients+ and Nutrients−) treatments.Chl a, chl c, phycoerythrin and phycocyanin contents are expressed in mg g −1 DW.Fuxocanthin, violaxanthin, antheraxanthin, zeaxanthin and β-carotene contents are expressed in µg g −1 depth) are shown in the first column and in bold for each depth.Uppercase letters denote significant differences after SNK test in algae collected at 2.0 m depth, nd: no data irradiance, at least in the short-term period analyzed.In any case, prolonged time can eventually reduce the values of ETR max due to less available energy at 2.0 m than that at 0.5 m in spite of photoinhibition.The ETR max of algae collected from 2.0 m depth when transplanted to 100% PAB increased only in non-

Table 1 .
ANOVA results after in situ experiment testing for the effect of irradiance and nutrients on C and N contents and C:N ratios of Cystoseira tamariscifolia and Ellisolandia elongata collected at 2 different depths.We used a significance level of α = 0.05, shown in bold

Table 2 .
ANOVA results after in situ experiment testing for the effect of irradiance and nutrients on photosynthetic parameters of Cystoseira tamariscifolia and Ellisolandia elongata collected at 2 different depths.We used a significance level of α = 0.05, shown in bold.F v /F m : maximal quantum yield, α ETR : photosynthetic efficiency, ETR max : maximal electron transport rate, Ek ETR : saturated irradiance of ETR, NPQ max : maximal non-photochemical quenching, Ek NPQ : saturated irradiance of NPQ (Table

Table 4 .
ANOVA results after in situ experiment testing for the effect of irradiance and nutrients on the photosynthetic pigment content of Cystoseira tamariscifolia and Ellisolandia elongata collected at 2 different depths.We used a significance level of α = 0.05, shown in bold; nd: no data

Table 7 .
. The response of the xanthophyll cycle and light absorption could re -ANOVA results after in situ experiment testing for the effect of irradiance and nutrients on total mycosporine-like amino acid (MAA) content, and percentages of shinorine and palythine of Ellisolandia elongata collected at 2 different depths.We used a significance level of α = 0.05, shown in bold