Macroalgae in temperate regions, such as the Mediterranean coast of Spain, are exposed to high daily integrated solar irradiance, both ultraviolet (UV) and photosynthetically active (PAR) (Häder and Figueroa 1997). The high irradiance and transparency of shallow water in this region suggest that macroalgae have developed efficient photoprotection mechanisms to tolerate light stress (Figueroa and Gómez 2001). In fact, intertidal macroalgae subject to high solar irradiance and desiccation can survive and grow under the stressful conditions of the intertidal system due to active photoprotection mechanisms such as dynamic photoinhibition, accumulation of UV screen substances and increase in antioxidant capacity (Häder and Figueroa 1997, Korbee et al. 2006, Bischof et al. 2006, Hanelt and Figueroa 2012).

Brown algae accumulate UV screen compounds (polyphenols) under high PAR and UVR (Pavia et al. 1997). In addition to the UV screen capacity, phenolic compounds also have strong antioxidant activity (Connan et al. 2006, Cruces et al. 2012), thus reducing DNA damage (Gómez and Huovinen 2010). Phlorotannins are phenolic compounds identified in brown algae constituting up to 25% dry weight (Targett et al. 1992). Concentrations of phlorotannins show phenotypic plasticity in response to changes in environmental parameters, such as salinity, nutrients, light quality and irradiance availability, and herbivory (Peckol et al. 1996, Pavia et al. 1997, Pavia and Toth 2000, Honkanen and Jormalainen 2002, Swanson and Druehl 2002, Abdala-Díaz et al. 2006). Phenolic compounds can also be released from the thalli into alkaline medium of seawater under stressful conditions, reacting rapidly with both proteinaceous and carbohydrate substances to form UV-absorbing complexes (Dujmov et al. 1996, Swanson and Druehl 2002, Koivikko et al. 2005). Hence, it is very important to determine both internal and released polyphenols in order to evaluate the photoprotective capacity of these compounds (Koivikko et al. 2005, Gómez and Huovinen 2010, Cruces et al. 2012).

Cystoseira species are considered to grow mainly in high-quality waters according to the Water Framework Directive of the European Union (WFD, 2000/60/EC) and they are indicator of waters with high-quality ecological status including the Andalusia Coast (Ballesteros et al. 2007, Arévalo et al. 2007). In our study, Cystoseira tamariscifolia (Hudson) Papenfuss, was selected as a model species because it is an abundant and is a key species on the southern shores of the Mediterranean Sea. The evaluation of photosynthetic and antioxidant activities in thalli collected at different places in short spatial scales such as rocky shores (RS, thalli exposed to the air during some time during the daily cycle) and rockpools (RP, thalli always immersed in the seawater and with a high rate of water renewal) can give information on the vulnerability and acclimation capacity of this species to environmental changes. In addition, it is important to analyse the relation between photosynthetic activity and energy dissipation by using in vivo chlorophyll a fluorescence and polyphenol content and antioxidant activity in thalli collected in summer and winter and submitted to an emersion/immersion regime (RS- versus RP-collected thalli) and incubated under outdoor conditions.

Two physiological indicators have been used to evaluate the physiological status of seaweeds (Figueroa and Korbee 2010): (1) maximum quantum yield of PSII (F_v/F_m) as an indicator of physiological status of macroalgae and photoinhibition (Schreiber et al. 1986) and (2) electron transport rate (ETR) as an indicator of photosynthetic capacity (Figueroa et al. 2003). Two biochemical indicators of stress conditions have also been used: (1) stoichiometric ratios (C:N) as an indicator of nutritional status and (2) the content of phenolic compounds, such as photoprotective and antioxidant substances in brown algae (Pavia et al. 1997, Connan et al. 2004, Abdala-Díaz et al. 2006).

The hypothesis is that thalli submitted to high stress conditions in the natural environment have a greater acclimation capacity and less vulnerability to increased solar irradiance in the short to medium term (3 and 7 days).

Photosynthetic capacity (ETR_max) and photosynthetic efficiency (α_ETR) were higher in C. tamariscifolia collected in summer than in winter and these differences were maintained after 7 days of incubation in cylindrical vessels under full solar radiation and the temperature of each season. In summer, during the experimental period, high daily integrated irradiance of PAR (99.77 MJ m^–2) and temperature (22°C day/17°C night) favoured photosynthetic activity in C. tamariscifolia compared with the winter period (54.26 MJ m^–2 and 18°C day/12°C night). These results are in agreement with its latitudinal and zonal distribution in the coastal areas (Lüning 1990, Thibaut et al. 2005). A positive correlation between the ETR_max calculated from the RLC and the in situ ETR_max from the daily cycles of ETRs (r=0.89, p<0.001, n=60) was found: the latter were always 4.5 times higher. Parameters derived from RLCs as ETR_max or Ek are sensitive to diurnal fluctuations as the effective and maximum quantum yields of PSIIs (Belshe et al. 2007).

ETRs calculated in daily cycle (in situ ETR) tend to be higher than in RLCs, as was described by Longstaff et al. (2002). The ETR was also higher under solar radiation (in situ measurements) than under the halogen lamp provided by the Diving-PAM (RLC determination). This result can be explained by the different light qualities of the radiation sources, i.e., the solar radiation has a much higher blue:red light ratio than the halogen lamp of the Diving-PAM, contributing to a higher electron flow by accessory pigments (carotenoids) to chlorophyll. Brown algae showed a high photosynthetic quantum yield in blue light according to the action spectra for photosynthesis reported by Lüning and Dring (1985).

The microhabitat of collection (RS or RP), in addition to the season, affected the photosynthetic pattern in field collected C. tamariscifolia. Higher ETR_max and α_ETR in RS than in RP field-collected algae was observed, but only in winter. One possible explanation for this pattern is that the emersion periods in RS algae favoured photosynthetic activity by direct incorporation of CO₂ from the air. C. tamariscifolia can grow in RP, where it is always submerged during the daily period. On the other hand, C. tamariscifolia growing in RS may be subjected to different cycles of desiccation and rewetting, increasing atmospheric CO₂ uptake and nutrient incorporation; the inorganic carbon uptake in C. tamariscifolia growing in rockpools depend mainly on the amount of dissolved HCO₃^– and carbon concentration mechanisms (CCMs) through carbonic anhydrase (CA), with the consequent energy cost (Falkowski 1997). It has been reported that intertidal algae under moderate desiccation conditions have higher nitrogen and phosphate uptakes (Lobban and Harrison 1994, Nygard and Dring 2008), photosynthetic rates (Dring and Brown 1982, Mercado et al. 1998). The carbon incorporation under emersion is higher than that under submerged conditions due to the direct uptake of CO₂ (Flores-Moya et al. 1998).

Internal N content was higher in winter- than in summer-collected algae, as is expected according to nitrate content in the water (Ramírez et al. 2005). Interestingly, these differences were maintained after 7 days of incubation in cylindrical vessels in spite of the nitrate enrichment (maximum level of 50 mM). Therefore, the nutritional state seems to be more favourable in winter- than in summer-grown algae as a lower C:N in thalli collected in winter was found. Additionally, thalli collected in summer seemed not to accumulate N compounds after nitrate enrichment due to a low uptake rate. It seemed that thalli accumulate N during winter as a reservoir. This result could be related to the high amount of energy that these macroalgae demand in summer, the period in which the activation of photoprotection and acclimation mechanisms may occur (Hanelt and Figueroa 2012).

This result, in combination with the higher photosynthetic rate (ETR_max) and efficiency (α_ETR) in thalli collected in summer and incubated for 7 days, suggests that C. tamariscifolia is not limited by N in summer and it can invest the photosynthetic energy in growth. Sales and Ballesteros (2012) reported higher growth rate in Cystoseira crinita from the northwestern Mediterranean in summer than in winter. This is also in accordance with the higher ETR_max in thalli collected from RS than from RP in both seasons. Moreover, Celis-Plá et al. (2014) found higher ETR_max in C. tamariscifolia collected from 0.5 m depth waters than from 2.0 m depth waters in summer after an in situ experimental period. As in this study, ETR_max at initial time was also higher in algae of the intertidal zone during the emersion than the submersion period (Nitschke et al. 2012).

Most of the differences between season and growing sites observed in the thalli collected from the field (field algae) remains after 7 days of incubation under immersed conditions in cylindrical vessels. These results indicate a high resilience of this species. The higher decay observed after 3 d of exposure in summer-grown algae and after 7 d of exposure in winter-grown algae indicates a possible accumulative inhibitory effect in winter and high photoacclimation capacity in summer-grown algae. Some authors have also shown that the dynamic photoinhibitory response may be related to acclimation responses to UV radiation (Häder and Figueroa 1997, Figueroa et al. 1997, Flores-Moya et al. 1998, Figueroa et al. 2003).

In fact, studies on daily photoinhibition and full recovery in intertidal Mediterranean algae suggest that photoinhibition is a photoprotective mechanism against high solar radiation, as in higher plants, and that the pattern of photoinhibition and recovery is affected by accumulative dose (Figueroa and Viñegla 2001). An enhanced capacity for dynamic photoinhibition and subsequent recovery has been previously reported in macroalgae, including brown macroalgae from southern Spain (Häder et al. 1998, Flores-Moya et al. 1999). In summer-grown thalli collected from RP, the ETR decay was delayed, as was observed in the daily cycle or in situ measurements. Our results suggest that photoinhibition can be a mechanism that protects C. tamariscifolia against high irradiance as observed in other intertidal seaweeds (Osmond 1994, Hanelt 1996). In addition to dynamic photoinhibition, another indicator of high photoacclimation capacity is the high energy dissipation that allows species to cope with excess excitation energy, as is the case of NPQ (Klughammer and Schreiber 2008). C. tamariscifolia specimens collected in summer and from RS showed higher values of NPQ than those collected in winter and from RP. High values of NPQ indicate active photoprotective mechanisms, which are highly related to the xanthophyll cycle (Demmig-Adams and Adams 2006).

Phenolic content was higher in winter than in summer. This could be an indicator of a good physiological status, i.e. accumulation of secondary metabolic compounds in nutrient-replete conditions (winter) to be used in nutrient-depleted conditions (summer) (Celis-Plá et al. 2014). Abdala-Díaz et al (2006) showed both seasonal and hourly variation in phenolic compounds depending on the daily integrated irradiance (dose) or hourly irradiances, respectively. It has been described that the variability in the phenolic content could be related to environmental factors such as herbivory, light, depth, salinity, nutrients and seasonality, as well as to intrinsic ones such as age, length and kind of tissue (see Amsler and Fairhead 2006, for review). Zubia et al. (2008) described a complexity of seasonal variations suggesting a stronger correlation between phenolic contents and local environmental factors (e.g. grazing intensity in different areas of the coral reef) than between large scale factors (i.e. months, seasons, latitude). The phenolic content and the antioxidant activity have been related to algal zonation (Connan et al. 2004). In the eulittoral and intertidal zone, some algae (Fucus spiralis, Fucus vesiculosus, Ascophyllum nodosum) show higher phenolic content than algae growing in the low intertidal or sublittoral zone (Fucus serratus, Bifurcaria bifurcata, Himathalia elongata and Laminaria digitata) (Connan et al. 2004). Also, contents are higher in summer when irradiance is the highest, as observed in several brown macroalgae from Brittany (Connan et al. 2004) and in C. tamariscifolia collected in Southern Spain (Abdala-Díaz et al. 2006). In contrast, in our study C. tamariscifolia showed higher phenolic content in winter than in summer, probably in relation to the high winter nitrate availability in Málaga (Ramírez et al. 2005). N can enhance the accumulation of phenolic compounds in some brown algae (Pavia and Toth 2000, Celis-Plá et al. 2014) as well as in Ulva rigida (Cabello-Pasini et al. 2011). In contrast to internal phenolic content, the release of phenolic compounds was similar in RP in winter and in summer, and higher in RS (Table 7), whereas the percentage of release to internal content was clearly higher in summer- than winter-collected algae, i.e. after 7 days of incubation the percentage of release was about 3 or 5 times higher in algae collected in RP and RS, respectively, in summer than in winter. Phlorotannins released to seawater from the tissues react with other substances to form UV-absorbing complexes (Craigie and McLachlan 1964, Carlson and Carlson 1984, Jennings and Steinberg 1994, Dujmov et al. 1996). However, a few data are available on quantities of released phlorotannins (Toth and Pavia 2000) or on their physiological and ecological function. Swanson and Druehl (2002) reported high excretion of phenols by increasing UV radiation. Although the effect of UV radiation on release rates was not directly examined in our study, there is a positive relationship between solar incident irradiance of PAR, UVA and UVB and rate of phenol release. The release rate of polyphenols in our study was about 3-5 times lower than that observed by Jennings and Steinberg (1994) in Eklonia radiata (10-24 mg g^–1 DW d^–1). The release rate can be related to the light history and the species, i.e. C. tamariscifolia is an intertidal species subject to higher daily integrated irradiance than the subtidal species Ecklonia radiata. High PAR irradiances and emersion have been associated with increasing phlorotannin release rates (Ragan and Jensen 1978, Carlson and Carlson 1984). In addition, phlorotannins in macroalgae are produced and released into seawater during periods of UVA stress and they are released but under UVB i.e at concentration of >0.84 g mL^–1, they reduce the impact of UVB exposure in UV-sensitive kelp meiospores (Roleda et al. 2006, Huovinen et al. 2010). Taking into account that phlorotannins exhibit absorption maxima at 200 and 270 nm, the putative shielding capacity of phlorotannins would be more efficient in the case of DNA damage (caused mainly by UV-B wavelengths) than photosynthesis, for example, which is normally also affected by wavelengths in the UV-A region (Huovinen el at. 2010). Koivikko et al. (2005) also described exudation of phlorotannins to the surrounding water, and the rate of exudation was not affected by nutrient shortage. Karban and Baldwin (1997) reported an indirect defence of phlorotannins in algae, i.e. increased excretion of these compounds into the water when algae were grazed.

C. tamariscifolia showed higher antioxidant capacity in thalli collected in summer than in winter and in thalli collected from RS than from RP in spite of the lower content of internal phenols. Therefore, high antioxidant activity is produced in algae submitted to high solar irradiances and low internal N content. Since the internal polyphenol content is lower in summer than in winter, we suggest that the antioxidant activity in summer could be related to other internal substances such as carotenoids.

Photoacclimation capacity of C. tamariscifolia was higher in thalli collected in summer than in winter and in thalli from RS than from RP, i.e. the algae are less vulnerable to increased solar exposure when subject to more stressful conditions (e.g. high solar irradiance and low nitrate level). In thalli collected in summer from RS, photosynthetic activity was higher and photoinhibition lower after 7 days of incubation than in thalli collected in winter from rockpools. This higher acclimation capacity could be explained by: (1) high dynamic photoinhibition, as is shown during daily cycles, i.e. fast and high increase of ETR_max and F_v/F_m in the afternoon (high recovery); (2) high NPQ_max, indicating an efficient energy dissipation and high photoprotection capacity (Celis-Plá et al. 2014); and (3) high antioxidant activity (low EC₅₀), related not to internal phenolic compounds but probably to other antioxidant substances such as carotenoids. A high acclimation capacity to increased UVB radiation of C. tamariscifolia has recently been shown based on the accumulation of UV screen substances, high release rates of polyphenols and high antioxidant activity (Figueroa et al. 2014).