| Summary: | In this thesis, the vulnerability and capacity of acclimation to environmental stress related to Climate change mainly in Cystoseira tamariscifolia and other species as Ellisolandia elongata, Cystoseira compressa and Padina pavonica are evaluated. The effects of physical stressors as elevated irradiance of Photosyntethic active radiation (PAR) (.=400-700 nm) and Ultraviolet radiation (UVR) (.=280-400 nm) and temperature and chemical stressors as nutrient, heavy metals and CO2, separately and in interaction with different levels were evaluated. Six experimental studies were conducted under varying irradiance, temperature, nutrient conditions, acidification and heavy metals. The common approach in these studies was the use functional bioindicators to evaluate the physiological state macroalgal species of Mediterranean Sea in studies conducted both in situ in ultra-oligotrophic (Cabo de Gata-Nijar Natural Park, Almeria, Southern Spain), oligotrophic waters (La Araña beach, Malaga, Southern Spain), in waters with natural acidification (Vulcano, Southern Italy) and with algae transported to controlled experimental systems under out-door conditions. In addition, an indoor experiment study was performed in C. tamariscifolia collected in the North Atlantic Ocean, the northern limit of distribution of this species. We observed in a seasonal study conducted in two subsequent years in oligotrophic coastal waters of Malaga that C. tamariscifolia presented the highest production and it was less vulnerable in spring than those in the rest seasons through the year due to environmental, temperature and nutrient were more favorable, temperature and nutrient environmental conditions. However, we also observed that the algae in summer could efficiently acclimate to increased stress conditions by the increase its antioxidant activity. Then, the vulnerability and capacity of acclimation of C. tamariscifolia were evaluated in outdoor experimental conditions in algae collected in La Araña (Malaga) in two periods of the year (summer and winter) and from two sites, rocky shores (algae with emersion phases during the daily cycle) and rockpools (algae always immersed during the daily cycle). The photosynthetic activity estimated as electron transport rate (ETRmax) at initial time (before the incubation) was higher in rocky shore than in rockpool collected algae but only wintertime. In rockpool-collected algae, ETRmax was higher in summer than that in wintertime. In contrast, the maximal quantum yield as indicator of photoinhibition (Fv/Fm) was lower in summer time respect to the winter time in algae collected from both sites. These responses indicated photoinhibition for C. tamariscifolia in summer time and high vulnerability respect to the winter periods as it was suggested in the seasonal study in the field. The highest production (ETRmax) in rocky shore algae is related to the use of CO2 from the air during emersion periods and higher nitrate incorporation during rehydration after drying periods compared alga growing in rockpools (always immersed). In addition to ETRmax obtained from the fitting of ETR-irradiance function from rapid light curves, ETR was determined during the daily cycle under solar radiation (in situ ETR). In summer time, in situ ETR was higher in algae collected than that winter in both rocky shore and rockpools collected algae. In situ ETR under solar radiation was higher than ETRmax fitted form rapid light curves. The in situ data explained better the physiological state than ETRmax from in situ ETR is an actual production whereas ETRmax is a potential production. Internal nitrogen content and phenolic compounds were higher in algae collected in winter than in summer, however the highest antioxidant activity was reached in summer time. The decrease of phenols in summer time is related to its higher release to the water, a process with photoprotective role, but in addition, it is not discarded the accumulation of other photoprotector compounds as carotenoids. The photoacclimation capacity in the short term to natural solar irradiance was evaluated by transplant approach in summer time in Cabo de Gata-Nijar (ultraoligotrophic waters) by using algae collected form 0.5 and 2.0 m and incubated in surface waters to 1 00% and 70% of surface irradiance that were covered with neutral mesh. In addition to C. tamariscifolia, the red calcareous macroalga, E. elongata, was studied. In this experiment, we tested interactive effects between light, nutrient and two different depths for both algae. The photosynthetic activity (i.e. ETRmax) in C. tamariscifolia collected from both 0.5 and 2.5 depth waters was higher in shade conditions without nutrient enrichment. These differences could be explained by the high penetration of UV radiation (high water transparency provoking photoinhibition in algae of the surface waters). In contrast in E. elongata, the photosynthetic activity (i.e. ETRmax), was also higher in shade conditions but with nutrient enrichment. The vulnerability was lower in algae collected from 2.0 depth in different light conditions, respect to the algae collected from shallow waters and full light conditions. Fv/Fm as expected was higher shaded that in full light conditions but under non-enriched nutrient condition. The elevated NPQmax indicates high photoprotection capacity. Sun type pattern is showed in transplanted algae from 2.0 to 0.5 m such as increase of ETRmax and Ek and decrease of €ETR. The nitrogen internal content, as indicator of the nutritional status in C. tamariscifolia was highest in algae collected from shallow waters with nutrient enrichment. The response to nutrient enrichment was very rapid. The photoprotection in C. tamariscifolia by phenolic compounds was higher in shallow water (0.5 m) under nutrient enrichment conditions independent of the light. In contrast, in depth waters (2.0) the photoprotectors content was higher in non-enrichment conditions independent of the light conditions. This could be explained because of the high irradiance in shallow waters, phenolic compounds can be released preventing the photodamage as a photoprotection strategy. The carotenoids were less influenced by irradiance or nutrients with the exception of violaxanthin that had higher content after nutrient enrichment. In C. tamariscifolia, the content of violaxanthin was higher in the simulated deeper irradiance. However, antheraxanthin and .-carotene were significantly affected by the interaction of factors, irradiance and nutrients. In this study, it is shown that the functional indicators, (1 ) physiological ETRmax, Fv/Fm and NPQmax, (2) nutritional indicator (C:N) and (3) biochemical indicator of stress in C. tamariscifolia collected in the northern geographical distribution (southern England, UK) were sensible to Copper and nitrate variations and they respond as interactive effect. The data show complex interaction effects between copper and nitrate on the ecophysiological variables. C. tamariscifolia presented a high resistance to copper with no apparent damage effects after 1 4 d culture even at 2.0 lM CuT. We found higher internal copper and in the water under exposed to high copper concentration, independent of the nutrient conditions. Phenols were released mainly in high copper levels and with nitrate enrichment conditions and positive correlation with antioxidant, activity was found. Thus, the highest antioxidant activity was produced in the highest stress treatment, i.e., high Cu but also at high N levels. Chla, c and fucoxanthin contents were higher in high coper levels independent of the nitrate conditions. In general, at the end of the experimental period, middle copper levels seems to be more favorable in low nitrate conditions, respect to the physiological status of C. tamariscifolia. In contrast, in high copper levels with high nitrate levels, the physiological status in C. tamariscifolia was also favorable. These results suggest that interaction between copper and nitrate can give a high resistance to copper of C. tamariscifolia with no apparent damage effects after 1 4 d culture. In the natural pH gradient in the Mediterranean Sea (Vulcano Island, Italy), interactive effects between light, CO2 and nutrient levels were studied in C. compressa and in the calcareous alga Padina pavonica. Both C. compressa and P. pavonica were benefited as physiological level by the increases of DIC (dissolved inorganic carbon), but the extent of the algal response, depends upon nutrient and light availability. The photosynthetic activity responses (i.e. ETRmax), in C. compressa and P. pavonica were higher in ambient CO2 with full light and ambient nutrient conditions. Maximal quantum yield (Fv/Fm), in C. compressa was higher in high CO2, shaded with enriched nutrient conditions; in contrast, in P. pavonica was higher in non-enrichment levels. In C. compressa, elevated CO2 treatments resulted in higher carbon content and antioxidant activity in shaded conditions both with and without nutrient enrichment they had more Chla, phenols and fucoxanthin with nutrient enrichment and higher quantum yield (Fv/Fm) and photosynthetic efficiency (€ETR) without nutrient enrichment. In P. pavonica, elevated CO2 treatments had higher carbon content, Fv/Fm, €ETR, and Chla regardless of nutrient levels they had higher concentrations of phenolic compounds in nutrient enriched, fully lit conditions and more antioxidants in shaded, nutrient enriched conditions. The nitrogen internal content, in C. compressa was higher in high CO2 in shaded and enrichment nutrient conditions; in contrast, in P. pavonica was higher in ambient CO2 in shaded with nutrient levels. Nitrogen content increased significantly in fertilized treatments, confirming that these algae were nutrient limited in this oligotrophic part of the Mediterranean. The phenolic compounds increase in C. compressa in high CO2 levels with nutrient enrichment independent of the light conditions. The increased temperature (4oC) provoked a decrease of polyphenols however only in algae grown under ambient CO2 conditions. Thus, the internal level of phenols remained constant with the increasing temperature under acidification conditions allowing maintaining the antioxidant capacity. In C. compressa and P. pavonica, antioxidant activity and EC50 were affected by the interactions between light levels and CO2. EC50 tended to be higher in shaded, high CO2 treatments with and without nutrient addition, suggesting a positive correlation with phenolic compounds and their use as antioxidants to prevent photodamage. Together, NPQmax, phenol production and EC50 indicate that in elevated CO2 conditions some species will have a higher capacity for photoprotection. Our findings strengthen evidence that brown algae can be expected to proliferate as the oceans acidify where physicochemical conditions, such as nutrient levels and light, permit. Temperature and CO2-dependent processes in two populations of the C. tamariscifolia collected from ultraoligotrophic and oligotrophic waters were studied in out-door experimental system to assess the interactive effects of temperature and CO2 expected levels in future scenarios. The photosynthetic activity (ETRmax) was higher in high CO2 levels in algae collected from both sites. After experimental period (28d), the maximal quantum yield (Fv/Fm) was higher in C. tamariscifolia from ultraoligotrophic waters under increased temperature (+4ºC) and ambient CO2 levels. In contrast, for oligotrophic waters, Fv/Fm was higher in high CO2 levels and ambient temperature. Thus, the interactive effect of temperature and CO2 was different in algae collected under different light and nutrient history grown conditions. We do not know if C. tamariscifolia from the two sites corresponds to different ecotypes but at least the results indicate the importance of light and nutrient history of the macroalgae in the responses to climate change factors. The nitrogen and carbon internal content for both sites was higher in high CO2 levels and ambient temperature, this indicate less vulnerability for this treatments and good state for this macroalgae. We found benefits of DIC (dissolved inorganic carbon) increase on growth rate in C. tamariscifolia being the physiological responses more accelerated in ultraoligotrophic than in oligotrophic harvested algae. Temperature increase has negative effect on growth rate only in algae from oligotrophic waters. Although the biomass in both C. tamariscifolia increased in elevated CO2 conditions, biomass of algae collected from ultraoligotrophic waters was higher than algae collected from oligotrophic waters. This suggest C. tamariscifolia had interactive effects between origin of populations and CO2 conditions, probability that algae collected in ultraoligotrophic waters maybe can capitalize carbon of the aquatic system when the nutrient is not limited in extreme. The phenolic compounds were higher in algae from ultraoligotrophic waters in high CO2 waters than that form oligotrophic waters in high CO2 waters in ambient temperature. The higher concentration of phenolic compounds in algae from ultraoligotrophic waters can be related to the photoacclimation to high irradiance levels in coastal waters with high transparency. A positive correlation between phenolic compounds and antioxidant activity expressed as EC50 indicates that phenolic compounds can prevent photodamage. In algae from oligotrophic waters the levels of phenols after 28 d incubation was lower, but carotenoids higher than that from oligotrophic grown algae suggesting more importance of carotenoid for photoprotection in oligotrophic grown algae. As it was suggested, the algae growing under more stress conditions i.e., ultraoligotrophic compared to oligotrophic waters seem to have more amplified physiological responses to the variations of physical chemical variables. In this study, the decrease of maximal quantum yield and electron transport rate , the increase of phenolic compounds and antioxidant activity or the increase of C:N ratio are produced in stress conditions and thus they are validated as stress indicator. In addition, it is possible to evaluate the direction of the physiological response i.e. positive or negative to expected changes under climate change factors or other anthropogenic impacts, as eutrophication (increased nitrate levels in the water column) or pollution by heavy metals. However, on the other hand, the increase of phenolic compounds is also produced under increased photosynthetic activity showing a link between antioxidant and algal production. This not a strange result since a high photosynthetic activity is related to a high oxygen production which can be produced oxidative stress. Non-photochemical quenching, oxygen consumption through Mehler reaction and increased antioxidant activities are down regulation mechanisms to survive under promoted oxygenic scenario. Phenolic accumulation under increased nitrate and CO2 levels or the release of phenols under increased irradiance in C. tamariscifolia shows us that this species has effective biochemical mechanisms to acclimate for the expected variations in climate change factors although this is limited by temperature. Phenolic compounds are related to secondary metabolism but in C. tamariscifolia but the direct positive relation found with photosynthetic activity and internal nitrogen in all experiments seem to link the phenols to primary metabolism. In summary, increased CO2 under high irradiance, but not photoinhibitory, conditions will be favorable for growth and physiological responses. The nitrate enrichment reduced stress provoked by irradiance or pollution by Cooper due to the photoprotection mechanisms are favored by nitrate increase. However, the positive effect of CO2 and nitrate is dependent on temperature, summer temperature in the field or 4oC increased temperature in outdoor experiments provoked physiological stress. Consequently, ocean acidification will be favorable for C. tamariscifolia only under no very high increase of temperature, less 2-3oC and without nutrient limitation. The oligotrophication produced in certain areas of Mediterranean Sea will be unfavorable for C. tamariscifolia communities in a climate change scenario. The data on vulnerability and acclimation to climate change factors of C. tamariscifolia, E. elongata, C. compressa and P. pavonica presented in this study can help the management of macroalgal communities, mainly in protected areas. The data of the physiological and biochemical will help to predict the effects of climate change on bioactive compounds with antioxidant capacity and their potential biotechnological uses as phenolic compounds, mycosporine like aminoacids and carotenoids.
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