Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions

Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions David Lopes Calvão Mestrado em Ecologia, Ambiente e Território Departamento de Biologia 2013/2014 Orientador Dr. Francisco Arenas, CIIMAR FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 2 Todas as correções determinadas pelo júri, e só essas, foram efetuadas. O Presidente do Júri, Porto, ______/______/_________ FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 3 Agradecimentos Quero agradecer ao Laboratório de Biodiversidade Costeira do CIIMAR por aceitar a minha presença nas suas instalações para poder concretizar esta tese de mestrado. Quero também agradecer ao meu orientador, Dr. Francisco Arenas, por todo o tempo dedicado e pelo apoio que me deu, por estar sempre presente quando necessitei dele, sempre com entusiasmo e paciência, e com boas ideias para ajudar a solucionar os problemas que surgissem. Agradeço também a todos os que trabalharam comigo no Laboratório de Biodiversidade Costeira. Para além de serem bons colegas de trabalho, são também amigos, e que tornavam o dia a dia do laboratório numa experiência única. Sem eles, este ano que passou não teria sido tão bom. Quero também agradecer à minha familia, sem eles não seria quem sou hoje. Obrigado à Diana, por me ter acompanhado na minha vida académica, obrigado à Susana pelos vários lanches partilhados ao longo do ano e obrigado a todos os meus amigos que de alguma forma me apoiaram ao longo da minha vida. Obrigado a todos os co-autores do artigo (submetido no Journal of Experimental Marine Biology and Ecology a 24 de Setembro de 2014) pelas sugestões para o melhorar. O suporte financeiro deste projeto foi providenciado pela Fundação para a Ciência e a Tecnologia (FCT), através do projeto CLEF (PTDC/AACAMB/102866/2008) FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 4 Abstract The simultaneous increase of ocean temperature and acidification caused by global climate change will have major consequences in marine ecosystems. However, community scale studies on this interaction are rare. In this study, we used mesocosms to assess the joint effects of elevated temperature and CO2 concentration on a synthetic intertidal rockpool community composed of Ulva lactuca, Chondrus crispus and Gammarus locusta. We examined the effects of both climate change related factors on some ecosystem functioning variables (i.e. biomass, photosynthetic efficiency, productivity, respiration and grazer’s mortality) using two pH levels (normal and low), three temperature levels (low, medium and high) and two grazers levels (present or absent). C. crispus, biomass increased with a higher temperature (20ºC) combined with low pH (7.9) as it did with low pH and grazer’s absence. U. lactuca’s biomass also increased with low pH, and at higher temperatures with no grazers. U. lactuca’s photosynthetic efficiency increased with medium temperatures (17ºC). GPP and NPP of the macroalgal assemblages increased in higher temperatures with both pH treatments. Respiration increased with increasing temperature and low pH. Low pH and high temperatures also led to an increase in mortality of G. locusta. In a future scenario of increasing temperature and CO2 concentration, rockpool communities could change due to a simultaneous decrease of potential herbivores and an increase in the biomass and functional activity of some macroalgal species. Keywords: Climate change, warming, acidification, macroalgal assemblages, primary productivity, seaweed-grazer interaction, Ulva lactuca, Chondrus crispus, Gammarus locusta FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 5 Resumo O aumento em simultâneo da temperatura dos oceanos e da acidificação causada pelas alterações climáticas globais irá ter efeitos nefastos nos ecosistemas marinhos. Estudos à escala da comunidade sobre estas interações são escassos. Neste trabalho, usamos mesocosmos para compreender os efeitos conjuntos do aumento da temperatura e da concentração de CO2 numa comunidade artificial de poças de maré da zona intertidal composta por Ulva lactuca, Chondrus crispus e Gammarus locusta. Examinamos os efeitos conjunto dos factores relacionados com as alterações climáticas em algumas variáveis influentes no funcionamento do ecossistema (biomassa, eficiência fotosintética, produtividade, respiração e mortalidade de herbívoros) usando dois níveis de pH (normal e baixo), três níveis de temperatura (baixa, media e alta) e dois níveis de herbivoria (presente e ausente). Para o C. crispus, uma temperatura elevada (20ºC) combinado com um baixo pH (7.9) levou a um aumento de biomassa, assim como nos tratamentos com pH baixo e ausência de herbívoros. A biomassa da U. lactuca também aumentou em pH baixo e a temperaturas altas. A capacidade fotosintética da U. lactuca aumentou em temperaturas médias (17ºC). O GPP e o NPP das comunidades de macroalgas aumentaram em temperaturas mais altas em ambos os tratamentos de pH. A respiração aumentou em temperaturas elevadas e em pH baixo. pH baixo e temperaturas elevadas também levaram a um aumento da mortalidade de G. locusta. Num futuro cenário de aumento da temperatura e da concentração de CO2 , as comunidades de poças de maré podem-se alterar devido à diminuição de potenciais herbívoros simultaneamente com um aumento de biomassa e atividade funcional de algumas espécies de macroalgas. Palavras-chave: Alterações climáticas, aquecimento, acidificação, comunidades, produtividade primária, interação alga-herbívoro, Ulva lactuca, Chondrus crispus, Gammarus locusta FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 6 Table of contents 1 – Introduction 9 2 – Material and methods 14 2.1 – Algae and animals collection 14 2.2 – Synthetic assemblage procedure 14 2.3 – Experimental setup 15 2.4 – Treatment conditions 17 2.5 – Functional responses 19 2.5.1 – Standing biomass changes 19 2.5.2 – Physiological parameters 19 2.5.3 – Functional measures: respiration and productivity 20 2.6 – Statistical analysis 21 3 – Results 22 3.1 – Biomass changes 22 3.2 – Respiration and productivity 24 3.3 – Physiological parameters 26 3.4 – Grazer’s mortality 27 4 – Discussion and conclusions 28 5 – References 32 FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 7 Lista de quadros e de figuras Figure 1 – Change in average surface temperature (1986-2005 left and 2081-2100 right)(IPCC 2013) 9 Figure 2 - Change in ocean surface pH (1986-2005 left and 2081-2100 right) (IPCC 2013) 11 Figure 3 – Ulva lactuca (left) and Chondrus crispus (right). ( http://www.seaweed.ie/ ) 13 Figure 4 – Mesocosms inside tanks 15 Figure 5 - Schematic diagram of the experimental setup used during the mesocosm experiment 16 Figure 6 – Hourly mean temperature values of the treatments ±SE 18 Figure 7 – Hourly mean pH values of the treatments ±SE 18 Figure 8 - Mean (+SE) change of C. crispus biomass in mesocosms treated with three different temperature levels (L: low, M: medium, H:high) and two pH levels (N:normal, L:low) after 28 days of experiment. Different letters represent significant differences and same letters represent no significant differences based on on SNK tests at p=0.05 level. 22 Figure 9Mean (+SE) change of C. crispus biomass in mesocosms treated with pH (N: normal, L: low) and herbivores (grazers, no grazers) after 28 days of experiment. Different letters represent significant differences and same letters represent no signicant differences based on SNK tests at p=0.05 level. 23 Figure 10 - Mean (+SE) change of U. lactuca biomass in mesocosms treated with two levels of pH (N:normal, L:low) after 28 days of experiment. Different letters represent significant differences and same letters represent no significant differences. 23 Figure 11 - Mean (+SE) change of U. lactuca biomass in mesocosms treated with three temperature levels (L:low, M:medium, H:high) and herbivores (grazers, no grazers) after 28 days of experiment. Different letters represent significant differences and same letters represent no significant differences based on SNK tests at p=0.05 level. 24 Figure 12 - Respiration and Net Primary Production (NPP) (mean±SE, n=48) of communities with three different temperature levels (L: low, M:medium, H:high) and two pH levels (N:normal, L:low) after 28 days of experiment. Means with a common letter do not differ significantly from each other based on SNK tests at p=0.05 level. 25 Figure 13 - Respiration and (mean±SE, n=48) of communities with two levels of grazers (presence and absence) after 28 days of experiment. Means with a common letter do not differ significantly from each other based on SNK tests at p=0.05 level. 25 Figure 14 - (mean±SE, n=48) of communities with three different temperature levels (L: low, M:medium, H:high) and two pH levels (N:normal, L:low) after 28 days of experiment. Means with a common letter do not differ significantly from each other based on SNK tests at p=0.05 level. 26 Figure 15 - Photosynthetic efficiency of U. lactuca (mean±SE, n=48) with three temperature levels (L:low, M:Medium, H:high) after 28 days. Means with a common letter do not differ significantly from each other based on SNK tests at p=0.05 level. 27 Figure 16 - G. locusta survival with two levels of pH (N:normal, L:low) after 28 days of experiment. Different letters represent significant differences and same letters represent no significant differences. 27 FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 8 Table 1 – Treatment values (mean SE, n=8) of temperature (ºC) in mesocosms and pH in head tanks 17 Table 2 - Summary of analysis of variance for Chondrus crispus and Ulva lactuca biomass change. pH (normal and low), temperature (low, medium, high) and grazers (He) (presence and absence) are fixed factors. Significant results in bold. 22 Table 3 - Summary of analysis of variance of Respiration, NPP and GPP of assemblages. pH (normal and low), temperature (low, medium, high) and grazers (He) (presence and absence) are fixed factors. Significant results in bold. NPP results transformed with Ln(x+1) and GPP results transformed with Ln(x) 24 Table 4 - Summary of analysis of variance on the photosynthetic efficiency of U. lactuca and C. crispus. pH (normal and low), temperature (low, medium, high) and grazers (He) (presence and absence) are fixed factors. Significant results in bold. 26 Table 5 - Summary of analysis of variance of mortality of G. locusta. pH (normal and low), Ttemperature (low, medium, high) and grazers (He) (presence and absence) are fixed factors. Significant results in bold. 27 FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 9 1 – Introduction Since the beginning of the industrial revolution and as a result of human activities, the atmospheric carbon dioxide (CO2) concentration rose from a pCO2 of 280 ppm to about 380 ppm in the first years of the century (Feely et al. 2004), nowadays it is close to 400 ppm (http://CO2now.org accessed 21st September 2014). CO2 is not just accumulating more and more but also rates of increase are rising (Canadella et al. 2007) and it is predicted that by the end of the century CO2 could reach up to 936 ppm with an associated drop of mean ocean pH levels (Figure 2) between 0.14 and 0.35 units (IPCC 2013). Despite, representing only a small percentage of the atmospheric gases (approximately 0.04%), CO2 is considered the major responsible of the greenhouse effect on Earth’s climate. Greenhouse effect is the phenomenon that happens when gases absorb the thermal radiation emitted by Earth’s surface and as a result the atmosphere heats up. In fact, average surface temperatures are projected to increase between 1.1ºC to 4.8ºC (Figure 1) in the next decades (IPCC 2013). While trends in temperature are somewhat variable (Lima et al. 2007), the overall warming trend is clear for virtually all parts of the Earth. Since there is a continual exchange of heat between the oceans and the atmosphere, oceans are also warming up. The average temperature of the upper layers of the ocean has increased by 0.6ºC in the past 100 years (IPCC 2013). This increase of temperature is potentially the most important change occurring in the oceans in the last centuries, as it influences physiological and ecological processes at all biological levels, from genes to ecosystems (Kordas et al. 2011). Today, it is widely accepted that human activities are causing these environmental changes (IPCC 2013), and that they have a high ecological impact on natural systems (Halpern et al. 2008). This impact is changing the global biodiversity at unprecedented rates (Dobson 2005). Figure 1 – Change in average surface temperature (1986-2005 left and 2081-2100 right) (IPCC 2013) FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 16 In order to prevent large changes in water temperature due to heat exchanges with the air, the tanks (64 litres) that contained the mesocosms with the assemblages were submerged in a water bath created by 300 litres tanks. Water temperature was regulated using heaters (titanium heaters) and seawater chillers simultaneously (Aqua Medic® Titan 2000) controlled electronically using a microprocessor controller (Aqua Medic® AT Control System) with temperature sensors. We used two head tanks (1000 litres) adjacent to the mesocosms to create the pH treatments. In those tanks sea water pH was regulated using individual pH probes connected to the Aqua Medic AT Control System that controlled solenoid valves connected to a CO2 bottle. In the low pH tank, CO2 was burbled in order to maintain a pH of 7.7. To reduce CO2 flux from the CO2 bottle to the tank and to prevent rapid changes in pH we used a rotameter (Aalborg®) Water from the head tanks flowed to the six experimental tanks where the mesocosm were submerged. Every 12 hours, water from each treatment tank was completely renovated, mimicking the water entering the intertidal pools during the high tide. This renovation took 2 hours. Using water pumps (Oceanrunner OR1200), water was lead into each treatment from its respective water deposit (normal pH deposit and 7.7 pH deposit). Figure 5 - Schematic diagram of the experimental setup used during the mesocosm experiment. In yellow are represented the assemblages with grazers. FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 17 To prevent nutrient limitation, every two days NaNO3 and NaH3PO4 was added until a final concentration of 50 µM of N and 5 µM of P was achieved. 2.4 – Treatment conditions The goal of the experiment was to examine how climate change related environmental drivers, temperature and pH, may shape the interaction between grazers and primary producers. Thus, three treatments were included in the experiments: (1) water temperature, with three levels (low 14.47ºC± 2.01, medium 17.49ºC±1.74 and high 19.97ºC±1.45; (2) water pH, with two levels (normal pH 8.31±0.16 and low pH 7.97±0.25; (3) herbivore (Gammarus locusta) presence or absence (Figure 5). pH and temperature levels were selected based on predictions for the end of the century (IPCC 2013). Each treatment combination was replicated four times. Thus we used a total of 48 mesocosms in the experiment. During the day, temperatures varied according to Figure 5. For the acidification treatments, the pH values varied as seen in Figure 6. Table 1 – Treatment values (mean SE, n=8) of temperature (ºC) in mesocosms and pH in head tanks Temperature ºC pH Treatment Low Mid High Normal Low Mean 15.47 17.49 19.97 8.31 7.97 SE 2.01 1.74 1.45 0.16 0.25 FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 18 13 14 15 16 17 18 19 20 21 22 23 0 3 6 9 12 15 18 21 24 Temperature (Cº) Time (h) Low Mid High Figure 6 – Hourly mean temperature values of the treatments ±SE Figure 7 – Hourly mean pH values of the treatments ±SE 7,6 7,7 7,8 7,9 8 8,1 8,2 8,3 8,4 8,5 8,6 0 3 6 9 12 15 18 21 24 pH Time (h) pH N pH L FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 19 2.5 – Functional responses 2.5.1 – Biomass changes Fresh weight of each algal frond was recorded at the beginning of the experiment, and then again 28 days later at the end of the experiment. Biomass change was calculated using the following formula: FWf / FW0 where FWf is the fresh weight in grams at the end of the experiment and FW0 is the fresh weight in grams at the end of the experiment. At the end of the experiment, all fronds were rinsed with fresh water to remove salt. Using a scalpel, the algae in the boulders were scrapped. Subsequently, the algae were dried at 50ºC for 48 hours, and then the dry weights were determined in order to adjust data to biomass. 2.5.2 – Physiological variables The algae physiological status was assessed through the photosynthetic capacity of their photosystem II, also known as Maximal Quantum Yield of Photosynthesis. The principle behind this assessment is that light that is absorbed by chlorophyll molecules can meet one of three fates: being used in photosynthesis (photochemistry), being dissipated as thermal energy or being re-emitted as light (chlorophyll fluorescence). These processes compete with each other, so when there is an increase in the efficiency of one of them, the yield of the other two will have a decrease. Using this competition between the processes, information about changes in the efficiency of photochemistry and heat dissipation can be obtained by measuring the yield of chlorophyll fluorescence. By exposing a frond of a darkness adapted algae to a defined wavelength light, and measuring the amount of light re-emitted at longer wavelengths, the fluorescence yield can be assessed (Maxwell & Johnson 2000). Each individual’s photosynthetic capacity was measured using a mini-PAM (pulse amplitude modulated) (Walz) chlorophyll fluorometer. Each frond was placed in a black box (in order to prevent any external light input) and submerged in its treatment’s respective water. There was a waiting time in the dark of 30 minutes so that the algae FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 20 adapted to darkness. After that, the chlorophyll fluorometer measured the fluorescence in the dark (F0) and the maximum fluorescence (Fm). Using these values, the Maximal Quantum Yield of Photosynthesis was calculated, according to the next equation: Fv/Fm = (Fm – F0)/Fm where Fv is the variable fluorescence (or the difference between Fm and F0). The Fv/Fm determines the maximum efficiency of the photochemical energy conversion in a darkness adapted plant (Maxwell 2000). 2.5.3 – Respiration & Primary productivity Assemblages’ functional measures, i.e. respiration, net, net primary productivity (NPP) and gross primary productivity (GPP) were estimated through the determination of the dissolved O2 fluxes in incubations at seven different light intensities. We used the 2.5 dm3 acrylic cylinders to incubate our assemblages in an incubator provided with fluorescent lamps (30W Osram Biolux®). Each assemblage was exposed to seven consecutive light intensities (0, 24, 164, 262, 345, 417 and 1578 μmol m-2 s-1). pH and temperature treatment conditions were controlled as in the experimental setup using by the AT Control system and coolers. Changes in the O2 concentrations were recorded every 30 seconds by luminescent oxygen probes connected to a data logger (HQ40D Hach Lang) and placed in each incubation chamber. Each light cycle lasted for 20 minutes, after which the next sets of lights were turned on. Respiration and productivity were estimated for each irradiance level through the measurement of oxygen fluxes by regressing oxygen concentration in the chamber (measured in μmol) through time (s–1). Fluxes were corrected by seawater volume inside the cylinder and assemblages biomass. To prevent the influence on the productivity and respiration of microalgae in the treatments, all the seawater used was filtered (5µm). Three ecosystem functioning surrogates were determined per assemblage: (1) maximum net primary productivity (max NPP), the maximum productivity (i.e. maximum slope for the oxygen concentration versus time relationship) recorded at any light intensity (μmol O2 s–1); (2) assemblage respiration, the oxygen consumption during the dark period of the incubation (μmol O2 s–1); and (3) maximum gross primary productivity as the sum of the two previous variables, i.e. maximum NPP+|Respiration|. FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 21 2.6 – Statistical analysis Changes in biomass, primary productivity and physiological parameters were assessed using a 3-factor orthogonal analysis of variance (ANOVA). The factors were temperature (3 levels: low, medium, high), pH (2 levels: natural and low) and grazers (2 levels: presence and absence). All factors were fixed. A posteriori multiple comparisons were done using Student-Newman-Keul’s (SNK) tests (p > 0.05). The homogeneity of variances was evaluated by using Cochran’s test and data was transformed using ln(x+1) when necessary. Univariate statistical analyses were performed with WinGMAV 5.0 (EICC, The University of Sidney) FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 22 3 – Results 3.1 – Standing biomass changes The analysis of variance for Chondrus crispus and Ulva lactuca biomass changes is presented in Table 2. Biomass changes of C. crispus and U. lactuca over time (28 days) are shown in the next Figures. C. crispus biomass increased in all treatments, and this increase was influenced by temperature, pH (Figure 7) and the presence of grazers (Figure 8). C. crispus biomass increased significantly with temperature (table 2), but this change was not consistent with pH treatments (significant pH x Te interaction; p < 0.01, table 2). The increase in C. crispus biomass with high temperature was significant only when low pH was also applied (7). Corallina officinalis data was not used due a bleaching effect that occurred in all treatments. Table 2 - Summary of analysis of variance for Chondrus crispus and Ulva lactuca biomass change. pH (normal and low), temperature (low, medium, high) and grazers (He) (presence and absence) are fixed factors. Significant results in bold. Figure 8 - Mean (+SE) change of C. crispus biomass in mesocosms treated with three different temperature levels (L: low, M:medium, H:high) and two pH levels (N:normal, L:low) after 28 days of experiment. Different letters represent significant differences and same letters represent no significant differences based on on SNK tests at p=0.05 level. C. Crispus U. Lactuca DF F p DF F p pH 1 1.37 0.2492 1 4.31 0.0452 Temperature (Te) 2 11.45 0.0001 2 1.19 0.3157 Herbivore (He) 1 0.3 0.5897 1 2.33 0.1353 pHXTe 2 6.75 0.0032 2 1.03 0.3680 pHXHe 1 5.35 0.0265 1 2.18 0.1487 TeXHe 2 1.11 0.3404 2 5.29 0.0097 pHXTeXHe 2 1.56 0.2247 2 1.76 0.1858 a a a a ab c 0,0 0,5 1,0 1,5 2,0 2,5 L M H Biomass change Temperature C. crispus pH N pH L FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 23 C. crispus biomass was significantly affected by the presence of grazers, but this pattern was not consistent over pH treatments (significant pH x He interaction; p < 0.05, Table 2). The change in biomass was significantly higher in those mesocosms with no grazers and low pH (Figure 8) Figure 9Mean (+SE) change of C. crispus biomass in mesocosms treated with pH (N: normal, L: low) and herbivores (grazers, no grazers) after 28 days of experiment. Different letters represent significant differences and same letters represent no signicant differences based on SNK tests at p=0.05 level. U. lactuca biomass increased significantly with low pH conditions (Table 2, Figure 9). U. lactuca biomass showed a significant change with temperature variation and the presence of grazers (i.e., significant Te x He interaction; p > 0.01, Table 2). U. lactuca biomass was significantly higher with high temperatures and no grazers (Figure 10). No change in biomass was found in those mesocosms with grazers (Figure 10). Figure 10 - Mean (+SE) change of U. lactuca biomass in mesocosms treated with two levels of pH (N:normal, L:low) after 28 days of experiment. Different letters represent significant differences and same letters represent no significant differences. ab a ab b 1,3 1,35 1,4 1,45 1,5 1,55 1,6 1,65 1,7 1,75 1,8 pH N pH L Biomass change C. crispus Grazers No Grazers a b 0 0,5 1 1,5 2 2,5 3 3,5 4 pH N pH L Biomass change U. lactuca FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 24 Figure 11 - Mean (+SE) change of U. lactuca biomass in mesocosms treated with three temperature levels (L:low, M:medium, H:high) and herbivores (grazers, no grazers) after 28 days of experiment. Different letters represent significant differences and same letters represent no significant differences based on SNK tests at p=0.05 level. 3.2 – Functional measures: productivity and respiration The analyses of variance for the functional measurements indicated significant and interactive effects of pH and temperature in the productivity and respiration of the assemblages (Table 3). Respiration in the macroalgal assemblages increased with low pH and high temperature treatments (Figure 11) In the normal pH treatments, medium temperature led to an increase in NPP, and in the low pH there were no significant differences between treatments (Figure 11). The presence of grazers had a significant effect on the respiration of the macroalgal assemblage (Table 3). Table 3 - Summary of analysis of variance for Respiration, NPP and GPP of macroalgal assemblages. pH (normal and low), temperature (low, medium, high) and grazers (He) (presence and absence) are fixed factors. Significant results in bold. NPP results transformed with Ln(x+1) and GPP results transformed with Ln(x) Respiration NPP GPP DF F p DF F p DF F p pH 1 0.62 0.4378 1 0 0.9574 1 0.04 0.8367 Temperature(Te) 2 14.4 0 2 0.73 0.4898 2 3.14 0.0556 Herbivore (He) 1 5.3 0.0272 1 0.7 0.4083 1 1.93 0.1736 pHXTe 2 7.55 0.0018 2 5.31 0.0096 2 8.05 0.0013 pHXHe 1 0.2 0.6569 1 0.73 0.3996 1 0.51 0.4777 TeXHe 2 1.17 0.3218 2 0.9 0.4151 2 1.08 0.3513 pHXTeXHe 2 0.64 0.5351 2 0.02 0.9797 2 0.13 0.8797 a ab b a a a 0 1 2 3 4 5 L M H Biomass change Temperature U. lactuca Grazers No Grazers FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 25 a b a a a a 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 L M H NPP (g O2DWg-1 hr-1) Temperature pH N pH L Figure 12 - Net primary production (NPP) and respiration and (mean±SE, n=48) of assemblages with three different temperature levels (L: low, M: medium, H:high) and two pH levels (N:normal, L:low) after 28 days of experiment. Means with a common letter do not differ significantly from each other based on SNK tests at p=0.05 level. Figure 13 - Respiration (mean±SE, n=48) of macroalgal assemblages with two levels of grazers (presence and absence) after 28 days of experiment. Means with a common letter do not differ significantly from each other based on SNK tests at p=0.05 level. Regarding gross primary production (GPP), there was a significant increase in GPP in normal pH and medium temperature conditions (Figure 14). Similarly, a significant increase in GPP was found in low pH and high temperature conditions (Figure 13) a b b b ab c -1,8 -1,6 -1,4 -1,2 -1,0 -0,8 -0,6 -0,4 -0,2 0,0 L M H Respiration (g O2DWg-1 hr-1) pH N pH L a b -1,2 -1,0 -0,8 -0,6 -0,4 -0,2 0,0 Grazers No Grazers Respiration (g O2 DWg-1 hr-1) FCUP Acidification and warming effects on a rockpool community: an experimental approach to understand stressor interactions 32 5 – References Alsterberg, C. et al., 2013. 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