Effects of ocean acidification in Mediterranean coral

Anexo 1 D. José Manuel Vergara Martín, SECRETARIO DEL DEPARTAMENTO DE BIOLOGÍA DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA, CERTIFICA, Que el Consejo de Doctores del Departamento en sesión permanente tomó el acuerdo de dar el consentimiento para su tramitación, a la tesis doctoral titulada "Effects of Ocean Acidification in Mediterranean Corals" presentada por el doctorando D. Juan Ignacio Movilla Martin y dirigida por los Doctores Caries Pelejero Bou y Eva Calvo Costa. Y para que así conste, y a efectos de lo previsto en el Artº 6 del Reglamento para la elaboración, defensa, tribunal y evaluación de tesis doctorales de la Universidad de Las Palmas de Gran Canaria, firmo la presente en Las Palmas de Gran Canaria, a 30 de Octubre de 2015. ªD DE C\S PALMAS DE GRAN CANARIA Departamento de Biología iCM Effects of ocean acidification in Mediterranean coral (Efectos de la acidificación en corales Mediterráneos) Juancho Movilla Tesis Doctoral presentada por D Juancho Movilla Martin para obtener el grado de Doctor por la Universidad de las Palmas de Gran Canaria, Departamento de Biología, Programa en Oceanografía (Bienio 2007-2009) Directores: Dr. Caries Pelejero y Dra. Eva Calvo Universidad de Las Palmas de Gran Canaria Institut de Ciencies del Mar (ICM-CSIC) En Barcelona, a 30 de Octubre de 2015 El Doctorando Juancho Movilla El Director Caries Pelejero El Codirector Eva Calvo A mi familia y amigos, A Clara, por ser mi arrecife ! 5 CONTENTS Resumen/ Summary 7 General introduction 11 Aims and outline of the thesis 32 Studied species 35 Chapter 1 Calcification reduction and recovery in native and non-native Mediterranean corals in response to ocean acidification 43 Chapter 2 Detrimental effects of ocean acidification on the economically important Mediterranean red coral (Corallium rubrum) 67 Chapter 3 Long-term response of two Mediterranean azooxanthellate temperate corals to low-pH and high-temperature conditions 93 Chapter 4 Response of Mediterranean cold-water corals to ocean acidification 4.1 Resistance of two Mediterranean cold-water coral species to low-pH conditions 117 4.2 Differential response of two Mediterranean cold-water coral species to ocean acidification 127 Synthesis of results and general discussion 151 Thesis summary (Spanish version) 173 References (Introduction, Discussion and Summary) 227 Acknowledgments 245 7 Resumen RESUMEN Desde el comienzo de la era industrial, los océanos han absorbido casi un tercio del dióxido de carbono (CO2) liberado a la atmósfera por las actividades antropogénicas, mitigando su efecto invernadero y su influencia en el clima de la Tierra. Sin embargo, una consecuencia de esta absorción es la acidificación del océano (OA), un proceso que ha surgido como potencialmente alarmante para los ecosistemas marinos y se ha convertido en una prioridad el la investigación durante los últimos años. El Mediterráneo es actualmente uno de los mares más afectados del mundo, enfrentándose a muchas presiones antropogénicas. Sin embargo, sigue siendo objeto de debate si el impacto de la OA en este mar semi-cerrado será más fuerte que en el océano global. En esta tesis, contribuimos al conocimiento aún limitado de los efectos de medio a largo plazo de la OA en nueve especies bentónicas clave de los dos ecosistemas calcáreos del Mediterráneo más importantes en términos de diversidad, el coralígeno somero y las comunidades de aguas profundas. Para ello, desarrollamos un sistema para la manipulación experimental del pH en acuarios que nos ha permitido exponer los organismos a diferentes condiciones de pH y temperatura, simulando así los valores actuales y los previstos para el año 2100. Se evaluó la respuesta en la tasa de calcificación, en el estado de la estructura carbonatada de soporte (microestructura, microdensidad y porosidad específica del esqueleto) y en el balance metabólico (contenido en materia orgánica, hidratos de carbono, lípidos y proteínas del tejido) en cada una de las especies. En términos de calcificación, la especie más afectada fue el octocoral Corallium rubrum, exhibiendo una disminución promedio en torno al 59%, seguido de los corales zooxantelados Cladocora caespitosa y Oculina patagonica, con una disminución del 35 y 32%, respectivamente. Curiosamente, los corales azooxantelados mostraron una mayor resistencia a las condiciones acidificadas esperadas para finales de siglo, tanto en el caso de las especies temperadas Astroides calycularis y Leptopsammia pruvoti, como en las especies de aguas frías Lophelia pertusa, Madrepora oculata y Dendrophyllia cornigera. La única excepción fue Desmophyllum dianthus que presentó una reducción significativa del 70% en la calcificación de los especímenes más jóvenes tras la exposición a condiciones acidificadas. Nuestros resultados también muestran un cierto grado de variabilidad especie-específico en la respuesta metabólica, sugiriendo que diferentes estrategias y habilidades para la translocación del balance energético podrían explicar parte de la variabilidad observada en la respuesta de estos organismos a las condiciones de pH bajo. Esta tesis ilustra los efectos variables que la acidificación y el calentamiento global pueden tener en organismos calcificadores marinos pertenecientes a ecosistemas bentónicos Mediterráneos y pone de manifiesto los posibles cambios en la composición de estas comunidades en un futuro próximo. 14 Ocean acidification in the Mediterranean Sea the study of the geological record and the measurement of long-time series to detect natural trends, experimentation in the laboratory and in situ to determine potential effects on certain organisms and ecosystems, as well as numerical model simulations to estimate longterm impacts. Since year 2000, the number of papers devoted to OA has increased by 35% every year (Riebesell and Gattuso 2015), and this topic has been the focus of a number of conferences, meetings and workshops, including the Symposium on the Ocean in a High-CO2 World (periodically held since 2004 and with an attendance of 540 experts from 37 countries in its third edition in Monterey, US, in 2012). A number of special volumes in scientific journals have also been devoted to OA (e.g. Marine Ecology Progress Series vol. 373, 2008; Oceanography vol. 22, 2009; vol. 27, 2014; vol. 28, 2015; ICES Journal of Marine Science, in press). In addition, several funding agencies have launched specific programs on OA research, including the National Science Foundation (US), the Federal Ministry of Education and Research (Germany) and the Natural Environment Research Council (UK). However, despite the impressive development of this field in the last few years, only the changes on the seawater chemistry are known with a high level of confidence, whereas the potential biological and biogeochemical responses, as well as the socio-economic impacts, are only now starting to be understood for some groups of species and live stages. Lessons from the past The study of the geological record can provide clues about what the future will hold as a result of changes in ocean chemistry and their potential effects on marine life. However, estimating atmospheric and/or seawater chemistry during past climates is difficult due to the very few methods available for obtaining records of these components. In recent years, a growing attention has been granted to certain corals as potential palaeoceanographic archives, capable of encoding the signature of seawater signals including pH and carbonate ion concentrations Methods of Analysis For a proper characterization of the inorganic carbon system in seawater and, the simultaneous measurement of at least two of all parameters of the CO2 system in seawater is required, together with temperature and salinity. The partial pressure of CO2 (pCO2) can be determined either using a gas chromatograph or an infrared analyzer. The pH is usually measured using glass electrodes or by spectrophotometry with an indicator dye (m-cresol purple). CT is often measured by a colorimetric method or through titration in a closed cell. Finally, a potentiometric titration is used to determine AT. The latter two are conservative parameters, meaning that their concentrations are unaffected by changes in pressure or temperature. Therefore they are the preferred parameters used in numerical models of the ocean’s carbon cycle. 15 Introduction in their carbonate skeleton, with records covering from centuries to millennia in some long-lived colonial cold water coral (CWC) species and ages from recent to up to millions of years in fossil individuals (e.g. Pelejero et al. 2005; Anagnostou et al. 2011, 2012; Trotter et al. 2011; McCulloch et al. 2012b; Robinson et al. 2014) Regarding atmospheric CO2, the most reliable estimates come from analysis of air trapped in ice cores. Using this approach, we know that over the past 800.000 years, the atmospheric CO2 concentration oscillated between 170-280 ppmv (parts per million by volume; Lüthi et al. 2008 and references therein). On the other hand, based on stable boron isotopes in foraminifera, Hönisch et al. (2009) confirmed that through natural glacialinterglacial climate cycles, the average surface water pH oscillated between 8.3 and 8.1 units during cold and warm periods, respectively (Fig. 1). Finally, Pearson and Palmer (2000) reconstructed surface ocean pH for most of the Cenozoic (last 65 million years) at very low temporal resolution, indicating that atmospheric CO2 concentrations have remained below 500 ppmv since the early Miocene (24 million years ago). The geological record also shows that there have been several CO2 perturbation events throughout Earth’s history, even resulting in extinction of benthic marine calcifiers (Zachos et al. 2005; Pelejero et al. 2010; Hönisch et al. 2012). However, an identical analogue to the current human-driven decline in oceanic pH and Ω from the geological record does probably not exist (Haywood et al. 2011). The Paleocene Eocene Thermal Maximum (PETM) could provide perhaps the best analogue to current OA. Having occurred 55 million years ago, the PETM involved the release of a quantity of CO2 comparable to that expected from anthropogenic sources in the next centuries, resulting in the extinction of several benthic organisms, attributed to deep-sea acidification and differences in oxygenation (Zachos et al. 2005; Kump 2011; Pälike et al. 2014 and references therein). The CO2 injection associated to the Figure 1. Glacial–interglacial variability in surface water pH, reconstructed from boron isotopes in foraminifera (filled blue symbols; Hönisch et al. 2009), superimposed on the record of atmospheric CO2 concentration during the last 800,000 years inferred from the composition of air bubbles trapped in Antarctic ice cores (magenta curve; Lüthi et al. 2008). From Pelejero et al. (2010). 16 Ocean acidification in the Mediterranean Sea PETM seems to have occurred over about 10 thousand years (Panchuk et al. 2008) and ocean carbon models and sedimentary records show that chemical recovery from CO2 emissions took up to a hundred thousand years (Zeebe 2013). Differently, current OA is taking place only in a few hundred years (Kump 2011), which limits its analogy to the PETM event and suggests that present-day changes may be more severe than perturbations observed in the geological record (Hoegh-Guldberg et al. 2007; Pelejero et al. 2010; Hönisch et al. 2012). Present trends in global ocean surface pH Over the industrial era, human activities have released large quantities of CO2 to the atmosphere. In 2014, the atmospheric CO2 level in Mauna Loa, Hawaii, reached an annual mean of 398.55 ppmv (397.15 ppmv globally) and exceeded 400 ppmv during a few months (Dr. Pieter Tans, NOAA/ESRL; www.esrl.noaa.gov/gmd/ccgg/trends/), which implies a rise of ~43% of the pre-industrial concentration (280 ppmv). This rapid increase in atmospheric CO2 has risen the global average temperature of the earth’s surface by 0.6°C and is already causing measurable changes in ocean carbonate chemistry (IPCC 2013). For nearly three decades, surface-ocean carbonate system variables have been measured in three subtropical ocean time-series stations, the central North Pacific station ALOHA of the Hawaii Ocean Time-Series (HOT) program (22.75°N, 158°W), the Bermuda Atlantic TimeSeries Station at the western North Atlantic (BATS; 31.72°N, 64.17°W) and the European Time Series in the Canary Islands at the eastern North Atlantic (ESTOC; 24.04°N, 15.50°W). While there are slight regional differences, the expected pHT declines (~0.0018 ± 0.0003 units yr–1; slope ± SE) based on the observed trend in atmospheric and oceanic pCO2 are not significantly different between the three subtropical ocean stations (Bates 2007; Dore et al. 2009; Gonzlez-Dvila et al. 2010), and this decline is similar to other long-time series recorded at higher latitudes (Bates et al. 2014; Fig. 2). These ocean time-series contribute to understanding the response of the ocean carbon Figure 2. Time series of surface seawater anomalies of pH (colored symbols) and observed pH (gray symbols) at the different ocean time-series stations located at different latitudes across the globe, with trends in top right-hand corner of each panel. From Bates et al. (2014). 17 Introduction cycle to natural variability and anthropogenic perturbation, and help to project scenarios about future CO2 emissions. On a global scale, the present ocean remains mildly alkaline, with a pH of surface waters typically in the range of 8.1 (Bates et al. 2014). Ocean circulation models have estimated that the uptake of anthropogenic CO2 has already resulted in a decrease of marine pH by 0.1 units compared to pre-industrial times (Feely et al. 2004; Sabine et al. 2004; Orr et al. 2005). Nevertheless, seawater carbonate chemistry varies naturally across temporal and spatial scales, driven largely by physical and biological processes. Some areas are influenced by upwelling of cold deep waters (e.g. Feely et al. 2008), which are rich in DIC and present low pH, such as the Eastern Equatorial Pacific, the Arabian Sea or the African west coast (Fig. 3a). On the contrary, there are other areas where the fixation of dissolved inorganic carbon by phytoplankton raises surface seawater pH. In the water column, the subsequent downward flux of this carbon fixed in the euphotic Figure 3. Horizontal and vertical variability of seawater pH. (a) Map of mixed surface layer (upper 50 m) of in situ pHT values in the oceans. (b) Depth profiles of in situ pHT for the Pacific, Atlantic and Indian Oceans, with color gradient indicating latitude. From Pelejero et al. (2010). 18 Ocean acidification in the Mediterranean Sea zone, the so-called ‘biological pump’, and its remineralization in deeper layers, creates a lowering pH trend with depth (Fig. 3b). On a local scale, some locations are characterized by extreme pH conditions, in the vicinity of volcanic CO2 vents (e.g. Hall-Spencer et al. 2008; Fabricius et al. 2011), under the influence of freshwater inputs in shelf areas (Blackford and Gilbert 2007) and estuaries (Salisbury et al. 2008) and due to seasonal stratification (e.g. Andersson et al. 2011) or poor flushing efficiency of reef waters (e.g. Pelejero et al. 2005). Increasingly, these naturally high CO2 sites are being used for experimental studies, which are taken as potential analogues to future conditions in a high CO2 world (e.g. Manzello et al. 2008; Hettinger et al. 2013; Nagelkerken et al. 2015). In these dynamic environments, it is expected that the lowering pH trend associated to anthropogenic OA will be superimposed to the natural oscillations of pH (Friedrich et al. 2012). Future projections Numerical modeling is essential to generate projections about future changes in ocean chemistry, an information that is needed to design the experimental studies, for example on the effects of OA on marine organisms and ecosystems. It is important to establish the levels of human interference with the climate system. To this end, the Fifth Assessment Report (AR5) of the Intergovernmental Panel on Climate Change (IPCC 2013) selected a set of four greenhouse gas concentration trajectories or Representative Concentration Pathways (RCPs). These RCPs correspond to radiative forcing levels of 2.6, 4.5, 6.0, and 8.5 W m-2 in the year 2100 and are equivalent to atmospheric pCO2 concentrations of 450, 650, 850, and 1370 ppmv, respectively (Fig. 4). This form the basis of a new set of climate change projections and replace the earlier emission scenarios used on previous IPCC reports. Under these RCP scenarios, global surface seawater pHT at the end of the century is projected to be 8.1 units for RCP2.6, 8.0 units for RCP4.5, 7.9 units for RCP6.0 and 7.7 units for the ‘business-as-usual’ CO2 emissions scenario RCP8.5. These scenarios of greenhouse gas emissions also consider the increase in the global average temperature, predicted to increase by another 1–6°C by the end of the present century. Changes in temperature and pH, such as those projected under RCP8.5 for Figure 4. Historical and projected total anthropogenic radiative forcing (RF) between 1950 and 2100 relative to preindustrial. Previous IPCC assessments (IS92 and SRES) are compared with representative concentration pathway (RCP) scenarios. From AR5, IPCC (2013). 19 Introduction the year 2100, could have synergistic negative effects on growth, reproduction, settlement, development and survival of several marine organisms (Riebesell and Gattuso 2015). While OA is a global-scale phenomenon, there are areas especially sensitive to a decrease in seawater pH. Predictions suggest that polar ecosystems and tropical reefs will be the first to suffer the effects of OA (Fig. 5). By the time atmospheric CO2 exceeds 490 ppmv (in 2040 according to the RCP8.5 case), more than half of the Arctic Ocean will be undersaturated with respect to aragonite (Steinacher et al. 2009), while the Southern Ocean will become undersaturated on average when atmospheric CO2 exceeds 580 ppmv (Orr et al. 2005). Organisms living at depth in areas with fast CO2 uptake such as the North East Atlantic will on average be exposed to undersaturated waters by 2080 (Guinotte et al. 2006). In the case of tropical areas, they are not expected to reach undersaturation conditions with respect to aragonite by the year 2100 (Feely et al. 2004; Orr et al. 2005). In absolute terms, however, the decrease in Ω in the tropics will be in general more important than at higher latitudes (Feely et al. 2009) and the combination with other Figure 5. Projected regional changes in ocean chemistry likely to be experienced by particularly vulnerable ecosystems and compared to global-scale surface ocean changes. From Turley et al. (2010). 20 Ocean acidification in the Mediterranean Sea stress factors such as increased temperature threaten to destroy warm-water coral reefs before the end of this century (Kleypas et al. 1999; Hoegh-Guldberg et al. 2007). The Mediterranean Sea: an acidified basin? The Mediterranean Sea is a mid-latitude semienclosed sea composed of two nearly equal size basins connected by the Strait of Sicily. In the western basin, surface temperature ranges from 12ºC in winter to 26ºC in summer, while a smaller temperature oscillation dominates the eastern side, with surface values ranging from 16 to 27ºC in winter and summer, respectively. A high stratification period on the upper layers occurs during the summer season in the whole Mediterranean area, with a thermocline depth increasing from the western to the eastern basin. Deep-water temperatures (from 250 m to maximum depths) are very homogeneous, with a mean value of ~ 12.8°C over the whole year (Durrieu de Madron et al. 2011). There are reasons to believe that the Mediterranean is already one of the most impacted seas in the world, being subject to many intense anthropogenic pressures (Calvo et al. 2011). Climate change is predicted to become progressively one of the dominant pressures (Ballesteros 2006; Coll et al. 2010; Marbà et al. 2015), but potential synergies with other additional stressors like overfishing, habitat destruction, settlement of alien species and pollution are still largely unknown. Temperature time-series at L’Estartit Station (western Mediterranean Sea) reveal a consistent warming pattern over the last 40 years, with impact on both, the uppermost waters (~1.1ºC increase between 0 to 50 m) and deeper layers below the seasonal thermocline (~0.7°C increase at 80 m). This change in temperature in the last decades is consistent with other data registered in the northwestern Mediterranean Sea (Vargas-Yez et al. 2010) and more pronounced than the 0.6ºC temperature increase experienced by the global ocean (IPCC 2013). The other remarkable anthropogenic pressure related with CO2 emissions is OA. In the Mediterranean Sea, certain characteristics make it especially sensitive to increasing atmospheric CO2. Its high total alkalinity levels (Schneider et al. 2007) increase the capacity to absorb large amounts of anthropogenic CO2 (Goyet et al. 2009) and the shorter residence time of the deep waters (Bethoux et al. 2005) favors a deeper penetration of this anthropogenic tracer compared with the open ocean. The issue lies not so much in the reduction of Ω, since Mediterranean waters will not reach undersaturation levels in the near future, but rather in the potential impact of OA on the calcification of marine organisms, the speciation of nutrients (higher limitation of phosphorus), the modification of its trophic status (higher oligotrophy) and the disruption of food web structure (CIESM 2008). The dynamics of the Mediterranean Sea regarding air-sea fluxes and the penetration of anthropogenic CO2 is complex. On one hand, basin-scale estimates of air–sea CO2 21 Introduction fluxes point that the Mediterranean Sea, as a whole, acts as a small-to-medium sink for atmospheric CO2, although with opposite behaviors in the two basins (D’Ortenzio et al. 2008). According to this study, while the western side acts as a sink of carbon (8.6 Gt C y-1), the eastern basin acts more like a source (8.4 Gt C y-1), being 0.2 Gt C y-1 the overall absorption rate. Regarding anthropogenic CO2, since it is difficult to differentiate it from the natural background of this greenhouse gas, its penetration into the Mediterranean Sea waters needs to be estimated indirectly from observable physical and biogeochemical quantities (Touratier and Goyet 2011; Palmiéri et al. 2015). A first estimation of the magnitude of this penetration suggested a pH decrease from ~0.05 units in the eastern basin to ~0.14 units in the western Mediterranean Sea since preindustrial times, and quite homogeneous with depth, affecting the whole water volume at least in the western basin (Touratier and Goyet 2011). When compared to the typical decrease in pH of ~ 0.1 for the global surface waters (Orr et al. 2005), the Mediterranean Sea appears to be thus more severely impacted by this pressure. According to a recent study, however, despite that the uptake of CO2 is larger in the Mediterranean due to its higher AT, the average surface pH decline seems to range from 0.08 to 0.10 units, quite similar to the global ocean (Palmiéri et al. 2015). Nevertheless, these are still preliminary results and further studies are necessary to reliably project future changes in the Mediterranean basin. Effects of ocean acidification on marine ecosystems A wide range of organismal functions can be affected by OA, including calcification, photosynthesis, growth, reproduction and survival (Howes et al. 2015). There is, therefore, growing concern about how future low-pH scenarios may impact marine organisms at different levels, from molecular and biochemical processes to abundance and distribution of species. Due to the direct effect of a decreased Ω, the calcification process is one of the most adversely affected, and thereby, organisms producing calcium carbonate shells and skeletons are thought to experience the strongest negative impacts from OA (Kroeker et al. 2010; Gattuso et al. 2015). However, there is considerable variation among the responses of different taxonomic groups. Most of the studies show a consistent decline in the calcification rates as a result of increasing CO2, but a few recent reports with a range of different calcifiers also show no response or an increase in calcification in a number of species (Ries et al. 2009; Hendriks et al. 2010; Fig. 6). Scleractinian reef-building corals and crustose coralline algae, with aragonitic and rich-magnesium skeletons, respectively, are amongst the organisms that exhibit the largest reduction in calcification rates as response to a lowering in pH (e.g. Kleypas et al. 1999, 2006; Hoegh-Guldberg et al. 2007; Kuffner et al. 2008; Martin and Gattuso 2009; Robbins et al. 2009; Comeau et al. 2014). Bivalve and gastropod mollusks with calcite structures 22 Ocean acidification in the Mediterranean Sea Figure 6. Calcification response patterns for 18 species of calcifying organisms subjected to CO2-induced reductions in ΩA. From Ries et al. 2009. 23 Introduction also show reduced calcification rates under high-CO2 conditions (Shirayama and Thornton 2005; Gazeau et al. 2010, 2011, 2013; Hendriks et al. 2010). Echinoderms are highly variable in their response (e.g. Wood et al. 2008) although early life stages show sensitivity to acidified conditions (Dupont et al. 2009; Vihtakari et al. 2013). Conversely, some crustaceans have been shown to exhibit a significant increase in growth rate under low-pH conditions, probably due to the presence of chitin coating their calcite exoskeleton (e.g. Ries et al. 2009; Kroeker et al. 2010; Dissanayake 2014). Evidence currently available suggests that OA will cause a decline in CaCO3 production in most planktonic calcifiers, both with calcite shells such as foraminifera (Bijma et al. 1999, 2002; Lombard et al. 2010) and aragonite shells such as pteropods (Feely et al. 2004; Orr et al. 2005; Comeau et al. 2009). Regarding coccolithophores, secreting shells made of calcite, a wide range of responses was observed (Meyer and Riebesell 2015). Calcification rate in these organisms was decreased (e.g. Riebesell et al. 2000; Delille et al. 2005; Engel et al. 2005; Zondervan 2007), unaffected (Langer et al. 2006, 2009) or stimulated (Iglesias-Rodriguez et al. 2008; Shi et al. 2009) under low pH conditions. This variable response to OA highlights that the solubility of the precipitated mineral is not the only driving factor, and in some groups, the biological control is also important in the calcification process. Predicting the impacts of OA on marine calcifiers as a whole is difficult since major groups carry out calcification via different routes and mechanisms. For coccolithophorids, for example, calcification occurs in intracellular vesicles (Brownlee and Taylor 2004), while in the case of foraminifera and corals this process is performed in enclosed extracellular compartments (Al-Horani et al. 2003; Cohen and McConnaughey 2003; Erez 2011). In both cases, precipitation of CaCO3 is thermodynamically favorable under high pH and Ω, achieved through energy-consuming ion transport processes (Mackinder et al. 2010; Allemand et al. 2011; McCulloch et al. 2012a). However, calcification mechanisms are poorly understood and they cannot fully explain the species-specific differences observed in CO2perturbation experiments. Wood et al. (2008) proposed that some organisms might be able to up-regulate their metabolism and calcification to compensate for the decrease in pH of seawater. It is therefore expected that, as the ocean acidifies and the availability of carbonate is reduced, the energetic cost for calcification will be increased (Cohen and Holcomb 2009). This extra energetic cost would divert energy from other essential processes as growth, reproduction or to counteract other environmental stresses (Hoegh-Guldberg et al. 2007; Brewer and Peltzer 2009). Only in some cases, full or partial compensation may be possible if the additional energy required to calcify under elevated CO2 can be supplied by food, nutrients and light, but even then, it would not be sustainable in the long term (Wood et al. 2008). From an ecological perspective, the relative differences in sensitivity to OA within 30 Ocean acidification in the Mediterranean Sea Table 1. Summary on the effects of ocean acidification on calcification of temperate and cold-water corals present in the Mediterranean sorted by increasing incubation time. Symbols meaning ‘é’ an increase; ‘ê’ a decrease; ‘✖’ no statistically significant change. Results included in this thesis are highlighted in grey. 31 Introduction Table 1. Summary on the effects of ocean acidification on calcification of temperate and cold-water corals present in the Mediterranean sorted by increasing incubation time. Symbols meaning ‘é’ an increase; ‘ê’ a decrease; ‘✖’ no statistically significant change. Results included in this thesis are highlighted in grey. 32 Ocean acidification in the Mediterranean Sea AIMS AND OUTLINE OF THE THESIS The main goal of this thesis is to determine the potential effects of the progressive acidification of the oceans on selected key marine calcifying organisms from two of the most important Mediterranean calcareous ecosystems in terms of species diversity, the shallow coralligenous and the deep-sea communities. In order to address this and the more specific questions listed below, we developed a system for experimental pH manipulation in aquaria that allowed us to expose the organisms to different pH (and temperature) conditions, simulating the present values and those expected at the end of this century. We evaluated the midto longterm response (months-year) to OA of eight key species of Mediterranean scleractinian corals, four of them typical of shallow-waters (Cladocora caespitosa, Oculina patagonica, Astroides calycularis and Leptopsammia pruvoti) and four deep-water species (Lophelia pertusa, Madrepora oculata, Desmophyllum dianthus and Dendrophyllia cornigera), as well as the most economically important octocoral, Corallium rubrum. This thesis is comprised of five self-contained research papers grouped in four main chapters that address the specific issues listed below. Chapter 1. Calcification reduction and recovery in native and non-native Mediterranean corals in response to ocean acidification. In this chapter, we investigate the response to OA of the two zooxanthellate coral species capable of constituting the main framework of the shallow benthic community, the endemic Cladocora caespitosa and the non-native Oculina patagonica, in order to estimate potential changes in the composition of the coralligenous communities in a near future. The main goals of this study were (1) to compare the response in calcification of both species and to determine whether the alien species had a higher tolerance to low-pH conditions than the native one; (2) to ascertain whether this response was homogeneous between specimens or there was a certain degree of intraspecific variability; (3) to evaluate the impact of acidified conditions in coral-associated zooxanthellae or in the coral skeleton microstructure; and (4) to assess the recovery potential and acclimation capacity of these species after an acidification event. Chapter 2. Detrimental effects of ocean acidification on the economically important Mediterranean red coral (Corallium rubrum). The valuable and endangered Mediterranean red coral was expected to be particularly susceptible to OA due to the elevated solubility 33 Introduction of its high-Mg calcite skeleton. In this context, we aimed at answering the following questions: (1) Does a lowering in pH affect calcification in C. rubrum? (2) What is the effect of OA on the morphology of the sclerites? (3) How does OA affect the metabolic balance of colonies in terms of total organic matter, carbohydrates, proteins, lipids and fatty acids? (4) Could OA lead to different physiological responses related with the organism’s capability to store energy? and (5) How will OA affect the harvestable stocks of red coral? Chapter 3. Long-term response of two Mediterranean azooxanthellate temperate corals to low-pH and hightemperature conditions. It has been suggested that OA could enhance the severity of the summer mortality events associated with global warming in the Mediterranean. However, up to date, only two studies have evaluated the potential synergistic effects of both factors in Mediterranean corals. In this chapter, we evaluate the response of two Mediterranean azooxanthellate temperate corals, Astroides calycularis and Leptopsammia pruvoti to the combined effects of high temperature and low pH over an annual cycle. We aimed to address how OA and temperature influenced (1) the skeletal growth rate, (2) the status of the carbonated supporting framework (specific microdensity and porosity) and (3) the metabolic balance (organic matter, lipid and protein content in the tissue) for each species. Chapter 4. Response of Mediterranean cold-water corals to ocean acidification. Deep-water ecosystems are characterized by relatively low Ω values and, due to OA, these habitats might be among the first to be exposed to undersaturated conditions in the forthcoming years. However, the responses of Mediterranean CWC to OA have hardly been evaluated. This question is addressed in the next two subchapters. 4.1 Resistance of two Mediterranean coldwater coral species to low-pH conditions. The goal of this subchapter was to investigate the mid-term effect of decreased pH on calcification of the two branching CWC species most widely distributed in the Mediterranean, Lophelia pertusa and Madrepora oculata. More specifically, we aimed at answering the following questions: (1) Does OA affect calcification rate or the carbonate skeleton microstructure in L. pertusa and M. oculata? (2) Is there a uniform pattern in their response? (3) Are the results consistent with the scarce available literature with similar species? and (4) Can we anticipate the possible long-term response of these organisms based on similar experiments with other Mediterranean corals, both temperate and CWC species? 4.2 Differential response of two Mediterranean cold-water coral species to ocean acidification. Considering the potential threat of OA and 34 Ocean acidification in the Mediterranean Sea the variability in the results observed in the experimental studies published so far on CWC, it was important to conduct further experiments, particularly on the other more abundant CWC species. In this context, the aim of this study was (1) to investigate, for the first time, the effect of OA on calcification of two Mediterranean CWC species, the yellow branching coral Dendrophyllia cornigera and the solitary cup coral Desmophyllum dianthus and (2) to determine the polyp age range from each species that shows greater sensitivity to acidified conditions. Moreover, it was also important to consider other variables that could affect their response (e.g. energetic reserve depletion). For that purpose, (3) we also investigated the long-term effect of a low pH scenario on the biochemical composition of these CWC species and (4) we speculated on the ecological implications of the higher energy demand expected for calcification in a highCO2 ocean. 35 Introduction STUDIED SPECIES In the following, we provide details on the main features of the coral species studied in this thesis. All of them are key species of the Mediterranean ecosystems characterized by the potential to form bioconstructions and/or for its large variety of associated fauna. Cladocora caespitosa (Linnaeus, 1767) is the only reef-forming endemic zooxanthellate coral in the Mediterranean (Morri et al. 1994; Aguirre and Jiménez 1998; Casado-Amezúa et al. 2011), with colonies being as large as a meter or more in diameter (Kružic and Pozar-Domac 2003). Usually, these colonies are phaceloid, with corallites about 8mm in diameter at the end of parallel branches separated by 2-3mm and forming compact masses, even though some colonies can show a more open branching structure as described by Zibrowius (1980). In the Mediterranean, it is a widespread species mostly found in turbid waters (Rodolfo-Metalpa et al. 2008), from shallow waters to depths of approximately 40 m (where light still allows photosynthesis by the symbiotic zooxanthellae) and exposed to different hydrodynamic conditions (Zibrowius 1982; Kružic and Benkovic 2008). The bioconstructions of this emblematic species along the Mediterranean Sea have been severely affected over the last years by mass mortality events related to global warming (Perez et al. 2000; Rodolfo-Metalpa et al. 2006; Coma et al. 2009; Garrabou et al. 2009; Lejeusne et al. 2010; Templado 2014). Oculina patagonica (De Angelis, 1908) is a colonial facultative zooxanthellate coral believed to have been introduced in the Mediterranean by shipping from the temperate southwest Atlantic (Zibrowius 1974; Fine et al. 2001). A new study has cast doubt to this view and, on the contrary, suggests that this species may have long remained isolated from the western Atlantic, and have only recently expanded due to environmental change (Leydet and Hellberg 2015). Its dissemination and proliferation in the Mediterranean has been favored by a combination of biological features, such as sexual and asexual reproduction, early reproductive age, high growth rate and high resistance to temperature, salinity, UV radiation, turbidity and strong wave energy. Although it was first reported in the Ligurian coast of Italy, it is now widely spread throughout the Mediterranean, being most common in Spanish and Israeli shores, where it is actually experiencing an increase in its distribution and abundance 36 Ocean acidification in the Mediterranean Sea (Fine et al. 2001; Sartoretto 2008; Coma et al. 2011; Serrano et al. 2013). O. patagonica form encrusting colonies up to one meter of diameter and less than one centimeter thick in the infralittoral, between surface and 12 m depth. Azooxanthellate colonies can also be found in dark caves and crevices at a depth of 1 to 6 m. Its widespread distribution includes different habitats, from pristine natural rocky substrate to disturbed areas such as artificial boulders, harbors and heavily polluted marinas. Thermal bleaching (loss of zooxanthellae symbionts) has been described in Israel waters but not in the western Mediterranean (Fine et al. 2001), which seems to be related with the different temperature maximum reached during the summer months between the eastern and western basins. Corallium rubrum (Linnaeus, 1758) is a long-lived, slowgrowing gorgonian endemic to the Mediterranean Sea and its neighboring Atlantic rocky shores, where it can be found between 10 and 600 m depth (Rossi et al. 2008; Costantini et al. 2010). Although it is predominantly found in the western basin, it is also present in some areas of the eastern basin and the AfricanAtlantic coast (Zibrowius et al. 1980), typically associated with coralligenous communities growing in small cavities, vertical cliffs and overhangs with dim light conditions. Due to its bright red skeleton, it is considered as one of the most valuable precious corals (Tsounis et al. 2010) and that is why it has been called the ‘Mediterranean Red Gold’. Shallow-water populations of C. rubrum have been harvested since ancient times, and nowadays it is considered to be an overexploited species (Santangelo et al. 2004). It is expected that the full recovery of these shallow-water populations may take several decades or even centuries (Garrabou and Harmelin 2002). Interestingly, its axial skeleton and the sclerites present in the tissue are made of high-Mg calcite (Grillo et al. 1993; Vielzeuf et al. 2008, 2010), the more soluble form of calcium carbonate. It is therefore expected that C. rubrum is highly susceptible to the decrease in pH conditions and among the first species to be affected by this pressure over the coming years. Leptopsammia pruvoti (Laze-Duthiers, 1897) is an ahermatypic, azooxanthellate and solitary coral distributed in the western Mediterranean basin and along the European Atlantic coast from Portugal to southern England (Zibrowius 1980). It is one of the most common organisms in shaded areas of bedrock as overhangs and crevices ranging in depth from the surface to 70 m depth, where they can reach densities of several thousand individuals per square meter (Zibrowius 37 Introduction 1980; Goffredo et al. 2006). Polyps can grow up to ~ 6 cm in height and ~3 cm in diameter and have a bright yellow color that makes this species attractive to recreational divers. Populations from the Italian coast have been extensively studied during the last ten years, and it has been observed that the latitudinal gradient in temperature and solar radiation do not significantly influence its biometry, population abundance and demographic traits (Goffredo et al. 2007; Caroselli et al. 2012). However, a positive correlation between temperature and the microdensity has been described on its calcium carbonate skeleton (Caroselli et al. 2011). Astroides calycularis (Pallas, 1766) is a colonial azooxanthellate coral distributed along the Atlantic coasts of Morocco and Spain and in the south-central part of the Western Mediterranean Sea (Zibrowius 1980, 1995; Bianchi 2007), with some recent records in the northeast Adriatic Sea (Kruzic et al. 2002; Grubelic et al. 2004, Casellato et al. 2007). A. calycularis typically inhabits low irradiance habitats (i.e. vertical walls and overhangs) mainly in the shallow infralittoral (0-15 m; Rossi 1971) although it can be found down to 50 m (Cebrian and Ballesteros 2004; Casado-Amezúa et al. 2012). The recent range expansion into the Adriatic Sea is thought to be due to seawater warming and to the Ionian cyclonic stream (Bianchi 2007), with the northward circulation that seems to have favored the flow of larvae along the Croatian coasts (Grubelic et al. 2004). Similarly, currents dispersing larvae out of the Gibraltar Strait (Ocaa et al. 2000) have expanded its distribution along the Atlantic coast of Morocco and Spain (Zibrowius 1995; Bianchi 2007). Lophelia pertusa (Linnaeus, 1758) is the most common reefbuilding CWC. Also known as ‘white coral’, this species forms tree-like colonies consisting of thousands of coral polyps, which can in turn form large bioherms similar in many ways to shallow water corals reefs (Rogers 1999). This coral has a cosmopolitan distribution (Zibrowius 1980) and has been reported in all major oceans usually at depths of 50-1000 m, and up to 3000 m in some locations (Rogers 1999; Roberts et al. 2006). The largest coverage of this species has been observed off the coast of Norway (Freiwald et al. 2004). While initially living samples of this species were rarely found in the Mediterranean (Zibrowius 1980; Rogers 1999; Tursi et al. 2004), recent explorations have discovered flourishing populations at the eastern (Tursi et al. 2004; Taviani et al. 2005; Freiwald et al. 2009) and western (Orejas et al. 2009; Gori et al. 2013) Medi- 38 Ocean acidification in the Mediterranean Sea terranean basins. This species, like the rest of CWC, is believed to be particularly vulnerable to rising temperature and changes in the global ocean circulation patterns that could reduce the amount of food available (Roberts 2009). Madrepora oculata (Linnaeus, 1758) is also a bioherms constructor (Freiwald et al. 2004), generally associated with other CWC such as L. pertusa. Commonly known as ‘zigzag coral’, this species grows forming branched colonies up to 50 cm high (Tsounis et al. 2010) at depths between 50 to 1000 m (Schroeder et al. 2005; Roberts et al. 2006), but there are records of specimens occurring deeper than 1900 m (Zibrowius 1980; Freiwald 2004). Like L. pertusa, it is a cosmopolitan coral and has been recorded in the North Atlantic (Tursi et al. 2004), Gulf of Mexico (Schroeder et al. 2005), along the Brazilian coast (Zibrowius 1980), the Pacific and the Indian Ocean (Tursi et al. 2004). In the Mediterranean, this species occurs more commonly than L. pertusa (Freiwald et al. 2004; Taviani et al. 2005) living between 80 and 1500 m depth (Zibrowius 1980). Desmophyllum dianthus (Esper, 1794) is a cosmopolitan CWC species with solitary large polyps between 5 to 10 cm in height and 15 to 30 mm in diameter, found at depths from 12 to 4000 m (Risk et al. 2002; Försterra et al. 2005; Jantzen et al. 2013). It is considered a slow growing, frame-builder coral usually associated with L. pertusa and M. oculata (Remia and Taviani 2005; Taviani and Freiwald 2005). It has a cosmopolitan distribution, with records in the North and West Atlantic (Sorauf and Jell 1977; Cogswell et al. 2009), the Chilean, South African and Australian coasts (Zibrowius 1980; Försterra et al. 2005). In the Mediterranean, D. dianthus (also known as Desmophyllum cristagalli; Milne Edwards and Haime 1848) is relatively common and widespread in the upper bathyal zone, between ~200 and 1200 m depth. Records include live samples from the Balearic Sea in Spain, Banyuls and Marseille in France, Santa Maria di Leuca in Italy and as far as Cyprus in the Eastern Mediterranean (Tursi et al. 2004; Taviani et al. 2005; Addamo et al. 2012). This species is of interest to paleoceanographers to reconstruct oceanographic and climatic variability by deciphering geochemical signals embedded within its aragonite skeleton (e.g. Anagnostou et al. 2011, 2012). 39 Introduction Dendrophyllia cornigera (Lamarck, 1816) forms large polyps of 20 to 40 mm in diameter and colonies larger than 50 cm in height (Brito and Ocaa 2004) at depths of 200 to 800 m (Zibrowius 1980), but locally as shallow as 30 m (Castric-Fey 1996). Unlike other CWC species, the distribution of D. cornigera is restricted to particular geographical areas in which temperature ranges from 11 to 17ºC (Zibrowius 1980; Cairns 1994; Roberts et al. 2009), including the Mediterranean Sea, the Eastern Atlantic from the south of Ireland to the Cape Verde Islands, the Bay of Biscay and the Canary Islands (Gori et al. 2014 and references therein). However, it is absent from the northeastern Atlantic, where temperatures range from 5 to 10ºC, suggesting that D. cornigera may have a preference for temperate environments (Gori et al. 2014). In the western Mediterranean, D. cornigera has been mostly observed at temperatures around 12 to 14ºC (Zibrowius 1980; Freiwald et al. 2009), as isolated colonies or in small patches in the submarine canyons of the Gulf of Lions, but it can also form extensive aggregations of colonies (Orejas et al. 2009; Gori et al. 2013). 46 Ocean acidification in Mediterranean zooxanthellate corals rocky infralittoral. Seawater temperature and light measurements from the area were obtained using Onset Stow Away data-loggers set up to register data at 1 h intervals over a full year cycle. The loggers were regularly either cleaned or replaced by scuba divers to prevent bio-fouling and for data downloading. The environmental conditions of the area are characterized by a marked seasonality, with temperatures ranging from 12 ºC in winter to 27 ºC in summer, and often with low irradiance due to the high turbidity of the water. The collected specimens were placed immediately in large seawater containers and transported to the Experimental Aquarium Zone (ZAE) at the Institute of Marine Sciences (ICM) in Barcelona. Colonies were placed in a 225 L acclimation tank with 50 µm filtered running seawater (pumped from 300 m offshore, 10 m depth, in front of the ICM). Temperature (14.5°C) and light conditions (~50 µmol photons m-2 s-1 on a 12:12 light:dark cycle) were chosen to simulate those at the collection site. Five nubbins (12 ± 5 polyps) were harvested from each of the 10 collected colonies from each species, carefully cleaned of encrusting organisms and sediment and glued onto labeled methacrylate holders with an inert mastic compound. The buoyant weight of each nubbin was carefully measured before gluing to be able to subtract the holder and glue weight from the total weight measurements (see below). Temperature at the acclimation tank was increased gradually (0.4°C per day) up to 20°C (simulating mean summer conditions at the area of collection) over a two weeks period and maintained for one further week before the beginning of the experiment. Experimental setup and carbonate system manipulation We implemented a pH-manipulative experimental system following the experimental design described by Reynaud et al. (2003) (Fig. 1). Seawater was continuously supplied to two 150 L tanks and pH was adjusted to values of ~8.09 and ~7.83 units (total scale) simulating, respectively, current and future pH levels predicted for year 2100 following A2 IPCC SRES (Plattner et al. 2008). These pH levels correspond to Mediterranean seawater in equilibrium with an atmosphere of ~390 ppm CO2 for the high pH condition, and ~800 ppm CO2 for the low pH treatment (Table 1). In the two large tanks, we bubbled CO2 (99.9% purity) or CO2-free air (using a home-made filter filled with soda lime, Sigma Aldrich) to either reduce or increase pH, respectively. Seawater pH was monitored continuously by glass electrodes (LL Ecotrode plus - Metrohm) connected to a pH controller (Consort R305, Topac Inc., USA), which automatically opened and closed the solenoid valves of CO2 or CO2free air when needed. To avoid drifts in the pH measurements, glass electrodes were calibrated on a daily basis with a Tris buffer, following standard procedures (SOP6a of Dickson et al. 2007). Water from every large tank was continuously transferred to two replicate 25 L methacrylate experimental aquaria where the corals were maintained. Seawater renewal rate in these aquaria was 10 times per day, and seawater was continuously mixed with HYDOR Koralia pumps (4.5W, 1500 L h-1). The aquaria were covered with a methacrylate wrap 47 Chapter 1 to reduce evaporation and minimize surfaceair gas exchange. Two HQI-lamps (T5 ATI Aquablue Special 4x24W), running on a 12:12 light:dark cycle, were adjusted to the required irradiance with a plastic grey mesh. Irradiance was measured using a Li-COR underwater spherical quantum sensor (Li-1935B; Lincoln, NE; USA) and adjusted to 95 µmol photons m-2 s-1, equivalent to the mean daylight irradiance at 5 meters depth in June at the area of collection, when mean water temperature is 20ºC (Onset Stow Away data-loggers). The pH-manipulative experimental set-up was installed inside a thermostated room, ensuring constant values (~20°C) during the whole experiment. Once a week, fresh Artemia salina nauplii (20 mg dry weight per coral fragment) were supplied by closing the seawater flow-through for 4 hours to ensure a proper feeding. Forty-eight out of the fifty initial nubbins for each species were distributed in the four aquaria, such that at least one representative from each colony in each aquarium. During the first week of the acidification experiment, the pH was gradually adjusted (0.03 units per day) up to the final selected values and maintained there for 92 days. After this period, we initiated the recovery experiment in which we gradually raised the pH of the experimental acidified aquaria to match the conditions of the control aquaria while maintaining the control aquaria as before (Fig. 2). The objective of this manipulation was to determine whether any detrimental effects observed in the corals exposed to acidic conditions could be reversed. The recovery experiment run from day 92 to day 216. Figure 1. Experimental setup used to control and modifies the seawater pH in each aquarium. A) and B) large 150 L tanks for seawater conditioning at pH ~7.83 and 8.09, respectively; C) glass electrodes for pH and PT100 probes for temperature measurements; D) pH controller and data logger; E) solenoid valves; F) soda lime filter; G) 50 kg CO2 bottle; H) seawater filtering system (sand, sediment trap and 50µm cartridge filter); I) and J) control and low pH experimental aquaria, respectively (two replicates per treatment); K) HQI-lamps; L) Microbubble diffusers. 48 Ocean acidification in Mediterranean zooxanthellate corals Measured and derived parameters of the CO2 system The temperature and pH of the large 150 L tanks were logged every 10 minutes using Pt100 probes and glass electrodes, respectively. In addition, temperature and salinity in the four experimental tanks were measured every 2-3 days, using an YSI-30M/10FT probe. Small volumes of water were also taken from the tanks (once a week during the first month and twice a month for the rest of the experiment) to analyze total alkalinity (TA) by potentiometric titration (Perez and Fraga 1987; Perez et al. 2000) and pH using spectrophotometry (Clayton and Byrne, 1993), which provides better precision than with electrodes. AT and pH (always reported on total scale) were used to calculate dissolved inorganic carbon (DIC), carbonate ion concentration, bicarbonate ion concentration, dissolved CO2, aragonite saturation state (ΩA) and atmospheric CO2 concentration in equilibrium, using the CO2calc software (Robbins et al. 2010), with dissociation constants for carbonate determined by Mehrbach et al. (1973) and refit by Dickson and Millero (1987). Chemical and physical conditions of both treatments during the experiment are shown in Table 1. Calcification rate Changes in coral calcification were assessed from measurements of buoyant weight (Davies 1989; Jokiel et al. 1978), using a 0.1 mg resolution balance (Mettler Toledo AB204 SFACT). Measurements during the acidification experiment were performed on days 15, 36, 49 and 92. A last weighing was performed at the end of the recovery experiment, on day 216. Table 1. Parameters of the seawater carbonate system in each treatment. Total alkalinity, pHT, salinity and temperature were used to calculate all the other parameters using the CO2calc software (USGS). For the four measured parameters we report the values as mean ± standard deviation (SD) and range (in brackets). All other calculated parameters are expressed as mean ± SD. N = 12 and 6 for the control and high-CO2 treatment, respectively. Treatment T 20.0 ± 0.6 (19.1 - 21.1) 19.8 ± 0.3 (19.5 - 20.2) Treatment pCO2χCO2DIC [CO2]aq [HCO3 - ] [CO3 2- ]ΩA Control 381 ± 9 390 ± 9 2211 ± 15 12.1 ± 0.5 1966 ± 20 232 ± 6 3.6 ± 0.1 High-CO2780 ± 40 800 ± 40 2362 ± 14 25 ± 1.4 2197 ± 18 140 ± 7 2.1 ± 0.1 Calculated parameters (37.0 - 37.7) High-CO2 7.830 ± 0.021 2539 ± 9 37.3 ± 0.2 (7.807 - 7.862) (2525 - 2550) (37.0 - 37.5) Measured parameters pHT TA Sal Control 8.092 ± 0.008 2534 ± 11 37.4 ± 0.2 (8.070 - 8.100) (2520 - 2550) “pHT” = pH in total scale; “TA” = total alkalinity (μmol/kg-SW); “Sal” = salinity; “T” = temperature (°C); “pCO2” = partial pressure of CO2 of air in equilibrium with seawater (ppm); “χCO2” = mole fraction of CO2 in dry air (ppm); “DIC” = dissolved inorganic carbon (μmol/kg-SW); “[CO2]aq” = CO2 concentration in seawater (μmol/kg-SW); “[HCO3 -]” = bicarbonate ion concentration (μmol/kg-SW); “[CO3 2-]” = carbonate ion concentration (μmol/kg-SW); “ΩA “= saturation state of seawater with respect to aragonite. 49 Chapter 1 Before each measurement, epiphytes were carefully removed with a soft brush from all holders to avoid the presence of micro bubbles that could alter the weight of the organisms. Since the buoyant and dry weights are linearly correlated (with the regression passing through the origin) making percent-changes in both weights equivalent (Ries et al. 2009, 2010), in this work, we calculate the calcification rate directly from buoyant weight data. To this end, we normalize the net buoyant weight of the corals (total coral weight minus the coral holder and glue) to their initial mass. Growth rate (G) is expressed as mg of mass increase per gram of initial weight per day, taking as a reference the initial day of each period (day 0 in the acidification section of the experiment and day 92 in the recovery stage). Density of symbionts Coral tissue was removed from the skeleton of five nubbins from each species at the beginning of the acidification experiment and after the exposure to the control and low pH treatment conditions by means of a jet of re-circulated filtered seawater using an oral irrigator (WaterPik™). The resulting slurry was homogenized with a glass pestle and the volume of the homogenate was recorded (~10 mL). Density of symbiotic dinoflagellates was determined by using 5 replicate counts on a haemocytometer (Neubauer chamber) using a Zeiss standard microscope. Algal size was recorded for 15-20 cells in each sample. After correcting for homogenate volume, the density of symbiotic dinoflagellates was normalized to skeletal surface area, which was calculated by using the aluminium foil technique (Marsh 1970). Figure 2. pH fluctuations during the two stages of the experiment. The solid gray line and the black dotted line represent the pH logged using glass electrodes in the acidified and control large conditioning tanks, respectively. Stars indicate corresponding discrete pH measurements performed by spectrophotometry in the aquaria. 50 Ocean acidification in Mediterranean zooxanthellate corals Scanning Electron Microscope (SEM) images of coral skeletons Only samples of C. caespitosa were analyzed by Scanning Electron Microscope. Three nubbins were randomly selected from each treatment and covered with a thin layer of gold-palladium (<200 Å) for morphology and microstructure SEM observations. A SEM Hitachi S3500N, working at 5 kV, was used. Observations focused on the amount and size of the septal flank spines. Statistical analyses The effect of both experiments (i.e. the acidification experiment conducted over the first 92 days and the recovery experiment conducted from day 92 to day 216) was examined separately for each species. For the acidification experiment, a two-way nested ANOVA was used for each species to examine whether calcification rate varied between treatment (i.e. exposure to low pH conditions and exposure to current pH conditions) and aquaria. Aquarium was considered as a random factor nested within treatment. A two-way ANOVA was also used to examine whether calcification rate varied between both species and aquaria in control conditions after 92 days of experiment. For the recovery experiment, a two-way nested ANOVA was used for each species to examine whether calcification rate varied between treatment (i.e. exposure to current pH conditions of colonies previously exposed to low pH and exposure to current pH conditions of colonies previously exposed to current pH conditions) and aquaria. Aquarium was considered as a random factor nested within treatment. Normality (KolmogorovSmirnov test) and heterocedasticity (Cochran’s test) of both species growth data were assessed after arctan-transformation. Calcification rate results are expressed as mean ± standard error of the mean (SE). Non-parametric Kruskal-Wallis ANOVA was used to examine differences between both treatments from the acidification experiment in the abundance and size of symbionts. These statistical analyses were performed using the software package Statistica 6.0 (StatSoft, Inc. 2001). Results Seawater chemistry Our experimental set-up allowed a precise adjustment of the selected pH conditions, which were maintained during the first three months at 8.09 ± 0.01 and 7.83 ± 0.02 pH units for the control and acidified pH treatment, respectively (Table 1; Fig. 2). Total alkalinity values remained constant in both treatments (2534 ± 11 and 2539 ± 9 µmol kg-1 for the control and acidified pH treatment, respectively) throughout the entire experiment. The average calculated ΩA and cCO2 (mole fraction of CO2 in dry air) for the control pH treatment were 3.6 and 390 ppm, respectively. In the acidified treatment, these values changed to 2.1 and 800 ppm, 51 Chapter 1 respectively. Average values of other parameters of the carbonate system are summarized in Table 1. Temperature and salinity were constant throughout the whole experiment (20.0 ± 0.6 ºC and 37.4 ± 0.2, respectively). Effects of low pH on coral calcification rates Significant differences were observed between the calcification rates of the two species reared under control conditions after 92 days of experiment (Table 2), being 37% higher in O. patagonica than in C. caespitosa (Fig. 3a). The effect of the low pH treatment was similar; both species showed a significant decrease in average calcification rate compared to control conditions: 32% lower for O. patagonica and 35% lower for C. caespitosa (Table 2, Fig. 3a). At the end of the acidification period the decrease in skeletal growth rate exhibited by both species was similar and no significant differences were observed between them (Table 2). The survivorship in each treatment was 100% and no tank effect was detected between aquaria replicates of the same treatment in any analysis. On the basis of the absence of differences between duplicate aquaria, we calculated the effect of low pH on the skeletal growth (difference in growth between the control and the low pH treatments) of each of the 10 distinct coral colonies used in the experiments. Source of variation DF MS F-ratio P-value Species 1 0.09 7.65 0.0083 Aquarium 1 0.00 0.06 0.80 Error 44 0.01 Effect of low pH in O. patagonica Treatment 1 0.05 5.91 0.0192 Aquarium (treatment) 2 0.01 1.32 0.28 Error 44 0.01 Effect of low pH in C. caespitosa Treatment 1 0.06 7.00 0.0113 Aquarium (treatment) 2 0.00 0.34 0.71 Error 44 0.01 Species 1 0.00 0.01 0.94 Error 18 0.42 Recovery in O. patagonica Treatment 1 0.01 2.17 0.15 Aquarium (treatment) 2 0.00 0.62 0.54 Error 44 0.01 Recovery in C. caespitosa Treatment 1 0.02 1.74 0.19 Aquarium (treatment) 2 0.00 0.44 0.64 Error 44 0.01 Species 1 0.00 0.69 0.42 Error 18 0.00 Comparison of growth in control conditions Comparison of low pH effect on both species Comparison of recovery potential on both species Table 2. Summary of the outputs ANOVA tests evaluating the effect of two levels of pH (8.09 and 7.83 units) on the growth of Cladocora caespitosa and Oculina patagonica and the subsequent recovery. Bold face numbers indicates P < 0.05. Figure 3. Skeletal growth rates of O. patagonica and C. caespitosa after the first three months under the two pH treatments (a) and during the following four months when the acidified treatment was progressively basified to match the control pH (b). White and grey bars represent control (~8.09 pH units) and treatment (~7.83 pH units during the first three months, brought to ~8.09 pH units during the following four months), respectively (N = 24, mean ± SE). 52 Ocean acidification in Mediterranean zooxanthellate corals As previously described, each colony was split in five and distributed among the 2 treatments (4 aquaria). A one-way ANOVA was performed to examine whether the effect of exposure to low pH differed between the two species. We observed that the decrease in calcification rates at the end of the acidification experiment was similar for both species (Table 2). However, when the mean growth reduction of each colony from both species in the acidified experiment (after 92 days) was compared with the average growth rate of the same colony in control conditions (Fig. 4), an interesting pattern arose: colonies exhibiting faster growth rates were more affected by the decreased pH. The difference in O. patagonica calcification between the treatment and control exposure exhibited a large spread of responses among the colonies during the first month, which later progressively diminished (i.e. there was a reduction of the variance along the length of the experiment; Fig. 5). The previously observed pattern of a larger detrimental effect of acidification on coral colonies that grew faster (Fig. 4) is contributing to this trend, because it causes an attenuation of the differences between the growth rate of the colonies over time. Although the same effect is observed in C. caespitosa (Fig. 4), the slow growth rate of this coral species prevents measurement of a clear reduction of the variance over time (Fig. 5). This observation highlights the importance of running experiments long enough to assess more realistically the effect of these environmental perturbations. During the recovery experiment, the nubbins of O. patagonica and C. caespitosa grown under low pH conditions gradually recovered after Figure 4. Correlation between the average growth rate of colonies in the control pH and the difference in growth between these colonies in the acidified and the control conditions, corresponding to C. caespitosa (solid line, grey dots) and O. patagonica (dashed line, white dots). Linear regressions are statistically significant in both cases (O. patagonica b = -0.57; R2 = 0.78; p = 0.0007; N = 10 and C. caespitosa b = -0.48; R2 = 0.75; p = 0.0011; N = 10). Figure 5. Variability in the differences in growth between colonies in the acidified treatment and the corresponding counterparts in the control conditions during all the time intervals when corals were weighted. White and grey boxes represent Horizontal dotted line represents identical growth of the colonies in each treatment. The corresponding box-and-whisker diagrams depict the median (solid line) and the 10th, 25th, 75th and 90th percentiles (N = 10). 53 Chapter 1 being returned to the current pH conditions of the control treatment. At the end of the recovery experiment (216 days), no significant differences in overall mean calcification rate were detected between the nubbins in control and those in recovered conditions for both species (Table 2, Fig. 3b). The recovery potential showed by both species was similar and no significant differences were detected between them (Table 2). Encouraged by the absence of aquaria effect, we then calculated the difference in skeletal growth between the control and recovery treatments for each of the 10 distinct coral colonies used in the experiment. At the end of the recovery experiment (216 days), we did not observe a significant variation in skeletal growth rate of the treatment colonies with respect to control between both species. In addition, the recovery experiment did not exhibit any significant relationship between the average calcification rate of each colony exposed to the recovery treatment and that of the same colony exposed to the control conditions (R2 = 0. 29, p = 0.11 in O. patagonica and R2 = 0.34, p = 0.08 in C. caespitosa; n = 10), that is, growth rate under control conditions did not predict growth rate under recovery conditions for a given colony. Density of symbionts The initial abundance of zooxanthellae was about 2-fold higher in O. patagonica (8.39 ± 6.23 x106 cells cm-2; mean ± SD) than in C. caespitosa (4.85 ± 2.46 x106 cells cm-2; mean Figure 6. Zooxanthellae density (A) and symbiotic dinoflagellate size (B) measured at the beginning and after the exposure to the control and acidified conditions. White and grey bars represent O. patagonica and C. caespitosa, respectively. Data are mean ± SD (n = 5). ± SD; Fig. 6a; Kruskal-Wallis, p = 0.03). In contrast, zooxanthellae were on average half a micron smaller in O. patagonica (6.8 ± 0.4 mm; mean ± SD) than in C. caespitosa (7.5 ± 0.6 mm; mean ± SD; Fig. 6b; Kruskal-Wallis, p = 0.05). Zooxanthellae density (O. patagonica, Kruskal-Wallis, p = 0.518; C. caespitosa, Kruskal-Wallis, p = 0.304), and cell size (O. patagonica, Kruskal-Wallis, p = 0.338; C. caespitosa, Kruskal-Wallis, p = 0.395) did not differ between treatments (control and acidified) for either species, indicating that zooxanthellae in the two Mediterranean coral species were not affected by the level of acidification to which they were subjected. 54 Ocean acidification in Mediterranean zooxanthellate corals Microimaging of coral skeleton SEM observations of C. caespitosa nubbins revealed no clear differences in the skeletal morphology and microstructure between treatments (Fig. 7). At a gross morphological level, slight differences were found in the appearance of the distal tips of the septa (thinner in nubbins grown under lower pH conditions) as well as on the number and size of the septal flank spines. However, at a much higher magnification, no differences were apparent in the size and arrangement of the microcrystalline units, and different fiber crystallization patterns were observed within the same corallite regardless of the treatment. Figure 7. Examples of Scanning Electron Microscope (SEM) images of C. caespitosa polyps at the end of the experiment, that were exposed to control (a, c, e) and acidified (b, d, f) conditions. Polyp distal view (a, b, scale bar represents 3mm); morphology of distal septal edge from enlarged view (c, d, scale bar represents 500 µm) and septal flank spine from at greater magnification (e, f; scale bar represents 5µm). 55 Chapter 1 Discussion Effects of acidification on corals from the Mediterranean Sea During the first stage of the experiment, colonies of O. patagonica and C. caespitosa reared in the low pH treatment (pH 7.83), suffered a decrease in calcification rates of 32 to 35% compared with colonies maintained in control conditions (pH 8.09; Fig. 3). Our experiment thus exhibited the expected result of detrimental effects on coral calcification. Previous experiments with Mediterranean corals testing the effect of acidification on calcification rates have revealed different responses, pointing to some complexity in the effect of low pH on temperate corals. Our results are consistent with those of the only experiment reported to date with O. patagonica and Madracis pharensis (Fine and Tchernov 2007). In that study, O. patagonica reared in aquaria under different pH treatments, showed a dissociation of the colony form and complete skeleton dissolution when exposed to seawater pH of 7.3 – 7.6. However, differences in the low pH treatment between Fine and Tchernov’s (2007) study and ours are substantial, preventing an exact comparison of both works. First, the pH in the acidified treatment was lower in the earlier study (pH ~7.4) than in our experiment (pH ~7.83), in which our goal was to mimic realistic projections for the year 2100. In addition, the pH adjustment in Fine and Tchernov’s (2007) experiment was performed by adding HCl (reducing alkalinity) whereas, in our case, we bubbled CO2 (maintaining alkalinity constant), a method that provides a more realistic approach. In any case, despite the differences between the studies, our results, together with those from Fine and Tchernov (2007), show decreased calcification rate of O. patagonica at lower pH and ΩA in seawater. In contrast, results for a temperate coral species from the same genera (Oculina arbuscula, not present in the Mediterranean), showed a nonlinear response of calcification rates to CO2induced ocean acidification (Ries et al. 2010) by exhibiting no changes until ΩA was reduced to 0.8 (equivalent to a pH of 7.48 units). These results suggest a greater resistance to low pH of O. arbuscula in comparison to O. patagonica as reported by Fine and Tchernov (2007) and our study. Regarding the endemic Mediterranean species, a previous study by Rodolfo-Metalpa et al. (2010) exhibited no significant difference in calcification rate of C. caespitosa when reared in aquaria under elevated pCO2 (700 ppm equivalent to ~7.88 pH units), pointing to a lower sensitivity of this temperate coral species to ocean acidification. However, further work with C. caespitosa and Balanophyllia europaea grown under the influence of natural high pCO2 vents (Rodolfo-Metalpa et al. 2011), showed that, whereas net calcification in B. europaea remained positive even at pH 7.3, net calcification rates of C. caespitosa became negative at pH 7.5. 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Mémoires de l’Institut Océanographique 11:1-284 67 Chapter 2 Chapter 2 Detrimental effects of Ocean Acidification on the economically important Mediterranean red coral (Corallium rubrum) Bramanti L, Movilla J, Guron M, Calvo E, Gori A, Dominguez-Carrió C, Grinyó J, López-Sanz A, MartínezQuintana A, Pelejero C, Ziveri P, Rossi S (2013) Global Change Biology 19:1897-1908 Abstract The mean predicted decrease of 0.3 to 0.4 pH units in the global surface ocean by the end of the century has prompted urgent research to assess the potential effects of ocean acidification on the marine environment, with strong emphasis on calcifying organisms. Among them, the Mediterranean red coral (Corallium rubrum) is expected to be particularly susceptible to acidification effects, due to the elevated solubility of its Mg-calcite skeleton. This, together with the large overexploitation of this species, depicts a bleak future for this organism over the next decades. In this study, we evaluated the effects of low pH on this species from aquaria experiments. Several colonies of C. rubrum were long-term maintained for 314 days in aquaria at two different pH levels (8.10 and 7.81 pHT). Calcification rate, spicule morphology, major biochemical constituents (protein, carbohydrates and lipids) and fatty acids composition were measured periodically. Exposure to lower pH conditions caused a significant decrease in the skeletal growth rate in comparison to control treatment. Similarly, the spicule morphology clearly differed between both treatments at the end of the experiment, with aberrant shapes being observed only under the acidified conditions. On the other hand, while total organic matter was significantly higher under low pH conditions, no significant differences were detected between treatments regarding total carbohydrate, lipid, protein and fatty acid composition. However, the smaller variability found among samples from the acidified treatment relative to those in control conditions suggests a possible effect of pH decrease on the metabolism of the colonies. Our results show, for the first time, evidence of detrimental ocean acidification effects on this valuable and endangered coral species. (Photo credits by E. Obis) 68 Ocean acidification in the Mediterranean red coral Introduction Ocean acidification (OA) is considered a major threat to the marine environment in the coming years (Doney et al. 2009). An average reduction of 0.1 pH units has already affected the surface waters of the world’s oceans since the preindustrial era (Orr et al. 2005) and future projections predict a decrease by 0.3 to 0.4 pH units by the end of the century, depending on the considered CO2 emission scenarios (e.g. Joos et al. 2011). Although OA acts at a global scale, its impact varies locally. In the case of the Mediterranean Sea, the fast turnover time of its waters (50-100 years; Bethoux et al. 2005) and the very high concentration and fast penetration of anthropogenic CO2 (Schneider et al. 2007, 2010; Touratier and Goyet 2009) makes it one of the world’s most sensitive regions to increasing atmospheric CO2 (Ylmaz et al. 2008; Calvo et al. 2011, Ziveri 2012). A recent study estimated a pH decrease of up to 0.14 units since the pre-industrial era (Touratier and Goyet 2011), larger than the global averaged surface ocean pH decrease of ~0.1 pH units recognised normally (e.g. Orr et al. 2005). The Mediterranean Sea is also characterized by other important environmental stressors (e.g. Calvo et al. 2011; Durrieu de Madron et al. 2011), which can impact marine organisms and ecosystems in different ways, in many instances synergistically (Crain et al. 2008; Lejeusne et al. 2010; Coll et al. 2010). Several studies examining the potential impact of the projected pH levels on marine organisms have shown a wide variety of responses (e.g. Doney et al. 2009; Guinotte and Fabry 2008; Ries et al. 2009; Wicks and Roberts 2012; Suggett et al. 2012). However, in the case of coral reef communities, most experiments indicate that the decreased concentration of carbonate ion associated to the reduction in pH has a detrimental effect, hindering the calcification process and the skeleton formation (e.g. Kroeker et al. 2010; Chan and Connolly in press and references therein). However the calcification process in corals is highly complex and the impact of OA on critical physiological/ biochemical mechanisms controlling the pH at the site of calcification is barely understood (Allemand et al. 2011; Cohen and Holcomb 2009). There is evidence that some species have the capacity to raise pH and [CO3 2-] at the calcifying site (Al-Horani et al. 2003; Cohen and McConnaughey 2003; Ries 2011; Trotter et al. 2011), yet this active physiological process requires a significant amount of energy and a high metabolic cost for the organism (Wood et al. 2008; Cohen and Holcomb 2009; Edmunds et al. 2012; Li and Gao 2012; McCulloch et al. 2012). It is likely that the rate of change and the magnitude of seawater pH decrease are key factors in determining the energy demand for this process, reducing its availability to undertake other activities such as locomotion, reproduction, tissue growth or to counteract other environmental stresses (Brewer and Peltzer 2009; Hoegh-Guldberg et al. 2007). Therefore, studies aiming to identify which metabolic pathways can be more influenced by OA may play a helpful role to understand such an effect. 69 Chapter 2 OA could also have clear regional socioeconomic ramifications such as those related with the reduction in the harvest of high commercial interest species (Cooley et al. 2009). One of these key species is the red coral, Corallium rubrum (Linnaeus 1758), the so-called “Mediterranean Red Gold”. It is a long-lived, slow-growing gorgonian endemic to the Mediterranean Sea and its neighbouring Atlantic rocky shores, where it can be found between 10 and 600 m depth (Costantini et al. 2010; Rossi et al. 2008). It is considered as one of the most valuable precious corals due to its bright red durable skeleton used as raw material in the jewellery industry (Tsounis et al. 2010). For these reasons, C. rubrum has been harvested since ancient times and it is now considered overexploited (Santangelo et al. 2004). Moreover, due to its rarity, cultural importance and landscape aesthetic value, red coral can be considered as a patrimonial or flagship species of the Mediterranean Sea (Bramanti et al. 2011). Red coral is composed of an axial skeleton and sclerites coated with living tissue (coenenchyma). The external surface of the coenenchyma is formed by the ectoderm, under which the mesoglea lies, a thick acellular layer of collagen containing small sclerites (30-50 μm). The axial skeleton has the same mineralogical composition than the free sclerites (Mg-rich calcite; Vielzeuf et al. 2008) but it is not the product of their fusion (Grillo et al. 1993). The main function of the sclerites is to provide mechanical protection against abrasion (Allemand 1993), although it has been suggested that they may act as temporary stocks of CaCO3 readily available for the formation of the axial skeleton (Vielzeuf et al. 2008). The solubility of Mg-rich calcite minerals is greater than that of aragonite or calcite (Plummer and Mckenzie 1974), and the seawater saturation state with respect to carbonate minerals decreases with increasing latitude (Andersson et al. 2008; Orr et al. 2005). Thus, it is conceivable that Mg-rich calcite, temperate calcifying organisms such as C. rubrum should be highly vulnerable to the decrease of pH conditions and among the first to be affected by these new environments. Owing to the demand for management and conservation plans for a sustainable harvesting of this precious octocoral (Brukner and Roberts 2009; Bussoletti et al. 2010; GFCM 2010, 2011), dynamic models have been developed, projecting demographic trends over time (Santangelo et al. 2007; Bramanti et al. 2009). However, there is a lack of information regarding future OA effects in these colonies with potential to be harvested. To shed light on this topic, we investigated the effect of OA on C. rubrum by simulating in aquaria the future pH conditions projected by the end of the century. The potential changes in calcification rate, sclerite morphology and biochemical composition of the colonies reared under different pH conditions were evaluated. The results provide valuable information on the response of C. rubrum to low pH conditions that will allow better understanding and forecasting the economic and ecological consequences of OA impacts on this precious species. 70 Ocean acidification in the Mediterranean red coral In this study, we aimed at answering the following questions: (1) Does a lowering in pH affect the CaCO3 deposition in C. rubrum? (2) What is the effect of OA on the morphology of sclerites? (3) How does OA affect the metabolic balance of colonies in terms of total organic matter, carbohydrates, proteins, lipids and fatty acids? (4) Could OA lead to different physiological responses related with the organism’s capability to store energy? (5) How will OA affect the harvestable stocks of red coral? Materials and methods Specimen collection and experimental setup Colonies of C. rubrum were carefully removed from rocky substrates at 35-40 m depth from the Marine Protected Area of Cap de Creus (Spain, NW Mediterranean Sea, 42°19’N; 003°19’E) in November 2010 when in situ temperature was 13ºC. The collected specimens were transported in large seawater containers at constant temperature (12.5 ± 0.5ºC) to the Institut de Ciències del Mar (ICM, CSIC) in Barcelona (Spain). Colonies were maintained in a 100 L acclimation tank with 50 μm filtered running natural seawater at in situ temperature and salinity conditions (12ºC and 37.6, respectively). As the red coral commercially harvested populations dwells between 80 and 100 meters depth (Tsounis et al. 2013), where the seasonal temperature variation are very small, colonies in the tanks were maintained at constant temperature and in complete darkness. For feeding, de-frozen Cyclops (Ocean Nutrition™) were supplied daily (~55 mg of dry weight per aquarium). After 1 month of acclimation, 48 colonies of C. rubrum (Fig. S1) were selected and randomly distributed among 6 aquaria (30 litres each). Aquaria were further subdivided into 2 treatments (3 replicates per treatment), control pH and low pH (see below). The experimental set up is shown in Fig. S2. Seawater pH was gradually adjusted (0.03 units per day) in two large tanks of 150 L up to ~8.10 and ~7.81 pH units (total scale) simulating, respectively, the Mediterranean seawater in equilibrium with an atmosphere of ~380 ppm CO2 (current levels) and ~800 ppm CO2 (future levels predicted for year 2100 following A2 IPCC SRES; Plattner et al. 2008). In order to achieve the desired pH levels, seawater was bubbled with CO2 (99.9% purity) or CO2free air (using a filter filled with soda lime absorber, Sigma Aldrich). Seawater pH was continuously monitored by glass electrodes (LL Ecotrode plus - Metrohm) connected Figure S1. Colony of Corallium rubrum in the experimental aquarium. 71 Chapter 2 to a pH controller (Consort R316, Topac Inc., USA). The glass electrodes were calibrated on a daily basis with a Tris buffer, following standard procedures (SOP6a of Dickson et al. 2007). In addition, small volumes of water were taken periodically (once a month during the first 3 months and bimonthly for the rest of the experiment) to analyse total alkalinity (AT) by potentiometric titration (Perez and Fraga 1987; Perez et al. 2000) and pH using spectrophotometry (Clayton and Byrne 1993), which provides better precision than electrodes. TA and pH were used to calculate the rest of parameters of the carbonate system in seawater in both treatments, using the CO2calc software (Robbins et al. 2010; Table S1). Water from the large tanks flowed continuously (12 L per hour) to the experimental aquaria where colonies were maintained. Water in each aquarium was mixed with a pump (HYDOR Koralia; 4.5W, 1500 l h-1) and a plastic wrap was used to reduce evaporation and surface-air gas exchange. The pH-manipulative experimental set-up was installed inside a temperature-controlled room, ensuring constant values (~12°C) during the whole experiment. Colonies of C. rubrum were sampled quarterly, from December 2010 (Time 0) to November 2011 (Time 3). During each sampling event, the buoyant weight of all the colonies was measured and 6 random colonies (1 from each experimental aquarium) were removed and kept at -80ºC, subsequently freeze-dried and then stored frozen at -20ºC until morphological and biochemical analyses were undertaken. Furthermore, at the end of the experiment, one Figure S2. Experimental setup used to control and modify seawater pH in each aquarium. A) and B) large 150 l tanks for seawater conditioning at pHT 7.81 and 8.10, respectively; C) glass electrodes for pH and PT100 probes for temperature measurements; D) pH controller and data logger; E) solenoid valves; F) soda lime filter; G) 50 kg CO2 bottle; H) and I) control and low pH experimental aquaria, respectively (three replicates per treatment); J) Microbubble diffusers. 78 Ocean acidification in the Mediterranean red coral Discussion Changes in the structural features of Corallium rubrum After 314 days of exposure, colonies of C. rubrum reared under lower pH conditions (pH 7.81) suffered a decrease in skeletal growth rate of 59% compared with those maintained in the control treatment (pH 8.10). This reduction confirms the expected detrimental effects on skeletal formation and it is consistent with previous experimental studies examining the response to OA of other benthic calcifying species. In the case of tropical scleractinian corals, the decline in the calcification rate ranges from 10% to 60% at double elevated pCO2 conditions (Guinotte and Fabry 2008; Kleypas et al. 2006), reaching up to 70% of reduction in the case of Porites rus at pH 7.80 (Muehllehner and Edmunds 2008). Some species, however, are able to calcify even under low saturation state conditions (Jury et al. 2010; Krief et al. 2010; Ries et al. 2010; Comeau et al. 2013), pointing to some complexity in the mechanisms controlling the intracellular calcification process (Ries 2011 and references therein). Similarly, a wide range of responses has been observed in Mediterranean coral species. From a total skeleton dissolution, as reported by Fine and Tchernov (2007) in Oculina patagonica at pH 7.4, to 35% decreased calcification rate in O. patagonica and Cladocora caespitosa ! Source DF SS MS F-value p-value Treatment 1 7566.6 7566.6 18.87 0.0005 *** Time 3 2442.2 814.1 2.03 0.15 Treatment x Time 3 6454.4 2151.5 5.37 0.009 *** Residuals 16 6414.6 400.9 Post-hoc Tukey’s HSD test pH x Time Diff Lwr Upr p adj T0: Control=Low pH 1.14E-13 -56.601 56.60 0.99 T1: Control=Low pH -5.77E+00 -62.369 50.83 0.99 T2: Control<Low pH -7.19E+01 -128.48 -15.27 0.008 *** T3: Control<Low pH -6.44E+01 -121.00 -7.80 0.02 * Control: T2=T3<T0=T1 -6.02E+01 -116.83 -3.62 0.03 * Low pH: T0=T1=T2=T3 4.17E+00 -52.42 60.77 0.95 Table 3. ANOVA and Post-Hoc test for total organic matter. Factors are Time (Time 0, Time 1, Time 2 and Time 3) and Treatment (Control and Low pH). Figure 2. Total organic matter (%) in the coenenchyma of Corallium rubrum colonies (N = 24) during the whole experiment. White and grey bars represent control (8.10 pHT units) and treatment (7.81 pHT units), respectively (mean ± SD). 79 Chapter 2 at pH 7.83 (Movilla et al. 2012) or a complete absence of effects on C. caespitosa subjected to pH 7.88 (Rodolfo-Metalpa et al. 2010a) have been described in aquaria experiments. In addition, the only experiment to date in which temperate corals were transplanted to a natural pH gradient influenced by volcanic CO2 vents, showed evidences of dissolution on the exposed skeleton of C. caespitosa at pH 7.5 environments, while no effects were observed in Balanophyllia europaea, which skeleton was completely covered by tissue, even at pH 7.3 (Rodolfo-Metalpa et al. 2011). Regarding Mediterranean bryozoans, Schizoporella errata transplanted to the same naturally acidified site showed skeletal corrosion and disruption in calcification at pH 7.76 (Lombardi et al. 2011a). However, the skeletal growth rate and appearance of Myriapora truncata were not affected until a pH of 7.43 (Lombardi et al. 2011b; Rodolfo-Metalpa et al. 2010b), suggesting that the presence of organic tissue enveloping the skeleton could play a key role in these organisms (Ries et al. 2009; RodolfoMetalpa et al. 2011), providing protection against the corrosiveness of lower pH seawater. It should be noted that, unlike scleractinian corals or bryozoans as S. errata, whose skeletons are made of calcite or aragonite (Cohen and McConnaughey 2003; Smith et al. 2006), the red coral skeletal structure consists entirely of Mg-rich calcite (Vielzeuf et al. 2008). As previously mentioned, the solubility of this latter form is much higher than the first two and, therefore, this species could be considered as one of the most susceptible organisms to OA in the Mediterranean. Our results also showed an effect of lower pH on the morphology of microscopic sclerites, with an overall shape different between treatments Figure 3. Total protein (a), lipid (b) and carbohydrate (c) concentration (µg mg-1 OM) in the coenenchyma of Corallium rubrum colonies (N = 24) during all the periods of time when colonies were sampled. White and grey bars represent control (pHT 8.10 units) and treatment (7.81 pHT units), respectively (mean ± SD). CV= coefficient of variation. 80 Ocean acidification in the Mediterranean red coral and anomalous forms observed only in acidified conditions. Sclerites abnormal skeletogenesis has been observed previously in sea urchin larvae exposed to high pCO2 conditions (Kurihara and Shirayama 2004). According to Allemand (1993), the main function of these sclerites in C. rubrum is to ensure the mechanical protection against abrasion, although it has been suggested that they may act as CaCO3 temporary stocks readily available for the axial skeleton formation through mechanisms of dissolution, transport, and recrystallization (Vielzeuf et al. 2008). Furthermore, in the order Alcyonacea, the skeleton is typically proteinaceous (Grillo et al. 1993) with elastic properties that allow the colonies to bend forward and back to an upright position (Jeyasuria and Lewis 1987). The morphology and abundance of sclerites in the tissue limit the extent of these movements, being a determinant factor that defines their overall structure (Lewis and von Wallis 1991). Therefore, our results suggest that, in the long term, OA will affect C. rubrum and other gorgonians in compromising the CaCO3 stocking capacity as well as the skeleton biomechanical properties. This study focused in evaluating the effect of OA on the growth of a Mediterranean anthozoan with high commercial value such as the red coral. However, in addition to OA, other environmental pressures may also be at play, perhaps with complex interactions between them in their cumulative effect to red coral populations. The increase in atmospheric CO2, for example, is also causing seawater warming which, in the Mediterranean Sea, has been shown to translate into longer stratification periods associated with mass mortality events (Coma et al. 2009; Garrabou et al. 2009). Although the synergistic effects of warming and ocean acidification could well affect shallow water red coral populations (Santangelo et al. 2012a), the present study has a particular focus on populations with harvest potential (i.e. below 60 meters depth; Rossi et al. 2008), where temperature changes are not as marked and tend to remain relatively constant throughout the year. Nevertheless, high temperatures induce increased metabolic and respiration rates and depress the polyp activity in red coral and other gorgonians (Previati et al. 2010). In addition, long-term exposure to high temperature can result in partial or total mortality of the colonies and recruits (Bramanti et al. 2005; Garrabou et al. 2001, 2009; Torrents et al. 2008). Thus, it is essential to perform future studies manipulating OA and temperature to assess the existence of possible interactions between multiple stressors and establish to which extent coral colonies inhabiting shallower ranges will be threatened. Figure 4. Total fatty acid concentration (µg mg-1 OM) in the coenenchyma of Corallium rubrum colonies (N=24). White and grey bars represent control (pHT 8.10 units) and treatment (7.81 pHT units), respectively (mean ± SD). CV= coefficient of variation. 81 Chapter 2 Metabolic response of red coral to a lowering in pH In gorgonians, the skeletal consists of internal formations (central axis and sclerites) protected by a thick layer of mesoglea gel (Alderslade and Fabricius 2008), a composite material of collagen-like fibrils randomly oriented in a hydrated polymer matrix (Lewis and von Wallis 1991). The presence of a tissue layer prevents the direct exposure of the skeleton to seawater and may modulate the effects of OA in coral and other organisms (Hoffman et al. 2010; Ries et al. 2009; Rodolfo-Metalpa et al. 2011). However, in our experiment, although the tissue of colonies remained intact, a clear effect of high pCO2 in coral growth was observed. In addition to minimizing growth, the colonies of C. rubrum reared under acidified conditions may have activated physiological processes to offset the new conditions, with the consequent increase in energy consumption (Al-Horani et al. 2003; Cohen and Holcomb 2009; Tsounis et al. 2012). Our results on Total Organic Matter tend to point in the direction of this hypothesis. For example, throughout the experiment, higher content of organic matter was observed in the coral colonies reared under lowered pH conditions in comparison to those in the control, which displayed a progressive lowering in organic matter content with time (Fig. 2). Similar responses were found in more drastic pH lowering (pH 7.4) in which the temperate corals O. Patagonica and Madracis pharencis were exposed (Fine and Tchernov 2007). In this case, polyps of these species suffered a complete dissolution of the skeleton, but their biomass was three times higher than those maintained in control conditions (Fine and Tchernov 2007). As it has been described in the case of gastropods, the energy allocation for the shell construction vs that needed to build the organic matrix is very different and changes under stress conditions (Palmer 1983, 1992). Under lowered pH conditions the metabolism is affected (Edmunds 2012; Edmunds et al. 2012) and the energetic cost of calcification is expected to be higher (e.g. Cohen and Holcomb 2009), thus C. rubrum may respond favouring the formation of organic matter to the detriment of calcification. Total carbohydrate, protein and lipid content in colonies grown under control conditions in the present work were similar to those observed by Rossi and Tsounis (2007) in natural conditions (Fig. 3), indicating that the biochemical composition of C. rubrum tissue in aquaria is comparable to that observed in situ. Protein and lipid content was also comparable to other gorgonians such as Paramuricea clavata and Leptogorgia sarmentosa (Rossi 2002; Rossi et al. 2006). Data on fatty acid concentration of C. rubrum are presented in this work for the first time. Regarding composition, a wide range of fatty acid profiles was observed (Fig. S4) but most of the fatty acid esters were quite similar to those described in the case of zooxanthellate anthozoans (Meyers and Quinn 1974; Latyshev et al. 1991; Gori et al. 2012b), although azooxanthellate organisms may have other physiological and metabolic mechanisms to counter the effect of external stress. As mentioned, a larger variability in the 82 Ocean acidification in the Mediterranean red coral total concentration of carbohydrate, protein and lipid content was observed throughout the experiment in the control colonies, whereas the variability was much more reduced in the colonies under the acidified conditions (Fig 3). This difference in response could perhaps be an indication of a more dimmed seasonal trend in the colonies under low pH pressure than those in the control treatment or the observed under natural conditions (Rossi and Tsounis 2007). This could be due to endogenous processes, dormancy or some kind of life cycle disruption experimented under certain environmental pressures, with the aim of saving energy by reducing or shutting down some physiological processes (e.g. respiration; Previati et al. 2010) and thus, survive. This conforms with the findings on sea urchins Hemicentrotus pulcherrimus and Echinodetra mathei where the fertilization success, developmental rates, larval size, and sclerite skeletogenesis also decreased in those specimens reared under low pH (Kurihara and Shirayama 2004) probably because a considerable part of the energy is invested in respiration and in protein synthesis related with basic metabolic paths as found in barnacle and copepods (Wong et al. 2011, Li and Gao 2012). In coral species, metabolism reduction has also been observed as a defensive mechanism to survive under adverse conditions (Rossi 2002; Previati et al. 2010). Nonetheless, this could translate into less available energy for processes such as growth or reproduction (Brewer and Peltzer 2009; Hoegh-Guldberg et al. 2007). Our study was focused on assessing the effect of lower pH on skeletal growth rates of C. rubrum. However, further research on the effects on reproduction and on the response in a natural seasonal environment should be undertaken to gain deeper insights on the response of red coral populations to OA. Economy and management Understanding the population dynamics of the precious Mediterranean red coral is essential to implement best management and conservation strategies for this species under the ongoing rapid climate and environmental change. In summer 1999 and 2003, shallow populations of C. rubrum, among other benthic suspension feeders in the northwestern Mediterranean Sea, suffered a mass mortality event due to a temperature anomaly (Cerrano et al. 2000; Bramanti et al. 2005; Coma et al. 0 10 20 30 40 50 60 70 TIME 1 TIME 2 TIME 3 TIME 1 TIME 2 TIME 3 PUFA MUFA SAFA CONTROL LOW pH mg FFAA per mg TOM Fatty acids composition: SAFA = total Saturated Fatty Acids; MUFA = Mono Unsaturated Fatty Acids; PUFA = Poly Unsaturated Fatty Acids. Figure S4. Fatty acids composition. SAFA: total Saturated Fatty Acids; MUFA: Mono Unsaturated Fatty Acids; PUFA: Poly Unsaturated fatty acids. 83 Chapter 2 2006; Santangelo et al. 2007; Garrabou et al. 2001; 2009; Cupido et al. 2012). These mass mortality events, coupled with the uncontrolled harvesting, could bring some populations to local extinction (Santangelo et al. 2012a). In fact, the distribution and size of red coral have been altered over time (Rossi et al. 2008, Linares et al. 2010). In Cap de Creus (NE Spain), shallow and deep population structures have been affected by management rules (Santangelo et al. 2012b) turning the ‘coral forest’ into ‘coral grass plain’ (Rossi et al. 2008, 2012; Tsounis et al. 2007). On the other hand, depending on the CO2 emission scenarios, seawater pH and carbonate saturation states could drop significantly by the end of the century (Joos et al. 2011). The present study shows, for the first time, a negative response of C. rubrum deep-water populations to OA. The observed decrease in the calcification rate together with some evidence of a potential reduction in the metabolism of red coral colonies as possible survival mechanism against low pH conditions, could have negative rebounds on the economy of the jewellery industry linked to this species and lead to a price deflation for years. The market price of the red coral colonies is currently very high: thin juvenile branches can be sold for 230-300 US$ kg-1 while colonies with diameter > 4cm can reach 50.000 US$ kg-1 (Tsounis et al. 2007, 2010). Under the increasing global environmental and economic pressures, causing limited raw material production and possible price deflation, the red coral industry may suffer a decline worldwide. In Europe, red coral industry has significantly declined over time and, today, only 30% of the coral processed in Torre del Greco (Italy) is C. rubrum, whereas 70-80% is represented by other species of precious corals imported from Japan and Taiwan (Nonaka and Muzik 2009; Tsounis et al. 2010; Chen 2012). These economic losses represent more than 230 million US$yr-1 (Tsounis et al. 2010). Furthermore, without proper protection and management plans, this slow-growing, endemic precious species of Mediterranean Sea may suffer local extinction in the near future. Young populations of C. rubrum may be unable to cope with combined pressures of heat waves, ocean acidification, overexploitation and other anthropogenic activities. Moreover, local extinction and changes in the population structure could affect the role that engineering species (Jones et al. 1994) as C. rubrum play in the benthic pelagic coupling and biogeochemical cycles (Rossi et al. 2012). Data from the present study provide experimental input that can be used in simulations of population trends overtime based on matrix models (Caswell 2001) that will allow making projections on population dynamics under the expected future acidification scenarios. Management and conservation actions should be then planned on the basis of the outcome of these simulations in order to preserve this precious species with the associated biodiversity and the economy linked to its exploitation. 84 Ocean acidification in the Mediterranean red coral Aknowledgements This research has been funded by the EC 7th FP (grant agreement 265103, Project MedSeA) and by the Spanish Ministry of Economy and Competitiveness (MINECO; CTM2009-08849, Project ACDC). The authors are grateful for the invaluable support of the Cap de Creus Marine Protected Area staff. We thank José Manuel Fortuño for assisting us with SEM imaging and the ZAE staff at the ICM for technical assistance. Thanks are also due to TDI and A. Ferrucci for the Megalodon CCR equipment used for underwater sampling and to Eduardo Obis for the pictures. L.B. was supported by a Marie Curie IEF fellowship (EC7th FP, Project nº 221072), J.M. by a FPI studentship (BES2007-16537), M.G. by of Erasmus Mundus scolarship (contract no. JEMES European UAB 2009/No.3) and S.R. by a Ramón y Cajal Contract (RyC-2007-01327) from MINECO. US National Science Foundation grant OCE 0844785 provided support to L.B. during part of the writing of manuscript. This is a contribution from the Marine Biogeochemistry and Global Change research group, funded by Generalitat de Catalunya through grant 2009SGR142. References Abràmoff M, Magalhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics International 11: 36-42 Alderslade P, Fabricius KE (2008) Octocorals. In: The Great Barrier Reef: biology, environment and management. 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Among them, coral reef communities have attracted special attention because their calcifying organisms may be severely affected by these two global stressors in the future, particularly under the pessimistic range of the projected IPCC Representative Concentration Pathway scenarios (IPCC 2013). Skeletal records from the Great Barrier Reef show, for instance, that coral calcification has declined over the last decades (Cooper et al. 2008; De’ath et al. 2009) and, although the causes of this decline remain unknown, increasing sea temperature and declining pH are amongst the most likely drivers (e.g. Brown 1997; Hoegh-Guldberg 1999; Anthony et al. 2007; Fabricius et al. 2011). Since both stressors act simultaneously, over the past decade a growing number of studies have started to evaluate the combined effect of increased temperature and declined pH on reef corals. However, outcomes have revealed diverse and often contrasting responses as a result of the interaction between both factors, with warming counteracting the adverse effects of acidification in some instances (e.g. Muehllenhner and Edmunds 2008; McCulloch et al. 2012) or leading to a synergy of both stresses in others (e.g. Reynaud et al. 2003; Anthony et al. 2008). The Mediterranean is a biodiversity hotspot (Coll et al. 2010; Templado 2014) that is highly vulnerable to global change (e.g. Calvo et al. 2011; Herrmann et al. 2014; Marbà et al. 2015) and where OA and global warming seem to be occurring more rapidly than in the global oceans (Vargas-Yáñez et al. 2010; Touratier and Goyet 2011; Touratier et al. 2012; but see Palmiéri et al. 2014). Furthermore, the additive synergistic and/or antagonistic character of the interactions of multiple processes that take place, particularly in coastal zones, is still poorly known, which makes difficult the assessment of the vulnerability and future trends of the organisms inhabiting these areas (Lejeusne et al. 2010; Calvo et al. 2011; Durrieu de Madron et al. 2011). However, and in contrast to the large number of studies including tropical species, only a small number of experiments have experimentally assessed the responses of corals inhabiting shallow temperate areas to OA (e.g. Ries et al. 2010; Holcomb et al. 2010, 2012) and, among them, only five have been conducted with Mediterranean species so far. These five Mediterranean studies have revealed a high variability between and within species in their 95 Chapter 3 response to acidified conditions, with results varying largely from a complete dissolution of the skeleton in Oculina patagonica (Fine and Tchernov 2007), to a decrease in calcification rate up to ~35% in O. patagonica and Cladocora caespitosa (Movilla et al. 2012) and ~18% in Balanophyllia europaea (Fantazzini et al. 2015). When the effect of low pH was further assessed in combination with warming, no detectable effects on the calcification of colonies of C. caespitosa (Rodolfo-Metalpa et al. 2010) or B. europaea (Rodolfo-Metalpa et al. 2011) were found at pH levels similar to those expected for the year 2100. In the case of temperate zooxanthellate corals, as well as in most tropical species, the complexity of the combined effects of pH and temperature on coral calcification can result, in part, from the tight relationship between photosynthesis and calcification (Gattuso et al. 1999). The absence of symbionts, in the case of azooxanthellate corals, should presumably facilitate to discern whether there is an interactive effect of OA and temperature on calcification, but so far all the previously mentioned studies have targeted temperate zooxanthellate species. Furthermore, it is important to point out that all these previous studies with temperate coral species assessed the responses of the organisms in terms of skeletal growth, photosynthesis, respiration, skeletal microdensity or porosity, yet we do not know how the two stressors may affect their biochemical composition. Similarly, it is still uncertain what are the effects of the increase in energy consumption that is needed by these organisms to calcify under adverse conditions (Cohen and Holcomb 2009; Allemand et al. 2011) and whether it may affect other physiological and biological processes (Hoegh-Guldberg et al. 2007; Brewer and Peltzer 2009; Pelejero et al. 2010). Therefore, studies assessing the effect of decreasing pH and increasing temperature in skeletal growth and metabolic balance are critical to fully understand the potential response of temperate corals in a changing environment. In this work, we aimed at addressing the cumulative effects of high temperature and low-pH on the calcification and biochemical composition of two temperate azooxanthellate corals from the Mediterranean, the colonial Astroides calycularis and the solitary Leptopsammia pruvoti, by simulating an annual temperature cycle in an aquarium experiment. The bright orange scleractinian A. calycularis (Pallas 1766) is a colonial azooxanthellate coral distributed along the Atlantic coasts of Morocco and Spain and in the south-central part of the Western Mediterranean Sea (Zibrowius 1980, 1995; Bianchi 2007), with some recent records in the north-east Adriatic Sea (Kruzic et al. 2002; Grubelic et al. 2004, Casellato et al. 2007). A. calycularis typically inhabits low irradiance habitats (i.e. vertical walls and overhangs) mainly in the shallow infralittoral (0-15 m; Rossi 1971) although it can be found down to 50 m (Cebrián and Ballesteros 2004). The sunset cup coral L. pruvoti (LacazeDuthiers 1897) is a solitary azooxanthellate coral distributed in the Mediterranean basin 96 Ocean acidification in azooxanthellate temperate corals and along the European Atlantic coast from Portugal to southern England, that also inhabits low irradiance habitats at 0-70 m depth (Zibrowius 1980, Goffredo et al. 2006). These two non-symbiotic coral species are known to be already affected by several anthropogenic factors, such as the destruction of rocky shores and habitat loss, seawater pollution, angling or recreational scuba diving, which, in the case of A. calycularis, has led to its recognition as a threatened species currently under protection (Moreno et al. 2008). To our knowledge, this is the first multi-seasonal assessment with these two coral species, where the potential response to future acidification with distinct thermal regimes is documented, encompassing the regional variation in sea surface temperature across the Western Mediterranean Sea. Materials and methods Specimen collection In January 2011, fifteen colonies of A. calycularis and sixty individuals of L. pruvoti were collected by scuba diving between 6 and 12 m depth in Cartagena (SE Spain, 37°38′N, 0°41′W) and in L’Estartit (NE Spain, 42°3’N, 3°13’E), respectively. Coral specimens were immediately transported to the Experimental Aquarium Zone (ZAE) at the Institute of Marine Sciences (ICM-CSIC) in Barcelona and placed in 225 L tanks with 50 μm-filtered running natural seawater. Shortly after arrival, four fragments (~3 cm in diameter) harvested from each of the 15 collected colonies of A. calycularis and the sixty individuals of L. pruvoti were carefully cleaned of epiphytes and sediment, and glued onto labeled methacrylate holders with an inert mastic compound. All the fragments/specimens were randomly distributed in 11 L experimental aquaria (12 aquaria, 5 fragments/specimens of each species per aquarium, 60 fragments/specimens of each species in total) with 20 times per day seawater renewal rate. Light intensity was adjusted to ~ 9 μmol photons m−2 s–1 in a 12:12 light:dark cycle to simulate the conditions in the sampling areas (measured with a Li-cor, Li-1935B; Lincoln, NE; USA). A mixed diet including freezedried Tetraselmis sp. and Mysidacea (Ocean Nutrition ™) and fresh Artemia salina nauplii was supplied three times a week. Treatment conditions and experimental setup An acclimation phase simulating winter conditions at the sampling areas (13.0 ± 0.5ºC) was conducted during the 3 subsequent months since the last handling of the specimens, thereby ensuring a proper healing of the tissue (Fig. 1). After this time and following a first weighing (T-1, see next section), the specimens were exposed during 99 days to the natural spring temperature rise from 13 to 20ºC, a period during which the basal calcification rate in the aquaria was estimated. At this point (July 2011), pH and temperature were gradually adjusted to the experimental values (see below) and remained during 369 days in these conditions. This experimental stage started coinciding with the beginning of the warm 97 Chapter 3 12 15 18 21 24 Control High Temp Medes Islands (5 meters depth) Acclimation Basal calcification Experimental phase T -1 T2T3T4T5T6T7T8 Adjustment of pH and Temp MayMarJan Jul Sep Nov Jan Mar May Jul Sep T0T1 Temperature (ºC) Figure 1. Experimental design including the different buoyant weight sampling times and the mean monthly temperature (ºC) over the whole experiment under control (open circles) and high-temperature conditions (filled circles). Grey squares and dashed line represent the mean monthly temperature registered at 5 meters depth in Medes Islands from January 2011 to September 2012. summer season, when higher growth rates were expected, in order to facilitate the detection of potential differences between treatments in terms of calcification. Four treatments crossed two temperatures with two pH levels, using triplicate aquaria in each treatment. The temperature was adjusted electronically with a Pt100 regulator (Delta Ohm HD9022) and achieved using a water cooler and 300 W heaters, to cool or warm, respectively, the water in the two baths where the experimental aquaria were submerged. Temperature in the control treatment was weekly adjusted according to their seasonal values measured at ca. 5 m depth at one of the sampling areas (Medes Islands, L’Estartit) using HOBO data-loggers, and the high temperature treatment was gradually adjusted (0.3ºC per day) at the beginning of the experimental phase and kept 3°C above the control, simulating a rise in temperature as expected in the Mediterranean by the end of the century (Solomon et al. 2007; Sokolov et al. 2009). Seawater pH was gradually adjusted (0.03 units per day) by bubbling CO2 and the 98 Ocean acidification in azooxanthellate temperate corals pH levels were continuously monitored by glass electrodes (LL Ecotrode plus - Metrohm) connected to two pH controllers (Consort R362; Topac Inc., USA). The control pHT (total scale at the in situ temperature) consisted of a value of 8.05 units, similar to the mean ambient seawater pH measured in the sampling area of Medes Islands. The decreased pHT level used was 7.72 units, following the ‘business-asusual’ RCP8.5 (Representative Concentration Pathway, 8.5 Watts m-2) scenario by the year 2100 (IPCC 2013). Analyses of total alkalinity (TA) by potentiometric titration (Pérez and Fraga 1987; Pérez et al. 2000) and seawater pH by spectrophotometry (Clayton and Byrne 1993) were carried out bimonthly. These values were used to calculate the rest of the parameters of the carbonate system in seawater using the CO2sys.xls software (Version 2.1; Pelletier et al. 2007) with the carbonic acid dissociation constants in seawater of Mehrbach et al. (1973), refitted by Dickson and Millero (1987). Calcification rates Skeletal calcification rate of all specimens was assessed by means of the buoyant weight (BW) technique (Jokiel et al. 1978, Davies 1989), using a 0.1 mg resolution balance (Mettler Toledo AB204 SFACT). An initial measurement of the BW was taken at the end of the acclimation phase (T-1); a second one right before setting the experimental pH and temperature conditions (T0) and subsequent weighing were conducted throughout the experiment (T1-T8; at 20, 49, 91, 143, 200, 258, 307 and 369 days; Fig. 1). During the measurements, temperature and salinity of seawater were constantly monitored using an YSI-30M probe. The BW of the corals was transformed to dry weight (DW) using the seawater density (δSW) and the specific value of the aragonite skeleton microdensity (δar) previously determined for each species (see next section), using the function: Calcification was normalized to the initial skeletal DW of coral fragments and expressed as mg CaCO3 g -1 d -1 using the exponential growth function: where G is the net calcification rate, W0 is the initial weight, Wn is the weight after n days and n is the time interval (in days) respect the beginning of the exposure (e.g. Reynaud et al. 2007; Maier et al. 2013). Specific skeleton microdensity and porosity One nubbin of each species and aquarium (3 replicates per treatment of each species) were randomly selected at the end of the experiment to estimate specific skeleton microdensity and porosity. For that, total enclosed volume, skeleton matrix volume and bulk density 99 Chapter 3 were also calculated for each sample using the equations described in Bucher et al. (1998). Coral nubbins were dipped in sodium hypochlorite during 2 days to remove the organic matter (OM) and then washed with distilled water. BW and DW of each sample were performed before and after their inclusion in molten paraffin wax (105–110ºC) to form a watertight barrier. In both cases, the BW was measured in distilled water at 20ºC with a specific gravity of ~1.00 g cm–3. Organic matter, protein and lipid content Also at the end of the experiment, two nubbins from each species and aquarium (6 replicates per treatment of each species) were randomly selected to assess potential effects of OA and temperature on the total OM of the coral, as well as on their protein and lipid content. Nubbins were frozen at -80ºC, subsequently freeze-dried, crushed and homogenised with a laboratory mortar and then stored frozen at -20ºC until biochemical analyses were conducted. The proportion of OM of each fragment was determined from ~ 250 mg of the homogenised material (including tissue and skeleton). To this end, samples were heated at 80ºC for 48 h and weighed to obtain the DW. Later, the samples were burned at 450ºC during 5 h and weighed again to obtain the ash weight (AW). OM was calculated as the difference between DW and AW (Slattery et al. 1995) and it is expressed as per cent with respect to the DW of the sample. For protein extraction, ~75 mg (DW) from each sample were homogenized in NaOH (1N) and heated to 90ºC during 30 min. Protein content was quantified colorimetrically using the BCA assay Kit (Interchim) with bovine serum albumin as standard. To determine the lipid content in the coral tissue, ~75 mg (DW) from each sample were homogenized in 3 ml of chloroform-methanol (2:1). The lipid content was quantified colorimetrically according to the method of Barnes and Blackstock (1973), using cholesterol as standard. All biochemical analyses were performed using spectrophotometry (Varian Cary 100 UV-Vis for lipids and a multimode microplate reader Tecan Infinite M200 for proteins). Results are presented as µg lipid or protein mg-1 OM. Statistical analyses Normality and homoscedasticity were tested in all cases using the Kolmogorov-Smirnov and Levene tests, respectively. A root squareor arctan-transformation was applied when normality was not fulfilled. A two-way ANOVA with pH and temperature as fixed factors was used for each species to examine differences between treatments in calcification rate at each sampling point, and in skeleton microdensity, porosity, OM, protein and lipid content of the tissue at the end of the experiment. Calcification rate results are expressed as mean ± standard error of the mean (SE), while the other parameters are expressed as mean ± standard deviation of the mean (SD). Statistical analyses were performed using the JMP 9.0.1 software (SAS Institute Inc., Cary, NC, USA). 100 Ocean acidification in azooxanthellate temperate corals Treatment Measured parameters Calculated parameters Temperature pH pHT TA Sal T pCO2 χCO2 DIC [CO2]aq [HCO3 -] [CO3 2-] Ωc ΩA Natural cycle 8.0 8.045 ± 0.012 2527 ± 16 37.9 ± 0.4 12.5 - 21.4 432 ± 13 441 ± 14 2249 ± 37 14.7 ± 1.5 2033 ± 54 201 ± 20 4.7 ± 0.5 3.1 ± 0.3 7.7 7.717 ± 0.016 2532 ± 15 37.9 ± 0.4 12.5 - 21.4 1028 ± 36 1049 ± 36 2414 ± 34 35.0 ± 4.3 2273 ± 44 106 ± 14 2.5 ± 0.3 1.6 ± 0.2 + 3ºC 8.0 8.017 ± 0.009 2526 ± 15 37.9 ± 0.3 15.8 - 23.9 466 ± 13 477 ± 13 2240 ± 37 14.6 ± 1.5 2017 ± 55 208 ± 20 4.9 ± 0.5 3.2 ± 0.3 7.7 7.706 ± 0.015 2531 ± 15 37.8 ± 0.3 15.8 - 23.9 1056 ± 38 1081 ± 42 2401 ± 29 33.4 ± 2.9 2255 ± 37 113 ± 12 2.6 ± 0.3 1.7 ± 0.2 pHT = pH in total scale; TA = total alkalinity (µmol/kg-SW); Sal = salinity; T = temperature (ºC); pCO2 = partial pressure of CO2 (µatm); χCO2 = mole fraction of CO2 in dry air (ppm); DIC = dissolved inorganic carbon (µmol/kg-SW); [CO2]aq = CO2 concentration in seawater (µmol/kg-SW); [HCO3 -] = bicarbonate ion concentration (µmol/kgSW); [CO3 2-] = carbonate ion concentration (µmol/kg-SW); ΩC = saturation state of seawater with respect to calcite; ΩA = saturation state of seawater with respect to aragonite. Table 1. Parameters of the seawater carbonate system in the aquaria for each treatment. Temperature results are expressed as a range and the rest of parameters as mean ± SD; N = 7. Results Carbonate system of seawater in the aquaria The seawater CO2 system conditions of all the treatments were monitored from periodical pHT and TA measurements in the laboratory (Table 1). Alkalinity and salinity values were similar in all treatments and remained constant throughout the experiment (with average values of ~2530 μmol kg-1 and 38, respectively). Temperature in the control ranged from 12.4°C in winter to 22.5°C in summer (similar to the natural cycle at 5 m depth at l’Estartit sampling site), and the high-temperature treatment was, on average, 2.9 ± 0.3°C above the control (Fig. 1). The values of pHT and TA in the control treatment were 8.045 ± 0.012 pH units and 2527 ± 16 μmol kg-1, respectively, and correspond to a partial pressure of CO2 (pCO2) around 430 μatm. The acidified treatments correspond to levels of pCO2 two and a half fold (~1030 μatm, pHT = 7.717 ± 0.016) with respect to the control. No aragonite undersaturation conditions were reached at any time throughout the experiment. Calcification rates During the previous phase to the adjustment of the experimental conditions (T-1 to T0), both species showed similar mean calcification rates (0.75 ± 0.04 and 0.88 ± 0.08 mg CaCO3 g-1day-1 for A. calycularis and L. pruvoti, respectively; mean ± SE, N = 57), coinciding with the 13º to 20ºC natural temperature rise associated to the spring season. No significant differences were detected between specimens from each species reared in the different aquaria on this preliminary phase (T0; Table S1). Over the experimental stage, the specimens from both species reared under control conditions displayed similar values of calcification throughout the warmest period (T3; Fig. 2a). However, when the whole annual cycle was considered, the mean calcification rate in control conditions decreased to 0.48 ± 0.06 and 0.52 ± 0.10 mg CaCO3 g-1 day-1 for A. calycularis and L. pruvoti, respectively (mean ± SE, N = 15; Fig. 2b). This indicates that about 2/3 of the annual growth occurs during the late spring-early fall period. Regarding the response of the organisms under different treatments, 101 Chapter 3 the effect of high temperature caused a ~25% significant reduction in the calcification rate of A. calycularis compared to control temperature at the end of the warmer season (ANOVA twoway, F(1,56) = 4.448, P = 0.040; Table S1; Fig. 2a), while no detrimental effect was observed due to low pH conditions or the combination of low-pH and high-temperature. Conversely, although L. pruvoti showed a decrease in the calcification rate in both acidified treatments regardless of temperature, these differences were not significant (ANOVA two-way, F(1,54) = 3.188, P = 0.080; Table S1; Fig. 2a). When the whole annual cycle was considered, the trends observed during the warm season were attenuated (Fig. 2b). In this case, no significant effect of temperature, pH or the combined effect was observed in any of the two species by integrating a full yearly cycle of growth (Table S1). None of the corals showed any signs of partial mortality during the experiment and no tank effect was detected between aquaria replicates of the same treatment in any of the analyses. Specific skeletal microdensity and porosity Mean skeletal microdensity was 2.3 ± 0.2 g cm-3 for A. calycularis and 2.5 ± 0.2 g cm-3 for L. pruvoti, (mean ± SD; N = 12). The mean skeleton porosity was 60 ± 5% for A. calycularis and 53 ± 5% for L. pruvoti (mean ± SD; N = 12). Microdensity values were not significantly affected by low-pH, high-temperature or the combination of both factors, either for A. calycularis or for L. pruvoti (Fig. 3a; Table S2). Conversely, the acidified treatment caused a significant increase in the porosity value of both tested species (ANOVA two-way, F(1,10) = 6.278, P = 0.041 and ANOVA two-way, F(1,11) = 8.484, P = 0.020 for A. calycularis and L. pruvoti, respectively; Fig 3b; Table S2). Figure 2. Calcification rate of Astroides calycularis and Leptopsammia pruvoti in all treatments after 91 (a) and 369 days (b) of exposure. Results are expressed as mean ± SE. 0.2 0.4 0.6 0.8 1(a) T3 (91 days) 0.2 0.4 0.6 0.8 Astroides calycularis Leptopsammia pruvoti (b) T8 (369 days) 102 Ocean acidification in azooxanthellate temperate corals 1.5 2 2.5 3(a) 30 40 50 60 70 (b) Astroides calycularis Leptopsammia pruvoti Organic matter, protein and lipid content A different pattern in the biochemical composition of the tissue was observed in both tested species at the end of the experiment, with L. pruvoti displaying high sensitivity to hightemperature conditions. While no significant variation between treatments was detected in the OM content in A. calycularis, the percentage of OM was significantly reduced to half in specimens of L. pruvoti reared under hightemperature conditions (ANOVA two-way, F(1,11) = 6.217, P = 0.037; Fig. 4a; Table S2). Both species showed a similar pattern in terms of the protein and lipid content of the tissue, with lower values displayed in specimens reared under high temperature conditions (Fig. 4b,c). However, these differences respect to control temperature conditions were significant only in L. pruvoti (ANOVA two-way, F(1,22) = 8.114, P = 0.010 and ANOVA two-way, F(1,22) = 11.806, P < 0.01 for proteins and lipid content, respectively; Table S2). Discussion Effects of OA and global warming on Calcification rates After 91 days of exposure to different experimental conditions and coinciding with the end of the warm season, both examined coral species were more resistant than presumed to low pH conditions expected by the year 2100. The calcification rate of A. calycularis at control conditions (low-temperature and high-pH) exhibited a mean value of 0.78 mg CaCO3 g-1 d-1, which was negatively affected by exposure to the high temperature treatment (+3ºC) but not to the low pH conditions (7.72 pHT units). Conversely, L. pruvoti exhibited a rather similar calcification at control temperature and pH treatment (mean: 0.77 mg CaCO3 g-1 d-1), and this rate was unaffected by the low pH or the high temperature conditions. These results are in agreement with the high thermal tolerance Figure 3. Skeletal microdensity (a) and porosity (b) of Astroides calycularis and Leptopsammia pruvoti under the different treatments at the end of the experiment. Results are expressed as mean ± SD. 103 Chapter 3 previously described for L. pruvoti (Caroselli et al. 2011, 2012) and with the lack of response to acidified conditions exhibited by other temperate corals species such as Cladocora caespitosa, although set pH was slightly higher (7.9 instead of 7.7; Rodolfo-Metalpa et al. 2010). However, further work has shown that the response in terms of calcification of temperate corals reared under low-pH seawater can be more variable than previously thought. The zooxanthellate C. caespitosa and O. patagonica reared in aquaria at pH of 7.8 and 20ºC exhibited a ~35% and 32% decrease in calcification rate, respectively (Movilla et al. 2012). Colonies of C. caespitosa transplanted for three months along a natural CO2 gradient exhibited a ~80% decrease in calcification rate at pH 7.8 and ~26ºC, whereas net calcification of this species became negative at 7.5 pH units (Rodolfo-Metalpa et al. 2011). Along a similar pH gradient, Balanophyllia europaea was more resistant to low pH conditions than C. caespitosa exhibiting a ~20% decrease in calcification rate at pH 7.8-7.7 (Rodolfo-Metalpa et al. 2011; Fantazzini et al. 2015) and a ~50% decrease at 7.5 pH units (Rodolfo-Metalpa et al. 2011). The effect of the high-temperature treatment on A. calycularis was not expected because it is considered a warm-water species with a narrow temperature tolerance and, on this basis, it has been suggested that its ability to spread to other areas could be related to rising temperatures (Bianchi 2007; Casado-Amezúa et al. 2012). However, our results suggest that, in the near future and at least during the warm period, calcification in this species Figure 4. Organic matter (a), protein (b) and lipid content (c) of Astroides calycularis and Leptopsammia pruvoti under the different treatments at the end of the experiment. 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Geobios 28:9-16 117 Chapter 4.1 Chapter 4.1 Resistance of Two Mediterranean Cold-Water Coral Species to LowpH Conditions Movilla J, Gori A, Calvo E, Orejas E, López-Sanz A, Domínguez-Carrió C, Grinyó J, Pelejero C (2014) Water 6:59-67 Abstract Deep-water ecosystems are characterized by relatively low carbonate concentration values and, due to ocean acidification (OA), these habitats might be among the first to be exposed to undersaturated conditions in the forthcoming years. However, until now, very few studies have been conducted to test how cold-water coral (CWC) species react to such changes in the seawater chemistry. The present work aims to investigate the mid-term effect of decreased pH on calcification of the two branching CWC species most widely distributed in the Mediterranean, Lophelia pertusa and Madrepora oculata. No significant effects were observed in the skeletal growth rate, microdensity and porosity of both species after 6 months of exposure. However, while the calcification rate of M. oculata was similar for all colony fragments, a heterogeneous skeletal growth pattern was observed in L. pertusa, the younger nubbins showing higher growth rates than the older ones. A higher energy demand is expected in these young, fast-growing fragments and, therefore, a reduction in calcification might be noticed earlier during long-term exposure to acidified conditions. (Photo credits by A. Gori) 118 Ocean acidification in cold-water corals 118 Introduction Due to the absorption by the ocean of a major part of the anthropogenic CO2 emitted to the atmosphere, the pH of global surface waters has already dropped by 0.1 units since the preindustrial era (Orr et al. 2005; Gattuso and Hansson 2011; Khatiwala et al. 2013). In the case of the Mediterranean Sea, this pH decrease seems to have been greater than in the global ocean (Touratier and Goyet 2011) and this area is actually considered one of the most sensitive regions to Ocean Acidification (OA) during the forthcoming years (Scheneider et al. 2007, 2010; Touratier and Goyet 2009; Calvo et al. 2011). On the other hand, it is expected that zones characterized by naturally low carbonate concentration values such as high-latitude and deep-water ecosystems will be amongst the first to experience undersaturated conditions (Guinotte et al. 2006; Steinacher et al. 2009; Yamamoto et al. 2012). This would be the case of cold-water coral (CWC) communities, which are found in areas characterized by very low aragonite saturation state values (ΩA) (Thresher et al. 2011; Form and Riebesell 2012). Therefore, studies evaluating the potential impact of OA on CWC populations are essential to determine the future of one of the most complex deep-sea habitats in the Mediterranean Sea. However, unlike the numerous experiments with tropical or temperate corals, where OA has been suggested to have diverse ecological and physiological impacts (Doney et al. 2009; Ries et al. 2009; Pelejero et al. 2010; Wicks and Roberts 2012; Parker et al. 2013), the responses of CWC to OA remain largely unexplored, with the few available studies showing contrasting results. For instance, the first short-term studies based on short incubations (between 24 h and 1 week-long) conducted on specimens of Lophelia pertusa and Madrepora oculata, found a reduction in the calcification rate ranging from 30% to 56% when the pH was dropped between 0.1 and 0.3 units (Form and Riebesell 2012; Maier et al. 2009, 2012). On the contrary, in the most recent short-term (between 24 h and 21 days) and in the two medium-term experiments (between 6 months and 9 months) conducted to date with the same species, no effects were observed when rearing corals at pH values similar to those expected by the end of the century (Form and Riebesell 2012; Maier et al. 2012, 2013; Hennige et al. 2013). Thus, despite the different results observed in the first short-term experiments, the outcomes so far from medium-term experiments evidence that some CWC species could be well adapted to possible changes in the chemical conditions of seawater. It is important to remark that, in these previous studies on possible effects of OA in the development of CWC, coral growth was exclusively assessed through measurements of calcification, with no studies so far reporting on possible effects on coral skeletal microdensity or porosity. In this context, we investigated the mid-term effect (6 months) of OA on the calcification rate, skeleton microdensity and porosity of two CWC species inhabiting deep Mediterranean waters, the branching corals L. pertusa and M. oculata. In this work, we compare our results with the two previous similar studies published with the same CWC species. We anticipate also possible long-term responses of these organisms based on experiments assessing the effect of OA on other Mediterranean corals, both temperate and CWC species. 119 Chapter 4.1 119 Materials and Methods Specimen Collection and Experimental Setup Colonies of L. pertusa and M. oculata (Fig. S1) were collected at 250 m depth in the Cap de Creus canyon (NW Mediterranean Sea) in July 2006 and September 2007 by means of the ROV “Phantom HD2 + 2” and the submersible “JAGO” (GEOMAR) respectively, on board of the Research Vessel “García del Cid”. Coral specimens were kept in aquaria with 50 μm filtered running natural seawater at salinity of 38, temperature of 12°C and in complete darkness as described by Olariaga et al. (2009). A mixed diet including frozen Cyclops, Mysis and Artemia (Ocean Nutrition™) were supplied 5 days a week. At the beginning of the experiment, 24 nubbins of L. pertusa (2–5 polyps) and 36 of M. oculata (8–20 polyps) were selected and randomly distributed and incubated together into six 30 liters aquaria subject to two pH treatments (8.10 and 7.81 for control and acidified conditions, respectively; 3 replicates per treatment). See Bramanti et al. (2013) for further details on the experimental setup. Discrete analyses of total alkalinity (TA) by potentiometric titration (Perez and Fraga 1987, Perez et al. 2000) and seawater pH by spectrophotometry (Clayton and Byrne 1993) were carried out periodically. The rest of the carbonate system parameters were calculated using the CO2calc software (v1.0.30 USGS). Skeletal Measurements Skeletal growth of all coral nubbins was assessed every two months by means of the buoyant weight technique (Jokiel et al. 1978; Figure S1. Colonies of Lophelia pertusa (left) and Madrepora oculata (right) in the experimental aquarium. 127 Chapter 4.2 Chapter 4.2 Differential response of two Mediterranean cold-water coral species to ocean acidification Movilla J, Orejas C, Calvo E, Gori A, López-Sanz A, Grinyó J, Dominguez-Carrió C, Pelejero C (2014) Coral Reefs 33:675-686 Abstract Cold-water coral (CWC) reefs constitute one of the most complex deep-sea habitats harboring a vast diversity of associated species. Like other tropical or temperate framework builders, these systems are facing an uncertain future due to several threats, such as global warming and ocean acidification (OA). In the case of Mediterranean CWC communities, the effect may be exacerbated due to the greater capacity of these waters to absorb atmospheric CO2 compared to the global ocean. Calcification in these organisms is an energy demanding process and it is expected that energy requirements will be greater as seawater pH and the availability of carbonate ions decrease. Therefore, studies assessing the effect of a pH decrease in skeletal growth and metabolic balance are critical to fully understand the potential responses of these organisms under a changing scenario. In this context, the present work aims to investigate the mediumlong term effect of a low pH scenario on calcification and the biochemical composition of two CWC from the Mediterranean, Dendrophyllia cornigera and Desmophyllum dianthus. After 314 days of exposure to acidified conditions, a significant decrease of 70% was observed in D. dianthus skeletal growth rate, while D. cornigera showed no differences between treatments. Instead, only subtle differences between treatments were observed in the organic matter amount (OM), lipid content, skeletal microdensity or porosity in both species, although due to the high variability of the results, these differences were not statistically significant. Our results also confirmed a heterogeneous effect of low pH on the skeletal growth rate of the organisms depending on their initial weight, suggesting that those specimens with high calcification rates may be the most susceptible to the negative effects of acidification. (Photo credits by E. Obis) 128 Ocean acidification in cold-water corals 128 Introduction The atmospheric CO2 concentration has been rising steadily since the beginning of the industrial era and has currently reached unprecedented levels which, according to predictions based on different scenarios, will be largely overcome in the coming decades (Peters et al. 2012). One effect of the increase in the atmospheric CO2 is the so-called Ocean Acidification (OA), which is related to the absorption of a major part of this CO2 by the oceans (Gattuso and Hansson 2011; Khatiwala et al. 2013). As a result, the surface pH of the global oceans has already dropped by 0.1 units compared to pre-industrial levels and it is expected that this decrease will reach 0.3 or 0.4 units by the end of the century (Orr 2011). Research on OA has expanded widely over the last years (Hoegh-Guldberg et al. 2007; Fabry et al. 2008; Gattuso and Hansson 2011), and declining ocean carbonate concentration associated to the decrease in pH has been suggested to have diverse ecological and physiological impacts on marine organisms (Doney et al. 2009; Ries et al. 2009; Pelejero et al. 2010; Wicks and Roberts 2012; Parker et al. 2013 and references therein). However, although OA is considered a global effect, it is likely that its severity will vary locally (Turley et al. 2010). For instance, highlatitude and deep-water ecosystems, which are characterized by naturally low carbonate concentration values, may be the first to be exposed to undersaturated conditions (Guinotte et al. 2006; Steinacher et al. 2009; Yamamoto et al. 2012). This is the case of the habitats of cold-water corals (CWC), which are typically characterised by aragonite saturation state (ΩA) values below 2.5 and, in some regions, close to 1 or even lower (Thresher et al. 2011; Form and Riebesell 2012). The highly productive conditions with elevated food availability that usually characterizes the zones inhabited by these CWC communities (Roberts et al. 2009; Thresher et al. 2011; Jantzen et al. 2013), such as the sampling area of this study (submarine canyon of Cap de Creus, NW Mediterranean; Company et al. 2008; Orejas et al. 2009; Gori et al. 2013a, 2013b), may offset the high costs involved in calcification and allow the presence of these organisms even when ΩA approaches 1 (Thresher et al. 2011). Nevertheless, models predict that the aragonite saturation horizons will become shallower as the oceans acidify (Orr et al. 2005), thus narrowing the depth range suitable for the correct development and calcification of CWC. In fact, it is predicted that most of these species will thrive exposed to waters undersaturated with respect to aragonite by the end of the century (Guinotte et al. 2006). In the case of the Mediterranean Sea, the high natural levels of total alkalinity (TA) (Schneider et al. 2007) and the fast turnover time of its waters (Bethoux et al. 2005) enhance the absorption of anthropogenic CO2. Because of that, the pH decrease since the preindustrial era in this semi-enclosed sea seems to have been even greater than the reduction in the global averaged surface ocean (Touratier and 129 Chapter 4.2 129 Goyet 2011), and it is considered to be one of the regions of the world that will show higher sensitivity to OA in the forthcoming years (Schneider et al. 2010; Touratier and Goyet 2009; Calvo et al. 2011). Unlike the numerous studies on tropical corals and, to a lesser extent on temperate corals, to date only a handful of studies have focused on evaluating the effects of OA in CWC. This is largely due to the difficulties of sampling in deep-sea areas, and only in recent years has it been possible to apply the available technology for this purpose (e.g. remotely operated vehicles (ROVs) or manned submersibles). To our knowledge, only six studies have experimentally assessed the calcification rates of CWC under different pH levels, all of them conducted with Lophelia pertusa and Madrepora oculata, with specimens from the Northeast Atlantic and the Mediterranean Sea. Initially, a first short-term study based on 24 h incubations conducted on specimens of L. pertusa, found a reduction in the calcification rate ranging from 30 to 56% after lowering the pH by 0.15 and 0.3 units, respectively (Maier et al. 2009). According to a subsequent study, despite the reduced growth observed during the first week of the experiment, L. pertusa was able to compensate the adverse effects of a lower pH treatment after a 6-month period, indicating certain plasticity to counter-balance the possible negative effects of acidification (Form and Riebesell 2012). However, in four more recent works on L. pertusa and M. oculata, no differences were observed in either short (between 24 h and 21 days) or long term (between 6 and 9 months) experiments when rearing them at pH values similar to those expected by the end of the century (Maier et al. 2012, 2013a; Hennige et al. 2013; Movilla et al. 2014). These studies suggested no effects of OA in CWC species and found evidence of acclimation in calcification to changes in carbonate chemistry. Regarding CWC species other than L. pertusa and M. oculata, their response to OA is still pending to be evaluated. Considering the potential threat of OA and the variability in the results observed in the experimental studies published so far on CWC, it is important to conduct further experiments, particularly on the other more abundant CWC species, to get a better understanding of the threats that OA could mean for them. Moreover, long-term experimental designs are desirable to detect the presence of mechanisms of acclimation (e.g. Dupont et al. 2012), and it is also important to consider other variables that could affect their response (e.g. energetic reserve depletion). In this context, the aim of this study was to investigate the effect of OA on two CWC species present in the Mediterranean: the yellow branching coral Dendrophyllia cornigera and the solitary cup coral Desmophyllum dianthus. This is the first time that the response of these two CWC species to OA is documented. We reared them in aquaria at lowered pH conditions and followed the evolution of skeletal growth rate, microdensity and porosity as well as the organic matter amount (OM) and lipid content after long-term exposure (314 days). 130 Ocean acidification in cold-water corals 130 Materials and methods Study species D. cornigera (Lamarck 1816) forms large polyps of 20–40 mm in diameter (Fig. S1) and colonies larger than 50 cm in height (Brito and Ocaña 2004). It can be found at depths of 200–800 m (Zibrowius 1980), but locally as shallow as 30 m (Castric-Fey 1996). In the western Mediterranean, D. cornigera has been mostly observed as isolated colonies or in small patches in the submarine canyons of the Gulf of Lions (Orejas et al. 2009; Gori et al. 2013a), but it can also form extensive aggregations of colonies (R. Aguilar, OCEANA pers. comm., G. Bavestrello pers. comm.). D. dianthus (Esper 1794) (also known in the Mediterranean as D. cristagalli; Milne Edwards and Haime 1848) is a cosmopolitan species with solitary polyps of 5 to 10 cm height and 15 to 30 mm in diameter (Fig. S1) found from 12 m to 4000 m depth (Risk et al. 2002; Jantzen et al. 2013 and references therein). In the Mediterranean, D. dianthus is relatively common and widespread between ~200 and 1200 m depth, both in the eastern and the western basins (Tursi et al. 2004; Taviani et al. 2005; Addamo et al. 2012). Specimen collection and experimental setup Colonies of D. cornigera were collected at 250 m depth in the Cap de Creus canyon (NW Mediterranean Sea) during the research cruises ‘Deep Coral I_Coral4’ and ‘HERMES IV_Coral8’ in July 2006 and September 2007 by means of the ROV ‘Phantom HD2+2’ and the submersible ‘JAGO’ (IFM-GEOMAR, Kiel, Germany) respectively, on board of the Research Vessel (RV) ‘Garcia del Cid’. Some specimens of D. dianthus were also sampled in the same area during these cruises, although most of them were collected in Malta Island at 300 m depth in September 2007 by means of an epibenthic sledge and kept alive on board of the RV ‘Urania’ during the cruise ‘MARCOS’ in April 2007. Coral specimens were transported to the Experimental Aquarium Zone (ZAE) at the Institut de Ciències del Mar (ICM-CSIC) in Barcelona and kept in aquaria with 50 μm filtered running natural seawater at ambient conditions of salinity and temperature for both CWC species (37.6 and 12ºC, respectively), and in complete darkness as described by Olariaga et al. (2009). A mixed diet including frozen Cyclops, Mysidacea and Artemia (Ocean Nutrition ™) was supplied 5 days a week. 49 days before the beginning of the experiment (carried out between December 2010 and November 2011; 314 days in total), 18 specimens of each species with a skeletal Figure S1. Specimens of Desmophyllum dianthus (left) and Dendrophyllia cornigera (right) in the aquaria (Photo credits by E. Obis). 131 Chapter 4.2 131 dry weight (DW) ranging between 2.8 and 30.6 g in D. cornigera and between 1.0 and 24.5 g in D. dianthus, were randomly distributed in 6 aquaria (30 litres each) subjected to two pH treatments (3 replicates per treatment). Treatment 1 consisted of a pH of 8.10 units (total scale), similar to the current natural pH value observed in the sampling area at similar depths and taken as control conditions, while treatment 2 consisted of a pH of 7.81 units, simulating the future Mediterranean decline predicted for the year 2100 following an A2 IPCC SRES scenario (Plattner et al. 2008). Seawater pH was gradually adjusted (0.03 units per day) to the desired pH levels by bubbling CO2 or CO2-free air for the acidic and control treatments, respectively (for further details on the experimental setup see Movilla et al. 2012; Bramanti et al. 2013). Analyses of TA by potentiometric titration (Pérez and Fraga 1987; Pérez et al. 2000) and seawater pH by spectrophotometry (Clayton and Byrne 1993) were carried out periodically (once a month during the first 3 months and every second month for the rest of the experiment). These values were used to calculate the rest of the parameters of the carbonate system in seawater using the CO2calc software (v1.0.30 USGS). Furthermore, to compare our control treatment with the natural range that the organisms experience in the field, temperature and salinity profiles were obtained with a Seabird CTD911 from surface to 400 m depth and water samples for pH and TA measurements were taken every 30 m with twenty-four 12-L Niskin bottles mounted in a rosette during the research cruise ‘FishJelly’ carried out in the sampling area in July 2011. Skeletal growth rates Skeletal growth of all coral nubbins was assessed by means of the buoyant weight (BW) technique (Jokiel et al. 1978; Davies 1989), using a 0.1 mg resolution balance (Mettler Toledo AB204 SFACT). An initial measurement of the BW was performed when the organisms were distributed in the aquaria (T-1), a second one when the pH conditions started to be adjusted (T0) and subsequent weighing were conducted every two months throughout the experiment (T1-T5). During the measurements, temperature and salinity of seawater were constantly monitored using a YSI-30M probe. The net BW of the corals (calculated as the total coral weight minus coral holder and glue weight) was transformed to DW using the specific value of the aragonite skeleton microdensity for each species previously determined (see next section). Calcification rates were calculated using an exponential growth function and normalized to the skeletal weight at the beginning of the experiment (T0) and at the beginning of each sampling period (T0-T4). Results are expressed as mg CaCO3 increase per gram per day. Specific skeleton microdensity and porosity After 314 days of incubation under experimental conditions (T5), one nubbin of 132 Ocean acidification in cold-water corals each species and aquarium (3 organisms from the control and 3 from the acidified treatment) were randomly selected to estimate specific skeleton microdensity and porosity (Bucher et al. 1998). Coral nubbins were dipped in sodium hypochlorite during 2 days to remove the OM and washed with distilled water. BW and DW of each sample were recorded before and after their inclusion in molten paraffin wax (105– 110ºC) to form a watertight barrier. In both cases, the BW was measured in distilled water at 20ºC with specific gravity of ~1.00 g cm–3. Total enclosed volume, skeleton matrix volume and bulk density were also calculated for each sample using the equations described in Bucher et al. (1998). Organic matter amount and lipid content At the end of the experiment (T5), one nubbin from each species and aquarium (again, 3 different organisms from each treatment) were randomly selected to assess potential effects of OA on the total coral OM, as well as on their lipid content. Nubbins were frozen at -80ºC, subsequently freeze-dried at -110ºC and 5 mbar of pressure, crushed and homogenised with a laboratory mortar and then stored frozen at -20ºC until biochemical analyses were conducted. The proportion of OM of each fragment was determined from ~300 mg of the homogenised material (including tissue and skeleton). To this end, samples were heated at 80ºC for 48 h and weighed to obtain the DW. Later the samples were burned at 450ºC during 5 h and weighed again to obtain the ash weight (AW). OM was calculated as the difference between DW and AW (Slattery et al. 1995) and is expressed as per cent with respect to the DW of the sample. To determine the lipid content in the coral tissue, ~71 mg (± 2 mg; DW) from each sample were homogenized in 3 ml of chloroform-methanol (2:1). The lipid content was quantified colorimetrically according to the method of Barnes and Blackstock (1973), using cholesterol as standard and a Varian Cary 100BIO spectrophotometer. Results are presented as µg of lipid per mg of OM. Statistical analyses Repeated-measures two-way ANOVA was used to test potential variations in skeletal growth rates between treatments and aquaria over time (aquarium was considered as a random factor nested within treatment) and means were compared between groups by post-hoc Tukey HSD test. A one-way ANOVA was performed to assess differences between treatments in the initial weight and the calcification rate of the nubbins, for each of the size ranges selected. Finally, a one-way ANOVA was also used to examine differences in skeleton microdensity, porosity, OM amount and lipid content between both treatments and both species. Normality and homoscedasticity were tested in all cases using the Kolmogorov-Smirnov and Levene tests, respectively. Statistical analyses were performed using JMP 9.0.1 software (SAS Institute Inc., Cary, NC, USA). 133 Chapter 4.2 Results Carbonate system of seawater in the aquaria The seawater CO2 system parameters of both treatments were calculated from periodical pHT and TA laboratory measurements (Table 1). In the control treatment, values of pHT and TA throughout the experiment (8.097 ± 0.021 pH units and 2536 ± 14 μmol kg-1, respectively) were very similar to those observed in the sampling area at similar depths during the ‘FishJelly’ cruise carried out in July 2011 (8.084 ± 0.017 pH units). The calculated χCO2 (mole fraction of CO2 in dry air) and ΩA values were 384 ± 24 ppm and 2.8 ± 0.1, respectively. For the acidified treatment, a decrease of pHT by ~0.3 units (7.808 ± 0.027 pH units) at a similar TA value (2543 ± 11 μmol kg-1) caused an increase in χCO2 up to 821 ± 53 ppm and a decrease of ΩA to 1.6 ± 0.1 (all parameters are expressed as mean ± SD; N=6). Temperature and salinity remained constant throughout the experiment (12.3 ± 0.3°C and 37.6 ± 0.1, respectively) and no undersaturation conditions with respect to the aragonite were reached at any time. Skeletal growth rates Under control conditions, the mean skeletal growth rate of D. cornigera was ~37 % lower than in D. dianthus (0.664 and 1.061 mg CaCO3 g -1 day-1, respectively). During the 49 days previous to setting the pH conditions (between T-1 and T0), no significant differences between treatments were observed in the calcification rates, neither for D. cornigera (repeated-measures ANOVA F1,12 = 1.895, p = 0.194; Table S1), nor for D. dianthus (F1,12 = 0.276, p = 0.609). Once subjected to the acidified conditions, D. cornigera did not show Measured parameters Treatment pH T TA S T Control 8.097 ± 0.021 2536 ± 14 37.6 ± 0.1 12.3 ± 0.2 (8.061 - 8.115) (2521 - 2551) (37.5 - 37.8) (11.9 - 12.5) High -CO2 7.808 ± 0.027 2543 ± 11 37.6 ± 0.1 12.3 ± 0.3 (7.787 - 7.854) (2524 - 2551) (37.4 - 37.7) (12.0 - 12.6) Calculated parameters Treatment pCO 2 χCO 2 DIC [CO 2 ] aq [HCO 3 -] [CO 3 2-] Ω C ΩA Control 379 ± 23 384± 24 2280 ± 19 15.2 ± 0.9 2080 ± 23 185 ± 7 4.3 ± 0.2 2.8 ± 0.1 High -CO2 810 ± 53 821 ± 53 2421 ± 15 32.4 ± 2.2 2284 ± 18 105 ± 6 2.5 ± 0.2 1.6 ± 0.1 pHT = pH in total scale at in situ temperature; TA = total alkalinity (µmol/kg-SW); S = salinity; T = temperature (ºC); pCO2 = partial pressure of CO2 of air in equilibrium with seawater (ppm); χCO2 = mole fraction of CO2 in dry air (ppm); DIC = dissolved inorganic carbon (µmol/kg-SW); [CO2]aq = CO2 concentration in seawater (µmol/kg-SW); [HCO3 -] = bicarbonate ion concentration (µmol/kg-SW); [CO3 2-] = carbonate ion concentration (µmol/kg-SW); ΩC = saturation state of seawater with respect to calcite; ΩA = saturation state of seawater with respect to aragonite. Table 1. Parameters of the seawater carbonate system in the aquaria for each treatment. Total alkalinity, pHT, salinity and temperature were used to calculate all other parameters using the CO2calc software (USGS). For the four measured parameters we report the values as mean ± SD (N = 6) and range (in brackets). All other calculated parameters are expressed as mean ± SD (N = 6). 134 Ocean acidification in cold-water corals any significant differences in the calcification rate between treatments either taking as reference the weight at the beginning of the experiment (Fig. 1a) or at each sampling time (Fig. 1b). Conversely, the response observed in D. dianthus under the acidified treatment was different depending on the method used to normalize the skeletal growth rate. While no differences between treatments were observed standardizing the calcification rate with respect to the initial weight (Fig. 1a), a significant reduction was detected during the second half of the experiment (Tukey test, F5,53 = 3.061, p = 0.018) when normalizing the average calcification rate with respect to the weight at each sampling time (Fig. 1c), with a decline of 72% compared with control conditions after 314 days of exposure to the acidified treatment (repeated-measures ANOVA F1,12 = 0.575, p = 0.022; Table S1). During the experimental period (T0-T5), an effect on the calcification was detected in both species regardless of the treatment (repeated-measures ANOVA F5,8 = 3.895, p = 0.012 and F5,8 = 3.096, p = 0.023 for D. cornigera and D. dianthus, respectively; Table S1). In addition, a strong correlation between the cumulative skeletal growth rate after 314 days of exposure and the initial weight of the nubbins at the beginning of the experiment (T0) was observed in both species, where smaller nubbins exhibited greater calcification rates than those with a higher initial weight (Fig. 2). This allowed us to clearly differentiate two trends depending on the initial size of the nubbins, keeping the same number of Figure 1. (a) Skeletal growth rates respect to the weight at the beginning of the experiment of D. cornigera (diamonds) and D. dianthus (dots) under control (white symbols, solid line) and acidified conditions (grey symbols, dashed line). (b, c) Discrete calcification rates respect to the weight at the beginning of each sampling period showed by D. cornigera (b) and D. dianthus (c) under control (white bars) and acidified treatment (grey bars). Shaded area indicates calcification during the acclimation period (49 days). Significant differences are indicated with an asterisk (p < 0.05). Mean ± SE; N = 9. 135 Chapter 4.2 individuals from both treatments (and both species) in each size range. Nubbins with an initial weight < or > than 7 g (DW) were classified as small or large fragments, respectively. No significant differences between treatments were detected in the initial weight of the nubbins for any of the size range selected (Table S2) and hence the effect of the acidified treatment on the calcification rate was analyzed separately for each trend. D. cornigera showed no significant differences between treatments during the last period of the experiment (T5; Fig. 3a), neither in small fragments with rapid growth rates, nor in larger fragments that showed a minimal growth. Conversely, a different result was observed in D. dianthus (Fig. 3b), supporting our previous findings during the last sampling time of the experiment (Fig. 1c). While the skeletal growth of the nubbins with a larger initial size was similar in both treatments, significant differences were detected in the calcification rate of the small fragments subjected to different pH levels (ANOVA, F1,11 = 8.642, p = 0.015), pointing out that the differences between treatments previously described are mainly due to differences in the calcification of the smaller nubbins and not to the larger ones. Specific skeletal microdensity and porosity No significant differences between treatments were observed in microdensity, neither for D. cornigera (ANOVA, F1,5 = 0.066, p = 0.814) nor for D. dianthus (F1,5 = 0.198, p = 0.680; Fig. Figure 2. Correlation between the initial weight of the fragments and their calcification rate computed for the total duration of the experiment (314 days), for Dendrophyllia cornigera (diamonds, solid line) and Desmophyllum dianthus (dots, dashed line) under control and acidified conditions (white and grey symbols, respectively). The shaded area indicates the distinction between small (< 7g) and large nubbins. 142 Ocean acidification in cold-water corals after stress episodes such as the presence of chemical compounds, intense light conditions or due to rough handling (Lang 1973; Bak and Elgershuizen 1976; Chadwick 1987), but it has never been associated before with a decrease in pH. This observation suggests that although growth rates did not change between treatments in D. cornigera and in the larger polyps of D. dianthus, other adverse effects associated with a lower pH could have been in play. Indeed, it is known that OA could affect benthic organisms in different ways, and that synergies with other stressors could make them more susceptible to other threats (e.g. Wicks and Roberts 2012). For instance, a suppression of metabolism and complex effects on genes involved in calcification have been detected in response to acidification, both in zooxanthellate corals (Kaniewska et al. 2012; Moya et al. 2012) and CWC (Carreiro-Silva et al 2014). Future experiments addressing changes in gene expression would add valuable information to the effects of OA on CWC species at different levels. Effect of OA on the energy reserves of CWC No significant differences between treatments were detected in the OM amount, or lipid content in the tissue at the end of the experiment neither for D. cornigera, nor for D. dianthus (Fig. 5). It might be possible that the absence of significant differences was due to the low number of samples available for the biochemical analyses (only 3 replicates per treatment) and the high natural variability observed in the individual response. However, since this is the first time that biochemical analyses of these species have been tested, these results should be taken to provide insight into the potential effect of OA on energy reserves of CWC. Our results suggest that on a long-term basis, the acidified conditions affected neither the amount, nor the composition of the coral tissue. The integrity of the external tissue has been suggested as a mechanism of passive protection, preventing direct exposure of the underlying skeleton to seawater and therefore, modulating the effects of OA (Ries et al. 2009; Hofmann et al. 2010). Conversely, in our experiment, although the tissue of the specimens remained intact, a clear effect of low pH on skeletal coral growth was observed in the smaller polyps of D. dianthus. A similar response has been observed in previous studies testing the effects of OA on other Mediterranean coral species, such as Cladocora caespitosa and Oculina patagonica (Movilla et al. 2012), as well as on the octocoral Corallium rubrum (Bramanti et al. 2013) where, despite the decreased skeletal growth rate, the external tissue remained undamaged during the entire exposure time. However, in the latter study and in contrast to the results showed in the present study, a significant increase in the OM amount was observed in those specimens reared under low pH conditions, which led the authors to suggest that this increase may occur at the expense of a reduction in the calcification rate (Bramanti et al. 2013). Total lipid content in tropical corals has been shown to fluctuate in response to seasonal changes in temperature, nutrient levels, depth, 143 Chapter 4.2 diurnal light variability and turbidity (Cooper et al. 2011 and references therein). Recent experiments have speculated on the possibility that CWC may, at least temporarily, maintain the growth rate under acidified environments by mobilizing the lipid reserves located in their tissue (Hennige et al. 2013; Maier et al. 2013b). However, as stated in Maier et al. (2013b), this mechanism should be successful only on a short-term basis, thereby maintaining the calcification rate until lipid reserves are consumed. Our results suggest that, if the adverse effect persists over a long-term, it is possible that a reduction in calcification rate ends up occurring, regardless of their lipid stock. These species-specific responses in the metabolic reserves may contribute to the differential resistance of corals under stressful conditions. Furthermore, the changes in ocean chemistry conditions expected by the end of the century will not occur in isolation, and it is likely that these organisms will have to cope with synergetic effects caused by the increase in atmospheric CO2. In particular, the rise in temperature associated with OA could largely reduce the distribution area of CWC (Wild et al. 2011). The higher metabolic rates of CWC expected under elevated temperatures (Dodds et al. 2007), together with lower prey availability due to longer stratification periods affecting vertical migration of mesopelagic zooplankton (Roberts et al. 2009), would place them in harsh conditions in the forthcoming decades. Moreover, as mentioned before, the Mediterranean Sea is believed to be one of the world’s most sensitive regions to global warming and OA (Calvo et al. 2011), so CWC communities inhabiting these waters might be amongst the first to respond to such effects. Further investigation is required to understand the biochemical balance response of CWC to synergetic effects of different stressors such as acidification, temperature or food availability in the near future. Acknowledgements We want to thank A. Olariaga for his suggestions during the experimental set-up, to M. Dalmau, F.X. Capdevila, and L. Pedret, for helping with coral maintenance, to M. Delgado (ZAE, ICM) for technical assistances and to the Marine Technology Unit (UTM, CSIC), M. Taviani (ISMAR-CNR, Bologna) and the crews of RVs ‘Urania’, ‘Garcia del Cid’ and the ‘JAGO-Team’ (IFM-GEOMAR) for their support during coral collection. We are also in debt with P. Siles for valuable advice in coral maintenance and behavior. This research was supported by the European Projects HERMES (Goce-CT-2005511234-I), HERMIONE (grant agreement number 226354), the Spanish Projects CTM2009-08849/MAR and CTM2012-32017 and by the Marine Biogeochemistry and Global Change research group (Generalitat de Catalunya, 2009SGR142). JM was funded by a FPI studentship (BES-2007-16537) and AG by an I3P studentship (Ref. I3P-BPD2005) from the Spanish Government. 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Oceanogr. 11:1–284 151 Synthesis and discussion SYNTHESIS OF RESULTS AND GENERAL DISCUSSION This thesis presents a first compilation of possible midto long-term responses of Mediterranean corals to the acidified and warming conditions expected by the end of this century. Ultimately, this study attempts to draw a picture of the ecological implications that the higher energetic costs of calcification will represent in a high-CO2 ocean. For that purpose, we applied a multidisciplinary approach to assess the effects (1) on calcification rate, (2) on the calcium carbonate supporting framework (skeleton microstructure, microdensity and porosity) and (3) on the biochemical balance (protein, carbohydrate, lipid and fatty acids content of the tissue) of several representative species from the shallower coralligenous and the deep-water benthic ecosystems present in the Mediterranean (Table 1). Chapter 1 presents the comparative response to OA and the subsequent resilience of the two zooxanthellate coral species capable of constituting the main framework of the Mediterranean coralligenous, the endemic Cladocora caespitosa and the nonnative Oculina patagonica, in order to estimate potential changes in the composition of the community in a near future. Chapter 2 shows the effects of acidification on the endangered Mediterranean red coral (Corallium rubrum) and assesses what the consequences would be for its harvestable stock. Chapter 3 focuses on the response of two Mediterranean azooxanthellate temperate corals, the colonial Astroides calycularis and the solitary Leptopsammia pruvoti to the combined effects of high temperature and low pH over an annual cycle. Finally, Chapter 4 investigates the response to low-pH conditions of four CWC species, Lophelia pertusa, Madrepora oculata, Dendrophyllia cornigera and Desmophyllum dianthus, and attempts to identify which members of their populations are likely to be the most affected. Overall, our results show some degree of variability, not only at speciesspecific level, suggesting that corals may use different physiological pathways to cope with OA, but also within the same species, which in some cases could be related with the age, seasonal cycle, metabolism or genetic conditions of the organisms. In this section, we present an overview of the main results obtained in the four chapters summarized above and an integrative discussion of all of them. Ocean acidification effects on coral calcification In this section, we examine the response in calcification rate and skeletal structure of different species of Mediterranean corals, including temperate zooxanthellate and azooxanthellate specimens, as well as deep-sea corals. Under control conditions, zooxanthellate corals showed higher calcification rates