(Supplemental Table 2) Plant characteristics, GHG fluxes, and soil chemical and microbial properties in experimental treatments in Alexandra Fiord

We evaluated above- and belowground ecosystem changes in a 16 year, combined fertilization and warming experiment in a High Arctic tundra deciduous shrub heath (Alexandra Fiord, Ellesmere Island, NU, Canada). Soil emissions of the three key greenhouse gases (GHGs) (carbon dioxide, methane, and nitrous oxide) were measured in mid-July 2009 using soil respiration chambers attached to a FTIR system. Soil chemical and biochemical properties including Q10 values for CO2, CH4, and N2O, Bacteria and Archaea assemblage composition, and the diversity and prevalence of key nitrogen cycling genes including bacterial amoA, crenarchaeal amoA, and nosZ were measured. Warming and fertilization caused strong increases in plant community cover and height but had limited effects on GHG fluxes and no substantial effect on soil chemistry or biochemistry. Similarly, there was a surprising lack of directional shifts in the soil microbial community as a whole or any change at all in microbial functional groups associated with CH4 consumption or N2O cycling in any treatment. Thus, it appears that while warming and increased nutrient availability have strongly affected the plant community over the last 16 years, the belowground ecosystem has not yet responded. This resistance of the soil ecosystem has resulted in limited changes in GHG fluxes in response to the experimental treatments.

Impact of ocean acidification and warming on respiration, heart rate and acid-base status of Mytilus edulis from the North Sea

Anthropogenic climate change confronts marine organisms with rapid trends of concomitant warming and CO2 induced ocean acidification. The survival and distribution of species partly depend on their ability to exploit their physiological plasticity during acclimatization. Therefore, in laboratory studies the effects of simulated future ocean acidification on thermal tolerance, energy metabolism and acid-base regulation capacity of the North Sea population of the blue mussel Mytilus edulis were examined. Following one month of pre-acclimation to 10 °C and control CO2 levels, mussels were exposed for two weeks to control and projected oceanic CO2 levels (390, 750 and 1120 µatm) before being subjected to a stepwise warming protocol between 10 °C and 31 °C (+ 3 °C each night). Oxygen consumption and heart rates, anaerobic metabolite levels and haemolymph acid-base status were determined at each temperature. CO2 exposure left oxygen consumption rate unchanged at acclimation temperature but caused a somewhat stronger increase during acute warming and thus mildly higher Q10-values than seen in controls. Interestingly, the thermally induced limitation of oxygen consumption rate set in earlier in normocapnic than in hypercapnic (1120 µatm CO2) mussels (25.2 °C vs. 28.8 °C), likely due to an onset of metabolic depression in the control group following warming. However, the temperature induced increase in heart rate became limited above 25 °C in both groups indicating an unchanged pejus temperature regardless of CO2 treatment. An upper critical temperature was reached above 28 °C in both treatments indicated by the accumulation of anaerobic metabolites in the mantle tissue, paralleled by a strong increase in haemolymph PCO2 at 31 °C. Ocean acidification caused a decrease in haemolymph pH. The extracellular acidosis remained largely uncompensated despite some bicarbonate accumulation. In all treatments animals developed a progressive warming-induced extracellular acidosis. A stronger pH drop at around 25 °C was followed by stagnating heart rates. However, normocapnic mussels enhanced bicarbonate accumulation at the critical limit, a strategy no longer available to hypercapnic mussels. In conclusion, CO2 has small effects on the response patterns of mussels to warming, leaving thermal thresholds largely unaffected. High resilience of adult North Sea mussels to future ocean acidification indicates that sensitivity to thermal stress is more relevant in shaping the response to future climate change.

Experimental data on photophysiology and growth rates of Pararotalia calcariformata

The eastern Mediterranean is a hotspot of biological invasions. Numerous species of Indo-pacific origin have colonized the Mediterranean in recent times, including tropical symbiont-bearing foraminifera. Among these is the species Pararotalia calcariformata. Unlike other invasive foraminifera, this species has been discovered only two decades ago and is restricted to the eastern Mediterranean coast. Combining ecological, genetic and physiological observations, we attempt to explain the recent invasion of this species in the Mediterranean Sea. Using morphological and genetic data, we confirm the species attribution to P. calcariformata McCulloch 1977 and identify its symbionts as a consortium of diatom species dominated by Minutocellus polymorphus. We document photosynthetic activity of its endosymbionts using Pulse Amplitude Modulated Fluorometry and test the effects of elevated temperatures on growth rates of asexual offspring. The culturing of asexual offspring for 120 days shows a 30-day period of rapid growth followed by a period of slower growth. A subsequent 48-day temperature sensitivity experiment indicates a similar developmental pathway and high growth rate at 28°C, whereas an almost complete inhibition of growth was observed at 20°C and 35°C. This indicates that the offspring of this species may have lower tolerance to cold temperatures than what would be expected for species native to the Mediterranean. We expand this hypothesis by applying a Species Distribution Model (SDM) based on modern occurrences in the Mediterranean using three environmental variables: irradiance, turbidity and yearly minimum temperature. The model reproduces the observed restricted distribution and indicates that the range of the species will drastically expand westwards under future global change scenarios. We conclude that P. calcariformata established a population in the Levant because of the recent warming in the region. In line with observations from other groups of organisms, our results indicate that continued warming of the eastern Mediterranean will facilitate the invasion of more tropical marine taxa into the Mediterranean, disturbing local biodiversity and ecosystem structure.

Impacts of ocean warming and acidification on the larval development of the barnacle Amphibalanus improvisus

The world's oceans are warming and becoming more acidic. Both stressors, singly or in combination, impact marine species, and ensuing effects might be particularly serious for early life stages. To date most studies have focused on ocean acidification (OA) effects in fully marine environments, while little attention has been devoted to more variable coastal ecosystems, such as the Western Baltic Sea. Since natural spatial and temporal variability of environmental conditions such as salinity, temperature or pCO2 impose more complex stresses upon organisms inhabiting these habitats, species can be expected to be more tolerant to OA (or warming) than fully marine taxa. We present data on the variability of salinity, temperature and pH within the Kiel Fjord and on the responses of the barnacle Amphibalanus improvisus from this habitat to simulated warming and OA during its early development. Nauplii and cyprids were exposed to different temperature (12, 20 and 27°C) and pCO2 (nominally 400, 1250 and 3250 µatm) treatments for 8 and 4 weeks, respectively. Survival, larval duration and settlement success were monitored. Warming affected larval responses more strongly than OA. Increased temperatures favored survival and development of nauplii but decreased survival of cyprids. OA had no effect upon survival of nauplii but enhanced their development at low (12°C) and high (27°C) temperatures. In contrast, at the intermediate temperature (20°C), nauplii were not affected even by 3250 µatm pCO2. None of the treatments significantly affected settlement success of cyprids. These experiments show a remarkable tolerance of A. improvisus larvae to 1250 µatm pCO2, the level of OA predicted for the end of the century.

Nicotinamide nucleotide and adenylate concentrations in mante, siphon and gill tissue of old and young Laternula elliptica individuals under control and experimental conditions

Future oceans are predicted to contain less oxygen than at present. This is because oxygen is less soluble in warmer water and predicted stratification will reduce mixing. Hypoxia in marine environments is thus likely to become more widespread in marine environments and understanding species-responses is important to predicting future impacts on biodiversity. This study used a tractable model, the Antarctic clam, Laternula elliptica, which can live for 36 years, and has a well-characterized ecology and physiology to understand responses to hypoxia and how the effect varied with age. Younger animals had a higher condition index, higher adenylate energy charge and transcriptional profiling indicated that they were physically active in their response to hypoxia, whereas older animals were more sedentary, with higher levels of oxidative damage and apoptosis in the gills. These effects could be attributed, in part, to age-related tissue scaling; older animals had proportionally less contractile muscle mass and smaller gills and foot compared with younger animals, with consequential effects on the whole-animal physiological response. The data here emphasize the importance of including age effects, as large mature individuals appear to be less able to resist hypoxic conditions and this is the size range that is the major contributor to future generations. Thus, the increased prevalence of hypoxia in future oceans may have marked effects on benthic organisms' abilities to persist and this is especially so for long-lived species when predicting responses to environmental perturbation.

Impact of ocean acidification and warming on the Mediterranean mussel (Mytilus galloprovincialis)

In order to assess the effects of ocean acidification and warming on the Mediterranean mussel (Mytilus galloprovincialis), specimens were reared in aquarium tanks and exposed to elevated conditions of temperature (+3°C) and acidity (-0.3 pH units) for a period of 10 months. The whole system comprised a factorial experimental design with 4 treatments (3 aquaria per treatment): control, lowered pH, elevated temperature, and lowered pH/elevated temperature. Mortality was estimated on a weekly basis and every 2 months, various biometrical parameters and physiological processes were measured: somatic and shell growth, metabolic rates and body fluid acid-base parameters. Mussels were highly sensitive to warming, with 100% mortality observed under elevated temperature at the end of our experiment in October. Mortality rates increased drastically in summer, when water temperature exceeded 25°C. In contrast, our results suggest that survival of this species will not be affected by a pH decrease of 0.3 in the Mediterranean Sea. Somatic and shell growth did not appear very sensitive to ocean acidification and warming during most of the experiment, but were reduced, after summer, in the lowered pH treatment. This was consistent with measured shell net dissolution and observed loss of periostracum, as well as uncompensated extracellular acidosis in the lowered pH treatment indicating a progressive insufficiency in acid-base regulation capacity. However, based on the present dataset, we cannot elucidate if these decreases in growth and regulation capacities after summer are a consequence of lower pH levels during that period or a consequence of a combined effect of acidification and warming. To summarize, while ocean acidification will potentially contribute to lower growth rates, especially in summer when mussels are exposed to sub-optimal conditions, ocean warming will likely pose more serious threats to Mediterranean mussels in this region in the coming decades.

Mesocosm experiment on warming and acidification effects in 2012: Zooplankton data

Concerns about increasing atmospheric CO2 concentrations and global warming have initiated studies on the consequences of multiple-stressor interactions on marine organisms and ecosystems. We present a fully-crossed factorial mesocosm study and assess how warming and acidification affect the abundance, body size, and fatty acid composition of copepods as a measure of nutritional quality. The experimental set-up allowed us to determine whether the effects of warming and acidification act additively, synergistically, or antagonistically on the abundance, body size, and fatty acid content of copepods, a major group of lower level consumers in marine food webs. Copepodite (developmental stages 1-5) and nauplii abundance were antagonistically affected by warming and acidification. Higher temperature decreased copepodite and nauplii abundance, while acidification partially compensated for the temperature effect. The abundance of adult copepods was negatively affected by warming. The prosome length of copepods was significantly reduced by warming, and the interaction of warming and CO2 antagonistically affected prosome length. Fatty acid composition was also significantly affected by warming. The content of saturated fatty acids increased, and the ratios of the polyunsaturated essential fatty acids docosahexaenoic- (DHA) and arachidonic acid (ARA) to total fatty acid content increased with higher temperatures. Additionally, here was a significant additive interaction effect of both parameters on arachidonic acid. Our results indicate that in a future ocean scenario, acidification might partially counteract some observed effects of increased temperature on zooplankton, while adding to others. These may be results of a fertilizing effect on phytoplankton as a copepod food source. In summary, copepod populations will be more strongly affected by warming rather than by acidifying oceans, but ocean acidification effects can modify some temperature impacts

Land based mesocosm experiment studying the simultaneous impact of warming and acidification on the planktonic food web of the Eastern Mediterranean

A land based mesocosm experiment focusing on the study of the simultaneous impact of warming and acidification on the planktonic food web of the Eastern Mediterranean took place in August-September 2013 at the mesocosm facilities of HCMR in Crete (CRETACOSMOS). Two different pCO2 (present day and predicted for year 2100) were applied in triplicate mesocosms of 3 m**3. This was tested in two different temperatures (ambient seawater T and ambient T plus 3°C). Twelve mesocosms in total were incubated in two large concrete tanks. Temperature was controlled by sophisticated, automated systems. A large variety of chemical, biological and biochemical variables were studied, including salinity, temperature, light and alkalinity measurements, inorganic and organic, particulate and dissolved, nutrient analyses, biological stock (Chla concentration, enumeration and community composition of microbial, phyto- and zooplankton organisms) and rate (primary, bacterial, viral production, copepod egg production, zooplankton grazing, N2 fixation, P uptake) measurements, bacterial DNA extraction and phytoplankton transcriptomics, calcifiers analyses. Twenty three scientists from 6 Institutes and 5 countries participated in this experiment.

Experiment: Adaptation of a globally important coccolithophore to ocean warming and acidification

Although oceanwarming and acidification are recognized as two major anthropogenic perturbations of today's oceanswe know very little about how marine phytoplankton may respond via evolutionary change.We tested for adaptation to ocean warming in combination with ocean acidification in the globally important phytoplankton species Emiliania huxleyi. Temperature adaptation occurred independently of ocean acidifcation levels. Exponential growth rates were were up to 16% higher in populations adapted for one year to warming when assayed at their upper thermal tolerance limit. Particulate inorganic (PIC) and organic (POC) carbon production was restored to values under present-day ocean conditions, owing to adaptive evolution, and were 101% and 55% higher under combined warming and acidification, respectively, than in non-adapted controls. Cells also evolved to a smaller size while they recovered their initial PIC:POC ratio even under elevated CO2. The observed changes in coccolithophore growth, calcite and biomass production, cell size and elemental composition demonstrate the importance of evolutionary processes for phytoplankton performance in a future ocean. At the end of a 1-yr temperature selection phase, we conducted a reciprocal assay experiment in which temperature-adapted asexual populations were compared to the respective non-adapted control populations under high temperature, and vice versa (1. Assay Data, Dataset #835336). Mean exponential growth rates ? in treatments subjected to high temperature increased rapidly under all high temperature-CO2 treatment combinations during the temperature selection phase (2. time series, Dataset #835339).