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The combination of ocean acidification (OA) and global warming is expected to have a significant effect on the diversity and functioning of marine ecosystems, particularly on calcifying algae such as rhodoliths (maërl) that form extensive beds worldwide, from polar to tropical regions. In addition, the increasing frequency of extreme events, such as heatwaves, threaten coastal ecosystems and may affect their capacity to fix blue carbon. The few studies where the simultaneous effects of both temperature and CO2 were investigated have revealed contradictory results. To assess the effect that high temperature spells can have on the maërl beds under OA, we tested the short-time effects of temperature and CO2 on the net photosynthesis, respiration and calcification of the recently described species Phymatolithon lusitanicum, the most common maërl species of southern Portugal. Photosynthesis, calcification and respiration increased with temperature, and the differences among treatments were enhanced under high CO2. We found that in the short term, the metabolic rates of Phymatolithon lusitanicum will increase with CO2 and temperature as will the coupling between calcification and photosynthesis. However, under high CO2, this coupling will favor photosynthesis over calcification, which, in the long term, can have a negative effect on the blue carbon fixing capacity of the maërl beds from southern Portugal. Data_SordoetalData of Net and gross production and respiration under control and high CO2 conditions (Excel sheet 1), Data of light and dark calcification under control and high CO2 conditions (Excel sheet 2), Carbonate system parameters in each treatment. Salinity, temperature (T), total alkalinity (TA) and pH (NBS scale) were measured, while dissolved inorganic carbon (DIC) and saturation state of seawater with respect to aragonite (ΩAR) were calculated using the programe CO2SYS (Excel sheet 3)

Mäerl/rhodolith beds are protected habitats that may be affected by ocean acidification (OA), but it is still unclear how the availability of CO2 will affect the metabolism of these organisms. Some of the inconsistencies found among OA experimental studies may be related to experimental exposure time and synergetic effects with other stressors. Here, we investigated the long‐term (up to 20 months) effects of OA on the production and calcification of the most common mäerl species of southern Portugal, Phymatolithon lusitanicum. Both the photosynthetic and calcification rates increased with CO2 after the first 11 months of the experiment, whereas respiration slightly decreased with CO2. After 20 months, the pattern was reversed. Acidified algae showed lower photosynthetic and calcification rates, as well as lower accumulated growth than control algae, suggesting that a metabolic threshold was exceeded. Our results indicate that long‐term exposure to high CO2 will decrease the resilience of Phymatolithon lusitanicum. Our results also show that shallow communities of these rhodoliths may be particularly at risk, while deeper rhodolith beds may become ocean acidification refuges for this biological community. In order to allow full comparability with other ocean acidification data sets, the R package seacarb (Gattuso et al, 2016) was used to compute a complete and consistent set of carbonate system variables, as described by Nisumaa et al. (2010). In this dataset the original values were archived in addition with the recalculated parameters (see related PI). The date of carbonate chemistry calculation by seacarb is 2018-08-06. Supplement to: Sordo, Laura; Santos, Rui; Barrote, Isabel; Silva, João (2018): High CO2 decreases the long-term resilience of the free-living coralline algae Phymatolithon lusitanicum. Ecology and Evolution, 8(10), 4781-4792

Most ocean acidification (OA) experimental systems rely on pH as an indirect way to control CO2. However, accurate pH measurements are difficult to obtain and shifts in temperature and/or salinity alter the relationship between pH and pCO2. Here we describe a system in which the target pCO2 is controlled via direct analysis of pCO2 in seawater. This direct type of control accommodates potential temperature and salinity shifts, as the target variable is directly measured instead of being estimated. Water in a header tank is permanently re-circulated through an air-water equilibrator. The equilibrated air is then routed to an infrared gas analyzer (IRGA) that measures pCO2 and conveys this value to a Proportional-Integral-Derivative (PID) controller. The controller commands a solenoid valve that opens and closes the CO2 flush that is bubbled into the header tank. This low-cost control system allows the maintenance of stabilized levels of pCO2 for extended periods of time ensuring accurate experimental conditions. This system was used to study the long term effect of OA on the coralline red algae Phymatolithon lusitanicum. We found that after 11 months of high CO2 exposure, photosynthesis increased with CO2 as opposed to respiration, which was positively affected by temperature. Results showed that this system is adequate to run long-term OA experiments and can be easily adapted to test other relevant variables simultaneously with CO2, such as temperature, irradiance and nutrients. In order to allow full comparability with other ocean acidification data sets, the R package seacarb (Gattuso et al, 2015) was used to compute a complete and consistent set of carbonate system variables, as described by Nisumaa et al. (2010). In this dataset the original values were archived in addition with the recalculated parameters (see related PI). The date of carbonate chemistry calculation is 2016-11-03. Supplement to: Sordo, Laura; Santos, Rui; Reis, Joao; Shulika, Alona; Silva, João (2016): A direct CO2 control system for ocean acidification experiments: testing effects on the coralline red algae Phymatolithon lusitanicum. PeerJ, 4, e2503