high pCO2: A future perspective

Science of the Total Environment 628–629 (2018) 375–383

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Photosynthesis and mineralogy of Jania rubens at low pH/high pCO2: A future perspective Lucia Porzio a,b,⁎, Maria Cristina Buia a, Viviana Ferretti c, Maurizio Lorenti a, Manuela Rossi d,e, Marco Trifuoggi c, Alessandro Vergara c,f, Carmen Arena b a

Integrative Marine Ecology Department, Stazione Zoologica Anton Dohrn, Center of Benthic Ecology-Villa Dohrn, Punta S. Pietro, 80077 Ischia, Naples, Italy Department of Biology, University of Naples Federico II, Via Cinthia, 80126 Naples, Italy Department of Chemical Sciences, University of Naples Federico II, Via Cinthia, 80126 Naples, Italy d Department of Earth, Environment and Resources Sciences, University of Naples Federico II, Via Cinthia, 80126 Naples, Italy e Royal Mineralogical Museum, Centro Musei delle Scienze Naturali e Fisiche, University of Naples Federico II, Via Mezzocannone 8, 80134 Naples, Italy f CEINGE Biotecnologie Avanzate scarl, Naples, Italy b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Calcifying red algae may show speciesspecific response to ocean acidification (OA). • Photosynthesis and mineralogy (biosphere) were assessed after a threeweek transplant. • Field carbon chemistry (hydrosphere) and irradiance (atmosphere) were also considered. • Photosynthesis decreased while calcification was maintained under future pH conditions. • The calcifying Jania rubens may survive but reducing the fitness under OA.

a r t i c l e

i n f o

Article history: Received 2 December 2017 Received in revised form 6 February 2018 Accepted 6 February 2018 Available online xxxx Editor: Elena PAOLETTI Keywords: Bleaching Calcifying algae Carbonates Chlorophyll a fluorescence Diving-PAM Ocean acidification

a b s t r a c t Corallinales (Rhodophyta) are high Mg-calcite macroalgae and are considered among the most vulnerable organisms to ocean acidification (OA). These sensitive species play fundamental roles in coastal systems as food source and settlement promoters as well as being involved in reef stabilization, and water carbonate balance. At present only a few studies are focused on erect calcifying macroalgae under low pH/high pCO2 and the contrasting results make difficult to predict the ecological consequences of the OA on the coralline algae. In this paper the physiological reasons behind the resistance of Jania rubens, one of the most common calcareous species, to changing ocean pH are analysed. In particular, we studied the photosynthetic and mineralogical response of J. rubens after a threeweek transplant in a natural CO2 vent system. The overall results showed that J. rubens could be able to survive under predicted pH conditions even though with a reduced fitness; nevertheless physiological limits prevent the growth and survival of the species at pH 6.7. At low pH (i.e. pH 7.5), the maximum and effective PSII efficiency decreased even if the increase of Rubisco expression suggests a compensation effort of the species to cope with the decreased light-driven products. In these circumstances, a pH-driven bleaching phenomenon was also observed. Even though the photosynthesis decreased at low pH, J. rubens maintained unchanged the mineralogical composition and the carbonate content in the cell wall, suggesting that the calcification process may also have a

⁎ Corresponding author at: Stazione Zoologica Anton Dohrn of Naples, Integrative Marine Ecology Department, ‘Villa Dohrn’, Punta S. Pietro, 80077 Ischia, Naples, Italy. E-mail address: [email protected] (L. Porzio).

https://doi.org/10.1016/j.scitotenv.2018.02.065 0048-9697/© 2018 Elsevier B.V. All rights reserved.

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physiological relevance in addition to a structural and/or a protective role. Further studies will confirm the hypotheses on the functional and evolutionary role of the calcification process in coralline algae and on the ecological consequences of the community composition changes under high pCO2 oceans. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Calcifying Corallinales play important ecological roles in marine ecosystems: they promote settlement of other benthic organisms (Asnaghi et al., 2015), they are food source for herbivores (Maneveldt et al., 2006), they are involved in the stabilization of reef networks, and contributes in the carbonate production (Amado-Filho et al., 2012). Thus, understanding the effects of ocean acidification on Corallinales could help scientists to assess consequences of global changes on marine communities and on the ecosystem services associated with the community itself. Coralline algae may present one of two habitus: geniculate (articulated), that is erect calcareous algae (ECA) with non-calcified articulations (genicula); or non-geniculate, that is crustose calcareous algae (CCA) without genicula (McCoy and Kamenos, 2015). Due to their high diversity, coralline algae are found in different habitats all around the world (Nelson, 2009). These algae are one of the most sensitive groups in future CO2-rich oceans, as their calcareous thallus may dissolve at low carbonate saturation state (Ω), predicted by 2100. A previous work on coralline algae and elevated CO2 showed decreased calcification at high pCO2 (Gao et al., 1993) whereas field studies showed low population densities of coralline algae at high pCO2/low pH vents in both temperate (Porzio et al., 2011) and tropical ecosystems (Fabricius et al., 2015) even before the carbonate is saturated in seawater (Ω ≥ 1). For the first time we investigate the physiological and the stress response associated with the mineralogy of the geniculate macroalga Jania rubens (Linnaeus) J.V. Lamouroux in a natural CO2 vent system. Jania rubens is a very common calcifying seaweed in shallow coastal habitats and lives as an epiphyte on other algae forming more or less thick cushions according to the hydrodynamic energy. In a future high pCO2 environment, thin and erect calcifying species will be more exposed and more sensitive to the changing ocean pH compared to crustose calcareous algae. The latter are less susceptible to ocean acidification as they live understory where the lowering of the seawater pH could be buffered by the neighbouring species (Hendriks et al., 2014). Along a natural pCO2 gradient, J. rubens density, together with that of other Corallines (Rhodophyta), is higher at the pH maximum (8.1) until it disappears at the lowest pH (6.7) (Porzio et al., 2011). Calcification and photosynthesis are key processes for the growth of this algal group. At present, the functional role of calcification is not yet well understood. Calcification may have a structural (Borowitzka, 1981) and/or a protective role against grazing or light (Littler and Littler, 1980). Other hypotheses state that calcification cannot be associated to any function, since this process is a secondary effect of the photosynthetic activity, and it is favoured by the basic pH of the marine environment (Borowitzka, 1982). It is known that the photosynthetic process should help the calcification by definition; the CO2 uptake for photosynthesis should increase the local pH favouring the carbonate deposition in the cell wall (Borowitzka, 1981). Several authors investigated the rate of photosynthesis and calcification to understand the physiological limits of calcareous algae at high pCO2. Both crustose and erect calcareous algae are involved in the ocean biogenic carbonate balance, but the data linking ocean acidification to ECA is more fragmented and even contrasting compared to CCA (McCoy and Kamenos, 2015). In future environmental management and conservation planning, the outcome of ocean acidification should be understood in terms of primary production and carbonate balance, and loss of biodiversity in the ocean buffering system. Thus, it is fundamental to explore the response to acidification at species and population level to understand the different physiological patterns linked to the global response. A variable response

among different coralline algae may evidence a species-specific response to OA even though other factors, such as high temperature, may negatively influence photosynthesis more than acidification itself (Nash et al., 2016). Some species decrease photosynthetic activity at low pH without any definite implication to the calcification process (Martin et al., 2013); some others increase the production at high pCO2 (i.e. ~1000 μatm) and decrease calcification (Gao et al., 1993; Noisette et al., 2013). More recent studies focusing on mineralogy showed that changes in the Mg and Ca ratio of their calcified skeleton might be considered as an attempt of the species to cope with ocean acidification. Some species are, in fact, able to lower the Mg/Ca ratio at decreasing aragonite saturation state (Ωaragonite) (Ries, 2011) to make the skeleton calcite less soluble whereas some others shift to less soluble calcium sulphate (Goffredo et al., 2014) or dolomite (Nash et al., 2012) at low pH. On the other hand, Kamenos et al. (2016) found that Lithophyllum, Titanoderma and Phymatolithon spp. maintained their mineralogy and size at pH 7.6. This paper aims to improve the knowledge on the response of one of the most common calcifying species to changing ocean pH and helps in understanding and quantifying the loss of ecosystem services in high pCO2 oceans. In particular, we have studied the physiological behaviour and mineral composition of the calcareous alga Jania rubens along a natural pH gradient. We hypothesized that the decrease of J. rubens density at low pH is the results of a reduced photosynthetic efficiency and calcification likely due to the energy costs to maintain and grow the calcite skeleton at low carbonate concentration (Ω b 1). 2. Material and methods 2.1. Study area and the experimental design The study area is at the Castello Aragonese (Island of Ischia, Italy) (40° 043.84′ N; 13° 57.08′ E) and it is characterized by underwater CO 2 vents of volcanic origin that cause a natural pH gradient (~300 m long) spanning between pH ~8.1 to lower values as low as pH 6.7 (Hall-Spencer et al., 2008). As regard as the environmental conditions of the study area, we referred to Frieder's (2013) data who monitored in the same season and year of our study (late summer 2009) for five days. Temperature was measured every 15 min; salinity, total alkalinity (A T ) and total dissolved inorganic carbon (CT) were measured at the beginning and at the end of the experimental period (n = 4). pH was measured daily by collecting two samples in the morning in each pH site for five consecutive days in early September. We refer to Frieder (2013) for further details on the sampling and the analytical methods. Mean salinity, temperature, total alkalinity (AT) and pHT, assessed by Frieder (2013), were used to calculate carbon chemistry with CO2SYS (Pierrot et al., 2006) (see Table A.1). During the experimental period the average PPFD (photosynthetic photon flux density) at noon was ~ 1600 μmol photons m−1 s−1 at the experimental depth (0.50 m) (Porzio et al., 2017). To assess the degree of physiological adaptation of J. rubens to the stress induced by the pCO2/pH gradient, we carried out transplants from the native pH 8.1, to pH 7.5 and 6.7. We also included data by Porzio et al. (2011) on changes in species abundance and frequency of the reproductive structures on the thallus (see Fig. A.1). We conducted a stratified experimental design: 120 thalli of J. rubens were randomly selected from the wild population at pH 8.1. 30 were used as control (8.1-W) while 90 were tied on 9 PVC nets of 10 ∗ 10 cm (10 thalli for each net), fastened to the rocky substrate. We

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covered the samples with a light mesh fabric (1 mm diameter) in order to avoid the “grazing” factor but not to limit the daylight. We put three nets at pH 6.7 (6.7-T) and three at pH 7.5 (7.5-T). To evaluate a possible stress due to the transplant, the last three nets were attached in the native environment, at pH 8.1 (8.1-T). The nets were deployed at 0.50 m depth, at least 10 m apart within each pH environment, with a distance of about 100 m between different pH environments (see Fig. A.2). Transplanted thalli were left to acclimatize in situ for three weeks since this is the minimum time needed to allow the acclimation of some traits, such as the photosynthetic pigment content (Ralph et al., 2002). At the end of the three-week transplant, the bleaching and the chlorophyll a fluorescence were assessed in situ. The bleaching of the species was estimated as percentage cover (%) of bleached thalli in each transplanted net area and in three additional 10 ∗ 10 cm quadrates, randomly selected in the wild population. Later all the algal samples were collected and brought to the laboratory. The collection of samples, as well as the fluorescence emission measurements, were carried out around noon (local time) to avoid possible effects related to the daily variation in the algal photo-response. Among the collected samples, four replicates per treatment were randomly chosen to assess biometry (i.e. thallus height, cm); the remaining samples were stored at −20 °C for further analyses: pigment contents, Rubisco expression and skeleton mineralogy. 2.2. Chlorophyll a fluorescence measurements, photosynthetic pigment content and Rubisco expression The chlorophyll a fluorescence emission was assessed by a diving pulse amplitude modulated fluorometer (diving-PAM, Walz, Effeltrich, Germany). Preliminary measurements on J. rubens were carried out before the transplant experiment in order to set the optimal range of Photosynthetic Photon Flux Densities (PPFDs), according to the light acclimation of this species. Then, fluorescence emission measurements were conducted in situ on transplanted thalli, at pH 8.1 (8.1-T) and pH 7.5 (7.5-T), and on natural populations (8.1-W) after three weeks from transplanting. Rapid light curves (RLCs) were performed to evaluate the photosynthetic capacity and potential over increasing light doses of 10 s following procedures described in Porzio et al. (2017). PPFDs ranging from 0 to 950 μmol photons m−2 s−1 were produced by the diving-PAM internal halogen lamp; these values were then converted to values of ambient PPFD by a factor 2.8 (Raniello et al., 2006) to adjust to the light levels experienced in situ by the algae: a PPFD range from 0 to 2600 μmol photons m−2 s−1 was obtained after conversion. To assess the status of the PSII, including the occurrence of photoinhibition, the maximum quantum yield of PSII (Fv/Fm ratio) was determined on 10 min dark-adapted thalli according to Beer et al. (2014). The quantum yield of PSII electron transport (ΦPSII), that represents the photosynthetic efficiency, was estimated according to Genty et al. (1989):
ΦPSII ¼ Fm 0 −Ft = Fm 0

ð1Þ

where Fm′ is the maximum fluorescence in the light while Ft is the fluorescence at the steady-state. The relative electron transport rate (rETR) and the non-photochemical quenching (qN) were also considered for the photochemical assessment. rETR was calculated as: rETR ¼ ΦPSII  PAR  0:5

ð2Þ

where the value 0.5 assumes an equal distribution of absorbed quanta between PSI and PSII.

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The relative maximum electron transport rate (rETRmax), the onset of saturating light intensity (Ek) (sensu Beer et al., 2014) of rETR (Ralph and Gademann, 2005) and the alpha (α = rETRmax/Ek) (i.e. the rate of photon conversion under low irradiances) were calculated by the SigmaPlot software (SPSS Inc.). Non-photochemical quenching (qN), an indicator of the light energy dissipated as heat, was obtained as follows:
qN ¼ 1− Fm 0 −F0 0 =ð Fm −F0 Þ

ð3Þ

where F0 and F0′ represent the ground fluorescence of thalli adapted to the dark (10 min) and the minimal fluorescence when all PSII reaction centres are open in the light-adapted state, respectively. Chlorophyll a content and total carotenoids were quantified according to Lichtenthaler (1987). They were extracted from four individuals, for both transplanted (T) and wild thalli (W), in ice-cold 100% acetone from 0.100 g fresh weight (FW) of frozen tissue and centrifuged at 3000 rpm for 7 min. Chlorophyll a and carotenoid content was estimated by VIS spectrophotometry (Cary 100 UV-VIS, Agilent Technologies, Santa Clara, CA, USA) at 662 nm and 470 nm wavelengths, respectively. The phycobiliproteins phycocyanin (PC) and phycoerythrin (PE) were extracted in aqueous solution of 0.1 M phosphate buffer (pH 6.5) and centrifuged at 10000 ×g for 20 min. PC and PE concentrations were obtained according to Sampath-Wiley and Neefus (2007) equations. Among the thalli stored at −20 °C, three replicates were randomly chosen in order to assess the Rubisco expression, following the method described in Porzio et al. (2017). The data were computed with Quantity One 1-D Analysis Software (Bio-Rad Laboratories S.r.l., Milan, Italy) after minimizing the background effect. Density values were expressed as arbitrary units and represented as bar diagrams that are pixel volumes of Rubisco bands. 2.3. Quantification of organic (TOC) and inorganic (IC) carbon Total Carbon (TC) is defined as the total amount of carbon present in a sample originating from both organic and inorganic components, and it is represented as the total mass of carbon per amount of sample. Inorganic Carbon (IC) is defined as the inorganic carbon content in a sample that after acidification, turns into carbon dioxide. The IC fraction is usually used as a proxy of the carbonate content within soils (Vicente et al., 2011) and calcareous organisms (Sciandra et al., 2003). Total Organic Carbon (TOC) is defined as the organic carbon of a sample that is converted into carbon dioxide after oxidation. Samples were analysed in triplicate by the carbon analyser Primacs SNC 100 (Skalar, The Netherlands). High temperature combustion with Non-Dispersive Infrared detection (NDIR) was used. Samples of ~250 mg were weighted, and TC was assessed by using ceramic vessels while IC was determined in glass vessels. TOC is obtained from the difference between TC and IC. 2.4. Determination of calcium and magnesium Calcium and magnesium content was determined through Microwave plasma atomic emission spectroscopy (MP-AES). Microwave plasma atomic emission spectroscopy is an atomic emission technique. Once an atom of a specific element is excited, it emits light in a characteristic pattern of wavelengths, an emission spectrum, as it returns to the ground state. MP-AES quantifies the concentration of an element in a sample by comparing its emission to known concentrations of that element, plotted on a calibration curve. The analyzer used was MP-AES 4210 (Agilent, United States). Samples before being introduced into the instrument, were subjected to acid digestion oxidative using mixture of 6 ml nitric acid (65%) and 2 ml of hydrogen peroxide at high temperature and pressure, assisted by the microwave Mars (CEM S.R.L.,

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Italy). Calcium and magnesium content was determined in triplicate. 2.5. Mineralogy assessment A confocal Raman microscope (Jasco, NRS-3100) was used to obtain Raman spectra. The 514 nm line of an air-cooled Ar + laser (Melles Griot, 35 LAP431 220), was injected into an integrated Olympus microscope and focused to a spot diameter of approximately 3 μm by a 20× objective with a final 4 mW power at the sample. A holographic notch filter was used to reject the excitation laser line. In order to measure the bandwidth Δν1 of the band around 1089 cm− 1 , Raman spectra have been collected using a diffraction lattice of 1200 grooves/mm with three different slits (10, 100 and 200 μm) achieving up to a resolution of 1 cm−1. Bandwidth has been obtained by extrapolating the value at zero slit width. Samples were pretreated in order to remove the organic part with NaClO 4% for 4 h,

then they were washed 5 times with milli-Q water, dried overnight plus 6 h in the oven at 40 °C (Smith et al., 2016). The color-less solid was investigated via Raman. It took 60 s to collect a complete data set by a Peltier-cooled 1024 × 128 pixel CCD photon detector (Andor DU401BVI). Raman measurements were at least in triplicate for the scope of reproducibility. Wavelength calibration was performed by using cyclohexane as a standard. Raman spectra of 11 algae samples were recorded along with those of reference crystals of calcite, magnesite and dolomite (provided by the Royal Mineralogical Museum of the University of Naples Federico II).

2.6. Statistical analyses Statistical differences in photochemical parameters, Rubisco expression, photosynthetic pigment content, percentage of thallus bleaching, thallus mineralogy and carbon content were assessed among different treatments by one-way ANOVA. Tukey's post hoc test was applied for all multiple comparison procedures based on a significance level of P b 0.05. Kolmogorov-Smirnov test was used to check normality and the data were transformed (y = log(y)) when this assumption was not satisfied. The statistical software package Prism 5.0 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com) was used for all analyses.

3. Results

Fig. 1. Light curves of relative electron transport rate (rETR) of Jania rubens in wild (8.1-W, white triangles) and transplanted thalli at pH 8.1 (8.1-T, black triangles) and 7.5 (7.5-T, white circles). Dashed line shows the saturating irradiance at 1845 μmol photons m−2 s−1. Data are mean ± SEM (n ≥ 8).

After a week of transplant, all the thalli placed at pH 6.7 completely disappeared. Therefore, all the presented results were reported to wild (8.1-W) and transplanted thalli at pH 8.1 (8.1-T) and 7.5 (7.5-T). In all experimental conditions, rETR reached the highest activity at saturating irradiance of 1845 μmol photons m−2 s−1 (Fig. 1). It is worth noting that no difference was observed at ambient pH (pH 8.1) between wild (8.1-W) and transplanted thalli (8.1-T) (Fig. 2 and Table 1). On the other hand, the quantum yield of PSII electron transport (ΦPSII) strongly decreased at pH 7.5 (0.086 ± 0.0057, mean

Fig. 2. Photochemical parameters in Jania rubens measured in situ at saturating irradiance (1845 μmol photons m−2 s−1). Quantum yield of PSII (ΦPSII) (A), relative electron transport rate (rETR) (B), non-photochemical quenching (qN) (C) and maximum PSII quantum yield (Fv/Fm) (D) in wild (8.1-W) and transplanted thalli (8.1-T and 7.5-T). Tukey's test showed that ΦPSII, rETR and Fv/Fm differ significantly at pH 7.5 (7.5-T) from pH 8.1 (8.1-W and 8.1-T). Significance values for 7.5-T are indicated by *: P b 0.05; **: P b 0.01; ***: P b 0.001. Bars represent mean ± SEM (n ≥ 8).

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ambient pH; on the contrary, the bleaching percentage increased significantly (N50%) at high pCO2/low pH (Table 1). In the same way, we observed that the expression of the enzyme Rubisco increased by 30% at pH 7.5 compared to pH 8.1 (ANOVA: F2,6 = 11; P = 0.0099) (Fig. 3A and B). Inorganic carbon percentage (IC) as well as total organic carbon (TOC) showed similar content at both pH 8.1 and 7.5 (Table 2). Similarly, absolute content of magnesium and calcium was comparable at different pH environments; a constant molar Mg/Ca ratio of 0.14 among treatments was observed. By a qualitative point of view, we found a vibrational frequency (ν1) at 1089 cm−1, ascribed to the presence of calcite (Table 2) whereas neither the bicarbonate (1014 cm−1) nor the gypsum (1008 cm−1) were detected. At the same way, the extrapolated bandwidth (FWHM) at 1089 cm−1 were in the range of 12,8 b Δν1 b 15,6 cm−1. The experimental values from independent measurements of both frequency and bandwidth were similar among the different treatments (Table 2).

Fig. 3. Rubisco protein expression in Jania rubens. A) Densitometric analysis of Rubisco content in arbitrary units. The bar diagram represents pixel volumes of Rubisco bands in wild (8.1-W) and transplanted thalli at pH 7.5 (7.5-T) and 8.1 (8.1-T). B) Western blot of Rubisco enzyme. ST represents the Rubisco protein standard. Each bar represents mean ± SEM (n = 3).

± SEM) compared to pH 8.1 (0.14 ± 0.0057, mean ± SEM) (ANOVA: F2,24 = 22; P b 0.0001) (Fig. 2A). Similarly, thalli transplanted at pH 7.5 showed a 40% decrease in rETR (23.77 ± 1.57, mean ± SEM) compared to thalli transplanted at pH 8.1 (38.89 ± 1.58, mean ± SEM) (ANOVA: F2,24 = 21.78; P b 0.0001) (Fig. 2B). The qN showed no difference among thalli at pH 8.1 and 7.5 (Fig. 2C) whereas the maximum PSII quantum yield Fv/Fm showed a decrease of 23% at pH 7.5 (0.36 ± 0.020, mean ± SEM) compared to pH 8.1 (0.47 ± 0.023, mean ± SEM) (ANOVA: F2,24 = 8.089; P = 0.0021) (Fig. 2D). Analysis of variance (ANOVA) and the post-hoc Tukey's test (Table 1) showed that the derived RLC curve parameters were affected by the pH condition. In particular, a significant decrease of Ek and rETRmax was observed in thalli of J. rubens at pH 7.5 when compared to that at pH 8.1. The photosynthetic pigment content (chlorophyll a and carotenoids), expressed per gram of fresh weight (FW), were significantly affected by pH, (Table 1); more specifically, chlorophyll a, total carotenoids and Chl a/total carotenoid ratio decreased at pH 7.5 compared to pH 8.1. Among phycobilines, Phycoerythrin (PE) was the most abundant pigment in J. rubens and decreased by 30% at low pH compared to pH 8.1; on the other hand, Phycocyanin (PC) was not affected by different pH (Table 1). Thallus size was N40% smaller at pH 7.5 compared to

4. Discussion In the perspective of a future environmental management and conservation, it is fundamental to understand the consequences of ocean acidification on biodiversity, primary production and carbonate balance in the ocean buffering system. The specific responses of common and widespread species to ocean acidification, such as coralline algae, is important in the evaluation of physiological patterns that lead to the overall community modification in the changing environments. In this paper, for the first time, the photosynthetic and mineralogical responses of Jania rubens in a natural pH gradient after a three-week transplant have been analysed. The transplant at pH 6.7 determined, after only one week, the complete disappearance of J. rubens thalli, indicating the physiological tipping point for this species. At pH 6.7, the absence of J. rubens may be the consequence of the calcareous skeleton dissolution at low pH that may have favoured the detachment of the remnants from the nets. Furthermore, the dissolution of calcareous skeleton may have made the naked thalli more sensitive to solar UV radiation (Gao and Zheng, 2010), causing their death. This hypothesis is consistent with the J. rubens population distribution along the pH gradient, at very shallow depth (i.e. 0.5 m) where UV radiation still penetrates. The absence of difference among wild (8.1-W) and transplanted thalli (8.1-T) within the same environment (pH 8.1) suggests that no stress was due to the transplanting. OA negatively affected the photosynthetic apparatus, causing a 40% decrease of the PSII quantum yield of the electron transport and electron transport rate at saturating irradiance, at noon local irradiance (Porzio et al., 2017). Even under low light, a deleterious effect on photosynthesis was observed at low pH despite the higher pCO2 supply that could enhance the photosynthesis.

Table 1 Rapid light curves (RLCs) parameters (Ek, rETRmax, alpha), pigment content (chl a, total carotenoids, chl a/total carotenoid ratio, PE, PC), thallus size and percentage of bleaching in Jania rubens wild (8.1-W) and transplanted thalli at pH 8.1 (8.1-T) and 7.5 (7.5-T). The number of replicates is reported in parentheses. Data are means ± SEM. Different letters indicate statistical differences among treatments. Ek, the onset of saturating irradiance; rETRmax, relative maximum electron transport rate; alpha, the rate of photon conversion under low irradiances; Chl a, Chlorophyll a; PE, Phycoheritrin; PC, Phycocyanin; d.f., degrees of freedom.

Ek (μmol photons m−2 s−1) rETRmax (μmol m−2 s−1) alpha Chl a (mg/g FW) Total carotenoids (mg/g FW) Chl a/total carotenoids (mg/g FW) PE (mg/g FW) PC (mg/g FW) Thallus size (cm) Bleaching (%)

8.1-W

8.1-T

7.5-T

F(d.f.)

P value

356 ± 19 (5) ac 31 ± 2.7 (5) a 0.24 ± 0.011 (5) a 0.15 ± 0.013 (5) ac 0.027 ± 0.0025 (5) a 0.0058 ± 9.7 ∗ 10−5 (5) a 0.51 ± 0.37 (5) a 0.066 ± 0.014 (5) a 2.1 ± 0.12 (4) a 0.0 ± 0.0 (3) a

443 ± 54 (5) a 40 ± 2.0 (5) a 0.26 ± 0.020 (5) a 0.18 ± 0.022 (6) a 0.032 ± 0.0039 (6) a 0.0056 ± 0.00011 (6) a 0.43 ± 0.29 (4) a 0.024 ± 0.0064 (4) a 2.2 ± 0.12 (4) a 0.67 ± 0.67 (3) a

308 ± 24 (7) bc 25 ± 2.4 (7) b 0.23 ± 0.0.14 (7) a 0.10 ± 0.0074 (6) bc 0.0047 ± 0.00067 (6) b 0.022 ± 0.0023 (6) b 0.11 ± 0.17 (6) b 0.035 ± 0.0085 (6) a 1.3 ± 0.10 (4) b 55 ± 5.0 (2) b

F2,14 = 4.2 F2,14 = 9.0 F2,14 = 1.0 F2,14 = 7.8 F2,14 = 30 F2,14 = 48 F2,12 = 63 F2,12 = 4.0 F2,9 = 19 F2,5 = 213

0.038 0.0031 0.40 0.0053 0.0001 0.0001 0.0001 0.048 0.0006 0.0001

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Table 2 Mineralogy and chemistry of Jania rubens: Wave number frequency (ν1), bandwidth of the ν1 mode of the carbonate anion (FWHM), Mg and Ca content, the relative molar ratio and the carbon content in wild (8.1-W) and transplanted thalli at pH 8.1 (8.1-T) and 7.5 (7.5-T). ν1 and FWHM are shown for mineral standards of calcite, magnesite and dolomite. Data are reported as mean ± SEM; the number of replicates is in parentheses. IC: inorganic carbon; TOC: total organic carbon. ν1 frequency (cm−1) FWHM (cm−1) 8.1-W 8.1-T 7.5-T CaCO3 (calcite) MgCO3 (magnesite) CaMg(CO3)2 (dolomite)

1088.2 ± 0.45 (4) 1088.8 ± 0.35 (3) 1087.9 ± 0.4 (4) 1085.3 1093.4 1095.4

Mg (g/kg)

Ca (g/kg)

mol Mg/Ca

IC (%)

TOC (%)

IC/TOC

13.2 ± 0.15 (4) 10.41 ± 0.55 (4) 126.17 ± 11.1 (4) 0.14 ± 0.01 (4) 4.40 ± 0.82 (3) 4.20 ± 1.42 (3) 1.9 ± 1.27 (3) 13.3 ± 0.17 (3) 10.16 ± 0.37 (4) 127.60 ± 6.15 (4) 0.14 ± 0.01 (4) 3.57 ± 0.23 (3) 3.05 ± 0.84 (3) 1.4 ± 0.46 (3) 12.9 ± 0.1 (4) 10.21 ± 0.45 (4) 116.22 ± 7.70 (4) 0.14 ± 0.005 (4) 3.30 ± 0.29 (3) 3.42 ± 0.39 (3) 0.99 ± 0.11 (3) 6.6 13.8 10.4

Generally, the decline in photochemistry is often associated to an increase of non-photochemical quenching (qN), that acts as a safety mechanism to dissipate the excess of light energy not utilised in photosynthesis, in order to avoid photodamages. Surprisingly at low pH, the decrease of PSII photochemical efficiency in J. rubens thalli was not concomitant with the qN increase. It has been hypothesized that the low pH may have enhanced the rate of PSII repair as observed by Li et al. (2012). A further explanation for the major sensitivity of J. rubens to low pH may be also ascribed to the effects of acidification on photosynthetic pigment content. In fact, at pH 7.5 a significant decrease of pigment amount was found together with a strong bleaching of thalli. It is possible that the combination low pH and solar ultraviolet radiation (UV) may have caused the bleaching in thalli at the shallow depth (i.e. 0.5 m) where this species lives. Consistently, other authors observed a synergistic effect of low pH and UV radiation on red algae (Gao and Xu, 2008; Gao and Zheng, 2010). Our experiment evidenced, for the first time, a low pH-driven bleaching in J. rubens, similar to the Coralline White Patch Disease (CWPD) (i.e. a diffusive white discoloration) observed in Jania sp. and other coralline algae exposed to high temperature (Hereu and Kersting, 2016; Quéré et al., 2015). From these results is evident that OA strongly affects the synthesis of photosynthetic pigment even though the action mechanism is still unknown. The bleaching phenomenon may be triggered by several factors such as an enhanced susceptibility of thalli to pathogens or allelopathic substances (Hereu and Kersting, 2016), and the combined effect pH/UV radiation, both responsible for the occurrence of oxidative stress (Gao and Zheng, 2010). Anyway, the action of the enzyme bromoperoxydase may be involved in the algal bleaching (Latham, 2008). In fact, one product of the enzyme activity can be the formation of allelopathic substances (Ohsawa et al., 2001) and unstable Bromonium ions (HOBr) that react with organic compounds to produce halogenated organic compounds (VHOCs). During a severe stress, HOBr could directly cause the degradation of the photosynthetic pigments (Latham, 2008). The increase in VHOC at low pH has been observed in other macroalgae (Mithoo-Singh et al., 2017), suggesting that bromoperoxydase activity may be involved under OA. VHOCs play a major role in shaping prey-predator interactions (Zupo et al., 2015) and, in a global change scenario, we suppose that the enhanced release of these compounds or other allelopathic substances by coralline algae may alter the chemical information network in the coastal ecosystems under OA. As regards photosynthesis, the significant decrease of photochemical efficiency found in thalli at pH 7.5, was unexpectedly concomitant with the increase of Rubisco expression level found in the same thalli. This results is in contrast with data in literature, since under high pCO2 a lower Rubisco content is generally expected due to the higher carboxylation efficiency of this enzyme under elevated CO2 (CarmoSilva et al., 2015). It has been demonstrated that the synthesis of Rubisco is generally proportional to the content of Rubisco activase (RCA) that, in turn, is prompted by the high amount of light-driven ATP production (Chen et al., 2010). In our case, the higher Rubisco expression level at pH 7.5 compared to pH 8.1, may be interpreted as a

compensation mechanism to counteract the decrease of the electron transport rate and the consequent lower ATP content at pH 7.5 (Carmo-Silva et al. (2015). The reduced photochemical efficiency as well as the increase of the pigment bleaching negatively affect the biomass production, causing a reduction of N40% in thallus size at pH 7.5 compared to ambient pH. Although the photosynthetic activity should be directly related to the calcification rate (McCoy and Kamenos, 2015), the calcareous skeleton of J. rubens was unaffected by OA in terms of content and characteristics of constitutive carbonates, despite photosynthetic decline. These results indicate that this species, at pH 7.5, invests more energy in preservation of its calcareous skeleton than in mechanisms avoiding damages to photosynthetic tissues. In literature, different calcification responses have been observed in other calcareous seaweeds at low pH: they may calcify less in order to face the stress induced by acidification (McCoy and Ragazzola, 2014) or they can modify the Mg content into the skeleton or even change the mineralogy to reduce the calcite solubility at low pH (Kamenos et al., 2016; Ries, 2011). In J. rubens, the carbonate as well as total organic carbon showed a similar content at both pH 8.1 and 7.5. Littler and Littler (1980) observed that branched coralline algae allocate much more energy to structural tissues than to photosynthetic tissues, highlighting the importance of the skeleton for the protection from high light, wave action and herbivore attacks. The calcareous skeleton may also have the fundamental role of protecting from UV rays (Gao and Zheng, 2010) in shallow populations of Jania and this important function could be one of the reasons associated to its maintenance, whatever the cost, under OA. From a qualitative point of view, Raman spectroscopy is a valuable technique to identify carbonates, bicarbonates and changes in the relative concentration of Mg++ ions (Pauly et al., 2015). The non-significant change in frequency ν1 is indicative of non-significant changes in the kind of carbonate crystals (Bischoff et al., 1985). Any change of the bandwidth (FWHM) at 1089 cm−1 would be positively related to the positional disorder of the carbonate ion and an increasing rotation of CO2– 3 out of the basal plane due to shorter Mg\\O bonds into the calcite at increasing Mg concentration (Bischoff et al., 1985). This evidence may indicate that OA does not affect the relative Mg content in the skeleton of J. rubens. Indeed, the absence of any trend in the relative Mg-concentration as a function of the pH variation, was further supported by the MP analysis that provides an absolute estimation of the Mg content and the Mg/Ca molar ratio (mMg/Ca). According to Ries (2011), the mMg/Ca ratio indicate that the solubility of carbonate crystals may be more related to the calcite (trigonal crystal system) or to the aragonite (orthorhombic crystal system), polymorph of the calcite. The skeletal mMg/Ca ratio of Jania (0.14) is more similar to aragonite than high-Mg calcite (Ries, 2011). The species Corallina sp., another articulated algae, showed no variation of Mg/Ca ratio at different pH (0.17) similar to Jania (Noisette et al., 2013). The crustose calcareous algae (CCA) had a higher Mg/Ca molar ratio (~0.20) (Noisette et al., 2013) and, among them, only Neogoniolithon sp. showed the ability to adjust the Mg/Ca ratio (0.20 b Mg/Ca b 0.23) according to the saturation state (Ω) and the seawater Mg/Ca (Ries, 2011). Calcification in a low pH ocean could be favoured in

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species with a lower Mg/Ca ratio compared to coralline algae characterized by the higher Mg calcite, more soluble than calcite itself and aragonite. On the basis of available literature data, the skeletal mMg/Ca ratio could be the result of the evolutionary history of the species, thus explaining the different mineralogical and physiological behaviour to OA. The seawater mMg/Ca ratio has changed significantly throughout the Phanerozoic: higher seawater Mg/Ca (N2), favouring higher Mg calcite, has alternated with lower seawater Mg/Ca ratio (b2) (Ries, 2010). By matching historical seawater data (Ries, 2010) with the phylogenetic tree of coralline algae by Aguirre et al. (2010), we observed that most of the CCAs have derived from a branching event between 120 and 130 Ma when the seawater chemistry favoured the mineralization of the high-Mg calcite in marine organisms (Ries, 2011). On the other hand, the branching event that led to the articulated Jania spp. and Corallina spp. has occurred after the last lower seawater Mg/Ca period (~60 Ma), implying that the mineralogy of these species may be advantaged at low pH compared to CCAs. Further studies on physiology and mineralogy of a higher number of coralline algae with a different speciation time will help to confirm our hypothesis.

competitiveness and fitness for this species compared to brown and turf algae that would become dominant at low pH (Porzio et al., 2017, 2013). At population level, the negative effect of OA is amplified compared to the individual response, being the decline of species abundance more than two-fold lower (i.e. 85%). Although the calcification yield could be not affected by the different pH conditions, the smaller individuals and their lower abundance at pH 7.5 will likely lead to a lower carbonate deposition in the whole population. As a consequence, the loss of the primary production and carbonate account, at scale of the global species distribution, might have unpredictable impacts on the coastal systems. Due to the importance of coralline algae, this paper confirms the potentiality of the natural CO2 vent systems in the study of the long-term OA impact on coastal ecosystems and highlights the need of an improved knowledge of the ecological consequences associated to the response of this specific algal group to OA. The acquired knowledge will help scientific community to get an accurate assessment of the ecosystem services loss in the next future.

5. Conclusions

Acknowledgements

On a long-term perspective (decades), our results suggest that the reduction in photosynthetic efficiency could not necessarily lead to the complete disappearance of J. rubens under OA but may determine a slowdown of growth rate (i.e. 40%) causing a decreased in

We thank the staff of SZN for the field assistance. We are grateful to the native English speaker Mrs. Rosanna Messina (SZN) for the manuscript language revision. The authors have no conflict of interests to disclose.

Appendix A. Appendix Table A.1 Carbon chemistry at the study area in early September 2009. From Frieder (2013), modified. Parameter

Salinity T (°C) pHT AT (μmol/kg) pCO2 (μatm) CO2 (μmol/kg) CO2− 3 (μmol/kg) HCO− 3 (μmol/kg) Ωcalcite Ωaragonite

Ambient pCO2/pH (8.1)

High pCO2/low pH (7.5)

Extreme high pCO2/low pH (6.7)

Mean ± SEM

Mean ± SEM

Mean ± SEM

38.1 ± 0.00 (2) 25.5 ± 0.1 8.04 ± 0.00 (10) 2564 (1) 448.8 (1) 12.3 (1) 248.6 (1) 1954.4 (1) 5.82 (1) 3.86 (1)

38.1 ± 0.00 (2) 25.4 ± 0.00 7.5 ± 0.005 (12) 2573 ± 5.5 (2) 1988.3 ± 20.3 (2) 54.8 ± 0.6 (2) 82.3 ± 1.05 (2) 2371.72 ± 3.01 (2) 1.93 ± 0.03 (2) 1.28 ± 0.02 (2)

38.1 ± 0.00 (2) 25.6 ± 0.00 6.84 ± 0.01 (12) 2575 ± 3.5 (2) 9105.1 ± 225.8 (2) 251 ± 6.2 (2) 20.4 ± 0.4 (2) 2525 ± 4.6 (2) 0.5 ± 0.03 (2) 0.3 ± 0.02 (2)

Fig. A.1. Coralline algae along the pH gradient. A) Distribution of coralline species. B) Frequency of the reproductive structures (i.e. conceptacles) of J. rubens at pH 8.1, 7.5 and 6.7. From Porzio et al. (2011), modified.

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Fig. A.2. Experimental design for Jania rubens at pH 8.1, 7.5 and 6.7.

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