Climate change and ocean acidification effects on seagrasses and marine macroalgae

Abstract

Although seagrasses and marine macroalgae (macro-autotrophs) play critical ecological roles in reef, lagoon, coastal and open-water ecosystems, their response to ocean acidification (OA) and climate change is not well understood. In this review, we examine marine macro-autotroph biochemistry and physiology relevant to their response to elevated dissolved inorganic carbon [DIC], carbon dioxide [CO₂], and lower carbonate [CO₃²⁻] and pH. We also explore the effects of increasing temperature under climate change and the interactions of elevated temperature and [CO₂]. Finally, recommendations are made for future research based on this synthesis. A literature review of >100 species revealed that marine macro-autotroph photosynthesis is overwhelmingly C₃ (≥ 85%) with most species capable of utilizing HCO₃⁻; however, most are not saturated at current ocean [DIC]. These results, and the presence of CO₂-only users, lead us to conclude that photosynthetic and growth rates of marine macro-autotrophs are likely to increase under elevated [CO₂] similar to terrestrial C₃ species. In the tropics, many species live close to their thermal limits and will have to up-regulate stress-response systems to tolerate sublethal temperature exposures with climate change, whereas elevated [CO₂] effects on thermal acclimation are unknown. Fundamental linkages between elevated [CO₂] and temperature on photorespiration, enzyme systems, carbohydrate production, and calcification dictate the need to consider these two parameters simultaneously. Relevant to calcifiers, elevated [CO₂] lowers net calcification and this effect is amplified by high temperature. Although the mechanisms are not clear, OA likely disrupts diffusion and transport systems of H⁺ and DIC. These fluxes control micro-environments that promote calcification over dissolution and may be more important than CaCO₃ mineralogy in predicting macroalgal responses to OA. Calcareous macroalgae are highly vulnerable to OA, and it is likely that fleshy macroalgae will dominate in a higher CO₂ ocean; therefore, it is critical to elucidate the research gaps identified in this review.

Introduction

Objectives of the review

The goal of this review is to provide a baseline upon which future research can build a more comprehensive understanding of climate change and carbon dioxide (CO₂) enrichment effects on ecosystems where seagrasses and macroalgae play important ecological roles, with a focus on the tropics and subtropics. These macro-autotrophs are taxonomically and metabolically more diverse than terrestrial plants, which makes generalizations about their responses to climate change difficult, and their responses potentially more varied. There are approximately 60 seagrass species worldwide, all monocots, and the major tropical genera include: Cymodocea, Enhalus, Syringodium, Thalassia, Halodule and Halophila (Short et al., 2007). There are far more species of marine macroalgae than seagrasses; estimates vary, but are in the many thousands (Thomas, 2002; Guiry & Guiry, 2012 ), and they encompass three major divisions: Chlorophyta (greens), Rhodophyta (reds), and Ochrophyta (browns).

Seagrass meadows are major coastal ecosystems and substantial attention has been paid to their ecology, because globally they are in serious decline with their loss accelerating from about 1% yr⁻¹ before 1940 to 7% yr⁻¹ presently (Waycott et al., 2009). However, the losses have been mainly attributed to development, overexploitation, pollution, and natural causes, rather than to the climate, with the possible exception of temperature-induced hypoxia and phytotoxin (hydrogen sulfide) production (Holmer & Bondgaard, 2001; Koch & Erskine, 2001; Koch et al., 2007; Höffle et al., 2011). But this could change as CO₂ concentration [CO₂] and temperatures rise and either ameliorate or compound the current stress on seagrasses. They, along with macroalgae, are the base of the food web and a primary food source for herbivores on coral reefs, lagoons, and other shallow habitats, particularly in the tropics where phytoplankton biomass is extremely low (Adey, 1998). They reduce sediment resuspension and their roots enhance sediment accretion in seagrass meadows, thus maintaining high water quality. Both seagrasses and macroalgae are critical constituents in nutrient cycling processes, nutrient retention, and sediment-water nutrient flux (Valiela, 1984). In terms of CO₂ mitigation and carbon storage, it is estimated that seagrasses may account for as much as 30% of marine net primary productivity (NPP) that is buried in sediments (Duarte & Cebrián, 1996).

Less attention has been paid to macroalgae even though they constitute some of the most significant biogenic producers of calcium carbonate (CaCO₃) and contribute to deep-sea productivity (Wefer, 1980; Littler et al., 1991; Nelson, 2009). They are sediment producers in tropical lagoons and create the matrices between corals that facilitate reef accretion (Hillis-Colinvaux, 1986a,b; Adey, 1998; Chisholm, 2003), an important process in the context of sea level rise. A few keystone species provide three-dimensional biomass structure for marine faunal habitats, including nursery areas for juvenile fish and shellfish, which reduce predation in clear tropical waters. Crustose coralline algae facilitate larval settlement of marine invertebrates including coral planulae (Ritson-Williams et al., 2009). They create foundations for entire ecosystems in algal-dominated reefs, lagoons, and patch reef areas, as well as open-ocean drift algal systems (e.g., Sargasso Sea). With the current rates of coral loss to bleaching and sea level rise, as well as threats to calcifying macroalgae by ocean acidification (OA), and the concomitant lowering of CaCO₃ saturation states (Hoegh-Guldberg et al., 2007), seagrasses and non-calcifying macroalgae may become community dominants on reefs and in other carbonate-based ecosystems. Thus, understanding their physiological and ecological responses to OA and climate change is merited.

Dissolved inorganic carbon in seawater

Before the industrial revolution, the oceans were a net source of CO₂ to the atmosphere, but over the last 250 years they have become a CO₂ sink (Sabine et al., 2004a,b; Sabine & Feely, 2007). Currently, the oceans sequester ~2 petagrams of carbon per year (1 Pg = 10¹⁵ g or 1 billion metric tons), and the anthropogenic uptake has been estimated to be 118 ± 19 Pg C from 1800 to 1994 (Sabine et al., 2004a, Sabine & Feely, 2007). Although there is no doubt that oceans sequester large amounts of anthropogenic CO₂, there is controversy over the potential rates of increase, due to variable estimates of future burning of fossil fuels, and strong positive and negative feedbacks. On the current trajectory, however, the present atmospheric [CO₂] of 394 ppm (NOAA-CCGG, 2012) is likely to approach or exceed 1000 ppm by the year 2100 under the ‘business as usual’ CO₂ emissions scenario (Meehl et al., 2007; Fabry et al., 2008). This [CO₂] has not been seen in the atmosphere over the last 800 000 years (Petit et al., 1999; Pearson & Palmer, 2000; Siegenthaler et al., 2005; Lüthi et al., 2008), and the current rate of rise is unprecedented over the last millennium (Doney & Schimel, 2007).

As ocean [CO₂] increases, the consequence is a lower pH or increase in the hydrogen ion concentration [H⁺] of seawater, termed OA. In fact, since the industrial revolution, the [H⁺] in the oceans has risen by ~30%, dropping the pH 0.1 units (Fig. 1; Orr et al., 2005; Raven et al., 2005b; Meehl et al., 2007). If atmospheric [CO₂] reaches 1000 ppm, it is anticipated that pH in ocean surface waters will decline by another 0.3–0.4 pH units by the end of the century (Orr et al., 2005). In seawater, pH changes control the carbonate equilibrium which is the pH-controlled distribution of dissolved inorganic C species: CO₂(aq), bicarbonate (HCO₃⁻) and carbonate (CO₃²⁻) ions (Fig. 1). Elevated [CO₂] in the oceans increases the total [DIC] and [H⁺], thereby lowering the pH, and shifting the relative proportion of each DIC species. Under current ocean pH (~8.04), CO₂ is the smallest pool of DIC, but will have the greatest percent increase (>250%) among the DIC constituents as the pH drops under the predicted rise in atmospheric [CO₂] for 2100 (Fig. 1). In contrast, the larger HCO₃⁻ pool will only increase by 24% by 2100; however, in absolute terms (mol kg⁻¹) HCO₃⁻ levels will rise more than CO₂ (e.g., Raven et al., 2005b). Perhaps most importantly for calcifying macroalgae is the predicted decline in [CO₃²⁻] by more than 50% (Fig. 1). These fundamental changes in ocean chemistry are likely to have cascading biological consequences in marine ecosystems (Hofmann et al., 2010).

Figure 1. The fate of atmospheric CO₂ as it exchanges into the oceans at the air-sea interface and becomes part of the aqueous carbonate system. The carbonate equilibrium equations are shown (left) and the concentrations (μmol kg⁻¹) of the dissolved inorganic C (DIC) forms (CO2SYS, Pierrot et al., 2006) under pre industrial, present, and 2100 levels of [CO₂] in the atmosphere in equilibrium with the ocean surface layer (Meehl et al., 2007; NOAA-CCGG, 2012). Inorganic C speciation was calculated using the following inputs to CO2SYS: 25 °C temperature, 2300 μmol kg⁻¹ alkalinity, 35 psu salinity, and pH shown with the CO₂ output given in μatm. The K₁ and K₂ carbonate dissolution constants used are from Mehrbach et al. (1973) refit by Dickson & Millero (1987). The saturation states of calcite (Ω_calcite) and aragonite (Ω_aragonite) CaCO₃ polymorphs were also generated using CO2SYS based on the above parameters. Percent change was calculated from pre industrial CO₂ levels (in ppm) to those predicted for 2100 (Raven et al., 2005a; Orr et al., 2005; Meehl et al., 2007) modified from Fabry et al. (2008).

The greater increase predicted for CO₂ than HCO₃⁻ has important implications for the responses of seagrasses and macroalgae, as a majority of autotrophs have a higher photosynthetic affinity for CO₂ than HCO₃⁻ (Bowes, 1985; Madsen & Sand-Jensen, 1991; Durako, 1993). An increase in CO₂ availability is also likely to enhance the competitive advantage of species with a greater dependence on and ability to rapidly sequester CO₂. The shift in proportion and concentration of DIC species, along with OA and warming, affects not only photosynthesis but also calcification in marine macroalgae. There is a wide range of macroalgal species that depend on the production of CaCO₃ for their growth and thalli (body) structure. Major impacts on calcifiers will cascade through marine ecosystems, as they are vital constructs forming 3-D complexity in benthic systems, including reefs. Thus, elevated atmospheric [CO₂] will affect marine plants and algae in unique ways compared to terrestrial species and these interactions warrant closer examination and a research focus.

Below we elaborate on what is known about marine plant and macroalgal biochemistry and physiology that is likely to influence their response to elevated [DIC], [CO₂] particularly, and a lowered [CO₃²⁻] and pH. We also examine how the responses are affected by elevated temperature with climate change, define tropical macro-autotroph thermal limits, and review mechanisms of thermal tolerance. Finally, we discuss the current experimental work on OA and climate change effects on calcifying macroalgae, including intriguing field studies from vent sites with CO₂ outgassing, and make recommendations for future research on these topics.

Photosynthetic carbon acquisition in seagrasses and macroalgae

Photosynthetic pathways

A key to understanding how marine autotrophs will adjust to changes in inorganic C speciation and elevated total DIC is their species-specific photosynthetic biochemistry and physiology. Some insight can be gained from work on terrestrial species, as photosynthetic pathways have a major bearing on plant responses to CO₂ and temperature (Long, 1991; Long et al., 2006; Sage & Kubien, 2007). Over the past 800 000 years of glacial and interglacial periods atmospheric [CO₂] has fluctuated (Lüthi et al., 2008) with terrestrial C₃ communities dominant when [CO₂] is high and C₄ species favored in the warm lower latitudes when [CO₂] declines (Ehleringer, 2005; Leakey & Lau, 2012). High [CO₂] benefits C₃ photosynthesis because ribulose bisphosphate carboxylase-oxygenase (Rubisco), the initial carboxylating enzyme in C₃ photosynthesis, is not substrate-saturated by the current [CO₂], due to the fact that its CO₂ fixation is inhibited by atmospheric O₂ (Bowes & Ogren, 1972). Furthermore, Rubisco catalyzes a reaction with O₂, which leads to around 35% of previously fixed carbon being lost as CO₂ through the photorespiratory pathway (Bowes et al., 1971; Ehleringer 2005). In contrast, the initial carboxylating enzyme in C₄ photosynthesis, phosphoenolpyruvate carboxylase (PEPC), is unaffected by O₂ (Bowes & Ogren, 1972). In C₄ species, cytosolic PEPC initiates a CO₂ concentrating mechanism (CCM) that provides saturating [CO₂] to Rubisco in the chloroplasts, thereby overcoming the deleterious effects of O₂. As a result, their photosynthesis is saturated at current atmospheric [CO₂] and photorespiration is minimized (Bowes, 1993; Bowes et al., 2002). Consequently, C₄ species show much less of a productivity response to rising [CO₂] and temperature than C₃ species. Plants with Crassulacean Acid Metabolism (CAM) also use PEPC, but fixation by this enzyme is at night with the subsequent refixation of CO₂ by Rubisco during the day (Dodd et al., 2002). This results in an increase in titratable acidity (mainly malic acid) during the night that decreases during the day, and this diel change is a marker for CAM activity. It is unlikely that CAM metabolism is dominant in marine macroalgae, but the basic physiology of most macroalgal species is not known (Keeley, 1998). For this review, we compiled the available literature on seagrass and macroalgal photosynthetic characteristics to determine if any patterns emerge to provide insight on how these important macro-autotrophs might respond to OA and climate change. Table 1 summarizes this information, including the possible photosynthetic pathway, the ability to use HCO₃⁻, whether or not an external carbonic anhydrase (CA) is present to catalyze HCO₃⁻ to CO₂ and make the latter more readily available, and whether or not photosynthesis is saturated at current seawater [DIC]. Based on these data, several trends are apparent. The first observation is that the great majority of species (≥85%) can be characterized as C₃, rather than C₄ or CAM species, albeit with notable exceptions. Among seagrasses, initial reports that Thalassia testudinum was a C₄ plant were discounted, because after exposure to ¹⁴CO₂ the greatest initial incorporation of ¹⁴C-label was in 3-phosphoglycerate of the Calvin cycle. Although C₄ acids became ¹⁴C-labeled, they did not turnover into Calvin cycle sugar phosphates, which is a hallmark of a typical C₄ system (Benedict & Scott, 1976; Benedict et al., 1980; Beer & Wetzel, 1982). The tropical species Cymodocea nodosa shows the most evidence for a C₄ pathway based on ¹⁴C incorporation into C₄ acids during photosynthesis and a Rubisco : PEPC activity ratio of 1 (Beer et al., 1980). Furthermore, 48% of the ¹⁴C label in ¹⁴CO₂ initially entered malate and within 45 s was ‘chased’ by ¹⁴CO₂ into phosphate esters and sucrose, similar to terrestrial C₄ species. Halophila stipulacea also exhibited ¹⁴C-labeling of malate and its PEPC activity was equivalent to that of Rubisco, but the label in malate accumulated during the short chase and did not appear to turnover. What effect this has on malate pool sizes was not determined (Beer et al., 1980). Thus, H. stipulacea has been classified as a C₃ species. However, species such as T. testudinum and H. stipulacea bear further investigation, as they may have a facultative C₄ system like the freshwater angiosperm Hydrilla verticillata in which the C₄ system is only induced at low [CO₂]. In dense seagrass meadows, high productivity and CO₂ uptake rates may significantly reduce the day-time [DIC] and [CO₂] in the canopy, and thus the possibility that facultative C₄ species exist in the marine environment cannot be ruled out. For terrestrial leaves, ¹³C/¹²C ratios (δ¹³ values) have been used to distinguish between C₃ and C₄ species because Rubisco discriminates against ¹³CO₂ to a much greater extent than PEPC (Farquhar et al., 1989). Seagrasses tend to have negative δ¹³ values reminiscent of C₄ photosynthesis. However, the photosynthetic use of both CO₂ and HCO₃⁻, which differ in δ¹³ values, as well as enhanced recycling of CO₂ due to aqueous DIC diffusion restraints, make δ¹³ isotopic C signatures an unreliable indicator for C₄ photosynthesis in the aquatic environment, and thus C₄ predictions based on such data should be viewed with caution (Andrews & Abel, 1979; McMillan et al., 1980; Carvalho & Eyre, 2011).

Table 1. Photosynthetic pathways (C₃, C₄, or CAM) and the ability to use HCO₃⁻ (Y) or only CO₂ (N) reported in the literature for marine seagrasses and macroalgal species. The presence of external carbonic anhydrase (External CA) activities (Y/N) and whether current seawater DIC concentrations saturate photosynthesis (Y/N) are also shown. Tropical species (*; tropical distribution in AlgaeBase [Guiry & Guiry, 2012]), calcareous species (Ca), unknown (−), uncertain (?) and references are noted

Division (Phylum) and species	Photosynthetic pathways	HCO3^− use	External CA	DIC saturation	Reference
Reviewed in: (1) Abel (1984); (2) Andrews & Abel (1979); (3) Andría et al. (1999); (4) Beer (1989); (5) Beer et al. (1980); (6) Beer & Eshel (1983); (7) Beer & Rehnberg (1997); (8) Beer & Waisel (1979); (9) Beer & Wetzel (1982); (10) Benedict et al. (1980); (11) Björk et al. (1997); (12) Borowitzka (1981); (13) Borowitzka & Larkum (1976); (14) Cook & Colman (1987); (15) Cook et al. (1986); (16) Cook et al. (1988); (17) Durako (1993); (18) Enríquez & Rodríguez-Román (2006); (19) Giordano & Maberly (1989); (20) Hellblom & Axelsson (2003); (21) Holbrook et al. (1988); (22) Invers et al. (1999); (23) Invers et al. (2001); (24) Johnston & Raven (1986); (25) Johnston & Raven (1987); (26) Johnston et al. (1992); (27) Kremer & Küppers (1977); (28) Kremer & Willenbrink (1972); (29) Kübler et al. (1999); (30) Larsson et al. (1997); (31) Maberly (1990); (32) McMillan et al. (1980); (33) Millhouse & Strother (1986a); (34) Millhouse & Strother (1986b); (35) Moulin et al. (2011); (36) Raven & Osmond (1992); (37) Reiskind & Bowes (1991); (38) Reiskind et al. (1988); (39) Sand-Jensen & Gordon (1984); (40) Schwarz et al. (2000); (41) Smith & Bidwell (1989); (42) Surif & Raven (1989); (43) Uku et al. (2005); (44) Zou et al. (2003).
Tracheophyta (Seagrasses)
Cymodocea nodosa*	C4	Y	Y	N	4, 5, 8, 22, 23
C. rotundata*	−	Y	Y	N	11, 43
C. serrulata*	−	Y	Y	N	11, 40, 43
Enhalus acoroides*	−	Y	Y	N	11, 43
Halodule uninervis*	C3	Y	Y	N	4, 5, 8
H. wrightii*	C3	Y	Y	N	9, 11, 43
Halophila beccarii*	C3	−	−	−	32
H. ovalis*	−	Y	Y	N	4, 8, 11, 40, 43
H. spinulosa*	C3	Y	Y	−	2
H. stipulacea*	C4?	Y	Y	N	4, 5, 8
H. tricostata*	C3	−	−	−	32
Phyllospadix torreyi	−	Y	Y	N	23
Posidonia oceanica	−	Y	Y	N	22, 23
Ruppia cirrhosa	−	Y	Y	N	20
R. maritima*	−	Y	−	Y	39
Syringodium isoetifolium*	C3	Y	Y	N	4, 5, 8, 11, 43
S. filiforme*	C3	−	−	−	9
Thalassia hemprichii*	C3	Y/N	Y	N	1, 2, 5, 11, 43
T. testudinum*	C3	Y/N	Y	N	9, 10, 17, 18
Thalassodendron ciliatum*	C3	Y/N	Y	N	4, 5, 11, 43
Zostera marina	C3	Y	Y	Y/N	7, 9, 23, 39
Z. muelleri	−	Y	Y	N	33, 34
Ochrophyta (Brown algae)
Alaria esculenta	−	Y	Y	N	19, 42
Ascophyllum nodosum	CAM-like?	Y	Y	N	19, 25, 26, 42
Dictyota (Dilophus) guineënsis*	C4?	Y	−	N	21
Fucus distichus ssp. edentatus	−	Y	N	−	15
F. serratus	C3	Y	Y	Y	19, 27, 42
F. spiralis	−	Y	Y	Y	19, 42
F. vesiculosus	CAM-like?	Y	Y	Y	19, 36, 39, 42
Halidrys siliquosa	−	Y	Y	N	19, 42
Himanthalia elongata	C3	Y	Y	−	19, 27
Hizikia fusiforme	C3	Y	Y	N	44
Laminaria digitata	−	Y	Y	N	19, 42
L. hyperborea	−	Y	Y	N	19, 42
L. saccharina	C3	Y	Y	N	19, 28, 42
Lobophora variegata*	C3	Y	Y	N	18, 21
Padina sanctae-crucis* (Ca)	−	Y	−	N	18
Pelvetia canaliculata	C3	Y	Y	N	19, 27, 42
Pelvetiopsis limitata	−	Y	N	−	14
Petalonia zosterifolia	C3	Y	−	−	27
Turbinaria turbinata*	C3	Y	−	N	21
Chlorophyta (Green algae)
Avrainvillea nigricans* (Ca)	C3	Y	−	−	21
Chaetomorpha linum*	−	Y	−	Y	39
Cladophora rupestris	C3	Y	Y	−	19, 26
Cladophoropsis membranacea*	C3	Y	−	Y	21
Codium decorticatum*	C3	Y	Y	N	37, 38
C. fragile*	−	Y	N	−	19
Enteromorpha compressa*	C3	Y	−	−	27
E. intestinalis*	−	Y	Y	−	30
E. linza*	C3	Y	Y	−	27
Enteromorpha sp.	−	Y	N	Y	19, 39
Halimeda discoidea* (Ca)	−	Y	Y	Y	13
H. macroloba* (Ca)	−	Y	Y	−	13
H. tuna* (Ca)	−	Y	Y	−	13
Monostroma fuscum	−	Y	N	−	19, 30
Prasiola stipitata*	−	Y	Y	−	19
Udotea flabellum* (Ca)	C4	Y	Y	N	37, 38, 39
Ulva lactuca*	C3	Y	Y	Y	19, 27, 31
Ulva sp.	−	Y	−	Y	6
Rhodophyta (Red algae)
Amphiroa anceps* (Ca)	C3	Y	−	N	12
A. foliacea* (Ca)	C3	Y	−	N	12
Bostrychia scorpioides*	C3	Y	−	−	27
Botryoglossum farlowianum	−	Y	N	−	15
Callithamnion tetragonum*	−	Y	Y	N	35
Calliblepharis jubata	C3	Y	−	−	27
Callophyllis marginifructa	−	Y	N	−	15
C. violacea	−	Y	N	−	15
Ceramium rubrum*	C3	Y	Y/N	Y	16, 19, 31, 35, 39
C. tenuissimum*	C3	Y	N	−	16
Chondracanthus acicularis*	−	Y	Y	−	35
Chondrus crispus	C3	Y	Y/N	−	19, 27, 28, 35, 41
Corallina officinalis* (Ca)	C3	Y	N	−	19, 27
Corallina sp.* (Ca)	−	Y	Y	−	35
Cystoclonium purpureum	−	Y	Y	Y	35
Delesseria sanguinea	C3	N	Y/N	Y/N	26, 31, 35
Dumontia contorta	−	Y	N	Y	19, 35
Erythrophyllum delesseroides	−	Y	N	−	15
Fauchea laciniata	−	Y	N	−	15
Furcellaria lumbricalis	−	Y	Y	Y	35
Gastroclonium ovatum*	C3	Y	Y	−	27
Gelidium crinale*	C3	Y	N	−	16
G. latifolium*	−	Y	Y	−	35
Gigartina exasperata	−	Y	N	−	15
G. papillata	−	Y	N	−	15
Gracilaria gaditana	−	Y	Y	−	3
G. gracilis*	−	Y	Y	Y	35
Gymnogongrus linearis	−	Y	N	−	15
Hymenena flabelligera	−	Y	N	−	15
Hypoglossum hypoglossoides*	−	Y	Y	N	35
Iridaea lineare	−	Y	N	−	15
I. cordata var. splendens	−	Y	N	−	15
Laurencia papillosa*	C4?	Y	N	N	19, 21
L. pinnatifida*	−	Y	Y	Y	19, 26
Lomentaria articulata*	C3	Y/N	Y/N	Y/N	19, 26, 29, 31, 35
Mastocarpus stellatus*	−	Y	Y/N	−	19, 35
Membranoptera alata	C3	N	Y	−	19, 29
Microcladia coulteri	−	Y	N	−	15
Palmaria palmata	−	Y	N	Y	14, 19, 26, 35
Phycodrys rubens	−	N	Y/N	N	19, 31, 35
Phyllophora pseudoceranoïdes	−	−	Y	−	35
Plocamium cartilagineum*	−	N	Y/N	−	19, 31, 35
Plumaria elegans	−	Y	Y	−	19
Polysiphonia elongata	−	Y	Y	−	35
P. fucoides*	−	Y	Y	−	35
P. lanosa	−	Y	N	−	19
P. stricta*	−	Y	Y	−	35
P. urceolata	−	Y	Y	Y	35
P. violaceae	−	Y	−	Y	39
Porphyra occidentalis	−	Y	N	−	15
P. purpurea	−	Y	Y	−	19, 35
P. umbilicalis*	−	Y	Y	Y	19, 35
Pterocladia capillacea*	C3	Y	N	−	16
Ptilota plumosa	−	N	−	−	31
Rhodoglossum affine	−	Y	N	−	15
Rhodymenia palmata	−	Y	N	−	15
Rhodomela confervoides	−	−	N	−	19
R. subfusca	C3	Y	−	−	27

Among marine macroalgae reviewed (Table 1), the presence of a single-cell C₄ pathway has been documented in the tropical chlorophyte Udotea flabellum (Reiskind et al., 1988; Reiskind & Bowes, 1991). This species showed C₄ gas-exchange characteristics and the initial carboxylation was via a cytosolic phosphoenolpyruvate carboxykinase (PEPCK) to yield C₄ acids that turned over in the light. Furthermore, upon inhibition of PEPCK, photosynthesis was reduced and C₃-like gas-exchange properties emerged. There is some evidence for a C₄ system in Dictyota guineënsis and Laurencia papillosa, but it is inconclusive as the distinction was solely based on high PEPC and PEPCK activities relative to Rubisco (Holbrook et al., 1988).

An extensive literature review on CAM photosynthesis in aquatic species indicates no CAM activity in the Chlorophyta and Rhodophyta species investigated, based on titratable acidity changes (Keeley, 1998). However, in the Ochrophyta (browns), particularly in the Fucaceae, several species show diel acidity changes, but the acid pool sizes are only a tenth of that reported for a true submersed CAM plant, Isoetes howellii, a freshwater lycophyte. Some CAM-like activity has been shown in the temperate intertidal ochrophyte Fucus vesiculosus, based upon small diel changes in titratable acidity and a high affinity for inorganic carbon, and similar findings have been reported for F. spiralis and F. serratus (Raven & Osmond, 1992; Keeley, 1998). Likewise, small diel fluctuations and dark fixation have been reported for Ascophyllum nodosum, and its photosynthetic gas-exchange responses appear to involve a carbon concentrating mechanism (CCM) although most likely based on HCO₃⁻ usage, rather than a C₄ or CAM system (Johnston & Raven, 1986, 1987; Johnston, 1991). The β-carboxylation of C₄ acids by the enzyme PEPCK and fixation in the dark has long been recognized among some brown macroalgae, but whether the role is in photosynthesis or just to recharge the supply of Krebs cycle acids (anaplerosis) remains unresolved (Kremer & Küppers, 1977; Kremer, 1980; Johnston, 1991).

Inorganic C species utilization

Based on this review (Table 1), a majority, or 95% of the marine macro-autotroph species examined, possess the ability to utilize HCO₃⁻, which is more than that estimated for freshwater macrophytes (Madsen & Sand-Jensen, 1991). A majority of the seagrasses appear to have the capacity to utilize HCO₃⁻ with a few exceptions and uncertainty for some species (Table 1). Tropical seagrasses (Cymodocea serrulata and Halophila ovalis) exhibit photosynthetic rates on par with macroalgae at high pH values where [CO₂] is low, presumably by utilizing HCO₃⁻ through active transport (Schwarz et al., 2000; Beer et al., 2002). Active transport and CCMs would be expected at high pH, as HCO₃⁻ dehydration to CO₂ and subsequent passive diffusion is dependent on a low pH. For this reason, DIC limitation can occur in high pH seawater even where [HCO₃⁻] is relatively high, if active transport systems are compromised.

Similar to seagrasses, most marine macroalgae use HCO₃⁻ for photosynthesis (Table 1). In a study of 35 temperate marine macroalgae, Maberly (1990) showed HCO₃⁻ use in 83% of the species. The few macroalgae restricted to CO₂ use were primarily represented by the Rhodophyta and tended to grow in low irradiance, subtidal environments (Maberly, 1990; Johnston et al., 1992; Raven et al., 1995, 2005a; Hepburn et al., 2011). It is notable that rhodophyte Rubisco has a greater specificity for CO₂ relative to O₂ compared to other species, and thus CO₂ loss from photorespiration should be less even without HCO₃⁻ use or a CCM (Badger et al., 1998; Reinfelder, 2011). Hepburn et al. (2011) used δ¹³C values below −30‰ to indicate HCO₃⁻ use in temperate brown macroalgae, and suggested the possible operation of a HCO₃⁻-based CCM. A recent review of algal CCMs indicates that they are likely present in all three major divisions of marine macroalgae, but CCMs are not ubiquitous in all classes or for species within a class (Raven et al., 2012). The ability to access the high [HCO₃⁻] in seawater enhances carbon acquisition where CO₂ diffusion is severely limiting. However, low photosynthetic K_0.5 values (i.e., the substrate concentration that drives the reaction at half maximal rate) show that CO₂ remains the ‘preferred’ form for photosynthesis in seagrasses and macroalgae. For example, K_0.5 values for CO₂ reported for Zostera marina (seagrass), Ceramium rubrum (Rhodophyta), Fucus vesiculosus (Ochrophyta), and Ulva lactuca (Chlorophyta) are twofold to threefold lower (0.26–0.28 mm) than that for HCO₃⁻ (0.54–0.8 mm; Sand-Jensen & Gordon, 1984).

Low light may energetically limit the ability of species to utilize HCO₃⁻ or to employ a CCM, increasing their reliance on CO₂ diffusion (Hepburn et al., 2011). Therefore, even species that use HCO₃⁻ or have CCMs may be dependent on CO₂ and positively respond to elevated [CO₂] at sub-saturating irradiance. Furthermore, there is evidence that HCO₃⁻ use and CCMs can be facultative, such that at high [CO₂], HCO₃⁻ use and CCM activity is down-regulated. This may decrease the ratio of internal to external CO₂ and/or the affinity for DIC, but it generally does not lower the absolute [CO₂] inside the cell (Hepburn et al., 2011). Cornwall et al. (2012) found that the proportion of CO₂ compared to HCO₃⁻ use in photosynthesis increased under short-term CO₂ enrichment in the fleshy chlorophyte Ulva sp. This facultative ability to change the degree of dependence from HCO₃⁻ to CO₂ use in photosynthesis may provide a competitive advantage at elevated [CO₂] because it reduces energy allocation to carbon acquisition (Raven et al., 2011).

Although the method and degree of HCO₃⁻ usage may well impact the response of a species to rising CO₂, the mechanisms and critical values that saturate Rubisco with CO₂ and lower their dependence on a CCM are not well understood (Giordano et al., 2005; Raven, 2010, 2011; Raven et al., 2011). Furthermore, the mode and phenotypic plasticity of HCO₃⁻ usage differs with species and conditions (Maberly & Madsen, 2002). It should be noted here that the ability to use HCO₃⁻ does not necessarily mean that the organism has a CCM. By definition, a CCM is an active transport process that raises the [CO₂], not just HCO₃⁻, in the vicinity of Rubisco above that in the external medium. Thus, to be a CCM and accumulate CO₂ around Rubisco, the active accumulation rate must exceed the Rubisco CO₂ fixation rate and the leakage of CO₂ out of the chloroplast. If the HCO₃⁻ use system operates at a lower rate than that of Rubisco it can still improve fixation without being a CCM by lessening the degree to which the [CO₂] limits Rubisco activity.

Uptake processes of inorganic carbon

Access to HCO₃⁻ can be achieved in several ways. The ion, potentially in co-transport with H⁺, may be actively transported across the plasma membrane into the cell. Alternatively, in the light H⁺ are actively secreted into localized regions of the leaf/thalli boundary layer to lower the pH and promote dehydration of HCO₃⁻, which increases the external [CO₂] and diffusion gradient into the cell. The localized secretion of H⁺ produces either alternating acid/alkaline zones, as along the filaments of the freshwater alga Chara corallina (Lucas & Berry, 1985; Shimmen et al., 2003), or a bipolar leaf in which the abaxial (lower) side becomes acidified and the adaxial (upper) side becomes alkaline, as in Hydrilla (van Ginkel et al., 2001). Boundary layer acidification and HCO₃⁻/H⁺ co-transport has been reported for the seagrasses Halophila ovalis and Ruppia cirrhosa using indirect inhibitor methods, but the acidification zones were not characterized, although a bipolar leaf scenario seems possible (Beer et al., 2002, 2006; Hellblom & Axelsson, 2003; Uku et al., 2005). They may also occur in some macroalgae, including Laminaria saccharina (Mercado et al., 2006). Exposure of the leaf or thallus to Tris buffer apparently negates the acidification process, and as a consequence decreases the CO₂-enhancement of photosynthesis that would otherwise result (Price & Badger, 1985; Beer et al., 2002, 2006).

Carbonic anhydrase secretion into the cell wall can minimize CO₂ depletion problems and provide access to HCO₃⁻ by catalyzing the interconversion of HCO₃⁻ to CO₂, but this is pH-dependent and is most effective when the boundary layer pH is low. This is because the enzyme only acts to accelerate the carbonate equilibrium shift from HCO₃⁻ to CO₂ in response to a lower pH; it should be noted that this is a reversible reaction if pH becomes elevated by photosynthetic uptake of CO₂. The evidence from a number of studies indicates that an external CA occurs in all the seagrasses and a majority of macroalgae (Table 1). Among the rhodophytes, however, it appears that the proportion lacking an external CA is greater (Table 1). As with HCO₃⁻ use, there are conflicting literature reports for the same species (Table 1). The inability to measure an external CA is not unequivocal evidence for its absence, given that assay methods differ and lack precision, and its secretion may be facultative. Surprisingly, there is not always a good correlation between the ability to use HCO₃⁻ and external CA (Giordano & Maberly, 1989), and in Table 1 some CO₂-only users show the presence of an external CA, and vice versa. However, it might be anticipated that an external CA should enhance CO₂ diffusion by catalyzing the conversion of HCO₃⁻ to CO₂ in the boundary layer.

Inorganic C saturation

Irrespective of the photosynthetic mechanisms, a key issue in predicting the response to rising [CO₂] is whether or not photosynthesis and growth are saturated by seawater [DIC] under the present-day conditions of inorganic C speciation, pH, [O₂] and temperature (Table 1). The literature reports are somewhat mixed in this regard, but it does appear that in many cases an increase in [DIC] results in higher photosynthetic and growth rates. Seagrass photosynthesis appears limited by current [DIC], due to the slow diffusive supply of CO₂ to the leaves, and possibly a less effective use of HCO₃⁻ when compared to many macroalgae (Beer, 1989, 1994), although this lower efficiency is not resolved (Beer et al., 2002). The majority of studies examining seagrasses showed they were limited by the current seawater [DIC] even though they are capable of using HCO₃⁻ (Table 1). Several seagrass species have also exhibited higher photosynthesis, increased reproduction, belowground biomass, and greater production of nonstructural carbohydrates, as well as, lower leaf-N and chlorophyll under elevated [CO₂] (Durako, 1993; Zimmerman et al., 1997; Beer & Koch, 1996; Palacios & Zimmerman, 2007; Jiang et al., 2010). Thus, CO₂ enrichment can shift C-allocation to carbohydrates and away from N-containing compounds, such as Rubisco, presumably resulting in a higher nitrogen use efficiency, similar to findings in terrestrial C₃ species (Leakey et al., 2009). Consequently, an increase in dissolved [CO₂] could be positive for photosynthesis and growth in seagrasses that are presently under-saturated with respect to DIC, regardless of their capacity to utilize HCO₃⁻.

The exception is under low irradiance which can alter the degree to which [DIC] limits photosynthesis and growth. This response can range from little change as light limitation ensues (Palacios & Zimmerman, 2007) to a greater sensitivity to elevated [CO₂] if plants rely on CO₂ diffusion, but are DIC limited. Schwarz et al. (2000) found that deep-water plants of Halophila ovalis and Cymodocea serrulata in the tropical waters of Zanzibar growing at low irradiance were far more limited by the seawater [DIC] than the same species in the high-light intertidal region. These responses were likely the result of a greater dependence on CO₂ under low light conditions because of the high energetic requirements of HCO₃⁻ use.

Similar findings apply to marine macroalgae (Reiskind et al., 1989; Maberly, 1990; Johnston et al., 1992) with notable exceptions (Israel & Hophy, 2002). Of five tropical species examined from Bahamian waters, only one showed saturation of photosynthesis at seawater [DIC] (Holbrook et al., 1988; Table 1). The same trend was reported by Wu et al. (2008) where a twofold to threefold increase in [CO₂] enhanced growth in macroalgae capable of using HCO₃⁻ (Gao et al., 1991, 1993; Table 1). Likewise, seawater [DIC] failed to saturate the photosynthetic rates of Codium decorticatum and Udotea flabellum, even though the latter has a single-cell C₄ system and is not inhibited by O₂ (Reiskind et al., 1988). These results are interesting, as many marine macroalgae have been reported to possess HCO₃⁻-based CCMs (Johnston, 1991; Raven, 1997; Larsson & Axelsson, 1999; Raven et al., 2011). Israel & Hophy (2002) suggest that such HCO₃⁻-based CCMs were the reason they observed no enhanced production, growth or change in Rubisco activity or amount in a diversity of macroalgal species from the Mediterranean grown under CO₂ enrichment. Although most of the experiments to date are of short-term, and there are species-specific and regional responses, the broader literature support the conjecture that seagrasses and fleshy macroalgae are likely to show positive responses to elevated [CO₂] consistent with terrestrial C₃ species, but the degree of response is not certain. A better understanding of macroalgal photosynthesis, the potential down-regulation of Rubisco and modulation of HCO₃⁻ use and CCMs for marine autotrophs is clearly warranted.

As a caveat, it should be noted that terrestrial C₃ species growth responses from decadal field CO₂ enrichment experiments are more modest than those predicted from short-term photosynthesis experiments, although growth is continuing to track increases in atmospheric [CO₂] (Long et al., 2006; Ainsworth et al., 2008; Leakey et al., 2009, 2012). During CO₂ enrichment, terrestrial C₃ plants undergo photosynthetic acclimation, primarily through down-regulation of Rubisco protein amount and hence leaf-N (Drake et al., 1997; Leakey et al., 2012). This appears to be a resource optimization process that results in a higher nitrogen use efficiency and C : N ratio, but a less than expected increase in growth and yield.

Although there are similarities, marine autotrophs will respond to rising [CO₂] with their own specificity, particularly because of HCO₃⁻ usage, which does not occur in terrestrial species, low [CO₂] at high pH, and steep [DIC] diffusion gradients. Marine macro-autotrophs will also be responding to changes in physical oceanographic processes, such as thermocline shifts, reworking of coastal zones with sea level rise, that will change nutrient availability and light with the potential to affect autotroph responses to DIC availability (Raven et al., 2011). Over the next few years, decades, and toward the end of the 21^st century, it is unclear to what extent marine species that use HCO₃⁻ and have a HCO₃⁻-based CCM will mimic the responses of terrestrial species and show modest, but continuing, responses to elevated [CO₂] and down-regulation of Rubisco (Leakey et al., 2012).

Climate change effects on seagrasses and macroalgae

Sea surface temperatures (SST)

Along with the rise in atmospheric [CO₂] (Fig. 1), mean global surface temperatures have increased by ~0.8 °C over the last century (Levitus et al., 2001; Hansen et al., 2006). Reconstructed temperature data from 35 million years ago indicate that tropical to subtropical SST ranged from 35 to 40 °C when atmospheric [CO₂] was ~1000 ppm (Kiehl, 2011), whereas modern day upper average temperature values are ~30 °C. By the end of this century temperatures are projected to increase by ~3–4 °C (Meehl et al., 2007); thus, the average SST could feasibly increase to those during the Eocene. Rising SST is already causing population shifts in temperate and tropical macroalgal species across various biogeographic regions (Wernberg et al., 2011), including economically and ecologically important species, such as kelp at the edges of their range (Liu & Pang, 2010). However, higher [CO₂] may ameliorate some of the negative effects of climate change on kelp through life history adaptations (Roleda et al., 2012). A study of over 20 000 herbarium records of macroalgae collected over 70 years from the Pacific and Indian oceans around the Australian coast shows that a poleward shift of several temperate species is already occurring (Wernberg et al., 2011). These changes are likely to continue, and therefore an understanding of species temperature thresholds and mechanisms for adaptation and interaction with elevated [DIC] would assist in predicting future community shifts.

Biochemical and physiological effects of elevated temperature

A climate warming of 3–4 °C increases photorespiration in terrestrial C₃ plants (Long, 1991; Sage & Kubien, 2007); however, CO₂ enrichment partially offsets high temperature effects because high [CO₂] promotes Rubisco carboxylation. As a result, elevated [CO₂] has been shown to raise the thermal optima in terrestrial C₃ species (Sage & Kubien, 2007). Furthermore, if photorespiration is ameliorated by high [CO₂], increasing temperatures within the range of 5–40 °C are likely to elevate photosynthetic rates by increasing electron transport capacity and sucrose/starch synthesis (Sage et al., 1995; Sage, 2002; Sage & Kubien, 2007). Relevant to net photosynthesis, tropical plants are known to acclimate to high temperatures by lowering their C loss to respiration as temperatures rise. So even when classical Q₁₀ relationships predict increases in enzymatic and other metabolic processes with climate change, including respiration, thermal acclimation may limit metabolic responses to temperature, particularly in plants from warm climates (Berry and Raison, 1981; Tjoelker et al., 2001). Thus, the interrelationship between increasing [CO₂] and temperature should be considered together, as these two variables fundamentally influence the biochemistry and physiology of plants. Consequently, it is these combined effects that are likely to control marine autotroph photosynthetic and growth responses, particularly those without CCMs or if CCMs are down-regulated. Although responses to elevated temperature have been studied at length in terrestrial plants, it is difficult to predict from these models how seagrasses and macroalgae will respond to elevated temperature, even though most appear to be C₃ species.

In particular, their potential for photosynthetic acclimation and Rubisco down-regulation is largely unknown. The primary inhibition of photosynthesis and decline in productivity of terrestrial species under moderate heat stress has been attributed to a reduction in the CO₂ fixation activity of Rubisco (Salvucci & Crafts-Brandner, 2004). This is a consequence of the thermal inactivation of Rubisco activase, a chloroplast protein that regulates the proportion of Rubisco enzyme that is catalytically active (Salvucci et al., 2001; Salvucci & Crafts-Brandner, 2004; Portis et al., 2008). Unfortunately, data on the role of Rubisco activase in the temperature responses of seagrasses or marine macroalgae are rare, and Rubisco activase does not appear to be universally present. No gene for Rubisco activase protein has been found in the genomes of the diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana, which utilize the form ID Rubisco gene family also present in red algae (Kroth et al., 2008; Raven et al., 2012). It is not known if tropical marine species have activase which is more thermally stable, as occurs in terrestrial species from high thermal regimes (Salvucci & Crafts-Brandner, 2004). Such information would have a bearing on which species may be able to survive future temperature extremes. Likewise, the effects of temperature on the facultative use of HCO₃⁻ are poorly documented. We do, however, have information on the thermal thresholds of some seagrass and macroalgal species, based on experimental and field studies, and are beginning to unravel their thermal tolerance mechanisms; these are reviewed below.

Thermal limits in tropical and subtropical seagrasses and macroalgae

Upper temperature thresholds – sustained

The temperature optima for photosynthesis in tropical seagrasses range from 27 to 33 °C and for temperate species from 21 to 32 °C, while their growing temperatures average ~3 and 8 °C lower, respectively (reviewed in Lee et al., 2007). Thus, tropical seagrasses are growing closer to their photosynthetic and physiological optimum in comparison to their temperate counterparts, making them highly vulnerable to climate warming (Tewksbury et al., 2008). Although tropical seagrasses are vulnerable to rising thresholds encountered during midday in the tropics, they can maintain photosynthetic and growth rates at relatively high temperatures compared to temperate species. For example, Halodule uninervis increased photosynthesis and growth from 27 to 33 °C with no photoinhibition, cellular damage or elevated respiration (Collier et al., 2011). This response, however, was dependent on light saturation (400 μmol photons m⁻² s⁻¹). In low light (40 μmol photons m⁻² s⁻¹) photosynthesis, growth and respiration did not respond to increasing temperature and all were low. The high thermal optima of tropical seagrasses in saturating light may be related to their ability to elevate sucrose synthesis in response to temperature (Sage et al., 1995; Touchette & Burkholder, 2000; Sage & Kubien, 2007). Elevated sucrose concentrations were found in leaves of two tropical seagrass species (Halodule wrightii, Thalassia testudinum) after 38 days at 34–35 °C during which relatively high maximum quantum yields (Fv/Fm-dark adapted) and live shoots were sustained (Koch et al., 2007). In addition, respiration in seagrasses has been shown to increase with temperature up to 40 °C (Touchette & Burkholder, 2000), thus the photosynthetic to respiratory ratio and C available for growth is likely to be a function of temperature but species specific (Zimmerman et al., 1989; Terrados & Ros, 1995; Collier et al., 2011). As shown by Collier et al. (2011) for Halodule, the respiration rates of some tropical species do not rise with temperature. Thus, thermal acclimation of respiration to temperature should be further investigated to advance our understanding of species-specific shifts in P : R ratios in response to climate change. It is likely that tropical species, and perhaps some temperate species, will down-regulate respiration in a warmer ocean similar to terrestrial plants up to their respective thermal threshold.

Tropical seagrass temperature thresholds (33–35 °C) determined experimentally (McMillan, 1984; Koch et al., 2007) correspond to those (<36 °C) where they are found in the field; thus, they may be predictive of future geographic distributions (Zieman, 1970, 1975; Thorhaug et al., 1978; Bulthuis, 1983; Ralph, 1998; Seddon & Cheshire, 2001; Massa et al., 2009). Temperate species are growing below their photosynthetic optima, but closer to their growth optima, and are likely to see range contractions as more thermally tolerant tropical species become competitively dominant. Even in the same geographic range, tolerance to elevated sustained temperatures will likely influence seagrass genus and species dominance. For example, along the NE coast of Australia, Zostera muelleri currently grows with Halodule uninervis, but Zostera is unable to tolerate sustained high temperatures of 33 °C (Collier et al., 2011). Thus, sustained elevated temperatures are likely to limit the distribution of Zostera, and to promote Halodule around the Great Barrier Reef (Campbell et al., 2006; Collier et al., 2011). Similarly, relatively low thermal tolerance of the dominant temperate seagrass species Posidonia oceanica in the Mediterranean Sea experiencing rapid warming is becoming a concern (Marbà & Duarte, 2010; Jordà et al., 2012). This species may be replaced by tropical species better adapted to high temperatures, although recruitment issues for tropical species could result in an overall decline of seagrasses in the Mediterranean (Jordà et al., 2012), and possibly a greater representation by macroalgae with a higher recruitment potential.

The majority of tropical macroalgae exhibit thermal limits ~2–3 °C lower than seagrasses, although many species have not been studied. The majority of macroalgal thermal limits that have been established are based on species shifts after El Niño Southern Oscillation (EÑSO) events and in response to anthropogenic thermal inputs (Zieman, 1970; Thorhaug, 1976; Carballo et al., 2002). Biber (2002) modeled thermal limits in south Florida macroalgae and showed significant declines in biomass at temperatures >31 °C. Anderson (2006) found fleshy branching tropical species (Dictyota menstrualis, Sargassum fluitans, and Laurencia chondroides) maintained relatively consistent net productivity rates at 32 °C, but at 34 °C, both D. menstrualis and S. fluitans net productivity fell below zero. In the same 28 day experiment, coralline crustose algal species (calcified prostrate rhodophyte forms) over the temperature range of 28–34 °C showed no change in maximum quantum yield (Fv/Fm-dark adapted), live tissue coverage, or mortality, indicating a high potential for thermal tolerance. In support of these data, Lirman & Biber (2000) observed a high relative abundance of coralline crustose algae during seasonally high temperatures (30–31 °C) along the Florida Keys reef tract. Thorhaug (1976) made the observation that many tropical marine macroalgae had maximum growth in the summer months when temperatures (31–32 °C) are surprisingly close to species lethal and sublethal temperatures (~32–38 °C), and found that thermal acclimation did not raise thermal limits. These temperatures also correspond to those disrupting (32 °C) and inhibiting (35 °C) photosynthesis in a tropical chlorophyte Codium edule on reefs in Nanwan Bay, southern Taiwan, which were causing summer die-back in the field (Lee & Hsu, 2009). Thus, climate change impacts on tropical marine macroalgae and seagrasses will be influenced by a sustained 3–4 °C rise by 2100, as the majority of these species are currently growing at their upper thermal limits.

Upper temperature thresholds – pulsed

The thermal limits established in longer term seasonal exposures are slightly lower than limits established in short-term pulsed experiments simulating midday or intertidal high temperature conditions (McMillan, 1984; Erftemeijer & Herman, 1994; Seddon & Cheshire, 2001; Campbell et al., 2006; Collier et al., 2011). Campbell et al. (2006) examined photosynthetic responses of seven Great Barrier Reef-associated tropical seagrass species to acute, 4 h pulses of high temperature exposures from 35 to 45 °C compared to controls (26 °C), including both intertidal and subtidal species. Five of the seven species maintained 80% of control-level effective quantum yield (ΔF/Fm′-light adapted) after 3 days of 4 h treatments at 40 °C. Four of these species showed no inhibitory effect on quantum yield and no change in photochemical or non-photochemical quenching relative to controls. Interestingly, these experiments were conducted at sub-saturating irradiance (100 μmol photons m⁻² s⁻¹), showing that tropical seagrass short-term thermal tolerance can occur under relatively low light. None of the species examined by Campbell et al. (2006) survived repetitive short exposures to 45 °C because of irreversible photosynthetic machinery damage.

Similarly for macroalgae, but at lower temperatures, Codium edule maintained photosynthetic function with photochemical quenching after 2 h at 35 °C, but at 4 h was replaced by non-photochemical quenching with a concomitant decline in effective quantum yield (ΔF/Fm′-light adapted; Lee & Hsu, 2009). The examination of ultrastructure also showed a loss of turgidity and a breakdown of the central vacuole, and after 8 h at 35 °C, the vacuole ruptured, which led to chloroplast decomposition. Thus, the period of exposure to high temperature is critical in both seagrasses and macroalgae. Climate change will elevate the current short-term maximum temperatures, such that midday sublethal and lethal extremes may become more frequent and sustained. Understanding the mechanisms of tolerance and differential species adaptations to these extremes is critical to understanding climate change impacts, particularly for species growing at their thermal limits (Collier et al., 2011).

Mechanisms of thermal stress tolerance

The mechanisms of thermal tolerance are not well studied in tropical seagrasses and macroalgae, but the studies that have been conducted indicate comparable responses to heat stress among temperate seagrasses, macroalgae, and terrestrial plants. For example, at upper threshold temperatures, thermal stress in seagrasses was identified by the breakdown of photosystem II function and elevated non-photosynthetic quenching (Ralph, 1998, 1999; Seddon & Cheshire, 2001; Campbell et al., 2006; Winters et al., 2011). Winters et al. (2011) measured photophysiology and gene expression simultaneously in Zostera marina, a temperate species, after exposure to thermal stress (25–27 °C). Initial up-regulation of superoxidase dismutase was found coincident with elevated effective quantum yield (10 s dark adaptation) and electron transfer rates, indicating that seagrass thermal stress is linked to photophysiology and that Z. marina has the capacity to up-regulate anti-oxidative machinery (Reusch et al., 2008). Although heat shock proteins were not shown to be expressed in Z. marina in response to elevated temperatures (Reusch et al., 2008), heat shock proteins (HSP70 family) were up-regulated by the intertidal seagrass Zostera noltii exposed to thermal stress (37 °C for 4 h, Massa et al., 2011). At the same time, photosynthesis-related genes were down-regulated with the exception of genes associated with non-photosynthetic quenching and oxidative stress. The expression of photosynthetic genes primarily encoding for chlorophyll (a-b)-binding proteins that play a role in the transfer of electrons through antennae and reaction centers was reduced. This was presumably in response to the down-regulation of photosynthesis and/or simply a consequence of programmed cell death.

In intertidal species, the negative compounding effects of desiccation and high temperature (32 °C for temperate species) can be lethal with a slow recovery depending on length of exposure to the air and the ability to reemerge through rhizome regrowth post disturbance (Seddon & Cheshire, 2001). This combined stress likely explains the lack of extensive intertidal seagrass meadows in the tropics. It also may explain the large-scale mortality events of the seagrasses Posidonia australis and Amphibolis antarctica in Spencer Gulf, South Australia (12 700 ha loss in 2000; Seddon & Cheshire, 2001). Intertidal species subjected to desiccation, high temperature and light are vulnerable to future conditions of climate change, particularly species that are less desiccation tolerant. Burritt et al. (2002) showed that the ability to tolerate desiccation in the macroalga Stictosiphonia arbuscula (Rhodophyta) growing in the upper intertidal zone was linked to its ability to limit the production of H₂O₂ and lipid hydroperoxides, thereby minimizing membrane and protein damage, as compared to individuals of the same species growing in the lower intertidal. The mechanism controlling oxidative stress was shown to be related to the up-regulation of antioxidant enzyme activities that maintained constitutive ascorbate and glutathione levels comparable to those under non-stress conditions, rather than by maintaining a large antioxidant pool; however, catalase activity may also have been important in limiting H₂O₂ accumulation (Burritt et al., 2002). The ability to rapidly up-regulate anti-oxidants and produce fewer reactive oxygen species in marine macro-autotrophs is critical for species subjected to stress on a daily basis, either in the intertidal zone or sub-tidally, where tissues are exposed to extreme high temperatures and other stressors for a few hours at midday (Murthy & Sharma, 1989; Collén & Davison, 1999; Dummermuth et al., 2003; Dring, 2006; Lesser, 2006; Ross & Van Alstyne, 2007). These species may be poised to tolerate elevated temperatures with climate change, up to some threshold. They could also serve as model systems to study potential mechanisms of upper thermal stress tolerance.

Both seagrasses and macroalgae possess diverse acclimation strategies to tolerate high temperatures and other stressors associated with climate change, but the sustained level and period of exposure is critical. Temperature and CO₂ interactive effects have been shown to be synergistic in phytoplankton studies with highly species-specific results (Fu et al., 2007; Hare et al., 2007; Feng et al., 2009; Torstensson et al., 2012); thus, further studies are needed to understand these interactions in seagrasses and macroalgae. Temperature has also been shown to exacerbate the negative impacts of OA on marine calcifying macroalgae (Anthony et al., 2008; Martin & Gattuso, 2009; Sinutok et al., 2011; Diaz-Pulido et al., 2012); however, the mechanisms for this synergy are not understood.

Calcification, elevated [CO₂], and temperature responses in calcareous macroalgae

Calcification in marine macroalgae and climate change

Marine calcareous macroalgae span all three major algal divisions, are represented by >100 genera (Hillis-Colinvaux, 1980) and are important calcifiers in most marine ecosystems, but particularly in the tropics. These calcareous algae have varied mineralogies and crystalline forms of CaCO₃ and species-specific sites of calcification. All calcifying macroalgae precipitate crystals outside of the cell or extracellularly. The dominant calcifiers form crystals in intercellular spaces (ICS), such as in the genus Halimeda, within cell walls, as in the corallines, or on the surface of their thalli, as represented by the genus Padina (Table 2). This diversity of calcification sites and their proximity to pH-elevating processes (e.g., photosynthesis, H⁺ pumping) relative to external seawater is likely to affect different species' potential to accommodate lower external pH with OA. However, we currently lack an understanding of how these diverse thalli morphology and their species-specific physiology control calcification and dissolution. This is in part a result of the high diversity of marine macroalgae and their complex mechanisms of organic and inorganic C cycling and recycling. That said, the abiotic controls on calcification are relatively well understood (Millero, 2007). Furthermore, there are specific genera for which we have a basic understanding of the structural and biological controls on biogenic calcification (reviewed in Borowitzka, 1982; Pentecost, 1985; Cabioch & Giraud, 1986; Borowitzka, 1987; Pentecost, 1990). There are also recent experimental and field studies that can be synthesized to make predictions on the potential consequences of OA and climate change on marine macroalgae.

Table 2. Dominant calcifying macroalgal division, order, and genera, their carbonate polymorph and site of extracellular calcification. ICS, intercellular spaces; CW, cell wall; Sh, sheath; Utr, utricular; B, crystals bundled; S, surface; Calcite (hexagonal-rhombohedral morph CaCO₃ crystal); Aragonite (orthorhombic morph CaCO₃ crystal); HMgCalcite-high magnesium calcite

Division/Order	Genus	Polymorpha^,b^,c	Sitee	Ref
Reviewed in: (1) Borowitzka et al. (1974); (2) Borowitzka (1982); (3) Borowitzka (1987); (4) Ries (2011b). ^a Aragonite/Calcite can vary with season in some species. ^b MgCO3 concentrations can vary with season and Mg : Ca ratios in seawater. ^c High-Mg calcite thalli ~7–30% MgCO3. ^d Intercellular spaces exhibit a range of connectivity with external seawater dependent on species and morphology. ^e Many sheath formers also possess calcium oxalate (CaC2O4) in vacuoles. ^f Udotea species range from 100% Aragonite to non-calcified.
Chlorophyta/	Halimeda	Aragonited	ICS/Utr	1, 2
Bryopsidales	Udotea	Aragonitef	ICS to Sh/B	2, 3
	Penicillus	Aragonite	ICS/Sh/B	1, 2, 3
	Rhipocephalus	Aragonite	ICS/Sh/B	2, 3
Dasycladales	Acetabularia	Aragonite	CW/Sh/B	2, 3
Cladophorales	Neomeris	Aragonite	ICS	1, 2
Ochrophyta/	Padina	Aragonite	S	1
Dictyotales
Rhodophyta/	Lithophyllum	HMgCalcite	CW	1, 2
Corallinales	Lithothamnion	HMgCalcite	CW	2
	Porolithon	HMgCalcite	CW	2
	Corallina	HMgCalcite	CW	1, 2
	Jania	HMgCalcite	CW	2
	Amphiroa	HMgCalcite	CW	2
	Neogoniolithon	HMgCalcite	CW	4
	Goniolithon	HMgCalcite/Brucite	CW	1, 2
Nemaliales	Liagora	Aragonite	ICS	1
	Galaxaura	Aragonite/Calcitea	ICS/CW/Sh	2, 3
Peyssonneliales	Peyssonnellia	Aragonite	CW	2

Biogeochemical controls on macroalgal calcification

Calcification and CaCO₃ saturation state

The chemical reaction of calcification is controlled by the saturation state (Ω) of CaCO₃ at in situ conditions of temperature, salinity, and pressure (Millero et al., 2006; Millero, 2007). The is a function of the ion concentration product of CO₃²⁻ and Ca²⁺ and the solubility product of the pure solid CaCO₃ mineral form according to the following equation:

• (Eqn 1)

where K*_sp is the unique stoichiometric solubility product with respect to the CaCO₃ mineral form. In seawater, Ca²⁺ concentrations are approximately conservative with salinity (UNESCO, 1987; Feely et al., 2004); thus, is primarily controlled by the ratio of CO₃²⁻ and K*_sp. Thus, the current decline in seawater [CO₃²⁻] lowers the ((Eqn 1), Fig. 1). The is primarily affected by changes in [CO₂] with temperature rise associated with climate change a second-order factor (Cao et al., 2007). Theoretically, when >1 seawater is considered super-saturated and precipitation of CaCO₃ is favored, whereas when <1 seawater is under-saturated and dissolution is favored.

The two dominant crystalline forms of CaCO₃ represented in marine macroalgae are calcite and aragonite. The calcite polymorph is primarily formed in the Rhodophyta, whereas aragonite crystals are primarily produced by the Chlorophyta (Table 2). If macroalgal thalli behaved as pure crystals, calcifiers precipitating calcite would be more resilient to OA given their respective K*_sp. However, other geochemical and biological processes influence calcification and dissolution in biological organisms in addition to , such that calcification and dissolution do not follow pure crystal dynamics (Feely et al., 2004; Ries et al., 2009).

Purity of mineral form

One major geochemical characteristic of the crystal polymorphs that influences calcification and dissolution in nature is their purity and physical structure. For example, biogenic calcite solubility increases with increasing magnesium (Mg) content (Morse et al., 2006). Macroalgae that form high-Mg calcite (Mg/Ca > 4 mol %) characterize the majority of coralline species within the Rhodophyta (Table 2), including those species important in the formation of reefs and free-living rhodoliths (Nelson, 2009). Mineral impurities, such as varying constituents (e.g., H₂O, OH⁻, SO₄²⁻, Sr, HCO₃⁻) and adsorbed organics, also influence the CaCO₃ solubility of biogenic carbonates (Morse et al., 2006). Therefore, the Mg : Ca ratios do not always correspond directly to the solubility of the CaCO₃ polymorphs (Moberly, 1968; Morse et al., 2006). Furthermore, as the sites of calcification are compromised (e.g., thalli aging, herbivory, dissolution-precipitation), crystal structure can change to unrecognizable forms (Borowitzka, 1982) to the point where they are not as predictable as their original polymorph structure.

Organic cellular effects on crystallization

Another major factor that drives precipitation of carbonates is the presence of organic substrates. These bind Ca²⁺ ions and provide sites for nucleation, thereby promoting calcification at relatively low (Borowitzka, 1987). Mineral formation in a solution is against a free-energy gradient; therefore, nucleating surfaces are required to lower the activation energy (Simkiss, 1986). It is further suggested that cellular organics in the cell wall of rhodophytes define the calcite polymorph (Table 2). The calcite crystals tightly align with the cell wall polysaccharide fibrils and are embedded in an organic matrix that results in a smooth demarcation between the interior cell and cell wall (Bilan & Usov, 2001) easily seen using electron microscopy (Borowitzka et al., 1974; Fig. 2c). Even in species that form aragonite in intercellular spaces, such as in the Chlorophyta (Table 2), crystals frequently occur at right angles adjacent to the cell walls, as opposed to being randomly oriented within the intercellular space (Borowitzka et al., 1974; Bilan & Usov, 2001). This conformation indicates cellular influence on initial crystallization at the cell wall surface (Borowitzka, 1984, 1987). After initial crystallization, which can be a multi-step process, calcification is thought to follow crystal growth influenced by biological processes (Borowitzka, 1987).

Figure 2. Three macroalgal calcification models are depicted: (a) intercellular space (ICS) represented by Halimeda, (b) extracellular cell surface represented by Chara, and (c) extracellular cell wall represented by Lithothamnion. In Halimeda (a), aragonite (see crystal image) ICS calcification (3) is enhanced by light-dependent CO₂ uptake for photosynthesis (1) and proton exchange in adjacent cells. Cellular CO₂ uptake (and possibly H⁺) increases ICS pH and shifts the carbonate equilibrium toward CO₃²⁻ and CaCO₃ precipitation. Photosynthesis is promoted by the dehydration of HCO₃⁻ to CO₂ catalyzed by carbonic anhydrase (CA). However, CA activity is low at high pH, thus active transport of HCO₃⁻, or efflux of H⁺ to lower pH at the outer cell boundary layer, is likely. CO₂ from respiration (2) is recycled into calcification. Under ocean acidification (OA), diffusion of CO₂ and H⁺ into the ICS from seawater and a preferential DIC uptake from the cell walls facing the external medium (S) may lower net calcification and increase CaCO₃ dissolution (4). In the Chara model (b), active H⁺ pumps (or OH⁻ efflux) create alkaline and acid zones. The alkaline zone promotes calcification (calcite) and the acid region increases dehydration of HCO₃⁻ to CO₂ and subsequent diffusion within cell plasmalemma invaginations. Calcification in Chara occurs toward the cell surface, thus Ca²⁺ is hypothesized to be acquired intercellularly as part of an ATPase Ca²⁺ pump and the C source CO₂ generated from photosynthesis (1). In coralline algae (c), organics produced by the cell wall influence nucleation, Ca²⁺ incorporation and calcite mineralogy, while Mg²⁺ is incorporated into the calcite crystals in correspondence with the Ca : Mg ratio in seawater. Photosynthesis (1) also stimulates calcification by controlling pH, potentially H⁺ flux and carbonate equilibrium, as in models (a) and (b). Studies on the subapical growth pattern of cell wall calcifiers show a highly controlled cellular mechanism, which can induce dissolution and recalcification of epithallial cells, presumably driven by polysaccharide production and H⁺ pumps, although these mechanisms and their response to OA are presently not known. Figures drawn after Hillis-Colinvaux (1980), Borowitzka (1987), McConnaughey & Falk (1991) and other references cited; further details are provided in the text. SEM images of Halimeda (a) and Lithothamnion (c) provided by J. Ries. Chara sp. illustration reproduced with permission from UF IFAS – Center for Aquatic and Invasive Plants.

Although cellular organics promote calcification in macroalgae, and control the alignment of crystals and their polymorph (Cabioch & Giraud, 1986), one hypothesis explaining a lack of calcification in a majority of marine macroalgae is their production of organics (Pentecost, 2004). A suite of organic compounds are known to inhibit CaCO₃ nucleation and crystallization (Borowitzka, 1982, 1984). Thus, identifying the organic compounds that induce vs. inhibit calcification is critical to understanding their calcification mechanisms and potential vulnerability to OA.

Seawater chemistry and mineralogy

Although organic production has been shown to induce calcification and control the polymorph characteristics of different macroalgal groups (Table 2), their ability to limit impurities that increase CaCO₃ solubility has limits. In fact, the polymorph dominance and purity has changed in response to ocean chemistry over geological time scales (Ries, 2010; Stanley et al., 2010). This shift in polymorphs with ocean chemistry is important to consider because the current paradigm is that the solubility of calcifiers to OA is based on their fixed mineralogy, whereas in fact for some species it is highly dependent on seawater chemistry, primarily Mg : Ca ratios, and even season (Borowitzka, 1982). Several studies have shown that macroalgal aragonite and high-Mg calcite producers are favored when the seawater Mg : Ca mole ratio is >2, and low Mg calcite producers are favored when Mg : Ca mole ratios are <2 (Füchtbauer & Hardie, 1976, 1980). This occurs because the Mg slows net calcite precipitation (Möller & Parekh, 1975; Davis et al., 2000). The mineralogy, production, and calcification of chlorophytes (Halimeda, Penicillus and Udotea) and rhodophytes (Neogoniolithon, Amphiroa) were examined in a series of experiments reviewed in Ries (2010) where seawater was modified to simulate current ocean ‘aragonite seas’ (Mg : Ca ratio 5.2) compared to ‘calcite seas’ (Mg : Ca ratio 1.0). Remarkably, under the ‘calcite sea’ conditions the chlorophytes modified their present-day relatively pure aragonite thalli composition to 22–46% Mg calcite. Their aragonite preference, however, was shown by a higher reproductive output, productivity rate, and carbonate production under high Mg : Ca ratios. In contrast, the corallines maintained their calcite polymorph across a range of Mg : Ca ratios (1.0–7.0), although the Mg content in their thalli tracked the Mg : Ca ratios. Thus, seawater chemistry can influence the polymorph of CaCO₃ in the thalli of chlorophytes and the Mg incorporation in rhodophytes (Ries, 2010), which would theoretically affect their solubility under OA. Macroalgal species with calcium-binding polysaccharides that produce low rather than high Mg²⁺ calcite or aragonite should be favored in a high CO₂ ocean. Whether or not this could be an acclimation response is highly uncertain, and perhaps unlikely given the results of the experiments above, unless seawater Mg : Ca ratios fall. Organisms also exert biological control on net calcification and biologically mediated mechanisms have been suggested to be more important than mineralogy in defining species-specific responses to OA (Ries et al., 2009; Ries, 2011b).

Models of calcification in macroalgae

The specific mechanisms controlling calcification and dissolution in the dominant calcareous macroalgae have not been well studied with the exception of a few genera (e.g., Halimeda, Chara, and a few coralline spp.). Their calcification mechanisms have been characterized as either ‘biologically induced’ by biological processes and external media or ‘organic matrix-mediated’ controlled by the cell wall organic matrix (Lowenstam, 1981; Borowitzka, 1987), but these classifications are not strict and primarily define the dominant mechanism, as both are biologically controlled by photosynthesis and organically influenced through nucleation. In synthesis, three models are illustrated (Fig. 2) to provide a starting point from which to ask questions about how climate change and OA will potentially affect calcifying macroalgae, two with ‘biologically induced’ calcification (Fig. 2a and b) and one with ‘organic matrix-mediated’ calcification (Fig. 2c). For Halimeda spp. that calcify in intercellular spaces (ICS), gases and ions exchange between the ICS, the adjacent cells and seawater (Fig. 2a; Borowitzka, 1987). Halimeda surface utricular cells are appressed resulting in ~75% of the cell walls interfacing the semi-enclosed ICS. Consequentially, DIC is exchanged amongst photosynthesis, respiration, and calcification (Fig. 2a). Isotopic δ¹³C signatures of thalli carbonate (Lee & Carpenter, 2001) and microsensor studies (de Beer & Larkum, 2001) support this conjecture. As CO₂, and potentially HCO₃⁻, are taken up from the ICS by the adjacent cells for photosynthesis, the ICS pH increases and shifts the carbonate equilibrium toward CO₃²⁻ and CaCO₃ precipitation (Fig. 2a). This in turn lowers ICS [Ca²⁺] and promotes diffusive uptake of Ca²⁺ (de Beer & Larkum, 2001). The precipitation reaction produces CO₂, which can be subsequently taken up by the cells and photosynthetically fixed (Fig. 2a).

In contrast to intracellular calcification, some calcareous macroalgae limit their calcification to the surface of the thalli, as exhibited by Chara (Fig. 2b). Although Chara is not classified as a marine alga, it can grow in low salinity environments and its calcification mechanism has been well studied (McConnaughey, 1991; McConnaughey & Falk, 1991). In the Chara model, distinct bands of acidified and alkaline zones are created along the algal surface giving rise to a banding of CaCO₃ crystals shown as a rough texture on the thalli surface (Fig. 2b). In the acid zone, H⁺ are pumped into plasmalemma invaginations (plasmalemmasomes) lowering the pH and shifting the inorganic C equilibrium from HCO₃⁻ to CO₂ catalyzed by CA which facilitates diffusive CO₂ uptake for photosynthesis (Fig. 2b). The alkaline zone is promoted by either H⁺ influx or OH⁻ efflux raising the pH and promoting calcification. Two models have been presented by McConnaughey & Falk (1991), H⁺ channel and Ca²⁺ ATPase, to account for the uptake of H⁺, with the latter producing 2CO₂. Both models result in a 1 : 1 ratio of photosynthesis to calcification and fundamentally link HCO₃⁻ utilization to extracellular calcification. Although active Chara-like alkaline-acid banding was initially postulated to explain calcified bands in Padina, one of the only marine calcifying brown algae (Borowitzka, 1984), the bands have been subsequently attributed to an ICS that develops as the apical cells grow with an organic pilose layer providing nucleation (Okazaki et al., 1986). Secondary calcification in Padina may be dependent on initial nucleation and/or correspond to zones of high photosynthesis associated with tetrasporangia (Pentecost, 1990). In the third model, dominated by the corallinales, calcification occurs in the cell wall controlled primarily by polysaccharide production and organic fibrils (Fig. 2c, discussed below).

The morphology and calcification sites of the model organisms illustrated in Fig. 2 are distinctive, but several of the biologically induced mechanisms influencing their calcification are shared among different macroalgae and are considered together below with model-specific mechanisms highlighted.

Photosynthesis-calcification coupling

Light enhancement of calcification has been shown in a wide range of temperate and tropical calcareous macroalgae from different divisions and varied forms (Goreau, 1963; Stark et al., 1969; Borowitzka & Larkum, 1976, 1977; Pentecost, 1978; Borowitzka, 1981; Jensen et al., 1985; Semesi et al., 2009a). Experimental evidence for photosynthesis-calcification coupling was provided by de Beer & Larkum (2001) using microelectrodes at the cell surface of Halimeda discoidea. They found that photosynthesis created the pH environment conducive for calcification via uptake of CO₂. Furthermore, they estimated a 1 : 2 ratio of Ca²⁺ uptake to O₂ produced in the light that supported earlier estimates (Borowitzka & Larkum, 1976). The relationship between calcification and photosynthesis is likely influenced by a rapid series of reactions: (1) CO₂ uptake by photosynthesis, (2) a subsequent increase in pH, (3) a shift in the carbonate equilibrium toward CO₃²⁻, and finally (4) CaCO₃ precipitation. Even in cell wall calcification common in the Rhodophyta (Fig. 2c), there exists a strong positive correlation between pH and net calcification (R² = 0.80–0.97), as well as net calcification and [CO₃²⁻] (R² = 0.90) (Pentecost, 1978; Gao et al., 1993; Semesi et al., 2009a).

It should be noted that attention needs to be given to the irradiance in experimental and field studies, as ion pumps (e.g., H⁺, HCO₃⁻) that presumably drive photosynthesis and calcification (Fig. 2), as well as their interaction, have thresholds for light that are presently not adequately understood. For example, a light-induced putative H⁺ pump at the cell surface of Halimeda is triggered at low light (~12 μmol m⁻² s⁻¹; de Beer & Larkum, 2001), perhaps as a mechanism to prime photosynthesis by lowering pH and elevating CO₂ availability through the dehydration of HCO₃⁻, part of a proton exchange system similar to Chara, or simply driven to maintain intracellular ion homeostasis. This low light-induced H⁺ pump enhanced dissolution of CaCO₃ (Ca²⁺ efflux); however, increases in irradiance to values closer to those that saturate photosynthesis caused calcification rates and pH to rise significantly. Thus, the importance of light control and measurements are critical when assessing OA effects on calcifying macroalgae, because putative pumps and metabolic reactions that affect biogenic calcification are coupled to the energetics of light reactions.

Although photosynthesis under high light has been shown to promote calcification, photosynthesis can become [DIC] limited as the [CO₂] and [HCO₃⁻] are lowered at elevated pH (Fig. 1). The dehydration of HCO₃⁻ by CA is also low at high pH (>9) requiring the algae to utilize HCO₃⁻ through active transport (Lucas & Berry, 1985). This DIC limitation accounts for the negative correlation between pH and photosynthesis (R² = 0.90; Semesi et al., 2009a) and maximum photosynthetic rates at low pH (6.5–7.5) (Borowitzka, 1981). Consequently, a rise in the [CO₂] and [HCO₃⁻] with OA will raise the photosynthetic rates of many macroalgae, which are [DIC] limited (see previous section), providing a positive feedback between elevated DIC-enhanced photosynthesis and calcification. However, there are several negative feedback processes that lower calcification. For example, raising [CO₂] enhanced the photosynthetic rate of the coralline macroalga Hydrolithon by 13%, but reduced calcification rates by 20% (Semesi et al., 2009a). These data suggest that an increase in photosynthesis does not necessarily counter the negative effects of elevated [CO₂] on calcification. We hypothesize that these metabolic processes likely become uncoupled under elevated [CO₂] and temperature.

Constituent diffusion and membrane ion transport

Intercellular calcification, such as in Halimeda (Fig. 2a), is achieved by having a slow diffusion rate of external CO₂ through appressed cells to the ICS, relative to the uptake of CO₂ from the ICS for photosynthesis, thereby maintaining CaCO₃ super-saturation in the ICS (Fig. 2a; Borowitzka & Larkum, 1976). As the oceans acidify, the [CO₂] and [H⁺] in external seawater will rise (Fig. 2); thus, external diffusion rates of CO₂ and H⁺ to the ICS will increase and potentially lead to a lower pH in the ICS (Fig. 2a). Increasing temperatures could also raise diffusion coefficients and affect membrane integrity and thus ion transport; however, will also lower the solubility of CO₂. Macroalgal cell wall surfaces exposed to seawater may also utilize external CO₂ preferentially over CO₂ generated in the ICS as external [CO₂] rise and diffusion through the outer cell wall presumably increases. At a lower pH, CA would also be more effective at increasing external HCO₃⁻ use, and thus elevate [CO₂] at the cell surface (Fig. 2a [S]) and increase diffusive flux into the cell, again competing with DIC uptake from the ICS.

Elevated H⁺ in seawater will also affect diffusion gradients across cell boundaries increasing external to internal [H⁺] ratios ([H⁺]_E/[H⁺]_I; Jokiel, 2011; Ries, 2011b). Calcifiers will have to efflux H⁺ across a much stronger diffusion gradient under OA with a concomitant energy cost (Ries, 2011b). Because proton pumps affect a diversity of ion exchange processes, including calcification (McConnaughey & Whelan, 1997), shifts in [H⁺] gradients across cell membranes and other boundaries isolating regions of calcification has been put forth to explain OA effects on corals (Jokiel, 2011). This ‘proton flux hypothesis’ proposes that fewer protons generated by CaCO₃ precipitation are exported out of the region of calcification, thereby lowering the pH and limiting further calcification (Jokiel, 2011). Ries (2011b) tested several models of H⁺ regulation and showed that [H⁺]_E/[H⁺]_I ratios were maintained under high external [H⁺] in a temperate coral, although this resulted in elevated internal [H⁺] and negatively affected calcification. In an alternative model, the [H⁺]_E/[H⁺]_I ratio would be raised through active H⁺ efflux that would stabilize the internal pH at the site of coral calcification, although this adaptation requires a higher energetic cost (Ries, 2011b). These studies on corals, the well-studied ion transport systems of terrestrial plants, and a few aquatic species that have been examined (McConnaughey, 1991; McConnaughey & Falk, 1991) suggest that the ability to control ion transport across membranes and pH, that affect photosynthesis and calcification, will likely be a major factor in defining species-specific responses to OA. One potential problem with measuring [H⁺] in the ICS of macroalgae, as was found in Halimeda, is their wound-healing response. de Beer & Larkum (2001) saw a rapid pH drop (8 to 4-5) when microelectrodes were inserted into Halimeda ICS. Thus, alternative methods of assessing [H⁺]_E/[H⁺]_I ratios may be required in some macroalgal species.

Organics and cell wall calcification

The focus of mechanistic research on cell wall calcifiers, such as the Corallinales, has been on the role of cell wall polysaccharides, because of their presumed control in binding Ca²⁺ and fostering nucleation (Borowitzka & Larkum, 1977; Borowitzka, 1984; Cabioch & Giraud, 1986; Bilan & Usov, 2001; Martone et al., 2010; Navarro et al., 2011). Calcifying species also appear to produce 10- to 30-fold less polysaccharides, especially at the calcification sites in the cell wall, although this may fluctuate during cell turnover. Calcifiers of the Corallinales produce very specific cell wall matrix polysaccharides, including unusual sulfated xylogalactans (corallinans) and alginic acids. Martone et al. (2010) recently compared polysaccharide structure in calcifying and non-calcifying segments in the same individuals of a coralline sp. (Calliarthron cheilosporioides). They identified a xylose-branching structure of xylogalactans that supported calcification and found methylated glycans inhibited calcification. These data indicate a specific spatial biochemical control by the alga define the alternating calcified and uncalcified segments in this species. These processes may also partially explain the calcifying-decalcifying mechanisms by which calcareous species shed cell layers at the epidermis to reduce epiphytes and to grow subapically. Subapical growth in corallines occurs through an intricate series of steps including demineralization of cell walls of epithallial cells initiated by wall ingrowths of underlying cells (Wegeberg & Pueschel, 2002; Pueschel et al., 2005). These subepithallial invaginations presumably promote H⁺ efflux involved in dissolving the calcified epidermal cells. This dissolution exposes a dense organic microfibrillar matrix into which underlying, newly divided cells grow, followed by cell wall recalcification (Wegeberg & Pueschel, 2002; Pueschel et al., 2005). The subepithallial invaginations have been compared to the characean charasomes (modified plasmalemma) that play a role in H⁺ efflux and DIC uptake (Pueschel et al., 2005).

During surface cell replacement in corallines, the thalli may be more vulnerable to OA, but this supposition needs evaluation. Furthermore, it will be important to understand the effects of OA and temperature on polysaccharide production and speciation. As discussed previously, elevated [CO₂] and temperature can increase carbohydrate synthesis; therefore, excess production of dissolved organics (including polysaccharides) within a species or by other primary producers, such as non-calcareous macroalgae and seagrasses, may affect calcification. There is at present very little understanding of how OA and rising ocean temperature will affect the release of organic compounds that promote or inhibit calcification in marine macroalgae (Pentecost, 2004) and their relative sensitivities during various life stages.

Dark calcification, respiration, and dissolution

In all macroalgal calcifiers, calcification and respiration are two important biochemical processes that generate CO₂ internally, which can promote dissolution of CaCO₃ and lower net calcification, particularly at night, unless CO₂ is recycled. CO₂ production at night from respiration and dark calcification release H⁺ and lower pH (de Beer & Larkum, 2001). Under dark conditions, [Ca²⁺] was lower at the thallus surface of H. discoidea than in seawater indicating dark calcification, albeit at slower rates than in the light (de Beer & Larkum, 2001). Isotopic analyses by Lee & Carpenter (2001) support the hypothesis of dark calcification and the importance of CO₂ generated from respiration incorporated into CaCO₃ (Fig. 2a). They concluded that 16–36% of the CaCO₃ in Halimeda and Amphiroa was derived from respiratory carbon input. These data support earlier ¹⁴C-labeling experiments in which respiratory CO₂ was estimated to account for ~30–50% of calcification, particularly during the light, and contributed to inorganic C for photosynthesis in chlorophytes and rhodophytes (Borowitzka & Larkum, 1976; Pentecost, 1978). The higher availability of CO₂ with OA may affect the need for the algae to recycle respiratory CO₂ from the ICS or other sites of calcification and result in CO₂ and H⁺ accumulation, lower pH, and subsequently, lower calcification rates. Certainly, a more in-depth understanding of calcification, particularly in a few keystone species, would assist in mechanistically understanding how OA and climate change affect calcification and dissolution in marine macroalgae.

Experimental results of OA effects on calcareous marine macroalgae

Although the evidence from short-term studies indicate stimulation of photosynthesis and growth in macroalgae with increased CO₂ availability, a majority of longer term experimental studies to date show a decrease in calcification and enhanced dissolution in calcifying species under elevated [CO₂] (Table 3). In 82% of the experiments reviewed, the [CO₂] predicted for 2100 (~700–1000 ppm) leads to a decline in calcification, growth and/or recruitment of macroalgae in the two dominant calcifying divisions, Chlorophyta and Rhodophyta (Tables 2 and 3). At elevated temperatures (+3 °C) predicted for 2100, the negative effects of elevated [CO₂] on net calcification were enhanced in species from both divisions and in 100% of the studies (Lithophyllum, Porolithon and Halimeda). Although high temperature lowered calcification in combination with elevated CO₂, at low Arctic temperatures (7–9 °C) summer calcification rates of Lithothamnion glaciale were positive up to ~1000 ppm [CO₂] under low light, suggesting differences in seasonal responses across latitudes.

Table 3. Ocean acidification (elevated CO₂) effects on marine calcifying macroalgae based on lab and mesocosm experiments. Experimental conditions: pH (seawater scale unless noted), CO₂ (ppm unless noted), temperature (°C) and irradiance (µmol photons m⁻² s⁻¹, PAR; UVR, ultraviolet radiation) and dominant responses noted: calcification, G_net; productivity, P_net (O₂ flux) or rETR (relative electron transport rate by fluorescence); carbonic anhydrase (CA); saturation state of aragonite (Ω)

Species	Experimental conditions				OA effects calcareous macroalgae	(Ref)
	pH	CO2	Temp	Light
^a Converted Pa to ppm based on std atm pressure; ^b Converted watt to µmol photons m^−2 s^−1 by dividing 0.3 and assuming PAR after Taiz & Zeiger (2010); ^c Median; ^d µatm; ^e Personal communication; ^f Median for range of temperatures at each pH; (−), not provided. Refs: (1) Gao & Zheng (2010); (2) Gao et al. (1993); (3) Hofmann et al. (2012); (4) Büdenbender et al. (2011); (5) Kuffner et al. (2008); (6) Jokiel et al. (2008); (7) Martin & Gattuso (2009); (8, 9) Russell et al. (2009, 2011); (10) Anthony et al. (2008); (11) Diaz-Pulido et al. (2012); (12) Semesi et al. (2009a); (13) Ries et al. (2009); (14) Price et al. (2011); (15) Sinutok et al. (2011).
Corallina sessilis	8.4	380	23–27	~1000	Gnet & Pnet low at high CO2 (UVR control)	(1)
	7.9	1000		(noon)	UVR increased CO2 and CO3^2− in the media
					(−) synergistic effects CO2 & UV
					High CO2 lower Chla & phycoerythrin
Corallina pilulifera	–	350	20	30	High CO2 no Ca^2+ uptake in the light	(2)
	–	1600			High CO2 Ca^2+ efflux in the dark
Corallina officinalis	8.30	384	15	50	Low relative growth at all high CO2 levels	(3)
	7.84	1313			Small decrease in inorganic content
	7.67	1939			No CO2 effect on Fv/Fm or rETR
					(−) correlation CA activity and CaCO3 of thalli
Lithothamnion glaciale	8.07c	387c	7–9	0–7	Summer Gnet was positive ≤975 ppm CO2	(4)
	7.78	819			Winter (dark conditions) low Gnet
	7.71	974			Winter Gnet negative ≥390 ppm CO2
	7.53	1568
Lithophyllum pallescens	8.17	365d	23–29e	~2000/	Gnet was negative at high CO2	(5, 6)
Hydrolithon sp.	7.91	765		(full sun)	Coralline recruitment low at high CO2
Porolithon sp.					Initially, fleshy algae dominant in high CO2
					Herbivores lowered fleshy algae in 10 months
Lithophyllum cabiochae	8.06c	417c	13–22	6–35	No high temp or CO2 effect on Gnet	(7)
	7.86	728	(Ambient +3 °C)		(−) synergistic effect high temp & CO2 on Gnet
					High necrosis/mortality at high temp & CO2
					Negative Gnet dead algae at high temp & CO2
Lithophyllum spp.(crust)	8.1	380	17	34	Crusts % cover declined under elevated CO2	(8, 9)
Feldmannia spp. (turf)	7.95	550			Turfs % cover increased under elevated CO2
					No significant effect of CO2 on rETR
					Interactive effects with CO2 & UVB, Nutrients
Porolithon onkodes	8.20c	298c	25.5c	1000	Highest level CO2 reduced Pnet & Gnet	(10, 11)
	7.90	613	28.5		Highest level CO2 caused bleaching
	7.65	1185	(Ambient +3 °C)		No effect Gnet at 613 ppm CO2, but lower Pnet
					(−) synergistic effects at high CO2 & Temp
					Endoliths lowered Gnet at high CO2 & Temp
Hydrolithon sp.	8.1	~400	25	150	No high CO2 effect on Gnet after 1 h	(12)
	7.8	900			High CO2 decreased Gnet after 5 days
					No high CO2 effect on Pnet after 5 days
					Gnet was (+) in the light 0 or (−) in the dark
Neogoniolithon sp.	8.19	409	25	~710b	Parabolic Gnet response with Ωa	(13)
Halimeda incrassata	8.05	606		1420	Gnet increased at 606 & 903 ppm CO2
	7.91	903		(213–426 W m^−2)	Gnet decreased at 2856 ppm CO2
	7.49	2856			Mineralogy did not control species responses
Halimeda opuntia	8.0	440	29	150	Gnet declined at high CO2 in both spp.	(14)
Halimeda taenicola	7.7	946			H. opuntia shed > segments high CO2 than H. taenicola
					H. opuntia > CaCO3 thalli high CO2 than H. taenicola
Halimeda macroloba	8.1c	340a^, f	28	300	Upper limit 997 ppm CO2 & 32 °C	(15)
Halimeda cylindracea	7.9	640	30		(−) synergistic effects CO2 & Temp
	7.7	1076	32		High CO2 reduced Gnet, Fv : Fm & Pnet
	7.4	2383	34		High CO2 reduced Chla,b, crystal width

The majority of experimental studies indicate a negative effect of high [CO₂] and lower pH on net calcification, however, the effects on photosynthesis are not as clear (Table 3). For example, negative effects of high [CO₂] and lower pH on photosynthesis (quantum yield) were found for Corallina sessilis, Porolithon onkodes, and Halimeda macroloba and cylindracea, whereas no effect was reported for L. glaciale, Hydrolithon spp., Lithophyllum spp., and Halimeda opuntia (Table 3). Within the Halimeda genus, CO₂ enrichment significantly lowered maximum photosynthetic quantum yield (Fv/Fm-dark adapted) in H. macroloba and H. cylindracea at pH 7.4 at ambient temperature (28 °C) and pH 7.7 and 7.9 at 32 °C, showing a clear pH effect and a negative synergism between pH and high temperature (Sinutok et al., 2011). However, in another study by Price et al. (2011), the maximum relative electron transport rate (rETR_max) was not significantly different (P = 0.06) between CO₂ enrichment (pH 7.7) and control (pH 8.0) treatments for Halimeda opuntia (28 and 33 μmol photons m⁻² s⁻¹, respectively) and H. taenicola (24 and 24 μmol photons m⁻² s⁻¹, respectively) at ambient temperature (29 °C), even though rETR was approximately 16% lower in elevated CO₂ for H. opuntia relative to controls.

The elevated CO₂ effect on Halimeda photosynthesis was modest compared to the highly significant negative effect of elevated CO₂ on net calcification (P < 0.01) and growth of calcified segments (Price et al., 2011). The relative differences in the photosynthetic responses of the different Halimeda species examined could be a result of experimental design (length of exposure, light levels and pH ranges) or differences in species thalli structure and/or physiology; clearly more comparative and mechanistic studies are required to be able to generalize across genera and species. However, results of these longer term studies indicate that photosynthesis in calcifying algae is not likely to increase significantly in response to elevated CO₂, as has been found in some fleshy species, because they will ultimately be constrained by negative OA impacts on calcification. Even when CA activities increased in response to higher [CO₂] in Corallina officinalis, likely as a result of lower pH, inorganic material (% dry wt) declined (Hofmann et al., 2012). These data indicate that greater CO₂ and HCO₃⁻ availability under OA may uncouple photosynthesis-calcification reactions and subsequently impede calcification and growth.

Net calcification is also dependent on dissolution, which increases under elevated [CO₂] (Table 3). Smaller and more abundant aragonite crystals have been documented in Halimeda from herbarium specimens comparing those from the 1960–1970s to 2007–2008 off the South Florida Shelf (Robbins et al., 2009). Robbins et al. (2009) proposed that small crystal size was a result of changing ocean chemistry, and indicated a shift toward greater dissolution. Further compounding the problem is that smaller crystals promote dissolution due to their larger surface-to-volume ratio. Because Robbins et al. (2009) herbarium samples were from the field, smaller crystals could have been due to OA, as suggested by the authors, but also greater net heterotrophy with coastal eutrophication over the last few decades, as greater heterotrophy also elevates [CO₂] and [H⁺]. Regardless of the cause, experimental data from Sinutok et al. (2011) support the above relationship between OA and shifting macroalgal crystal morphology. They found that crystal width significantly declined (~0.04 μm) as pH was lowered from 8.1 to 7.7 and 7.4 in both H. macroloba (~2.5 μm) and H. cylindracea (~3.5 μm) (Table 3). However, in contrast to Robbins et al. (2009), the density of crystals was not greater in acidified individuals. Johnson et al. (2012) also found a positive relationship between pH and crystal width, but a negative correlation between crystal density and pH, in the brown calcifier Padina at CO₂ vent sites indicative of greater dissolution or lower net calcification.

In addition to live macroalgae, high dissolution rates were found for dead calcified thalli under high [CO₂], indicating the potential for habitats dominated by marine calcifyers to change from being net calcifying systems to ones where dissolution dominates under OA as live tissue cover declines. Thus, net calcification at the ecosystem or community scale can result from lower net calcification in macroalgae, and from an increase in dead : live calcified thalli exposed to seawater under lower pH conditions.

Field studies: scaling up to communities and ecosystems

Additional insight into OA effects on macroalgal communities has come from CO₂ vent surveys (Hall-Spencer et al., 2008; Martin et al., 2008; Fabricius et al., 2011; Porzio et al., 2011; Johnson et al., 2012). These studies provide valuable information on OA effects in the field even though they have some disadvantages. Limitations include high [CO₂] variances, an inability to control [CO₂] at any particular site, an incomplete understanding of historical biogeochemistry, and biotic relationships that are not simulated, including recruitment from non-OA affected areas. Although the biotic issues are difficult to resolve, a thorough characterization of the water chemistry dynamics at vent sites can determine those that best represent OA future conditions, including natural diel DIC dynamics (Yates & Halley, 2006a) not overwhelmed by vent fluxes of extremely high [CO₂] (Riebesell, 2008; Kerrison et al., 2011). With these caveats in mind, we present macro-autotroph data from two vent systems: a site proximate to Ischia Island, Italy, and a site in Milne Bay, Papua New Guinea (Table 4). While the vents from Italy incorporate sites with a very broad range in pH, which in itself could be a stress, the vent survey data clearly support the experimental results presented above. Furthermore, they provide insight into the species that are highly competitive at high CO₂. The other benefit to examining vents from the tropics and temperate climates is that it allows OA responses to be compared across systems and geographic regions. The two studied vent systems represent a range of pH, CO₂ and temperatures, and even though they are geographically distinct, the effect of OA on macro-autotroph communities was found to be quite similar (Table 4).

Table 4. Effects of ocean acidification on marine macro-autotroph communities in the field at cold water CO₂ seeps in Italy and Papua New Guinea. Abbreviations are as follows: CCA, crustose coralline algae, Fv/Fm, photosynthetic efficiency, ETR_max, electron transport rate, pH in total scale and CO₂ in ppm unless otherwise noted

Vent site	Field conditions				OA effects macro-autotroph communities	(Ref)
	Stn	pH	CO2	Temp
^a ppm. ^b µatm. ^c Data from Hall-Spencer et al. (2008) supplemental. ^d Annual range. Refs: (1) Martin et al. (2008); (2) Hall-Spencer et al. (2008); (3) Porzio et al. (2011); (4) Fabricius et al. (2011).
Ischia Island, Italy	St1	8.15–8.18	298–324a	13–25d	St1-4 CCA 20–70% cover; St5 CCA absent	(1)
	St2	8.13–8.20	280–349		St4 50% < CCA seagrass epiphytes than St1
	St3	8.05–8.15	325–435		CCA and epiphytes (+) correlated with pH
	St4	7.67–8.16	319–1.2 K		Low non-CCA epiphytes St5 = 5%
	St5	6.98–8.14	335–6.3 K		Seagrass density = St1-4; > density St5
Ischia Island, Italy	S1	8.13–8.17	304–346b^, c	13–25d	Calcareous algae declined S1 to S2, 0 at S3	(2, 3)
	S2	7.35–8.16	314–2.6 K		Non-calcareous algae dominate S3
	S3	6.07–7.37	2.5–52 K		72% loss macroalgal spp. richness at S3
	P1	8.15–8.18	296–322		Two spp. of calcified crusts higher at S2 than S1
	P2	8.13–8.20	281–347		Fleshy algae species shift across S1–S3
	P3	7.67–8.16	315–1.2 K		CCA seagrass epiphytes dissolved at P4
	P4	6.98–8.14	334–6.3 K		Similar seagrass Fv/Fm and ETRmax P1–4
					Seagrass shoot density and growth > at P4
Milne Bay, Papua	Control (C)	7.97–8.14	296–494a	~23–29	Fleshy macroalgae two times > at S vs. C sites	(4)
New Guinea	Seep (S)	7.73–8.00	444–953		Seagrass eightfold higher at S vs. C sites
					CCA other calcareous algae sevenfold lower at S
					S > 500 CO2 had 3–4 times > seagrass shoots & biomass
					S > 500 CO2 no CCA seagrass epiphytes ~0%
					Elevated alkalinity at S indicated CaCO3 dissolution

For example, they both showed a loss of crustose coralline algal epiphytes on seagrass leaves and fewer calcareous macroalgal species close to the vent source, as well as fleshy macroalgal dominance and greater seagrass density closer to the seeps where the CO₂ bubbling is visible (Table 4). More specifically, at the Italian vent, Porzio et al. (2011) quantified higher species richness at the control site with an average pH of 8.14 (S₁), 5% fewer species at pH of 7.83 (S₂) and a further 72% drop at pH 6.57 (S₃) (Table 4). The abundance of calcified algae was also clearly influenced by this pH gradient. Twenty calcified species were encountered at S₁, whereas there was a 25% drop at S₂ and no calcified macroalgae were found at S₃. Erect forms of algae also dropped in numbers by 18 and 59% from S₁ to S₂-S₃, respectively. Similarly, the number of crustose coralline algal species dropped by 5% at S₂ and 89.5% at S₃; however, two species of crustose algae (Hydrolithon cruciatum, Peyssonnelia squamaria) increased their dominance from S₁ to S₂, perhaps resulting from less competition by species more susceptible to OA or lower grazing at the higher [CO₂] sites. Certainly, these two calcareous species should be examined for their apparent OA resilience compared to a majority of the other coralline species. Fleshy algae that grew as short turfs were also surprisingly sensitive to OA, dropping by 6% at S₂ and 72% at S₃. In contrast, a few larger fleshy macroalgal species, particularly ochrophytes, were notably resilient to high pH and some were enhanced around the seeps at S₃. Two fleshy species (Dictyota dichotoma, Hildenbrandia rubra) were found along the entire pH gradient, whereas others (Sargassum vulgare, Cladostephus spongiosus, Chondracanthus acicularis) were only dominant at the most acidic site (S₃).

While currently not as studied in detail, data from a vent in Papua New Guinea that were integrated from three seep sites (Fabricius et al., 2011) showed a reduction in the Shannon diversity index for seagrass species from 138 to 83 at the control and seep sites, respectively (Table 4). In the same comparison, seagrass shoot density increased from 1357 to 3641 shoots m⁻², resulting in an increase in belowground biomass from 342 to 1628 g m⁻². This increase in seagrass belowground carbohydrate storage of photosynthates would presumably enhance the competitive advantage of seagrass species at higher [CO₂]. Such C-allocation could lead to a competitive advantage of seagrasses over fleshy macroalgae under elevated [CO₂]; however, macroalgae may allocate greater C gains to reproduction. Seagrass epibionts, organisms living on their leaf surfaces, did not follow seagrass trends and were reduced from ~22% in the control site to 1–0.3% at seep sites. The percent cover of calcareous crustose coralline algae and other red calcareous algae also declined from 6.2% to 0.3%. Changes in the fleshy macroalgal cover were not as substantial as from the Italian vents, only increasing from 1.6% to 3.3% from control to seep sites, and were highly variable. Along a gradient at one of the seep sites (Upa-Upasina Reef), and across a broad range of pH, crustose coralline algae typically had a higher percent cover at pH >8.0. However, there were observations of crustose coralline algae >10% at pH of ~7.8. It is these outlier species that would be most important to examine to understand the potential for calcifying macroalgae to tolerate some level of OA.

Synthesizing results from experiments and CO₂ vent systems, we conclude that fleshy macroalgae and seagrasses, most of which are C₃ species, are likely to be competitively dominant under OA and climate change due to their resilience to dissolution and ability to sequester additional DIC under elevated CO₂. Even calcified species, such as the brown macroalgae Padina, which do not depend on a highly calcified thalli have recently been shown to increase their rETR_max, chlorophyll content, and percent cover under elevated [CO₂](pH ~7.0–8.2) relative to controls (pH ~7.8–8.4), regardless of the fact that their CaCO₃ composition declined (Johnson et al., 2012). Thus, there will certainly be winners and losers. However, the mechanisms driving these changes and more detail on species-specific responses at modest, controlled elevated [CO₂] are needed to develop models of how OA will change marine macroalgal- and seagrass-dominated communities over the next few decades and into the next century. Elevated temperature and [CO₂] interactive effects need to be resolved at the mechanistic, individual, and community level.

Although not addressed in this review, critical ecosystem-level processes, such as changes in ‘top-down’ control, sediment biogeochemistry and water quality (nutrients and light) are also likely to be significant drivers of marine macro-autotroph community changes going forward (Russell et al., 2009, 2011). For example, Arnold et al. (2012) observed seagrass (Cymodocea nodosa) with greater signs of grazing at sites proximate to vent seeps (Island of Vulcano, Italy), which appear to be related to a down-regulation of phenolics; however, the significance of this effect was only found at very low pH (7.32) at the vent sites and further corroboration is required. Although the direct effects of OA are a result of chemical changes in pH and , certainly biotic and top-down interactions need to be explored further at field sites and in field and mesocosm experiments.

Also, ecosystem diurnal shifts in pH and that control net dissolution or precipitation of calcifiers in a community also need further consideration given that autotrophs, such as seagrasses and algae, can significantly modulate external pH with impacts on net calcification (Beer et al., 2006; Yates & Halley, 2006a,b, 2007; Semesi et al., 2009b). Highly productive tropical seagrasses (e.g., Thalassia hemprichii) that raise external pH during the day to ~9 through uptake of CO₂ for photosynthesis (Beer et al., 2006; Semesi et al., 2009b) have been shown to elevate calcification rates approximately two- to six-fold in calcifying algae growing in their vicinity (Hydrolithon sp., Mesophyllum sp., Halimeda renschii; Semesi et al., 2009b). While seagrasses can raise during the day, their leaves, along with highly organic sediments, have high respiratory demands, particularly under coastal eutrophication, which can depress pH at night to values (7.8; Semesi et al., 2009b) that would promote dissolution. Because of the strong diel variation in coastal , there is a need to understand the balance of calcification and dissolution in these autotroph-driven systems and the drivers of their dynamics, which can vary considerably between carbonate and siliceous-based ecosystems and different habitat types (Yates & Halley, 2006a, 2007), as well as be influenced by light, temperature, and autotrophic stress (Yates & Halley, 2006b). Diel, seasonal, and inter-annual shifts in inorganic C chemistry are likely to control the long-term conditions that promote either net calcification or dissolution, and the presence of calcified macro-autotrophs and other calcifiers in coastal ecosystems with species-specific tolerances. Similarly, but over annual and decadal time frames, shifts in will impact tropical open-ocean drift and deep-sea calcareous macro-autotrophs as a result of changes in currents, thermoclines, upwelling, and depth of the carbonate compensation depth, as the chemistry of these systems is modified under climate change and ocean acidification.

Conclusions and future research needs

Photosynthetic carbon acquisition in seagrasses and macroalgae

The seagrasses and marine macroalgae examined overwhelmingly show C₃ photosynthetic characteristics, but the capacity of most to utilize HCO₃⁻ differentiates them from terrestrial C₃ species. Some species appear to possess a HCO₃⁻- or C₄-based CCM, but for most this may not be the case. However, the facultative nature of HCO₃⁻ use may mask the potential induction of a CCM that might occur under low [DIC] conditions. Regardless of whether a CCM operates, the ability to use HCO₃⁻, the dominant DIC form in seawater, assists photosynthesis in an aquatic environment where CO₂ diffusion is severely limiting. However, CA-catalyzed dehydration of HCO₃⁻ to CO₂ is dependent on low pH. Furthermore, the capacity to utilize HCO₃⁻ does not indicate that photosynthesis is saturated with respect to CO₂. In fact, the photosynthesis of the majority of the species examined was not saturated at the current levels of [DIC] in the ocean (~2 mm) and responded to an increase in CO₂, as confirmed for a few species in growth studies under CO₂ enrichment. Although marine macroalgae from all three divisions utilize HCO₃⁻, the Rhodophyta has the most obligate CO₂ users with a high affinity for CO₂ and includes some that lack external CA, suggesting that this group as a whole may respond differently to elevated [CO₂]. These results, and the presence of CO₂-only users, lead us to conclude that seagrasses and many marine macroalgae have the potential to respond positively, in terms of photosynthesis and growth, under elevated ocean [CO₂] and OA, similar to that found in terrestrial plant systems. It is possible that seagrasses with the capacity to sequester the additional carbohydrates in belowground organs may respond more than macroalgae that lack substantial sinks for carbohydrate storage; however, macroalgae could allocate more C to reproduction.

Research needs

1. The majority of tropical marine macroalgae still need to be classified with respect to their photosynthetic pathways and have the potential to influence their response to OA; very few tropical species have been investigated to date and even fewer calcifiers.
2. Although there is evidence for a CCM in some species, a better understanding is needed of the regulation and degree to which CO₂ can be concentrated at the Rubisco fixation site. Information as to how light and temperature impact the operation of the CCMs is largely lacking, along with how such interactions modulate the expected effect of elevated CO₂. This is critical as many fleshy macroalgal species are strong competitors and can become nuisance species.
3. More evidence is needed about the facultative nature of photosynthetic processes in tropical marine macro-autotrophs, especially in regard to HCO₃⁻ use, to more clearly understand how seagrasses and macroalgae will respond to elevated [DIC] and its speciation. Also, if CA becomes more effective at a lower pH, will the availability and use of HCO₃⁻ increase.
4. Finally, long-term studies of tropical species exposed to elevated [CO₂], [H⁺] and temperature, particularly in the field, will assist in determining how short-term physiological studies scale to the community and ecosystem level.

Climate change effects on seagrasses and macroalgae

Although elevated temperature can lower carbon acquisition in terrestrial C₃ plants through photorespiration, elevated [CO₂] has been shown to partially ameliorate this loss, and raise the thermal optima of photosynthesis and growth. These interactions have not been widely examined in marine systems. In the tropics, seagrasses and macroalgae grow close to their thermal limits. Thus, for species to survive they will have to up-regulate stress-response systems to tolerate more frequent and longer sublethal and lethal temperature exposures. The interaction of elevated [CO₂] on thermal acclimation in these species is not well understood, but based on terrestrial research, it is possible that an increase in [CO₂] may offset to some degree the deleterious effects of high temperatures. Temperate species not adapted to high temperature and with steno-thermal ranges are likely to see range contractions and competition from tropical species. Temperature affects photosynthesis, calcification, growth, and most biochemical and biogeochemical processes; thus, ocean warming is likely to influence species, communities, and ecosystems in ways that presently cannot be predicted with any certainty.

Research needs

1. The fundamental biochemical linkages between elevated temperature and [CO₂] with climate change support the notion that more studies should be conducted examining their combined interactions.
2. A better understanding of the stress-response mechanisms, rather than just general growth responses, will assist in articulating which macro-autotroph species are likely to tolerate ocean warming in the tropics. Genetic diversity and ability to up-regulate moderating compounds of stress should also be considered. Intertidal species exposed to UV, desiccation, and thermal stress would be excellent candidates for model species to examine thermal stress mechanisms with elevated [CO₂] as many of the stress responses are not stressor specific.
3. Elevated temperature and [CO₂] interactions will also likely impact reproductive output, as occurs in terrestrial species, and thus longer term and field studies on this aspect of the life cycle need to be considered.
4. Biogeographic surveys of important species should be ongoing to document range shifts of economically and ecologically critical macro-autotroph species.

Calcification and elevated [CO₂], and temperature responses in calcareous macroalgae

At present, there is a better understanding of pure carbonate mineral geochemistry than biogenic calcification and dissolution, highlighted by the fact that organisms with different CaCO₃ mineralogy exhibit similar susceptibilities to OA. Furthermore, the response to OA and climate change in calcareous macroalgae is more complex than that for seagrasses and fleshy macroalgae. Although photosynthesis will likely be enhanced with an increase in [CO₂] and [H⁺], the calcification process will be adversely affected, and the degree may differ among species with different mechanisms of calcification. How these competing responses will influence growth and survival is difficult to predict. There is evidence that biological processes, location of calcification, and mechanism of calcification may be more important than the mineralogy in understanding OA effects on net calcification. However, we have very little understanding of the mechanisms that control calcification and even less on dissolution in most of the calcifying macroalgal species, including the dominant species on reefs. Although photosynthesis clearly promotes calcification, respiration and calcification produce CO₂ which can have a negative feedback on CaCO₃ precipitation. There is also a complex interchange between inorganic and organic carbon cycling in these species that affect calcification. When species are exposed to elevated DIC and H⁺ under OA, diffusion gradients of ions and active membrane-transport processes are likely to change and potentially negatively influence calcification and promote dissolution, as well as uncouple calcification-photosynthesis reactions. The negative synergistic effects of elevated temperature on net calcification further suggest that temperature-controlled processes (e.g., respiration and/or membrane transport) are affected by OA. Although elevated [CO₂] stimulates photosynthesis, it typically lowers calcification in calcareous macroalgae, particularly at high temperature and in the dark. This supposition is supported by the few experimental studies conducted to date. In addition, field surveys at CO₂ vent sites depict macroalgal community shifts from calcified to total dominance by fleshy macroalgae and seagrasses that are not dependent on calcification. However, vent sites where no calcifiers are found have extreme ranges of pH and CO₂, whereas at moderate pH sites, some calcifiers persist, although at present their acclimation mechanisms are elusive.

Research needs

1. The majority of experiments on calcification mechanisms in macroalgae were conducted several decades ago; thus, new tracer technologies could be applied to gain a more contemporary in-depth understanding of how tropical marine macroalgae calcify.
2. Furthermore, the OA effects on calcification and dissolution need to be studied across a range of temperatures and light conditions in long-term experiments with a priority on dominant and ecologically important macro-autotroph species, those acting as foundation species, and those from ocean regions with low CaCO₃ saturation states.
3. There is an immediate need to conduct longer term mesocosm and in situ field studies to examine the mechanisms of calcification and the vulnerability of tropical macroalgal calcifiers under moderate sustained elevated [CO₂].
4. A priority should also be given to calcareous species that currently occupy habitats with high [CO₂] across a range of light, depth and temperature regimes to ascertain their mechanisms of resilience to OA.
5. Finally, it is critical to consider community and ecosystem level processes, such as biogeochemistry and nutrient cycling, as well as reproduction, recruitment, and grazer susceptibility, as all these factors will interact to define the growth and survival of seagrasses and macroalgae as CO₂, DIC, H⁺, and temperature increase in the ocean with climate change.

Acknowledgements

We acknowledge all the scientific contributors to the literature that made this review possible. We also thank graduate students Kate Peach and Anthony Denardo, particularly the former, who assisted in compiling references and reviewing tables and text. The efforts by the lead author were supported in part by FAU's Climate Change Research Initiative. We sincerely thank three anonymous reviewers for their critique and suggestions that significantly improved the manuscript. SEM images of macroalgal calcification were kindly provided by Justin Ries. None of the authors have any conflicts of interest with the publication of the information in this manuscript.