Contrasting effects of ocean acidification on tropical fleshy and calcareous algae

Introduction

Changes in ocean chemistry associated with anthropogenic carbon dioxide (pCO₂) emissions, a process known as ocean acidification (OA) (Kleypas et al., 1999; Orr et al., 2005), have raised widespread concern for the survival and persistence of marine biota (Kleypas et al., 1999; Hoegh-Guldberg et al., 2007). Identifying the groups of organisms that will be susceptible to rapid OA versus those that may be resistant has prompted numerous studies (Ries, Cohen & McCorkle, 2009; Kroeker et al., 2010; Kroeker et al., 2013). To date, research has focused on understanding how reductions in the saturation state (Ω) of calcium carbonate (CaCO₃) and seawater pH associated with OA will impact the growth and physiology of commercially important calcifying organisms or entire ecosystems, such as coral reefs, that build carbonate platforms (Kleypas et al., 1999; Andersson & Gledhill, 2013). However, examination of a wider taxonomic representation, including those that calcify and those that do not, within and across ecosystems is critical to developing ecological predictions of community-level responses to OA.

The changes in the carbonate system have important implications for marine calcifiers, namely that OA may inhibit the ability of these species to grow, develop, reproduce and sustain themselves within a community, although plasticity in organismal responses indicates that some species may have wider tolerance limits (Doney et al., 2009; Kroeker et al., 2010; Kroeker et al., 2013; Johnson, Moriarty & Carpenter, 2014). Mounting evidence from coral reefs suggests that decreasing carbonate saturation (Ω) has negative effects on calcification (Langdon & Atkinson, 2005; Doney et al., 2009; Andersson & Gledhill, 2013), reproductive success (Albright, 2011), and competitive strength (Diaz-Pulido et al., 2011) of scleractinian corals. However, less attention has been given to the response of tropical marine primary producers to rising oceanic CO₂, particularly fleshy and calcareous benthic macroalgae which are also among the most dominant constituents of the coral reefs benthos.

The future trajectory of coral reefs may be influenced by concurrent effects of OA on both fleshy and calcified algae (reviewed in Koch et al., 2013), which serve key functional roles in reef systems in addition to competing with corals for space and resources. Calcareous algae contribute to framework development and some are active reef builders that account for up to 90% of living benthic cover on reefs (Tribollet & Payri, 2001). Crustose coralline algae (CCA) serve important ecological functions on reefs by contributing to primary production and carbonate production (Chisholm, 2003), producing settlement cues for coral larvae (Harrington et al., 2004; Price, 2010), and maintaining structural integrity of the framework by acting as reef cement (Camoin & Montaggioni, 1994). Calcareous green algae, such as Halimeda spp., are a major source of primary production and CaCO₃ (Rees et al., 2007) due to their fast growth and turnover rates (Smith et al., 2004), and are a preferred food source for many coral reef fishes (Mantyka & Bellwood, 2007; Hamilton et al., 2014). Fleshy macroalgae include a highly diverse group of seaweed species that act as a source of food for higher trophic levels and directly compete with corals for space (McCook, Jompa & Diaz-Pulido, 2001) on the reef benthos. Some fleshy macroalgae produce toxic allelochemicals which can kill corals upon contact (Rasher et al., 2012) while others may transmit coral disease (Nugues et al., 2004) or affect microbial assemblages associated with the coral holobiont via release of dissolved organic carbon (Smith et al., 2006; Haas et al., 2013; Nelson et al., 2013). Furthermore, the relative balance of calcifiers to fleshy macroalgae is important for reef resilience (Hughes et al., 2010). Increased cover of fleshy macroalgae, associated with anthropogenic disturbances such as poor water quality (Fabricius, 2005) and overfishing, is often used as an indicator of deteriorating reef health (Hughes, 1994). Given the important roles that calcareous and fleshy algae serve in the structure and function of coral reef ecosystems, it is critical to identify the potential differential effects of OA on these functionally distinct groups.

Increased CO₂ has the potential to have disparate effects on physiological processes for calcareous and fleshy algae, namely on photosynthesis and biomineralization. In terrestrial systems, rising atmospheric CO₂ can fertilize primary producers and enhance production (Ainsworth & Long, 2005), but in marine ecosystems, photosynthesizers have access to other relatively abundant carbon species, such as bicarbonate (HCO₃⁻), that can be used for photosynthesis. The potential for CO₂ fertilization of marine primary producers is likely contingent on species–specific mechanisms of carbon acquisition, influenced by the activity of carbon concentrating mechanisms (CCMs) (Giordano, Beardall & Raven, 2005; Raven et al., 2011; Koch et al., 2013). Laboratory manipulations and field studies from temperate and Mediterranean ecosystems (Hall-Spencer et al., 2008; Porzio, Buia & Hall-Spencer, 2011) suggest that OA may enhance carbon fixation (Kroeker et al., 2010; Cornwall et al., 2012; Kroeker et al., 2013) and photosynthesis in fleshy algae resulting in increases in algal growth rates (Gao et al., 1991; Kubler, Johnston & Raven, 1999; Cornwall et al., 2012). However, variations in interspecific responses may depend on the extent to which a species is presently carbon-limited (Harley et al., 2012; Koch et al., 2013). The photosynthetic response of seaweeds to OA is poorly understood in part because data on the presence, absence, or activity level of CCMs is often lacking for many tropical species (Hurd et al., 2009; Raven et al., 2011). Although much of the literature on OA effects on marine algae has shown that CO₂ enrichment enhances photosynthesis in phytoplankton and phanerograms (Riebesell et al., 1997; Palacios & Zimmerman, 2007; Gattuso & Hansson, 2011), the photosynthetic response of seaweeds to OA has been highly variable across experiments (Koch et al., 2013) and sometimes negative for calcified species (Anthony et al., 2008; Sinutok et al., 2011; Sinutok et al., 2012).

Conversely, OA effects on skeletal production in calcareous algae have been studied in more detail and changes in carbonate chemistry (i.e., lower pH, lower carbonate availability, and decreased CaCO₃ saturation state) have been shown to inhibit calcification in many species. The effects of OA on calcification in marine organisms may be influenced by the ability for a species to control carbonate chemistry at the intracellular or extracellular site of calcification (Ries, Cohen & McCorkle, 2009). A decrease in Ω in the external environment associated with OA could make biogenic CaCO₃crystal precipitation more difficult. When Ω decreases below the saturation horizon (<1) CaCO₃ dissolution is thermodynamically favored (Milliman et al., 1999). This saturation horizon is influenced by temperature, pressure, and mineralogy (Feely et al., 2004; Orr et al., 2005), and net dissolution and calcification can occur both above and below this threshold, respectively, depending on the organism and the environment (Milliman et al., 1999). CCA may be some of the most sensitive calcifiers to OA because they secrete high Mg-calcite, the most soluble polymorph of CaCO₃ (Morse, Andersson & Mackenzie, 2006; Andersson, Mackenzie & Bates, 2008). Other studies have found that OA decreases CCA calcification (Semesi, Kangwe & Bjork, 2009; Johnson & Carpenter, 2012; Comeau, Carpenter & Edmunds, 2012; Comeau et al., 2013; Johnson, Moriarty & Carpenter, 2014), structural integrity (Ragazzola et al., 2012) and increases mortality and that these effects that may be exacerbated by warming temperatures (Anthony et al., 2008; Martin & Gattuso, 2009; Diaz-Pulido et al., 2012; Martin et al., 2013) or increased solar UV radiation (Gao & Zheng, 2010). The articulated calcareous green algae Halimeda spp. have been shown to be both sensitive (Sinutok et al., 2011; Sinutok et al., 2012) and insensitive to OA (Comeau et al., 2013), where the direction and magnitude of the response of Halimeda to OA varies among species (Ries, Cohen & McCorkle, 2009; Price et al., 2011; Comeau et al., 2013). Negative effects of OA on calcification of CCA and Halimeda spp. may have serious implications for carbonate production and framework stability on coral reefs because they are often common members of ‘intact’ benthic reef communities (Sandin et al., 2008; Williams et al., 2013).

The primary objective of our study was to determine if there are consistent, differential responses of fleshy and calcareous tropical marine algae to OA using parallel, replicated experimental manipulations. On Palmyra Atoll in the northern Line Islands, five common species of fleshy algae and six species of calcareous algae were exposed to CO₂ levels expected by the year 2100 under a business-as-usual carbon emissions scenario (Meinshausen et al., 2011). In particular, the hypotheses tested were that, even with variation in species–specific physiological responses, elevated CO₂, (1) reduces net calcification across calcareous algae but, (2) stimulates growth of fleshy algae by enhancing photosynthesis. This study provides one of the first efforts to quantify OA effects on multiple species of both calcareous and fleshy algae from a coral reef environment, and provides insight into the effects of OA on a suite of algae that are important in the structure and function of coral reefs.

Materials and Methods

Study site and species

All experiments were conducted on Palmyra Atoll in the Northern Line Islands, central Pacific, in the recently established Pacific Remote Island Areas Marine National Monument (PRIAMNM) protected by the US Fish and Wildlife Refuge. Due to its isolation (∼1,700 km south-southwest of Hawaii) and lack of permanent human residence, Palmyra’s coral reefs are considered relatively healthy and are dominated by reef builders (Sandin et al., 2008; Williams et al., 2013). The remote nature of the field station limits research excursions to a few weeks at a time. Due to the absence of potentially confounding local anthropogenic impacts, Palmyra provides a unique setting for global change experiments.

To explore the effects of OA on different algal functional groups, eleven common species of algae were used in CO₂ enrichment experiments (see Sandin et al., 2008 and Williams et al., 2013 for relative abundances). Algae were categorized into three functional groups: fleshy macroalgae (Acanthophora spicifera, Caulerpa serrulata, Dictyota bartayresiana, Hypnea pannosa, and Avrainvillea amadelpha), upright calcareous algae (Halimeda taenicola, Halimeda opuntia, Galaxaura rugosa, and Dichotomaria marginata), and crustose coralline algae (CCA: Lithophyllum prototypum, formerly Titanoderma prototypum, and Lithophyllum sp.) (Fig. 1, Table S1). Specimens were collected via SCUBA at a depth of ∼5 m from the shallow western terrace (5°53.1696′N, 162°7.5756′W), excluding L. prototypum. L. prototypum was collected at a depth of ∼10 m from the southern fore reef (5°53.7906′N, 162°7.6859′W) where the species is abundant. Except for the corallines, which were collected as free-living rhodoliths, individuals were removed at the holdfast or from rhizoids in order to minimize stress. Coralline rhodoliths were comprised of 100% live coralline cover, and no bare carbonate was exposed to the potentially corrosive conditions. Samples were cleaned carefully of epiphytes with a soft-bristled brush and allowed to acclimate for at least one day in fresh, ambient seawater.

Results

Species–specific effects of CO₂ enrichment on calcification and growth

High CO₂ conditions decreased net calcification rates in 4 of the 6 calcareous species, and potentially enhanced net growth in 2 of the 5 fleshy species (Fig. 1; Table 2). CO₂ enrichment significantly decreased calcification in the red calcareous macroalga D. marginata (by 98%), and the two CCA Lithophyllum sp. (by 185%) and T. prototypum (by 190%) relative to controls (Table 2). The response of the green calcareous algae in the genus Halimeda was species–specific: the effect of CO₂ enrichment on net calcification rates was negative for H. opuntia (when repeated experiments were pooled) but negligible for H. taenicola (Table 2). CO₂ enrichment significantly increased growth in the fleshy red macroalga H. pannosa (by 93%) relative to controls (Table 2). The fleshy brown macroalga D. bartayresiana showed slight but non-significant increases in growth in high CO₂ likely due to small sample size and lack of power (β = 0.46; Table 2).

Table 2:

Results of pooled growth and photosynthetic parameters in response to treatment conditions.

The results of independent t-tests to analyze the effect of CO₂ enrichment on response variables for each species. Responses of species used in multiple experiments (different years) were pooled and averaged across years by treatment to calculate an overall mean for each species. CO₂ treatment was treated as a fixed, independent factor. Degrees of freedom (df) are the same for all photosynthetic parameters. Each experimental replicate (n) consisted of one aquarium containing one algal individual. Statistically significant differences (p < 0.05) are emphasized in bold.

	Growth			rETRMax			α		β
Species	df	t	p	df	t	p	t	p	t	p
Fleshy macroalgae
A. spicifera	10	1.15	0.275	9	2.55	0.031	0.341	0.741	0.088	0.932
A. amadelpha	18	3.12	0.006
C. serrulata	22	0.066	0.948	19	0.282	0.781	0.350	0.730	0.356	0.726
D. bartayresiana	8	2.13	0.066	8	1.55	0.159	0.274	0.791	1.14	0.292
H. pannosa	5	4.90	0.004	5	0.186	0.556	0.602	0.60	0.624	0.560
Upright calcareous algae
D. marginata	8	3.83	0.005	8	0.440	0.83	0.092	0.929	0.823	0.434
G. rugosa	10	1.63	0.134	10	4.10	0.002	1.71	0.118	0.760	0.465
H. opuntia	16	2.59	0.020	16	0.046	0.964	1.43	0.171	0.223	0.827
H. taenicola	38	0.21	0.832	18	0.193	0.849	2.23	0.039	1.62	0.123
Crustose coralline algae
Lithophyllum sp.	16	5.28	<0.0001	16	0.582	0.569	0.280	0.783	1.0	0.332
L. prototypum	6	2.79	0.032	6	0.357	0.733	0.404	0.700

DOI: 10.7717/peerj.411/table-2

In addition to across species variability in the growth response, there was intra-specific variation to CO₂ enrichment across different years of experiments (Fig. 2). The trends and absolute magnitude in growth responses remained the same for 2 of the 4 species tested over multiple years. Irrespective of year, the calcareous green alga H. opuntia calcified significantly less (by 14.55 mg g⁻¹ d⁻¹ in 2009 and 12.97 mg g⁻¹ d⁻¹ in 2011) under high CO₂ conditions (Table 3), although the relative response varied by year. H. opuntia calcified 200% less at high CO₂ than ambient conditions and even experienced net dissolution in 2009, but only calcified 50% less in 2011 and experienced net growth, despite the same high CO₂ conditions (Table S4). Lithophyllum sp. showed a consistent response to CO₂ treatment in direction and absolute and relative magnitude across years. Lithophyllum sp. calcified 185% less at high CO₂ in both 2009 and 2011 (Table S4). H. taenicola calcified 89% less at high CO₂ relative to controls in 2009, but there was no significant difference in calcification during the 2010 and 2012 experiments (Table 3). C. serrulata grew significantly more at high CO₂ in the 2010 experiment, however in 2011 C. serrulata grew less in the CO₂ enrichment treatment than in ambient conditions (Table 3).

Several fleshy macroalgal species became reproductive in CO₂ treatments over the course of our study, as evidenced by the presence of fertile tissue which eventually released gametes or spores leaving behind only a small portion of the vegetative thallus. All samples of A. spicifera and A. amadelpha released spores or gametes, respectively, upon exposure to treatment conditions. In 2011, C. serrulata also reproduced, causing tissue loss in both ambient and CO₂ treatments; 40% of Caulerpa individuals in the ambient treatment reproduced, whereas 100% of Caulerpa samples in the CO₂ enrichment treatments reproduced.

Species–specific effects of CO₂ enrichment on photophysiology

Exposure to CO₂ treatments had no detectable effect on relative photophysiology of the 9 species tested, with a few exceptions (Fig. 3). CO₂ enrichment significantly increased the maximum photosynthetic capacity (rETR_Max) in the calcareous red alga G. rugosa (Fig. 3A, Table 2) relative to the control. In the fleshy red alga A. spicifera, rETR_Max was significantly lower following exposure to high CO₂, however, these individuals had reproduced during the experiment and the remaining vegetative tissue following gamete release was not representative of healthy algal tissue. In the calcareous green alga H. taenicola, the initial slope of the RLC (α) was significantly depressed after CO₂ enrichment (Fig. 3B, Table 2). There was no evidence of photoinhibition (β) in any of the species tested (Fig. 3C, Table 2).

Table 3:

Results of growth/calcification by species and year.

The mean growth and calcification rates of species tested in multiple experiments were examined using independent t-tests for each species by year; CO₂ treatment was treated as a fixed, independent factor. Each experimental replicate (n) consisted of one aquarium containing one algal individual. Statistically significant differences (p < 0.05) are emphasized in bold.

		Growth
Species	Year	df	t	p
Fleshy macroalgae
C. serrulata	2010	10	4.28	0.002
	2011	8	1.75	0.119
Upright calcareous algae
H. opuntia	2009	6	7.32	0.0003
	2011	8	3.62	0.007
H. taenicola	2009	6	5.93	0.001
	2010	10	0.224	0.827
	2012	18	0.612	0.548
Crustose coralline algae
Lithophyllum sp.	2009	6	4.10	0.006
	2011	8	3.43	0.009

DOI: 10.7717/peerj.411/table-3

Meta-analysis of experiments across years

Experimental effects were combined across species to assess the consistency of physiological responses to CO₂ enrichment within different algal functional groups using meta-analyses. The mean effect size for calcification and growth was significantly greater than zero for fleshy species, but significantly less than zero for both groups (upright and encrusting) of calcareous species (Table 4; Fig. 4A). There was no overall effect of CO₂ enrichment on photophysiology (rETR_Max, α, β) relative to the control for algal functional groups (Fig. 4, Table 4). The variation between experiments was never significantly different from 0 (Q ≤ 2.04, p > 0.05 for each functional group and response variable; Table 4), indicating that the inconsistencies in PAR did not influence the overall response of fleshy versus calcareous algae to OA. Due to the significant effect of CO₂ enrichment on growth and calcification rates across experiments, and the lack of significant variation in the strength of this response, we pooled species across years to show overall trends in treatment responses (Fig. 1).

Table 4:

Meta-analysis results.

Heterogeneity (Q_T) in overall analyses and results from a random effects model of standardized mean differences for response variables pooled by functional group: fleshy macroalgae, upright calcareous algae, or crustose coralline algae (CCA). Statistically significant values (p < 0.05) are emphasized in bold. rETR_Max, maximum relative electron transport rate (µM photon m⁻² s⁻¹); α, photosynthetic efficiency or initial slope of the rapid light curve (µM electrons µM photons⁻¹); β, photoinhibition (µM electrons µM photons⁻¹).

Response	df	QT	p	k	Mean effect size	Z	p
Fleshy macroalgae
Growth	19	0.07	>0.05	3	16.1 ± 12.5	2.11	0.017
rETRMax	12	0.18	>0.05	3	0.454 ± 6.19	0.144	0.886
α	12	0.07	>0.05	3	−0.005 ± 0.13	0.073	0.471
β	12	0.16	>0.05	3	−0.0004 ± 0.0009	0.632	0.2248
Upright calcareous algae
Growth	4	2.04	>0.05	7	−10.8 ± 4.7	3.80	0.0001
rETRMax	4	1.62	>0.05	6	0.031 ± 3.56	0.017	0.987
A	4	0.72	>0.05	6	−0.020 ± 0.06	0.722	0.470
B	4	0.60	>0.05	6	0.001 ± 0.01	0.756	0.4333
Crustose coralline algae
Growth	6	0.08	>0.05	3	−0.405 ± 0.35	1.90	0.029
rETRMax	6	0	>0.05	3	0.693 ± 27.4	0.339	0.735
α	6	0.05	>0.05	3	−0.002 ± 0.09	0.053	0.941
β	6	0	>0.05	3	−0.001 ± 0.004	0.169	0.2637

DOI: 10.7717/peerj.411/table-4

Discussion

This series of experimental manipulations indicate that tropical algae respond differently to CO₂ enrichment depending on species and whether or not they are calcified. When combining data from multiple experiments, calcareous algae experienced a reduction in biomineralization while fleshy algae became more productive. The magnitude of algal growth and calcification responses to OA conditions varied by species, and occasionally, within a species over multiple experiments. In contrast, there was no effect of CO₂ enrichment on algal photophysiology relative to controls as measured by short illumination RLCs. Furthermore, exposure to OA conditions initiated sexual reproduction in 3 out of 5 species of fleshy macroalgae tested. These results support the hypothesis that OA has differential effects on the growth of fleshy macroalgae and the calcification of calcareous algae.

Biomineralization by seaweeds substantially contributes to carbonate production on tropical reefs and these results suggest that OA may decrease reef formation and cementation services provided by these often over-looked ecosystem engineers. In these experiments, OA decreased calcification of calcareous green algae (H. opuntia and H. taenicola) and caused net dissolution of calcareous red macrophytes and CCA (D. marginata, G. rugosa, Lithophyllum sp., and L. prototypum). Many other studies have reported decreased calcification as a consequence of simulated OA for tropical (Table 5) and temperate calcareous algae even in milder acidification scenarios than used in our study (see Koch et al., 2013 for review). However, much of the previous work exploring OA effects on calcareous algae across ecosystems has focused on the crustose coralline algae (family Corallinaceae) and this study is among the first to expand to different taxonomic entities such as the lightly calcified red algae D. marginata and G. rugosa (Table 5).

The results of this study indicate that calcareous algae calcified less after two weeks of exposure to CO₂ enrichment than ambient controls, but the response varied by functional group. CCA, which deposit the more soluble high Mg-calcite (12–18% MgCO₃; Milliman, Gastner & Muller, 1971), experienced net dissolution in the OA treatments, where Ω_Mg-calcite was ≤1 (using the solubility constant estimated by Lueker, Dickson & Keeling, 2000), despite assuming our samples deposited the conservative lower range of 8% Mg mole fraction. Intracellular dolomite (CaMg[CO₃]₂), a stable form of carbonate, can be the source of Mg in other species of CCA and actually reduces net thallus dissolution at higher skeletal mole fractions (Nash et al., 2012). The exact mineral composition of the carbonate in our CCA species is unknown, but was not robust to our treatment conditions. The calcareous upright algae all deposit aragonite and calcified less under OA, but only experienced net dissolution in one instance. Differences in the magnitude of effects between calcareous species may be influenced by species–specific mechanisms of calcification (Price et al., 2011; Comeau, Carpenter & Edmunds, 2012; Koch et al., 2013), mineralogy of CaCO₃ deposited (Ries, Cohen & McCorkle, 2009), and potential compensatory or antagonistic effects of high CO₂ on photosynthesis (Table 5). Differences in within-species susceptibility to OA demonstrate the complexity of how ocean acidification may influence biological and chemical interactions in tropical marine primary producers. Within-species responses across years of experiments may have been driven by changes in dissolution versus calcification or by net growth rate, and the relative contribution of dissolution versus calcification in influencing net effects of OA on organisms should be a focus in future studies.

Understanding the effects of OA on algal physiology is difficult because photosynthesis and calcification are inextricably coupled. In the process of fixing carbon, algal photosynthesis alters the intracellular environment in favor of CaCO₃ precipitation (Borowitzka & Larkum, 1976). In the external environment, photosynthesis also has the potential to alter carbonate chemistry and to create conditions more favorable for calcification (Gattuso, Pichon & Frankignoulle, 1995; Anthony, Kleypas & Gattuso, 2011; Smith et al., 2013). Fleshy macroalgae that are currently carbon limited are hypothesized to be affected positively by increasing CO₂ concentrations (Gao et al., 1991), which is demonstrated here, but these effects are species and condition specific. Previous studies have documented both positive and negative effects of CO₂ enrichment on growth in fleshy macroalgae (Table 5). Enhanced algal growth also has been documented in situ in ecosystems near underwater volcanic vents where conditions of low pH and high CO₂ facilitate communities dominated by fleshy organisms (Hall-Spencer et al., 2008; Fabricius et al., 2011). It has been hypothesized that higher concentrations of dissolved CO₂ would enhance fleshy macroalgal growth by stimulating photosynthesis. However, despite the fact that fleshy algae grew more with high CO₂ there was not a concurrent response in photosynthetic parameters measured from chlorophyll fluorescence. While the fluorescence technique is used widely to monitor algal photophysiology, it can be highly variable (Edwards & Kim, 2010) and provides only an instantaneous snapshot of photophysiological function. Short illumination RLCs (<1 min) are not comparable to estimates obtained using oxygen evolution from photosynthesis-irradiance curves because there is not sufficient time with RLCs for organisms to reach steady-state flow of electrons (Enriquez & Borowitzka, 2010). Thus, it may not be the most suitable technique to assess the cumulative effects of CO₂ enrichment on algal photophysiology and more direct measures of photosynthesis are preferred.

Predicting the response of primary producers to high CO₂ is complex and may depend on resource acquisition strategies that are species–specific and potentially plastic over time. The primary substrate for the photosynthetic enzyme Rubisco in all marine algae is dissolved CO₂. Seaweeds must compensate for the slow rates of CO₂ diffusion through seawater, as opposed to air, as well as the higher concentration of HCO₃⁻ compared to CO₂. Some primary producers have developed carbon concentrating mechanisms (CCM) that increase the concentration of CO₂ in the proximity of Rubisco (Raven, 1970). Thus, the presence or absence of CCMs may influence species–specific responses to CO₂ enrichment (Hurd et al., 2009; Koch et al., 2013), and changes to CCM activity levels may explain the mixed responses of photosynthesis in the literature, as well as the growth results documented here. One possible mechanism that may have facilitated increased algal productivity under high CO₂ in the present study, without concurrent increases in rETR_Max or α, may have been an increase in algal energy reserves through down regulation of energetically costly CCMs, noted in another tropical green macroalga (Liu, Xu & Gao, 2012) and phytoplankton (Eberlein, Van de Waal & Rost, in press). An additional alternative hypothesis is that nitrate reductase activity, an enzyme that reduces nitrate to nitrite, can be stimulated by CO₂ (Hofmann, Straub & Bischof, 2013), potentially releasing seaweed from nitrogen resource limitation in oligotrophic coral reef ecosystems. Furthermore, photophysiology should be assessed using more direct techniques in addition to RLCs such as measuring oxygen evolution rates, in order to accurately quantify photosynthetic rates. Predicting changes in enzymatic activity is critical to understanding mechanisms behind species–specific responses to OA, yet basic physiological descriptions are lacking for the majority of tropical algae, including the species used in the present study.

This and other studies have documented high variability among species in response to OA. However, there also was within species variability across years, suggesting that species–specific responses to OA may be context dependent. For example, due to logistical constraints experiments conducted in 2009 and 2010 had substantially lower daily mean irradiances than in 2011 and 2012 (ESM Table 2). Although mixing rates were consistent from year to year, flow rates in experimental aquariums were relatively low. Thus, care should be taken when extrapolating these biological responses to OA under higher water flow regimes. Few studies have experimentally tested the effects of both water flow and OA on coral reef algae, although flow rate has been shown to be an important factor influencing pH gradients within the diffusive boundary layer (DBL) (Hurd et al., 2011) and the response of some reef calcifiers to high CO₂ (Anthony et al., 2013). Increasing DBL thickness, with decreasing water flow, may buffer organisms against changes in the carbonate chemistry of bulk seawater by providing a metabolically mediated microenvironment of higher pH within the DBL. Furthermore, the biological variability in carbonate conditions introduced by algal photosynthesis and respiration in the contained, aerated volume of water likely created a diel cycle in pH that may have approximated carbonate chemistry variability on a shallow reef flat (Hofmann et al., 2011). Variability in pH conditions has been shown to influence growth rates of coralline algae (Johnson, Moriarty & Carpenter, 2014), therefore care should be taken when extrapolating the results from the present study to other systems. Diel cycles in carbonate chemistry were not characterized in this experiment, and have been infrequently included in descriptions of experimental conditions in many OA studies. However, the variability in all experimental conditions across this suite of experiments is far less than that of experiments combined in several recent meta-analyses (Hendriks, Duarte & Alvarez, 2010; Kroeker et al., 2010; Kroeker et al., 2013)). With the meta-analysis approach used here to explore effects of OA across experiments, we accounted for the variability within and across experiments and found that OA treatment was a significant driver of enhanced growth in fleshy macroalgae, and loss of calcified biomass in upright calcareous algae and CCA.

Some within-species variability in response to OA treatment was due to the induction of sexual reproduction following exposure to treatment conditions. Higher irradiance levels can modulate the negative effects of high CO₂ on algal responses to OA (Sarker et al., 2013; Yildiz et al., 2013), including potentially triggering reproduction and may explain the inconsistent results from year to year, specifically for C. serrulata. In 2011 the decrease in C. serrulata growth likely was a result of the loss of algal tissue in individuals that became sexually reproductive upon exposure to high CO₂ and relatively higher irradiance. Similar reproductive responses to treatment conditions were also noted for related A. amadelpha and for a red macroalga, suggesting that there may be an interactive effect between irradiance levels and CO₂ concentrations. Sexual reproduction in these taxa has been observed in Hawaii and the Caribbean during the spring (Clifton & Clifton, 1999; Smith, Hunter & Smith, 2002). In all of these species, a large portion of the algal thallus senesced after sexual reproduction, and for the green algae the reproduction was clearly visible due to the loss of pigmentation following release of heavily pigmented gametes or spores (Clifton & Clifton, 1999). The typical progression of sexual reproduction in Bryopsidales ranges from 1–2 days (Clifton & Clifton, 1999), and the specimens did not show signs of reproduction prior to the experiment. Furthermore, gametogenesis in C. serrulata has been shown to be induced either as a coping mechanism (Williamson, 2010) or to maximize favorable conditions (Brawley & Johnson, 1992). Experiments were conducted outside the potential seasonal reproductive cycle of tropical algae, and there was no evidence of sexual reproduction before experiments, thus it is likely that sexual reproduction was induced by experimental conditions. An alternative explanation is that the reproductive response may have been an artifact of experimental manipulation and stress associated with rapid exposure to high pCO₂. The rate of exposure to high pCO₂ has been shown to be an important determinant in the response of coralline algae to CO₂ enrichment (Kamenos et al., 2013). Future work should explore the effects of both rate and magnitude of CO₂ enrichment on reproduction in fleshy macroalgae.

OA poses an ever-increasing global threat (Kleypas et al., 1999; Hoegh-Guldberg et al., 2007) to the ecological balance and stability of tropical reef systems via disparate effects on calcareous versus fleshy taxa (Hall-Spencer et al., 2008; Fabricius et al., 2011; Porzio, Buia & Hall-Spencer, 2011). It is difficult to predict the specific responses of macroalgal taxa to CO₂ enrichment; however, the patterns of response presented here suggest that growth of fleshy macroalgae on coral reefs may be stimulated by OA, while calcareous species may be depressed. Given that numerous other human impacts (overfishing, pollution, warming) negatively affect corals and other calcifying reef builders while enhancing the abundance of fleshy algae, our results suggest that OA may potentially exacerbate community shifts towards fleshy macroalgal dominated states. However, little is known about how reef species or communities will respond to the interactive effects of multiple stressors including OA. Given the importance of coral reefs for supporting biodiversity (Knowlton, 2001), as well as human populations and economies of coastal nations (Moberg & Folke, 1999), it is imperative that we understand the scope of species responses to impending rapid climate change.

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