CHARACTERIZING THE RESPONSE OF CORALLINE ALGAE TO

OCEAN ACIDIFICATION

_______________

A Thesis

Presented to the

Faculty of

San Diego State University

_______________

In Partial Fulfillment

of the Requirements for the Degree

Master of Science in Biology

with a Concentration in

Ecology

_______________

by

Brenna Elizabeth Bulach

Summer 2012

iii

Copyright © 2012

by

Brenna Elizabeth Bulach

All Rights Reserved

iv

DEDICATION

I would like to dedicate my thesis to my parents, Virgil and Marsha Bulach, without

whom none of this would have been possible.

v

ABSTRACT OF THE THESIS

Characterizing the Response of Coralline Algae to Ocean Acidification

by Brenna Elizabeth Bulach

Master of Science in Biology with a Concentration in Ecology San Diego State University, 2012

Future levels of atmospheric carbon dioxide, currently at 380ppm are predicted to

reach 1000ppm by the year 2100 and the accompanying increase of dissolved CO2 in the oceans will result in an overall decrease in seawater pH. Most research on ocean acidification has found reduced calcification in animals that create calcium carbonate (CaCO3) structures, but few have focused on photosynthetic coralline algae, such as those prevalent in kelp forest ecosystems. Here, we compare photosynthesis and calcification in three of the dominant species of geniculate coralline algae along the coast of California, Bossiella californica, Calliarthron tuberculosum and Corallina officinalis, with the purpose of identifying how they will respond to elevated pCO2. Specifically we conducted short-term exposure experiments by subjecting algae to two pCO2 levels, 380ppm and 1000ppm, under a range of irradiances for 2.5 hours during bottle incubations. Construction of photosynthesis-irradiance curves indicated the algae suffered reductions in both maximum rates of photosynthesis and saturation irradiances. However, all three species exhibited higher photosynthetic efficiencies under non-saturating irradiances (those below 30 μmol photons·m-2·s-1). In contrast, calcification rates under elevated pCO2 varied among the species, declining by 18% in B. californica and 49% in C. officinalis, but increasing by 20% in C. tuberculosum. Likewise, percent CaCO3 in the algal thalli decreased in B. californica and C. officinalis, but increased in C. tuberculosum under elevated pCO2, suggesting that B. californica and C. officinalis may be more strongly adversely impacted by climate change than C. tuberculosum.

Assessing only the immediate responses to elevated CO2 neglects the possibility of phenotypic plasticity, which may result in variable abilities to acclimate to changing environmental conditions. To examine this in the three species of geniculate coralline algae, a longer four-week mesocosm experiment was conducted to measure changes in photosynthesis and calcification under elevated pCO2 in all three species. Here, the algae were exposed to one of three pCO2 levels, 500ppm, 1000ppm and 1500ppm, and algal photosynthetic rates, net calcification rates, and CaCO3 thallus contents were measured every seven days. Bleaching was observed in the 1000ppm and 1500ppm mesocosms starting on day 14 and continued to the end of the experiment. Photosynthetic rates when averaged across species decreased with increasing pCO2 by approximately 74% in the 1500ppm mesocosm relative to rates at 380ppm. Calcification rates for all species decreased under elevated pCO2 to an average of -0.896±0.307 μmol C·g-1·h-1 at 1500ppm, indicating net dissolution of CaCO3. Despite these negative values, net photosynthesis and an eventual stability of calcification rates occurred in all treatments by the end of the experiment, indicating a possible acclimation to elevated CO2.

vi

The in situ effects of ocean acidification are challenging to study due to methodological constraints and difficulties in maintaining experiments in aquatic environments. The third component of this research assessed the impacts of elevated pCO2 on photosynthesis and calcification of all three species of geniculate coralline algae in field incubations. Here, the coralline algae were incubated in translucent bags filled with seawater at two pCO2 levels, 500ppm and 1000ppm. The bags were placed within the kelp forest at 10 meters depth for a 2.5-hour incubation period, during which algae experienced natural light and wave energy (hereafter ‘immediate incubation’). A second collection of algae was brought back to the laboratory and placed in mesocosms (500ppm and 1000ppm) for two weeks. Following this acclimation period, the algae were then incubated for 2.5 hours in the field as described above (hereafter ‘acclimated incubation’). Two immediate incubations and two acclimated incubations were conducted. The three species averaged 45% higher photosynthesis rates under 1000ppm pCO2 than under 500ppm pCO2 during the immediate incubations, but only 30% higher during the acclimated incubations. This suggests carbon-fertilization, yet also suggests the algae can acclimate to elevated pCO2. Further, calcification rates decreased in all species at 1000ppm during both immediate and acclimated incubations; immediate incubations resulted in a 44% average decrease in calcification under elevated CO2, whereas acclimated incubations resulted in an 81% average decrease in calcification. This indicates that photosynthesis in these algae has the potential to acclimate to elevated CO2, whereas calcification does not but rather declines rapidly. The response of calcifying autotrophs will be dependent on the ability of calcification rates to maintain CaCO3 structures and less on the potential fertilization effects of CO2 on photosynthesis. Together, the results from all three experiments suggest negative impacts of future levels of CO2 on temperate coralline algae. The variable responses among species were not substantial enough to give one species a physiological advantage over another and this study stresses the importance of understanding the impacts of ocean acidification on a group of species that fulfill similar ecological roles.

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TABLE OF CONTENTS

PAGE

ABSTRACT ...............................................................................................................................v

LIST OF TABLES ................................................................................................................. viii

LIST OF FIGURES ................................................................................................................. ix

INTRODUCTION .....................................................................................................................1

MATERIALS & METHODS ....................................................................................................7

Target species.......................................................................................................................7

Site .......................................................................................................................................7

Characterization of Photosynthesis and Calcification of Coralline Algae Under Elevated pCO2 ......................................................................................................................8

Assessing the Response of Coralline Algae to Short-Term Exposure to Elevated pCO2 via Photosynthesis-Irradiance Response Curves ...................................8

Assessing the Response of Coralline Algae to Intermediate-Term Exposure to Elevated pCO2 via Mesocosm Experiments ................................................................10

Assessing the Response of Coralline Algae to Elevated pCO2 via In Situ Incubations ...................................................................................................................12

RESULTS ................................................................................................................................15

Photosynthesis-Irradiance Response Curves .....................................................................15

Four-Week Mesocosm Experiments ..................................................................................21

Field Experiments ..............................................................................................................26

DISCUSSION ..........................................................................................................................30

ACKNOWLEDGEMENTS .....................................................................................................35

REFERENCES ........................................................................................................................36

APPENDIX ..............................................................................................................................43

viii

LIST OF TABLES

PAGE

Table 1. Photosynthesis Parameters Based on P-I Curves for TIC. Means ± SE of Two Incubations per Treatment ...................................................................................17

Table 2. Two-Way PERMANOVA Table of Results for Parameters of P-I Curves Based on TIC with Factors pCO2 and Species (n = 252). Boldface* Denotes Significant Differences ................................................................................................17

Table 3. Pair-Wise Comparison Between Species for Both CO2 Levels. Boldface* Denotes Significant Differences ..................................................................................17

Table 4. Three-Way PERMANOVA Table of Results for Calcification Rates With Fixed Factors pCO2 and Species, and Irradiance as a Random Factor. Boldface* Denotes Significant Differences .................................................................19

Table 5. Three-Way PERMANOVA Table of Results for % CaCO3 With Fixed Factors pCO2 and Species and Irradiance as a Random Factor. Boldface* Denotes Significant Differences ..................................................................................20

Table 6. Average % CaCO3 After Incubations for Two pCO2 Levels (Ambient = 380ppm; Elevated = 1000ppm). Boldface* Denotes Significant Differences .............20

Table 7. Three-Way PERMANOVA Table of Results for TIC With Fixed Factors Day, pCO2 and Species (n = 100). Boldface* Denotes Significant Differences .........21

Table 8. Three-Way PERMANOVA Table of Results for Calcification in 3 Mesocosms for 28 Days With Fixed Factors Day, pCO2 and Species (n = 100). Boldface* Denotes Significant Differences .......................................................25

Table 9. Three-Way PERMANOVA Table of Results for TIC Under Two Treatments (Immediate and Acclimated) and Two CO2 Levels (500ppm and 1000ppm) for All Three Species. Boldface * Denotes Significant Differences .................................28

Table 10. Three-Way PERMANOVA Table of Results for Calcification Rates Under Two Treatments (Immediate and Acclimated) and Two CO2 Levels (500ppm and 1000ppm) for All Three Species. Boldface* Denotes Significant Differences ...................................................................................................................29

ix

LIST OF FIGURES

PAGE

Figure 1. Mesocosm set-up at the Coastal and Marine Institute Laboratory with PVC pipes to supply 12oC seawater at three pCO2 levels (500ppm, 1000ppm and 1500ppm) and shut-off valves to control flow to each mesocosm. .............................11

Figure 2.One 2.5'x1' PVC field frame during an incubation at 10m depth at New Hope Rock; four incubation bags contain 500ppm seawater and four contain 1000ppm seawater; all three species are represented at each pCO2 along with two controls ..................................................................................................................13

Figure 3. Photosynthesis-Irradiance response curves of B. californica, C. tuberculosum, and C.officinalis under two pCO2 levels: (a) ambient at 380ppm and (b) elevated at 1000ppm ........................................................................................16

Figure 4. Calcification rates for each species at increasing irradiance under ambient pCO2 and elevated pCO2 for (a) B. californica, (b) C. tuberculosum, and (c) C. officinalis......................................................................................................................18

Figure 5. Average calcification rates for each species under ambient pCO2 (light bars) and elevated pCO2 (dark bars). ....................................................................................19

Figure 6. Photosynthesis rates over a 28-day span in three different mesocosms (pCO2 of 500ppm, 1000ppm, and 1500ppm) for (a) B. californica, (b) C. tuberculosum, and (c) C. officinalis. ............................................................................23

Figure 7. Calcification rates over a 28-day span in three different mesocosms (pCO2 of 500ppm, 1000ppm, and 1500ppm) for (a) B. californica, (b) C. tuberculosum, and (c) C. officinalis. ............................................................................24

Figure 8. Average percent CaCO3 measured across species on days 1 and 28 in the three mesocosms (500ppm, 100ppm & 1500ppm) (* denotes significant differences, p = 0.028, 0.006). .....................................................................................26

Figure 9. Comparisons of photosynthesis and calcification rates under two levels of CO2 during the Immediate Incubations (a and c) and Acclimated Incubations (b and d). Letters denote significant differences. .........................................................27

Figure 10. Measurements of ambient CO2 levels in the Point Loma kelp forest from seawater collected at 10m depth on 9/24/11 (samples 1, 2, 3), 9/26/11 (4, 5, 6), 10/8/11 (7, 8, 9) and 10/10/11 (10, 11, 12). .................................................................44

1

INTRODUCTION

There has recently been an increase in scientific research examining the effects of

climate change on both terrestrial and marine organisms. Current changes in the Earth’s

climate are indirectly affecting many environmental and biological processes, including

shifts in species phenology and distributions, nutrient fluxes, and growth and productivity

(Parmesan & Yohe, 2003). One of the main drivers of this change is anthropogenic increases

in atmospheric greenhouse gases, namely carbon dioxide (CO2). In the past 250 years the

global concentration of CO2 has increased by nearly 40%, from 280 parts per million (ppm)

to 384ppm (Solomon et al., 2007). As the world’s greatest carbon reservoirs, the oceans are

responsible for absorbing at least one-third of the added CO2, whereas the terrestrial

biosphere is responsible for taking up about one-fifth (Feely et al., 2004). It takes

approximately one year for the partial pressure of CO2 (pCO2) of the ocean to equilibrate

with that of the atmosphere, indicating fairly rapid diffusion (Doney et al., 2009). Once this

CO2 has diffused into the surface waters it undergoes speciation through a series of

dissociation reactions:

CO2(aq) + H2O ↔ H2CO3 ↔ H+ + HCO3- ↔ 2H+ + CO3

2- (1)

These reactions are near equilibrium and due to the natural buffering of the ocean, it

is impossible to vary the concentration of one species of CO2 without affecting the others

(Millero et al., 2002; Hurd et al., 2009). However, when in equilibrium, approximately 90%

of the inorganic carbon occurs in the form of bicarbonate (HCO3-), while ~9% occurs as

carbonate (CO32-), and only ~1% as dissolved CO2 (Doney et al., 2009). Consequently, as

more CO2 is dissolved into the oceans, its dissociation causes an increase in free hydrogen

ions (H+), resulting in a decrease in oceanic pH, a process referred to as ocean acidification

(OA). As a consequence of this process, the average pH of the ocean has shown a decrease

from 8.21 to 8.1 since the Industrial Revolution (Royal Society, 2005; IPCC

(Intergovernmental Panel on Climate Change), 2007). Further, the Intergovernmental Panel

on Climate Change (IPCC) reports that atmospheric pCO2 levels may reach 800-1000ppm by

the end of the century if current carbon-producing methods continue, and this is expected to

result in a decrease in average ocean pH by 0.3-0.4 units (IPCC, 2007).

2

An increase in OA is expected to have significant impacts on many marine taxa. For

example, current OA studies predict losses to biodiversity and shifts in species compositions

(Harrison, 2000; Hale et al., 2011; Porzio et al., 2011; Wernberg et al., 2011), changes to

photosynthesis and ecosystem productivity (Martin & Tortell, 2006; Anthony et al., 2008),

altered recruitment and growth rates of individual taxa (Kübler et al., 1999; Kuffner et al.,

2008), and reduced calcification in CaCO3-producing species (Hall-Spencer et al., 2008;

Comeau et al., 2009; Martone, 2010; and Büdenbender et al., 2011). However, while studies

on the impacts of OA on marine animals have become increasingly common, studies

examining the impacts of OA on photosynthetic organisms (plants and algae) remain

comparatively lacking. As the base of all marine food webs, autotrophs are necessary in

marine systems for energy production, habitat, maintaining structure and stability and

nutrient cycling. Their responses to OA are extremely important to understand, particularly

because photosynthesis can be greatly affected by changes in CO2 concentrations.

Ecologists have observed varied responses to elevated CO2 in photosynthetic

organisms in terrestrial ecosystems. For example, terrestrial free-air CO2-enrichment (FACE)

studies have shown an increase in photosynthesis under higher levels of pCO2, indicating

carbon fertilization positively influences plant productivity (Ainsworth & Long, 2005; Norby

et al., 2005). These experiments are ideal systems to study elevated pCO2 over time. They

allow for natural wind and diffusion of gases and reduce the “chamber effect” that may cause

down-regulation of photosynthesis (Hendry, 1993; Lewin et al., 1994; Morgan et al., 2001).

They also allow for migration of organisms throughout these habitats and interactions of

multiple species. FACE studies are continually improving in design and many scientists have

discovered effective ways to reduce edge effects and homogenize CO2 throughout the plots.

Natural CO2-enrichment studies are valuable and research has been able to deduce much

about the physiological limits of organisms. These experiments have led to an increase in the

use of CO2 fertilization in greenhouses, hydroponics and biofuels. Likewise, there have been

a few aquatic studies that have demonstrated success in methods of CO2-enrichment by

utilizing open-top chambers, such as those constructed by Campbell and Fourqurean (2011).

In a six-month experiment in the shallow seagrass beds in the Florida Keys, the internal

chamber spaces maintained relatively stable pH levels despite larger fluctuations outside of

the chambers, demonstrating a promising method to study elevated pCO2 in shallow

3

environments. Unfortunately, these methods are not as feasible in deeper subtidal habitats

and research in marine ecosystems has not been able to easily replicate the technology and

methodology of terrestrial FACE studies. However, small-scale marine experiments have

demonstrated similar results (Behrenfeld et al., 2006; Martin & Tortell, 2006; Connell &

Russell, 2010; Gao & Zheng, 2010). For example, some non-calcifying algae and seagrasses

have exhibited higher photosynthesis (e.g. Spijkerman, 2008; Porzio et al., 2011) and growth

(e.g. Zou & Gao, 2009) under elevated pCO2. Such high rates of photosynthesis in select

organisms could potentially lead to shifts in their distribution, abundance and/or competitive

abilities (e.g. Swanson & Fox, 2007; Connell & Russell, 2010; Connell et al., 2011). While

up-regulation of photosynthesis is likely in many primary producers, there are many marine

macroalgae that produce CaCO3 structures for protection and support, and these structures

are predicted to be negatively impacted by OA. However, while this has been observed in

CaCO3-producing animals, it remains unclear how it will impact organisms that also

photosynthesize.

Calcification is the biogenic formation of CaCO3 by many organisms to create shells,

skeletons and/or structural crusts. CaCO3 synthesis is limited by the bioavailability of CO32-

(Borowitzka, 1981; Borowitzka & Larkum, 1987) and should therefore be dramatically

impacted as the equilibrium states of dissolved CO2 reduce the concentration of CO32-

(Doney et al., 2009). Although the mechanism of calcification is not well understood in most

organisms, CaCO3 is known to be synthesized in several different forms, including calcite,

aragonite, and high-magnesium calcite, the latter of which is the most water-soluble and

therefore most susceptible to decreases in ocean pH (Mucci, 1983; Adey, 1998). Due to the

chemical requirements for calcification, rates have been shown to decrease under elevated

pCO2 in most calcifying marine organisms (Comeau et al., 2009; Feng et al., 2009;

Büdenbender et al., 2011; Basso & Granier, 2012; Diaz-Pulido et al., 2012) while dissolution

of existing CaCO3 structures can occur with severe acidification (Feely et al., 2004). Most

studies have focused on the calcification of tropical corals. Corals have exhibited bleaching

and mortality in many tropical reef ecosystems due to increases in water temperature and

have therefore been of great concern to marine researchers. Many species of corals have also

shown decreased calcification under elevated pCO2 (Langdon et al., 2000; Reynaud et al.,

2003; Anthony et al., 2008), further exacerbating the problem. These studies may help with

4

predictions for coralline algae, but corals are symbionts in which photosynthesis and

calcification occur in separate organisms. This is difficult to compare to an organism that

does both of these processes simultaneously. An example of organisms that do both calcify

and photosynthesize are the calcifying phytoplankton, such as Emiliana huxleyi and

Calcidiscus leptoporus, for which studies have also shown a decrease in calcification under

elevated CO2, despite the potential for carbon fertilization to increase photosynthesis

(Riebesell et al., 2000). Many studies, however, have investigated calcification as a single

mechanism without addressing the combined effects of elevated pCO2 on both

photosynthesis and calcification. Consequently, the impacts on net photosynthesis of

calcifying algae remain unclear (Martin & Gattuso, 2009; Ries et al., 2009; Semesi et al.,

2009; Gao & Zheng, 2010; Büdenbender et al., 2011). It will be important to determine if the

predicted increases in photosynthesis under elevated pCO2 are sufficient to overcome the

expected declines in calcification. Therefore, the focus of this research is to assess the how

OA may cause changes in the physiological responses of coralline algae.

Most OA research has attempted to define the effects of elevated pCO2 over relatively

short time periods. The difficulties of studying elevated pCO2 in real-time makes predictions

extremely challenging. It is also important to understand species-specific responses before

attempting to address responses of entire ecosystems, and these may require longer time

periods. This will allow researchers to determine the potential for species to acclimate to

changing pCO2 and the role of phenotypic plasticity in mediating the response to OA.

Morphological and physiological acclimation in algae has been shown to occur during

intermediate-term OA studies (hereafter defined as those conducted over two or more

weeks), for example, with increasing irradiance levels (Falkowski & LaRoche, 1991), metal

concentrations (Bossuyt & Janssen, 2004), temperature and depth (Duarte & Ferreira, 1995),

and/or salinity (Eggert et al., 2007). Non-calcifying macroalgae have also shown overall

increases in photosynthesis (Kübler et al., 1999; Swanson & Fox, 2007; Connell & Russell,

2010). These increases have lead to higher growth rates and higher abundances of algae.

However, the opposite response to elevated pCO2 has also been observed. For example,

Björk et al. (2004) demonstrated that the green alga Ulva may decrease the use of HCO3- and

increase their maximum rates of photosynthesis (Pmax) under high pCO2 conditions upon

initial exposure, but will acclimate to these conditions and reduce photosynthesis within just

5

7-8 days. Likewise, a cold-water coral, Lophelia pertusa, was shown to acclimate to elevated

pCO2 and actually exhibited higher calcification rates after six months at a pH decrease of

0.1 (Form & Riebesell, 2012). Acclimation studies on calcifying algae have resulted in

varying responses, most concluding that elevated pCO2 has a negative effect on the ability to

calcify and that acclimation is prevented by simple seawater chemistry and a reduction in the

saturation state of CaCO3 (Gao et al., 1993; Jokiel et al., 2008; Martin & Gattuso, 2009;

Büdenbender et al., 2011; Diaz-Pulido et al., 2012). A few acclimation studies have used

crustose coralline algae and have measured reductions in recruitment and percent cover

(Kuffner et al., 2008), as well as decreases in photosynthesis and calcification (Anthony et

al., 2008, Büdenbender et al., 2011). Due to very similar life history strategies and ecosystem

functions (Littler, 1972; Steneck, 1986; Nelson, 2009), geniculate coralline algae may

similarly show an initial decrease in physiological functioning as a result of prolonged

exposure to elevated pCO2. Nonetheless, the ability to acclimate will play a significant role in

the survival and distribution of calcifying organisms.

While laboratory acclimation studies are useful, they are not often ecologically

relevant when certain spatial and temporal factors are ignored due to laboratory limitations

(e.g. Foster, 1990; Carpenter, 1998). The majority of marine in situ CO2-enrichment studies

have taken place at natural CO2 seeps and volcanic vents. Jason Hall-Spencer and Riccardo

Rodolfo-Metalpa have conducted extensive studies on organisms at natural CO2 vents off the

Island of Ischia in Italy. By conducting surveys and transplanting calcifying organisms, they

have been able to look at abundances and calcification rates over a pH gradient (Hall-Spencer

et al., 2008; Martin et al., 2008; Rodolfo-Metalpa et al., 2010). Studies at those vents have

also measured species richness and spatial distributions, noting shifts toward non-calcifying

communities as pH levels decrease (Porzio et al., 2011; Johnson et al., 2012). However,

these studies analyze systems that respond much differently from algal-dominated systems

that do not naturally experience such high pCO2 levels. Although such studies are useful in

determining calcification limits, the ability to maintain CaCO3 structures is ultimately

controlled by seawater chemistry and available CO32-. Given these differences in natural

pCO2 exposure, understanding how coralline algae that naturally experience variable levels

of elevated pCO2 will respond and acclimatize to future pCO2 levels is essential.

6

The order Corallinales (Rhodophyta) is one of the most prominent groups of

calcifying algae, exhibiting up to 100% of the benthos in some temperate kelp forests along

the coast of California (Reed & Foster, 1984; Edwards, 1998; Nelson, 2009). As a significant

carbon reservoir, they are important for carbon cycling and productivity, as well as

maintaining habitat and biodiversity, playing an influential role in recruitment processes,

preserving reef structure and stabilizing substrates (Nelson, 2009). All forms of CaCO3 are

found abundantly in nature and have been detected in many intra-, inter- and extracellular

spaces of algal thalli (Milliman, 1974; Borowitzka & Larkum, 1987; Lobban & Harrison,

1994; Lee & Carpenter, 2001). However, these coralline algae deposit high-Mg calcite into

their cell walls (Littler & Littler, 1984; Payri, 1997; Adey, 1998), suggesting high

susceptibility to OA. While their ecological importance to the temperate kelp forests is well

documented (Hicks, 1971; Jones et al., 1994; Pedley & Carannante, 2006; Nelson, 2009), the

effects of OA on coralline algae remain poorly understood. Additionally, their unique

physiology, i.e. being able to both photosynthesize and calcify, makes them model organisms

to test processes that are predicted to respond inversely to OA.

Undoubtedly, the net effect of elevated pCO2 on coralline algae is extremely complex

and the outcome difficult to predict. Both photosynthesis and calcification are processes

essential to survival and capable of being drastically affected by changes in pCO2. Therefore,

the objective of this research is three-fold: (1) to assess the short-term effects of elevated

pCO2 on photosynthesis and calcification of coralline algae by comparing differences in their

photosynthesis-irradiance response curves, (2) to assess the intermediate-term effects of

elevated pCO2 on coralline algae by measuring photosynthesis and calcification throughout a

four-week mesocosm experiment, and (3) to assess both short- and intermediate-term

responses to elevated pCO2 during in situ incubations in the Point Loma kelp forest. These

three components will allow us to make better predictions about the responses of temperate

coralline algae to impending climate change and can set the stage for future studies

incorporating other organisms and anthropogenic interferences.

7

MATERIALS AND METHODS

TARGET SPECIES

The three most abundant geniculate coralline algal species in the Point Loma kelp

forest (see below), namely Bossiella californica ssp. schmittii (Manza), Calliarthron

tuberculosum (Postels & Ruprecht), and Corallina officinalis var. chilensis (Decaisne), were

collected for use in laboratory and field experiments. These three species exhibit different

morphologies and although they co-occur within the kelp forest, they exhibit different depth

distributions. Specifically, C. tuberculosum has smooth intergenicula, can grow to over 20

cm in length and is found subtidally (15-20 meters) to low intertidal pools. C. officinalis has

fronds that tend to lie in one plane reaching only 15 cm in length and has a similar depth

distribution as C. tuberculosum. B. californica forms fan-like tufts to 12 cm long and is

commonly found within Macrocystis pyrifera forests to as deep as 40 meters (Abbott &

Hollenberg, 1976).

SITE

Field collections of the three target species were done at approximately 10-15m depth

at New Hope Rock in the southern half of Point Loma kelp forest (approximately

32o687’N/117o266’W), located off the coast of San Diego, California. The Point Loma kelp

forest is one of the largest continuous giant kelp forests in the world with a natural history

that has been thoroughly examined (Dayton et al., 1984, 1992, 1998; Tegner et al., 1997;

Graham et al., 2008). The habitat is characterized by both cross-shore and longshore

gradients in the physical landscape and biological composition. Temperatures in Point Loma

ranged from 12-15oC during experiments and average benthic irradiances (at 10-20 m)

ranged from 5 μmol photons·m-2·s-1 in heavily shaded areas to 30-50 μmol photons·m-2·s-1in

canopy openings. The primary canopy-forming alga is Macrocystis pyrifera, which grows on

a broad, gently sloping shelf paralleling the shoreline (Turner et al., 1968). Several other kelp

species, namely Laminaria farlowii, Pterogophora californica, and Eisenia arborea, create a

subsurface canopy layer. Fleshy turf algae, both geniculate and non-geniculate (crustose)

coralline algae, are common understory species.

8

CHARACTERIZATION OF PHOTOSYNTHESIS AND

CALCIFICATION OF CORALLINE ALGAE UNDER

ELEVATED PCO2

The following sections describe the methods used to assess the responses of the three

target species to elevated pCO2 via three experiments: (1) short-term bottle incubations to

measure short-term responses to elevated pCO2, (2) mesocosm experiments to observe

responses after four weeks of exposure to elevated pCO2, and (3) in situ incubations under

elevated pCO2 to measure both short-term responses and changes after two-weeks of

acclimation to elevated pCO2.

Assessing the Response of Coralline Algae to Short-Term Exposure to Elevated pCO2 via Photosynthesis-

Irradiance Response Curves

To characterize impacts of elevated pCO2 on the photosynthetic performance of each

species, photosynthesis-irradiance (P-I) response curves were constructed for all three

species under two pCO2 levels; 380ppm and 1000ppm. Specifically, this allowed for the

comparison of photosynthetic efficiency (α) under non-saturating irradiances, maximum rates

of photosynthesis (Pmax) under saturating irradiances, and the saturation irradiance itself (IK)

under the two pCO2 levels. Specimens of Bossiella californica, Calliarthron tuberculosum,

and Corallina officinalis were collected on SCUBA and transferred to a laboratory holding-

tank set at 12oC and 15 μmol photons·m-2·s-1 (approximate benthic irradiance) for a period no

longer than three days. Seawater was collected in 5-gallon airtight plastic containers using a

12-volt bilge pump and filtered through a 60μm filter for use in the lab incubations.

P-I curves were constructed for both dissolved O2 production (in μmol O2 produced

per gram of dry weight algae per hour) and total inorganic carbon (TIC) uptake (in μmol C

taken up per gram of dry weight algae per hour) for each species under each pCO2 level.

Pmax, α and IK were calculated for each curve. In addition, net calcification (Cnet) was

calculated from changes in total alkalinity (TA) as μmol C incorporated per gram dry weight

algae per hour. Specifically, dissolved O2 was measured using a Clark-type oxygen electrode

with a guard cathode (Revsbech, 1989) connected to a picoammeter (PA2000, Unisense,

Denmark). To calibrate the electrode, a 2-point calibration was done using 100% air-

saturated seawater and oxygen-free seawater; the 100% air-saturated seawater was created by

9

bubbling ambient air through 12oC seawater overnight and anoxic seawater was created by

adding 1 g·L-1 of sodium sulfite (Na2SO3) to 250 mL of seawater. Total μmoles of dissolved

O2 could then be determined from known standardized tables (Dickson et al., 2007).

Seawater TIC and TA were measured using potentiometric titration (Millero et al., 1993).

Our titration system consisted of a Metrohm 765 Dosimat titrator and Orion 920A pH meter

connected to a PC by RS232 communication cable. A jacketed beaker supplied by a 25oC

water bath allowed samples to be run at a constant temperature (within 0.1oC). Titration

custom software based on Q-basic determined the volume of acid added during titration and

the resulting electromotive force (EMF) through an RS-232 communication cable. TIC and

TA could then be entered into CO2sys software (http://cdiac.ornl.gov/ftp/co2sys/) to

calculate the pH and pCO2 at a constant temperature and salinity (Lewis & Wallace, 1998;

Dickson et al., 2007).

The following incubations were conducted using ambient seawater (~380ppm) and

seawater with an elevated pCO2 of approximately 1000ppm for all three species. To begin

each incubation, 27-500mL BOD bottles were filled with the filtered seawater, three of

which were used as references to measure initial dissolved O2, TIC and TA in the water at

12oC. Following this, pCO2 levels in the seawater were manipulated by bubbling pure CO2

gas through the water using a diffuser. pCO2 levels were verified prior to the start of the

incubations by potentiometric titration. All algae were cleaned of visible epiphytes by hand

and approximately 2.0g (±1.0g) of thallus were added to each BOD bottle. The bottles were

sealed and immediately placed into a 12oC water bath where they were exposed to one of

seven different irradiance levels (0, 5, 10, 15, 30, 75 and 100 μmol photons·m-2·s-1) with three

replicates each. The incubations were conducted for 2.5 hours, with the bottles agitated

periodically to break up boundary layer formation. At the conclusion of the incubation

period, the algae were removed, dried for 48 hours at 65oC, and weighed. Each individual

was then immersed in a 1N hydrochloric acid (HCl) solution overnight to allow complete

dissolution of the CaCO3 structure. The algae were again dried for 48 hours, reweighed, and

the mass of CaCO3 in their thalli was determined by subtracting the decalcified dry mass

from the original dry mass (Martone, 2010). The average percent CaCO3 per gram of dry

weight thallus was then determined. The seawater in each bottle was analyzed according to

the protocols described above in order to determine changes in dissolved O2, TIC and TA

10

over the 2.5-hour incubation period. Two replicate incubations were conducted per species

for each pCO2 level.

TIC was found to be a much more sensitive measurement of photosynthesis, and the

oxygen electrode used was not sensitive enough to detect the small changes in dissolved O2

within the bottles. Therefore oxygen measurements, while still measured, were omitted from

this analysis. Pmax, α and IK were consequently based on the changes in TIC, and were

compared between the two pCO2 levels and three species using a two-way Fixed Model

PERMANOVA (PRIMER v6), followed by permutation post-hoc tests when appropriate.

Calcification and percent CaCO3 were both analyzed using separate three-way mixed-model

PERMANOVAs, with species and pCO2 as fixed factors and irradiance as a random factor.

Permutation post-hoc tests were done when significant differences were found.

Assessing the Response of Coralline Algae to Intermediate-Term Exposure to Elevated pCO2 via

Mesocosm Experiments

Mesocosm experiments were conducted in the laboratory to assess changes in

photosynthesis and calcification within the three species of coralline algae to elevated pCO2

over a four-week time period. The mesocosm system consisted of three 100-gallon

cylindrical high-density polyethylene tanks that were used as reservoirs for seawater that was

collected at Scripps Institute of Oceanography in La Jolla, California. Each reservoir was

connected to a system of insulated PVC pipes that brought fresh seawater from the reservoirs

to the flow-through mesocosms (Figure 1). Flow-control valves that allowed regulation of

flow volumes were placed along the PVC pipes from which low-density polyethylene tubing

carried seawater to the mesocosms. Seawater entered the 12”x12”x8” translucent

polycarbonate mesocosms at the bottom left-hand corner and flowed to the top right-hand

corner where a tube returned the seawater back to the PVC pipe leading to the reservoir. This

ensured all seawater was cycled through the chambers. Each mesocosm was airtight and

sealed with neoprene gaskets in order to prevent gas exchange with the atmosphere. Seawater

in the reservoirs was manipulated to reflect three CO2 concentrations, an ambient level at

500ppm (Appendix, Figure 10) and two elevated levels at 1000ppm and 1500ppm. Ambient

11

Figure 1. Mesocosm set-up at the Coastal and Marine Institute Laboratory with PVC pipes to supply 12oC seawater at three pCO2 levels (500ppm, 1000ppm and 1500ppm) and shut-off valves to control flow to each mesocosm.

seawater was created by continuously bubbling atmospheric air into the reservoir using a

standard aquarium air pump connected to a venturi injector. Elevated CO2 levels were

created by bubbling certified CO2 mixtures for 1000ppm and 1500ppm (Praxair, San Diego).

Verification of pCO2 within the seawater was done by daily potentiometric titrations. All

reservoirs and mesocosms were kept at a constant 12oC under a 12:12 light regime at

approximately 15 µmol photons·m-2·s-1 to approximate benthic irradiance at the collection

site within the Point Loma kelp forest.

Collections for Bossiella californica, Calliarthron tuberculosum, and Corallina

officinalis were conducted according to the methods described above. Algae were acclimated

in holding tanks to laboratory conditions for 48 hours at 12oC before the mesocosm

experiment began. Following this, approximately 50g of each species was placed into one

mesocosm at 500ppm, one at 1000ppm, and one at 1500ppm and held for four weeks. To

assess changes in photosynthesis and calcification throughout this period, samples of the

algae were collected after 1, 3, 7, 14, 21 and 28 days, and bottle incubations were conducted

for each species under each pCO2 level. Specifically, on the designated incubation day, 18-

12

500mL BOD bottles were filled with filtered seawater from the mesocosm system at the

appropriate pCO2 (six bottles per mesocosm). Eighteen 2.0±1.0g algal pieces without visual

epiphytes were placed into sealed bottles. The 2.5-hour incubation was conducted in a 12oC

water bath under 15 μmol photons·m-2·s-1 and the bottles were agitated periodically to break

up the boundary layer surrounding the algae. After 2.5 hours, the algae were removed from

the bottles, dried for 48 hours at 65oC and decalcified and weighed to determine CaCO3

content on Day 1 and Day 28. The seawater in each bottle was reanalyzed to determine

changes in TIC and TA using potentiometric titration.

Variances of these data were unequal, so ANOVAs could not be done, therefore

photosynthesis rates, calcification rates and CaCO3 content were compared between the three

pCO2 levels, the three species, and six days using independent three-way Fixed Model

PERMANOVA (PRIMER v6). These analyses were followed by permutation post-hoc tests

when appropriate.

Assessing the Response of Coralline Algae to Elevated pCO2 via In Situ Incubations

The responses of coralline algae to elevated pCO2 under natural conditions were

assessed via in situ incubations within the Point Loma kelp forest. Photosynthesis and

calcification rates were compared among the three species (B. californica, C. officinalis and

C. tuberculosum) and between two pCO2 levels (500ppm and 1000ppm). This experiment

also assessed acclimation to these conditions by comparing incubations of algae done

immediately after collection with incubations done after two weeks of acclimation to

elevated pCO2 in the laboratory mesocosms. Field frames were assembled to allow for in situ

incubations. Each frame was composed of a weighted PVC rectangle (approximately 2.5’x1’)

with 8-500mL translucent plastic incubation bags (Ziplock brand) attached to the frame by

zip ties (Figure 2). Four bags were filled with 500ppm seawater for separate incubations of

all three species plus a control bag. The other four bags were filled with 1000ppm seawater,

one bag for each species and a control. Each field incubation was therefore comprised of

13

Figure 2. One 2.5’x1’ PVC field frame during an incubation at 10m depth at New Hope Rock; four incubation bags contain 500ppm seawater and four contain 1000ppm seawater; all three species are represented at each pCO2 along with two controls.

three of these frames for a total of 24 bags, resulting in three replicates for each species at

each pCO2 level.

Seawater for the incubations was collected via research boat from just outside the

Point Loma kelp forest at 10m depth in 5-gallon airtight plastic containers using a 12 volt

bilge pump. The seawater was filtered in the laboratory with a 60μm filter and pCO2 was

manipulated by bubbling pure CO2 gas through the seawater using a diffuser. pCO2 was

verified using potentiometric titration. Afterwards, twelve incubation bags were each filled

with 500mL of 500ppm seawater and twelve bags with 1000ppm seawater. These bags were

then transported in coolers at 12oC on the research boat until commencement of the

incubation in the field. Upon arrival at New Hope Rock in the Point Loma kelp forest

(32o687’N/117o266’W), algae were collected on SCUBA at approximately 10 meters depth

and brought to the surface in dark bags. Half of the algae collected were immediately used in

an incubation (hereafter “Immediate Incubation”) and half of the algae were brought back to

the lab where they were split evenly between two mesocosms, one at ambient pCO2 seawater

(~500ppm) and one at elevated pCO2 seawater (~1000ppm) to be used later in a second

incubation (hereafter “Acclimated Incubation”). For the latter, all three species were exposed

14

to these pCO2 levels for two weeks within the same mesocosm system described above. After

two weeks the algae were returned to the field and subjected to an in situ incubation

following the protocols used in the Immediate Incubation. Each incubation began on the deck

of the research boat by placing one 2.0±1.0g piece of algae into the appropriate bag on the

PVC frame. The bags were sealed and the frame was placed on the substrate at

approximately 10m depth. This process was repeated for all three frames. Time was carefully

monitored to ensure each unit was on the benthos for 2.5 hours. Irradiance, temperature and

depth were recorded at the benthos next to each frame. A water sample was also collected

near each frame to measure in situ total inorganic carbon (TIC) and total alkalinity (TA).

After 2.5 hours, the frames were retrieved, the algae were immediately removed from the

bags, and both the algae and bags were brought back to the lab for analysis. The algae were

dried and weighed. The seawater was analyzed TIC and TA using potentiometric titration.

Two Immediate Incubations and two Acclimated Incubations were conducted, and data was

averaged to compare the two treatments.

The data had unequal variances that could not be fixed by transformations, therefore

ANOVAs could not be done. Consequently, photosynthesis and calcification rates were

compared using independent three-way Fixed Model PERMANOVAs (PRIMER v6) using

Species, pCO2, and Treatment (Immediate vs. Acclimated) as factors. This was followed by

permutation post-hoc tests when significant differences were found.

15

RESULTS

The following three sections describe the changes in photosynthesis and calcification

rates as a result of elevated pCO2 for Bossiella californica, Calliarthron tuberculosum, and

Corallina officinalis measured in: (1) laboratory incubations and the construction of

photosynthesis-irradiance response curves, (2) a four-week laboratory mesocosm experiment

and (3) field incubations from an in situ acclimation study conducted within the Point Loma

kelp forest.

PHOTOSYNTHESIS-IRRADIANCE RESPONSE CURVES

Incubations under both ambient (380ppm) and elevated (1000ppm) pCO2 resulted in

P-I curves exhibiting similar patterns for all three species (Figure 3, Table 1). Specifically,

each species exhibited decreased rates of maximum photosynthesis (Pmax) under elevated

pCO2, indicating less carbon uptake via photosynthesis, despite the increased availability of

carbon. The curves also showed increases in photosynthetic efficiency under non-saturating

irradiances (α). Finally, the curves showed evidence of decreases in saturating irradiances

(IK), which indicates that these species are reaching maximum photosynthesis rates at a much

lower irradiance under elevated pCO2. However, despite the overall trends revealed in the

curves, increases in pCO2 did not result in significant overall changes to the P-I parameters (p

= 0.117; Table 2). In fact, the only statistically significant differences observed were between

two of the species; B. californica and C. tuberculosum produced different photosynthesis-

irradiance curves (p = 0.045, Table 3). Specifically, average Pmax was 15.58±1.68 μmol C·g-

1·h-1 in B. californica but only 8.88±1.28 μmol C·g-1·h-1 in C. tuberculosum. In contrast, B.

californica had the highest α (0.86±0.03), but the lowest average Ik (18.10±1.56 μmol

photons·m-2·s-1), suggesting it was more efficient at low irradiances, but its photosynthetic

mechanisms were saturated at a much lower irradiance.

Calcification rates were impacted by pCO2 level when examined across all species (p

= 0.040), causing lower average rates at 1000ppm by almost 23% and exhibiting a saturation

point at approximately 20-30 μmol photons·m-2·s-1.

16

Fig

ure

3. P

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osyn

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is-I

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ian

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esp

onse

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of

B. c

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ube

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nd

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: (a

) am

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ated

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17

Table 1. Photosynthesis Parameters Based on P-I Curves for TIC. Means ± SE of Two Incubations per Treatment

Species pCO2 Pmax α IK

B. californica 380 16.00 ± 1.80 0.84 ± 0.01 18.95 ± 2.25 1000 15.15 ± 3.65 0.86 ± 0.07 17.25 ± 2.85

C. tuberculosum 380 8.95 ± 4.20 0.26 ± 0.03 32.95 ± 6.35 1000 8.80 ± 1.80 0.39 ± 0.01 22.50 ± 4.10

C. officinalis 380 15.75 ± 1.25 0.50 ± 0.01 31.55 ± 1.75 1000 15.60 ± 3.80 0.67 ± 0.24 24.35 ± 3.25

Table 2. Two-Way PERMANOVA Table of Results for Parameters of P-I Curves Based on TIC with Factors pCO2 and Species (n = 252). Boldface* Denotes Significant Differences

PERMANOVA Table of Results

Source df SS MS Pseudo-F P (perm) pCO2 (pC) 1 125.280 125.280 3.431 0.117 Species (Sp) 2 375.030 187.510 5.136 0.020* pCxSp 2 39.463 19.732 0.469 0.733 Res 6 252.630 36.512 42.105

Table 3. Pair-Wise Comparison Between Species for Both CO2 Levels. Boldface* Denotes Significant Differences

Groups t P (perm) Bossiella, Corallina 2.556 0.057 Bossiella, Calliarthron 2.389 0.045*Corallina, Calliarthron 1.386 0.229

These saturation points were similar for all three species (Figure 4). However, these

differences were driven mainly by the calcification pattern seen in C. officinalis (Table 4,

Figure 5). Post-hoc comparisons of the pCO2xSpecies interaction indicated that C. officinalis,

unlike the other two species, incorporated significantly different amounts of carbon between

the two pCO2 levels (p = 0.003). Specifically, C. officinalis calcified at an average rate of

4.31±0.98 μmol C·g-1·h-1 at 380ppm, and 2.20±1.10 μmol C·g-1·h-1 at 1000ppm, indicating a

49% decrease under elevated pCO2. However, while calcification was susceptible to elevated

pCO2 in C. officinalis, it was not significantly different in the other two species. B.

californica had the highest average calcification rates (4.80±0.89 μmol C·g-1·h-1 at 380ppm

18

Figure 4. Calcification rates for each species at increasing irradiance under ambient pCO2 and elevated pCO2 for (a) B. californica, (b) C. tuberculosum, and (c) C. officinalis.

‐4.0

‐2.0

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Cal

cifi

cati

on

(μm

ol C

·g-1

·h-1

)

Irradiance (μmol photons·m-2·s-1)

(a) B. californica

Ambient

Elevated

0             5             10           15           30 75           100

‐4.0

‐2.0

0.0

2.0

4.0

6.0

8.0

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0 5 10 15 30 75 100

Cal

cifi

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on

(μm

ol C

·g-1

·h-1

)

Irradiance (μmol photons·m-2·s-1)

(b) C. tuberculosum

Ambient

Elevated

‐4.0

‐2.0

0.0

2.0

4.0

6.0

8.0

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Cal

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mol

C·g

-1·h

-1)

Irradiance (μmol photons·m-2·s-1)

(c) C. officinalis

Ambient

Elevated

0             5             10           15           30 75           100

19

Table 4. Three-Way PERMANOVA Table of Results for Calcification Rates With Fixed Factors pCO2 and Species, and Irradiance as a Random Factor. Boldface* Denotes Significant Differences

PERMANOVA Table of Results

Source df SS MS Pseudo-F P (perm) pCO2 (pC) 1 15.071 15.071 6.814 0.040* Species (Sp) 2 62.479 31.240 4.643 0.030* Irradiance (Irr) 6 612.830 102.14 57.721 0.001* pC x Sp 2 22.201 11.101 41.047 0.001* pC x Irr 6 13.271 2.212 1.250 0.326 Sp x Irr 12 80.741 6.728 3.802 0.003* pC x Sp x Irr 12 3.245 0.270 0.153 1 Res 42 884.160

Figure 5. Average calcification rates for each species under ambient pCO2 (light bars) and elevated pCO2 (dark bars).

and 3.95±0.98 μmol C·g-1·h-1 at 1000ppm) but incubations did not demonstrate significant

differences between ambient and elevated pCO2, despite the slightly lower calcification rate

under elevated pCO2. Likewise, C. tuberculosum exhibited an entirely different pattern,

increasing calcification by 20% under elevated pCO2 (from 2.06±0.38 to 2.47±0.47 μmol

C·g-1·h-1), although absolute rates of calcification in C. tuberculosum were lower than the

other two species.

20

Measurements of thallus CaCO3 exhibited similar trends to those observed for

calcification rates, although CaCO3 was not affected by irradiance level over the short 2.5-

hour incubation. There were overall differences in tissue CaCO3 among species (p = 0.003,

Table 5), with B. californica exhibiting a higher percent tissue CaCO3 than either C.

tuberculosum or C. officinalis. Additionally, a significant pCO2xSpecies interaction indicated

the same pattern that was observed from calcification rate measurements, namely CaCO3

decreased under elevated pCO2 by 0.78% for B. californica (p = 0.148, Table 6) and by

2.89% for C. officinalis (p = 0.023), but was 1.8% higher in C. tuberculosum (p = 0.022).

The lack of differences between pCO2 levels in percent thallus CaCO3 in B. californica is

consistent with the responses of photosynthesis and calcification rates.

Table 5. Three-Way PERMANOVA Table of Results for % CaCO3 With Fixed Factors pCO2 and Species and Irradiance as a Random Factor. Boldface* Denotes Significant Differences

PERMANOVA Table of Results

Source df SS MS Pseudo-F P (perm) pCO2 (pC) 1 12.229 12.229 3.946 0.102 Species (Sp) 2 40.742 20.371 9.582 0.003* Irradiance 6 24.533 4.089 0.835 0.562 pCxSp 2 115.790 57.897 10.817 0.002* pCxIrr 12 18.596 3.099 0.633 0.717 SpxIrr 12 25.513 2.126 0.434 0.945 pCxSpxIrr 84 64.230 5.353 1.093 0.365 Res 114 519.590 4.896

Table 6. Average % CaCO3 After Incubations for Two pCO2 Levels (Ambient = 380ppm; Elevated = 1000ppm). Boldface* Denotes Significant Differences

Species pCO2 Average % CaCO3 % Change

Bossiella californica 380 88.42±0.27 - 0.78

1000 87.64±0.33

Calliarthron tuberculosum 380 85.76±0.41 +1.79

1000 87.55±0.39

Corallina officinalis 380 88.60±0.79 -2.89

1000 85.71±0.42

21

FOUR-WEEK MESOCOSM EXPERIMENTS

After the algae were exposed to 500ppm, 1000ppm and/or 1500ppm for four weeks,

changes in photosynthesis and calcification were much more conspicuous than those

observed in the short-term incubations described above. Specifically, the algae in the 500ppm

(ambient) mesocosm appeared healthy and remained dark red in color throughout the entire

experiment, while most of the specimens in the 1000ppm and 1500ppm mesocosms appeared

unhealthy by day 14 and exhibited bleaching and discoloration. Also, their fronds on these

became brittle and broke easily at the genicula. As a result, B. californica suffered 100%

mortality in the 1500ppm mesocosm by day 21, and in all mesocosms by day 28. The other

two species were alive for bottle incubations throughout the four-week period to allow for

measurements of photosynthesis and calcification.

There were significant differences in photosynthesis among the six sample days,

pCO2 levels, and the three species, as well as interactions between these factors (Table 7).

Together, photosynthesis was negatively affected by increasing pCO2 with each species

exhibiting lower rates of photosynthesis under pCO2 levels of 1500ppm (p = 0.001).

Specifically, photosynthesis averaged 1.253±0.614 μmol C·g-1·h-1 across all three species in

the 1500ppm mesocosm, which increased to 4.591±1.412 μmol C·g-1·h-1 in the 1000ppm

mesocosm, and 5.019±1.576 μmol C·g-1·h-1 in the 500ppm mesocosm.

Table 7. Three-Way PERMANOVA Table of Results for TIC With Fixed Factors Day, pCO2 and Species (n = 100). Boldface* Denotes Significant Differences

PERMANOVA Table of Results

Source df SS MS Pseudo-F P (perm) Day 5 1755.400 351.080 141.260 0.001* pCO2 2 251.410 125.700 50.577 0.001* Species 2 85.961 42.981 17.293 0.001* DayxpCO2 10 536.730 53.673 21.595 0.001* DayxSpecies 9 224.210 24.913 10.024 0.001* pCO2xSpecies 4 17.070 4.268 1.717 0.153 DayxpCO2xSpecies 17 94.070 5.534 2.226 0.017* Res 50 124.270 2.485

22

Carbon uptake also differed throughout the four-week period (p = 0.001, Figure 6)

and those differences were caused primarily by low photosynthesis rates in C. tuberculosum

in the 1500ppm mesocosm (p = 0.001). At the onset of the experiment (day 1),

photosynthesis was significantly lower in the 1500ppm mesocosm and there were no

detectable differences between photosynthesis rates in the 500ppm and 1000ppm mesocosms

(p = 0.93). Likewise, day 3 revealed no significant differences in photosynthesis rates

between all three mesocosms. In contrast, day 7 resulted in a drastic increase in

photosynthesis in all three species in the 500ppm and 1000ppm mesocosms. This was not

observed in the 1500ppm mesocosm, which stayed at a fairly low constant rate for all three

species throughout the 4-week period. Day 14 was indicative of a return to lower rates of

photosynthesis in all three mesocosms, although rates were still higher than measurements

taken before the Day 7 increase. Photosynthesis in the 1500ppm mesocosm was significantly

lower than photosynthesis in the 500ppm mesocosm (p = 0.025), although C. tuberculosum,

exhibited slightly higher photosynthesis in the 1000ppm mesocosm. By day 21 B. californica

suffered 100% mortality in the 1500ppm mesocosm and photosynthesis dropped further for

C. tuberculosum and C. officinalis, however, there were no significant differences between

the 500ppm and 1000ppm mesocosms (p = 0.117). On day 28 at the conclusion of the

experiment, only C. tuberculosum and C. officinalis were available for bottle incubations and

photosynthesis was not significantly different between these two species in any of the

mesocosms.

All three species demonstrated a similarly negative response to elevated CO2 in

calcification rates. Calcification rates were less variable than photosynthesis throughout the

four weeks and species differences were not significant (p = 0.092, Figure 7, Table 8).

Throughout the experiment, calcification was significantly lower in the 1500ppm pCO2

mesocosm (p = 0.001). Calcification rates decreased as pCO2 increased with rates averaging

1.326±0.297 μmol C·g-1·h-1 in the 500ppm mesocosm, 1.299±0.343 μmol C·g-1·h-1 at

1000ppm, and -0.896±0.307 μmol C·g-1·h-1 at 1500ppm, indicating dissolution of CaCO3.

However, despite the lower average rates of calcification with higher pCO2, the decrease

from the 500ppm to the 1000ppm was not significantly different (p = 0.762). The increase at

day 7 that was observed for photosynthesis was not as noticeable for calcification, and in

fact, calcification seemed to increase on average toward the end of the four-week period.

23

Figure 6. Photosynthesis rates over a 28-day span in three different mesocosms (pCO2 of 500ppm, 1000ppm, and 1500ppm) for (a) B. californica, (b) C. tuberculosum, and (c) C. officinalis.

‐5.0

0.0

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)

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(a) B. californica 500ppm

1000ppm

1500ppm

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1                    3                    7                   14                  21                 28

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(c) C. officinalis 500ppm

1000ppm

1500ppm

1                    3                    7                   14                  21                 28

24

Figure 7. Calcification rates over a 28-day span in three different mesocosms (pCO2 of 500ppm, 1000ppm, and 1500ppm) for (a) B. californica, (b) C. tuberculosum, and (c) C. officinalis.

‐5

‐3

‐1

1

3

5

Cal

cifi

cati

on

(μm

ol C

·g-1

·h-1

)

Day

(a) B. californica 500ppm

1000ppm

1500ppm

1                    3                    7                   14                  21                 28

‐5

‐3

‐1

1

3

5

Cal

cifi

cati

on

(μm

ol C

·g-1

·h-1

)

Day

(b) C. tuberculosum

1                    3                    7                   14                  21                 28

‐5

‐3

‐1

1

3

5

Cal

cifi

cati

on

(μm

ol C

·g-1

·h-1

)

Day

(c) C. officinalis

1                    3                    7                   14                  21                 28

25

Table 8. Three-Way PERMANOVA Table of Results for Calcification in 3 Mesocosms for 28 Days With Fixed Factors Day, pCO2 and Species (n = 100). Boldface* Denotes Significant Differences

PERMANOVA Table of Results

Source df SS MS Pseudo-F P (perm) Day 5 49.395 9.879 9.459 0.001* pCO2 2 81.274 40.637 38.912 0.001* Species 2 5.174 2.587 2.477 0.092 DayxpCO2 10 29.424 2.942 2.818 0.008* DayxSpecies 9 18.900 2.100 2.011 0.057 pCO2xSpecies 4 15.121 3.780 3.619 0.015* DayxpCO2xSpecies 17 36.541 2.149 2.058 0.021* Res 50 52.216 1.044

Species differences were seen between C. officinalis and C. tuberculosum, but only in the

500ppm (p = 0.026) and the 1000ppm mesocosms (p = 0.007) where C. tuberculosum had

lower calcification rates. Similar responses to the high pCO2 level in the 1500ppm mesocosm

masked any species-specific responses.Measurements of percent thallus CaCO3 was similar

to measurements of calcification rates in that CaCO3 content decreased with increasing pCO2

(Figure 8). There was an increase from Day 1 to Day 28 in CaCO3 in all specimens in the

500ppm mesocosm (by 0.7%), but decreases in both the 1000ppm (-2.38%) and the 1500ppm

mesocosms (-1.93%). C. tuberculosum also had the highest percent CaCO3 throughout the

experiment (p = 0.001), averaging 87.73±1.77% at 500ppm, 86.61±0.67% at 1000ppm and

85.49±0.93% at 1500ppm. Percent CaCO3 was the highest for all species in the 1000ppm

mesocosm, averaging 86.56±0.73% CaCO3 on day 1 and the 84.50±1.15% at the end of the

experiment. However, despite the higher overall percent CaCO3 at 1000ppm, algae in

seawater at an elevated pCO2 had less CaCO3 at the end of the four weeks than at the

beginning.

FIELD EXPERIMENTS

Photosynthesis rates responded similarly to elevated pCO2 during both the Immediate

Incubations and the Acclimated Incubations. All incubations resulted in higher

photosynthesis under elevated pCO2, both for algae incubated directly after collection (p =

0.014) and those incubated after a two-week acclimation period (p = 0.02). Specifically,

26

Figure 8. Average percent CaCO3 measured across species on days 1 and 28 in the three mesocosms (500ppm, 100ppm & 1500ppm) (* denotes significant differences, p = 0.028, 0.006).

during the Immediate Incubations, rates in B. californica increased under elevated pCO2 by

39%, in C. tuberculosum by 66%, and in C. officinalis by 41% (Figure 9). During the

Acclimated Incubations, however, the increase in carbon uptake under elevated pCO2 was

slightly lower than the Immediate Incubations, although differences were not statistically

significant (p = 0.78, Table 9). During the Acclimated Incubations, rates in B. californica

increased under elevated pCO2 by 20%, in C. tuberculosum by 36%, and in C. officinalis by

43%. These increases in photosynthesis suggest a carbon-fertilization effect. Species

differences also accounted for some variability for both incubations (p = 0.002). B.

californica had consistently higher rates of photosynthesis, while C. tuberculosum had the

lowest rates in both treatments. B. californica and C. tuberculosum also both exhibited a

decline in the percent increase of photosynthesis rates under elevated pCO2 in the Acclimated

Incubations. C. officinalis, however, actually had a greater up-regulation of photosynthesis

under elevated pCO2 during the Acclimated Incubations.

Calcification rates were negatively affected by the increase in pCO2 in both the

Immediate and the Acclimated incubations (p = 0.001, see above Figure 9, Table 10). During

the Immediate Incubations, calcification rates decreased under elevated pCO2 and there were

76.0

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28

Table 9. Three-Way PERMANOVA Table of Results for TIC Under Two Treatments (Immediate and Acclimated) and Two CO2 Levels (500ppm and 1000ppm) for All Three Species. Boldface * Denotes Significant Differences

PERMANOVA Table of Results

Source df SS MS Pseudo-F P (perm) Species (Sp) 2 166.920 83.462 9.733 0.002*pCO2 (pC) 1 108.540 108.540 12.658 0.003*Treatment (Tr) 1 0.695 0.695 0.081 0.780 SpxpC 2 0.671 0.336 0.039 0.971 SpxTr 2 21.433 10.716 1.249 0.313 pCxTr 1 5.575 5.575 0.650 0.424 SpxpCxTr 2 3.820 1.910 0.223 0.816 Res 59 505.920 8.575

Table 10. Three-Way PERMANOVA Table of Results for Calcification Rates Under Two Treatments (Immediate and Acclimated) and Two CO2 Levels (500ppm and 1000ppm) for All Three Species. Boldface* Denotes Significant Differences

PERMANOVA Table of Results

Source df SS MS Pseudo-F P(perm) Species (Sp) 2 5.3604 2.6802 2.8322 0.072 pCO2 (pC) 1 64.786 64.786 68.46 0.001*Treatment (Tr) 1 1.592 1.592 1.6823 0.185 SpxpC 2 8.2505 4.1252 4.3591 0.012*SpxTr 2 1.2498 0.62489 0.66032 0.509 pCxTr 1 14.064 14.064 14.861 0.001*SpxpCxTr 2 1.2553 0.62763 0.66321 0.517 Res 59 55.834 0.94634

29

no significant differences in calcification among species (p = 0.072). Averaged among all

three species, algae incubated at 1000ppm exhibited 44% lower calcification rates.

Specifically, average calcification under ambient levels of pCO2 was 2.263±0.183 μmol C·g-

1·h-1 but decreased to 1.267±0.251 μmol C·g-1·h-1 under 1000ppm. However, the acclimated

incubations resulted in calcification rates that were much lower under elevated pCO2 than

those measured in the immediate incubations. Averaged across species, rates declined under

elevated pCO2 by almost 81%, decreasing from 3.480±0.258 μmol C·g-1·h-1 at 500ppm to

0.676±0.276 μmol C·g-1·h-1 at 1000ppm. However, calcification under ambient pCO2 in the

Acclimated Incubations was much higher than calcification under ambient pCO2 in the

Immediate Incubations (p = 0.001), such that calcification rates under elevated pCO2 did not

differ between treatments (p = 0.154). Differences in calcification rates between species for

the Acclimated Incubations confirmed C. tuberculosum did not respond as negatively as the

other two. It maintained lower calcification rates at ambient pCO2, averaging 2.422±0.446

μmol C·g-1·h-1, and endured a 50% decrease at 1000ppm to 1.233±0.720 μmol C·g-1·h-1.

30

DISCUSSION

This study identified several patterns in the photosynthesis and calcification responses

of coralline algae to elevated pCO2. Further, it is clear from these experiments that duration

of exposure to elevated pCO2 is extremely important to consider. The short-term laboratory

incubations for these three species of coralline algae resulted in no significant differences in

photosynthesis under elevated pCO2. However, when the algae were exposed to elevated

pCO2 for longer time periods, differences between the mesocosms became apparent, such

that algae in the 1500ppm mesocosm exhibited significantly lower rates of photosynthesis

and calcification. However, the algae in the 1000ppm mesocosm did not respond differently

from those under ambient pCO2, suggesting that 1000ppm is not as physiologically taxing as

is often predicted. In fact, Feely et al. (2008) have measured pCO2 levels over 1000ppm

along the coast of California due to upwelling events. Other recently published data indicate

that some of the larger fluctuations in pH occur in southern California by as much as 0.259

units with a fairly high rate of change (Hofmann et al., 2011). Diel fluctuations in pH levels

have also been documented within the Point Loma kelp forest (Edwards, unpublished data)

as respiration rates increase pCO2 levels in the kelp forests at night and photosynthesis

reduces these levels during the day. These studies strongly suggest that 1000ppm is not

outside the levels that these algae already experience. This was confirmed in the in situ

incubations during which photosynthesis rates did not increase as much at 1000ppm after the

algae had been exposed to elevated pCO2 for two weeks in the laboratory mesocosms. Such

results indicate that algae are acclimating to higher levels of pCO2 and are able to do so

within a short period of time. In contrast, calcification rates did not show any signs of

acclimating to elevated pCO2. Generally speaking, all experiments demonstrated negative

effects of high pCO2 on calcification. Each experiment exhibited varying degrees of

reductions in calcification, but the underlying outcome of each experiment is that

calcification heavily relies on seawater chemistry. Specifically, an increase in pCO2 results in

an increase in OA and a decrease in the available CO32-, and despite any other factors that

may favor calcification, creating CaCO3 is difficult if CO32- is reduced (Borowitzka, 1981;

Borowitzka & Larkum, 1987).

31

One significant cause of variation in all of the experiments was interspecific

differences in the way the species responded to elevated pCO2 in regards to irradiance.

Lower photosynthesis rates coincided with lower saturation irradiances under elevated pCO2

during the 2.5-hour incubations and the mesocosm experiment. Photosynthetic saturation at

lower irradiances may explain why the algae incubated at elevated pCO2 levels did not reach

the photosynthesis rates that were observed under ambient pCO2. Additionally, these three

species were collected on the benthos at low levels of irradiance, which suggests they are

already light-limited (Alaback, 1982; Hart & Chen, 2006). However, the spatial distribution

of the three target species may explain some of the variation in photosynthetic responses to

elevated CO2. B. californica did not exhibit significant differences in its maximum

photosynthesis, saturation irradiance, or photosynthetic efficiency. This may be a result of

varying depth and irradiance preferences. C. tuberculosum and C. officinalis are found on

rocky ledges and in brighter clearings within the kelp forest. In fact, Corallina spp. and C.

tuberculosum are often found in intertidal areas along the Southern California coast (Abbott

& Hollenberg, 1976), indicating that they may be more light-adapted species. B. californica,

on the other hand, is found in the darker recesses of the kelp forest, often underneath dense

Macrocystis pyrifera canopies and rocky overhangs. Such shading limits the available

irradiance but does not necessarily mean that it decreases its primary production (Alaback,

1982; Miller et al., 2012). Lower irradiances would necessitate the ability to be as efficient as

possible during photosynthesis. Consequently, the differences in photosynthesis that were

seen in the more light-adapted species (lower maximum photosynthesis and saturation

irradiance and higher efficiency under lower irradiances) would not be expected for B.

californica. Irradiance preferences may have come in to play as well during the four-week

mesocosm experiments, during which B. californica suffered 100% mortality, yet C.

tuberculosum and C. officinalis survived throughout the entire experiment. Ultimately these

results demonstrate that differences in photosynthetic capabilities under elevated pCO2 may

be influenced by their adaptations to specific irradiance and depth microhabitat preferences.

In the same way, calcification rates increased as irradiance increased, up to the point where

photosynthesis was light saturated. This is consistent with other literature on calcification

(Chalker, 1981; Zou & Gao, 2009; Gao & Zheng, 2010). This saturation indicates that

32

calcification is also light-limited at low irradiances and with the added constraints under

elevated pCO2, more energy will be necessary to maintain and build CaCO3 structures.

While duration and irradiance are strong drivers of the differences in photosynthesis

and calcification, the physiological mechanisms within the algae themselves may result in

varying responses to elevated pCO2. The decreases in photosynthesis rates contrast many

studies that show carbon-fertilization effects on photosynthesizing plants and non-calcifying

algae (Ainsworth & Long, 2005; Spijkerman, 2008; Porzio et al., 2011). This suggests these

coralline species may already be carbon-saturated at current pCO2. Differences in

photosynthetic rates may be due to the fact that many coralline algae likely have carbon-

concentration mechanisms (CCMs) (Borowitzka, 1981; Gao et al., 1993; Hepburn et al.,

2011). CCMs use energy to allow organisms to concentrate inorganic carbon in their cells at

concentrations higher than the surrounding environment for use in photosynthesis

(Reinfelder, 2011). Such high intracellular levels of carbon suggest they are already saturated

at current pCO2 levels, so an increase in pCO2 should not evoke an immediate photosynthetic

response. However, CCMs can respond to changes of many abiotic factors, including

temperature, PAR, nutrients, and UV irradiation (Raven et al., 2011). Since the three species

in this study exhibit higher photosynthesis rates under elevated pCO2 during the field

incubations, it may be that they possess low-efficiency CCMs. Several processes, including

the slow diffusion rate of HCO3- into algal cells and the dehydration of HCO3

- into the usable

form of CO2, require extra time and energy, and some CCMs are more efficient than others

(Reinfelder, 2011). Low-efficiency CCMs would allow these species to maintain a small pool

of intracellular CO2, but would also benefit from a higher concentration of extracellular CO2.

In this study, these low-efficiency CCMs would be able to up-regulate photosynthesis as

pCO2 increased to 1000ppm. Algae with CCMs are also light-limited under present-day

conditions (Hepburn et al., 2011), indicating that at low light levels (such as those in the

Point Loma kelp forest), algae with diffusive uptake of CO2 may have the potential

outcompete those with CCMs. Many of the fleshy red turf algae in Point Loma do not use

CCMs, so diffusive uptake of CO2 may allow for proliferation of these turf algae over the

coralline algae (Hepburn et al., 2011). Such advantages could cause large-scale species

composition changes in the benthic habitat, influencing invertebrate recruitment, substrate

stability, and herbivory. Additionally, algae may down-regulate CCMs and switch to passive

33

CO2 uptake in the future under elevated pCO2 (Hurd et al., 2009; Raven et al., 2011). CCMs

may also play a role in calcification as CCMs are an energy-requiring process. In algae where

the necessity of CCMs may decrease, the energy that was previously used to sequester

carbon may be used in the calcification process. This would allow these algae to survive

under pCO2 levels where we would expect the low saturation point of high-Mg calcite to

cause net dissolution of CaCO3. C. tuberculosum exhibited the lowest average photosynthetic

rates and calcification rates, indicating that it is a species that operates physiologically at a

low-energy level and is not drastically affected by changes in light and pCO2. Ultimately, the

physiological machinery that operates photosynthesis and calcification responds fairly

quickly to changing pCO2, such that the pCO2 levels predicted for the near future may not be

as detrimental as previously thought.

The mesocosm system used during the acclimation experiments would benefit from

improvements to better control for nutrient levels, oxygen concentrations, and other

mesocosm artifacts. The declines in photosynthesis during the four-week acclimation period

in all three species were likely due to mesocosm limitations. For example, nutrient levels

were difficult to maintain, despite fairly regular additions of fresh seawater. By the end of the

four-week period, nitrate had reached very low levels, which potentially had negative effects

on photosynthesis. However, photosynthesis did not cease completely and mortality in C.

tuberculosum and C. officinalis did not occur (only in B. californica). This suggests that even

with the stress imposed by the mesocosms and pCO2, algae were still able to maintain net

photosynthesis in all three mesocosms for four weeks. As this study was as much a

methodological experiment as it was a physiological experiment, results suggest this

mesocosm design was sufficient to answer the research questions at hand and allow for the

logical transition to field experiments allowing for exposure to wave energy and natural light

levels, including light-flecking. Mesocosm studies in the future will benefit from assessing

species interactions, including competition and herbivory, to determine if the ecological role

of coralline algae is negatively affected by OA despite negligible differences in

photosynthesis and calcification. Additionally, increases in pCO2 will not happen overnight,

and thus it is reasonable to use the conclusions from the longer-term experiments to better

predict how these species will respond to climate change. Experiments designed for

predictive purposes cannot truly assess changes at the current rate of increase in pCO2. This

34

being said, all three experiments can tell us something about the phenotypic plasticity of

coralline algae to temporal changes in pCO2 and can be useful for future experimental

designs.

This research strongly suggests that future pCO2 levels will have negative effects on

photosynthesis and calcification of coralline algae. This is one of the first studies to examine

the effects on a multi-species suite of calcifying organisms. It is important to understand the

negative effects of elevated pCO2 on a group of organisms, particularly those that constitute

an entire guild within the kelp forest. There were differences in photosynthesis and

calcification rates between species, but results for the dominant coralline algae in Point Loma

suggest the health and species richness of these algae will decline. It seems that

photosynthesis and calcification are tied to each other by the energy requirements of the

species. Differences among species will play important roles in the response to elevated

pCO2 as well, for example coralline algae operating with low rates of photosynthesis and

calcification may be less affected by pCO2 changes. However, overall reductions in

photosynthesis and calcification among coralline algae as a result of elevated pCO2 will

likely cause a decrease in productivity and abundance. This could have adverse effects on

invertebrate recruitment, herbivory by grazers, susceptibility to breakage, and substrate

stability and reef structure. Future studies will need to assess the ramifications of lower

CaCO3 content on fragility, epibionts, and benthic composition and to assess the validity of a

reduced role of coralline algae in kelp forests under elevated pCO2.

35

ACKNOWLEDGEMENTS

There are a number of people I would like to acknowledge that made this project

possible. First and foremost I’d like to thank my advisor, Matt Edwards, for putting so much

time and expertise into my project so that I was able to be a part of a new line of research

within the lab. The San Diego State University BEERPIGS were also invaluable, particularly

Andrea Pesce, who was there by my side every step of the way and helped me keep my

sanity and motivation over the past three years. Kyle Van Meter and Kayla Kleinsasser were

also extremely helpful SDSU undergraduate students who provided much assistance both in

the lab and the field. Several other students in the Ecology department, namely Renee

Dolecal, Ryan Driscoll, and Kim Miller were also influential throughout this process.

I would also like to acknowledge the funding I received over the last three years,

including the Council on Ocean Affairs, Science, and Technology (COAST) Student Award,

the Mabel Myers Memorial Scholarship, the Jordan D. Covin Memorial Scholarship, and

several travel awards that allowed me to present my research at scientific meetings.

36

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APPENDIX

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Figure 10. Measurements of ambient CO2 levels in the Point Loma kelp forest from seawater collected at 10m depth on 9/24/11 (samples 1, 2, 3), 9/26/11 (4, 5, 6), 10/8/11 (7, 8, 9) and 10/10/11 (10, 11, 12).

0

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1 2 3 4 5 6 7 8 9 10 11 12

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