Understanding individual and combined effects of ocean
acidification, coastal runoff and warming on marine calcifying
organisms on tropical coral reefs
Nikolas Vogel
Dissertation submitted in fulfillment of the requirements for the degree of
Doctor of Natural Science
-Dr. rer. nat.Faculty of Biology/Chemistry
University of Bremen
January 2015
Statement of originality
I, Nikolas Vogel hereby certify that I am the sole author of this thesis and any assistance received is fully
acknowledged. To the best of my knowledge this thesis does not contain any material, which has been
previously published or written by others. Any content of other work utilized in this thesis is cited in
accordance with the standard referencing procedures.
Date:
Signed:
This thesis was conceived and written at the Australian Institute of Marine Science (AIMS) in Townsville,
Australia and at the Leibniz Center for Tropical Marine Ecology (ZMT) in Bremen, Germany between
January 2012 and January 2015. This thesis was conducted under the supervision of Dr. Sven Uthicke
(AIMS) and Prof. Dr. Christian Wild (ZMT). Research for this thesis was funded by the Australian Institute of Marine Science and was conducted with the support of funding from the Australian Government’s
National Environmental Research Program (NERP).
First Examiner:
Prof. Dr. Christian Wild
Leibniz Center for Tropical Marine Ecology, Bremen
Second Examiner:
Prof. Dr. Kai Bischof
University of Bremen
Additional Examiners:
Prof. Dr. Claudio Richter
Alfred Wegener Institute, Bremerhaven
Dr. Mirta Teichberg
Leibniz Center for Tropical Marine Ecology, Bremen
Student Members:
PhD Student Ines Stuhldreier
Leibniz Center for Tropical Marine Ecology, Bremen
Master Student Hagen Buck-Wiese
University of Bremen
Date of Defense:
23.03.2015
Acknowledgments
Many thanks to Sven Uthicke who invited me to AIMS in the first place and gave me the opportunity
to set the course to conduct this thesis. His great supervision on professional and friendly basis made
me come back to AIMS several times and contributed to the lovely environment that I have experienced
in our working group. Thanks for giving me the opportunity to experience places that I would not have
seen otherwise. It has been a great time.
Many thanks to Christian Wild, who offered to be my doctoral thesis supervisor and supported the
idea to conduct this thesis as collaboration between the AIMS and the ZMT. His great supervision and
impulsion considerably helped me to conduct this thesis. Thanks for inviting me to the ZMT and to the
welcoming and friendly Coral Reef Ecology working group.
I am very grateful to Kai Bischof who offered to be the second examiner of this thesis. Many thanks to
Claudio Richter, Mirta Teichberg, Ines Stuhldreier and Hagen Buck-Wiese to be part of the examination
committee.
Thanks to Sam Noonan, who contributed with corrections to the Abstract, General Introduction and
General Discussion of this thesis.
I thank the Water Quality Group at AIMS, Katharina Fabricius, Sam Noonan, Yan Ow, John Pfitzner,
Britta Schaffelke, Julia Strahl, Lindsay Trott, Sven Uthicke and Irena Zagorskis, it has been a privilege
working with you.
Gratitude to my co-authors Line Bay, Neal Cantin, Catherine Collier, Katharina Fabricius, Florita Flores,
Paulina Kaniewska, Friedrich Meyer, Sam Noonan, Yan Ow, Julia Strahl, Sven Uthicke, Christian Wild
and all my other colleagues in Australia and Germany, it has been a pleasure working with you.
Many thanks to Julia and Sam and all my other friends in Australia who made me feel like home. It has
been a sensational time and I am grateful for all the fantastic things that we have done together. Many
thanks also to all my friends in Germany, who reminded me on home and gave me a great time when I
was visiting Germany.
Thank you Lisa for your support, motivation and understanding. I am glad that we came to the right
decision in the beginning.
Last but not least, I want to thank my family, in particular my mother and father, who always supported
my ideas and dreams. I love you and I am very lucky to have you.
Table of contents
1
Zusammenfassung der Doktorarbeit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
Thesis abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
General introduction
4
1.1
Coral reef ecosystems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
1.2
Coral reefs: sociological and economic importance . . . . . . . . . . . . . . . . . . . .
5
1.3
Global threats to coral reefs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
1.3.1
Ocean acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
1.3.2
Ocean warming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
1.4
2
Local threats to coral reefs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.4.1
Coastal runoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.4.2
Other local stressors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.5
Combinations of global and local stressors . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.6
Knowledge gaps & research questions . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.7
Experimental species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.8
Introduction to study sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1.9
Publication outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Calcification and photobiology in symbiont-bearing benthic foraminifera and responses to
a high CO2 environment
34
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3
2.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.3
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.4
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Calcareous green alga Halimeda tolerates ocean acidification conditions at tropical carbon
dioxide seeps
60
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.2
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.3
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.4
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
i
4
Decreased light availability can amplify negative impacts of ocean acidification on calcifying coral reef organisms
86
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
5
4.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4.2
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
4.3
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
4.4
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Effects of elevated dissolved inorganic carbon and nitrogen on the physiology of scleractinian corals and calcareous macroalgae under ocean acidification and eutrophication conditions
112
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
6
5.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
5.2
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
5.3
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
5.4
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Interactive effects of ocean acidification and warming on coral reef associated epilithic algal
communities under past, present and future ocean conditions
140
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
7
6.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
6.2
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
6.3
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
6.4
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
General Discussion
165
7.1
Responses of coral reef organisms to ocean acidification . . . . . . . . . . . . . . . . . 165
7.2
Responses of coral reef organisms to combinations of ocean acidification and decreased
light availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
7.3
Responses of coral reef organisms to combinations of elevated dissolved inorganic carbon and nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
7.4
Responses of coral reef organisms and communities to past and future OA and OW
conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
7.5
Ecological implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
7.6
Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
ii
Appendix
181
Abbreviations and glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
List of tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
List of figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
iii
Zusammenfassung der Doktorarbeit
Anthropogen verursachte Treibhausgas-Emissionen führen zu zwei wesentlichen Umweltveränderungen von globalem Ausmaß für Korallenriffe: Ozeanversauerung (OA) und Ozeanerwärmung (OW).
Zusätzlich kann steigender terrestrischer Oberflächenabfluss, welcher Düngemittel, Abwässer, Sedimente und andere Verunreiniger in küstennahe Gebiete zuführt, die Wasserqualität auf lokaler Ebene
verschlechtern. Folglich sind photosynthese-betreibende und kalkbildende Korallenriff-Lebewesen von
OA, OW und küstennahem Oberflächenabfluss betroffen, aber interaktive Auswirkungen dieser Stressfaktoren auf Schlüssel-Lebewesen des Korallenriffs sind weitestgehend unbekannt. Das Ziel dieser Doktorarbeit war es, zu erforschen, wie sich OA einzeln und in Kombination mit OW oder lokalen Stressfaktoren (d. h. verringerter Lichtverfügbarkeit und anorganischer Eutrophierung) auf wichtige kalkbildende
Korallenriff-Lebewesen auswirkt. Eine Reihe von feld- und labor-basierten Experimenten wurde am
Great Barrier Reef und an natürlichen vulkanischen Kohlendioxid-Quellen in Papua Neuguinea durchgeführt. Eine Auswahl von Reaktionsfaktoren, einschließlich Wachstum, Kalzifizierung und Photosynthese, wurden auf der Arten- und Artengemeinschafts-Ebene untersucht. OA zeigte keine negativen
Kurzzeit-Auswirkungen auf drei große benthische Foraminiferen-Arten (Kapitel 2) und Langzeit-Auswirkungen auf mehrere kalkbildende Grünalgen-Arten der Gattung Halimeda (Kapitel 3). OA in Verbindung mit verringerter Lichtverfügbarkeit führte nach kurzer Zeit zu additiven negativen Auswirkungen auf die Koralle Acropora millepora (Kapitel 4), während OA in Verbindung mit Eutrophierung
nach kurzer Zeit keine signifikanten Auswirkungen auf die Korallen Acropora tenuis und Seriatopora
hystrix, sowie auf die kalkbildende Grünalge Halimeda opuntia zeigte (Kapitel 5). Die Kombination aus OA und OW führte in einem Langzeit-Experiment zu verringertem Wachstum und reduzierter
Kalzifizierung von epilithischen Algen-Gemeinschaften, insbesondere von Krustenrotalgen (Kapitel 6).
Die unterschiedliche Sensitivität der untersuchten Arten gegenüber globalen und lokalen Stressfaktoren
deutet auf Veränderungen von Korallenriff-Gemeinschaftsstrukturen in naher Zukunft hin. Verringerte
Lichtverfügbarkeit könnte die negativen Auswirkungen von OA auf Korallen verstärken und dadurch zu
Verschiebungen von Korallen- zu Algen-dominierten Gemeinschaften, bei Riffen, die von küstennahem
Oberflächenabfluss betroffen sind, beitragen. Die wissenschaftlichen Erkenntnisse der vorliegenden
Doktorarbeit deuten darauf hin, dass OA in Verbindung mit anderen Stressfaktoren den Reichtum von
kalkbildenden Lebewesen reduzieren und dadurch die Herstellung von Kalziumkarbonat der Korallenriffe in der Zukunft verringern könnte. Dies könnte zu gemindertem Riffwachstum, erhöhter Zerbrechlichkeit und reduzierter Erholungsfähigkeit nach akuten Störungen führen. Letztlich wird rückläufiges
Riff-Habitat wahrscheinlich zu verringerter Biodiversität führen, was Auswirkungen auf die Menschen
haben könnte, die auf Korallenriffe angewiesen sind, da sie ihre Lebensgrundlage bilden.
1
Thesis abstract
Anthropogenically induced greenhouse gas emissions result in two major environmental changes on
the global scale for coral reefs: ocean acidification (OA) and ocean warming (OW). Additionally, increasing levels of terrestrial runoff, that introduce fertilizer, sewage, sediments and other contaminants
into coastal areas, can decrease water quality on the local scale. Consequently, photosynthesizing and
calcifying coral reef organisms are affected by OA, OW and coastal runoff, but knowledge about the
interactive effects of these stressors on key coral reef organisms is scarce. The aim of this thesis was
to investigate how OA individually, and in combination with OW or local stressors (i.e. decreased light
availability and inorganic eutrophication), affects important calcifying coral reef organisms. A series of
field- and laboratory-based experiments were conducted on the Great Barrier Reef and at natural volcanic carbon dioxide seeps in Papua New Guinea. A range of response parameters, including growth,
calcification and photosynthesis, were investigated at the species and community level. OA showed no
negative impact on three large benthic foraminiferal species in the short term (Chapter 2) and several
calcifying green algae species of the genus Halimeda in the long term (Chapter 3). OA combined with
decreased light availability resulted in additive negative effects on the coral Acropora millepora in the
short term (Chapter 4), while OA combined with inorganic eutrophication did not exhibit any significant effects on the corals Acropora tenuis and Seriatopora hystrix and on the calcifying green alga
Halimeda opuntia in the short term (Chapter 5). In the long term, the combination of OA and OW
resulted in decreased growth and calcification of epilithic algal communities, particularly in crustose
coralline red algae (Chapter 6). The different sensitivity of the species investigated to global and local
stressors, suggests that changes will occur in coral reef community structures in the near future. Reduced light availability may amplify negative effects of OA on corals and thereby contribute to shifts
from coral to algae dominated communities on reefs affected by coastal runoff. The scientific findings
of the present thesis indicate that OA in combination with other stressors may reduce the abundance
of calcifying organisms and thus lower the calcium carbonate production on coral reefs in the future.
This may lead to reduced reef growth, increased brittleness and reduced recovery potential after acute
disturbances. Ultimately, declining reef habitat will likely lead to a reduction in biodiversity and may
thus have implications on the people, who are dependent on coral reefs for their livelihood.
2
Chapter 1
General introduction
1.1 Coral reef ecosystems
Coral reef ecosystems are based upon living framework builders, which particularly consist of scleractinian (stony) corals (Spalding et al. 2001). However, other calcifying organisms, such as coralline
algae, mollusks and many invertebrate species, also contribute to the ‘cementation’ and structural complexity of coral reefs (Fagerstrom 1987; Chisholm 2000). Reef-building organisms are able to secret
calcium carbonate (CaCO3 ). They precipitate CaCO3 as skeletons or shells and build layer upon layer
of limestone and by that lay the structural foundation for coral reefs. The abiotic factors temperature,
light availability and aragonite saturation determine the global distribution of coral reefs and hence are
considered as ‘first-order-determinants’ (Kleypas et al. 1999). On a local scale other factors, such as
wave exposure, storm frequency or biodiversity, limit coral reef development and are thus considered
‘second-order-determinants’ (Kleypas et al. 1999). The building of coral reefs is a constant and ongoing
process in which reef-building as well as reef-eroding organisms are involved. Moreover, disturbances,
such as storms and other extreme weather events, lead to alterations of coral reefs on a regular basis.
According to the non-equilibrium hypothesis an intermediate frequency of disturbances is needed to sustain the high biodiversity found on coral reefs. However, as the frequency declines, or in other words if
disturbances become chronic, diversity will decline (Connell 1978). Tropical coral reefs occur at water
depths less than 100 m in tropical regions and many coral reef organisms rely on sunlight to conduct
photosynthesis for autotrophic nutrition. With an estimated global area of 255,000 km2 (Spalding and
Grenfell 1997; Spalding et al. 2001) coral reefs cover less than 0.1% of the world’s oceans and less than
1.2% of the continental shelf area (Spalding et al. 2001). At the same time, they provide habitat for about
93,000 (34%) of currently 274,000 described marine species (Reaka-Kudla et al. 1996). Thus, similar to
rainforests on land coral, reefs are the ocean’s ‘biodiversity hotspots’ and are presumed to host at least
950,000 species in total with estimations ranging from 600,000 to 9 million species (Reaka-Kudla et al.
4
1 - General introduction
1996). This would also suggest that only ∼10% of all coral reef species have been currently described.
Tropical coral reefs are among the most productive ecosystems in the world (Sorokin 1993). Gross
primary productivity of coral reefs is exceptionally high compared with other marine and terrestrial
ecosystems (Odum and Odum 1955; Lewis 1977). This is often considered as a paradox since tropical waters are oligotrophic meaning they are naturally poor in nutrients (Muscatine and Porter 1977;
Crossland 1983). The high productivity can only be sustained by a complex and efficient mechanism of
nutrient recycling within the coral reef ecosystem (Muscatine and Porter 1977).
1.2 Coral reefs: sociological and economic importance
Coral reefs play a critical role in human societies. Approximately 500 million people are dependent on
coral reefs for food, coastal protection, building materials and income (Wilkinson 2008). This includes
30 million people, who are completely dependent on coral reefs for their livelihood (Wilkinson 2008).
The physical structure of coral reefs provides habitat and protection for reef fish and pelagic fish juveniles as well as many invertebrate species. Thus, coral reef organisms are an important food source
for people inhabiting coastal areas in tropical regions and significantly contribute to the sociological
structure of island communities (Wilkinson and Buddemeier 1994). Moreover, coral reefs protect the
coast line from waves and erosion, and calcifying coral reef organisms produce the reef and reef-sand
which constitute the fundament of coral islands (Wilkinson and Buddemeier 1994). They also provide
building material, such as sand and limestone rocks, utilized for the construction of houses. In addition, many people inhabiting coastal areas in tropical regions rely on coral reefs as source of income by
offering recreational activities for tourists or by exporting goods produced by the reefs (Wilkinson and
Buddemeier 1994). Moreover, bioactive compounds of many of the coral reef organisms are essential
for conventional medicine and many more have, yet unknown, pharmaceutical potential (Moberg and
Folke 1999). The possible net benefit streams per year of coral reefs worldwide are estimated with 29.8
billion US$ in total, including 12.7 billion US$ for the Southeast Asia region and 6.3 billion US$ for
Australia (Cesar et al. 2003). Not least, coral reefs have a priceless aesthetic value. All-embracing, coral
reefs have a tremendous importance for humans: socially, economically as well as culturally.
1.3 Global threats to coral reefs
Worldwide coral reefs are in decline (Bellwood et al. 2004; Hughes et al. 2003). In particular, anthropogenically induced environmental changes on both global and local scales put pressures on coral reef
ecosystems and lead to the observed declines (De’ath et al. 2012; Wilkinson 2008). Global stressors are
environmental factors which affect coral reefs on a global scale. Yet, regional pollution (air pollution
5
1 - General introduction
in particular) is responsible for the changes happening worldwide. Global stressors are generally more
difficult to manage than local stressors, since the consequences are not necessarily seen in regions where
the pollution takes place, which makes it easier for polluters to disclaim responsibility. Ironically, often
small island countries, which are the least responsible for the causation of these problems, suffer the
most under the associated environmental changes (Wilkinson 2008).
1.3.1
Ocean acidification
Anthropogenic carbon dioxide (CO2 ) emissions from burning fossil fuels, cement production and fire
clearance are increasing the CO2 partial pressure (pCO2 ) in the atmosphere. Latest projections of the
Intergovernmental Panel on Climate Change (IPCC 2013) assume global mean pCO2 will rise twoor three-fold, compared to pre-industrial levels (∼280 µatm), within the present century due to anthropogenic greenhouse gas (GHG) releases (Fig. 1.1). Four possible ‘representative concentration
pathways’ (RCP2.6, RCP4.5, RCP6.0 and RCP8.5) have been suggested to estimate future atmospheric
GHG concentrations (Moss et al. 2010). The different RCPs include the possibilities from immediate
and drastic reduction of GHG emissions (RCP2.6) to business-as-usual GHG emissions (RCP8.5). Depending on the RCP followed, future atmospheric CO2 concentrations are predicted to reach 420-936
µatm (RCP2.6-RCP8.5, respectively) by the year 2100 (Ciais et al. 2013).
Atmospheric CO2
2000
RCP 8.5
RCP 6.0
RCP 4.5
RCP 2.6
µatm
1500
1000
500
2000
2050
2100
2150
2200
2250
2300
Year
Figure 1.1: Trends in atmospheric CO2 concentrations with long-term projections following RCP2.6RCP8.5 (modified from IPCC 2013)
Atmospheric CO2 is in constant exchange with the surface ocean, and additional CO2 in the atmosphere leads to an increase of CO2 in the ocean. By now, the world’s oceans already took up one third
of the anthropogenic CO2 which has been introduced to the atmosphere (Sabine et al. 2004). When CO2
diffuses into seawater the reaction of both is described in the following series of equilibria:
Equation 1.1-1.4 Equilibria in the inorganic system H2 O−CO2 −CaCO3 , whereas g = gaseous, aq =
aqueous and l = liquid
6
1 - General introduction
−
CO2 (g) −
↽
−⇀
− CO2 (aq)
(1.1)
−−
CO2 (aq) + H2 O(l) ↽
−⇀
− H2 CO3 (aq)
(1.2)
+
−
−
H2 CO3 (aq) −
↽
−⇀
− H (aq) + HCO3 (aq)
(1.3)
+
2−
−−
HCO3− (aq) ↽
−⇀
− H (aq) + CO3 (aq)
(1.4)
(Equation 1.1) Atmospheric CO2 (g) diffuses into seawater and appears as dissolved CO2 (aq) in
aqueous solution. (Equation 1.2) Dissolved CO2 reacts with H2 O forming carbonic acid (H2 CO3 ).
(Equation 1.3) In the following reaction, H2 CO3 dissociates into hydrogen ions (H+ , first ionization)
plus bicarbonate ions (HCO3– ). (Equation 1.4) In the next step, HCO3– dissociates into an H+ ion
(second ionization) and a carbonate ion (CO32– ). Additional H+ ions decrease the pH of the seawater
(since pH = −log10 [H+ ]) making the water more acidic. This process was termed ‘ocean acidification’
(OA), and since the industrial revolution a reduction of 0.1 units has already occurred. According to
projections of the IPCC (2013), future oceans will experience a further pH reduction of 0.2-0.3 units by
the year 2100 (following RCP2.6-RCP8.5, respectively) (Fig. 1.2).
Global ocean surface pH
pHtotal
8.20
8.00
7.80
7.60
1850
historical
RCP2.6
RCP4.5
RCP6.0
RCP8.5
1900
1950
2000
2050
2100
Year
Figure 1.2: Trends in global ocean surface pH with long-term projections following RCP2.6-RCP8.5
(modified from IPCC 2013)
In turn, a decrease in pH leads to a shift of the equilibria in the seawater carbonate system to the left,
from CO32– towards HCO3– (see Equation 1.4), which reduces the CO32– ion concentration and thus
leads to a reduction of the calcium carbonate saturation state (Ω, Equation 1.5). Due to a decrease in Ω
many marine calcifying organisms, such as corals, mollusks, calcareous algae and foraminifera, become
impaired in building up their calcium carbonate (CaCO3 ) skeletons (Gattuso et al. 1998; Langdon et al.
2000; Orr et al. 2005; Ries et al. 2009).
7
1 - General introduction
Equation 1.5 Calcium carbonate saturation state (Ω), whereas X is the metal involved in calcification
(Mg or Ca) and Ks is the solubility constant of the carbonate mineral (aragonite, high Mg-, or low
Mg-calcite)
Ω−[X2+ ] × [CO32− ] × Ks−1
(1.5)
Calcifying organisms can deposit CaCO3 in different naturally occurring polymorphs. The three
main minerals of CaCO3 are calcite and aragonite as well as calcite with high magnesium content (highMg-calcite). Calcite is the most stable, aragonite is less stable and high-Mg-calcite is the most soluble
form of CaCO3 . Thus, organisms depositing CaCO3 in form of aragonite and high-Mg-calcite are assumed to be the most vulnerable to OA (Kuffner et al. 2007).
Photosynthesis and respiration of organisms play a crucial role in calcification (Equation 1.6-1.8).
By taking up CO2 during photosynthesis, the consequential increase in Ω alters the carbonate chemistry
of the intra- and extracellular environment and thus facilitates the deposition of CaCO3 . Vice versa, during darkness respiratory CO2 release reduces Ω and thus alters the organisms’ environment to conditions
unfavorable for calcification.
Equation 1.6-1.8 Chemical reaction of photosynthesis, respiration and calcification
CO2 + H2 O −−→ CH2 O + O2
(1.6)
CH2 O + O2 −−→ CO2 + H2 O
(1.7)
Ca2+ + 2 HCO3− −−→ CaCO3 + CO2 + H2 O
(1.8)
Besides effects on growth and calcification rates, OA can have negative impacts on other physiological parameters. For instance, OA can have impacts on responses of fish towards olfactory cues impairing
their predator and habitat recognition and thus reducing their chance of survival in future environments
(Munday et al. 2009; Dixson et al. 2010). In addition, coral recruitment can be implicated as a consequence of disrupted larval-algal interactions due to OA (Doropoulos et al. 2012). Moreover, OA can
have negative impacts on egg fertilization and early development of marine invertebrates (Kurihara and
Shirayama 2004; Havenhand et al. 2008; Uthicke et al. 2013).
Nevertheless, there are also studies that observed several calcifying species are not impacted, but
show resilience (Comeau et al. 2014) or even benefit under future OA conditions (Fabricius et al. 2011).
One common example is seagrass which is assumed to be limited in dissolved inorganic carbon (DIC)
concentrations under ambient pCO2 conditions. Seagrass will likely overcome this limitation under
8
1 - General introduction
future OA (i.e. elevated DIC) environments by increasing photosynthetic capacity and performance in
comparison with calcareous epiphytes (Fabricius et al. 2011). Thus, the emerging paradigm of OA being
harmful for marine life should be considered with caution, and impacts of OA should be evaluated at the
species level.
1.3.2
Ocean warming
Next to OA, greenhouse gas emissions have another major impact on many marine organisms on the
global scale. Due to the greenhouse effect, the mean global surface temperature is predicted to rise between 1.0 and 3.7 °C depending on the RCP followed (RCP2.6 and RCP8.5, respectively) by the year
2100 (Collins et al. 2013). Part of this heat energy is absorbed by the oceans, which consequently leads
to an increase in sea surface temperatures with highest warming in tropical and subtropical regions. Projections estimate an ocean warming (OW) in the top one hundred meters of about 0.6-2.0 °C (RCP2.6RCP8.5, respectively) (Collins et al. 2013). Seasonal fluctuations in water temperature are natural, and
organisms generally benefit from warmer summer months by increasing their metabolic- and growth
rates (Pörtner 2001). However, many tropical and subtropical species today already live close to their
thermal limits and an additional increase of 2 °C above summer maxima may have deleterious effects
on marine life (Hoegh-Guldberg 1999). With higher frequencies of temperature anomalies bleaching
of corals living in symbiosis with zooxanthellae will become more common (Hoegh-Guldberg 1999;
Hallock et al. 2006). A rise in temperatures of only 1-2 °C disrupts the coral-zooxanthellae symbiosis
and leads to an expulsion of the endolithic algae by the host, or to the loss of pigments by endolithic
algae (Buddemeier and Fautin 1993), leaving the corals bleached and colorless. Similar mechanisms
have been reported in other organisms living in symbiosis with algae such as soft corals, anemones,
giant clams and foraminifera (Buddemeier and Fautin 1993; Hallock et al. 2006; Schmidt et al. 2011).
Bleaching in corals depends on the zooxanthellae type, the coral species and is region specific. Some
species show higher sensitivity towards temperature stress than others, and corals from some regions
show less tolerance than the same species in other regions (Berkelmans and Willis 1999; Baker et al.
2004). Moreover, recovery is possible after minor bleaching events, but without these vital symbionts
and their photosynthetically derived carbon source the host organisms may starve (Hoegh-Guldberg
1999; Harriott 1985). Subsequently, mass bleaching events can lead to complete loss of live corals in
entire regions and indicate substantial alterations to coral reefs in future (Hoegh-Guldberg 1999).
Besides implications on symbioses, OW also affects other physiological responses in a range of
marine organisms. Calcifying organisms show a linear increase in calcification rates with warming seawater temperatures (Lough and Barnes 2000; De’ath et al. 2009). However, studies also show non-linear
responses, leading to declined calcification rates at high temperatures (Cooper et al. 2008; Marshall and
9
1 - General introduction
Clode 2004; Cantin et al. 2010). Thus, interim benefits for calcifiers from OW will be removed under
heat stress and temperature anomalies. In addition, heat stress can have negative implications on photophysiology (Jones et al. 1998; Middlebrook et al. 2010; Schmidt et al. 2011) and metabolism (Pörtner 2001) of coral reef organisms. In turn, physiological stresses compromise host resistance towards
pathogens. Consequently, warmer water promotes the dispersal of diseases and pathogens in marine
biota, leading to increased mortality of coral reef organisms (Harvell et al. 2002; Harvell et al. 1999).
1.4 Local threats to coral reefs
Local stressors affect coral reef ecosystems on a smaller spatial scale and in contrast to global stressors
only occur in confined areas. Often, local stressors occur seasonally or over short temporal scales, but
in some cases they can become chronic, too. Local stressors are generally easier to manage than global
stressors since environmental action plans can be implemented in particular regions in order to improve
the local water quality. Several of these local stressors are interconnected and global environmental
changes may have an influence on local stressors, as well. For instance, OW may lead to an increasing
frequency and destructiveness of tropical cyclones in the future (Knutson et al. 2010; Emanuel 2005)
which affect coral reefs locally. In addition, warming oceans are predicted to increase mean precipitation
and promote heavy precipitation events, particularly in tropical regions, with associated increases in
future coastal runoff (IPCC 2013).
1.4.1
Coastal runoff
The term runoff incorporates a range of land derived substances which get transported into the ocean
by freshwater from precipitation and thereby decrease the water quality on a local scale. In tropical
regions, particularly during the summer months, enhanced precipitation leads to increased land- and
riverine runoff with related implications on near-shore reef communities. For one thing, freshwater from
precipitation acts as a ‘means of transport’ by carrying nutrients, sediments and other contaminants into
the ocean. For another thing, freshwater on its own can alter the seawater quality by reducing salinity.
Rapid decreases in salinity have been shown to cause coral death (Hoegh-Guldberg and Smith 1989) and
extensive mortality after flood events (Goreau 1964; Egana and DiSalvo 1982). Steadily declining water
quality along the populated coast next to the Great Barrier Reef (GBR), since the European settlement,
has been well documented (Wooldridge et al. 2006; Devlin and Brodie 2005; Furnas and Mitchell 2001).
The main impacts of coastal runoff on water quality are seen in elevated dissolved inorganic nutrients
(DIN), increased suspended particulate organic matter (POM), reduced light availability from turbidity
and increased sedimentation (Fabricius 2005).
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1 - General introduction
Agricultural land-use requires fertilizer; mostly nitrogen and phosphorous. The production and use
of fertilizers have steadily increased in the past decades and are predicted to rise in the future (IPCC
2013; Galloway et al. 2008). But only a fraction of the dispensed fertilizer gets taken up by the plants,
while the residuals get carried into the ocean leading to increased DIN concentrations at locations affected by coastal runoff. In combination with organic nutrients, mostly derived from sewage in populated
areas, the seawater gets artificially enriched with nutrients, a term named eutrophication. As outlined
earlier, coral reef organisms are adapted to oligotrophic conditions, and artificially increased DIN concentrations have been shown to affect coral reef ecosystems on the local scale. While corals respond with
an increase in photosynthesis and zooxanthellae numbers (Fabricius 2005), macroalgae are relieved from
nutrient limitation by enhancing their productivity and growth rates under elevated DIN conditions (Pedersen and Borum 1997; Larned 1998; Schaffelke 1999; Schaffelke et al. 2005). Ultimately, prolonged
or repeated exposure to increased DIN may lead to a shift in the community composition from coral to
macroalgae dominated reefs (Bell 1992; Fabricius et al. 2005; Schaffelke et al. 2005; Lapointe 1997).
In addition, elevated DIN concentrations lead to increased chlorophyll in the water column, enhanced
growth of phytoplankton and are responsible for algae blooms in eutrophic areas (Fabricius 2005; Lapointe 1997; Beman et al. 2005). In turn, the latter alterations lead to increased turbidity and decreased
light availability accompanied by negative implications for photosynthesizing coral reef organisms.
Many coral reef organisms rely on sunlight to conduct photosynthesis for autotrophic energy acquisition. Decreased light availability from turbidity reduces their productivity, calcification and subsequently
growth rates (Rogers 1979; Telesnicki and Goldberg 1995; Anthony and Hoegh-Guldberg 2003). Light
requirements are species specific with some being able to sustain positive growth rates in low light conditions, while others are not (Fabricius 2005). Intermediate light availability has been shown to support
the highest species richness by balancing the abundance between fast- and slow-growing species (Cornell and Karlson 2000). Moreover, in the short term organisms are able to acclimatize to reduced light
availability by increasing the size and the amount of chloroplasts (Fabricius 2005). However, eventually
reef development is limited as a function of light availability from > 40 m in clear water to < 4 m in
turbid conditions (Birkeland 1987; Yentsch et al. 2002).
Another component affecting near-shore reef communities on the local scale is sedimentation. Sediments from land erosion get carried into the ocean mainly by rivers. While larger grain sizes are
deposited within the first few kilometers of river catchments, smaller particles can travel over longer
distances (Fabricius 2005). Direct impacts of sedimentation can be seen in reduced photosynthetic efficiency of organisms (Philipp and Fabricius 2003) and increased metabolic costs for the removal of
particles (Telesnicki and Goldberg 1995). Heavy sedimentation can have severe effects on coral reefs,
leading to the death of reef building corals and the subsequent collapse of the reef framework (Risk and
11
1 - General introduction
Edinger 2011; Rogers 1990). Next to these direct effects, sedimentation also affects coral reefs indirectly by increasing turbidity and reducing the light availability with the associated negative impacts as
outlined above.
The limited data available on the effects of POM on coral reef organisms suggests some organisms
may benefit from increased POM availability, while others do not (Fabricius 2005). Recent experiments
indicate calcareous algae experience reduced productivity, while corals showed increased growth under
elevated POM (Meyer et al. pers. comm.). This may further contribute to changes in community
compositions.
1.4.2
Other local stressors
Besides coastal runoff, other disturbances can have severe impacts on coral reef ecosystems on the local
scale. For instance, in the past 27 years the GBR has lost half of its coral cover, and tropical cyclones
accounted for 48% of the observed decline (De’ath et al. 2012). Keeping in mind that the frequency and
intensity of tropical cyclones are predicted to rise with climatic change (Knutson et al. 2010; Emanuel
2005), coral reefs are facing increased structural damage and less time to recover in the future.
The coral eating ‘crown of thorns seastar’ Acanthaster planci accounted for 42% of the coral losses
observed over the past few decades (De’ath et al. 2012). In small numbers, crown of thorns seastars
(COTS) are part of the natural disturbance cycle and contribute to the high biodiversity found on coral
reefs (Sebens 1994). However, large population outbreaks are increasing in frequency with destructive
effects on coral reefs worldwide (Fabricius et al. 2010; Wilkinson 2008). Outbreaks of COTS have
been linked to increasing eutrophication of inshore waters promoting phytoplankton growth which is the
preferred natural food of COTS larvae (Bell 1992; Fabricius et al. 2010).
Industrial developments, such as dredging of shipping channels as well as dredging and blasting
for port development increase sedimentation on coral reefs with associated negative impacts (Risk and
Edinger 2011; Fabricius 2005; Nelson 2009). Recent studies also linked sedimentation from dredging
activity to increased coral disease (Pollock et al. 2014).
Unsustainable tourism, particularly structural damage from anchoring and destructive fishing techniques, such as trawling and dynamite fishing, put additional pressures on local coral reefs (Wilkinson 2008). Furthermore, overfishing can change the ecological communities by decreasing herbivore
abundance and thus facilitating proliferation of algae, causing widespread changes in reef ecosystems
(Anthony et al. 2011; Hughes et al. 2003; Jackson et al. 2001).
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1 - General introduction
1.5 Combinations of global and local stressors
In the coming decades, coral reef ecosystems will be increasingly affected by global and local stressors.
Yet, coral reefs are not only facing one stressor in isolation, but combinations of global and/or local
stressors which may have additive, synergistic or antagonistic effects. While additive effects are characterized by the sum of the individual stressors, synergistic effects result in larger effect sizes than the
sum of individual stressors combined and antagonistic effects result in smaller effects than the sum of
individual stressors (Dunne 2010). The exposure to one stressor may make coral reef organisms more
susceptible to other stressors, leading to synergistic effects.
With increasing atmospheric carbon, tropical coral reef organisms are facing the inevitable combination of both OA and OW, regardless of the presence of local stressors. Studies indicate interactive
effects of OA and OW on calcification, growth, or photophysiology of a vast range of marine organisms
such as scleractinian corals (Edmunds and Moriarty 2012; Reynaud et al. 2003), crustose coralline algae
(Johnson and Carpenter 2012; Anthony et al. 2008; Martin and Gattuso 2009) and benthic foraminifera
(Schmidt et al. 2014) as well as on many life history stages of invertebrates (Gibson et al. 2011).
The presence of local stressors may put additional pressure on coral reef organisms, compromising
their potential to withstand global environmental changes. However, the combined effects of many
stressors are unknown. For instance, few studies have investigated the interactive effects of OA and light
availability on corals and calcareous algae (Comeau et al. 2013; Marubini et al. 2001; Dufault et al.
2013), the interactive effects of OW and eutrophication on benthic foraminifera (Uthicke et al. 2011) as
well as the interactive effects of OW and herbicides on scleractinian corals (Negri et al. 2011).
Furthermore, multifactorial experiments have been conducted on scleractinian corals with more than
two stressors interacting (Comeau et al. 2014; Langdon and Atkinson 2005) including global and local
stressors.
1.6 Knowledge gaps & research questions
Previous studies have particularly concentrated on the responses of scleractinian corals to the global
stressor OA. Many other organism groups, such as calcareous algae, coralline algae or foraminifera
which also play a critical role in coral reef ecosystems, have been less intensely studied and their responses to altered environments are not fully understood. While some coral reef organisms are negatively impacted, others seem to be able to acclimatize to environmental changes. Individual studies also came to contradictory conclusions potentially due to species- and/or region-specific responses.
Moreover, different methodologies implemented in the experimental setup, such as the duration of experiments, or the method to simulate OA conditions, may have a substantial effect on how organisms
13
1 - General introduction
respond to this particular stressor. For instance, in the early years of OA research experimental studies often utilized hydrochloric acid (HCl) in order to reduce seawater pH and Ω. But HCl addition
only reduces pH and Ω without increasing the DIC concentrations (Riebesell et al. 2010), as under future rises of atmospheric pCO2 . Elevated DIC offers higher CO2 and HCO3– availability for auto- and
mixotrophic organisms, which may be DIC limited in their photosynthesis and/or calcification under
present-day environmental conditions and may benefit under elevated DIC concentrations. Thus, it is
important to investigate the effects of stressors at the species level and to use methods in experiments
that closely resemble natural environments, to try to understand future changes to coral reef ecosystems.
Coral reef organisms are increasingly affected by OA and coastal runoff, but the effects of many
of these interactions on coral reef calcifiers are unknown. For instance, photosynthesis is an important
factor in OA research, since photosynthetic CO2 uptake in the light and respiratory CO2 release in
darkness alter the seawater carbonate chemistry internally and externally. Photosynthesis may buffer
against OA as long as enough light is available, but may exacerbate negative OA effects in the dark or
when not enough light is available. But knowledge about the mechanisms of OA effects in the light
and in the dark is scarce. Reduced light availability, which indirectly results from coastal runoff, may
affect photosynthesizing and calcifying organisms and their responses to OA, but so far this important
interaction has been widely overlooked.
Photosynthesizing and calcifying organisms can be negatively affected by OA and eutrophication,
but at the same time their photosynthesis and calcification can be limited by the supply of DIC and
DIN. Increased DIN under eutrophication conditions and increased DIC under OA conditions may have
negative, but potentially also positive effects. Yet, these interactions are barely investigated. Moreover,
much of the previous work, that has studied the effects of elevated DIN, has used treatment levels beyond
realistic concentrations which may lead to biased outcomes that are not environmentally relevant. Field
studies offer an alternative to investigate the effects of eutrophication between areas with high and low
DIN concentrations. However, high DIN concentrations in the field co-occur with other stressors, such
as reduced light availability, and are difficult to study in combination with OA. Thus, investigating the
effects of increased DIC and DIN under manipulated conditions with naturally occurring concentrations
may help to understand the impacts of OA and eutrophication on coral reef organisms.
In future, all coral reef organisms will be affected by a combination of OA and OW, regardless of
the presence of local stressors. Knowledge gaps still exist in the responses of organisms and particularly
their community composition to combinations of OA and OW. In addition, most experimental designs
included future environmental scenarios, but only few studies included past pCO2 and temperature treatments similar as seen on coral reefs a few decades ago. By including past treatments in experimental
designs, organisms’ performance can be investigated under past, present-day and future environmental
14
1 - General introduction
conditions.
In summary, the objectives of the present thesis are to investigate (see also Fig. 1.3):
1. how photosynthesizing and calcifying coral reef organisms are affected by future ocean acidification scenarios, and whether they respond differently to ocean acidification conditions.
2. how decreased light availability affects the response of photosynthesizing and calcifying coral reef
organisms to ocean acidification.
3. whether increased dissolved inorganic carbon and -nitrogen have interactive effects on photosynthesizing and calcifying coral reef organisms.
4. how photosynthesizing and calcifying coral reef organisms and their communities respond to combinations of past and future ocean acidification and warming scenarios.
Turbidity
Eutrophication
Ocean
Warming
Ocean
Acidification
+
Ocean
Acidification
+
Ocean
Acidification
+
Ocean
Acidification
foraminifera,
calcareous algae
corals,
calcareous algae
corals,
calcareous algae
epilithic
communities
Figure 1.3: Illustration of research objectives with individual and interactive effects of global and local
stressors in sequence as covered in this thesis
1.7 Experimental species
Scleractinian corals Acropora millepora, Acropora tenuis and Seriatopora hystrix (Fig. 1.4a-c) are three
common and widespread species found on the GBR and throughout the tropical Indo-Pacific Region
(Veron 2000). They host endosymbiotic algae, are important primary- and carbonate producers and account for reef development. With their complex, three-dimensional structure they also provide habitat
for a vast range of invertebrates and reef fish. Corals use aragonite, a more soluble form of the carbonate
minerals, for calcification and thus are assumed to be more susceptible to OA than calcite depositing
organisms. Responses of the coral A. millepora towards combinations of OA and decreased light availability were investigated in Chapter 4. Responses of A. tenuis and S. hystrix towards combinations of
OA and eutrophication were studied in Chapter 5.
15
1 - General introduction
(a)
(b)
(c)
1cm
(d)
1 cm
(e)
1 cm
1 cm
(f)
1 cm
(g)
(h)
1 mm
(h)
1 mm
(i)
1 mm
(j)
1 mm
1 mm
1 mm
Figure 1.4: Experimental species (a) Acropora millepora, (b) Acropora tenuis, (c) Seriatopora hystrix,
(d) Halimeda digitata, (e) Halimeda opuntia, (f) crustose coralline algae, (g) Peyssonnelia spp., (h)
Amphistegina radiata, (i) Heterostegina depressa and (j) Marginopora vertebralis
The calcareous green algae Halimeda opuntia and Halimeda digitata (Fig. 1.4d, e) are crucial primary producers commonly found on tropical coral reefs (Littler and Littler 2003; Littler and Littler
2000). Halimeda spp. meadows considerably contribute to carbonate production and habitat formation
16
1 - General introduction
and provide habitat for many invertebrate species (Wefer 1980; Rees et al. 2007; Freile et al. 1995;
Fukunaga 2008). Halimeda spp. also utilize aragonite as skeletal mineral, suggesting higher OA sensitivity than calcite depositing organisms. H. opuntia and H. digitata were investigated in regard to their
ability to grow under natural OA conditions at tropical CO2 seeps in Chapter 3. Responses of H. opuntia
towards combinations of OA and decreased light availability were investigated in Chapter 4. Moreover,
responses of H. opuntia towards combinations of increased DIC and DIN were investigated in Chapter
5.
Epilithic algae, including crustose coralline algae (CCA) and Peyssonnelia spp. (Fig. 1.4f, g), play
a critical role in the benthic community. They provide the substrate and olfactory cues for coral larvae
settlement and metamorphosis (Heyward and Negri 1999; Harrington et al. 2004). In addition, they
drive reef cementation and thus are important for reef development and reinforcement (Chisholm 2000;
Chisholm 2003). CCA deposit high-Mg-calcite, the most soluble carbonate mineral and are presumed
to be highly sensitive to OA related reduced Ω. Peyssonnelia spp. vary in their calcification intensity
(Littler and Littler 2003) and deposit aragonite, which is more stable compared with high-Mg-calcite,
potentially offering advantages over CCA. Responses of latter organisms towards combinations of OA
and OW were studied in Chapter 6.
Large benthic foraminifera Amphistegina radiata, Heterostegina depressa and Marginopora vertebralis (Fig. 1.4h-j) are unicellular organisms hosting endosymbiotic algae. Benthic foraminifera are
utilized as biological indicators for ecosystem health (Hallock et al. 2003; Uthicke and Nobes 2008;
Uthicke et al. 2010) and provide important information about water quality. Foraminifera are widespread
over tropical coral reefs and play a crucial role in the production of carbonate sand for beaches and sand
cays (Fujita et al. 2009; Doo et al. 2014). A. radiata and H. depressa deposit low-Mg-calcite, while M.
vertebralis deposits high-Mg-calcite and thus is considered to be more sensitive to OA. Responses of
the latter foraminifera were investigated in regard to OA in Chapter 2.
1.8 Introduction to study sites
Field-studies and collections of specimens for laboratory experiments of this thesis have been conducted
at four main sites from tropical Papua New Guinea (PNG) to tropical Australia (Fig. 1.5). Study sites
were located at reefs next to Normanby Island in PNG, at Lizard Island in the northern GBR and around
the Palm Islands in the central section of the GBR.
The particular feature of the study site in PNG (S 9° 49.446’, E 150° 49.055’) is that pure volcanic
carbon dioxide is bubbling out of the seafloor (Fabricius et al. 2011). This changes the carbonate chemistry of the surrounding seawater according to predictions for coral reefs of the entire globe by the end
of this century (IPCC 2013). This ‘window to the future’ provides a unique opportunity to study coral
17
1 - General introduction
reef organisms living in their natural environment in OA conditions happening already today. The PNG
study site is included in Chapter 3 of this thesis.
With low to medium background levels of DIN concentrations (Table 1.1) the Lizard Island study site
(S 14° 40.768’, E 145°26.753’) was selected to conduct a multifactorial tank experiment under OA and
eutrophication conditions. Specimens were collected in the Lizard Island lagoon and transplanted into
experimental aquaria facilities on the island. In the experimental tanks CO2 and nitrate concentrations
were manipulated to mimic interactive effects of OA and eutrophication on coral reef calcifiers. This
study site is included in Chapter 5 of this thesis.
Table 1.1: Dissolved inorganic nutrient (phosphate, ammonium, nitrate + nitrite and nitrite) concentrations of the study sites
Site
Upa-Upasina (PNG)
Lizard Island
Palm Islands
Davies Reef
PO43[µmol L-1]
0.10 (0.04)
0.05 (0.01)
0.05 (0.01)
0.08 (0.06)
NH4+
[µmol L-1]
0.38 (0.13)
0.70 (0.14)
0.46 (0.14)
0.59 (0.19)
NO3- + NO2[µmol L-1]
0.27 (0.07)
0.44 (0.10)
0.38 (0.27)
0.30 (0.09)
NO2[µmol L-1]
0.04 (0.01)
0.14 (0.02)
0.02 (0.01)
0.01 (0.01)
The Palm Islands (S 18° 37.549’, E 146° 29.246’) study site was primarily utilized to collect specimens for manipulative tank experiments which were conducted at the Australian Institute of Marine
Science (AIMS) in Townsville. The Palm Islands are considered as an inshore location of the GBR.
Particularly in the summer months, they are exposed to land-runoff with associated increases in dissolved inorganic nutrients, decrease in light availability, reduced salinity and increased sedimentation
from suspended solids. Specimens were collected here for experiments in Chapter 2 and Chapter 4.
A fourth study site was located mid-shelf off the Palm Islands at Davies Reef (S 18° 49.072’, E
147° 38.959’). A multifactorial tank experiment was conducted at the AIMS in Townsville and required
initial growth of epilithic communities on artificial substrates. This substrate was deployed on Davies
Reef over a period of five months. This study site is included in Chapter 6 of this thesis.
1.9 Publication outline
Included in this thesis are five data chapters of which two have been published as research articles in
international, peer-reviewed journals. One article is accepted for publication and currently in press, and
two articles are in preparation for submission.
Publication 1
Calcification and photobiology in symbiont-bearing benthic foraminifera and responses to a high
CO2 environment
18
1 - General introduction
0º
9.5ºS
Dobu Island
Upa−Upasina
10.0ºS
Normanby Island
Alotau
Port
Moresby
10.0ºS
Milne Bay
10.5ºS
Alotau
150.5ºE
151.0ºE
14.5ºS
Cooktown
Lizard Island
Cairns
Townsville
20.0ºS
15.0ºS
Brisbane
Cooktown
15.5ºS
145.5ºE
30.0ºS
18.5ºS
Pelorus Island
Orpheus Island
Sydney
Fantome
Island
19.0ºS
40.0ºS
Townsville
146.5ºE
140.0ºE
150.0ºE
147.0ºE
160.0ºE
Figure 1.5: Map of study sites for experiments in Papua New Guinea and Australia
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1 - General introduction
Vogel N., Uthicke S.
Published in the Journal of Experimental Marine Biology and Ecology (2012) 424-425: 15-24
In Chapter 2 ‘Calcification and photobiology in symbiont-bearing benthic foraminifera and responses to a high CO2 environment’ we conducted a six week tank experiment at the AIMS in Townsville
in order to investigate the individual effects of OA on large benthic foraminifera. At the time this experiment was conducted the available information on how foraminifera will react to OA was scarce. With
this experiment we contribute to the understanding of how future OA conditions may affect photobiology and calcification of the three large benthic foraminifera species Amphistegina radiata, Heterostegina
depressa and Marginopora vertebralis. This experiment has been the first OA experiment at the AIMS
using a CO2 -injection system under flow-through conditions and was utilized to establish the technology
and methodology for many upcoming experiments with the same experimental system. Chapter 2 has
been published in the Journal of Experimental Marine Biology and Ecology (Vogel and Uthicke 2012).
Contributions: This project was initiated by N. Vogel and S. Uthicke. The experimental design was
developed by N. Vogel and S. Uthicke. A field trip for the collection of experimental specimens was
organized and realized by S. Uthicke and N. Vogel. Data sampling, analyzing and the writing of the
manuscript was conducted by N. Vogel with improvements by S. Uthicke.
Publication 2
Calcareous green alga Halimeda tolerates ocean acidification conditions at tropical carbon dioxide
seeps
Vogel N., Fabricius K. E., Strahl J., Noonan S. H. C., Wild C., Uthicke S.
Published in Limnology and Oceanography (2015) 60.1: 263-275
Chapter 3 ‘Calcareous green alga Halimeda tolerates ocean acidification conditions at tropical carbon dioxide seeps’ investigates the individual effects of OA on the ecology, physiology and skeletal
characteristics of Halimeda spp. grown at tropical carbon dioxide seeps. This field study was conducted
in Upa-Upasina, PNG where natural, unheated CO2 seeps change the carbonate chemistry of the seawater according to global projections for the upcoming decades due to OA. By now this is the only tropical
CO2 seep site worldwide and thus presents a unique opportunity to study the effects of OA on tropical
coral reef organisms in their natural environment. This chapter has been published in Limnology and
Oceanography (Vogel et al. 2015a).
Contributions: This project was initiated by N. Vogel, K. Fabricius and S. Uthicke. The experimental
design for this study was developed by N. Vogel, with the help of J. Strahl, S. Noonan and S. Uthicke.
A field trip to PNG was organized and realized by K. Fabricius, S. Noonan, S. Uthicke, N. Vogel and
J. Strahl. Data sampling was conducted by N. Vogel with the help of S. Uthicke, J. Strahl and S.
20
1 - General introduction
Noonan. Sample analyzing was conducted by N. Vogel. The manuscript was written by N. Vogel with
improvements from all contributing authors.
Publication 3
Decreased light availability can amplify negative impacts of ocean acidification on calcifying coral
reef organisms
Accepted for publication in Marine Ecology Progress Series (2015) doi:10.3354/meps11088
In Chapter 4 ‘Decreased light availability can amplify negative impacts of ocean acidification on
calcifying coral reef organisms’ we conducted a two week tank experiment in order to investigate the
individual and combined effects of OA and decreased light availability, which is a byproduct of coastal
runoff, on the coral Acropora millepora and the calcareous green alga Halimeda opuntia. Because
photosynthetic activity of organisms can alter the internal as well as external carbonate chemistry of
the seawater, it is important to understand how a decrease in light availability will affect the response
of organisms to OA. With this experiment we provide information on how organisms living inshore
(susceptible to coastal runoff) may be affected in future OA conditions when light availability is reduced
at the same time. This chapter is accepted for publication in Marine Ecology Progress Series (Vogel
et al. 2015b).
Contributions: This project was initiated by N. Vogel, C. Wild and F. Meyer. The experimental
design was developed by all contributing authors. A field trip for collection of experimental specimens
was organized and realized by N. Vogel and S. Uthicke. Data sampling and analyzing was conducted by
N. Vogel and F. Meyer. The manuscript was written by N. Vogel with improvements from S. Uthicke
and C. Wild.
Publication 4
Effects of elevated dissolved inorganic carbon and nitrogen on the physiology of scleractinian
corals and calcareous macroalgae under ocean acidification and eutrophication conditions
Vogel N., Ow Y., Flores F., Collier C., Wild C., Uthicke S.
Chapter 5 ‘Effects of elevated dissolved inorganic carbon and nitrogen on the physiology of scleractinian corals and calcareous macroalgae under ocean acidification and eutrophication conditions’
investigates the individual and combined effects of OA and increased inorganic nitrate on corals Acropora tenuis and Seriatopora hystrix and the calcareous green alga H. opuntia. We conducted a three week
tank experiment in a controlled manipulated environment on Lizard Island, Australia. Experiments investigating nutrient effects are often characterized by high nutrient background levels and even higher
nutrient treatments which are unnaturally high and prevent an extrapolation of results to natural inshore
21
1 - General introduction
environments as found at the GBR. In this study nutrient background levels were low and were elevated
to naturally relevant concentrations. This article is in preparation.
Contributions: This project was initiated by N. Vogel, Y. Ow and S. Uthicke. The experimental
design was developed by N. Vogel, Y. Ow, S. Uthicke and C. Collier. A field trip to Lizard Island
was organized by N. Vogel, Y. Ow and S. Uthicke. Data sampling was conducted by N. Vogel, Y.
Ow, S. Uthicke and F. Flores. Data analyzing and manuscript writing was conducted by N. Vogel with
improvements from all contributing authors.
Publication 5
Interactive effects of ocean acidification and warming on coral reef associated epilithic algal communities under past, present and future ocean conditions
Vogel N., Cantin N., Strahl J., Kaniewska P., Bay L., Wild C., Uthicke S.
In Chapter 6 ‘Interactive effects of ocean acidification and warming on coral reef associated epilithic
algal communities under past, present and future ocean conditions’ we conducted a long-term experiment over six months with four temperature and four CO2 treatments. In this experiment, we investigated
the individual and combined effects of OA and OW on early assemblages of epilithic algal communities.
This article is in preparation.
Contributions: This project was initiated by N. Vogel, N. Cantin and S. Uthicke. The experimental
design was developed by N. Cantin, J. Strahl, P. Kaniewska, L. Bay and N. Vogel. Field trips were
organized and realized by N. Cantin and P. Kaniewska. Data sampling was conducted by N. Vogel, N.
Cantin and S. Uthicke. Data analyzing was conducted by N. Vogel. The manuscript was written by N.
Vogel with improvements from all contributing authors.
22
1 - General introduction
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32
Chapter 2
Calcification and photobiology in
symbiont-bearing benthic foraminifera
and responses to a high CO2 environment
Nikolas Vogel 1,2,3 , Sven Uthicke 1
(1)
Australian Institute of Marine Science, PMB 3, Townsville MC, Queensland 4810, Australia
(2)
Leibniz Center for Tropical Marine Ecology, Fahrenheitstraße 6, 28359 Bremen, Germany
(3)
Faculty of Biology and Chemistry (FB 2), University of Bremen, 28359 Bremen, Germany
Keywords: Calcification, climate change, foraminifera, ocean acidification, photosynthesis
This chapter has been published in the Journal of Experimental Marine Biology and Ecology (2012)
424-425:15-24
34
2 - Foraminifera and ocean acidification
Abstract
The present study investigates impacts of ocean acidification on calcification rates and light responses of
large benthic foraminifera (LBF). Studies were conducted on diatom-bearing Amphistegina radiata and
Heterostegina depressa and dinoflagellate-bearing Marginopora vertebralis in controls and manipulated
seawater pCO2 conditions (467-1925 µatm pCO2 ). In a six week experiment, calcification and photobiology were investigated for all three species. Additionally, short-term experiments were carried out
on H. depressa and M. vertebralis to determine photosynthetic rates in several pCO2 environments and
impacts of elevated pCO2 in increasing light intensities (photosynthesis irradiance ‘P-I’ curves) on M.
vertebralis. In the long-term experiment, positive growth (inferred through cross-sectional surface area)
was measured in all control and acidification conditions but growth rates of A. radiata and H. depressa
were not affected by increased pCO2 (linear models, p > 0.05). However, M. vertebralis displayed
significantly (planned comparison t = 2.61, p < 0.05) increased calcification rates (63%) in elevated
pCO2 regimes. Increased pCO2 did not affect maximum quantum yield (measured by pulse amplitude
modulation ‘PAM’ fluorometry) and chlorophyll a content in any species investigated. Photosynthetic
measurements (oxygen evolution) on H. depressa and M. vertebralis revealed positive net photosynthesis under experimental light conditions (10 and 29 µmol photons m−2 s−1 , respectively), however no
significant effect of elevated pCO2 on net photosynthesis and dark respiration after both long- and shortterm exposure was observed. M. vertebralis measured under nine different light conditions displayed
typical P-I curves with light saturation points of app. 500 µmol photons m−2 s−1 . However, P max and E k
did not vary under different pCO2 conditions (496 and 1925 µatm). Thus, foraminiferal species investigated in the present study did not show negative effects in exposures up to 1925 µatm pCO2 . However,
previous field studies from natural CO2 vents showed that LBF disappear at pCO2 conditions predicted
for the near future (pH total = 7.9). This indicates that the short-term ability of the holobiont or symbiont
to cope or even benefit from elevated pCO2 is no guarantee for their survival in the long-term.
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2 - Foraminifera and ocean acidification
2.1 Introduction
Anthropogenic carbon dioxide emissions, from the burning of fossil fuels, cement production, and fire
clearance are changing the climate of our planet and contribute considerably to ocean acidification (OA).
Energy-related carbon emissions reached a record of 30.6 Gtonnes in the year 2010 (IEA 2011). Atmospheric carbon dioxide partial pressure (pCO2 ) is predicted to climb from present values of 390 µatm
(NOAA 2011) to 730-1020 µatm by the year 2100 (Meehl et al. 2007). Annual fluctuations in atmospheric pCO2 , as well as oscillations over longer periods of the earth’s history are common. However,
present day levels are higher than those seen over the past 650 thousand years or longer (Jansen et al.
2007). The rapid annual rate of increase is especially concerning, reaching 1.94 µatm in 2011 (NOAA
2011), and might not leave time for organisms to adapt or acclimatize to changing environmental conditions.
Approximately one third of all the CO2 introduced to the atmosphere to date has been taken up by the
world’s oceans (Sabine et al. 2004). Increased carbon dioxide changes the biogeochemistry of seawater,
which ultimately becomes more acidic (Zeebe and Wolf-Gladrow 2001). Model projections indicate a
further decrease in the current surface ocean pH (OA) between 0.3 and 0.5 units by 2100 (Caldeira and
Wickett 2005), adding to the 0.1 reduction already experienced since the industrial revolution. This in
turn leads to a shift in the participating carbonate species (Golubik et al. 1979) and thus to a reduction
in calcium carbonate saturation state Ω.
Manipulative experimental studies on an array of marine organisms have illustrated impacts on calcification rates with decreasing pCO2 levels predicted for the end of this century. For instance, a reduction
in calcification has been observed for a range of scleractinian, zooxanthellate corals (Gattuso et al. 1998;
Langdon et al. 2000), crustose coralline algae (Kuffner et al. 2007) and mollusks (Gazeau et al. 2007).
Other studies indicate reduced calcification rates and a threshold of aragonite saturation state (Ωar ) for
corals, below which calcification becomes impaired (Fabricius et al. 2011; Hoegh-Guldberg et al. 2007;
Ries et al. 2010). Ries et al. (2010) measured reduced calcification rates for temperate corals, sea urchins,
bivalves and many more taxa. Additionally, reduced shell weight of recent planktonic foraminifera was
observed when cultured in low [CO32– ] conditions (Lombard et al. 2010) or when compared to shell
sizes in sediment cores (de Moel et al. 2009; Moy et al. 2009). Impacts on calcification rates of living
benthic foraminifera are presented in the present study.
Besides reductions in calcifications, OA appears to have impacts on photobiology. Anthony et al.
(2008) showed reductions in productivity of coral photo-system and increased rates of bleaching (disruption in host-algae symbiosis) as a result of increased pCO2 levels. Such results suggest that OA could
have even more severe impacts on photobiology than on calcification. However, it remains to be seen
how other symbiont-bearing taxa (e.g., foraminifera) respond to such conditions.
36
2 - Foraminifera and ocean acidification
Foraminifera are single-celled organisms and several groups incorporate algal symbionts, which
provide energy to the host through photosynthesis. Different foraminifera taxa host a great diversity
of symbionts, such as green algae, red algae, dinoflagellates and diatoms (Lee 2006). Foraminifera
are major calcium carbonate producers (Langer et al., 1997). Several studies highlight the importance
of large benthic foraminifera (LBF) as biological indicators for water quality and ecosystem health
(Hallock et al. 2003; Uthicke and Nobes 2008; Uthicke et al. 2010).
Impacts of ocean acidification on LBF physiology are not well studied. In previous experiments,
calcification rates of some species generally decreased with lower pH. However, they showed non-linear
trends with decreasing pH and elevated calcification at intermediate pH conditions (Dissard et al. 2010;
Fujita et al. 2011; Kuroyanagi et al. 2009). Field studies from volcanic CO2 vents of the Mediterranean
Sea (Dias et al. 2010) and Papua New Guinea (Fabricius et al. 2011) have demonstrated changes in
foraminifera assemblages in pH/CO2 gradients. Fabricius et al. (2011) showed that LBF disappear
at levels elevated not much beyond predictions for the end of this century (pH total ∼7.9/pCO2 ∼800900 µatm). This suggests the threshold of pCO2 which LBF can tolerate chronically is close to levels
predicted for the end of this century.
Knowledge gaps still exist in the photobiology and metabolism of LBF. Only a few studies have
been conducted using pulse amplitude modulation (PAM) fluorometry measurements (Nobes et al. 2008;
Schmidt et al. 2011; Ziegler and Uthicke 2011) and fewer still have investigated respiration dynamics
(Uthicke et al. 2011). To date these parameters have not been examined in response to increased CO2
conditions.
The aim of the present study was to investigate the influence of increased CO2 on calcification and
photobiology of LBF hosting different types of photosynthetic endosymbionts. We chose to investigate
several dinoflagellate- and diatom-bearing species with different types of calcite skeleton (hyaline: lowMg, miliolid: high-Mg) to test if responses to elevated pCO2 depend on the symbiont or calcite type.
The test of these hypotheses was achieved in one six week flow-through experiment and a series of
shorter (acute exposure) experiments.
2.2 Methods
Experimental species
The foraminifera Amphistegina radiata, Heterostegina depressa and Marginopora vertebralis were sampled from two reefs of the Great Barrier Reef (GBR) on several field trips between October 2010 and
April 2011. Sample sites were located in the Orpheus Island National Park in the central GBR (GPS
coordinates: S 18° 39.083’, E 146° 29.183’ and S 18° 34.133’, E 146° 28.917’). Specimens used here
37
2 - Foraminifera and ocean acidification
are from the same locations and have the same morphology and genotype as those described in Reymond
et al. (2011) and Uthicke et al. (2011). Specimens were collected while SCUBA diving (A. radiata, H.
depressa, 10-14 m depth) or snorkelling (M. vertebralis, 0.5-1.5 m depth) and sorted into 250 mL plastic
containers. Foraminifera were retained in low light, low temperature conditions (∼10 µmol photons m−2
s−1 , 24-26 °C). Water in containers was exchanged for every 1-2 days using fresh filtered seawater (< 1
µm particle size).
Experimental setup
A flow-through (400 mL min−1 ) experiment was carried out over a period of six weeks, in order to
identify potential differences in calcification rates and photobiology of three LBF species (A. radiata,
H. depressa and M. vertebralis). Foraminifera were exposed to four different seawater CO2 conditions
(467, 784, 1169 and 1662 µatm pCO2 ; details are given in Table 2.1). Twelve glass aquaria with three
replicates for each treatment (working volume 17.5 L) were installed in a temperature controlled room
(25 ± 1 °C) and supplied with fresh filtered (< 0.5 µm) seawater from four CO2 header tanks. Treatments were alternated between tanks to eliminate any potential environmental effects within the aquaria
room. pH levels in the header tanks were regulated by a feedback control, CO2 gas injection system
(AquaMedic) and monitored via eight potentiometric sensors (± 0.01 pH unit) calibrated on the NIST
scale. Additional pH readings were conducted daily with a hand held potentiometric pH probe (console:
Oakton, pH probe: Eutech) on the NIST scale and compared to the Dickson seawater standard. Water
temperature was monitored throughout the experiment and remained constant (see Table 2.1). Water
movement and mixing of CO2 was provided by pumps and diffusers installed in mixing tanks and experimental aquaria. Water samples for carbonate system analyzes were taken 10 times (for each treatment)
during the long-term experiment, haphazardly (Table 2.1). Illumination was delivered by white light (55
W, 10,000 K, manufacturer: CA), covering the full sunlight spectrum. Light levels were determined by
a light-meter (LICOR) and were homogeneous among the aquaria.
LBF specimens were cultured in two six-well-plates (Nunc) per aquarium. To provide water exchange in these plates, lids were drilled open (Ø 3.3 cm) and covered with plankton meshnet to prevent
specimens from escaping. Each plate was wrapped in a layer of shade cloth to decrease irradiance levels. One plate with ‘shallow’ species M. vertebralis was covered in wide-meshed cloth, receiving 29-34
µmol photons m−2 s−1 ; while the second plate with ‘deep’ species A. radiata and H. depressa was covered in close-meshed shading cloth, gaining 8-12 µmol photons m−2 s−1 . Irradiance levels chosen were
similar to previous experiments conducted by Ziegler and Uthicke (2011) and Nobes et al. (2008). A
total of 72 individuals per species and treatment (24 per plate) were cultured over a period of six weeks.
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2 - Foraminifera and ocean acidification
Table 2.1: Mean carbonate system parameters of long- and short-term experiments. Carbonate system parameters were calculated from measured TA, DIC, salinity, temperature and pressure. Standard
deviations are given in brackets. SW = seawater
Measured parameters
pCO2
pHtotal
treatment
Temp
[°C]
TA
[µmol kgSW-1]
DIC
[µmol kgSW-1]
Calculated parameters
pHtotal
pCO2
[µatm]
Ωca
[µmol kgSW-1]
Long-term experiment
High
7.57-7.65
Intermediate 7.74-7.84
Low
7.88-7.96
Control
8.06-8.19
27.5 (0.5)
27.2 (0.4)
27.2 (0.4)
27.3 (0.4)
2281 (30)
2282 (31)
2282 (32)
2280 (31)
2192 (27)
2155 (33)
2100 (20)
1999 (24)
7.66 (0.07)
7.79 (0.05)
7.95 (0.03)
8.14 (0.01)
1662 (275)
1169 (143)
784 (47)
467 (16)
1.8 (0.3)
2.5 (0.3)
3.3 (0.2)
4.8 (0.2)
Short-term experiment
High
7.44-7.56
Intermediate 7.60-7.80
Low
7.82-8.94
Control
8.01-8.14
27.0 (0.0)
27.0 (0.0)
27.0 (0.0)
27.0 (0.0)
2184 (90)
2169 (70)
2214 (55)
2137 (64)
2151 (89)
2085 (68)
2067 (43)
1909 (45)
7.58 (0.03)
7.73 (0.03)
7.90 (0.05)
8.10 (0.02)
1925 (157)
1307 (118)
878 (106)
496 (26)
1.5 (0.1)
2.0 (0.2)
2.9 (0.3)
4.1 (0.3)
Determination of calcification rates
Growth rates were determined by increases in cross-sectional surface area. This method has been used
in previous studies (Schmidt et al. 2011; Uthicke and Altenrath 2010; Uthicke et al. 2011). Additionally,
weight-surface correlations (mg/mm2 ) of individual foraminifera were calculated to determine calcification rates and investigate potential changes in shell density due to OA. Prior to experimental treatments
digital photographs were taken with a Leica MS 5 stereo-microscope combined with a Leica (× 2.5)
stereoscope to Canon AF lens. Final pictures were taken after 40, 41 and 43 days (H. depressa, M.
vertebralis and A. radiata respectively). Cross sectional surface areas (accuracy: 10 -4 mm2 ) were then
quantified using digital analyzing software (Optimas) with reference to calibrations obtained using a microscope ruler. Foraminifera from initial pictures were individually recognized in final pictures and the
difference between final and initial surface area was calculated as daily percentage growth (described by
ter Kuile and Erez (1984). Negative values due to shell breakage were recognized and excluded from
the dataset.
Determination of chlorophyll a content
For chlorophyll a (Chl a) determination individual foraminifera were frozen (−80 °C) at the end of the
experiment and processed following methods in Schmidt et al. (2011). Absorbencies of Chl a extracts
were read on 750 and 665 nm in a microplate reader (BIO-TEK) and Chl a content was calculated
(normalized to wet weight of foraminifera).
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2 - Foraminifera and ocean acidification
Determination of chlorophyll a fluorometry
Pulse amplitude modulation (PAM) fluorometry measurements were conducted with an imaging PAM
(Walz), prior to experimental treatment and after six weeks of experimentation. Several studies (e.g.,
Schmidt et al. 2011; Uthicke and Nobes 2008; Ziegler and Uthicke 2011) previously used this method to
investigate photosynthetic performance in LBF. The maximum quantum efficiency [Fv /Fm ] was calculated by the initial fluorescence[F0 ] and the maximum fluorescence [Fm ] after dark adaptation (Schreiber
et al. 1986). Mortality was recorded weekly, defined by bleaching and lack of Chl a fluorescence.
Determination of photosynthetic rates
Photosynthetic rates of H. depressa and M. vertebralis were determined after six weeks of experimental culture. Additionally, impacts of ocean acidification (OA) on photosynthesis in H. depressa and M.
vertebralis were investigated after acute exposure. Photosynthetic rates were determined by change of
oxygen content in incubation chambers equipped with oxygen optodes (PreSens, see electronic supplementary material in Uthicke et al. (2011) for detailed description). Experimental runs were conducted
with average pCO2 levels of 496, 878, 1307 and 1925 µatm (see also Table 2.1) on 27 °C controlled in a
flow-through water bath (4.2 ± 0.1 L min−1 , Lauda).
Approximately 3 L of freshly filtered seawater (< 0.5 µm) were collected in a clean plastic bucket
and 100% CO2 from a gas cylinder (BOC) was bubbled manually into the water, while mixing slowly. A
temperature-controlled pH meter (console: Oakton, pH probe: Eutech) was used to read instantaneous
pH levels. Carbon dioxide bubbling was stopped once the pH of the seawater reached the target level.
Such treated water stayed stable for at least 30 min. Water samples for DIC and total alkalinity (TA)
determination were taken for six times (each treatment) haphazardly during the experiments.
Glass incubation chambers (6.62 mL) were placed into the prepared seawater and three foraminifera
were transferred into the glass chambers. Perspex lids were screwed onto the chambers under water, to
assure no air pocket remained inside. Experimental runs were initiated with a 30 min light (production)
cycle, followed by 30 min dark (respiration) cycle. Illumination was delivered by white light (55 W,
10,000 K, manufacturer: CA), covering the full sunlight spectrum. Similar to the conditions in the longterm experiment, irradiance was adjusted to 29 ± 3 µmol photons m−2 s−1 for M. vertebralis and 10
± 1 µmol photons m−2 s−1 for H. depressa. To normalize oxygen production and respiration rates to
wet weight, individual LBF were weighted (accuracy: 0.01 mg) at the end of each run. P/R ratios were
calculated on a daily basis (12 × net photosynthesis/24 × respiration).
In addition photosynthesis-irradiance (P-I) curves were measured for M. vertebralis to investigate
potential short-term effects of increased pCO2 on maximum photosynthetic capacity (P max, initial slope
(α) and minimum saturation irradiance (E k = P/α). Nine experimental runs were carried out for each
40
2 - Foraminifera and ocean acidification
treatment in the control condition (496 µatm) and an increased pCO2 condition (1925 µatm pCO2 ) at 27.0
°C. The light source for this experiment we used a light emitting diode (LED, Aqua Illumination) with
manually adjustable light intensities. Irradiance levels (white light) were determined by a light-meter
(LI-COR). Light curves were initiated by a dark cycle, followed by eight increasing light intensities (48,
75, 91, 129, 179, 239, 327 and 397 µmol photons m−2 s−1 ) and 10 min each level. The light curves
were plotted as gross photosynthesis values, calculated by adding initial respiration rate to measured net
photosynthesis rate.
Determination of carbonate system parameters
Water samples, which were collected during the long-term and short-term experiments, were analyzed
for dissolved inorganic carbon (DIC) and total alkalinity (TA) concentrations by AIMS Laboratory Services (determination of total inorganic carbon and titration for alkalinity by a Vindta 3C). Carbonate
system parameters (Table 2.1) were calculated by measured values of TA, DIC, temperature and salinity
by USGS CO2calc software (Robbins et al. 2010).
Statistical analyzes
Statistical analyzes were carried out with NCSS software to evaluate impacts of different pCO2 treatments on different response variables of LBF. Prior to analyzes growth rates and PAM data were arc-sine
transformed, as they represent percentages. Chl a content and photosynthetic rates were log transformed
before analyzes to satisfy the assumptions of homoscedasticity and Gaussian distribution. Linear Model
ANOVAs (analyzes of variances) were carried out with pCO2 conditions as fixed factor and replicate
tanks of the long-term experiment as a nested factor within. Similarly, the different experimental runs
in the short-term experiment were regarded as a nested factor and pCO2 as fixed factor. To increase
statistical power, planned comparisons of treatment groups (e.g., the two lowest against the two highest
levels) were carried out in some instances.
P-I curves were fitted with a hyperbolic tangent function following P = P max tanh (α E k/P max). The
functions showed high fit (R2 > 96) and allowed calculation of maximum photosynthesis (P max), light
saturation point (E k) and α.
2.3 Results
After 40-43 days of experimental treatment mortality rates were low for M. vertebralis (5%) and H.
depressa (3%). A somewhat higher mortality (20%) was recorded for A. radiata.
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2 - Foraminifera and ocean acidification
Carbonate system parameters
Parameters determined from water samples during the long- and short-term experiments of controls
showed somewhat higher pCO2 levels for seawater than expected with current atmospheric levels of app.
390 µatm (NOAA 2011). However, levels of pCO2 in seawater controls in the present experiments are
comparable with diurnal ranges in reef water determined by Kayanne et al. (1995) and in experiments
conducted, for example, by Anthony et al. (2008). Carbonate system parameters of different pCO2
treatments in the experiments were consistent and remained in the intended range.
Response in calcification
Mean growth rates (% surface area per day) of control treatments were 0.175 (SE ± 0.016) for A. radiata, 0.186 (SE ± 0.015) for H. depressa and 0.052 (SE ± 0.007) for M. vertebralis. The latter showed a
trend towards increased calcification rates under higher CO2 dosing (Fig. 2.1). However, Linear Model
ANOVAs (Table 2.2) revealed that growth rates did not vary significantly between single experimental
treatments for the three species. Additionally, the weight/surface-area ratios of 30 specimens per treatment and species showed no significant difference between pCO2 concentrations in H. depressa (Linear
Model ANOVA, p = 0.524) or M. vertebralis (Linear Model ANOVA, p = 0.874). Therefore the elevated pCO2 levels examined showed no significant effect on the calcification of any of the LBF species
investigated compared to control conditions.
However, a planned comparison analysis showed that the growth rate (in surface area) of M. vertebralis was significantly increased with elevated pCO2 (planned comparison t = 2.61, p < 0.05), when
comparing the two highest pCO2 treatments (1169 and 1662 µatm) with the two lowest (467 and 784
µatm). Mean calcification rates were 63% higher in the high-CO2 group compared to the low-CO2 group
with averages of 0.079 (SE ± 0.006) and 0.050 (SE ± 0.005) % surface area per day, respectively.
Planned comparisons conducted with A. radiata and H. depressa, revealed no significant difference
in calcification rates between low and high pCO2 groups (t = 0.574, p = 0.582 and t = 0.485, p = 0.641,
respectively).
Observed growth rates are comparable to previous experimental studies in aquaria and in situ for
A. radiata (range: 0.15-0.48% surface area d−1 ), H. depressa (range: 0.03-0.56% surface area d−1 )
(Uthicke and Altenrath 2010), and M. vertebralis (range: 0.057-0.060% surface area d−1 ) (Uthicke et al.
2011). Growth rates were generally lowest in M. vertebralis compared with other species investigated.
Response in chlorophyll a content
Initial Chl a values per mg foraminifera were 172 ng mg−1 (SE ± 12 ng mg−1 ) for A. radiata, 215 ng
mg−1 (SE ± 39 ng mg−1 ) for H. depressa and 97 ng mg−1 (SE ± 20 ng mg−1 ) for M. vertebralis. Com42
2 - Foraminifera and ocean acidification
H. depressa
M. vertebralis
0
Chl a [ng mg (ww)-1]
100
200
300
400
Maximum quantum efficiency
0.50 0.55 0.60 0.65 0.70 0.75 0.80
Growth [% surface area d-1]
−0.1 0.0 0.1 0.2 0.3 0.4 0.5
A. radiata
467
784 1169 1662
pCO2 [µatm]
467
784 1169 1662
pCO2 [µatm]
467
784 1169 1662
pCO2 [µatm]
Figure 2.1: Growth (n = 40-60), maximum quantum efficiency (n = 40-70) and chlorophyll a content
(n = 27-30) of A. radiata, H. depressa and M. vertebralis after six weeks of experimental treatment.
Whiskers represent upper and lower extremes. Data shown in graphs are untransformed
parisons of initial and final measurements of control treatments showed that Chl a content of A. radiata
and H. depressa remained constant during the experiment. However, Chl a content approximately doubled in M. vertebralis from initial to final measurements (Fig. 2.1). Linear Model ANOVAs (Table 2.2)
revealed no significant variation of Chl a content for any of the three species, between pCO2 treatments
43
2 - Foraminifera and ocean acidification
Table 2.2: Linear Model ANOVA results for growth rate, maximum quantum yield and chlorophyll a
content of A. radiata, H. depressa and M. vertebralis in the long-term experiment
Response variable
Growth-rate
Species
A. radiata
H. depressa
M. vertebralis
Maximum quantum
yield
A. radiata
H. depressa
M. vertebralis
Chlorophyll a
content
A. radiata
H. depressa
M. vertebralis
Source of variation
pCO2
aquaria
residual
pCO2
aquaria
residual
pCO2
aquaria
residual
pCO2
aquaria
residual
pCO2
aquaria
residual
pCO2
aquaria
residual
pCO2
aquaria
residual
pCO2
aquaria
residual
pCO2
aquaria
residual
df
3
8
167
3
8
184
3
8
148
3
8
167
3
5
199
3
6
227
3
4
101
3
4
100
3
4
100
F
3.84
1.6
p
0.057
0.128
1.18
0.58
0.376
0.794
2.48
2.64
0.136
<0.05*
2.55
9.39
0.169
<0.001*
1.3
19.28
0.372
<0.001*
0.97
3.6
0.465
<0.05*
0.56
0.92
0.668
0.454
1.1
1.07
0.447
0.374
0.83
1.96
0.543
0.107
after six weeks of experimentation. This indicates that increased CO2 dosing had no effect on total Chl
a content.
Response in maximum quantum yield
Initial values of controls showed mean maximum quantum yields of 0.622 (SE ± 0.009) for A. radiata,
0.684 (SE ± 0.002) for H. depressa and 0.612 (SE ± 0.003) for M. vertebralis. Initial values were on
same levels of energy conversion efficiency as final PAM measurements after six weeks experimentation
(Fig. 2.1). Linear Model ANOVAs (Table 2.2) revealed that the maximum quantum yield of A. radiata,
H. depressa and M. vertebralis did not vary significantly between different pCO2 treatments after six
weeks of experimentation. Therefore, the species utilized in this experiment experienced no measurable
change in energy conversion efficiency of PS II under increased CO2 dosing.
Response in photosynthesis
Photosynthetic rates were determined after chronic exposure (six weeks, Fig. 2.2) and short-term exposure (acute, Fig. 2.3) to several pCO2 environments for H. depressa and M. vertebralis. Linear Model
ANOVAs (Table 2.3) revealed that oxygen production and respiration for both species did not vary sig44
2 - Foraminifera and ocean acidification
nificantly between single experimental treatments, either in the long-term or acute.
M. vertebralis
Respiration [µg O2 L-1 h-1 mg(ww)-1]
0.00
−0.20 −0.15 −0.10 −0.05
Net photosynthesis [µg O2 L-1 h-1 mg(ww)-1]
0.15
0.00
0.10
0.20
0.05
H. depressa
496
878 1307 1925
pCO2 [µatm]
496
878 1307 1925
pCO2 [µatm]
Figure 2.2: Net photosynthesis and dark respiration (n = 9) of H. depressa and M. vertebralis after six
weeks of experimental treatment. Whiskers represent upper and lower extremes. Data shown in graphs
are untransformed
Photosynthetic rates after long-term exposure showed a mean net photosynthetic rate of 0.079 (SE ±
0.008) [µg O2 L−1 h−1 mg−1 ] and a mean respiration rate of 0.038 (SE ± 0.004) [µg O2 L−1 h−1 mg−1 ]
for H. depressa in control treatments. The average P/R ratio was 1.54. Photosynthetic rates showed
a trend towards decreased net photosynthesis with increasing pCO2 , however this was not significant
(planned comparisons t = 1.121, p = 0.299).
The mean net photosynthetic rate of M. vertebralis after long-term exposure in control treatments
was 0.071 (SE ± 0.011) [µg O2 L−1 h−1 mg−1 ] and the mean respiration rate was 0.098 (SE ± 0.005)
[µg O2 L−1 h−1 mg−1 ]. The average P/R ratio under the chosen light intensities was 0.86. Photosynthetic rates displayed a trend towards increased net photosynthesis and respiration in increasing pCO2
conditions, however planned comparisons revealed no significant difference between treatment groups
on net photosynthesis or respiration (t = 1.600, p = 0.148 and t = 0.055, p = 0.958, respectively). Al45
2 - Foraminifera and ocean acidification
M. vertebralis
Respiration [µg O2 L-1 h-1 mg(ww)-1]
−0.20 −0.15 −0.10 −0.05
0.00
Net photosynthesis [µg O2 L-1 h-1 mg(ww)-1]
0.00
0.05
0.10
0.15
0.20
H. depressa
496
878 1307 1925
pCO2 [µatm]
496
878 1307 1925
pCO2 [µatm]
Figure 2.3: Net photosynthesis and respiration n =6-9 of H. depressa and M. vertebralis after short-term
exposure. Whiskers represent upper and lower extremes. Data shown in graphs are untransformed
together, results indicate that pCO2 had no significant impacts on the production or respiration of H.
depressa and M. vertebralis after long-term exposure.
Results from short-term experiments showed mean net photosynthetic rates of 0.048 (SE ± 0.004)
[µg O2 L−1 h−1 mg−1 ] and mean respiration rates of 0.048 (SE ± 0.009) [µg O2 L−1 h−1 mg−1 ], for H.
depressa in control conditions. Net photosynthesis was marginally increased in 878 µatm and decreased
under higher CO2 dosing. However, planned comparisons showed no significant difference between
treatments (t = 1.447, p = 0.222). The P/R ratio for H. depressa in the short-term experiment was 1.
Net photosynthesis and respiration in M. vertebralis showed means of 0.053 (SE ± 0.009) and
0.087 (SE ± 0.004) [µg O2 L−1 h−1 mg−1 ], respectively. M. vertebralis did not show any trends in net
photosynthesis and respiration in differing pCO2 conditions in the short-term experiment. The P/R ratio
under experimental light conditions was 0.80.
Regression parameters of P-I curves (P max and α) were highly significant (p < 0.001) in both exper-
46
2 - Foraminifera and ocean acidification
Table 2.3: Linear Model ANOVA results for oxygen production and respiration rates of H. depressa and
M. vertebralis after long- and short-term exposition
Response variable
Long-term exposition
Net photosynthesis
Species
Source of variation
df
F
p
H. depressa
pCO2
run
residual
pCO2
run
residual
pCO2
run
residual
pCO2
run
residual
3
7
21
3
8
23
3
7
21
3
8
24
1.73
2.99
0.248
<0.05*
0.93
1.61
0.469
0.177
0.98
2.22
0.454
0.075
2.23
0.68
0.162
0.704
pCO2
run
residual
pCO2
run
residual
pCO2
run
residual
pCO2
run
residual
3
4
16
3
12
28
3
4
16
3
12
32
2.05
1.33
0.250
0.302
1.11
4.2
0.384
<0.001*
1.67
0.53
0.310
0.720
0.97
1.69
0.440
0.115
M. vertebralis
Respiration
H. dep.
M. vert.
Short-term exposition
Net photosynthesis
H. dep.
M. vert.
Respiration
H. dep.
M. vert.
imental treatments. The response curve model chosen (Fig. 2.4) explained 96 and 98% of the variation
for ambient and elevated pCO2 , respectively. Gross photosynthesis at light saturation (P max) was app.
400 µmol photons m−2 s−1 in both treatments. Mixed model ANOVA (Table 2.4) however, showed that
production parameters (P max, α and E k) did not vary significantly between the two pCO2 treatments in
the short term. Therefore, an acute increase in pCO2 had no significant impact on the shape of the P-I
curve in M. vertebralis. Thus, photosynthesis in M. vertebralis was not impacted by increased pCO2
with increasing light intensities.
2.4 Discussion
Experiments were conducted to determine acute and chronic effects of OA on calcification and photobiology in large benthic foraminifera. This study investigated whether different types of endosymbionts (dinoflagellates or diatoms) as well as different calcite compositions (low Mg or high Mg) in
foraminifera in experiments were influenced by OA. High Mg/Ca ratio calcite, which is deposited by
M. vertebralis, is the least stable type of calcium carbonate in the ocean (Kleypas and Langdon 2006)
and therefore the most vulnerable to decreased Ω/increased pCO2 . However, contrary to expectations,
results suggested that M. vertebralis displayed significantly increased calcification at elevated pCO2
47
Gross photosynthesis [µg O2 L-1 h-1 mg(ww)-1]
2 - Foraminifera and ocean acidification
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
100
200
300
400
-2
500
-1
Light intensity [µmol photons m s ]
Figure 2.4: Photosynthesis irradiance (P-I) curve for M. vertebralis in two different experimental treatments. Black symbols represent the control treatment (pCO2 = 496 µatm); n = 9, R2 = 0.96, p < 0.001.
White symbols represent the high CO2 treatment (pCO2 = 1662 µatm); n = 9, R2 = 0.98, p < 0.001.
Data are given as means ± 1 SE of 9 replicates in 3 experimental runs, each treatment. Data shown in
graph are untransformed
conditions. Investigations on energy conversion efficiency (maximum quantum yield) and Chl a content showed no effect of increased CO2 dosing after six weeks of experimentation in any species. After
long-term (chronic) exposure, photosynthetic rates showed a trend towards decreased net photosynthesis
and respiration for H. depressa and an increased net photosynthesis and respiration for M. vertebralis in
elevated CO2 conditions, however these trends were non-significant. Net photosynthesis and respiration
rates after short-term exposures displayed no-significant changes in different pCO2 treatments in either
species.
Low mortality, a Chl a content comparable to field conditions, photosynthetic rates and PAM mea-
Table 2.4: Regression parameters and Linear Model ANOVA results of light response experiments
Regression Parameters
pCO2 Treatment
R2
Pmax
p
518 µatm
0.96 0.5051 <0.001
2130 µatm
0.98 0.5098 <0.001
Mixed Model ANOVA
Response variable
Source of variation
pCO2
Pmax
run
residual
α
pCO2
run
residual
pCO2
Ek (P/α)
run
residual
α
3.3×10-3
3.8×10-3
p
<0.001
<0.001
Ek (Pmax/α)
153.1
134.2
df
1
4
12
1
4
12
1
4
12
F
0.06
3.44
p
0.820
<0.05
3.97
0.36
0.117
0.832
0.5
1.41
0.520
0.291
48
2 - Foraminifera and ocean acidification
surements showed that LBF performed well in the aquarium system during the long-term experiment.
Effects on calcification rates
Despite an average Ω ca of 1.8 [µmol kgSW−1 ] in the highest pCO2 treatments, the hyaline species A.
radiata and H. depressa as well as the miliolid species (de Nooijer et al. 2009) M. vertebralis experienced positive shell growth after six weeks of experimental treatment. Therefore, foraminiferal species
investigated were able to deposit their calcite skeleton in pCO2 conditions (730-1020 µatm) predicted
for the year 2100 and beyond.
Foraminifera in this study showed variable calcification rates within each species and within treatment replicates. This observation is in agreement with a study by Fujita et al. (2011) and most likely
resulted from different individual microenvironments, varying light gain in experimental cages and/or
varying symbiont densities (i.e. Chl a content) and therefore variable nutrition for individual LBF.
Experiments reducing pH by HCl addition (Kuroyanagi et al. 2009) showed the highest increase in
shell diameter of the LBF Amphisorus kudakajimensis at pH NIST 7.9 compared to control and pH NIST 7.7
treatment, after 10 weeks of experimentation. However, shell weight decreased with decreasing pH in
that study. In contrast, weight/surface ratios of H. depressa and M. vertebralis in the present experiment
displayed no trend in different pCO2 conditions, suggesting LBF experienced no shell-thinning, due to
decreased Ω ca.
Similar to the present study, Fujita et al. (2011) conducted acidification experiments on several LBF
by CO2 bubbling, in static incubators. The authors determined the shellweight and shell diameter of two
populations of Baculogypsina sphaerulata, Calcarina gaudichaudii and Amphisorus hemprichii. This
study revealed variability between populations, and in almost all cultures, weight and diameter peaked
at intermediate levels of pCO2 between 580 and 770 µatm (compared to 360 and 970 µatm pCO2 ).
Moreover, many treatment comparisons in Fujita et al. (2011) displayed no differences in calcification
and differences between treatments were often small. Thus, the present study concurs with previous
work that shows that short-term (up to 12 weeks) aquarium experiments indicate no major vulnerability
in growth rates of LBF to OA.
A potential explanation for different results of the present study compared to results from Kuroyanagi
et al. (2009) is the different experimental setups employed, namely that CO2 bubbling was used in the
present study instead of HCl addition. HCl addition does not alter the DIC concentration and only results
in a small increase of [HCO3– ], whereas CO2 addition increases DIC, mainly in form of HCO3– (Hurd et
al. 2009), which may be a limiting factor (ter Kuile et al. 1989a). Moreover, flow-through aquaria were
used instead of static incubators (Kuroyanagi et al. 2009). The flow-through setup in the present study
provided the host LBF with continuous nutrition. Additionally, static incubations result in artificially
49
2 - Foraminifera and ocean acidification
large boundary layers between ambient seawater and foraminifera (Köhler-Rink and Kühl 2000) and
carbonate parameters are subject to higher fluctuations between light and dark phase. Moreover, lower
irradiance levels were chosen in the present study (app. 35 µmol compared to 60 and 190 µmol photons
m−2 s−1 ). As discussed below, high irradiance can cause lower tolerance towards an additional stressing
agent, such as increased pCO2 , and therefore can damage the photosystem. Other taxa have displayed
variable responses in calcification to elevated pCO2 including the coccolithophore Emiliania huxleyi,
where calcification has been seen to increase (Iglesias-Rodriguez et al. 2008) and decrease (Riebesell
et al. 2000; Sciandra et al. 2003) under elevated pCO2 regimes.
In a manipulative experimental study carried out with a large taxonomic range and a total of 18 marine calcifying organisms, Ries et al. (2009) discovered increased calcification under intermediate CO2
regimes (606 and 903 µatm pCO2 ) in four experimental species. Interestingly, this group (Crepidula fornicata (limpet), Arbacia punctulata (purple urchin), Neogoniolithon sp. (coralline red algae) and Halimeda incrassata (calcifying green algae) involved a variety of phototrophic and heterotrophic species
with different types of calcium carbonate skeletons. Thus, a generalization of impacts on calcification
on organisms is difficult to draw, referring to calcium carbonate type or trophic level. However, these
factors may influence the response to acidification.
A potential explanation for the phenomenon of increased calcification in intermediate pCO2 is that
foraminiferal calcification and algal symbiont photosynthesis may be limited by DIC availability and
therefore compete for this factor at present pCO2 conditions. Thus, increased pCO2 levels result in
higher HCO3– concentrations available for photosynthesis and calcification. As shown in a previous
study on Amphistegina lobifera (similar symbionts as H. depressa and A. radiata) calcification and photosynthetic rates can be limited in lower DIC concentrations and calcification can profit from increased
external DIC levels (ter Kuile et al. 1989a). Moreover, ter Kuile et al. (1989b) showed a linear increase
in calcification rates of imperforate species A. hemprichii (similar symbionts as M. vertebralis) as a
function of external DIC concentrations between 0 and 4000 µmol kgSW−1 .
Although, here, LBF were exposed to more than two-fold decreased Ω ca conditions compared to
controls, M. vertebralis was able to increase calcification in those conditions. Another potential explanation for this phenomenon arises from Köhler-Rink and Kühl (2000), who showed in microsensor
studies on M. vertebralis, A. lobifera and A. hemprichii that the algal symbionts of foraminifera create
a microenvironment in the boundary layer around the surface of the holobiont. During the light phase
symbionts increased pH on the shells surface by up to 0.4 units and during the dark phase pH decreased
to ambient seawater conditions. Increasing the pH of the surrounding seawater may therefore facilitate carbonate deposition. Similarly, Rink et al. (1998) showed that algal symbionts from planktonic
foraminifera Orbulina universa were capable of creating a microenvironment surrounding the organism.
50
2 - Foraminifera and ocean acidification
The pH level on the shell surface increased up to 0.5 units compared to ambient seawater. During the
dark phase, however, the pH dropped to 7.9 units. This shows that foraminifera change the carbonate system parameters of seawater at a closer range and have to compensate for diurnal fluctuations of
almost one pH unit (Rink et al. 1998).
Moreover, de Nooijer et al. (2009) revealed that foraminifera increase their intracellular pH at the site
of calcification by up to one pH unit above ambient seawater conditions. This mechanism allows LBF
to control and increase Ω ca at sites of calcification, which in turn facilitates the precipitation of calcium
carbonate for the skeleton. This experiment also showed that both miliolid and hyaline species possess
the ability to form intracellular high pH vesicles, which are used for calcification. The mechanism
of increasing intracellular pH in decreased pH of ambient seawater however means that LBF have to
provide more energy for active transport mechanisms to produce such cytoplasmic vesicles (de Nooijer
et al. 2009).
Effects on photobiology
Responses of LBF in long-term exposure showed an increase in mean net photosynthesis of M. vertebralis in the two highest pCO2 treatments. However, this increase was not statistically significant.
Experiments conducted on corals showed slightly decreased photosynthesis with increasing pCO2
and no effect on dark respiration (Reynaud et al. 2003). Anthony et al. (2008) observed productivity
loss in high CO2 conditions in coralline algae and corals up to levels of zero productivity. Contrary, A.
intermedia showed increased production at intermediate pCO2 dosing. Similarly, Crawley et al. (2010)
observed increased maximum photosynthetic capacity (P max) of corals in intermediate pCO2 regimes
and no change in P max was found at highest CO2 concentrations compared to controls. Experiments by
Schneider and Erez (2006) revealed no significant impact of acidification on photosynthesis in Acropora
eurystoma. Studies have also demonstrated that E. huxleyi showed increased photosynthetic activity in
elevated pCO2 conditions (Fukuda et al. 2011; Iglesias-Rodriguez et al. 2008).
Moreover, CO2 enrichment stimulated photosynthesis in seagrass as described by Jiang et al. (2010).
In addition, the authors conducted rapid light curves (RLC) with PAM fluorometry and observed an increase in relative maximum electron transport rate (rETR max) and minimum saturating irradiance (E k)
with increasing CO2 . P-I curves based on oxygen measurements, which were carried out on M. vertebralis in the present short-term experiment, showed the expected shape of a saturation curve as in other
primary producers. Experiments on free living diatoms showed positive effects of acidification in form of
increased photosynthetic carbon fixation, yet other negative effects on carbon concentrating mechanisms
were observed (Wu et al. 2010). Experimental studies on different phylotypes of Symbiodinium (free living and symbiotic) showed mixed responses to OA, which vary from no effects to increased growth and
51
2 - Foraminifera and ocean acidification
photosynthetic activity with increasing CO2 (Brading et al. 2011). Since photosynthesis experiments
with diatom-bearing H. depressa and dinoflagellate-bearing M. vertebralis showed no significant trend,
symbionts in foraminifera might not have been affected differently in present experiments.
Experiments from ter Kuile et al. (1989b) indicate that photosynthetic DIC incorporation of LBF
display optima around pH = 8.2 and DIC incorporation in the skeleton show optima above 8.2 pH units.
However, the authors also found, that at constant pH, DIC levels can be limiting. Considering that algal
symbionts of LBF are capable of increasing pH at the shells surface and foraminifera increase pH at sites
of calcification, it is possible that increased pCO2 , despite reducing pH of ambient seawater, provides
higher DIC levels available for calcification and photosynthetic activity.
Carbon dioxide dosing showed no effect on maximum quantum yield, and thus on the energy conversion efficiency of PS II, after six weeks of experimental treatment. Previous studies revealed that
maximum quantum yield can be a reliable indicator for health or stress of the photosystem in diatomand dinoflagellate-bearing foraminifera (Schmidt et al. 2011; Uthicke et al. 2011, respectively).
Comparing initial Chl a content with final values after six weeks of experiment showed that total Chl
a content of LBF was not negatively affected by decreased pH conditions. However, Chl a content of
M. vertebralis increased two-fold when comparing initial with final measurements. Since M. vertebralis
was collected in shallow depths, this species was experiencing higher light gain in the field. Increasing
Chl a content in the experiment therefore might be a compensatory reaction towards lowered light levels.
Conclusions
The present study illustrated that species investigated were still able to build up their calcite skeleton in
pCO2 conditions predicted for the year 2100 and beyond. Calcification rates were not reduced compared
to control treatments. Contrary to expectations, M. vertebralis showed significantly increased growth
rates in elevated CO2 dosing. Foraminifera possess the capability of changing intra- and extracellular
carbonate chemistry to their advantage (de Nooijer et al. 2009; Köhler-Rink and Kühl 2000), however
this is associated with energy costs. Expected DIC limitation in photosynthetic rates was not present in
chosen experimental conditions. In field studies, Dias et al. (2010) showed a gradient of foraminifera
diversities and assemblages in decreasing pH conditions at volcanic CO2 vents in the Mediterranean Sea.
This indicates some high sensitivity of LBF to decreasing pH. Similarly, benthic foraminifera at coral
reefs on volcanic CO2 vents in Papua New Guinea also showed a clear reduction of foraminiferal densities along pH gradients and total extinction at near future (pH total ∼7.9) pH levels (Fabricius et al. 2011).
Thus field studies clearly indicate a negative impact of increased CO2 on foraminiferal populations and
diversity. Overall, this suggests that the pCO2 threshold that foraminifera can tolerate in the long-term
is not far from levels expected in the near future towards the end of the present century. However, this
52
2 - Foraminifera and ocean acidification
also indicates that care needs to be taken when inferring on impacts of OA alone.
Considering that future foraminifera not only have to cope with one stressor, but also with increasing
temperature and other factors such as land runoff, more research has to be conducted to assess the
impacts of interacting components under the changing conditions predicted for upcoming decades.
Acknowledgments
We are grateful for the support of the crew of the research vessel Cape Fergusson. We thank Florita Flores for her assistance in the long-term experiment. Stephen Boyle contributed through processing water
samples for carbonate system parameters. This research was supported by the Australian Government’s
Marine and Tropical Sciences Research Facility, implemented in North Queensland by the Reef and
Rainforest Research Centre Ltd. The International Office of Ludwig-Maximilians University Munich
supported NV financially for his travel expenses, with the PROSA scholarship.
53
2 - Foraminifera and ocean acidification
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58
Chapter 3
Calcareous green alga Halimeda tolerates
ocean acidification conditions at tropical
carbon dioxide seeps
Nikolas Vogel 1,2,3 , Katharina Elisabeth Fabricius 1 , Julia Strahl 1 , Sam Hamilton Croft Noonan 1 , Christian Wild 2,3 and Sven Uthicke 1
(1)
Australian Institute of Marine Science, PMB 3, Townsville MC, Queensland 4810, Australia
(2)
Leibniz Center for Tropical Marine Ecology, Fahrenheitstraße 6, 28359 Bremen, Germany
(3)
Faculty of Biology and Chemistry (FB 2), University of Bremen, 28359 Bremen, Germany
Keywords: Carbon dioxide, vent, calcification, photosynthesis, carbon content, stable isotope signature
This chapter has been published in Limnology and Oceanography (2015) 60.1: 263-275
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3 - Halimeda growing at CO2 seeps
Abstract
We investigated ecological, physiological and skeletal characteristics of the calcifying green alga Halimeda grown at CO2 seeps (pH total ∼7.8) and compared them to those at control reefs with ambient CO2
conditions (pH total ∼8.1). Six species of Halimeda were recorded at both the high CO2 and control sites.
For the two most abundant species H. digitata and H. opuntia we determined in-situ light and dark oxygen fluxes and calcification rates, carbon contents and stable isotope signatures. In both species, rates
of calcification in the light increased at the high CO2 site compared to controls (131 and 41%, respectively). In the dark, calcification was not affected by elevated CO2 in H. digitata, whereas it was reduced
by 167% in H. opuntia, suggesting nocturnal decalcification. Calculated net calcification of both species
was similar between seep and control sites, i.e. the observed increased calcification in light compensated for reduced dark calcification. However, inorganic carbon content increased (22%) in H. digitata
and decreased (−8%) in H. opuntia at the seep site compared to controls. Significantly lighter carbon
isotope signatures of H. digitata and H. opuntia phylloids at high CO2 (1.01 and 1.94h, respectively)
indicate increased photosynthetic uptake of CO2 over HCO3– potentially reducing dissolved inorganic
carbon limitation at the seep site. Moreover, H. digitata and H. opuntia specimens transplanted for 14
days from the control to the seep site exhibited similar δ 13C signatures as specimens grown there. These
results suggest that the Halimeda spp. investigated can acclimatize and will likely still be capable to
grow and calcify in pCO2 conditions exceeding most pessimistic future CO2 projections.
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3.1 Introduction
Anthropogenic emissions are increasing the carbon dioxide partial pressure (pCO2 ) in the atmosphere
(IPCC 2013). The present-day level of ∼395 µatm) (Dlugokencky and Tans 2014) has already exceeded
historic pCO2 levels observed over the last two million years (Hönisch et al. 2009) and is predicted
to double or triple from present-day levels within this century (Collins et al. 2013; Meinshausen et al.
2011; Moss et al. 2010). Increased pCO2 consequently leads to a decrease in ocean pH and aragonite
saturation state Ωar , a process called ocean acidification (OA). According to the Intergovernmental Panel
on Climate Change (IPCC 2013) the surface ocean will experience a further reduction of 0.203-0.310
pH units (‘representative concentration pathway’, RCP6.0-RCP8.5) by the year 2100 (Ciais et al. 2013).
Potential impacts of OA on life history traits, such as survival, growth, reproduction and recruitment
of marine organisms have been recently reported. It is becoming apparent that tropical coral reefs in
particular are facing major ecological changes in the upcoming decades (Pandolfi et al. 2011). An
emerging paradigm suggests that marine organisms will be negatively affected by OA. Indeed, some taxa
may be strongly impeded and may even become extinct in future environmental conditions (Carpenter
et al. 2008; Uthicke et al. 2013). However, studies also suggest species specific responses to OA and that
not every organism will be affected in future OA environments, and that some taxa may also be able to
cope with, or even thrive, under projected CO2 conditions (Fabricius et al. 2011; Johnson and Carpenter
2012; Ries et al. 2009).
Most conclusions of impacts of OA on organisms and consequent extrapolations to ecosystem level
are derived from laboratory experiments. While experiments control environmental factors allowing
comparisons between studies, they mostly do not account for intra- and interspecific interactions, natural
supply of nutrition and natural fluctuation of parameters, such as light, temperature and pH. Therefore,
investigating organisms in-situ in their natural environment, exposed to pCO2 conditions projected for
the near future could be the key in understanding acclimatization processes on organisms and changes
of coral reefs at the ecosystem level.
Natural volcanic CO2 seeps provide a unique opportunity to study the responses of organisms to
increased CO2 conditions, in their natural habitat. Benthic organisms growing close to CO2 seeps have
been exposed to these conditions throughout their life time and some may have been there for many
generations. Hence, organisms living at natural volcanic seeps are acclimatized (i.e. physiologically
adjusted to a changed environment) and in some cases potentially adapted (i.e. genetically changed
traits over several generations) to elevated CO2 environments. Volcanic CO2 seeps thus provide an
opportunity to identify which organisms are capable of living in CO2 conditions projected globally in a
few decades time and to investigate how these organisms are able to do so.
Volcanic CO2 seeps in Milne Bay, Papua New Guinea (PNG) provide unique natural CO2 conditions
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in shallow tropical waters (Fabricius et al. 2011), without additional freshwater or nutrient upwelling.
Detailed studies thus far have also identified no other stressors, such as elevated temperatures or heavy
metal concentrations, interfering with interpretation as OA as the only stressor. CO2 from the ascending
bubbles changes the carbonate chemistry of the seawater close to the seeps and establishes a pH gradient
from ambient pH (pH total ∼8.1), over predicted future pH (pH total ∼7.9), to extremely low pH (pH total
< 7) conditions. Areas of moderate seep activity are characterized by water quality parameters which are
likely to be reached worldwide in a few decades time, following RCP6.0-RCP8.5 (Moss et al. 2010). The
seep sites in PNG have been active for at least the last 80 years, as confirmed by oral communication with
traditional inhabitants, and possibly much longer (Fabricius et al. 2011). Therefore, the organisms living
on the reefs impacted by those seeps are acclimatized to a high CO2 environment for many decades.
Volcanic CO2 seeps and areas of CO2 upwelling have been described worldwide in temperate (Calosi
et al. 2013; Cigliano et al. 2010; Hall-Spencer et al. 2008; Inoue et al. 2013; Johnson and Carpenter
2012; Porzio et al. 2011) and tropical (Fabricius et al. 2014; Fabricius et al. 2011; Johnson and Carpenter 2012; Noonan et al. 2013; Russell et al. 2013; Uthicke and Fabricius 2012; Uthicke et al. 2013)
regions. So far, studies suggest reduced pH at CO2 seeps in PNG lead to a decline in coral diversity with
structurally complex species being particularly affected, and reduced taxonomic richness and density of
coral juveniles, and low cover of crustose coralline algae (Fabricius et al. 2014; Fabricius et al. 2011).
Next to direct physiological impacts on organisms, a loss of habitat complexity at CO2 seeps indirectly
leads to decreased densities of macroinvertebrate taxa (Fabricius et al. 2014). Densities and diversity
of large benthic foraminifera decrease at seep sites and are absent at pH total < 7.9, which is only a 0.2
unit reduction to present-day levels (Uthicke and Fabricius 2012; Uthicke et al. 2013). In contrast, cover
of some calcareous and non-calcareous macroalgae and seagrasses increased at CO2 seeps compared to
controls (Fabricius et al. 2011; Johnson and Carpenter 2012), indicating tolerance or acclimatization of
some organisms to future pCO2 conditions and possible gains in rates of photosynthesis.
Halimeda, a genus of calcareous green algae are important, fast growing primary producers associated with coral reefs. Their calcium carbonate (CaCO3 ) skeletons contribute significantly to carbonate
production and sediment formation (Freile et al. 1995; Rees et al. 2007; Wefer 1980). Halimeda deposit
aragonite, which is the more soluble form of the most common CaCO3 minerals. Moreover, Halimeda
spp. provide important habitat for invertebrate communities (Fukunaga 2008). However, impacts of low
pH on Halimeda spp. have not been investigated at tropical CO2 seeps. Findings from volcanic seeps
in Mediterranean showed that temperate Halimeda spp. were absent at mean Ωar ≤ 2.5 (Hall-Spencer
et al. 2008). Laboratory experiments revealed mixed responses of OA on Halimeda spp., with some
species being negatively impacted, while others are not (Hofmann et al. 2014; Koch et al. 2013; Price
et al. 2011; Ries et al. 2009; Sinutok et al. 2011; Vogel et al. 2015). Moreover, the calcareous brown
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algae Padina spp. are thriving at seeps in the Mediterranean and in PNG with increased abundance at
CO2 seep sites compared to controls (Johnson and Carpenter 2012), suggesting that some calcareous
organisms can benefit from increased CO2 availability. It is therefore not clear how calcifying algae,
among the most important organism groups in coral reefs, respond to OA.
This study investigates for the first time in-situ ecological, physiological and skeletal characteristics
of calcareous green algae of tropical Halimeda after a lifetime exposure to high levels of CO2 . The
distribution of six species of Halimeda was investigated in relation to the seawater carbonate chemistry
from water samples collected at the site of occurrence. For the two most abundant Halimeda spp.
(Halimeda cuneata f. digitata and Halimeda opuntia) in-situ rates of oxygen fluxes and calcification in
light and in darkness, organic- and inorganic carbon content and carbon isotopic signatures (δ 13C) were
compared between CO2 seep and control site.
3.2 Methods
Site description
At several locations in the Milne Bay Province, PNG (Fig. 3.1a), volcanic CO2 is seeping out of the
seafloor (Fabricius et al. 2011). The seep sites are located at Dobu Island and Upa-Upasina (Normanby
Island) close to the shore in shallow water of ∼1-15 m depth and extend over an area of ∼20 by 100 m
with different intensities of bubble activity within this area. Control reefs were allocated several hundred
meters away from the seep sites with no impact of the seep activity on their seawater carbonate system
(Table 3.1). The bubbles, which consist of pure CO2 , ascend to the surface and mix with the ambient
seawater, changing the carbonate chemistry. This study was confined to areas where seawater chemistry
was altered to levels projected for a vast part of the globe for the end of this century (RCP6.0-RCP8.5
scenarios) (Moss et al. 2010) (Table 3.1).
Sample collection
Water samples (Table 3.1) for occurrence/OA tolerance of Halimeda species were collected at Dobu Island control and seep site (S 9° 45.125’, E 150° 51.248’ and S 9° 44.199’, E 150° 52.060’, respectively)
and Upa-Upasina control and seep site (S 9° 49.693’, E 150° 49.231’ and S 9° 49.446’, E 150° 49.055’,
respectively) in April/May 2012 and May/June 2013. Physiological characteristics and skeletal properties of Halimeda cuneata f. digitata (referred as H. digitata) and Halimeda opuntia were determined at
Upa-Upasina control and seep site in April, May 2012. Specimens of H. digitata and H. opuntia (Fig.
3.1b and 3.1c) were sampled between 4-6 meters water depth at the control and CO2 seep sites. Samples
for inorganic carbon content and carbon isotopic signatures (δ 13C) were rinsed in freshwater and dried
64
(a)
−9.5
−9.0
3 - Halimeda growing at CO2 seeps
Dobu Island
Latitude [°]
−10.0
Upa−Upasina
Normanby Island
−10.5
Milne Bay
−11.0
Papua New
Guinea
150.0
(b)
150.5
(c)
151.0
Longitude [°]
151.5
152.0
(d)
Figure 3.1: (a) Map of Papua New Guinea, Milne Bay Province and Normanby Island with locations of
seep sites at Dobu Island and Upa-Upasina. (b) H. digitata growing at the CO2 seep site (Upa-Upasina).
(c) H. opuntia growing at the CO2 seep site (Upa-Upasina). (d) H. opuntia growing next to CO2 bubbles
(Dobu Island)
65
pHNIST
pHtotal
In-situ samples/ occurrence
Control
8.23 (0.03) 8.12 (0.01)
Impact
7.81 (0.30) 7.66 (0.30)
Incubation experiment
Control
8.26 (0.02) 8.17 (0.01)
7.87 (0.10) 7.77 (0.07)
Impact
Transplant experiment
Control
8.25 (0.03) 8.14 (0.03)
Impact
7.90 (0.09) 7.83 (0.07)
Treatment
2282 (32)
2249 (18)
2277 (30)
2330 (27)
2287 (27)
2332 (24)
29.0 (1.4)
29.1 (1.5)
29.2 (1.1)
29.1 (1.2)
TA
[µmol kgSW-1]
29.8 (0.6)
29.2 (0.6)
Temp
[°C]
1915(28)
2142 (40)
1900 (26)
2168 (34)
1907 (28)
2106 (129)
DIC
[µmol kgSW-1]
323 (32)
848 (166)
306 (27)
962 (125)
327 (12)
1544 (1384)
pCO2
[µatm]
1646 (39)
1973 (58)
1629 (40)
2014 (42)
1636 (24)
1941 (161)
HCO3[µmol kgSW-1]
261 (18)
147 (26)
263 (22)
129 (16)
263 (4)
125 (65)
CO32[µmol kgSW-1]
8.38 (0.80)
22.01 (4.72)
8.06 (0.86)
25.28 (3.50)
8.30 (0.20)
40.02 (35.92)
CO2
[µmol kgSW-1]
6.38 (0.46)
3.61 (0.64)
6.43 (0.56)
3.16 (0.40)
6.45 (0.12)
3.07 (1.60)
Ωca
4.25 (0.32)
2.41 (0.43)
4.28 (0.39)
2.10 (0.27)
4.31 (0.09)
2.04 (1.07)
Ωar
Table 3.1: Carbonate system parameters of water samples from in-situ collections (n total = 86) (Dobu Island and Upa-Upasina, 2012, 2013), incubations (n total
= 30) (Upa-Upasina, 2012) and transplant experiment (n total = 50) (Upa-Upasina, 2012). Data is given as mean and standard deviation
3 - Halimeda growing at CO2 seeps
66
3 - Halimeda growing at CO2 seeps
for 48 h at 40 °C for subsequent analyzes.
Occurrence/OA tolerance
To determine OA tolerance, water samples (n total = 86) were collected 5-10 cm above Halimeda spp.
thalli, growing at the control and seep sites of Dobu and Upa-Upasina by snorkeling and scuba diving.
Water samples were analyzed for pH NIST, temperature and voltage in millivolts (mV) with a temperature corrected bench top pH meter (OAKTON) and a refillable pH probe (Eutech), calibrated on NIST
(National Institute of Standards and Technology) scale. Additional pH readings were performed with
Tris-buffer in artificial seawater supplied by A. Dickson (Scripps Institute for Oceanography) to determine the accuracy of pH measurements in 2012 and 2013 (n = 19, pH = 8.15 ± 0.05 SD, temperature
= 29.3 ± 1.2 °C). Millivolt and temperature measurements were utilized to convert pH values to total
scale (pH total). In some instances conversion to total scale, lowered the variance of pH readings, as indicated in Table 3.1. Water collections were repeated on several days in 2012 and 2013 in the mornings
and evenings, to incorporate diurnal pH fluctuations, over a total of six sampling events.
Seawater carbonate system parameters
Subsamples (50 mL) of seawater were directly titrated for total alkalinity (TA) on a Metrohm 855 robotic
titrosampler by gran titration, using 0.5 mol L−1 HCl as described in Uthicke and Fabricius (2012). Total
alkalinity was calculated by non-linear regression fitting of hydrogen ion concentration and the volume
of titrant between pH 3.5 and pH 3.0, following the Standard Operating Procedure SOP3b outlined in
the ‘Guide to Best Practices for Ocean CO2 Measurements’ (Dickson et al. 2007). Acid concentration
was corrected by titrating Certified Reference Material (CRM Batch 106, A. Dickson, Scripps Oceanographic Institute). The accuracy of TA measurements was determined by CRM titrations in 2012 and
2013 (n = 38, TA = 2218 ± 11 SD). Carbonate system parameters (Table 3.1) of incubations and field
samples were calculated utilizing pH total and TA measurements by CO2calc software (Robbins et al.
2010) using CO2 constants from Lueker et al. (2000).
Calcification and photosynthesis
Calcification rates in the light and dark, as well as net photosynthesis and respiration rates, were measured in-situ at control (pH total = 8.17) and seep sites (pH total = 7.77, see Table 3.1 for carbonate chemistry). Branches 5-8 cm in height and with ∼20 phylloids of H. digitata and H. opuntia were collected
and retained at the site of collection until incubations commenced. Light incubations were conducted
in-situ at 5 m water depth at midday. Specimens were placed into 0.5 L clear Perspex chambers, simultaneously at control and seep sites, by two separate SCUBA diving teams. After ∼3 h incubation under
67
3 - Halimeda growing at CO2 seeps
ambient light, incubation chambers were retrieved and a water subsample was directly analyzed for TA
(as described above). Oxygen concentration was determined in each incubation chamber including two
blank incubations per treatment (to correct for seawater production/respiration) with a hand-held dissolved oxygen meter (HQ30d, Hach) as described elsewhere (Uthicke and Fabricius 2012; Witt et al.
2012). Light intensities of incubation conditions were recorded by two light loggers (Odyssey) each at
control and seep site. Photosynthetically available radiation (PAR) was dependent on weather conditions
and averaged 34 and 39 µmol photons m−2 s−1 at the control and seep site for H. digitata and 259 and
281 µmol photons m−2 s−1 for H. opuntia incubations. Dark incubations were conducted on board the
research vessel for ∼3 h in the evening. The incubation chambers were filled with water from the site of
origin of the plants (control vs. seep site). Chambers were placed in black plastic bins (45 L) with lids
for darkening and flow-through seawater for temperature control. Rates of calcification were determined
with the alkalinity anomaly technique (Chisholm and Gattuso 1991). Calcification rates (in µM C h−1
gFW−1 ) and oxygen fluxes (in µg O2 L−1 h−1 gFW−1 ) were calculated in relation to blank incubations
and standardized to the fresh weight (FW) of the plants. Daily net calcification rates were calculated by
12 h of daylight and 12 h of darkness.
C and N contents and stable isotope signatures
Apical phylloids of dried Halimeda spp. were crushed with mortar and pestle and the homogenate was
analyzed for total carbon (C tot) and total nitrogen (N) on a Flash EA 1112 elemental analyzer (Thermo
Fisher Scientific). In addition, organic carbon (C org) contents were measured after acidifying the sample
with 150 µL concentrated HCl to drive out C inorg. Inorganic carbon content was calculated by subtracting
C org from C tot. Stable isotope signatures were measured in a subset of these samples using a Delta S
mass spectrometer (Thermo Fisher Scientific) coupled with the elemental analyzer.
Transplant experiment
A transplant experiment was carried out at Upa-Upasina in 2012 over a period of 14 days. Branches
(∼20-30 phylloids) of H. digitata and H. opuntia were collected at the control and seep sites and attached
onto plastic trays, assuring specimens were physically separated. Three replicate trays were deployed at
each site in 5 m of water. Six individuals of each species were transplanted from control to control (CC)
and control to impact (CI) site. Two light loggers (Odyssey) were deployed next to each experimental
site, to record photosynthetically available radiation (PAR) throughout the course of the experiment.
Recordings of daily light sums averaged 5.31 and 4.34 mol photons m−2 d−1 for control and seep site,
respectively, with light maxima of 667 and 707 µmol photons m−2 d−1 . One layer of thin wire mesh
(∼3 cm mesh size) was wrapped around each tray to assure protection from large herbivore fish. After
68
3 - Halimeda growing at CO2 seeps
two weeks, specimens were sampled, rinsed in fresh water and dried for 48 h at 40 °C for subsequent
carbon, nitrogen and stable isotope signature analyzes as described above.
Statistical analyzes
To determine significant differences of responses between controls and seep sites, statistical analyzes
were conducted with the software R (R Development Core Team 2014). For each response variable
measured, we performed Linear Models with location (control and seep site) as fixed factor. To test data
for equal variance and homogeneity, we performed Levene’s tests on each response variable. In case the
null hypothesis was rejected (i.e. the variance between groups was unequal), we transformed (log10 or
arcsine dependent on variable) the data prior to subsequent analyzes.
3.3 Results
In-situ samples
At control and seep site a total of six different Halimeda species were identified with either lightly
calcified (LC), calcified (C) and heavily calcified (HC) phylloids. Species included H. cylindracea (C),
H. cuneata f. digitata (LC), H. cuneata f. undulata (LC), H. hederacea (HC), H. macroloba (LC) and
H. opuntia (HC) (Littler and Littler 2003). Water samples above the thalli ranged from pH total = 8.14 at
the control sites to pH total = 7.05 at the seep sites (Fig. 3.2, Table 3.1). Water samples collected above
Halimeda spp. at the control sites yielded mean pH total of 8.12 and samples from the seep sites yielded
mean pH total of 7.66 (Table 3.1). Calculated mean pCO2 and Ωar from collected water samples were
327 (± 12 SD) µatm and 4.31 (± 0.09 SD) at the control site and 1544 (± 1384 SD) µatm and 2.04 (±
1.07 SD) at the seep site, respectively. Based on personal observations during ∼47 dive hours the most
abundant species at both control and seep sites appeared to be H. digitata and H. opuntia.
Mean rates of light calcification of both H. digitata and H. opuntia, were significantly increased at
the seep site (131 and 41%, respectively) compared to the control site (Fig. 3.3, Table 2, Linear Models
p = 0.020 and p = 0.049, respectively). Rates of calcification in the dark of H. digitata was not affected
by CO2 , while rates of H. opuntia were significantly decreased (−167%) at the seep site resulting in
CaCO3 dissolution in the dark (Fig. 3, Table 2, p = 0.013). Calculated net calcification rates of both H.
digitata and H. opuntia were not significantly affected by CO2 (Fig. 3.3, Table 3.2).
Net photosynthesis of both species did not differ between seep and control site under experimental
light conditions (Fig. 3.4, Table 3.2), but mean respiration rate of H. digitata was significantly increased
(96%) at the seep site compared to the control site (Table 3.2, p = 0.029). Gross photosynthesis of both
H. digitata and H. opuntia was not affected by elevated CO2 (Table 3.2).
69
3 - Halimeda growing at CO2 seeps
Table 3.2: Linear Model ANOVA results for physiological and skeletal parameters of H. digitata and
H. opuntia with control and seep site as source of variation. Asterisks indicate significant differences of
response variables between control and seep site
Response variable
Light calcification
Species
H. digitata
H. opuntia
Dark calcification
H. digitata
H. opuntia
Net calcification
H. digitata
H. opuntia
Net photosynthesis
H. digitata
H. opuntia
Respiration
H. digitata
H. opuntia
Gross photosynthesis
H. digitata
H. opuntia
Ctot
H. digitata
H. opuntia
Corg
H. digitata
H. opuntia
Cinorg
H. digitata
H. opuntia
Corg : Cinorg
H. digitata
H. opuntia
δ13C
H. digitata
H. opuntia
δ13C (transplant)
H. digitata
H. opuntia
Source of variation
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
Site
Residuals
df
1
21
1
22
1
21
1
22
1
21
1
22
1
21
1
22
1
21
1
22
1
21
1
22
1
20
1
20
1
20
1
20
1
20
1
20
1
20
1
20
1
21
1
20
1
14
1
16
F
6.34
p
0.0200*
4.32
0.0495*
0.59
0.4497
7.33
0.0129*
0.46
0.5060
0.14
0.7126
3.19
0.0886
1.09
0.3084
6.16
0.0216*
0.23
0.6362
0.25
0.6197
1.23
0.2786
8.15
0.0098*
2.22
0.1520
12.36
0.0022*
3.11
0.0930
11.83
0.0026*
5.00
0.0369*
13.59
0.0015*
3.56
0.0737
9.50
0.0056*
19.07
0.0003*
9.07
0.0093*
8.16
0.0114*
70
3 - Halimeda growing at CO2 seeps
Species occurrence
ambient
ambient
7.50
RCP8.5
3
1600
Ωar
7.75
4
3200
pCO2 [µatm]
pHtotal
8.00
RCP8.5
2
ambient
1
800
7.25
400
yl
.c
H
Species
d
H rac
. d ea
H ig
. h ita
e
H der ta
.m a
ac cea
r
H olob
.o a
H pun
. u ti
nd a
ul
at
a
in
d
H rac
.
d
H ig ea
. h it
H ede ata
. m ra
ac cea
r
H olo
. o ba
H pu
. u nt
nd ia
ul
at
a
in
yl
.c
H
H
.c
yl
in
d
H rac
. d ea
H ig
. h it
a
H ede ta
. m ra
c
ac ea
r
H olo
. o ba
H pu
. u nt
nd ia
ul
at
a
7.00
Figure 3.2: Carbonate system parameters of water samples collected above Halimeda species growing
at Dobu Island and Upa-Upasina control and seep site. Each dot represents a water sample collected
above the corresponding species (green = control site, red = seep site). Dotted lines indicate ambient
(green) levels and predicted future (red) levels following the most pessimistic ‘representative concentration pathway’ RCP8.5. Solid lines (red) represent mean values of water samples for each species,
collected at the seep site
Total carbon content of H. digitata tissue was significantly higher (15%) at the seep, compared to
the control site (Fig. 3.5, Table 3.2, p = 0.010), while organic carbon content was significantly lower
(−29%) at the seep compared to the control site (Fig. 3.5, Table 3.2, p = 0.0022). H. digitata grown at
the seep site showed significantly higher inorganic carbon content (22%), compared to the control site
(Fig. 3.5, Table 3.2, p = 0.003). Total- and organic carbon content of H. opuntia tissue did not differ
between sites. However, inorganic carbon content of H. opuntia was significantly lower (−8%) at the
seep compared to the control site (Fig. 3.5, Table 3.2, p = 0.037). Moreover, C org : C inorg ratio of H.
digitata was significantly lower at the seep compared to control site (Fig. 3.5, Table 3.2, p = 0.0015),
while C org : C inorg ratio of H. opuntia did not significantly differ between sites.
Stable carbon isotope signatures of both H. digitata and H. opuntia specimens were significantly
lower (5 and 8%, respectively) at the seep compared to the control site (Fig. 3.6, Table 3.2, p = 0.006
and p < 0.001, respectively). Thus, both species showed proportionally increased fixation of lighter 12 C
at the seep compared to the control site.
Transplant experiment
After 14 days, stable carbon isotope signatures of newly grown phylloids of both, H. digitata and H.
opuntia were significantly lower (9 and 15%, respectively) in thalli that were transplanted from the
control to the seep site (Fig. 3.6, Table 3.2, p = 0.010 and p = 0.011, respectively). δ 13C values became
more negative and matched with carbon isotope signatures from Halimeda spp. that originally grew at
the seep site. As negative control, thalli transplanted from the control to the control site matched with
71
3 - Halimeda growing at CO2 seeps
Net calcification (µM C d-1 gFW-1)
Dark calcification (µM C h-1 gFW-1)
Light calcification (µM C h-1 gFW-1)
H. digitata
H. opuntia
40
*
30
20
*
10
0
15
*
10
5
0
−5
−10
500
400
300
200
100
0
Seep
Control
Site
Seep
Control
Site
Figure 3.3: In-situ light-, dark- and net calcification rates of H. digitata and H. opuntia grown at control
and CO2 seep site. Brackets indicate significant differences in ANOVAs, with significance levels ∗p <
0.05, ∗∗p < 0.001, ∗ ∗ ∗p < 0.0001
the carbon isotope signatures of Halimeda spp. that originally grew at the control site.
72
Net photosynthesis [µg O2 L-1 h-1 gFW-1]
3 - Halimeda growing at CO2 seeps
H. digitata
H. opuntia
1000
800
600
400
200
0
Seep
Control
Seep
Control
Site
Site
Figure 3.4: In-situ rates of net photosynthesis of H. digitata and H. opuntia grown at control and CO2
seep site
3.4 Discussion
We investigated ecological, physiological and skeletal characteristics of Halimeda spp. acclimatized
to elevated CO2 environments at volcanic seep sites and compared these to control reefs. Notably, we
recorded six different Halimeda species growing within areas close to CO2 seeps and at control sites at
Dobu Island and Upa-Upasina, with all species observed down to a pH total level of at least ∼7.7 (pCO2
∼1000 µatm). At several locations we observed Halimeda spp. growing directly next to ascending
CO2 bubble streams (Fig. 3.1d). Water parameters showed some Halimeda spp. were still capable to
grow in occasional extreme pH conditions (pH total < 7) and Ωar under-saturation (Ωar < 1) (Fig. 3.2).
Thus, Halimeda spp. at seep sites were growing in pCO2 conditions that exceed the most negative
‘representative concentration pathway’ RCP8.5 (IPCC 2013; Moss et al. 2010). This observation stands
in contrast to observations at CO2 seeps in the Mediterranean, where temperate Halimeda spp. were
absent at the site impacted by CO2 seeps (Hall-Spencer et al. 2008).
Why temperate Halimeda spp. are absent in elevated CO2 conditions, while tropical Halimeda spp.
are not, is unclear. Potentially, different oceanographic conditions between sites contributed to observed
differences. For instance water temperature affects the solubility of CaCO3 with favorable conditions
for organisms in tropical regions. However, the saturation state of aragonite in the present study was
lower (Ωar ∼2) compared to the seep site in the Mediterranean where Halimeda spp. were absent (Ωar
≤ 2.5). Potentially more stable conditions throughout the year in PNG compared to the Mediterranean
led to observed differences. Similarly, hard corals were absent under high CO2 conditions at temperate
seeps in Japan (Inoue et al. 2013), while coral cover was not impacted at seeps in PNG but the diversity
of species changed with increasing CO2 (Fabricius et al. 2011). Laboratory experiments investigating
73
3 - Halimeda growing at CO2 seeps
H. digitata
H. opuntia
Ctot [%]
26
22
18
14
**
Corg [%]
20
15
10
5
**
0
Cinorg [%]
12
10
**
8
*
6
4
Corg : Cinorg
4
3
2
1
**
0
Control Seep
Site
Control Seep
Site
Figure 3.5: Total-, organic- and inorganic carbon content and C org : C inorg ratio of H. digitata and H.
opuntia grown at control and CO2 seep site. Brackets indicate significant differences in ANOVAs, with
significance levels ∗p < 0.05, ∗∗p < 0.001, ∗ ∗ ∗p < 0.0001
the impacts of OA on Halimeda spp. also arrived at varying conclusions, with some suggesting that
growth and calcification of several Halimeda spp. may be impacted under future CO2 conditions (Price
et al. 2011; Ries et al. 2009; Sinutok et al. 2011), while others suggest that several others are unlikely to
be impacted by OA alone (Comeau et al. 2013; Hofmann et al. 2014; Vogel et al. 2015). Morphological
distinctions, such as surface area to volume ratio of phylloids may contribute to different responses
of different Halimeda species to OA where thicker phylloids may reduce OA impacts. In addition,
different morphologies affect diffusion of inorganic carbon to sites of calcification and photosynthesis.
74
3 - Halimeda growing at CO2 seeps
H. digitata
H. opuntia
−18
δ13C
−20
***
−22
−24
**
−26
−28
Seep
Control
In−situ
Seep
Control
In−situ
−18
*
δ13C
−20
−22
−24
**
−26
−28
CC
CI
Transplant
CC
CI
Transplant
Figure 3.6: δ 13C and δ 15N signatures of H. digitata and H. opuntia grown at control and CO2 seep site
and transplanted from control to control and control to seep site. Brackets indicate significant differences
in ANOVAs, with significance levels ∗ p < 0.05, ∗∗ p < 0.001, ∗ ∗ ∗ p < 0.0001
Moreover, different organisms possess different mechanisms of calcification. While aragonite deposition
in Halimeda takes place in the interutricular spaces (Borowitzka 1989), Padina calcification is initiated
intracellular (Okazaki et al. 1986) and corals deposit CaCO3 at their calicoblastic epithelium (Allemand
et al. 2004). However, in this study Halimeda growing at the seep sites did not show any pattern related
to their morphology, and included lightly and heavily calcifying species, as well as rock-anchoring
and sand-dwelling species. Our measured seawater carbonate system parameters provide evidence for
the existence of Halimeda in high CO2 environments, suggesting several tropical Halimeda spp. can
acclimatize to future OA conditions. This observation is in agreement with a previous study on the
slightly calcareous brown algae Padina sp., which occurs at volcanic CO2 seep sites in PNG and the
at the Mediterranean (Johnson and Carpenter 2012). However, seep sites investigate the effects of OA
in isolation and it is possible that other co-occurring factors predicted for the future (e.g. warming or
increase of terrestrial runoff) may interact to affect Halimeda spp.
We investigated H. digitata and H. opuntia physiology in detail since they were most abundant
75
3 - Halimeda growing at CO2 seeps
at both control and seep site. By selecting the most abundant species the potential of a bias towards
more resilient species cannot be excluded. Nevertheless, occurrence of H. cylindracea, H. hederacea,
H. macroloba and H. undulata at the seep sites suggests that several other species can tolerate this
particular environment. Net- and gross photosynthesis of both, H. digitata and H. opuntia, did not
differ between control and seep site. Increased dissolved inorganic carbon (DIC) availability did not
positively affect the photosynthesis of Halimeda spp. grown at volcanic seep sites incubated in otherwise
present environmental conditions (i.e. present light conditions). In contrast, previous studies observed
increased productivity of benthic foraminifera at the Upa-Upasina seep site, suggesting endosymbiotic
algae hosted by foraminifera may be carbon limited and thus benefit from increased DIC availability
(Uthicke and Fabricius 2012). Similar results were observed in an experiment with coral Acropora
eurystoma, which showed increased photosynthesis in elevated DIC concentrations, presuming carbon
limitation of zooxanthellae in ambient water conditions (Chauvin et al. 2011). As shown by Borowitzka
and Larkum (1976b) Halimeda tuna photosynthesis saturates at DIC ≤ 3 mmol L−1 (DIC ∼1900 µmol
kgSW−1 in present study, kgSW−1 = per kg seawater). Halimeda photosynthesis utilizes dissolved CO2
as the primary carbon source however HCO3– can also be used, but at a reduced rate (Borowitzka and
Larkum 1976b). Moreover, in experiments H. tuna photosynthesis saturated at 27 µmol L−1 CO2 and
2274 µmol L−1 HCO3– (Borowitzka and Larkum 1976b), both indicating photosynthesis should be DIC
limited under present environmental conditions at control sites (CO2 = 7.78 µmol kgSW−1 , Table 3.1).
Potentially, ambient PAR level of experimental incubations for H. digitata and H. opuntia (39 and 281
µmol photons m−2 s−1 , respectively) were below light saturation and organisms were subjected to light
limitation before DIC limitation could be observed.
In-situ calcification rates showed that both H. digitata and H. opuntia had increased calcification
rates in the light at the seep compared to the control site. This is an indication that calcification of some
Halimeda spp. may benefit from increased DIC availability. Increased bicarbonate concentrations at
the seep site may thus have relieved the organisms of limiting conditions for calcification. Borowitzka
and Larkum (1976b) showed that H. tuna calcification is saturated at about 5 mmol L−1 ∑CO2 , indicating carbon limitation at control conditions of the present study (DIC = 1.892 mmol kgSW−1 , Table
3.1). Calcification in Halimeda spp. is dependent on diffusion of CO32– and Ca2+ into the intercellular
space, suggesting the supply of DIC can become limiting (Borowitzka and Larkum 1977; Borowitzka
and Larkum 1976a; Borowitzka and Larkum 1976b). Thus, elevated DIC concentrations at seep sites
(DIC = 2163 µmol kgSW−1 , Table 3.1) may explain increased calcification rates of H. digitata and H.
opuntia, compared to control sites. However, low water motion in chambers may also have increased
the thickness of boundary layers on the organisms’ surface and thus exacerbated the positive effect of
elevated DIC on calcification as discussed by Langdon and Atkinson (2005) and seen for coral photosyn-
76
3 - Halimeda growing at CO2 seeps
thesis (Chauvin et al. 2011). Therefore, potentially a combination of DIC under-saturation at ambient
seawater conditions (1.892 mmol kgSW−1 ) and increased boundary layers in incubation chambers may
have resulted in increased calcification rates at the seep site, as presumed by Chauvin et al. (2011).
In contrast incubations in darkness showed calcification rates of H. opuntia were strongly and negatively impacted by decreased pH leading to decreased calcification and dissolution at the seep compared
to the control site. Positive calcification rates were still observed at the control site in darkness, despite
respiratory CO2 release. While Borowitzka (1986) showed some decalcification in ambient seawater
conditions due to respiratory CO2 during the night, he also showed much of the DIC, which is released
into the intracellular space, can be refixed in the morning. A potential reason why H. opuntia showed
significant impacts of elevated pCO2 during darkness, but H. digitata did not may emerge from the different morphology of both species. H. opuntia phylloids have a larger surface area to volume ratio and
thus calcified areas are more exposed to their physical environment. This may have an advantage during the day, when a proportionally larger surface area facilitates diffusion processes and thus increases
productivity and calcification. However, at night this property may be a disadvantage, where a higher
exposure to elevated CO2 conditions, may increase negative impacts, as seen in the present study. The
observed CaCO3 dissolution of H. opuntia in the present study is in agreement with a laboratory experiment, which showed no negative effect of OA on two photosynthesizing and calcifying organisms
(Acropora millepora and H. opuntia) in the light, but during the dark (Vogel et al. 2015). The latter
study also observed this phenomenon in incubation conditions with water movement, suggesting low
water motion did not exacerbate dissolution in darkness in the present study. Moreover, this observation
agrees with results from Borowitzka and Larkum (1976b), which showed that respiration can inhibit
calcification of Halimeda by decreasing pH and [CO32– ] and presumed that respiratory CO2 production
could lead to CaCO3 dissolution. In contrast, during light no negative impacts of OA on calcification
could be observed. Photosynthesis may thus offset impacts of OA by buffering pH during light, increase
Ωar and therefore facilitate deposition of CaCO3 (Al-Horani et al. 2003; Borowitzka and Larkum 1976b;
Goreau 1959; Vogel et al. 2015).
Calculated net calcification rates did not differ between control and seep site for neither H. digitata, nor H. opuntia. Increased light calcification and decreased dark calcification rates at the seep site
cancelled out each other to no difference of net calcification rates between sites. This observation is
in agreement with results derived from laboratory experiments on H. opuntia (Vogel et al. 2015), and
re-emphasizes our in-situ observations that show Halimeda spp. are capable to grow and calcify at high
CO2 .
Elevated CO2 showed opposite effects on inorganic carbon content of the two species with increased
C inorg in H. digitata, but decreased values in H. opuntia at the seep site, compared to controls. CaCO3
77
3 - Halimeda growing at CO2 seeps
dissolution during the dark may lead to a marginally lowered C inorg content of H. opuntia. Similarly,
increased C inorg content of H. digitata at the seep site may be explained by elevated calcification rates
during the light at the seep site. Decreased C inorg content (despite unaffected net calcification rates) of
H. opuntia was previously observed by Hofmann et al. (2014). Moreover, a previous study on Padina
showed lower CaCO3 content at PNG seep sites compared to controls (Johnson and Carpenter 2012).
Increased C inorg content of H. digitata is in contrast to previously discussed observations, but is in agreement with the calcification rates measured, showing a trend (non-significant) towards slightly increased
net calcification rates at the seep compared to control site. Decreased C org and increased C inorg of H.
digitata also reflected in a decreased C org : C inorg ratio at the seep site compared to controls. Notably,
despite changes in C inorg of H. digitata and H. opuntia, both were still capable to grow and to deposit
CaCO3 even in conditions temporary corrosive to aragonite (Ωar under saturation).
Both H. digitata and H. opuntia tissues showed increased negative δ 13C signatures (i.e. increased
fractionation of carbon isotopes) at the seep compared to the control site, indicating either 13 C depletion
or proportionally higher 12 C in tissues. In addition, tissues of both species showed depletion in 13 C after
14 day transplantation to the seep site, while thalli that remained at the control site showed the same
isotopic signatures as originally determined. Thus, we showed that the environment at the seep site led
to a depletion of 13 C and an increased fractionation of carbon isotopes in Halimeda spp. tissue compared
to controls and that these changes are detectable after as little as 14 days. This was most likely due to
increased CO2 availability at the seep site. Since CO2 is isotopically light compared to HCO3– (∼10h)
(Laws et al. 2002) an increased fractionation of carbon isotopes indicates an increased utilization of
CO2 over HCO3– at the seep site. This observation is an indication that Halimeda spp. may benefit
from increased CO2 availability at the seep site for photosynthetic carbon acquisition and organic carbon
assimilation in their tissue. In Halimeda spp. photosynthesis utilizes CO2 as primary source of inorganic
carbon. Therefore, elevated CO2 availability at the seep sites may facilitate the diffusion process and thus
the uptake of CO2 compared to the control sites. This observation has also been demonstrated for noncalcifying algae (Carvalho et al. 2010). Theoretically, calcification may alter fractionation of δ in organic
tissue due to supply of CO2 for photosynthesis derived from heavier HCO3– during calcification (Ca2+ +
2 HCO3– −−→ CaCO3 + CO2 + H2 O) (Laws et al. 2002). However, Laws et al. (2002) also provide
evidence that calcification does not supply heavier CO2 from calcification for photosynthesis (Buitenhuis
et al. 1999; Riebesell and Wolf-Gladrow 1995). Unaltered rates of net photosynthesis suggested that
both species did not benefit from elevated CO2 at the seep site and thus were not DIC (i.e. CO2 ) limited
under the experimental conditions. However, as discussed above, it is possible that the light conditions
during incubations were below saturation explaining why DIC limitation in net photosynthesis was not
detected. Nonetheless, carbon isotope signatures from transplants indicate Halimeda spp. may benefit
78
3 - Halimeda growing at CO2 seeps
from increased CO2 at the seep site, when integrated over several days.
With this study we provide evidence that several Halimeda spp. are tolerant of increasing pCO2 .
Some species (e.g. H. opuntia) that are found at the seep site are reported to be sensitive to OA. However,
this conclusion is derived from laboratory experiments in artificial conditions, while the results from the
present study are based on long-term exposure in a natural environment with natural light, nutrient and
flow regimes. Therefore, we suggest re-evaluating the impact of OA as single stressor on Halimeda spp.
However, in future environmental conditions, organisms will not only have to deal with OA, but also
with other environmental stressors, such as ocean warming and land runoff, which may have additive
or synergistic effects. Additional investigations are necessary to evaluate impacts of several stressors
combined.
Acknowledgments
We thank the crew of the M.V. Chertan for their sincere hospitality and their professional help in conducting this study. We are grateful to the local families at Dobu Island and Upa-Upasina for approving
our work in their neighborhood. Many thanks to Craig Humphrey for his support during the field work.
We thank Peter Davern, Mick Donaldson and Peter Coumbis for their help concerning the shipment of
our experimental equipment and legal advice. We thank the Leibniz Center for Tropical Marine Ecology, Dorothee Dasbach and Friedrich Meyer for helping with elemental and stable isotope analyzes.
The study was funded by the Australian Institute of Marine Science and conducted with the support of
funding from the Australian Government’s National Environmental Research Program.
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3 - Halimeda growing at CO2 seeps
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84
Chapter 4
Decreased light availability can amplify
negative impacts of ocean acidification on
calcifying coral reef organisms
Nikolas Vogel 1,2,3 , Friedrich Wilhelm Meyer 2,3 Christian Wild 2,3 and Sven Uthicke 1
(1)
Australian Institute of Marine Science, PMB 3, Townsville MC, Queensland 4810, Australia
(2)
Leibniz Center for Tropical Marine Ecology, Fahrenheitstraße 6, 28359 Bremen, Germany
(3)
Faculty of Biology and Chemistry (FB 2), University of Bremen, 28359 Bremen, Germany
Keywords: pH, turbidity, calcification, dissolution, photosynthesis, corals, algae, Acropora millepora,
Halimeda opuntia
This chapter is accepted for publication in Marine Ecology Progress Series (2015) doi: 10.3354/meps11088
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Abstract
Coral reef organisms are increasingly and simultaneously affected by global and local stressors, such as
ocean acidification (OA) and reduced light availability. However, knowledge of the interplay between
OA and light availability is scarce. We exposed two calcifying coral reef species (the scleractinian coral
Acropora millepora and the green alga Halimeda opuntia) to combinations of ambient and increased
pCO2 (427 and 1073 µatm, respectively), and two light intensities (35 and 150 µmol photons m−2 s−1 )
for 16 days. We evaluated the individual and combined effects of these two stressors on weight increase,
calcification rates, O2 fluxes and chlorophyll content for the species investigated. Weight increase of A.
millepora was significantly reduced by OA (48%) and low light intensity (96%) compared to controls.
While OA did not affect coral calcification in the light, it decreased calcification in the dark by 155%,
leading to dissolution of the skeleton. H. opuntia weight increase was not affected by OA, but decreased
(40%) at low light. OA did not affect algae calcification in the light, but decreased calcification in the
dark by 164%, leading to dissolution. Low light significantly reduced gross photosynthesis (56 and
57%), net photosynthesis (62 and 60%) and respiration (43 and 48%) of A. millepora and H. opuntia,
respectively. In contrast to A. millepora, H. opuntia significantly increased chlorophyll content by 15%
over the course of the experiment. No interactive effects of OA and low light intensity were found on any
response variable for either organism. However, A. millepora exhibited additive effects of OA and low
light, while H. opuntia was only affected by low light. Thus, this study suggests that negative effects of
low light and OA are additive on corals, which may have implications for management of river discharge
into coastal coral reefs.
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4.1 Introduction
Anthropogenically increased carbon dioxide (CO2 ) introduced into the atmosphere is changing the
earth’s climate. In addition to aggravating the greenhouse effect and thus driving global warming, approximately one-third of the atmospheric CO2 is taken up by the oceans (Sabine et al. 2004). CO2
which is added to the oceanic carbonate system, increases hydrogen ion concentrations and thus leads
to a reduction of seawater pH (ocean acidification, OA) (Golubik et al. 1979; Kleypas and Langdon
2006). Depending on the ‘representative concentration pathways’ (RCPs) followed, atmospheric CO2
is predicted to rise from ∼395 µatm at present (Dlugokencky and Tans 2014) to between 850 and 1370
µatm by the year 2100 (RCP6.0 and RCP8.5, respectively), which is correlated with a further decrease in
ocean pH unless drastic reductions in output and/or an increase in carbon capture are achieved (RCP2.6
and RCP4.5) (Moss et al. 2010). In turn, a reduction of pH leads to a shift in the oceanic carbonate
system, which results in a decreased calcium carbonate (CaCO3 ) saturation state (Ω) of seawater. Recent studies revealed negative effects of decreased Ω on growth/calcification of a vast range of coral reef
organisms, leading to predictions of shifts in community structures, loss of framework builders and loss
of coral reef biodiversity under future conditions (Gattuso et al. 1998; Langdon et al. 2000; Orr et al.
2005; Ries et al. 2009; Fabricius et al. 2011; Uthicke and Fabricius 2012; Fabricius et al. 2014).
In addition to increasing sea surface temperature (SST) and OA, often local disturbances (such as
elevated organic and inorganic nutrients, increased turbidity or decreased salinity) present additional
pressures on coral reef organisms at inshore reefs exposed to land-runoff (Bell 1992; Fabricius 2005;
Wooldridge et al. 2006; Fabricius 2011; Uthicke et al. 2011). Water quality is known to affect inshore
reef communities, leading to declines in hard coral diversity and increased macroalgae richness (Fabricius 2005; Schaffelke et al. 2005; De’ath and Fabricius 2010). Decrease in water quality on the Great
Barrier Reef (GBR) has been linked to anthropogenic activities associated with land-use, and has been
in decline since European settlement (McCulloch et al. 2003; Roff et al. 2013). Predominantly during
summer months, an increase in precipitation and hence riverine runoff results in more severe consequences on near-shore reef communities. Combinations of global and local stressors may have additive
or synergistic effects and may push organisms closer to tolerance thresholds. For instance, interactive
effects of OA and irradiance, OA and eutrophication, ocean warming (OW) and herbicides, or OW
and eutrophication have all been shown to have impacts on several coral reef organisms (Langdon and
Atkinson 2005; Chauvin et al. 2011; Negri et al. 2011; Uthicke et al. 2011; Comeau et al. 2014).
For many calcareous reef organisms photosynthesis is essential for energy supply, calcification
and/or survival, either because they are autotrophic primary producers or exhibit mixotrophic carbon
acquisition. Scleractinian corals host photosynthetically active dinoflagellates as endosymbionts, which
provide important energy to the host (Goreau 1959; Wainwright 1963). Moreover, by fixing CO2 from
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the environment during light, they increase cellular, surface and boundary layer pH levels and therefore facilitate the precipitation of CaCO3 by elevating the aragonite saturation state (Ωar ) (Goreau 1959;
Al-Horani et al. 2003). For the calcifying green alga genus Halimeda, photosynthesis is important for
calcification, since some species do not possess active calcification mechanisms. In fact, in some Halimeda species, calcification is a byproduct of increased intracellular pH from photosynthesis, which
results in abiotic precipitation of aragonite needles (de Beer and Larkum 2001). By increasing the pH
levels in the environment, photosynthesis may even protect organisms against OA, as long as sufficient
light is available (de Beer et al. 2000; Al-Horani et al. 2003).
At reefs susceptible to land runoff, increased turbidity leads to reduced light availability and therefore decreased photosynthetically available radiation (PAR) for photosynthesizing organisms. While
sediment from rivers and dredging activities directly increase turbidity, elevated nutrient levels from
agricultural land runoff increase turbidity indirectly. Inshore eutrophication can enhance the abundance
of chlorophyll, phytoplankton and microalgae blooms in the water column (Bell 1992; Devlin and Schaffelke 2009), which in turn leads to a reduction of PAR. Consequently, reduced PAR due to increased
turbidity with increasing OA may have additional negative effects on growth, calcification and other responses of coral reef organisms. As shown in previous studies, calcification and photosynthesis in corals
decrease with increasing turbidity (Kendall Jr et al. 1983; Kendall Jr et al. 1985) and decreasing light
intensity (Marubini et al. 2001; Mass et al. 2007). However, knowledge of the interaction between OA
and low light conditions is scarce, even though the interplay between these stressors may be crucial. Local stressors that affect light availability are generally easier to manage than global stressors; therefore,
it is important to understand these interactions, as findings from manipulative experiments can be used
to take action via environmental management plans aiming to reduce stressors on coral reef organisms.
Given that photosynthesis plays a crucial role in calcification, and OA has impacts on calcification of
many organisms, it is surprising that the present study is one of the first to investigate this interaction.
The aim of the present study was to investigate the individual and interactive effects of OA and
decreased PAR on two different coral reef taxa, the scleractinian coral Acropora millepora and the calcifying green alga Halimeda opuntia. A. millepora is common and widespread over tropical coral reefs
and contributes to primary productivity, carbonate production and reef development. H. opuntia is a crucial, fast growing primary producer, commonly found on tropical coral reefs. Halimeda spp. contribute
considerably to carbonate production, sediment formation and play an important role in the benthic community by providing habitat for many invertebrate species (Wefer 1980; Freile et al. 1995; Rees et al.
2007; Fukunaga 2008). Thus, we conducted a laboratory experiment using controlled conditions and determined the response parameters, growth rates (measured by buoyant weight), calcification rates in light
and in dark (measured by alkalinity anomaly), O2 fluxes (productivity and respiration) and chlorophyll
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a (Chl a) content.
4.2 Methods
Specimen collection and preparation
Colonies of the coral Acropora millepora were collected from an inshore fringing reef next to Pelorus
Island (central section of the GBR; S 18° 33.001’, E 146° 29.304’) between 2-4 m below lowest astronomical tide (LAT). After colonies were fragmented, individual coral nubbins were glued onto stubs
and kept at the Australian Institute of Marine Science (AIMS, Townsville) in flow-through (recirculating
flow ∼1200 L h−1 ) aquaria facilities under plasma light (150 µmol photons m−2 s−1 ) for more than three
months. Nubbins were transferred into experimental tanks two weeks prior to the start of experiment to
acclimate to experimental control conditions. Specimens of the calcifying green alga Halimeda opuntia
were collected from a fringing reef next to Orpheus Island, also an inshore reef in the central section of
the GBR (S 18° 36.737’, E 146° 29.110’) between 0.5-1.0 m below LAT. Alga fragments (each with 50
to 100 phylloids) were acclimated in experimental aquaria under control conditions for two weeks until
the start of the experiment.
Light regimes at the collection site of A. millepora were similar to control light conditions in the
experiment. H. opuntia was subjected to lower light conditions in the experiment than occurred in the
field, due to collection from shallower depths. However, both organisms were acclimated to the same
‘control light’ conditions for two weeks before the start of the experiment. Light levels chosen as the
control and low light conditions were well within average ranges found between 3-6 m below LAT at
mid-shelf and inshore reefs on the GBR, respectively (Uthicke and Altenrath 2010). Moreover, light
data were collected with light loggers (Odyssey) simultaneously at a mid-shelf location Rib Reef (S
18° 28.785’, E 146° 52.256’) and an inshore location Orpheus Island (S 18° 38.949’, E 146° 29.183)
at 5 m below LAT over a period of 18 days in February 2013. At Rib Reef, daily light sums averaged
10.45 mol photons m−2 d−1 ranging from 4.58 to 13.35 mol photons m−2 d−1 . Daily light sums at
Orpheus Island averaged 1.95 mol photons m−2 d−1 ranging from 0.22 to 4.66 mol photons m−2 d−1 .
Hence, experimental conditions (daily light sums of 6.48 mol photons m−2 d−1 for controls and 1.51
mol photons m−2 d−1 for low light regimes) were well within naturally occurring light intensities at ∼5
m below LAT at mid-shelf and inshore locations of the GBR.
Experimental setup
The manipulative aquaria experiment was carried out in flow-through conditions over a period of 16
days between July and August 2012 at AIMS. After a two weeks acclimation period, four nubbins of A.
90
4 - Ocean acidification and low light
millepora and two fragments of H. opuntia were allocated to each of the twelve experimental aquaria.
Four treatments with three replicate tanks (working volume 17.5 L) were placed in alternating order.
Treatments consisted of combinations of ambient pCO2 (427 µatm), high pCO2 (1073 µatm), low light
(35 µmol photons m−2 s−1 ) and control light (150 µmol photons m−2 s−1 ). High pCO2 conditions
corresponded to projections between the RCP6.0 and RCP8.5 scenario for the year 2100 (Moss et al.
2010). Light intensities were chosen from average PAR readings from an inshore and mid-shelf reef
at ∼5 m below LAT, present during the summer months. Water flow was provided with fresh filtered
(0.5 µm) seawater at 25 °C, with a salinity of 34.5 ppt, at a flow rate of 150 mL min−1 . Irradiance
was delivered by white light LED (6000 K, Aqua Illumination), covering the full color spectrum. Light
levels were set to a 12 h:12 h light:dark cycle. Additional aquaria pumps (250 L h−1 , AquarWorld)
were fitted into each tank to assure water movement. Target pH levels were achieved by an automatic
CO2 injection system (Aqua Medic) controlled by potentiometric pH sensors, as described in Vogel and
Uthicke (2012).
Carbonate system parameters
Total alkalinity (TA) was determined by gran titration with a Metrohm 855 robotic titrosampler using
0.5 M HCl (see also Uthicke and Fabricius 2012). Total alkalinity was calculated by non-linear regression fitting between pH 3.5 and 3.0 and was corrected to certified reference material (CRM Batch
106, A. Dickson, Scripps Oceanographic Institute). Seawater pH, temperature and millivolts (mV) were
measured daily (including early morning and evening measurements to incorporate diurnal fluctuations)
with a temperature corrected, hand-held pH meter (WTW), calibrated on the NIST (National Institute
of Standards and Technology) scale. Millivolt and temperature readings were utilized to calculate pH
on a total (pH total) scale. Carbonate system parameters (Table 4.1) were calculated with CO2calc software (Robbins et al. 2010) utilizing TA and pH total values and CO2 constants from Lueker et al. (2000).
Carbonate system parameters were calculated from measurements in each aquarium and three sampling
events over the course of the experiment. Calculated pCO2 levels yielded averages of 427 µatm for controls and 1073 µatm for future scenario conditions (Table 4.1). Ωar yielded averages of 3.3 and 1.7 for
controls and high pCO2 treatments, respectively.
Growth rates
Growth of organisms was determined by the buoyant weight technique. Individual specimens were
single-weighted (accuracy: 0.1 mg, Mettler Toledo) in a custom built buoyant weight setup with water
jacket and seawater of constant temperature (25 °C) and salinity (34.5 ppt) at the start and at the end of
the experiment. Growth of organisms was expressed as daily percentage of change.
91
Temp
[°C]
25.4 (0.2)
25.3 (0.1)
25.7 (0.8)
25.5 (0.3)
pHtotal
8.038 (0.031)
7.707 (0.038)
8.008 (0.015)
7.693 (0.016)
Treatment
control pCO2
+ control light
high pCO2
+ control light
control pCO2
+ low light
high pCO2
+ low light
2288 (5)
2278 (7)
2281 (7)
TA
[µmol kgSW-1]
2276 (13)
2164 (8)
1983 (11)
2160 (5)
DIC
[µmol kgSW-1]
1990 (17)
1076 (18)
433 (15)
1069 (71)
pCO2
[µatm]
421 (19)
2029 (9)
1762 (17)
2026 (8)
HCO3[µmol kgSW-1]
1774 (21)
106 (2)
209 (7)
104 (6)
CO32[µmol kgSW-1]
203 (5)
30 (1)
12 (1)
30 (2)
CO2
[µmol kgSW-1]
12 (1)
Table 4.1: Carbonate system parameters of experimental conditions. Data is given as means and standard deviations
1.7 (0.0)
3.4 (0.1)
1.7 (0.1)
3.2 (0.1)
Ωar
4 - Ocean acidification and low light
92
4 - Ocean acidification and low light
Calcification in light and dark, net photosynthesis and respiration
After 16 days in experimental conditions, two individuals of each species and replicate tank were incubated for 1 h in the light and thereafter 1 h in the dark to determine calcification and photosynthetic
rates. Light intensity and seawater pH of incubations corresponded to treatment condition of each organism. One experimental run consisted of 12 parallel incubations in 200 mL incubation chambers,
including two blanks per treatment. To assure constant water temperature during incubation, chambers
were placed into a flow-through water bath at 25 °C. Additionally, magnetic stirrer bars ensured water
movement within the incubation chambers.
Calcification rates in light and dark were determined by the alkalinity anomaly technique (Chisholm
and Gattuso 1991). A subsample of 50 mL was pipetted from the incubation seawater and directly
titrated for TA on a Metrohm 855 (as described above). CaCO3 precipitation or dissolution in µM
C h−1 was calculated following Gao and Zheng (2010) and standardized to organism surface area (A.
millepora) or buoyant weight (H. opuntia). Daily net calcification was calculated by 12 h of daylight and
12 h of darkness. We determined surface areas of coral nubbins using the wax-weight method (Veal et al.
2010) and chose buoyant weight as standardization for the algae due to their highly three dimensional
structures and lowest variability in data.
Net photosynthesis in the light or dark respiration were monitored consecutively during the incubations by three Firesting 4-channel oxygen meters (Pyroscience), which were connected to each chamber
with fiber optic cables. Gross photosynthesis, net photosynthesis and respiration rates were expressed
as µM O2 h−1 and standardized to organism surface area (A. millepora) or buoyant weight (H. opuntia).
Pigment content
Chl a content of algae tissue was determined spectrophotometrically. Organisms were frozen to −80
°C after incubations. Similar to the Chl a extraction described in Schmidt et al. (2011) and Vogel and
Uthicke (2012), apical segments of algae were placed in 15 mL Falcon tubes on ice and 4 mL of cold
ethanol (95% EtOH) was added. After crushing the segments with a homogenizer, extracts were heatshocked in a water bath (78 °C for 5 min), and left in a fridge for 24 h extraction. Absorbencies on 750
and 664 nm were read on a Powerwave microplate reader (BioTek). Chl a content was calculated with
equations by Mush (1980) and standardized to segment fresh weight.
Chl a content of coral A. millepora was determined after coral tissue was stripped from the skeleton
with an air gun utilizing fresh, ultra-filtered (0.2 µm) seawater. Zooxanthellae were isolated from the
host tissue and re-suspended in 2 mL of ethanol (EtOH 95%), heat-shocked and extracted for 24 h in the
cold. Absorbencies were read (as described above) and Chl a contents were calculated standardized to
nubbin surface area.
93
4 - Ocean acidification and low light
Statistical analysis
We statistically tested growth rates, net-, light- and dark-calcification rates, gross photosynthesis, net
photosynthesis, respiration, and Chl a content for significant differences between experimental treatment
conditions. Levene’s tests for equal variances were performed on datasets in the software program R
(R Development Core Team 2014). If necessary, response variables were log10 transformed prior to
analyzes, to fulfill assumptions of equal of variances. Mixed Linear Model ANOVAs were conducted on
datasets with NCSS software (Hintze 2007) with pH and light treatment as fixed factors. Replicate tanks
were considered as nested (random) factor. To distinguish significantly differing groups we conducted
Tukey-Kramer multiple comparison tests.
4.3 Results
The interaction between pCO2 and light intensity was not significant for any treatment parameter (Table
4.2). However, the coral A. millepora exhibited additive negative effects of high pCO2 and low light
conditions on growth rates and calcification rates in the dark (Table 4.3).
Mean growth rates (Fig. 4.1) of A. millepora were significantly (Table 4.2, p = 0.032) reduced in
high pCO2 by 48% compared to controls, while the growth rate of H. opuntia was not impacted by high
pCO2 . Low light significantly (p < 0.001) reduced growth rates of A. millepora by 96% compared to
controls, while growth of H. opuntia was not significantly reduced (p = 0.069).
Net calcification rates (Fig. 4.1) of A. millepora and H. opuntia (measured by alkalinity anomaly)
followed similar trends as growth rates measured by buoyant weight. Both buoyant weight method
(integrated over longer term) and alkalinity anomaly method (determined in the short term) showed
compatible results, and a similar pattern for treatment effects.
Elevated pCO2 had no effect on organisms’ calcification during the light (Fig. 4.1). While low light
significantly (Table 4.2, p = 0.006) reduced light calcification of A. millepora by 83%, H. opuntia light
calcification was not reduced in low light conditions.
The most distinct effect of elevated pCO2 however, was observed during dark incubations (Fig. 4.1).
Elevated pCO2 significantly reduced calcification of A. millepora and H. opuntia (Table 4.2, p = 0.002
and p < 0.001, respectively) by 155 and 164%, respectively, with decalcification of their skeletons
occurring in high pCO2 conditions. Reduction of calcification by more than 100% indicates decalcification. Moreover, low light conditions significantly (Table 4.2, p = 0.002) reduced dark calcification of
A. millepora by 155% compared to controls.
High pCO2 did not show any effect on gross photosynthesis, net photosynthesis or respiration of A.
millepora or H. opuntia (Fig. 4.2). However, low light levels significantly reduced gross photosynthesis
94
4 - Ocean acidification and low light
H. opuntia
3
***
p = 0.060
***
**
427 [µatm]1073 [µatm]150 [µmol] 35 [µmol]
Treatment
Net calcification [µM C d-1 gBW-1]
0
50 100 150 200 250
0
**
Light calcification [µM C h-1 gBW-1]
0
5
10
15
20
**
Dark calcification [µM C h-1 gBW-1]
−4 −3 −2 −1 0 1 2 3 4
Net calcification [µM C d-1 cm-2]
−2 0
2
4
6
8
Light calcification [µM C h-1 cm-2]
−0.2
0.0
0.2
0.4
0.6
Dark calcification [µM C h-1 cm-2]
−0.2 0.0
0.2
0.4
0.6
p = 0.069
Growth [% change d-1]
1
2
*
−0.2
Growth [% change d-1]
0.0
0.2
0.4
0.6
A. millepora
***
427 [µatm]1073 [µatm]150 [µmol] 35 [µmol]
Treatment
Figure 4.1: Growth rates, net-, light- and dark calcification rates of A. millepora and H. opuntia after
16 days exposure to experimental conditions. Data was pooled across pCO2 and light treatment because
there was no significant interaction. X-axes represent OA treatments in µatm pCO2 and light treatments
in µmol photons m−2 s−1 . Whiskers represent lower and upper extremes. Brackets indicate significant
differences in ANOVAs, with significance levels ∗ p < 0.05, ∗∗ p < 0.001, ∗ ∗ ∗ p < 0.0001
95
4 - Ocean acidification and low light
Table 4.2: Mixed Model ANOVA results for A. millepora and H. opuntia
Response
variable
Growth
Net
calcification
Light
calcification
Dark
calcification
Gross
photosynthesis
Net
photosynthesis
Respiration
Chlorophyll a
Source
of variation
pCO2
Light
pCO2:Light
Tank
Residual
pCO2
Light
pCO2:Light
Tank
Residual
pCO2
Light
pCO2:Light
Tank
Residual
pCO2
Light
pCO2:Light
Tank
Residual
pCO2
Light
pCO2:Light
Tank
Residual
pCO2
Light
pCO2:Light
Tank
Residual
pCO2
Light
pCO2:Light
Tank
Residual
pCO2
Light
pCO2:Light
Tank
Residual
df
1
1
1
8
29
1
1
1
8
11
1
1
1
8
11
1
1
1
8
11
1
1
1
8
12
1
1
1
8
12
1
1
1
8
12
1
1
1
8
12
A. millepora
F
p
6.78
64.98
0.17
2.44
0.032*
< 0.001*
0.692
0.037*
4.78
25.95
1.02
1.09
0.060
0.001*
0.342
0.434
0.14
13.72
0.18
1.61
0.722
0.006*
0.684
0.227
21.45
21.36
2.54
0.41
0.002*
0.002*
0.150
0.892
0.00
208.60
0.00
1.07
1.000
<0.001*
1.000
0.441
0.00
267.99
0.21
0.77
1.000
< 0.001*
0.661
0.636
0.0
31.31
0.23
4.13
0.947
0.001*
0.646
0.014*
0.34
2.23
0.02
3.17
0.577
0.174
0.895
0.035*
df
1
1
1
8
8
1
1
1
8
12
1
1
1
8
12
1
1
1
8
12
1
1
1
8
11
1
1
1
8
11
1
1
1
8
11
1
1
1
8
12
H. opuntia
F
p
0.60
4.41
0.05
1.71
0.460
0.069
0.833
0.231
3.59
1.09
0.31
2.26
0.095
0.328
0.594
0.098
0.01
0.48
0.45
2.14
0.910
0.509
0.523
0.113
57.53
2.81
0.09
4.15
< 0.001*
0.132
0.772
0.014*
3.51
38.76
0.41
1.53
0.098
0.003*
0.542
0.252
3.08
35.85
0.47
1.40
0.117
< 0.001*
0.512
0.297
2.85
25.38
0.06
3.89
0.130
0.001*
0.817
0.020*
1.09
6.69
1.10
1.16
0.326
0.032*
0.324
0.398
by 56 and 57% (both p < 0.001), net photosynthesis by 63 and 60% (both p < 0.001), and respiration
by 43 and 48% (both p = 0.001) for A. millepora and H. opuntia, respectively (Table 4.2).
Chl a content (Fig. 4.2) of H. opuntia was significantly (Table 4.2, p = 0.032) increased by 15%
in low light conditions compared to controls. Chl a within the coral A. millepora was not significantly
different among treatments (Table 4.2).
96
Respiration [µM O2 h-1 cm-2]
−0.6
−0.4
−0.2
0.0
***
***
***
***
2
Chlorophyll a [µg cm-2]
4
6
8
10
Chlorophyll a [µg gFW-1]
200
300
400
500
***
Net photosynthesis [µM O2 h-1 gBW-1] Gross photosynthesis [µM O2 h-1 gBW-1]
0
5
10 15 20 25 30
0 5 10 15 20 25 30 35
***
H. opuntia
Respiration [µM O2 h-1 gBW-1]
−8
−6
−4
−2
0
A. millepora
Net photosynthesis [µM O2 h-1 cm-2]
0.0
0.5
1.0
1.5
2.0
Gross photosynthesis [µM O2 h-1 cm-2]
0.0
0.5
1.0
1.5
2.0
4 - Ocean acidification and low light
*
427 [µatm]1073 [µatm] 150 [µmol] 35 [µmol]
427 [µatm]1073 [µatm] 150 [µmol] 35 [µmol]
Treatment
Treatment
Figure 4.2: Gross-, net photosynthesis, respiration and Chl a content of of A. millepora and H. opuntia
after 16 days exposure to experimental conditions. Data was pooled across pCO2 and light treatment
because there was no significant interaction. X-axes represent OA treatments in µatm pCO2 and light
treatments in µmol photons m−2 s−1 . Whiskers represent lower and upper extremes. Brackets indicate
significant differences in ANOVAs, with significance levels ∗ p < 0.05, ∗∗ p < 0.001, ∗ ∗ ∗ p < 0.0001
97
4 - Ocean acidification and low light
Table 4.3: Summary of effects of treatment variables on response parameters for A. millepora and H.
opuntia. Decreases > 100% are possible due to decalcification. ‘ns’ indicates no significant treatment
effect, ‘measured additive effects’ represent differences of means between the control pCO2 /high light
and high pCO2 /low light treatment
Response parameter
Growth
Net calcification
Light calcification
Dark calcification
Gross photosynthesis
Net photosynthesis
Respiration
Chlorophyll a content
Species
A. millepora
H. opuntia
A. millepora
H. opuntia
A. millepora
H. opuntia
A. millepora
H. opuntia
A. millepora
H. opuntia
A. millepora
H. opuntia
A. millepora
H. opuntia
A. millepora
H. opuntia
pCO2
- 48%
ns
- 57%
ns
ns
ns
- 155%
- 164%
ns
ns
ns
ns
ns
ns
ns
ns
Light
- 96%
ns
- 99%
ns
- 83%
ns
- 155%
ns
- 56%
- 57%
- 62%
- 60%
- 43%
- 48%
ns
- 15%
Predicted additive effect
- 144%
ns
- 156%
ns
ns
ns
- 310%
ns
ns
ns
ns
ns
ns
ns
ns
ns
Measured additive effect
- 114%
ns
- 127%
ns
ns
ns
- 204%
ns
ns
ns
ns
ns
ns
ns
ns
ns
4.4 Discussion
Negative effects of OA on a range of marine calcifying organisms have been well documented (e.g.
Orr et al. 2005; Guinotte and Fabry 2008; Kleypas and Yates 2009; Hendriks et al. 2010; Pandolfi et
al. 2011; Fabricius et al. 2014). The present study demonstrated that A. millepora in particular was
negatively affected by elevated pCO2 , and that decreased light availability can have an additional impact
on both organisms. Although the factors were not synergistic (i.e. higher than the effect of individual
stressors added together, see Table 4.3), additive effects on some response parameters clearly suggest that
some corals may better cope with global OA if PAR is not reduced at the same time. With increasing
OA, many corals will experience lower growth rates in future. If PAR is reduced at the same time,
the inhibitors (elevated pCO2 and reduced PAR) are additive (Dunne 2010) and growth rates of corals
impacted by reduced PAR at inshore reefs will be more compromised than those of corals on mid-shelf
reefs. Therefore, by improving water quality the additional stressor of low light availability for inshore
corals can be reduced.
In the present study we observed a significant reduction of growth rates for A. millepora in high
pCO2 conditions after 16 days in experimental conditions. To date, experiments have revealed mixed responses of coral reef calcifiers towards altered pCO2 conditions, showing decreased growth/calcification
in elevated pCO2 , or no effect at all (e.g. Ries et al. 2009; Comeau et al. 2013b). Yet, the present study
revealed that A. millepora belongs to the group which is likely to experience negative impacts under future environmental conditions. Presumably, reduced Ωar is the driving factor of decreased calcification
98
4 - Ocean acidification and low light
of corals in a high pCO2 environment (Schneider and Erez 2006; Marubini et al. 2008). Due to a decrease of Ωar in OA conditions, many organisms become impaired in building CaCO3 skeletons (Raven
et al. 2005; Kleypas and Langdon 2006; Hoegh-Guldberg et al. 2007). In contrast, H. opuntia showed no
significant trend on growth rates in relation to Ωar /pCO2 . Some previous studies suggest that Halimeda
spp. may be impacted in future OA conditions, showing reduced growth in elevated pCO2 (Ries et al.
2009; Price et al. 2011; Sinutok et al. 2011). However, similar to corals, Halimeda spp. exhibit different
growth forms with associated morphological distinctions. Halimeda spp. occur as heavily calcified and
less calcified species, sand-dwellers and rock-anchored species as well as species with different sizes
and shapes of phylloids. Halimeda spp. with smaller phylloids have a higher surface to volume ratio
than with larger phylloids and hence have a higher exposure to their physical environment. As shown
by Comeau et al. (2013b), Halimeda macroloba showed no impact of increased pCO2 on calcification,
but Halimeda minima showed reduced calcification in elevated pCO2 . However, different outcomes may
also arise from different methodologies implemented such as flow conditions, nutrient availability, size
of organisms, level of pCO2 condition implemented, or combinations of different stressors (e.g. OA
and OW). The impact of elevated pCO2 on growth of H. opuntia in Price et al. (2011) compared to
the lack of response to elevated pCO2 in the present study is unclear. We propose that different results
mainly arose due to different methodologies being implemented. The present study used flow-through
conditions with constant supply of fresh filtered seawater and associated nutrients, while in Price et al.
(2011), 0.7 L tanks were utilized with water exchange every 48 h, not accounting for nutrient depletion.
Moreover, daily light sums of control light in the present study were considerably higher and closer to
natural light conditions than reduced natural light regimes in Price et al. (2011), where light maxima
at mid-day averaged 150 µmol photons m−2 s−1 . In the present study we chose a pCO2 level which
is likely to be reached by the year 2100 under projections between the RCP6.0 and RCP8.5 scenario
(Moss et al. 2010), the experimental setup provided flow-through conditions with continuous supply of
nutrients and light regimes naturally found on mid-shelf and inshore locations of the GBR, at 5 m below
LAT. One potential explanation for why H. opuntia is capable of growth in a high pCO2 environment
while A. millepora is not may be that calcification rates in Halimeda spp. are generally higher than in
corals (when both standardized to either surface area or buoyant weight). For H. opuntia, daytime calcification rates were approximately one order of magnitude higher than dissolution rates in the dark under
elevated pCO2 conditions. Thus, even when some dissolution is taking place in the dark, higher light
calcification rates sum up to positive net calcification rates. In contrast, for A. millepora dark dissolution
and light calcification rate were in a similar range, indicating net dissolution if impacts of future OA and
low light conditions become additive (Table 4.3).
Notably, light calcification rates of both organisms, determined using the alkalinity anomaly tech-
99
4 - Ocean acidification and low light
nique, were unaffected by increased pCO2 . To our knowledge, this is the first study to show that calcification of A. millepora and H. opuntia in elevated pCO2 is not impacted during the light and supports
the assumption that during the light, photosynthetic activity can counteract negative impacts of OA by
increasing intracellular, surface and boundary-layer pH. By utilizing CO2 , photosynthesis increases pH
and Ωar (de Beer et al. 2000; de Beer and Larkum 2001; Glas et al. 2012). As shown by Al-Horani et al.
(2003), pH increases under the calcioblastic layer of corals in light, which elevated the super saturation
of Ωar from 3.2 up to 25, facilitating deposition of CaCO3 (Goreau 1959; Al-Horani et al. 2003).
However, the present study also suggested that for dark calcification rates, the opposite effect is the
case. During respiration in the dark, additional CO2 further reduces pH and Ωar (already lowered by
OA) and impedes deposition of CaCO3 . Hence, in the absence of light, both organisms were strongly
negatively impacted by high pCO2 conditions, leading to dissolution of their skeleton. In contrast, under
present-day conditions both organisms can calcify in the dark (i.e. in the absence of photosynthesis).
This observation is in agreement with a previous study, showing decalcification of Acropora eurystoma
in high pCO2 and darkness, while CaCO3 was still deposited under control conditions or in high pCO2
during light (Schneider and Erez 2006). Previous studies have also shown that reef communities can
change the diurnal local seawater carbonate chemistry by photosynthesis, respiration, calcification and
dissolution and that CaCO3 dissolution is primarily taking place during the dark (Chisholm 2000; Langdon and Atkinson 2005; Kleypas et al. 2011; Anthony et al. 2013). While respiration is taking place
during the dark, additional CO2 is added to the carbonate system and already lowered pH levels from OA
are further reduced, leading to an additional reduction of Ωar as already provoked by OA. Consequently,
A. millepora and H. opuntia were incapable of depositing CaCO3 in the dark and even experienced
dissolution of their skeletons under these conditions.
Considering the negative impacts of OA in darkness, we demonstrated that low light conditions may
likewise result in additional negative implications on organisms, once PAR is reduced below a level at
which photosynthesis cannot buffer reduced pH by OA. Presumably, this threshold level is below 35
µmol photons m−2 s−1 as tested in the present study. As shown in the present study by measurements
during the light, OA showed no impact on calcification rates of either organism. Thus, the availability
of sufficient light, associated with photosynthetic activity and apparent buffer capacity, mitigated negative effects of OA during light incubations. However, turbidity decreases PAR and therefore reduces
the capability of the organisms’ photosynthesis to buffer the negative impacts of OA, even during the
day. As shown by our O2 flux measurements, low light regimes significantly decreased gross- and net
photosynthesis in both organisms. This enhances negative impacts of OA during the day, especially at
inshore reefs, where riverine runoff leads to reduced PAR (Devlin and Schaffelke 2009). Light data from
mid-shelf and inshore GBR reefs at 5 m below LAT show that light availability can be extremely reduced
100
4 - Ocean acidification and low light
at inshore reefs, considerably impacting organisms’ photosynthetic capacities. Moreover, reduced photosynthetic activity of organisms experiencing reduced light availability may also change DIC/carbonate
chemistry on inshore reefs compared to mid-shelf locations. Under low light conditions, mean growth
rates of both the coral and algae were reduced compared to higher light. Light enhanced photosynthesis
and calcification of coral and algae is a well-documented phenomenon (Goreau 1959; Chalker and Taylor 1975; Chalker 1981; de Beer et al. 2000; de Beer and Larkum 2001). With increasing OA and the
additive negative effects of low light on coral growth, as demonstrated in the present study, the mechanism of light enhanced calcification may gain in importance. Moreover, under lower light conditions,
when photosynthetic activity is reduced, organisms obtain less energy supply, thus reducing the scope
for growth.
Photosynthesis of algae and coral can be limited by dissolved inorganic carbon (DIC) availability (Borowitzka and Larkum 1976; de Beer and Larkum 2001; Marubini et al. 2008; Crawley et al.
2010; Chauvin et al. 2011). Carbonic anhydrase can utilize elevated bicarbonate availability to increase the CO2 pool available for photosynthetic activity. Thus we assumed that photosynthesis could
be enhanced under higher pCO2 . However, photosynthesis of the organisms investigated here could not
benefit from increased DIC concentrations. This may have two different reasons: (1) the organisms
were not DIC-limited in experimental control conditions; (2) under present light conditions, photosynthesis/calcification of organisms was not saturated and hence there was no detectable benefit from
increased DIC availability. Studies indicating DIC limitation conducted by Marubini et al. (2008) and
Crawley et al. (2010) both utilized higher light intensities than the present study (∼300 and ∼1000 µmol
photons m−2 s−1 respectively). This suggests that under present experimental conditions (i.e. present
light intensities) calcification and photosynthesis were not DIC-limited.
Moreover, we showed that in decreased PAR, H. opuntia increased its tissue Chl a content in order
to compensate for less light availability, while the coral was not able to do so over the period of the
experiment. By adjusting its Chl a content the alga might acclimatize to reduced light availability in
the short term, and increase its photosynthetic capacity in low light. Increased productivity changes the
carbonate chemistry to the advantage of the algae, by facilitating deposition of CaCO3 . In contrast, A.
millepora did not have this advantage because it could not increase Chl a over the 16 days experimental
period and thus may not be able to acclimate in the short term to decreased light availability. As shown
by previous studies, corals alter their Chl a content by having either, a higher number of zooxanthellae
per unit area, or by an increase of Chl a content in the zooxanthellae (Coles and Jokiel 1978; Chauvin
et al. 2011). Field data suggest that A. millepora show increased pigmentation with decreasing water
clarity from mid-shelf to inshore (Fabricius 2006). However, this might also be a response towards
a more chronic exposure to low light conditions and other water quality parameters (i.e. increased
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nutrient availability at inshore reefs). Therefore, the algae may show more immediate responses towards
changing light regimes and thus have an advantage in acclimatization compared to the coral.
A. millepora and H. opuntia did not exhibit significant interactive effects on response parameters
measured, which is an indication that effects were not synergistic (Dunne 2010). Similarly, Comeau et
al. (2013a) and Comeau et al. (2014) found no interactive effects of OA and irradiance on calcification
rates of Porites rus and Acropora pulchra, respectively after three weeks exposure to experimental conditions. In contrast, a study on Pocillopora damicornis recruits presented interactive effects of OA and
light after 5 days of experimentation (Dufault et al. 2013). However, the responses were non-linear and
impacts of OA on calcification rates were only found at intermediate light intensities (70 µmol photons
m−2 s−1 ) and not at lower or higher light levels (31, 41, 122 and 226 µmol photons m−2 s−1 ). Moreover,
a study on Acropora horrida and Porites cylindrica showed impacts of OA on calcification that were
greatest during light calcification of corals grown in lower light conditions (100 µmol photons m−2 s−1 )
compared to corals grown in higher light (400 µmol photons m−2 s−1 ) after 5 weeks (Suggett et al.
2013). This is unexpected and in contrast to the present study, where the impact of OA on calcification
was not significant in the light, but was strong in the dark. In the present study A. millepora did not
show interactive effects of OA and light; however, there were additive effects of both stressors. We
detected reduced growth rates (−48%) when exposed to high pCO2 conditions and also reduced growth
rates when exposed to low light regimes (−96%), which resulted in a predicted additive growth rate of
−144% (which is similar to the measured −114% growth rate) (Table 4.3). This may have an ecological
implication for corals inhabiting inshore reefs susceptible to land-runoff and thus decreased light availability. Turbidity changes the attenuation of light penetrating the water column decreasing PAR with
increasing depth more rapidly than in clear water conditions. This, in turn, may lead to a stronger depth
limitation for corals and thus to potential habitat decline in future OA conditions, because they gain less
light with lower water depth, compared to clear water habitats.
Conclusions
In the present experiment, we confirmed that the marine calcifiers investigated are negatively impacted
by OA, with A. millepora showing more negative impacts than H. opuntia. As long as sufficient light
is available during the day, photosynthesis aids organisms to counteract negative impacts of OA. However, if there is not sufficient light available (e.g. due to high turbidity), there may be impacts of OA on
calcification also during the day. Thus, low light conditions inshore remove this advantage from photosynthesizing organisms. As suggested by the dark incubations, respiration potentially aggravates the
impacts of OA on the organisms, leading to dissolution of their skeleton. This highlights the importance
of considering light-dependent impacts of OA on photosynthesizing calcifiers. Moreover, we showed
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that decreased light availability is an additive stressor with OA, particularly for the coral A. millepora,
because the coral exhibits reduced calcification in OA conditions as well as in low light conditions. H.
opuntia, on the other hand, grows marginally less in low light, but was not negatively impacted by OA
in its overall growth. Consequently, the combination of OA and low light conditions may contribute to
a changing coral reef ecosystem with even less hard corals as framework builders and more macroalgae
on inshore reefs of the future. Potential acclimatization to environmental stressors in the long term could
lead to different responses of organisms. Therefore, further investigations are needed to test the effects
of OA in combination with light availability on coral reef organisms. Management of coastal runoff
could also play an important role, as by improving water clarity on inshore reefs the additional stressor
of low light availability for corals would be reduced.
Acknowledgments
The authors thank the SeaSim team at AIMS for providing the coral nubbins and their general assistance. We thank Michelle Liddy for her assistance in the field and laboratory. Many thanks for general
assistance from Florita Flores. We acknowledge the National Environmental Research Program, which
provided the stipend for Nikolas Vogel. This study was conducted with the support of funding from
the Australian Government’s National Environmental Research Program and an Australian Research
Council Discovery Grant.
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110
Chapter 5
Effects of elevated dissolved inorganic
carbon and nitrogen on the physiology of
scleractinian corals and calcareous
macroalgae under ocean acidification and
eutrophication conditions
Nikolas Vogel 1,2,3 , Yan Ow 1,4 , Florita Flores 1 , Catherine Collier 4 , Christian Wild 2,3 and Sven Uthicke
1
(1)
Australian Institute of Marine Science, PMB 3, Townsville MC, Queensland 4810, Australia
(2)
Leibniz Center for Tropical Marine Ecology, Fahrenheitstraße 6, 28359 Bremen, Germany
(3)
Faculty of Biology and Chemistry (FB 2), University of Bremen, 28359 Bremen, Germany
(4)
School of Marine and Tropical Biology, James Cook University, Townsville, Australia
Keywords: Nitrate, dissolved inorganic nutrients, Acropora tenuis, Seriatopora hystrix, Halimeda opuntia
This publication is in preparation
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Abstract
Global (i.e. ocean acidification, OA) and local (i.e. eutrophication) stressors can have deleterious effects
on coral reef organisms and their communities. On the other hand, calcifying and photosynthesizing
organisms can be limited by dissolved inorganic carbon (DIC), or nutrient (DIN) concentrations in seawater. Thus, increases of DIC under OA conditions and increases of DIN under eutrophication conditions may differentially affect the physiology of calcifying organisms. In a three week tank experiment,
we investigated the individual and combined effects of DIC (∼400, 700, 1100 µatm pCO2 ) and DIN
(0.4 and 1.9 µmol L−1 NO2 + NO3 ) on growth, calcification, photosynthesis, nutrient uptake, pigment
content, protein content and carbon and nitrogen content of the scleractinian corals Acropora tenuis and
Seriatopora hystrix and the calcareous green alga Halimeda opuntia. Contrary to expectations, elevated
DIC did not significantly affect any response parameter. We propose that the lack of responses to high
DIC observed in corals, as opposed to some previous experiments, may be due to slower response times
under close-to-natural experimental conditions as those used in the present experiment. Elevated DIN
increased photosynthesis, nitrate uptake rates and pigment contents of the organisms investigated. Moreover, for S. hystrix and H. opuntia we observed an increased organic fraction, indicating an imbalanced
growth between organic tissue and inorganic skeleton under elevated DIN. No significant interactions
between elevated DIC and DIN were observed. Meanwhile, all experimental species showed strong
physiological responses towards elevated DIN. Thus, organisms living in habitats affected by coastal
runoff may rapidly respond to short-term pulses in DIN concentrations, accompanied by physiological
alterations which may make some of them more susceptible to other environmental stressors.
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5 - Ocean acidification and eutrophication
5.1 Introduction
Carbon dioxide (CO2 ) from anthropogenic emissions leads to decreases in ocean pH and calcium carbonate saturation state Ω (IPCC 2013). This phenomenon is named ocean acidification (OA) and affects
many marine calcifying organisms which become impaired in building-up their skeletons made out of
the mineral aragonite. Atmospheric CO2 partial pressure (pCO2 ) is predicted to increase two- or threefold within the present century if emissions are not instantly and drastically reduced (Collins et al. 2013).
Consequently, further increases in pCO2 are directly associated with OA and further reductions in pH
and aragonite saturation state Ωar . Reports show deleterious effects of OA on many marine organisms
(Orr et al. 2005; Hoegh-Guldberg et al. 2007; Kleypas and Yates 2009), but recent studies also indicated resilience (Comeau et al. 2014; Vogel et al. 2015a) or even benefits to some organisms (Fabricius
et al. 2011) under predicted future OA conditions. These results suggest species- and region-specific
responses towards OA.
Besides decreasing pH/Ωar , increasing pCO2 also elevates dissolved inorganic carbon (DIC) concentrations in the seawater. The DIC species HCO3– and CO2 are essential for organisms’ calcification
and photosynthesis, and previous studies have shown that corals and calcareous algae (i.e. Halimeda)
can be DIC limited in ambient seawater conditions (Vogel et al. 2015a; Uthicke and Fabricius 2012;
Koch et al. 2013). Consequently, some organisms may benefit from increased DIC under future OA
scenarios. Thus, it is important to investigate the balance between negative effects of decreased pH/Ωar
and potential positive mechanisms of elevated DIC on organisms in order to try to understand future
impacts on coral reefs.
Many coral reefs are located in populated coastal areas and are not only affected by the global stressor OA, but also by degradation in local water quality. Anthropogenic impacts to water quality on coral
reefs can primarily be seen in increased land-use, land-erosion and coastal runoff. The use of fertilizer
has steadily increased in the past decades and is predicted to rise in the future (Galloway et al. 2008;
IPCC 2013), while ongoing population growth in coastal regions continues to intensify the risk to water
quality from point sources including sewage. On the Great Barrier Reef (GBR), coastal runoff leads
to increased dissolved inorganic nutrient (DIN) concentrations at inshore areas. Water residence time
in the inshore prolongs the effect from pulsed riverine discharge (Fabricius et al. 2014). The water
quality at inshore locations of the GBR has thus steadily declined since European settlement (Devlin
and Brodie 2005; Wooldridge et al. 2006). This eutrophication of naturally oligotrophic environments
can have severe impacts on coastal coral reef ecosystems (Fabricius 2005; Fabricius et al. 2005; De’ath
and Fabricius 2010; Fabricius 2011). Research shows direct effects of increased DIN on coral calcification, zooxanthellae density/pigment concentrations and productivity (Marubini and Davies 1996;
Ferrier-Pages et al. 2000). Moreover, nitrate enrichment in particular can negatively affect coral fecun114
5 - Ocean acidification and eutrophication
dity, fertilization, larvae settlement and embryo development (Hunte and Wittenberg 1992; Humphrey et
al. 2008). Many manipulative experiments utilized unnaturally high concentrations of DIN (5-20 µmol
L−1 NO3– ) which have little or no relevance to naturally occurring concentrations at inshore reefs on
the GBR (Bell 1992; Devlin and Schaffelke 2009). Field experiments investigating nutrient effects on
marine organisms are complicated by other runoff effects, such as reduced salinity, increased sedimentation and reduced light availability. On the other hand, increases in DIN may relieve organisms from
nitrogen limitation in oligotrophic environments as previously shown for zooxanthellae hosted by corals
(Marubini and Davies 1996; Ferrier-Pages et al. 2000).
Coral reefs are increasingly affected by DIC and DIN, particularly at inshore locations of the GBR
that are affected by coastal runoff. The interactions between elevated DIC and DIN are barely investigated, especially under environmentally relevant concentrations, but combined effects could range from
potentially destructive (e.g. causing mortality) to potentially promoting (e.g. increasing growth). Thus,
we conducted a three week tank experiment on Lizard Island Research Station on the GBR to comparatively investigate the individual and combined effects of OA and eutrophication on physiological
features of the scleractinian corals Acropora tenuis and Seriatopora hystrix, and the calcareous green
alga Halimeda opuntia.
5.2 Methods
Collection and preparation of specimens
We collected three colonies of each coral species A. tenuis and S. hystrix at the Lizard Island Lagoon (S
14° 41.370’, E 145° 27.392’) in 2-5 m water depth and prepared ∼80 coral branches (∼5 cm in height)
for each species. Branches were cut from colonies with pliers and attached to plastic stubs with food safe
epoxy putty (KNEADit aqua, Selleys). Prepared specimens were retained in holding tanks supplied with
natural seawater under flow through (∼100 L min−1 ) conditions and natural light regimes (50% reduced
by shading cloth) at the aquaria facility of the Lizard Island Research Station until commencement of
the experiment. H. opuntia specimens (∼4 × 4 cm fragments) were collected in 1-3 m water depth and
retained in holding tanks until commencement of the experiment, as described above. At the start of
the experiment, four branches of each coral species A. tenuis and S. hystrix as well as three pieces of
H. opuntia were randomly transferred into each experimental tank (total of 72, 72 and 54 specimens,
respectively).
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5 - Ocean acidification and eutrophication
Experimental setup
To investigate the interactive effects of OA and eutrophication on corals and the calcareous green alga,
we conducted a 24 day tank experiment in March/April 2014 on the Lizard Island Research Station
(S 14° 40.725’, E 145° 26.896’) in the northern section of the GBR. To simulate different DIC (i.e.
OA) scenarios, we enriched ambient seawater with pure CO2 (gas code 082, BOC) by an automatic,
pH controlled gas injection system (Aquamedic), as described elsewhere (Vogel and Uthicke 2012;
Vogel et al. 2015b). To simulate eutrophication, we enriched ambient seawater with sodium nitrate
solution (NaNO3 , Sigma-Aldrich). The experimental design consisted of three CO2 treatments (ambient,
moderate and high, i.e. ∼400, 700 and 1100 µatm pCO2 ) and two DIN treatments (ambient and high,
i.e. ∼0.4 and 1.9 µmol L−1 NO2 + NO3 ). CO2 treatments were mixed in three ∼400 L sump tanks
and the seawater was pumped into the respective experimental tanks. Experimental tanks (20 L) were
provided with a flow rate of ∼24 L h−1 of fresh unfiltered seawater. Additional aquaria pumps (2.5 L
min−1 , Hailea) were placed in each tank and ensured mixing of seawater within the experimental tanks.
Eutrophication treatments were provided with 2 mM stock solution of NaNO3 , delivered by peristaltic
pumps (Masterflex, Cole Parmer), at a flow rate of 30 mL h−1 . Complete carbonate chemistry is given in
Table 5.1 and complete water quality parameters are given in Table 5.2. Eighteen tanks (three replicates
each treatment) were arranged in alternating order and received natural sunlight, reduced to 50% by
a transparent rooftop. Two light loggers (Odyssey) were randomly allocated to experimental tanks to
record photosynthetically available radiation (PAR) and were transferred to different tanks three times
throughout the experiment. Mean daily light sums averaged 3.8 mol photons m−2 d−1 , ranging from 1.2
to 5.5 mol photons m−2 d−1 , with midday maxima of ∼538 µmol photons m−2 s−1 , over the course of
the experiment.
Determination of growth rates
Growth of the organisms was determined by the buoyant weight technique. Therefore, corals and algae
were single-weighted (accuracy: 0.1 mg, Mettler Toledo) in a custom built buoyant weight setup with
water jacket and seawater of constant temperature (25 °C) and salinity (34.5 ppt) at the start and at the
end of the experiment. Growth of organisms was expressed as daily percentage of change.
Incubation experiments
After 19-21 days in experimental conditions, two individuals of each experimental species and tank (6
each treatment) were transferred into 0.6 L Perspex chambers and incubated for ∼1 h in light around
midday and for ∼2 h in darkness in the evening. Closed incubation chambers were placed into black
plastic bins (45 L) provided with flow-through seawater for temperature control. Additionally, magnetic
116
pHtotal
8.02 (0.03)
8.03 (0.03)
7.83 (0.05)
7.83 (0.05)
7.65 (0.06)
7.66 (0.06)
Treatment
400/ 0.4
400/ 1.9
700/ 0.4
700/ 1.9
1100/ 0.4
1100/ 1.9
Temp
[°C]
28.6 (1.0)
28.3 (1.1)
28.2 (0.9)
28.6 (0.9)
28.5 (1.0)
28.3 (0.9)
TA
[µmol kgSW-1]
2223 (14)
2222 (11)
2224 (13)
2221 (13)
2223 (12)
2227 (11)
DIC
[µmol kgSW-1]
1917 (23)
1911 (25)
2016 (21)
2013 (18)
2095 (20)
2096 (23)
pCO2
[µatm]
409 (35)
401 (39)
690 (87)
691 (86)
1129 (182)
1097 (179)
HCO3[µmol kgSW-1]
1690 (31)
1682 (37)
1843 (30)
1841 (27)
1958 (27)
1958 (30)
CO32[µmol kgSW-1]
216 (11)
219 (14)
155 (13)
155 (13)
108 (12)
109 (12)
CO2
[µmol kgSW-1]
11 (1)
10 (1)
18 (2)
18 (2)
29 (4)
29 (4)
3.5 (0.2)
3.5 (0.2)
2.5 (0.2)
2.5 (0.2)
1.8 (0.2)
1.8 (0.2)
Ωar
Table 5.1: Mean (± SD) carbonate system parameters of experimental treatment conditions. Treatments consisted of 400, 700 and 1100 µatm pCO2 and 0.4 and
1.9 µmol DIN
5 - Ocean acidification and eutrophication
117
5 - Ocean acidification and eutrophication
Table 5.2: Mean (± SD) water quality parameters of experimental treatment conditions. Treatments
consisted of 400, 700 and 1100 µatm pCO2 and 0.4 and 1.9 µmol NOx
Treatment
400/ 0.4
400/ 1.9
700/ 0.4
700/ 1.9
1100/ 0.4
1100/ 1.9
NH4
[µmol L-1]
0.75 (0.12)
0.78 (0.17)
0.79 (0.21)
0.81 (0.06)
0.68 (0.22)
0.70 (0.17)
PO4
[µmol L-1]
0.04 (0.01)
0.04 (0.01)
0.04 (0.01)
0.04 (0.00)
0.04 (0.00)
0.04 (0.01)
NO2 + NO3
[µmol L-1]
0.45 (0.04)
1.91 (0.12)
0.42 (0.04)
1.88 (0.05)
0.40 (0.03)
1.97 (0.06)
NO2
[µmol L-1]
0.14 (0.00)
0.17 (0.01)
0.13 (0.00)
0.16 (0.00)
0.13 (0.01)
0.16 (0.01)
spin bars in chambers prevented the build-up of oxygen and alkalinity gradients. Light incubations
were conducted under white light LED (450 µmol photons m−2 s−1 , AquaIllumination), while during
dark incubations bins were equipped with black plastic lids to ensure complete darkening. During each
experimental run, incubation chambers without specimens were included to each plastic bin to correct
the final measurements for oxygen-, alkalinity- and nutrient fluxes derived from the seawater itself. After
each experimental run, the water in the incubation chambers was analyzed for oxygen content with a
hand-held dissolved oxygen meter (HQ30d, Hach), as described in Vogel et al. (2015b) and Vogel et
al. (2015a). Photosynthesis and respiration rates of specimens were calculated by change in oxygen
content of the seawater (referred to blank incubations), time of incubation and volume of the incubation
chamber, normalized to coral surface area or algal buoyant weight. In addition, a subsample of 50 mL
from each incubation chamber was directly pipetted and analyzed for total alkalinity (TA), as described
below and elsewhere (Uthicke and Fabricius 2012; Vogel et al. 2015a). Calcification rates in light and in
darkness (alkalinity anomaly technique) were calculated by change in TA (referred to blank incubations),
duration of the incubation and volume of the incubation chamber, normalized to coral surface area or
algal buoyant weight. Net calcification rates were calculated by 12 h light and 12 h dark calcification.
Moreover, we collected samples for seawater DIN concentrations from each incubation chamber. For
this, a 20 mL subsample was filtered (0.45 µm) with a 50 mL syringe into individual nutrient tubes
and was directly frozen to −20 °C until subsequent analysis of DIN content (NH4+ , PO43– and NO2+
and NO3– ) by Segmented Flow Analysis (Seal Analytical). DIN fluxes in light and in darkness were
calculated by change of NOx (NO2 + NO3 ) (referred to blank incubations), the duration of incubation
and volume of the incubation chamber, normalized to coral surface area or algal buoyant weight.
We determined surface areas of coral nubbins using the single wax-dipping method (Veal et al.
2010) and chose buoyant weight as standardization for the algae due to their highly three dimensional
structures and lowest variability in data.
118
5 - Ocean acidification and eutrophication
Determination of pigment content
At the end of the experiment, specimens were directly frozen in liquid nitrogen (−196 °C) and transferred into a −80 °C freezer at the Australian Institute of Marine Science (AIMS) in Townsville, until
analysis. Pigments (Chl a, Chl b and total carotenoids) were determined spectrophotometrically. Coral
and algal pigments were extracted in 95% EtOH as described in Vogel et al. (2015b). Pigment extracts
were pipetted into a microplate and absorbences were measured on a microplate reader (Powerwave,
BioTek) at 664, 649, 470 and 750 nm wavelengths. Pigment contents were calculated by equations from
Ritchie (2008) and Lichtenthaler (1987) and normalized to coral surface area or fresh weight of algal
segments.
Determination of coral protein content
Total protein contents of A. tenuis and S. hystrix were analyzed with the Bio-Rad protein assay kit.
Applying the method described in Leuzinger et al. (2003), coral tissue slurry was digested with 1 M
NaOH for one hour at 90 °C in a sealed deep-well plate. Cell-debris was separated from solution (1500
g for 10 min). Dilutions of protein standard (bovine serum albumin, BSA, Sigma Aldrich) and samples
were transferred into a 96-well microtiter plate and protein assay reagents were added. After 15 minutes,
absorbence was read on 750 nm wavelength in a microplate reader (Powerwave, BioTek). Total protein
content was calculated in correlation to the protein standard regression and was normalized to the surface
area of coral branches.
Determination of algal C and N content
After finalizing the experiment, algae were dried at 50 °C for 48 h and algal segments were pulverized
with a bead-beater. Homogenate was analyzed for total carbon (Ctot ), total nitrogen (N) and organic carbon (Corg ) on an elemental analyzer (Shimadzu). Total inorganic carbon (Cinorg ) content was calculated
by subtracting Corg from Ctot .
Determination of carbonate chemistry
Three sets of water samples were collected for each experimental tank throughout the course of the experiment. For each water sample we determined pHtotal with the indicator dye m-cresol purple, according
to the standard operating procedure (SOP 6b) (Dickson et al. 2007) and TA by directly titrating a subsample of 50 mL seawater on a robotic titrosampler (Metrohm) utilizing 0.5 M HCl, as described elsewhere
(Uthicke and Fabricius 2012; Vogel et al. 2015a). Carbonate chemistry was calculated by pH and TA
measurements with the software CO2calc (Robbins et al. 2010), using CO2 constants from Lueker et al.
(2000). Full carbonate chemistry of experimental treatments is given in Table 5.1. Twice weekly, we
119
5 - Ocean acidification and eutrophication
performed additional pH measurements for each aquaria with a bench top pH meter (OAKTON) and a
refillable pH probe (Eutech), calibrated on NIST (National Institute of Standards and Technology) scale,
to verify functionality of the CO2 injection system (data not presented). Moreover, six pH probes connected to the CO2 injection system constantly monitored (and logged) pH of each experimental treatment
(data not presented).
Statistical analyzes
We statistically tested calcification rates, oxygen- and nutrient fluxes, pigment content, coral protein
content and algal carbon/nitrogen content for significant differences between treatment conditions. We
tested datasets for equal variance with the Levene’s test and the software R (R Development Core Team
2014), and in case data was not equally distributed it was transformed prior to statistical analysis to
fulfill the assumption of equal variances. Mixed Linear Models were conducted with the software NCSS
(Hintze 2007), using pCO2 and nutrient condition as fixed factor and experimental tank as nested random factor. Significant treatment effects were further investigated with Tukey-Kramer post-hoc-tests, to
determine which treatments were significantly differing from others.
5.3 Results
After 24 days in experimental conditions mortality was generally low for A. tenuis (0%), S. hystrix (3%)
and H. opuntia (4%). Treatments for moderate and high pCO2 scenarios (Table 5.1, ∼700 and 1100
µatm pCO2 , respectively) were in the intended range of future predictions according to RCP6.0 and
RCP8.5 of the IPCC (2013). Nutrient additions for eutrophication treatments were calculated to reach
2.5 µmol L−1 NOx and were slightly lower (Table 5.2, 1.9 µmol L−1 NOx ) than the intended values.
Linear Model results showed elevated DIC/CO2 (in isolation) did not significantly affect growth
(Fig. 5.1), calcification rate (Fig. 5.2), photosynthetic rate (Fig. 5.3), nutrient uptake rate (Fig. 5.4), Chl
a content (Fig. 5.5), total protein content (Fig. 5.6) or carbon and nitrogen content (Fig. 5.7), but NOx
uptake of S. hystrix in light (Fig. 5.4), which was 104% decreased (Table 5.3, p = 0.027) at moderate
compared to ambient pCO2 .
Linear Model results indicated significant effects of elevated nitrate concentrations on several response variables of organisms. While photosynthetic rates of A. tenuis were not significantly affected,
net photosynthesis of S. hystrix was significantly (Table 5.3, p = 0.001) increased by 39% under elevated
nitrate availability (Fig. 5.3). Similarly, net photosynthesis of H. opuntia was increased (p = 0.011) by
43% at elevated compared to ambient nitrate concentration. Respiration of S. hystrix was significantly
(p = 0.010) lower (13%) at elevated compared to ambient nitrate concentrations. Moreover, gross photo-
120
5 - Ocean acidification and eutrophication
S. hystrix
H. opuntia
0
0.0
0.2
2
0.4
4
0.6
0.8
Growth [% change d-1]
0.0 0.2 0.4 0.6 0.8
6
A. tenius
ol
µm
9 l
1. mo
µ m
4
0. µat
00
11 atm
µ
0
70 atm
µ
0
40
ol
µm
9 l
1. mo
µ m
4
0. µat
00
11 atm
µ
0
70 atm
µ
0
40
ol
µm
9 l
1. mo
µ
4 tm
0. µa
00
11 atm
µ
0
70 atm
µ
0
40
Figure 5.1: Growth of A. tenuis, S. hystrix and H. opuntia after three weeks experimental treatment.
X-axes represent pCO2 treatments in µatm and DIN treatments in µmol L−1 NO x. Whiskers represent
upper and lower extremes. Plots illustrate pooled data, due to non-significant interactions
synthesis of S. hystrix was significantly (p = 0.002) increased by 32% under elevated nitrate availability.
Similarly, gross photosynthesis of H. opuntia was significantly (p = 0.018) elevated by 40% under increased nitrate concentrations.
NOx uptake rates in the dark of A. tenuis, S. hystrix and H. opuntia were significantly (Table 5.4,
p = 0.002, p = 0.001 and p = 0.028, respectively) increased by 349, 406 and 116%, respectively when
higher nitrate concentrations were available (Fig. 5.4). In addition, net NOx uptake of A. tenuis and
H. opuntia (p = 0.025 and p = 0.036, respectively) was increased by 173 and 97%, respectively under
higher nitrate availability.
Pigments Chl a, Chl b and total carotenoids of all three organisms, A. tenuis, S. hystrix and H.
opuntia were significantly increased under elevated nitrate availability (Fig. 5.5; only Chl a is presented
in detail). Chl a contents of A. tenuis, S. hystrix and H. opuntia were significantly (Table 5.4, p < 0.001,
p < 0.001 and p = 0.007, respectively) increased by 27, 45 and 28%, respectively at elevated compared
to ambient nitrate concentrations.
While protein content of A. tenuis was unaffected, protein content of S. hystrix significantly (Table
5.4, p = 0.047) increased by 14% under elevated compared to ambient nitrate conditions (Fig. 5.6).
Moreover, organic carbon and nitrogen contents of H. opuntia were significantly (Table 5.4, p =
0.024 and p = 0.001, respectively) increased by 27 and 56%, respectively at elevated compared to
ambient nitrate concentration (Fig. 5.7). Calculated C:N ratios of H. opuntia significantly (p < 0.001)
decreased by 19% under elevated nitrate availability.
Linear Model results indicated no significant interactive effects of pCO2 and nitrate concentration
on any response parameters but Chl a content of S. hystrix (Table 5.4, p = 0.004), which was lowest at
moderate pCO2 /low nutrients and highest at moderate pCO2 /high nutrients.
121
5 - Ocean acidification and eutrophication
0.8
0.6
0.4
0.2
0.0
Dark calcification [µM C h-1 gBW-1]
0.0
2.5
5.0
0.8
20
Net calcification [µM C d-1 gBW-1]
0
200
400
600
0.0
0.2
0.4
0.6
Dark calcification [µM C h-1 cm-2]
0.0
0.2
0.4
0.6
0.8
5
10
15
Net calcification [µM C d-1 cm-2]
0
5
10
15
20
0
Treatment
ol
µm
9 l
1. mo
µ
4 tm
0. µ a
00
11 atm
µ
0
70 atm
µ
0
40
ol
µm
9 l
1. mo
µ m
4
0. µat
00
11 atm
µ
0
70 atm
µ
0
40
ol
µm
9 l
1. mo
µ
4 tm
0. µa
00
11 atm
µ
0
70 atm
µ
0
40
Treatment
H. opuntia
Light calcification [µM C h-1 gBW-1]
0
10
20
30
40
50
S. hystrix
1.0
Light calcification [µM C h-1 cm-2]
0.0 0.2 0.4 0.6 0.8 1.0
A. tenius
Treatment
Figure 5.2: Light-, dark- and net calcification of A. tenuis, S. hystrix and H. opuntia after three weeks
experimental treatment. X-axes represent pCO2 treatments in µatm and DIN treatments in µmol L−1
NO x. Whiskers represent upper and lower extremes. Plots illustrate pooled data, due to non-significant
interactions
122
S. hystrix
0
20
40
60
80
−16
−12
−8
−4
Gross photosynthesis [µg O2 L-1 h-1 cm-2]
Respiration [µg O2 L-1 h-1 cm-2]
0
20
40
60
80 −16
−12
−8
−4
0
0
0
10
20
30
40
50
60
Net photosynthesis [µg O2 L-1 h-1 cm-2]
0 10 20 30 40 50 60
A. tenius
ol
µm
9 l
1. mo
µ
4 tm
0. µa
00
11 atm
µ
0
70 atm
µ
0
Treatment
H. opuntia
40
ol
µm m
4
0. µat
00
11 atm
µ
0
70 atm
µ
0
40
ol
µm
9 l
1. mo
µ
4 tm
0. µa
00
11 atm
µ
0
70 atm
µ
0
40
Treatment
Gross photosynthesis [µg O2 L-1 h-1 gBW-1] Respiration [µg O2 L-1 h-1 gBW-1] Net photosynthesis [µg O2 L-1 h-1 gBW-1]
0
1000 2000 3000 4000 −500 −400 −300 −200 −100 0
0
1000 2000 3000 4000
5 - Ocean acidification and eutrophication
Treatment
Figure 5.3: Net photosynthesis, dark respiration and gross photosynthesis of A. tenuis, S. hystrix and H.
opuntia after three weeks experimental treatment. X-axes represent pCO2 treatments in µatm and DIN
treatments in µmol L−1 NO x. Whiskers represent upper and lower extremes. Plots illustrate pooled data,
due to non-significant interactions
123
5 - Ocean acidification and eutrophication
−10
−20
−30
Dark NOx uptake [nM h-1 gFW-1]
−500
−250
0
−40
0
200
Net NOx uptake [nM d-1 gFW-1]
−15000 −10000 −5000
0
−20
−15
−10
−5
0
Dark NOx uptake [nM h-1 cm-2]
−20 −15 −10 −5
0
Net NOx uptake [nM d-1 cm-2]
−800 −600 −400 −200 0
200
−800 −600 −400 −200
Treatment
ol
µm
9 l
1. mo
µ
4 tm
0. µa
00
11 atm
µ
0
70 atm
µ
0
40
ol
µm
9 l
1. mo
µ m
4
0. µat
00
11 atm
µ
0
70 atm
µ
0
40
ol
µm
9 l
1. mo
µ
4 tm
0. µa
00
11 atm
µ
0
70 atm
µ
0
40
Treatment
H. opuntia
Light NOx uptake [nM h-1 gFW-1]
−750 −500 −250
0
10
S. hystrix
0
Light NOx uptake [nM h-1 cm-2]
−40 −30 −20 −10
0
10
A. tenius
Treatment
Figure 5.4: Light-, dark-, and net NO x uptake of A. tenuis, S. hystrix and H. opuntia after three weeks
experimental treatment. X-axes represent pCO2 treatments in µatm and DIN treatments in µmol L−1
NO x. Whiskers represent upper and lower extremes. Plots illustrate pooled data, due to non-significant
interactions
124
5 - Ocean acidification and eutrophication
H. opuntia
500
S. hystrix
0
Chl a [µg gFW-1]
200 300 400
0
4
8
12
Chl a [µg cm-2]
4
8
12
16
16
A. tenius
ol
µm
9 l
1. mo
µ
4 tm
0. µa
00
11 atm
µ
0
70 atm
µ
0
Treatment
40
ol
µm
9 l
1. mo
µ m
4
0. µat
00
11 atm
µ
0
70 atm
µ
0
40
ol
µm
9 l
1. mo
µ
4 tm
0. µa
00
11 atm
µ
0
70 atm
µ
0
40
Treatment
Treatment
Figure 5.5: Chlorophyll a content of A. tenuis, S. hystrix and H. opuntia after three weeks experimental
treatment. X-axes represent pCO2 treatments in µatm and DIN treatments in µmol L−1 NO x. Whiskers
represent upper and lower extremes. Plots illustrate pooled data, due to non-significant interactions
5.4 Discussion
We tested the individual and combined effects of OA and eutrophication on corals A. tenuis and S. hystrix
and on calcareous green alga H. opuntia. Low mortality after 24 days in experimental tanks indicated
organisms performed well under manipulated environments. Nitrate concentrations in eutrophication
treatments were increased by ∼5 times to ∼2 µmol L−1 NOx compared to control conditions. The mean
nitrate concentration was in range of high nitrate reefs (Kleypas et al. 1999) and in range of average
nitrate concentrations seen during flood plume events on the GBR (Devlin et al. 2001).
Effects of DIC
Increased DIC in OA treatments showed no effect on photosynthesis of corals A. tenuis and S. hystrix
and alga H. opuntia in the present study. As shown by several other studies, increased DIC availability
can elevate net photosynthesis in corals and algae (Langdon and Atkinson 2005; Crawley et al. 2010),
presuming DIC limitation of photosynthesis in ambient seawater. These apparent inconsistencies might
result from methodological differences such as O2 evolution in the present study vs. pulse amplitude
modulation (PAM) fluorometry measurements (Crawley et al. 2010) or different acclimatization time
of corals in the present tank experiment vs. life-long acclimatization at CO2 seeps (Strahl et al. pers.
comm.). Similar to results here, several other studies reported no significant effect of elevated DIC on
net O2 production (Leclercq et al. 2002; Reynaud et al. 2003; Langdon and Atkinson 2005), but at the
same time increases in net carbon production, indicating changes in the photosynthetic quotient between
O2 evolved and CO2 fixed (Langdon and Atkinson 2005).
After 24 days in experimental conditions, we did not see any OA effect on growth and net calci-
125
5 - Ocean acidification and eutrophication
S. hystrix
0.0
0.4
0.8
1.2
Total protein content [mg cm-2]
0.0
0.4
0.8
1.2
A. tenius
ol
µm
9
1. ol
µm
4
0. atm
µ
00
11 atm
µ
0
70 atm
µ
0
40
ol
µm
9
1. ol
µm
4
0. µatm
00
11 atm
µ
0
70 atm
µ
0
40
Treatment
Treatment
Figure 5.6: Total protein content of A. tenuis and S. hystrix after three weeks experimental treatment.
X-axes represent pCO2 treatments in µatm and DIN treatments in µmol L−1 NO x. Whiskers represent
upper and lower extremes. Plots illustrate pooled data, due to non-significant interactions
fication of A. tenuis, S. hystrix, or H. opuntia. While this observation agrees with previous studies on
calcareous alga Halimeda spp., including H. opuntia (Comeau et al. 2013; Hofmann et al. 2014; Vogel
et al. 2015b; Vogel et al. 2015a), this result was somewhat unexpected for corals A. tenuis and S. hystrix.
Previous literature indicated impacts of OA on calcification of several scleractinian corals, including
the genera Acropora, after short-term (4 weeks) exposure to ∼800 µatm pCO2 (e.g. Renegar and Riegl
2005) and Seriatopora after life-long exposure to ∼800 µatm pCO2 (Strahl et al. pers. comm.). Why we
did not see any impacts of OA on corals is unclear, but perhaps A. tenuis and S. hystrix do not respond
to OA in the short term. Several important differences exist between other studies and ours, such as
artificial vs. natural light regimes, filtered vs. unfiltered seawater, or high vs. low nutrient background
levels. For instance, temperatures in the present experiment were higher than in the study by Renegar
and Riegl (2005). Calcification increases with temperatures and Ωar (Silverman et al. 2007) which may
have contributed to the observed differences between the two studies. In addition, experimental tanks
in the present study were supplied with flow-through unfiltered reef seawater, while the other study utilized filtered seawater without continuous exchange (Renegar and Riegl 2005). Heterotrophic nutrition
from unfiltered reef seawater used in the present experiment may have enhanced the nutritional status
of corals. Higher energy levels may have enabled the corals to actively regulate internal pH levels to
protect themselves against OA impacts, which is consistent with previous studies showing that energy
levels can alleviate responses to other stressors such as warming (Fabricius et al. 2013). Moreover, the
light intensity has been shown to affect responses coral reef organisms to OA (Vogel et al. 2015b). Natural variable light regimes in the present study may have contributed to the high variability in growth/net
calcification data, hence weakening the OA effects. Thus, other environmental factors, may affect the
126
5 - Ocean acidification and eutrophication
8
10
C:N ratio
12 14
16
5.0
Cinorg
7.5 10.0 12.5
Total nitrogen
0.4
0.8
1.2
1.6
4
8
Corg
12
16
H. opuntia
9
1.
ol
µm
ol
µm
tm
µa
tm
µa
tm
µa
00
4
0.
11
0
70
0
40
Treatment
Figure 5.7: Organic carbon, nitrogen, inorganic carbon and C:N ratio of H. opuntia after three weeks
experimental treatment. X-axes represent pCO2 treatments in µatm and DIN treatments in µmol L−1
NO x. Whiskers represent upper and lower extremes. Plots illustrate pooled data, due to non-significant
interactions
127
5 - Ocean acidification and eutrophication
Table 5.3: Mixed Model ANOVA results of OA and elevated DIN on response parameters of organisms
investigated. Asterisks indicate significant treatment effects
Response
variable
Growth
Light
calcification
Dark
calcification
Net
calcification
Net
photosynthesis
Respiration
Gross
photosynthesis
Source
of variation
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
df
2
1
2
12
44
2
1
2
12
18
2
1
2
11
17
2
1
2
11
17
2
1
2
12
18
2
1
2
12
17
2
1
2
11
17
A. tenuis
F
p
1.89
0.31
0.23
3.03
0.194
0.588
0.798
0.004
0.14
2.12
0.42
0.88
0.875
0.171
0.663
0.580
0.67
0.13
0.27
2.13
0.531
0.725
0.772
0.078
0.22
1.39
0.36
0.99
0.807
0.264
0.707
0.497
2.19
3.21
1.25
1.12
0.154
0.098
0.321
0.400
0.22
0.18
0.23
2.35
0.806
0.677
0.795
0.053
1.72
2.31
1.04
1.07
0.223
0.157
0.387
0.438
df
2
1
2
12
46
2
1
2
12
18
2
1
2
12
17
2
1
2
12
17
2
1
2
12
18
2
1
2
12
17
2
1
2
11
17
S. hystrix
F
p
0.69
1.30
2.12
1.08
0.518
0.277
0.163
0.401
0.14
0.10
0.08
2.37
0.873
0.757
0.921
0.047*
0.81
0.10
0.05
8.45
0.469
0.761
0.949
< 0.001
0.46
0.00
0.06
4.59
0.642
0.999
0.945
0.002*
0.20
18.11
0.28
1.23
0.818
0.001*
0.759
0.338
0.85
9.23
0.73
0.89
0.451
0.010*
0.503
0.575
0.11
17.57
0.30
1.11
0.899
0.002*
0.745
0.412
df
2
1
2
12
31
2
1
2
12
18
2
1
2
12
17
2
1
2
12
16
2
1
2
12
16
2
1
2
12
17
2
1
2
11
15
H. opuntia
F
p
2.63
0.42
0.24
0.75
0.113
0.529
0.793
0.696
3.11
3.27
0.65
1.65
0.082
0.095
0.538
0.164
2.75
0.00
0.94
3.01
0.104
0.974
0.419
0.020*
2.48
2.05
0.70
1.69
0.125
0.178
0.515
0.161
2.61
8.93
0.81
0.90
0.114
0.011*
0.466
0.563
1.84
2.90
2.00
2.05
0.201
0.114
0.178
0.085
2.35
7.70
0.74
1.03
0.141
0.018*
0.497
0.464
responses of calcifying coral reef organisms to OA. But recent studies also reported resilience of several
scleractinian coral species to OA, suggesting species-specific and regional scale variation in responses
of organisms towards OA (Comeau et al. 2014).
Effects of DIN
As nitrate concentrations were elevated, the investigated organisms generally showed increased NOx
uptake in darkness and in calculated net NOx uptake over 24 h. These results, accompanied with nitrate
effects on other response parameters, suggest that the investigated organisms were nutrient limited in
ambient concentrations and were able to utilize the additionally available nitrate in elevated concen-
128
5 - Ocean acidification and eutrophication
Table 5.4: Continuation of Mixed Model ANOVA results of OA and elevated DIN on response parameters of organisms investigated. Asterisks indicate significant treatment effects
Response
variable
Light
NOx flux
Dark
NOx flux
Net
NOx flux
Chl a
Protein
content
Corg
Cinorg
N
C:N
Source
of variation
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
pCO2
DIN
pCO2:DIN
Tank
Residuals
A. tenuis
p
df
F
2
1
2
11
17
2
1
2
12
17
2
1
2
12
16
2
1
2
12
36
2
1
2
12
36
2.37
1.55
1.26
5.88
0.135
0.238
0.320
< 0.001*
0.31
16.05
0.24
1.19
0.741
0.002*
0.790
0.362
2.61
6.56
1.42
4.27
0.115
0.025*
0.279
0.004*
0.48
50.61
1.21
0.59
0.628
< 0.001*
0.331
0.838
1.68
0.27
2.00
0.50
0.227
0.613
0.178
0.899
df
2
1
2
12
16
2
1
2
12
16
2
1
2
12
16
2
1
2
12
35
2
1
2
12
34
S. hystrix
F
p
4.95
2.23
2.82
3.53
0.027*
0.161
0.099
0.010*
0.16
20.00
1.11
1.12
0.856
0.001*
0.362
0.411
1.54
2.09
1.03
3.52
0.255
0.174
0.386
0.010*
0.76
28.93
9.24
1.06
0.491
< 0.001*
0.004*
0.424
0.23
4.92
1.58
1.04
0.795
0.047*
0.247
0.440
df
2
1
2
12
18
2
1
2
12
16
2
1
2
12
18
2
1
2
12
33
2
1
2
12
33
2
1
2
12
33
2
1
2
12
33
2
1
2
12
33
H. opuntia
F
p
1.54
4.14
0.39
2.96
0.253
0.065
0.686
0.019*
1.95
6.22
0.14
5.06
0.185
0.028*
0.873
0.002*
1.64
5.58
0.36
4.14
0.235
0.036*
0.703
0.003*
1.48
10.36
0.61
2.21
0.266
0.007*
0.560
0.035*
1.65
6.66
0.92
1.43
0.232
0.024*
0.424
0.201
0.28
4.45
3.38
0.87
0.763
0.057
0.068
0.582
1.12
17.71
1.51
1.62
0.358
0.001*
0.261
0.135
1.78
30.45
2.38
1.31
0.210
< 0.001*
0.135
0.257
129
5 - Ocean acidification and eutrophication
trations. Coral nitrate uptake rates were in a similar range as determined in a previous study in-situ
for Acropora palmata by Bythell (1990). The latter study also showed a linear trend between nitrate
concentrations and uptake rates, suggesting corals/zooxanthellae are nitrate limited under naturally occurring concentrations (0.22-1.72 µmol L−1 NO3– ). Moreover, limited nutrient uptake rate of H. opuntia
in ambient conditions agrees with a study by Abel, Drew, et al. (1985), who showed nitrate uptake of H.
opuntia saturated around 13 µM NO3– .
An increase in photosynthesis under elevated nitrate, as observed for A. tenuis and H. opuntia in the
present study, has also been reported for several other scleractinian corals (Tanaka et al. 2007; Chauvin
et al. 2011) and calcareous algae including H. opuntia (Littler et al. 1988; Delgado and Lapointe 1994).
These studies attributed increased zooxanthellae density and pigment content to increased light capture
and photosynthetic rates. Contrary, results from Ferrier-Pagès et al. (2001) showed no nitrate effects
(< 1 µM and 2 µM) on zooxanthellae density or photosynthesis of the coral Stylophora pistillata, indicating concentration-specific or rather species-specific responses towards elevated nitrate, since NO3–
concentrations were low in the present study (∼2 µmol L−1 ). Despite increased photosynthetic rates of
organisms here, we did not see any elevation of calcification rates, indicating that additional available
energy is primarily utilized in other ways. Most notably, we observed increasing organic carbon and host
protein, results that are consistent with Tanaka et al. (2007), who observed an increase in organic tissue of Acropora pulchra at elevated nitrate. Other potential sinks include proliferation of zooxanthellae
density, zooxanthellae pigment concentration, and excess carbon may also lead to increased excretion
of organic components into the water column (Haas et al. 2010; Naumann et al. 2010).
In the present study we did not see any significant effect of elevated nitrate on calcification rates of
organisms. Previous studies have shown mixed effects including enhanced, neutral or negative effects
of elevated DIN on growth rates of corals. A study by Atkinson et al. (1995) suggested no effects of
increased nitrate on coral growth with concentrations of up to 5 µmol L−1 . However, the majority of
studies showed contradictory results with decreased coral calcification under elevated DIN, including
nitrate (Stambler et al. 1991; Marubini and Davies 1996; Ferrier-Pages et al. 2000; Renegar and Riegl
2005). The latter studies presume competition between photosynthesis and calcification about the internal DIC pool, and that enhanced photosynthesis under elevated nutrients may exhaust the internal
DIC pool for calcification. In contrast, Tanaka et al. (2007) reported an increase of coral calcification
under elevated nitrate, suggesting several possibilities for this nutrient enhanced calcification: (1) light
enhanced calcification may have directly been raised by increased photosynthesis in elevated nutrient
concentrations; (2) elevated nutrients may have promoted the synthesis of an organic matrix, which is a
required step for calcification; (3) increased supply of metabolic CO2 in the calicoblastic layer may have
promoted calcification rates (Tanaka et al. 2007). In the present study, the effects of nutrients on calcifi-
130
5 - Ocean acidification and eutrophication
cation were not significant, indicating neither light enhanced calcification by increased photosynthesis,
nor competition for the DIC pool between photosynthesis and calcification did take place under present
experimental nitrate concentrations. Notably, in the present study nitrate concentrations were considerably lower, but at the same time more similar to naturally occurring concentrations at inshore reefs on
the GBR compared to most other studies. In addition, non-significant effects of elevated nutrients on
calcification of H. opuntia agrees with previous results by Delgado and Lapointe (1994).
Chlorophyll a content of the investigated organisms increased under elevated nitrate concentrations.
Increased pigment content in corals can either derive from increased algal population density and/or increased pigment content per algal cell, which has been previously demonstrated for several corals under
elevated nitrate (Marubini and Davies 1996; Chauvin et al. 2011) and other nutrient species (Tanaka
et al. 2007). As demonstrated by Fabricius (2006) the warming of the coral surface is increased by up
to 1.5 °C in darker coral colonies (i.e. corals with increased pigmentation) compared to the ambient
seawater. Thus, even when higher pigment content does not seem to have a direct negative effect on
corals, the indirect effects of darker coloration may make them more susceptible to other stressors such
as warming accompanied by coral bleaching. Similarly, pigment content of H. opuntia was significantly
increased under elevated nutrients. Thus, zooxanthellae in both corals and pigments in H. opuntia were
nitrate limited under ambient seawater conditions.
Nitrate addition led to an imbalance between organic and inorganic growth. While host protein
content of coral A. tenuis was unaffected by elevated nitrate, it was significantly increased for coral S.
hystrix most likely due to an increase in nitrogen biomass of the host tissue, as presumed by Tanaka et al.
(2007). This increase in host protein content may lead to an imbalanced growth between organic tissue
and the inorganic carbonate skeleton, as demonstrated by Tanaka et al. (2007). Interestingly, respiration
of S. hystrix significantly increased under elevated nitrate which most likely arose from the increased
host-protein content and algae pigments.
In addition, organic carbon content of H. opuntia was significantly higher in nutrient treatments compared to controls. However, this elevation negatively correlated with inorganic carbon content, indicating
organic components (e.g. pigments) increased at the expense of inorganic components. Additionally,
total nitrogen content was increased with DIN, further contributing to the evidence that H. opuntia is
nitrate limited in ambient conditions. This result is also supported by accumulated nitrate of Halimeda
in nutrient up-welling locations on the GBR (Wolanski et al. 1988). Consequently, the calculated C:N
ratio considerably decreased in high nutrient concentrations.
131
5 - Ocean acidification and eutrophication
Interactive effects of pCO2 and elevated nitrate
In the present study, we tested whether elevated DIN affects responses of organisms to elevated DIC, but
we did not observe any significant interactive effects on the physiology of the organisms investigated.
Against our hypothesis, simultaneous increases in DIC and DIN did not show any synergistic effects on
productivity or growth rates of coral or algae. The present study showed that the physiology of organisms investigated responded more strongly to increased DIN than DIC after three weeks under present
experimental conditions. Elevated DIN reduces the thermal limit of corals due to an imbalanced supply
of other nutrients. This leads to phosphate starvation of coral zooxanthellae, changes in the lipid composition of the algal membranes and ultimately to the breakdown of the coral-zooxanthellae symbiosis
(Wooldridge 2009; Wiedenmann et al. 2013). Organisms inhabiting reefs susceptible to coastal runoff
may rapidly and strongly respond to DIN pulses in naturally occurring concentrations. These can lead
to associated effects on organisms’ physiology and can increase their susceptibility to other stressors,
which can ultimately lead to alterations in the community composition. It is unclear however, whether
or how fast organisms’ physiology will return to the original state in low nutrient conditions, a question
that should be addressed in future experimental approaches. Nevertheless, reducing fertilizer discharge
by coastal runoff will increase the organisms’ chances to cope with global environmental changes.
Acknowledgments
We want to thank the staff of the Lizard Island Research Station, Anne Hoggett, Lyle Vail, Cassy Thompson and Bruce Stewart for their support in conducting this experiment and their hospitality on the island.
Thanks to Stephen Boyle, Cassie Payn and Jane Wu Won from AIMS analytical services, for their help
with analyzing samples. Thanks to Jason Doyle for providing the protocol for pigment analyzes. This
study was funded by the Australian Institute of Marine Science, the Great Barrier Reef Foundation and
was conducted with the support of funding from the Australian Government’s National Environmental
Research Program.
132
5 - Ocean acidification and eutrophication
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138
Chapter 6
Interactive effects of ocean acidification
and warming on coral reef associated
epilithic algal communities under past,
present and future ocean conditions
Nikolas Vogel
1,2,3 ,
Neal Cantin 1 , Julia Strahl 1 , Paulina Kaniewska 1 , Line Bay 1 , Christian Wild
2,3
and Sven Uthicke 1
(1)
Australian Institute of Marine Science, PMB 3, Townsville MC, Queensland 4810, Australia
(2)
Leibniz Center for Tropical Marine Ecology, Fahrenheitstraße 6, 28359 Bremen, Germany
(3)
Faculty of Biology and Chemistry (FB 2), University of Bremen, 28359 Bremen, Germany
Keywords: Keywords: carbon dioxide, climate change, crustose coralline algae, Peyssonnelia spp.
This publication is in preparation
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Abstract
Epilithic algal communities play critical ecological roles on coral reefs, but their response to individual
and interactive effects of ocean warming (OW) and ocean acidification (OA) is still largely unknown. We
investigated growth, photosynthesis and calcification of early epilithic community assemblages exposed
to four temperature profiles (−1.1, ±0, +0.9, +1.7) that were crossed with four carbon dioxide partial
pressures (360, 440, 650, 940 µatm pCO2 ) under flow-through conditions and natural light regimes over
a six month period. Additionally, we compared the cover of heavily calcified crustose coralline algae
(CCA) and lightly calcified red algae of the genus Peyssonnelia. Increase in epilithic community cover
was higher at moderately increased than at high temperature and it was higher at present-day (440 µatm)
compared to reduced and elevated pCO2 conditions. Community level light-, dark- and net calcification
decreased with increasing pCO2 . Interactive effects resulted in lowest net calcification at low temperature/high pCO2 and highest net calcification at high temperature/past pCO2 . Final CCA cover was
higher at moderately increased than at all other temperatures and higher at present-day compared to all
other pCO2 conditions. Peyssonnelia cover was higher at high compared to ambient and moderate temperature, while it was lowered in past, but not affected by elevated pCO2 . Interactive effects resulted in
highest Peyssonnelia cover at low temperature/present-day pCO2 and lowest cover at moderate temperature/moderate pCO2 . Thus, CCA experienced additive negative effects from high temperature and high
pCO2 , while Peyssonnelia showed benefits in conditions where CCA were most affected, potentially due
to a different calcification intensity or -mineral. The lower cover of CCA and Peyssonnelia under past
pCO2 conditions suggests that acclimatization occurred from past to present-day pCO2 . If organisms
have no potential to further acclimatize, interacting OW and OA may change epilithic communities in
the future, leading to reduced reef stability and recovery.
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6.1 Introduction
Despite increasing awareness of climate change and its potential negative implications for species and
ecosystems, current anthropogenically induced greenhouse gas (GHG) emissions are higher than ever
before and predicted to rise further under the latest ‘representative concentration pathways’ (RCPs)
(Kirtman et al. 2013; Moss et al. 2010). The emission of GHGs has two well-documented impacts on
marine organisms. For one thing, the greenhouse effect leads to ocean warming (OW). The Intergovernmental Panel on Climate Change (IPCC 2013) predicts the strongest OW will occur in subtropical
and tropical regions with an increase in mean sea surface temperatures between 0.6 and 2.0 °C over the
course of this century following the RCP2.6 and RCP8.5, respectively (Collins et al. 2013). Temperature
changes within this range are already having detrimental effects on tropical species. For instance, coral
bleaching occurs one to two degrees above long-term summer maxima and result in increased mortality
and decreased coral cover on reefs around the globe (e.g. Hoegh-Guldberg 1999). Thus, tropical coral
reefs will be more frequently impacted by temperature extremes that leave less time for organisms and
communities to recover from the impacts of a warming ocean (Donner et al. 2005).
For another thing, increasing atmospheric carbon dioxide partial pressure (pCO2 ) leads to a decrease
in ocean pH, a term called ocean acidification (OA). OA results in a reduced saturation state of calcium
carbonate (Ω), the mineral required for the deposition of skeletons and shells of many marine species. To
date, atmospheric pCO2 has already exceeded historic peaks within the last two million years (Hönisch
et al. 2009) and is predicted to continuously rise from present-day levels of ∼395 µatm (Tans and Keeling
2015) to 670-936 µatm by the year 2100 with a consequent reduction of surface ocean pH of 0.2-0.3 units
following RCP6.0-RCP8.5, respectively (Ciais et al. 2013). OA has been shown to negatively impact
many coral reef organisms of a broad taxonomic range (e.g. Ries et al. 2009). Calcareous organisms are
of particular concern, since OA impedes the deposition of calcium carbonate (CaCO3 ) skeletons (Doney
et al. 2009).
Coral reef ecosystems are predicted to be negatively impacted by OW, OA and their interaction,
facing major ecological transformations in the upcoming decades (Hoegh-Guldberg and Bruno 2010;
Hoegh-Guldberg et al. 2007; Orr et al. 2005; Pandolfi et al. 2011). With current rates of increasing
emissions, marine organisms are facing unprecedented environmental changes (Masson-Delmotte et al.
2013) and their ability to acclimatize or adapt is challenged. The question to be answered is whether
organisms have a chance of such rapid acclimatization or adaptation or whether they will disappear.
Crustose coralline algae (CCA) are among the most important epilithic organisms and a key functional group in coral reef ecosystems (Fabricius and De’ath 2001). Epilithic communities, including
CCA and the calcareous red alga Peyssonnelia spp. significantly contribute to primary productivity, carbonate production and reinforce reef structures by CaCO3 deposition (Chisholm 2000; Chisholm 2003).
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Both, CCA and Peyssonnelia spp. provide the substrate and olfactory cues for invertebrate (i.e. corals)
settlement and metamorphosis (Harrington et al. 2004; Heyward and Negri 1999). CCA deposit calcite with high magnesium content (high-Mg-calcite) as the primary mineral of their heavily calcified
skeletons. High-Mg-calcite is the most soluble mineral of the carbonate species in seawater and makes
CCA specifically susceptible to OA (Anthony et al. 2008; Diaz-Pulido et al. 2012; Kuffner et al. 2007).
In contrast, CaCO3 content of Peyssonnelia spp. varies from lightly to heavily calcified tissues (Littler
and Littler 2003) with the more stable aragonite (James et al. 1988) which may create a competitive
advantage over CCA under future OA.
The few publications available generally observed increasing necrosis and decreasing calcification of
CCA under combinations of OW and OA (Diaz-Pulido et al. 2012; Johnson and Carpenter 2012; Martin
and Gattuso 2009). But the latter studies did not investigate the community composition of epilithic
algae, the interactions between functional groups, or their potential to acclimatize to changing pCO2 .
Thus, the long-term effects of changing environments on epilithic communities and their potential to
acclimatize to future environmental conditions are unknown. In the present study, we investigated these
questions by evaluating the performance of epilithic algae under past, present-day, and future OW and
OA conditions. We conducted a long-term aquaria experiment which included three experimental stages:
(1) colonization of substrates on a coral reef, (2) acclimatization to experimental aquaria facility and (3)
treatment phase with four temperature profiles that each included an anomaly of −1.1, ±0.0, +0.9 and
+1.7 °C, fully crossed with four fixed pCO2 conditions (360, 440, 650, and 940 µatm). After six months
exposure to different treatment combinations, we analyzed the effects of OW and OA on change in cover,
photosynthesis and calcification of the epilithic communities. We also compared the final spatial cover
of CCA and Peyssonnelia spp. which formed the main part of the communities on the experimental
substrates after six months of exposure.
6.2 Methods
Experimental design
PVC pipes were utilized as substrate for epilithic algal communities (Fig. 6.1a) and were deployed on
aluminum fragment racks inside the lagoon at Davies Reef (4 m water depth; S 18° 49.963’, E 147°
38.001’), within the central Great Barrier Reef, for five months between January and May 2012. Field
established epilithic algal communities were transferred into outdoor aquaria facilities at the Australian
Institute of Marine Science (AIMS) in Townsville. Organisms were acclimatized to the AIMS aquaria
system in ambient seawater conditions and natural sunlight (reduced by 75% with green shade cloth;
∼10 mol photons m−2 d−1 ), over a period of four months, between May 2012 and September 2012.
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In October 2012, 96 PVC pipes were distributed among 48 individual aquaria, and the temperature and
acidification treatments were implemented. CO2 concentration was reduced (360 µatm ± 36 SD) for past
pCO2 treatment, was left unmodified (440 µatm ± 21 SD) for present-day pCO2 treatment, was increased
(650 µatm ± 70 SD) for moderate pCO2 treatment (∼corresponding to RCP6.0) and was increased
(940 µatm ± 96 SD) for high pCO2 treatment (∼corresponding to RCP8.5). Similarly, temperature
was cooled by 1.1 °C ± 0.5 SD for low temperature treatment (25.0 °C average), followed long-term
averages on Davies Reef for the ambient temperature treatment (26.1 °C average), was increased by 0.9
°C ± 0.3 SD for moderate temperature treatment (26.5 °C average) and was increased by 1.7 °C ± 0.5
SD for high temperature treatment (27.7 °C average). Temperature peaks were reached in November
2012 with 25.9, 27.9, 29.4 and 30.7 °C for the low, ambient, moderate and high temperature treatment,
respectively. Thereafter, temperatures were continuously reduced again and communities were allowed
to recover from December to March 2013 (see Fig. 6.2 for experimental temperature profile and Table 1
for carbonate system parameters).
(a)
(b)
(c)
1 mm
360 440 650 940
µatm µatm µatm µatm
25.0/ 25.9 °C
360 440 650 940
µatm µatm µatm µatm
26.1/ 27.9 °C
(d)
1 mm
1 cm
360 440 650 940
360 440 650 940
µatm µatm µatm µatm µatm µatm µatm µatm
26.5/ 29.4 °C
27.7/ 30.7 °C
Figure 6.1: (a) Images of PVC substrates with encrusting communities taken after six months in experimental conditions. The bottom row shows one PVC rack out of each experimental tank. The top row
shows one close-up image of one substrate from each treatment (utilized to determine surface areas of
communities and organisms). (b) Microscopic image of CCA and (c) Peyssonnelia spp. (d) Example of
image utilized for surface area determination of different organisms. CCA and Peyssonnelia spp. were
manually color coded prior to automated surface area determination
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Temperature Treatment
Davies Reef Daily Avg (1991-2012)
Low
Moderate
High
Seasonal Average
1
2
3
32
Temperature [°C]
30
28
26
24
22
Jan
2012
Mar
2012
May
2012
Jul
Sep
2012 2012
Date
Nov
2012
Jan
2013
Mar
2013
Figure 6.2: Temperature profile of the experiment: (1) pre-experimental phase (for initial growth on
PVC substrates) on Davies Reef, (2) acclimatization phase to AIMS aquaria facilities and (3) temperature/acidification treatments
Experimental setup
Forty-eight aquaria (70 L) were evenly distributed on four tables, where individual temperature control
was achieved through submersion of aquaria in freshwater temperature controlled baths (EvoHeat DHP
40 heater/chiller pump, Evo Industries) supplied from four large sump tanks (5500 L; Gough Plastics),
one for each temperature treatment. Within each freshwater temperature control sump tank, four smaller
seawater sump tanks were fitted (575 L; Gough Plastics) in which the seawater carbonate chemistry was
individually manipulated with automated CO2 air injections bubbled into the sump tanks with solenoid
valves (SMC pneumatics) to pH set points. ISFET pH probes (Endress Hauser CPS-471D) in the sump
tanks continuously monitored seawater pH and the solenoid valves to maintain target pH values. Past
pCO2 conditions were achieved by passing atmospheric air through two soda lime canisters and mixing the low CO2 scrubbed air with the incoming seawater in a counter current exchange tower prior to
flowing into each experimental tank. The ambient pCO2 treatment was left unmodified. Experimental
aquaria were provided with recirculating (7 L min−1 ) ultra-filtered seawater (0.2 µm) seawater. Recirculating seawater was replaced with new filtered (0.2 µm) seawater at a flow rate of 0.3 L min−1 assuring
a turnover in the experimental tanks twice daily.
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Determination of carbonate chemistry
Experimental manipulation of the seawater carbonate chemistry, pH and pCO2 was monitored by continuous records of pH, using ISFET pH probes (Endress Hauser CPS-471D) and weekly measurements
of seawater pCO2 (LiCor LI-840A CO2 /H2 O Analyzer) from each experimental aquarium. Additionally, water samples were drawn from individual aquaria, according to the standard operating procedure
(SOP1) described in Dickson et al. (2007), ten times over the course of the experiment on 2-3 week
sampling intervals to determine total alkalinity (TA) and dissolved inorganic carbon (DIC). Each 250
mL sample was fixed with 125 µL saturated mercuric chloride solution and was subsequently analyzed
for TA and DIC content on a VINDTA 3C titrator (Marianda) at AIMS, as described elsewhere (Uthicke
et al. 2014). Carbonate system parameters (Table 6.1) were calculated as a function of measured salinity, temperature, TA and DIC using the software CO2SYS (Lewis and Wallace 1998) and dissociation
constants for carbonate system parameters as determined by Mehrbach (1973).
Change in community cover and final cover of CCA and Peyssonnelia spp.
To determine the surface area of the entire algal epilithic communities, as well as CCA and Peyssonnelia
spp., high resolution images of PVC substrates were taken with a copy stand mounted digital single
lens reflex (SLR) camera (Nikon D300), a 40 mm micro Nikkor lens and a ring flash (Nikon). Images
for initial and final cover of the epilithic communities were taken in October 2012 and March 2013,
respectively. The software ImageJ was used to determine the surface areas of epilithic communities and
organisms relative to standard reference within the images. CCA and Peyssonnelia spp. in each image
were manually selected and color-coded (Fig. 6.1d), before surface area (in cm2 ) was determined by
an automated macro function. For each experimental tank four images (each covering ∼33 cm2 ) from
two PVC pipes (192 images in total) were analyzed for community, CCA and Peyssonnelia spp. surface
area. Growth of the epilithic communities was expressed as change in cover (in percent) from initial to
final measurements. Final cover of CCA and Peyssonnelia spp. (Fig. 6.1b, c) was expressed as surface
area on the substrate (in percent) covered by the organisms.
Oxygen fluxes and calcification rates of the epilithic community
In April 2013, after six months of experimental treatment, oxygen fluxes and calcification rates of the
epilithic communities were determined under light and dark conditions. PVC substrates were divided
into subsamples (∼67 cm2 ) to fit the 445 mL incubation chambers. The bottom area of the substrates
was cleaned to remove all organisms to ensure that only the top light exposed area was contributing to
physiological measurements. After 24 h substrates were placed in individual incubation chambers filled
with water from the source experimental tank. Incubation chambers were placed in black plastic bins
146
Treatment targets
[°C/ µatm]
min/ 300
min/ 400
min/ 685
min/ 950
average/ 300
average/ 400
average/ 685
average/ 950
moderate/ 300
moderate/ 400
moderate/ 685
moderate/ 950
high/ 300
high/ 400
high/ 685
high/ 950
Av. temp
[°C]
25.2 (0.5)
24.8 (0.5)
24.9 (0.4)
25.1 (0.5)
26.2 (0.9)
25.9 (0.9)
26.1 (0.9)
26.2 (0.9)
27.0 (1.2)
27.0 (1.1)
26.8 (1.1)
27.0 (1.1)
27.6 (1.5)
27.6 (1.3)
27.7 (1.3)
27.8 (1.3)
Max. temp
[°C]
26.1
25.8
25.7
26.0
28.1
27.7
27.8
27.9
29.6
29.5
29.2
29.3
30.7
30.5
30.8
30.6
8.08 (0.02)
8.00 (0.03)
7.86 (0.02)
7.75 (0.03)
8.09 (0.02)
7.98 (0.02)
7.86 (0.03)
7.75 (0.03)
8.08 (0.03)
7.97 (0.02)
7.86 (0.08)
7.77 (0.07)
8.07 (0.03)
7.98 (0.02)
7.84 (0.06)
7.76 (0.03)
pHtotal
TA
[µmol kgSW-1]
2313 (35)
2295 (42)
2308 (40)
2308 (36)
2312 (38)
2299 (45)
2310 (40)
2317 (35)
2306 (41)
2306 (39)
2303 (41)
2307 (39)
2310 (37)
2303 (40)
2315 (38)
2316 (39)
DIC
[µmol kgSW-1]
1984 (41)
2012 (33)
2098 (34)
2150 (29)
1962 (29)
2020 (33)
2092 (34)
2151 (30)
1951 (39)
2020 (31)
2077 (49)
2125 (41)
1958 (42)
2006 (30)
2093 (44)
2134 (28)
pCO2
[µatm]
362 (35)
432 (19)
666 (68)
931 (108)
352 (33)
438 (11)
639 (55)
925 (67)
342 (28)
439 (35)
604 (80)
991 (93)
384 (46)
445 (18)
686 (78)
917 (115)
HCO3[µmol kgSW-1]
1742 (43)
1798 (33)
1924 (31)
2000 (25)
1706 (28)
1807 (28)
1913 (32)
1996 (30)
1693 (43)
1802 (30)
1892 (64)
1962 (49)
1701 (45)
1781 (28)
1909 (52)
1971 (24)
CO32[µmol kgSW-1]
232 (6)
201 (15)
156 (8)
125 (9)
246 (14)
200 (13)
162 (10)
131 (11)
249 (16)
205 (13)
168 (26)
141 (20)
248 (9)
212 (14)
166 (19)
141 (12)
CO2
[µmol kgSW-1]
10 (1)
12 (1)
18 (1)
25 (2)
9 (1)
13 (1)
18 (1)
24 (2)
9 (1)
13 (1)
18 (4)
23 (4)
10 (1)
12 (1)
18 (3)
23 (2)
3.67 (0.09)
3.19 (0.24)
2.47 (0.12)
1.98 (0.14)
3.92 (0.22)
3.19 (0.22)
2.58 (0.16)
2.09 (0.18)
3.98 (0.25)
3.28 (0.21)
2.69 (0.43)
2.25 (0.32)
3.98 (0.15)
3.41 (0.24)
2.66 (0.31)
2.27 (0.19)
Ωar
Table 6.1: Temperature (n ∼ 200) and carbonate system parameters (n = 10) of experimental tanks. Data is given as means (± SD)
6 - Ocean acidification and warming
147
6 - Ocean acidification and warming
(45 L) provided with recirculating water from the corresponding sump tank to maintain the intended
temperature target (low and high = 24.8 and 27.9 °C, respectively) for the incubation run. During net
photosynthesis and light calcification measurements (2 h), two lights (4 × 55 W, 10000 K, CA) provided
a constant light intensity of 250 µmol photons m−2 s−1 . During dark runs (3 h), black plastic lids were
fitted onto the bins to fully darken the incubation chambers. After each incubation run, O2 content (mg
L−1 ) of the seawater in the incubation chamber was determined with a hand-held dissolved oxygen meter
(HQ30d, Hach). Net photosynthesis and respiration, expressed in µg O2 L−1 h−1 m−2 , was calculated
in relation to blank incubations (seawater only) and normalized to tile specific epilithic algal cover.
Gross photosynthesis was calculated by net photosynthesis and dark respiration rates. Calcification
rates in light and dark were determined with the alkalinity anomaly technique (Chisholm and Gattuso
1991). Total alkalinity (TA) was determined in fresh subsamples (50 mL) of the incubation seawater
on a Metrohm 855 robotic titrosampler by gran titration, using 0.5 M HCl as described in Uthicke and
Fabricius (2012). Total alkalinity was calculated by non-linear regression fitting between pH 3.5 and 3.0.
Total alkalinity was corrected to Certified Reference Material (CRM Batch 106, A. Dickson, Scripps
Oceanographic Institute). Relative calcification rates were expressed in µM C h−1 m−2 calculated in
relation to blank incubations and normalized epilithic algal cover. Net calcification rates were calculated
from 12 h light- and 12 h dark calcification rates and expressed in mM C d−1 m−2 .
Statistical analyzes
To test for confirmation to the assumptions of Generalized Linear Models we used Levene’s test implemented within the R environment (R Development Core Team 2014). For CCA cover, variances were
equal when data were arc-sine transformed. Generalized Linear Models were undertaken with NCSS
software (Hintze 2007) on response variables change in epilithic community cover, photosynthesis, calcification and final cover of CCA and Peyssonnelia spp. with temperature and pCO2 as fixed factors
and experimental tank as nested random factor. To determine significantly differing treatment groups,
we conducted Tukey-Kramer Multiple Comparison tests with NCSS software (Hintze 2007). To test
for significant correlations between CCA and Peyssonnelia spp., Pearson Product-Moment Correlation
Tests were performed on surface areas with R software (R Development Core Team 2014).
6.3 Results
Change in cover of epilithic algal communities
During six months of exposure to experimental conditions, temperature and pCO2 significantly affected
growth of the epilithic communities investigated (Fig. 6.3, Table 6.2). The interaction of both stressors
148
6 - Ocean acidification and warming
did not show any significant effect (Table 6.2). Growth measured at moderate temperature (+0.9 °C
above ambient) was 87.6% higher as compared to growth at high temperature (Tukey-Kramer < 0.05),
but was not significantly different to low and ambient temperature (Table 6.3). At present-day pCO2
concentrations (440 ± 36 µatm) growth of the epilithic communities was 104.9, 57.8 and 103.3% higher
than at past, moderate and future pCO2 levels, respectively (Tukey-Kramer < 0.05, Table 6.3).
pCO2 [µatm]
Community growth [%]
0
100 200 300
Temperature [°C]
25.0/ 26.1/ 26.5/ 27.7/
360
25.9 27.9 29.4 30.7
Treatment
440
650
940
Figure 6.3: Percent change in cover of epilithic communities over six month of temperature and pCO2
treatment
Photosynthesis- and calcification rates of epilithic communities
After six months of experimental treatment temperature, pCO2 and the interaction of both factors significantly affected relative net photosynthesis of the epilithic community (Fig. 6.4, Table 6.2). Net photosynthesis was 8.8% lower at high compared to low temperature and 14.2% lower at present-day than
at past pCO2 (Tukey-Kramer < 0.05). The interaction of temperature and pCO2 resulted in lower net
photosynthesis at high temperature/present-day pCO2 compared to all other treatments (Tukey-Kramer
< 0.05). Dark respiration was 26.2% higher at high compared to low temperature (Tukey-Kramer
< 0.05, Fig. 6.4, Table 6.2), but was not affected by pCO2 or interactions. Gross photosynthesis was
significantly affected by pCO2 and the interaction of temperature and pCO2 (Fig. 6.4, Table 6.2). Gross
photosynthesis was lowest at present-day pCO2 (40.6 µg O2 h−1 m−2 ), which was significantly lower
compared to past pCO2 condition (Tukey-Kramer < 0.05, 46.3 µg O2 h−1 m−2 ). In addition, interactive
effects resulted in the lowest gross photosynthetic rates at high temperature/present-day pCO2 compared
to all other treatment combinations (Tukey-Kramer < 0.05, Fig. 6.4, Table 6.2).
Moreover, light calcification of the epilithic community was 76.5 and 81.8% lower at high compared
to past and present-day pCO2 , respectively (Tukey-Kramer < 0.05, Fig. 6.4, Table 6.2). Temperature,
pCO2 and the interaction of both significantly affected dark calcification of the epilithic community (Fig.
149
6 - Ocean acidification and warming
Table 6.2: Generalized Linear Model results for effects of temperature, pCO2 and the interaction of both
on response parameters of the epilithic community and CCA and Peyssonnelia spp. Tukey contrasts
represent significantly different treatment groups (1, 2, 3, 4) from temperature (25.0, 26.1, 26.5 and 27.7
°C) and pCO2 (360, 440, 650, 940 µatm)
Response variable
Community growth [%]
(change in cover from initial to
final measurement)
Community net photosynthesis
Community dark respiration
Community gross photosynthesis
Community light calcification
Community dark calcification
Community net calcification
CCA cover [%]
Peyssonnelia cover [%]
Mixed Linear Model
Source of variation
Temp
pCO2
Temp:pCO2
Tank
Residual
Temp
pCO2
Temp:pCO2
Tank
Residual
Temp
pCO2
Temp:pCO2
Tank
Residual
Temp
pCO2
Temp:pCO2
Tank
Residual
Temp
pCO2
Temp:pCO2
Tank
Residual
Temp
pCO2
Temp:pCO2
Tank
Residual
Temp
pCO2
Temp:pCO2
Tank
Residual
Temp
pCO2
Temp:pCO2
Tank
Residual
Temp
pCO2
Temp:pCO2
Tank
Residual
df
3
3
9
28
40
1
2
2
12
54
1
2
2
12
54
1
2
2
12
54
1
2
2
12
51
1
2
2
12
51
1
2
2
12
51
3
3
9
32
144
3
3
9
32
144
Tukey contrasts
1
2
3
4
ab ab a
b
a
b
a
a
F
3.70
13.94
1.94
1.46
p
0.0233*
< 0.0001*
0.0872
0.1343
5.86
6.01
8.40
2.64
0.0323*
0.0155*
0.0052*
0.0074*
a
a
ab
-
b
b
12.83
1.25
2.08
3.42
0.0038*
0.3206
0.1680
0.0009*
a
-
-
b
2.25
4.41
5.84
2.89
0.1597
0.0367*
0.0170*
0.0038*
a
b
-
ab
1.51
26.06
3.29
1.08
0.2429
< 0.0001*
0.0730
0.3960
a
a
-
b
31.05
33.24
5.03
0.53
0.0001*
< 0.0001*
0.0259*
0.8858
a
a
b
-
b
c
0.79
41.20
5.48
0.82
0.3901
<0.0001*
0.0204*
0.6302
a
a
-
b
13.26
9.83
1.43
9.08
<0.0001*
0.0001*
0.2184
<0.0001*
a
a
a
b
b
a
a
a
5.18
4.71
2.73
3.03
0.0049*
0.0078*
0.0175*
<0.0001*
ab
a
a
b
a
ab
b
ab
150
6 - Ocean acidification and warming
Table 6.3: Summary of individual and interactive effects of temperature and pCO2 on growth of the
investigated epilithic community and final cover of CCA and Peyssonnelia spp. Results are given as
means (± SD). Asterisks indicate significant treatment effects
Treatment
n
48
Community
growth [%] ± SE
31.06 (14.32)*
CCA cover
[%] ± SE
11.3 (2.2)*
Peyssonnelia
cover [%] ± SE
20.7 (2.1)*
Low temp
Ambient temp
48
29.23 (16.09)*
10.0 (2.2)*
15.6 (2.1)*
Moderate temp
48
82.33 (15.69)*
24.0 (2.2)*
13.2 (2.1)*
High temp
48
10.21 (15.31)*
4.7 (2.2)*
23.7 (2.1)*
Past pCO2
48
-5.59 (15.31)*
10.4 (2.2)*
12.8 (2.1)*
Present pCO2
48
114.11 (14.63)*
22.0 (2.2)*
23.8 (2.1)*
Moderate pCO2
48
48.11 (16.09)*
12.6 (2.2)*
19.1 (2.1)*
High pCO2
48
-3.80 (15.31)*
5.1 (2.2)*
17.4 (2.1)*
Low temp,
past pCO2
Low temp,
present pCO2
Low temp,
moderate pCO2
Low temp,
high pCO2
Ambient temp,
past pCO2
Ambient temp,
present pCO2
Ambient temp,
moderate pCO2
Ambient temp,
high pCO2
Moderate temp,
past pCO2
Moderate temp,
present pCO2
Moderate temp,
moderate pCO2
Moderate temp,
high pCO2
High temp,
past pCO2
High temp,
present pCO2
High temp,
moderate pCO2
High temp,
high pCO2
12
-8.70 (28.64)
5.4 (4.5)
11.4 (4.2)*
12
119.74 (28.64)
22.0 (4.5)
37.0 (4.2)*
12
28.59 (28.64)
9.9 (4.5)
22.8 (4.2)*
12
-15.37 (28.64)
7.7 (4.5)
11.5 (4.2)*
12
-29.69 (28.64)
8.6 (4.5)
11.5 (4.2)*
12
129.07 (31.37)
23.3 (4.5)
11.5 (4.2)*
12
18.12 (35.08)
4.7 (4.5)
21.7 (4.2)*
12
-0.60 (35.08)
3.6 (4.5)
17.6 (4.2)*
12
42.75 (40.50)
23.6 (4.5)
10.6 (4.2)*
12
99.73 (28.64)
31.1 (4.5)
14.5 (4.2)*
12
176.97 (31.37)
33.2 (4.5)
9.2 (4.2)*
12
9.88 (28.64)
8.4 (4.5)
18.4 (4.2)*
12
26.74 (28.64)
4.0 (4.5)
17.6 (4.2)*
12
107.90 (28.64)
11.6 (4.5)
32.4 (4.2)*
12
-31.23 (35.08)
2.7 (4.5)
22.7 (4.2)*
12
-9.10 (31.37)
0.7 (4.5)
22.1 (4.2)*
151
6 - Ocean acidification and warming
6.4, Table 6.2). Dark calcification was 62.8% lower at low than at high temperature, was 86.0% lower at
high than at present-day pCO2 and was 72.2% higher at past than at present-day pCO2 (Tukey-Kramer <
0.05). Interactions led to decalcification of the epilithic community in darkness at low temperature/high
pCO2 and highest calcification at high temperature/past pCO2 (Tukey-Kramer < 0.05, Fig. 6.4, Table
6.2). Furthermore, pCO2 and the interaction of temperature and pCO2 significantly affected relative net
calcification rate of the epilithic communities (Fig. 6.4, Table 6.2). Net calcification rates were 82.0
and 82.8% lower at high compared to past and present-day pCO2 , respectively (Tukey-Kramer < 0.05,
Fig. 6.4, Table 6.2). The interaction of temperature and pCO2 resulted in highest net calcification at
high temperature/present-day pCO2 compared to lowest net calcification at low temperature/high pCO2
(Tukey-Kramer < 0.05, Fig. 6.4, Table 6.2).
Final cover of CCA and Peyssonnelia spp.
The relative cover of CCA (lm, df = 15, F = 1.53, p = 0.1555) and Peyssonnelia spp. (lm, df = 15,
F = 0.77, p = 0.6967) did not differ between treatments when the experiment was commenced. Thus,
all significant treatment effects on final organism cover were derived from respective temperature and
pCO2 exposure. CCA and Peyssonnelia spp. formed the main part of the communities with an average
cover of 31% on the substrates, representing 59% of the communities, crossed over all experimental
treatments.
Temperature and pCO2 significantly affected final cover of CCA (Fig. 6.5, Table 6.2). At moderate temperature CCA cover was 112.4, 140.0 and 410.6% higher compared to low, ambient and high
temperatures, respectively (Tukey-Kramer < 0.05, Table 6.3). CCA cover was 111.5, 74.6 and 331.4%
higher at present-day than at past moderate and high pCO2 , respectively (Tukey-Kramer < 0.05, Table
6.3).
Temperature, pCO2 and the interaction of both significantly affected the cover of red algae Peyssonnelia spp. (Fig. 6.5, Table 6.2). At high temperature, Peyssonnelia spp. cover 51.9% higher than at
ambient temperature and 79.5% higher than at moderate temperature (Tukey-Kramer < 0.05, Table 6.3).
Lowest cover of Peyssonnelia spp. was measured at past pCO2 which was 46.2% lower compared to
present-day pCO2 treatment, but not significantly different to moderate and high pCO2 (Tukey-Kramer
< 0.05, Table 6.3). Interactive effects of temperature and pCO2 resulted in the highest cover of Peyssonnelia spp. at low temperature/present-day pCO2 and high temperature/present-day pCO2 and lowest
cover at moderate temperature/moderate pCO2 (Tukey-Kramer < 0.05, Table 6.3).
Pearson’s Product-Moment Correlation tests revealed a significant negative correlation between final
cover of CCA and Peyssonnelia spp. (t = −2.1307, df = 46, p < 0.0385, R2 = 0.30). Peyssonnelia spp.
were generally more abundant at temperatures and pCO2 treatments where CCA decreased in cover.
152
24.8 °C
27.9 °C
24.8 °C
27.9 °C
Net calcification [mM C d-1 m-2] Dark calcification [µM C h-1 m-2] Light calcification [µM C h-1 m-2]
0
2
4
6 −50 0
50 100 150 200 −100 0 100 200 300 400
Gross photosynthesis [µg O2 L-1 h-1 m-2] Respiration [µg O2 L-1 h-1 m-2] Net photosynthesis [µg O2 L-1 h-1 m-2]
0
20
40
60 −8
−6
−4
−2
0 0
20
40
60
6 - Ocean acidification and warming
360
440
940
360 440
pCO2 [µatm]
940
360
440
940
360 440
pCO2 [µatm]
940
Figure 6.4: Net photosynthesis, respiration and gross photosynthesis as well as light-, dark- and net
calcification of epilithic communities after six months of temperature and acidification treatments. Data
are normalized to the surface area of the epilithic communities
6.4 Discussion
Results of the present study showed that epilithic algal communities and organisms are affected by combinations of OW and OA. Potentially, they have acclimatized from past to present-day conditions, but
high increases of OW and OA in future, as predicted under the RCP6.0 and RCP8.5, indicate reduced
community productivity and calcification and severe shifts in the community compositions with associated implications for coral reefs on the global scale.
153
25.0/ 25.9 °C
26.1/ 27.9 °C
26.5/ 29.4 °C
27.7/ 30.7 °C
Peyssonnelia
CCA
0
Organism cover [%]
20 40 60 80
6 - Ocean acidification and warming
360 440 650 940
360 440 650 940
360 440 650 940
pCO2 [µatm]
360 440 650 940
Figure 6.5: Percent final cover of CCA and Peyssonnelia spp. after six months exposure to temperature
and acidification treatments
Effects of temperature
The observed increase in cover of the epilithic communities and the final cover of CCA under the moderate temperature profile (Fig. 6.3) indicates beneficial effects under future OW with GHG concentrations following RCP2.6-RCP4.5 (+0.9 °C). These results agree with elevated growth rates of epilithic
communities and coralline algae observed in summer months, under increased temperature and irradiance, compared to winter months (Klumpp, McKinnon, et al. 1992; Martin et al. 2006; Martin and
Gattuso 2009). Potentially, enhanced physico-chemical and metabolic processes promote growth rates
under moderately increased temperatures. Additionally, a higher saturation state of calcium carbonate
(CaCO3 ) minerals at warmer temperatures may facilitate growth of calcifying organisms.
We measured significantly lower growth of epilithic communities and cover of CCA in the high
temperature treatments, supporting prediction and previous data that CCA will be negatively affected
by climate change (e.g. Anthony et al. 2008; Diaz-Pulido et al. 2012; Johnson and Carpenter 2012).
Our data also indicate that epilithic algal communities may already live close to their upper temperature
tolerance limits, as it is the case in other coral reef organisms (Hoegh-Guldberg 1999), and an additional increase of only 1.7 °C (RCP8.5) above present-day summer maxima removes the advantages of
increased growth under moderate OW (+0.9 °C; RCP2.6-RCP4.5).
Peyssonnelia spp. cover was increased at highest compared to ambient and moderately increased
temperature. Thus, Peyssonnelia spp. were most abundant at temperatures at which CCA were less
abundant, indicating Peyssonnelia spp. may benefit from OW under GHG concentrations at which CCA
growth is impaired (i.e. RCP8.5, +1.7 °C). This hypothesis was additionally supported by significant
negative correlations between cover of Peyssonnelia spp. and CCA. Potentially, space limitation and
competitive advantages of CCA over Peyssonnelia spp. at ambient and moderate temperature were
removed at low and high temperature, so Peyssonnelia spp. could increase their relative abundance
within the community. Thus, under future OW, Peyssonnelia spp. may increase in cover at the expense
154
6 - Ocean acidification and warming
of the more temperature sensitive CCA.
Effects of pCO2
Reduced growth and calcification of epilithic communities at elevated pCO2 , as observed in the present
study, has been well documented for a range of calcareous organisms including CCA species (Comeau
et al. 2013; James et al. 2014; Kuffner et al. 2007; Orr et al. 2005). Decreases of light-, dark- and
net calcification under increasing pCO2 , as measured in the present study, may have been additionally
exacerbated by a relative increase of lightly calcified Peyssonnelia over heavily calcified CCA under
moderate and high pCO2 conditions. A reduction in pH, accompanied by a lowered availability of
free carbonate ions, increases the energetic demands for calcification. Moreover, an altered carbonate
chemistry with reduced Ω is believed to impair calcification particularly for high-Mg-calcite organisms
such as CCA (Kuffner et al. 2007). Reduced calcification rates under elevated pCO2 also reflect trends
in growth rates of the epilithic communities and final cover of CCA, sensitive to OA, as measured in the
present study.
Interestingly, net calcification rates were not lowered under past pCO2 condition which is coherent
with regards to carbonate chemistry (i.e. increased Ω), but is contrary to findings of lowered longterm growth of the epilithic communities as well as lowered final cover of CCA and Peyssonnelia spp.
in this particular treatment. The observation of reduced growth rates under past pCO2 is novel and
unexpected, particularly for CCA, since the carbonate chemistry (i.e. elevated Ω) in this treatment should
facilitate calcification and consequently growth rates and cover. Results from a short-term study (two
weeks) by Comeau et al. (2013) did not observe any effects of reduced CO2 on Hydrolithon onkodes
or Lithophyllum flavencens, while results from Diaz-Pulido et al. (2011) indicate mineral changes in
CCA under reduced CO2 conditions after an eight week experiment. So far, most studies, investigating
the effects of OA on calcareous organisms, considered present-day and future (i.e. elevated) pCO2 ,
but did not account for past (i.e. reduced) pCO2 conditions. As shown by Johnson et al. (2014) CCA
are able to acclimatize to oscillating pCO2 conditions. But previous studies disregard the potential for
acclimatization of organisms that may have already taken place from past to present-day conditions and
the possibility that they may have potential to further acclimatize under rising pCO2 .
Here, epilithic communities and CCA performed best under present-day pCO2 conditions, despite
it being a less favorable condition for calcification, compared to past pCO2 treatments (Table 6.1). As
shown by Albright et al. (2013) reef water from the GBR has substantial fluctuations in pCO2 due to diurnal changes in net carbon production of organisms and due to seasonal changes in seawater temperature
with values well above global average (∼397 µatm; Tans and Keeling (2015)). Moreover, particularly
at inshore locations of the GBR, pCO2 concentrations increased two- to three times faster and are al-
155
6 - Ocean acidification and warming
ready higher than the global average, potentially due to anthropogenically induced trophic changes in
the water column and benthos, as suggested by Uthicke et al. (2014). Thus, CCA may already have
physiologically acclimatized to higher pCO2 in their natural habitat. Potentially, CCA prefer a DIC optimum for photosynthesis, calcification and growth which appears to be in the range of the present-day
treatment of this study. Changes in carbonate chemistry diverging from present-day condition may have
implications on other cellular processes than calcification and may negatively influence growth rates, as
observed here. Nevertheless, epilithic communities are likely to reduce calcification, carbon fixation,
and carbonate production in future high pCO2 environments following RCP8.5. Consequently, high
pCO2 may have implications on carbonate production by epilithic communities, on structural properties of future coral reefs and their resilience against- as well as their recovery after disturbances such
as tropical cyclones. The question remains to which extent CCA are capable to acclimatize to further
increasing pCO2 .
Non-linear calcification responses of CCA have been reported elsewhere (Johnson and Carpenter
2012; Ries et al. 2009) and CCA may be able to acclimatize and to protect themselves from decreasing
Ω and associated dissolution under OW and OA conditions by increasing the more stable dolomite, over
the more soluble high-Mg-calcite, as their skeletal mineral (Diaz-Pulido et al. 2014). Dolomite-rich
CCA have been shown to better resist OA conditions (Nash et al. 2013). These findings agree with
the observed enhanced performance and thus potential acclimatization of CCA from past to present-day
pCO2 conditions, in the present study. However, findings from tropical CO2 seeps indicate reduced
abundance of CCA in OA conditions of ∼750 µatm pCO2 , suggesting no acclimatization after life-long
exposure to high levels of seawater CO2 (Fabricius et al. 2011). Results from the latter study, combined
with findings of the present study, indicate acclimatization is possible to a certain extent (i.e. past to
present-day and potentially to moderate pCO2 ), but not to high pCO2 . These results emphasize the
efforts to reduce anthropogenic GHG emissions in order to keep future GHG concentrations below the
RCP2.6–RCP4.5 to preserve CCA on future coral reefs.
Because Peyssonnelia spp. cover was not significantly lowered at high compared to present-day
pCO2 , they may be less impacted under pCO2 concentrations following RCP8.5, compared to CCA.
Potential reasons for the advantage gained by Peyssonellia spp. over CCA include the overcoming
of space limitation and interspecific competition. Additionally, the deposition of lightly compared to
heavily calcified skeletons, as well as the more stable aragonite compared to the more soluble high-Mgcalcite of CCA, may lead to competitive advantages of Peyssonnelia spp. over CCA in a high pCO2
world.
156
6 - Ocean acidification and warming
Interactive effects of temperature and pCO2
The increase in community net calcification at high temperature/present-day pCO2 and the decrease in
low temperature/high pCO2 agree with theoretical and observed optima of carbonate chemistry (i.e. Ω)
for calcification (Table 6.1). Community net calcification is improved under higher temperature and
lower pCO2 levels compared to lower temperatures and higher pCO2 . Similarly, dark calcification rates
were lowest at low temperature/high pCO2 and highest at high temperature/past pCO2 , as anticipated
from the particular carbonate chemistry in these treatments. Additional respiratory CO2 release from the
organisms in the dark may have exacerbated the negative effects of reduced Ω due to low temperature and
OA, leading to drastic declines in calcification if the epilithic communities. Peyssonnelia spp. cover was
promoted by high temperature and affected by past, but not increased pCO2 , indicating Peyssonnelia spp.
may benefit in future environments under increasing OW and OA following RCP8.5. Thus Peyssonnelia
spp. may better acclimatize and subsequently have competitive advantages over other organisms, such
as CCA, under future conditions. In addition, decreased cover of CCA under both factors, OW and
OA, suggest additive negative effects on CCA in future environmental conditions following RCP4.5RCP8.5, resulting in decreased abundance of CCA. The observed decrease in gross photosynthetic rates
of the epilithic community under high temperature and present day pCO2 treatment was unexpected
and potentially derived from normalization to relative surface area of organisms with relatively high
community cover of these substrates. This pattern was not seen in absolute gross photosynthetic rates
(Fig. 6.6 in supplementary material).
Epilithic communities were affected by temperature, pCO2 and the interaction of both and are likely
to drastically change under future environmental conditions. Acclimatization of CCA and Peyssonnelia
spp. may have been happening from past to present-day environmental conditions, as suggested in the
present experiment, but it is uncertain to what extent acclimatization may occur in the future. Heavily
calcified CCA were performing best under present-day (pCO2 ) and moderate (temperature) environmental conditions and may transitionally gain advantages by warming in the near future, but they are likely
to be negatively impacted under high GHG concentrations in the long term. Considerable implications
on their calcification rates and abundance will additionally reduce future reef resilience and recovery.
Lightly calcified Peyssonnelia spp. showed higher resilience to changing conditions and may benefit
under high GHG concentrations in the long term. However, it is unlikely that Peyssonnelia spp. will
be able to fill ecological gaps emerging from decreasing CCA. Peyssonnelia spp. have been shown to
induce settlement and metamorphosis of larvae from some coral species (Heyward and Negri 1999),
but studies also showed that Peyssonnelia are less suitable as settlement substrate compared to CCA
(Diaz-Pulido et al. 2010). Peyssonnelia may constitute a less stable substrate for larvae settlement and
may impede access of settled corals to the calcified reef substrate (Littler and Littler 1984). Lowered
157
6 - Ocean acidification and warming
absolute calcification and productivity (see Figure 6.6 in supplementary material) of the epilithic communities under high temperature and high pCO2 indicates reduced reef stability, productivity, nutrient
recycling and consequently biodiversity on future coral reefs.
Acknowledgments
We want to thank Jordan Hollarsmith, Emmett Clarkin, Camille Domy, Cassy Thompson, Patrick
Buerger, Kathryn Berry, Laura Arthur and Caroline Assailly for their great help in maintaining the
aquaria system. Many thanks to the staff at the SeaSim facility and the AIMS workshop, Andrea
Severati, Tom Barker, Paul Boyd, Craig Humphrey, Eneour Puill-Stephan, Grant Milton, Justin Hochen,
Niall Jeeves, Michael Kebben and Gary Brinkman who contributed to the aquarium design, control systems and monitoring of the experimental conditions. Thanks to Lindsay Harrington for her help with
the identification of CCA and epilithic organisms. Thanks to Michelle Liddy and Florita Flores for their
help with the incubation experiments and general assistance. This study was funded by the Australian
Institute of Marine Science, the Australian Government’s National Environmental Research Program
and a Super Science Fellowship grant from the Australian Research Council.
158
6 - Ocean acidification and warming
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27.9 °C
24.8 °C
Light calcification [µM C h-1 m-2]
0
50 100 150 200
24.8 °C
27.9 °C
Net calcification [mM C d-1 m-2] Dark calcification [µM C h-1 m-2]
0
1
2
3
−20
0
20
40
60
Gross photosynthesis [µg O2 L-1 h-1 m-2] Respiration [µg O2 L-1 h-1 m-2] Net photosynthesis [µg O2 L-1 h-1 m-2]
0
10
20
30
40 −6
−4
−2
0 0
10
20
30
40
Supplementary material
360
440
940
µatm
360
440
940
360
440
940
360
µatm
440
940
Figure 6.6: Net photosynthesis, dark respiration, gross photosynthesis and light-, dark- and net calcification of epilithic communities after six months of temperature and acidification treatment. Data are
normalized to the total surface area of the substrate
163
Chapter 7
General Discussion
The overall objective of this thesis was to fill current knowledge gaps in ocean acidification (OA)
research in order to gain a better understanding of future coral reef ecosystems under environmental
change. To achieve this, several field- and laboratory-based studies were conducted to investigate the
effects of OA and the effects of OA in combination with other stressors, namely decreased light availability, increased dissolved inorganic nutrients (DIN) and ocean warming (OW), on photosynthesizing and
calcifying coral reef organisms. The summarized results (Table 7.1 and 7.2) of individual and interactive
environmental stressors are utilized to predict effects on coral reef ecosystems in this rapidly changing
environment and to provide suggestions for future research and global and coastal management plans.
The particular objectives of this thesis were to investigate:
1. how photosynthesizing and calcifying coral reef organisms are affected by future ocean acidification scenarios, and whether they respond differently to ocean acidification conditions.
2. how decreased light availability affects the response of photosynthesizing and calcifying coral reef
organisms to ocean acidification.
3. whether increased dissolved inorganic carbon and -nitrogen have interactive effects on photosynthesizing and calcifying coral reef organisms.
4. how photosynthesizing and calcifying coral reef organisms and their communities respond to combinations of past and future ocean acidification and warming scenarios.
7.1 Responses of coral reef organisms to ocean acidification
As shown in the studies of the present thesis, the organisms of the different experiments varied in their
responses to OA conditions between, but also within organism groups (Table 7.2). Coral Acropora millepora was negatively affected by OA in its growth rates and net calcification (Chapter 4), while corals
165
7 - General Discussion
Table 7.1: Summary of experimental results from the different studies presented in this thesis
Ocean acidification
Foraminifera:
- increased growth under
OA in M. vertebralis
- no effects of OA on
photobiology of
foraminifera investigated
Halimeda spp.:
- no effect of OA on net
calcification
- increased light
calcification under OA in
H. digitata and H.
opuntia
- reduced dark
calcification under OA in
H. opuntia
- increased Cinorg content
under OA in H. digitata
- reduced Cinorg content in
H. opuntia
- reduced δ13C under OA
in H. digitata and H.
opuntia
Ocean acidification and
decreased light
availability
Corals:
A. millepora
- reduced growth and net
calcification under OA and
low light (additive effects)
- no effect of OA on light
calcification
- reduced light
calcification in low light
- reduced dark calcification
under OA and low light
(additive effects)
- reduced net- and gross
photosynthesis and
respiration in low light
Algae:
H. opuntia
- no OA effect on growth
and net calcification
- reduced growth in low
light
- no effect of OA on
calcification in light
- reduced dark calcification
under OA
- reduced net- and gross
photosynthesis and
- increased pigment
content in low light
Ocean acidification and
eutrophication
Ocean acidification and
warming
Corals:
A. tenuis
- no effects on
calcification and
photosynthetic rates
- increased dark and net
NOx uptake in DIN
treatments
- increased pigment
content in DIN treatments
Epilithic community:
- highest growth under
moderate temperature
increase
- lowest growth under pCO2
other than present-day
conditions
- reduced photosynthesis
under high temperature
- reduced photosynthesis at
present-day compared to
past pCO2
- reduced calcification under
high pCO2
- highest net calcification at
high temperature/presentday pCO2
- lowest net calcification at
low temperature/high pCO2
- changes in community
composition
S. hystrix
- no effects on
calcification rates
- increased photosynthetic
rates in DIN treatments
- increased NOx uptake in
light and dark in DIN
treatments
- increased pigment
content in DIN treatments
- increased protein
content in DIN treatments
Algae:
H. opuntia
- increased net
photosynthesis in DIN
treatments
- increased light NOx
uptake in DIN treatments
- increased pigment
content in DIN treatments
- increased Corg and N
content and reduced C:N
ratio in DIN treatments
CCA:
- highest cover under
moderate temperature
increase
- reduced cover under pCO2
other than present-day
conditions
Peyssonnelia spp.:
- increased cover at high
temperature
- reduced cover at past pCO2
- cover negatively correlated
to CCA
Acropora tenuis and Seriatopora hystrix did not show any negative effects on the response parameters
measured (Chapter 5). Similarly, previous literature indicates varying responses of coral calcification to
OA. Some studies showed negative effects (e.g. Renegar and Riegl 2005; Marubini et al. 2008; Comeau
et al. 2013b), while others showed no impacts of OA on different coral species (e.g. Cohen and Fine
2012; Comeau et al. 2013a; Comeau et al. 2014b). These different effects of OA on corals indicate
species-specific responses. As previously shown, branching corals were particularly reduced and thus
impacted by OA conditions at tropical CO2 seeps, while massive species were less affected (Fabricius
et al. 2011; Fabricius et al. 2014). This could also indicate that the growth form/morphology influences
166
7 - General Discussion
Table 7.2: Summary of significant treatment effects on response variables of experimental species from
the different studies presented in this thesis. Green arrows indicate an increase, red arrows a decrease
and circles no significant treatment effect on response parameters compared to control conditions
Organism/s
Corals:
A. millepora
A. tenuis
S. hystrix
Halimeda spp.:
H. digitata
H. opuntia
Epilithic algae
Community
CCA
Peyssonnelia spp.
Foraminifera
M. vertebralis
Response variable
OA
Low light
High DIN
OW
Growth
Net calcification
Light calcification
Dark calcification
Net photosynthesis
Dark respiration
Gross photosynthesis
Dark NOx flux
Net NOx flux
Pigment content
Net photosynthesis
Dark respiration
Gross photosynthesis
Dark NOx flux
Pigment content
Protein content
↓
↓
o
↓
o
o
o
o
o
o
o
o
o
o
o
o
↓
↓
↓
↓
↓
↓
↓
-
↑
↑
↑
↑
↑
↑
↑
↑
↑
-
Light calcification
Ctot
Corg
Cinorg
Corg:Cinorg
δ13C
Growth
Light calcification
Dark calcification
Net photosynthesis
Dark respiration
Gross photosynthesis
Light NOx flux
Dark NOx flux
Net NOx flux
Pigment content
Corg
Cinorg
N
C:N
δ13C
↑
↓
↓
↑
↓
↓
o
↑/o
↓/o
↑/o
o
o
o
o
o
o
o
↓
o
o
↓
↓
o
o
↓
↓
↓
↑
-
o
o
o
↑
o
↑
o
↑
↑
↑
↑
↓
↑
↓
-
-
Growth
Light calcification
Dark calcification
Net calcification
Final cover
Final cover
↓
↓
↓
↓
↓
o
-
-
↓
o
↑
o
↓
↑
Growth
↑
-
-
-
the responses of corals to OA. Branching species have a higher surface area to volume ratio compared to
massive species and thus are more exposed to their physical environment. Higher exposure may make
167
7 - General Discussion
them more susceptible to OA conditions and explain the different responses among corals. Yet, studies
also showed different responses within corals of the same genera and similar morphology. While A.
tenuis and S. hystrix were unaffected by OA conditions in the study of the present thesis (Chapter 5),
Acropora cervicornis was negatively affected in a study by Renegar and Riegl (2005), and S. hystrix
was impacted by OA at tropical CO2 seeps (Strahl et al. pers. comm.). This indicates that other factors may also contribute to the responses of corals to OA. The differences in the experimental setup
implemented between the present study and the study by Renegar and Riegl (2005) indicate environmental/experimental factors, such as temperature, or seawater supply, contributed to the different results
of the two experiments. Both, generally higher calcification rates and higher Ωar in warmer temperatures
(28.4 vs. 25.3 °C) may have resulted in no observed effects of OA in the present compared to the other
study (Renegar and Riegl 2005). The continuous supply of nutrition by flow-through conditions may
have increased the nutritional status of the corals in the present study and thus lowered its responses to
OA compared to the other study (Renegar and Riegl 2005). Moreover, other experimental factors such
as duration, light regimes, or the method used to change the carbonate chemistry, potentially contribute
to organisms’ responses to OA. Thus, it is important to choose experimental conditions which closely
resemble natural environments in order to extrapolate experimental results to coral reef ecosystems.
The calcifying green alga Halimeda opuntia was not negatively impacted in its growth or net calcification, under OA conditions, in the studies of the present thesis (Chapter 3, 4 and 5). In addition, several
other Halimeda species were still able to grow and calcify under OA conditions at tropical volcanic CO2
seeps in Papua New Guinea (PNG) (Chapter 3). While these results concur with some previous literature
(e.g. Comeau et al. 2013b; Hofmann et al. 2014), other studies suggest negative impacts of OA on some
Halimeda species (e.g. Hall-Spencer et al. 2008; Price et al. 2011; Sinutok et al. 2011). As discussed
above, experimental factors may contribute to the variable responses of organisms to OA, suggesting
that Halimeda spp. respond differently in artificial compared to natural experimental conditions. Yet,
the finding that Halimeda spp. were absent at temperate CO2 seeps in the Mediterranean (Hall-Spencer
et al. 2008), while they were unaffected at tropical CO2 seeps in PNG (Chapter 3), indicates regionalspecific responses of Halimeda spp. to OA may occur. Warmer and more constant water temperatures
throughout the year in the tropics may contribute to the observed differences. Overall, these results
suggest that several tropical Halimeda species are unlikely to be impacted by OA alone in the future.
Previous results from manipulative experiments, which showed negative OA effects on these Halimeda
species, may have to be re-evaluated with respect to their extrapolation potential to natural environments. Nevertheless, comparisons between similar studies also indicated regional-specific responses of
Halimeda spp. towards OA.
Epilithic communities that were investigated in the present thesis (Chapter 6) showed negative ef-
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7 - General Discussion
fects of OA on growth and calcification rates. Particularly, crustose coralline algae (CCA) showed
reduced cover, while other red algae of the genus Peyssonnelia were unaffected by high pCO2 conditions. The few studies available on CCA responses to OA suggest decreased calcification at high pCO2
(Diaz-Pulido et al. 2012; Comeau et al. 2013b), but also non-linear responses with higher calcification
rates at moderately increased pCO2 (Ries et al. 2009; Johnson and Carpenter 2012), or even signs of
acclimatization to elevated pCO2 conditions (Nash et al. 2013; Diaz-Pulido et al. 2014). The fact that
CCA were impacted by high pCO2 , while Peyssonnelia spp. were not, may be explained by utilization
of the less stable mineral for calcification (high-Mg-calcite vs. aragonite) or the generally higher calcification intensity of CCA compared to Peyssonnelia spp. Notably, growth of the community and cover
of CCA were reduced under lowered pCO2 as well as under increased pCO2 compared to present-day
conditions. The observed reduced growth in reduced pCO2 was novel and unexpected since the carbonate chemistry in this treatment should have facilitated calcification and growth of calcareous organisms.
This observation indicates: (1) the community and CCA are acclimatized to ambient pCO2 conditions,
and alterations in seawater carbonate chemistry (regardless if reduced or increased) lead to decreased
growth/calcification rates; (2) potential acclimatization has been happening from past to present-day
pCO2 conditions; (3) there are optimal conditions for the interplay of photosynthesis and calcification
in slightly elevated pCO2 environments, as experienced in the present-day compared to the past pCO2
treatment. These findings are supported by other studies that observed non-linear calcification responses
(Johnson and Carpenter 2012) and acclimatization of CCA by changes in the carbonate mineralogy
(Nash et al. 2013; Diaz-Pulido et al. 2014). Yet, observations from CO2 seeps in PNG indicate reduced
CCA abundance at high CO2 . Overall, the results from the present thesis and literature suggest that acclimatization of CCA may have been happening from past to present-day pCO2 and that it may happen
to some extend in the future. But acclimatization to high pCO2 , as projected under the RCP6.0-RCP8.5
by the end of this century, is unlikely.
The large benthic foraminiferal species were not negatively affected by elevated pCO2 (Chapter 2).
In the present study, the investigated species experienced positive shell growth and thus were still able
to calcify under future OA conditions after short-term (six weeks) exposure to elevated pCO2 . Contrary
to expectations, M. vertebralis could even increase its growth rates under elevated pCO2 which agrees
with another study that showed increased growth of several other foraminiferal species in intermediate
pCO2 treatments (Fujita et al. 2011). But the ability of these foraminifera to withstand or benefit from
short-term exposure to elevated pCO2 is no guarantee for their survival in the long term. Results from
field studies showed decreasing diversity of foraminifera under lowered pH at temperate CO2 seeps in
the Mediterranean (Dias et al. 2010) and absence of foraminifera at tropical CO2 seeps in PNG (Uthicke
et al. 2013). Thus, results from short-term and long-term studies have to be distinguished.
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7 - General Discussion
The different experimental species from the different studies of this thesis varied in their responses
to OA conditions (Table 7.1 and 7.2). While some species were negatively affected, others tolerated
elevated pCO2 supporting the hypothesis that photosynthesizing and calcifying organisms differently
respond to future OA scenarios. These different responses of coral reef organisms presumably lead to
shifts in community compositions on future coral reefs under OA conditions. Results from the present
thesis and literature also indicate that other factors, such as temperature, light regimes, or nutritional
status, can influence the response of photosynthesizing and calcifying coral reef organisms to OA.
7.2 Responses of coral reef organisms to combinations of ocean acidification and decreased light availability
One pattern that was seen in several species and experiments of this thesis was that the effects of OA
on calcification were primarily seen in the dark, while OA had no effects on organisms’ calcification in
the light (Table 7.2). This mechanism was explained by photosynthetic CO2 uptake in the light which
increases intra-, extra cellular and boundary layer pH and Ω. This in turn, buffers against negative OA effects and facilitates the deposition of calcium carbonate (CaCO3 ). As previously shown, photosynthetic
CO2 uptake of corals in the light can increase their intracellular super saturation of Ωar from ∼3 to ∼25
(Al-Horani et al. 2003). But in the dark, the opposite effect was the case. Missing buffering capacity of
photosynthesis and additional respiratory CO2 release, allowed and exacerbated a reduction in pH and
Ω which in turn decreased the calcification rates and in some cases led to decalcification of organisms’
skeletons. This finding led to the hypothesis that decreased light availability, derived from coastal runoff,
may affect the responses of photosynthesizing and calcifying coral reef organisms to OA. The study of
the present thesis, investigating this interaction, showed that growth and net calcification rates of the
coral A. millepora were reduced under OA and under low light, leading to additive negative effects of
both stressors (Chapter 4). In contrast, the calcareous green alga H. opuntia did not experience reduced
growth or net calcification under OA, but only under low light. Similarly, previous results showed that
net calcification rates of H. opuntia were unaffected by OA at tropical CO2 seeps (Chapter 3). Moreover,
both organisms investigated did not show any negative effect of OA in the light, supporting the hypothesis that coral (i.e. zooxanthellae) and algae photosynthesis buffers against OA as long as enough light
is available. In contrast, both A. millepora and H. opuntia experienced nocturnal decalcification under
OA, while positive calcification rates were still observed under control conditions which concurs with
results for H. digitata and H. opuntia from CO2 seeps (Chapter 3). As presumed, the missing buffering
capacity of photosynthesis and respiratory CO2 release led to the observed negative impacts of OA in the
dark. Furthermore, A. millepora experienced reduced light calcification rates under low light conditions
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7 - General Discussion
compared to the control light treatment, while H. opuntia did not. Previous literature also showed that
light intensity can affect coral responses to OA (Suggett et al. 2013) or warming (Comeau et al. 2014a),
but the latter studies did not show that low light intensity, as seen on inshore reefs, can amplify the negative effects of OA on coral reef organisms, as shown in the present study. Overall, the coral A. millepora
experienced additive negative effects of both OA and low light, suggesting inferior performance under
changing environmental conditions compared to the calcareous green alga H. opuntia. Thus, decreased
light availability may additionally contribute to shifts in coral reef community compositions away from
coral to algae dominance on inshore reefs under future OA scenarios.
7.3 Responses of coral reef organisms to combinations of elevated dissolved inorganic carbon and nitrogen
Photosynthetic carbon assimilation and calcification of H. digitata and H. opuntia were enhanced in elevated dissolved inorganic carbon (DIC) concentrations at tropical CO2 seeps in PNG (Chapter 3). Similarly, DIC limitation was observed in photosynthesis of corals at the PNG seep site (Strahl et al. pers.
comm.) and in laboratory experiments (Langdon and Atkinson 2005). In addition, coral-zooxanthellae
and algae can be limited in their supply with dissolved inorganic nutrients (DINs) (e.g. Langdon and
Atkinson 2005; Smith et al. 2004). This led to the hypothesis that elevated DIC under OA conditions
and elevated DIN under eutrophication conditions may have negative effects, but may also have positive
interactive effects on photosynthesis and calcification of organisms.
But against expectations, the measured physiological parameters in the corals A. tenuis and S. hystrix
and the calcareous green alga H. opuntia were not affected by elevated DIC or the interaction between
DIC and DIN in the present study (Chapter 5). This disproved the hypothesis that the coral reef organisms investigated may be positively or negatively affected by concurrent rises in DIC and DIN under
present experimental conditions. The alga showed trends towards the expected increases of photosynthesis and calcification in the light and decreases of calcification in the dark, but these were non-significant.
As presumed, the present experimental conditions, such as natural, variable light regimes, continuous
supply with nutrition and/or the duration of the experiment, led to the lack of organisms’ responses to
elevated DIC.
In contrast, the effects of elevated DIN were visible on photosynthetic rates, nutrient uptake rates
and pigment contents of the organisms investigated, which agrees with results from previous experiments
(e.g. Smith et al. 2004; Tanaka et al. 2007; Chauvin et al. 2011). Moreover, a shift between growth of
organic tissue and inorganic skeleton was observed for S. hystrix and H. opuntia; a result which has also
been observed for another coral (Tanaka et al. 2007). Thus, the organisms investigated responded more
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7 - General Discussion
rapidly and more pronounced to changes in DIN than DIC under experimental conditions that closely
resemble natural environments. These physiological alterations have been shown to have substantial
consequences for some coral reef organisms and the community composition (Fabricius 2005; Fabricius
et al. 2005). For instance, darker pigmentation can increase the temperature on the surface of coral
colonies by up to 1.5 °C above ambient seawater temperature (Fabricius 2006). Thus, darker coral
colonies will be more susceptible to bleaching which in turn has implications on their survival. Another
mechanism described is that zooxanthellae of corals, exposed to increased nitrate, suffer phosphate
starvation which ultimately leads to the breakdown of the coral-zooxanthellae symbiosis (Wiedenmann
et al. 2013) and increased mortality of the coral. In contrast, no such indirect responses of DIN have
been seen in H. opuntia, suggesting the algae may show no apparent negative effects to elevated DIN.
This would further contribute to changes in benthic communities of inshore reefs from coral to algal
dominance as also expected under decreased light availability (Chapter 4).
7.4 Responses of coral reef organisms and communities to past and future OA and OW conditions
Epilithic communities and CCA were negatively affected by high pCO2 and temperature treatments
(Chapter 6), suggesting they are likely to be negatively affected under future environmental conditions
by global stressors alone, regardless of the presence of local disturbances. Similarly, previous studies
showed that combinations of these stressors can have interactive effects leading to increased mortality
or decreased calcification of CCA (Martin and Gattuso 2009; Diaz-Pulido et al. 2012; Johnson and Carpenter 2012) and corals (Reynaud et al. 2003; Edmunds and Moriarty 2012). Growth of the epilithic
community and final cover of CCA were highest under moderately increased temperatures potentially
due to enhanced metabolic rates and improved carbonate chemistry for calcification. Notably, Peyssonnelia spp. showed highest cover at the highest temperature, suggesting superior performance under OW
compared to other organisms and potential relieve from space limitation. Additional analyzes indicated
cover of CCA and Peyssonnelia spp. were negatively correlated with higher abundance of Peyssonnelia
spp. in treatments with lower CCA cover. This suggests changes in competitiveness of organisms and alterations in future community composition under changing environments. Results of this study indicate
that CCA will experience additive negative effects under increasing OA and OW, while Peyssonnelia
spp. showed no effect under increasing pCO2 , but beneficial effects from high temperature. Notably,
both calcareous organisms CCA and Peyssonnelia spp. may have acclimatized from former to ambient
pCO2 conditions and they may be able acclimatize to low increases in pCO2 in future.
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7 - General Discussion
7.5 Ecological implications
The studies in the present thesis illustrate that the coral reef organisms investigated responded differently to rising OA and to combinations of OA with other stressors. The fact that some organisms were
negatively affected, while others tolerated OA conditions indicates future alterations in community compositions of coral reef ecosystems under environmental change are likely to occur. With reduced abundance of calcifying organisms, such as some corals, CCA and foraminifera, carbon fixation and CaCO3
production will likely be decreased on future coral reefs. CaCO3 skeletons and shells constitute a major
part of beaches and reef sand and thus contribute to the fundament of coral reef islands. With reduced
production of CaCO3 and slower reef growth, coral reef islands may be less protected from waves and
future land erosion may increase. In addition, reduced light availability can exacerbate the negative effects of OA which may restrict the potential habitat of affected organisms to shallower depths on future
coral reefs that are susceptible to coastal runoff. No interactive effects of elevated DIC and DIN were
observed, but strong and rapid physiological responses of organisms to elevated DIN concentrations
may affect corals in their response to other stressors. Particularly, corals and CCA were directly and
indirectly affected by OA in combination with other stressors, while Halimeda spp. and Peyssonnelia
spp. were less affected. This suggests that increasing CO2 and increasing coastal runoff will lead to further community shifts with less corals and more algae, particularly on inshore reefs. Moreover, reduced
cementation of the reef framework, due to decreased calcification of CCA, will make coral reefs more
fragile and more vulnerable to extreme weather events such as tropical cyclones. Since cyclones are predicted to increase in frequency and destructiveness in the future, OA and warming may have synergistic
effects on coral reef ecosystems.
Overall, OA by itself and co-occurring with other stressors will cause declines in growth rates of
many marine calcifying organisms, including important framework-builders, which will slow down reef
construction and will make coral reefs more fragile and subsequently more vulnerable to disturbances.
As a consequence, the balance between reef construction and destruction may shift towards destruction.
Thus, future coral reefs may be unable to recover between storm damages or other disturbances, leading
to diminishing reef structures which in turn causes fewer habitats for reef organisms including fish and
other invertebrates. Declining fish populations will have impacts on people who are dependent of them
as a source of nutrition. Moreover, reduced growth of coral reefs may make them unable to keep up
with future sea-level rise. This in turn, reduces their protective function against waves accompanied
by erosion of coral reef islands, subsequently affecting humans inhabiting coastal areas. Because not
all organisms will be negatively impacted under predicted environmental changes, competitiveness and
composition of organisms within the coral reef ecosystem will also change.
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7 - General Discussion
7.6 Conclusions and future perspectives
As shown in the studies of the present thesis different organisms varied in their responses to OA conditions. These differences were attributed to species-specific OA effects, but also to other environmental
or experimental factors that can influence the effects of OA on the response parameters measured. For
instance, the duration of experiments (short- vs. long-term), the seawater supply of experimental tanks
(filtered vs. unfiltered), the experimental light regimes (natural and parabolic vs. artificial and linear),
or the experimental treatments (past, present-day and future) all may have effects on the organisms’
responses to OA and the conclusions able to be drawn. Ultimately, the setup that is best to implement
will depend on the research question and the organisms under investigation. Thus, the experimental
design should be chosen carefully to make studies comparable and to allow for extrapolations to natural
environments in order to improve the knowledge about impacts of OA and other stressors on future coral
reef ecosystems. Suggestions for future research are to consider the different mechanisms of OA effects
in the light and in the dark. As shown in the present thesis, coral reef organisms and their community
composition will be increasingly affected by OA and OW. But inshore communities will also be exposed to increasing coastal runoff in the future. Thus, future research is encouraged to investigate the
interactive effects of all three factors. Moreover, several local stressors appear rather acute than chronic,
but knowledge about the potential of organisms to recover to the original state after short-term (weekly)
pulses of certain stressors, such as reduced light availability or elevated DIN, is rare and warrants further
investigation.
In conclusion, evidence from studies of this thesis suggests that coral reef ecosystems will change
under the projected environmental shifts during the 21st century. These anthropogenically induced ecological changes are already happening today and will continue to happen in the future. The question
whether organisms are able to withstand or go extinct depends not only on their ability to acclimatize to
the rapidly changing environmental conditions, but also on their ability to find their place in the ecosystem changing around them. Even if creatures (including humans) are not directly impacted by OA, OW
or coastal runoff they may be affected indirectly by ecological changes through loss of habitat, food
sources, or sources for income. If carbon emissions are drastically reduced within the next few years
a time-delayed effect of already stored greenhouse gases in the atmosphere will lead to future rises in
extreme temperature events (Ortiz et al. 2014). Hence, immediate and drastic reductions in carbon emissions and coastal pollution are encouraged to increase chances of future survival of coral reef organisms.
In the present thesis, it was shown that coastal runoff can have additional negative effects in combination with global stressors on coral reef organisms. Thus, by reducing runoff effects organisms will gain
some time or might be better able to acclimatize to inevitable environmental alterations on the global
scale. Environmental action plans, such as management of fertilizer usage and sedimentation sources,
174
7 - General Discussion
should be implemented at the regional scale to decrease the pressure from global stressors on coral reef
organisms which may help to preserve future coral reefs.
175
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Appendix
Abbreviations and glossary
AIMS
Australian Institute of Marine Science
ANOVA
Analysis of variance
AOI
area of interest
CaCO3
calcium carbonate
CCA
crustose coralline algae
Chl a
chlorophyll a
CO2
carbon dioxide
CO32–
carbonate
C inorg
inorganic carbon
C org
organic carbon
COTS
crown of thorns seastar
C tot
total carbon
DIC
dissolved inorganic carbon, DIC = [CO2 ] + [HCO3– ] + [CO32– ]
DIN
dissolved inorganic nutrients (ammonium, phosphate, nitrate and nitrite)
Ek
minimum saturation irradiance
GBR
Great Barrier Reef
GHG
greenhouse gases
H2 CO3
carbonic acid
HCl
hydrochloric acid
HCO3–
hydrogen carbonate, bicarbonate
IEA
International Energy Agency
IPCC
Intergovernmental Panel on Climate Change
LAT
lowest astronomical tide
LBF
large benthic foraminifera
mya
million years ago
181
Appendix
µM
micro mol per liter
NBS
National Institute of Standards and Technology
NERP
National Environmental Research Program
NH4+
ammonia
NIST
National Institute of Standards and Technology
NO2–
nitrite
NO3–
nitrate
NOAA
National Oceanic & Atmospheric Administration
OA
ocean acidification
OW
ocean warming
Ω
calcium carbonate saturation state
Ωar
aragonite saturation state
Ωca
calcite saturation state
PAM
pulse amplitude modulation
PAR
photosynthetically available radiation
pers. comm.
personal communication
PO43–
phosphate
POM
particulate organic matter
P-I
photosynthesis vs. irradiance
P max
maximum photosynthetic capacity
P net
net oxygen production
pCO2
carbon dioxide partial pressure
PNG
Papua New Guinea
ppt
parts per thousand
RCP
representative concentration pathway
SRES
Special Report on Emission Scenarios
SST
sea surface temperature
SD
standard deviation
SE
standard error
TA
total alkalinity, TA = [HCO3− ] + 2[CO32− ] + [B(OH)4− ] + [OH− ] − [H+ ]
UIPAC
International Union of Pure and Applied Chemistry
ZMT
Leibniz Center for Marine Tropical Ecology
182
Appendix
List of Tables
1.1
Dissolved inorganic nutrient (phosphate, ammonium, nitrate + nitrite and nitrite) concentrations of the study sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.1
Mean carbonate system parameters of long- and short-term experiments. Carbonate
system parameters were calculated from measured TA, DIC, salinity, temperature and
pressure. Standard deviations are given in brackets. SW = seawater . . . . . . . . . . . 39
2.2
Linear Model ANOVA results for growth rate, maximum quantum yield and chlorophyll
a content of A. radiata, H. depressa and M. vertebralis in the long-term experiment . . . 44
2.3
Linear Model ANOVA results for oxygen production and respiration rates of H. depressa
and M. vertebralis after long- and short-term exposition . . . . . . . . . . . . . . . . . . 47
2.4
Regression parameters and Linear Model ANOVA results of light response experiments . 48
3.1
Carbonate system parameters of water samples from in-situ collections (n total = 86)
(Dobu Island and Upa-Upasina, 2012, 2013), incubations (n total = 30) (Upa-Upasina,
2012) and transplant experiment (n total = 50) (Upa-Upasina, 2012). Data is given as
mean and standard deviation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.2
Linear Model ANOVA results for physiological and skeletal parameters of H. digitata
and H. opuntia with control and seep site as source of variation. Asterisks indicate
significant differences of response variables between control and seep site . . . . . . . . 70
4.1
Carbonate system parameters of experimental conditions. Data is given as means and
standard deviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
4.2
Mixed Model ANOVA results for A. millepora and H. opuntia . . . . . . . . . . . . . . 96
4.3
Summary of effects of treatment variables on response parameters for A. millepora and
H. opuntia. Decreases > 100% are possible due to decalcification. ‘ns’ indicates no
significant treatment effect, ‘measured additive effects’ represent differences of means
between the control pCO2 /high light and high pCO2 /low light treatment . . . . . . . . . 98
5.1
Mean (± SD) carbonate system parameters of experimental treatment conditions. Treatments consisted of 400, 700 and 1100 µatm pCO2 and 0.4 and 1.9 µmol DIN . . . . . . . 117
5.2
Mean (± SD) water quality parameters of experimental treatment conditions. Treatments consisted of 400, 700 and 1100 µatm pCO2 and 0.4 and 1.9 µmol NOx
5.3
. . . . . . 118
Mixed Model ANOVA results of OA and elevated DIN on response parameters of organisms investigated. Asterisks indicate significant treatment effects . . . . . . . . . . . 128
183
Appendix
5.4
Continuation of Mixed Model ANOVA results of OA and elevated DIN on response
parameters of organisms investigated. Asterisks indicate significant treatment effects . . 129
6.1
Temperature (n ∼ 200) and carbonate system parameters (n = 10) of experimental tanks.
Data is given as means (± SD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
6.2
Generalized Linear Model results for effects of temperature, pCO2 and the interaction
of both on response parameters of the epilithic community and CCA and Peyssonnelia
spp. Tukey contrasts represent significantly different treatment groups (1, 2, 3, 4) from
temperature (25.0, 26.1, 26.5 and 27.7 °C) and pCO2 (360, 440, 650, 940 µatm) . . . . . 150
6.3
Summary of individual and interactive effects of temperature and pCO2 on growth of the
investigated epilithic community and final cover of CCA and Peyssonnelia spp. Results
are given as means (± SD). Asterisks indicate significant treatment effects . . . . . . . . 151
7.1
Summary of experimental results from the different studies presented in this thesis . . . 166
7.2
Summary of significant treatment effects on response variables of experimental species
from the different studies presented in this thesis. Green arrows indicate an increase,
red arrows a decrease and circles no significant treatment effect on response parameters
compared to control conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
List of Figures
1.1
Trends in atmospheric CO2 concentrations with long-term projections following RCP2.6RCP8.5 (modified from IPCC 2013) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2
Trends in global ocean surface pH with long-term projections following RCP2.6-RCP8.5
(modified from IPCC 2013) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3
6
7
Illustration of research objectives with individual and interactive effects of global and
local stressors in sequence as covered in this thesis . . . . . . . . . . . . . . . . . . . . 15
1.4
Experimental species (a) Acropora millepora, (b) Acropora tenuis, (c) Seriatopora hystrix, (d) Halimeda digitata, (e) Halimeda opuntia, (f) crustose coralline algae, (g) Peyssonnelia spp., (h) Amphistegina radiata, (i) Heterostegina depressa and (j) Marginopora
vertebralis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.5
Map of study sites for experiments in Papua New Guinea and Australia . . . . . . . . . 19
184
Appendix
2.1
Growth (n = 40-60), maximum quantum efficiency (n = 40-70) and chlorophyll a content (n = 27-30) of A. radiata, H. depressa and M. vertebralis after six weeks of experimental treatment. Whiskers represent upper and lower extremes. Data shown in graphs
are untransformed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.2
Net photosynthesis and dark respiration (n = 9) of H. depressa and M. vertebralis after
six weeks of experimental treatment. Whiskers represent upper and lower extremes.
Data shown in graphs are untransformed . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.3
Net photosynthesis and respiration n =6-9 of H. depressa and M. vertebralis after shortterm exposure. Whiskers represent upper and lower extremes. Data shown in graphs are
untransformed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.4
Photosynthesis irradiance (P-I) curve for M. vertebralis in two different experimental
treatments. Black symbols represent the control treatment (pCO2 = 496 µatm); n = 9,
R2 = 0.96, p < 0.001. White symbols represent the high CO2 treatment (pCO2 = 1662
µatm); n = 9, R2 = 0.98, p < 0.001. Data are given as means ± 1 SE of 9 replicates in
3 experimental runs, each treatment. Data shown in graph are untransformed . . . . . . . 48
3.1
(a) Map of Papua New Guinea, Milne Bay Province and Normanby Island with locations
of seep sites at Dobu Island and Upa-Upasina. (b) H. digitata growing at the CO2 seep
site (Upa-Upasina). (c) H. opuntia growing at the CO2 seep site (Upa-Upasina). (d) H.
opuntia growing next to CO2 bubbles (Dobu Island) . . . . . . . . . . . . . . . . . . . . 65
3.2
Carbonate system parameters of water samples collected above Halimeda species growing at Dobu Island and Upa-Upasina control and seep site. Each dot represents a water
sample collected above the corresponding species (green = control site, red = seep site).
Dotted lines indicate ambient (green) levels and predicted future (red) levels following
the most pessimistic ‘representative concentration pathway’ RCP8.5. Solid lines (red)
represent mean values of water samples for each species, collected at the seep site . . . . 71
3.3
In-situ light-, dark- and net calcification rates of H. digitata and H. opuntia grown at
control and CO2 seep site. Brackets indicate significant differences in ANOVAs, with
significance levels ∗p < 0.05, ∗∗p < 0.001, ∗ ∗ ∗p < 0.0001 . . . . . . . . . . . . . . . 72
3.4
In-situ rates of net photosynthesis of H. digitata and H. opuntia grown at control and
CO2 seep site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
3.5
Total-, organic- and inorganic carbon content and C org : C inorg ratio of H. digitata and H.
opuntia grown at control and CO2 seep site. Brackets indicate significant differences in
ANOVAs, with significance levels ∗p < 0.05, ∗∗p < 0.001, ∗ ∗ ∗p < 0.0001 . . . . . . . 74
185
Appendix
3.6
δ 13C and δ 15N signatures of H. digitata and H. opuntia grown at control and CO2 seep
site and transplanted from control to control and control to seep site. Brackets indicate
significant differences in ANOVAs, with significance levels ∗ p < 0.05, ∗∗ p < 0.001,
∗ ∗ ∗ p < 0.0001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.1
Growth rates, net-, light- and dark calcification rates of A. millepora and H. opuntia
after 16 days exposure to experimental conditions. Data was pooled across pCO2 and
light treatment because there was no significant interaction. X-axes represent OA treatments in µatm pCO2 and light treatments in µmol photons m−2 s−1 . Whiskers represent
lower and upper extremes. Brackets indicate significant differences in ANOVAs, with
significance levels ∗ p < 0.05, ∗∗ p < 0.001, ∗ ∗ ∗ p < 0.0001 . . . . . . . . . . . . . . 95
4.2
Gross-, net photosynthesis, respiration and Chl a content of of A. millepora and H. opuntia after 16 days exposure to experimental conditions. Data was pooled across pCO2
and light treatment because there was no significant interaction. X-axes represent OA
treatments in µatm pCO2 and light treatments in µmol photons m−2 s−1 . Whiskers represent lower and upper extremes. Brackets indicate significant differences in ANOVAs,
with significance levels ∗ p < 0.05, ∗∗ p < 0.001, ∗ ∗ ∗ p < 0.0001 . . . . . . . . . . . . 97
5.1
Growth of A. tenuis, S. hystrix and H. opuntia after three weeks experimental treatment. X-axes represent pCO2 treatments in µatm and DIN treatments in µmol L−1 NO x.
Whiskers represent upper and lower extremes. Plots illustrate pooled data, due to nonsignificant interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
5.2
Light-, dark- and net calcification of A. tenuis, S. hystrix and H. opuntia after three weeks
experimental treatment. X-axes represent pCO2 treatments in µatm and DIN treatments
in µmol L−1 NO x. Whiskers represent upper and lower extremes. Plots illustrate pooled
data, due to non-significant interactions . . . . . . . . . . . . . . . . . . . . . . . . . . 122
5.3
Net photosynthesis, dark respiration and gross photosynthesis of A. tenuis, S. hystrix and
H. opuntia after three weeks experimental treatment. X-axes represent pCO2 treatments
in µatm and DIN treatments in µmol L−1 NO x. Whiskers represent upper and lower
extremes. Plots illustrate pooled data, due to non-significant interactions . . . . . . . . . 123
5.4
Light-, dark-, and net NO x uptake of A. tenuis, S. hystrix and H. opuntia after three weeks
experimental treatment. X-axes represent pCO2 treatments in µatm and DIN treatments
in µmol L−1 NO x. Whiskers represent upper and lower extremes. Plots illustrate pooled
data, due to non-significant interactions . . . . . . . . . . . . . . . . . . . . . . . . . . 124
186
Appendix
5.5
Chlorophyll a content of A. tenuis, S. hystrix and H. opuntia after three weeks experimental treatment. X-axes represent pCO2 treatments in µatm and DIN treatments in
µmol L−1 NO x. Whiskers represent upper and lower extremes. Plots illustrate pooled
data, due to non-significant interactions . . . . . . . . . . . . . . . . . . . . . . . . . . 125
5.6
Total protein content of A. tenuis and S. hystrix after three weeks experimental treatment. X-axes represent pCO2 treatments in µatm and DIN treatments in µmol L−1 NO x.
Whiskers represent upper and lower extremes. Plots illustrate pooled data, due to nonsignificant interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
5.7
Organic carbon, nitrogen, inorganic carbon and C:N ratio of H. opuntia after three weeks
experimental treatment. X-axes represent pCO2 treatments in µatm and DIN treatments
in µmol L−1 NO x. Whiskers represent upper and lower extremes. Plots illustrate pooled
data, due to non-significant interactions . . . . . . . . . . . . . . . . . . . . . . . . . . 127
6.1
(a) Images of PVC substrates with encrusting communities taken after six months in
experimental conditions. The bottom row shows one PVC rack out of each experimental
tank. The top row shows one close-up image of one substrate from each treatment
(utilized to determine surface areas of communities and organisms). (b) Microscopic
image of CCA and (c) Peyssonnelia spp. (d) Example of image utilized for surface area
determination of different organisms. CCA and Peyssonnelia spp. were manually color
coded prior to automated surface area determination . . . . . . . . . . . . . . . . . . . . 144
6.2
Temperature profile of the experiment: (1) pre-experimental phase (for initial growth on
PVC substrates) on Davies Reef, (2) acclimatization phase to AIMS aquaria facilities
and (3) temperature/acidification treatments . . . . . . . . . . . . . . . . . . . . . . . . 145
6.3
Percent change in cover of epilithic communities over six month of temperature and
pCO2 treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
6.4
Net photosynthesis, respiration and gross photosynthesis as well as light-, dark- and net
calcification of epilithic communities after six months of temperature and acidification
treatments. Data are normalized to the surface area of the epilithic communities . . . . . 153
6.5
Percent final cover of CCA and Peyssonnelia spp. after six months exposure to temperature and acidification treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
6.6
Net photosynthesis, dark respiration, gross photosynthesis and light-, dark- and net calcification of epilithic communities after six months of temperature and acidification treatment. Data are normalized to the total surface area of the substrate . . . . . . . . . . . . 163
187