University of Southern California Dissertations and Theses

Thermal acclimation and adaptation of key phytoplankton groups and interactions with other global change variables

(USC Thesis Other)

Thermal acclimation and adaptation of key phytoplankton groups and interactions with other global change variables

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Content THERMAL ACCLIMATION AND ADAPTATION OF KEY
PHYTOPLANKTON GROUPS AND INTERACTIONS WITH
OTHER GLOBAL CHANGE VARIABLES

by
Pingping Qu

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
For the Degree
DOCTOR OF PHILOSOPHY
(MARINE BIOLOGY AND BIOLOGICAL OCEANOGRAPHY)
UNIVERSITY OF SOUTHERN CALIFORNIA

August, 2019

Advised by: Professor David Hutchins and Fei-Xue Fu

Approved by Dissertation Committee:
Professor David A. Hutchins (Chair)
Professor Douglas G. Capone
Professor Sergio Sanudo-Wilhelmy
Professor Sarah J. Feakins

Copyright 2019 Pingping Qu
This document was formatted in accordance to the author guidelines specified for the Limnology
and Oceanography.

i
Table of Contents
DEDICATION ........................................................................................................................................................... III
ACKNOWLEDGEMENTS ...................................................................................................................................... IV
LIST OF FIGURES ..................................................................................................................................................... V
LIST OF TABLES .................................................................................................................................................... VII
ABSTRACT OF THE DISSERTATION ............................................................................................................. VIII
CHAPTER 1: INTRODUCTION OF THE DISSERTATION ................................................................................ 1
1.1 CLIMATE CHANGE VARIABLES AND THEIR IMPACTS ON MARINE PHYTOPLANKTON ........................................... 1
1.1.1 Ocean Acidification ..................................................................................................................................... 1
1.1.2 Warming and Increased Thermal Variability .............................................................................................. 3
1.1.3 Reduced nutrient availability ....................................................................................................................... 5
1.1.4 Other Climate Change Variables ................................................................................................................. 7
1.1.5 The Interactive Effects of Multiple Climate Change Variables ................................................................... 7
1.2 KEY PHYTOPLANKTON FUNCTIONAL GROUPS: DIATOMS AND DIAZOTROPHIC CYANOBACTERIA ....................... 8
1.2.1 Diatoms ........................................................................................................................................................ 8
1.2.2 Diazotrophic Cyanobacteria ...................................................................................................................... 10
1.3 SUMMARY ........................................................................................................................................................... 12
REFERENCES ............................................................................................................................................................. 14
CHAPTER 2: RESPONSES OF THE LARGE CENTRIC DIATOM COSCINODISCUS SP. TO
INTERACTIONS BETWEEN WARMING, ELEVATED CO2, AND NITRATE AVAILABILITY ............... 30
ABSTRACT ................................................................................................................................................................ 31
2.1 INTRODUCTION ................................................................................................................................................... 32
2.2 METHODS ........................................................................................................................................................... 35
2.2.1 Culturing and Incubation Methods ............................................................................................................ 35
2.2.2 Physiological and Biogeochemical Analyses ............................................................................................. 36
2.2.3 Statistics ..................................................................................................................................................... 39
2.3 RESULTS ............................................................................................................................................................. 39
2.3.1 Maximum growth rates (μmax) and half saturation constants (K1/2) ........................................................... 39
2.3.2 Chl a contents ............................................................................................................................................ 40
2.3.3 Carbon fixation rates and iron uptake rates .............................................................................................. 41
2.3.4 Elemental stoichiometry ............................................................................................................................. 42
2.3.5 Interactive effects ....................................................................................................................................... 43
2.4 DISCUSSION ........................................................................................................................................................ 43
2.4.1 Effects of temperature and CO2 on growth and half saturation constants ................................................ 44
2.4.2 Effects of nitrate availability on Chl a content .......................................................................................... 46
2.4.3 Responses of carbon fixation rates and iron uptake rates to the three variables ...................................... 47
2.4.4 Cellular C, N and P contents and stoichiometry under three variables .................................................... 49
2.4.5 Cellular Si contents and stoichiometry under three variables ................................................................... 50
2.4.6 Interactive effects of warming, CO2 and nitrate availability ..................................................................... 51
2.5 CONCLUSIONS ..................................................................................................................................................... 53
REFERENCES ............................................................................................................................................................. 55
CHAPTER 2-TABLES AND FIGURES ........................................................................................................................... 65
CHAPTER 2-SUPPLEMENTARY MATERIALS .............................................................................................................. 73
CHAPTER 3: DISTINCT RESPONSES OF THE NITROGEN-FIXING MARINE CYANOBACTERIUM
TRICHODESMIUM TO A THERMALLY-VARIABLE ENVIRONMENT AS A FUNCTION OF
PHOSPHORUS AVAILABILITY ............................................................................................................................ 76
ABSTRACT ................................................................................................................................................................ 77
3.1 INTRODUCTION ................................................................................................................................................... 78
ii
3.2 MATERIALS AND METHODS ................................................................................................................................ 80
3.2.1 Cultures and Incubation Methods .............................................................................................................. 80
3.2.2 Physiological and Biogeochemical Analyses ............................................................................................. 82
3.2.3 Statistics ..................................................................................................................................................... 84
3.3 RESULTS ............................................................................................................................................................. 84
3.3.1 Thermal performance curves (TPC) for growth and nitrogen fixation rates ............................................ 84
3.3.2 Growth rates of constant and variable temperature treatments ................................................................ 85
3.3.3 Nitrogen and carbon fixation rates in thermal variation experiments ...................................................... 86
3.3.4 Elemental stoichiometry and Chl a to carbon ratio ................................................................................... 87
3.3.5 Phosphorous use efficiency (PUE) ............................................................................................................ 88
3.4 DISCUSSION ........................................................................................................................................................ 88
3.4.1 P availability and phosphorus use efficiency (PUE) ................................................................................. 89
3.4.2 Impacts of intensity of temperature variation ............................................................................................ 91
3.4.3 Interactions between temperature variability and P availability in two seasons ...................................... 91
3.4.4 Implications for Trichodesmium growth and biogeochemistry under climate change .............................. 93
3.5 CONCLUSIONS ..................................................................................................................................................... 95
REFERENCES ............................................................................................................................................................. 97
CHAPTER 3-TABLES AND FIGURES ......................................................................................................................... 105
CHAPTER 3-SUPPLEMENTARY MATERIALS ............................................................................................................ 114
CHAPTER 4: THE MARINE NITROGEN-FIXING CYANOBACTERIA TRICHODESMIUM AND
CROCOSPHAERA EMPLOY CONTRASTING ACCLIMATION AND ADAPTATION STRATEGIES
DURING LONG-TERM THERMAL SELECTION ............................................................................................ 116
ABSTRACT .............................................................................................................................................................. 117
4.1 INTRODUCTION ................................................................................................................................................. 118
4.2 METHODS AND MATERIALS .............................................................................................................................. 120
4.2.1 Cultures and experimental evolution ....................................................................................................... 120
4.2.2 Temperature performance curves (TPCs) Determination ....................................................................... 121
4.2.3 Switch experiments ................................................................................................................................... 121
4.2.4 Physiological measurements .................................................................................................................... 122
4.2.5 Statistics ................................................................................................................................................... 124
4.3 RESULTS ........................................................................................................................................................... 124
4.3.1 Growth rates in temperature response curves (TPCs) ............................................................................ 124
4.3.2 Nitrogen and carbon fixation rates .......................................................................................................... 125
4.3.3 Elemental stoichiometry and Chl a to POC ratio .................................................................................... 126
4.3.4 Trichome and cell size ............................................................................................................................. 127
4.4 DISCUSSION ...................................................................................................................................................... 127
4.4.1 Plasticity versus adaptation in thermally-selected Trichodesmium and Crocosphaera ......................... 127
4.4.2 The link between physiological plasticity and evolutionary adaptation .................................................. 129
4.4.3 Genetic backgrounds and lifestyles of the two diazotrophs ..................................................................... 130
4.4.4 Possible impacts of future warming on cellular stoichiometry and biogeochemistry ............................. 132
4.4.5 Future N2 fixation and species competition between the two diazotrophs .............................................. 133
4.4.6 Size change, growth and carbon export ................................................................................................... 134
4.5 CONCLUSION .................................................................................................................................................... 134
REFERENCES ........................................................................................................................................................... 136
CHAPTER 4-FIGURES .............................................................................................................................................. 147
CHAPTER 4-SUPPLEMENTARY MATERIALS ............................................................................................................ 155
CHAPTER 5: DISSERTATION CONCLUSION ................................................................................................. 161
5.1 MAJOR ACHIEVEMENTS AND IMPLICATIONS FOR FUTURE STUDIES ................................................................. 161
5.2 CONTRIBUTIONS AND BROADER IMPACTS ........................................................................................................ 163
REFERENCES ........................................................................................................................................................... 165

iii
Dedication
For my grandmother De-Xian, who was always watching over me.

iv
Acknowledgements
I would like to give many thanks to my co-advisors Prof. David Hutchins and Fei-Xue
Fu. They have been very supportive and encouraging during my Ph.D. stage and always
provided great suggestions for my study. They also helped me overcome the difficulties in my
courses and research. I learned lots of valuable qualities of a good researcher from them. Their
dedication and help made all my achievements in the past five years possible. I could never have
achieved a Ph.D. without the support and help from them.
I also really appreciate the help of the faculty and staff in Marine Biology and Biological
Oceanography and Earth Science, especially Prof. Douglas Capone, Sergio Sanudo-Wilhelmy,
Eric Webb, James Moffett and Sarah Feakins. They guided me in my qualification examination
and research and provided generous help for my experiments.
I also want to give thanks to the funding from National Sciences Foundation, and the
Provost Fellowship and Sonosky Fellowship. I appreciate the help of all the co-authors of the
publications and manuscripts to be submitted that arise from this thesis. Thanks to all my lab
mates, visiting scholars in our lab and my friends for your friendship and help. I really cherish
the time spent with them and they are all in my best memories at USC.
I am very grateful to my grandparents for raising me up and devoting their best to me. I
would appreciate their all-enduring love and care forever. I also thank my father and families for
being encouraging and understanding. Special thanks to my husband and also my best friend and
partner, Xianglong Zhang, who tried so hard in our long-distance relationship that lasted my
entire Ph.D. stage and supported me in all the aspects of my life.

v
List of Figures
Chapter 2 Figure 1. Relationship between specific growth rates μ (day
-1
) and nitrate concentrations
(μmol/L) in the four temperature / CO2 treatments.........................................................................69
Chapter 2 Figure 2. Relationships between cellular Chl a content (ng cell
-1
) and nitrate availability
in four temperature / CO2 treatments..............................................................................................70
Chapter 2 Figure 3. Carbon fixation rate (10
-13
mol C/h/cell), Iron uptake rate (10
-17
mol Fe/h/cell)
and Iron: Carbon uptake ratio (10
-3
mol: mol) of Coscinodiscus sp. at three nitrate concentrations
(5, 50 and 100 μmol/L) under four CO2 / temperature treatments...................................................71
Chapter 2 Figure 4. Cellular P contents (pmol cell
-1
), cellular Si contents (pmol cell
-1
), PON: POP
ratios (mol: mol) and BSi: POC ratios (mol: mol) at three nitrogen concentrations (5, 50 and 100
μmol/L) in four temperature / CO2 treatments................................................................................72
Chapter 2 Figure S1. Cellular C content (pmol cell
-1
), N content (pmol cell
-1
), and POC: PON
ratios (mol: mol) at three nitrogen concentrations (5, 50 and 100μM) in four temperature / CO2
treatments.......................................................................................................................................75
Chapter 3 Figure 1. Measured thermal response curves (TPC) based on growth rates of
Trichodesmium erythraeum strain GBRTRLI101 under P-replete and P-limited
conditions.....................................................................................................................................109
Chapter 3 Figure 2. Non-linear averaging model-predicted TPCs for the ± 2°C and ± 4°C
treatments.....................................................................................................................................110
Chapter 3 Figure 3. Average nitrogen-specific fixation rates and carbon-specific fixation rates
normalized to PON and POC, respectively, of Trichodesmium GBRTRLI101 in five constant and
variable temperature treatments under two P conditions (10 and 0.2 μmol/L) in summer and winter
experiments..................................................................................................................................111
Chapter 3 Figure 4. Chl a to C ratios of Trichodesmium GBRTRLI101 at five constant and variable
temperature treatments and two P conditions in summer and winter experiments........................112
Chapter 3 Figure 5. Phosphate Use Efficiency (PUE) for nitrogen and carbon fixation under P-
replete and P-limited conditions (10 and 0.2 μmol/L) in five different constant and variable
temperature treatments in summer and winter experiments..........................................................113
Chapter 4 Figure 1. The growth rates of Trichodesmium and Crocosphaera at 22
°
C, 28
°
C and 32
°
C,
showing rates of the ancestral strain just before the initial transfer to long-term cultivation at the
three temperatures, as well as the three thermally-selected cell lines at the end of the ~2-year
selection period............................................................................................................................149
Chapter 4 Figure 2. Measured thermal response curves (TPC) based on growth rates of
Trichodesmium erythraeum IMS101 and Crocosphaera WH0005 in the two weeks after
transferring cell lines thermally selected at three temperatures (22
°
C, 28
°
C and 32
°
C) for ~2 years
to the TPC.....................................................................................................................................150
vi
Chapter 4 Figure 3. Nitrogen and carbon fixation rates of long-term thermally-selected
Trichodesmium erythraeum IMS101, respectively normalized to PON and POC at five
temperatures at the end of the two week TPC incubation..............................................................151
Chapter 4 Figure 4. Nitrogen and carbon fixation rates of long-term thermally-selected
Crocosphaera WH0005, respectively normalized to PON and POC at five temperatures after the
two week TPC incubation and one month’ switch experiment.....................................................152
Chapter 4 Figure 5. POC: PON, PON: POP, POC: POP and Chl a: POC ratios of long-term
thermally-selected Trichodesmium erythraeum IMS101 and Crocosphaera WH0005 cell lines at
their selection temperatures..........................................................................................................152
Chapter 4 Figure 6. Trichome length and width of long-term Trichodesmium erythraeum IMS101
and cell radius of long-term Crocosphaera WH0005 at their selection temperatures...................153
Chapter 4 Figure S1. Chl a to POC ratio of long-term thermally-selected Trichodesmium
erythraeum IMS101 and Crocosphaera WH0005 two weeks after transfer to the thermal
performance curve from 18-34°C.................................................................................................160

vii
List of Tables
Chapter 2 Table 1. Seawater carbonate buffer system values- pH, total dissolved inorganic carbon
(DIC) and pCO2 values calculated from the former two parameters ..............................................65
Chapter 2 Table 2. Maximum specific growth rates μmax, half saturation constants K1/2 and μmax /
K1/2 ratios of the four CO2/temperature treatments relative to nitrate concentration ......................66
Chapter 2 Table 3. Significance of interactive effects of warming, CO2 and nitrate availability on
12 growth and physiological parameters of Coscinodiscus sp. ......................................................67
Chapter 2 Table S1. Coscinodiscus sp. cell volume (μm
3
) in the four CO2 / temperature treatments
at three nitrate concentrations (5, 50 and 100 μM) .........................................................................73
Chapter 2 Table S2. Coscinodiscus sp. cell density (ml
-1
) in the four CO2 / temperature treatments
at three nitrate concentrations (5, 50 and 100 μM) for the last dilution interval .............................74
Chapter 3 Table 1. Thermal performance curve (TPC) parameters at constant and two variable
temperature treatments (± 2°C and ± 4°C), all at two P concentrations (10 and 0.2 μmol/L) .......105
Chapter 3 Table 2. Elemental stoichiometry in five constant and variable temperature treatments
at two P concentrations (10 and 0.2 μmol/L) in summer and winter experiments ........................106
Chapter 3 Table S1. Experimental temperature and phosphate availability treatments. LTP: low
temperature phase; HTP: high temperature phase .......................................................................114
Chapter 3 Table S2. Elemental stoichiometry during the low temperature phase (LTP) and high
temperature phase (HTP) in three variable temperature treatments at two P concentrations (10 and
0.2 μmol/L) .................................................................................................................................115
Chapter 4 Table S1. Elemental stoichiometry (C: N, N: P, and C: P) of long-term Trichodesmium
erythraeum IMS101 and Crocosphaera WH0005 two weeks after transfer to the TPC from 18-
34°C ............................................................................................................................................158

viii
Abstract of the Dissertation
Several functional groups of marine phytoplankton groups play especially critical roles in
global primary productivity, carbon export and biogeochemistry. In particular, diatoms
substantially contribute to primary production, carbon sequestration and the food web.
Diazotrophic cyanobacteria are equally important as a source of new nitrogen (N) through
nitrogen fixation. The relationship between environmental forcing and these key marine
phytoplankton groups needs more attention, especially in the context of future global change.
Elevated atmospheric concentrations of the greenhouse gas carbon dioxide (CO2) are
bringing about ocean acidification and giving rise to sea surface warming. By the end of this
century, atmospheric CO2 concentration is predicted to rise from current levels of ~415 parts per
million (ppm) to 800-1150 ppm, while average surface seawater temperatures will increase by 2-
4°C. Global warming will also result in indirect changes, such as intensified stratification of
seawater, increased temperature variability and decreased nutrient inputs from the interior of
oceans. Phytoplankton communities in the euphotic zone will be forced to accommodate to all of
these concurrent climate change trends.
The three research chapters of this dissertation aim to increase our understanding of the
possible responses of marine diatoms and diazotrophic cyanobacteria to this changing ocean
environment. The first project investigated the physiological responses of the widespread centric
diatom Coscinodiscus sp. to interactions between elevated CO2, warming, and nitrogen
availability. The growth and physiology of Coscinodiscus were found to be regulated by both
individual and interactive effects of these three variables. Growth stimulation by warming was
coupled with a decreased growth affinity for nitrate, indicating increased vulnerability to nitrate-
limitation. Rising CO2 promoted carbon fixation rates, which may provide a negative feedback to
increasing atmospheric CO2. Cellular phosphorus (P) and silicon (Si) contents declined and
consequently N: P and C: Si ratios were elevated at high temperature, especially at ambient CO2
level. Moreover, numerous mutual interactions among the three climate change variables were
observed for growth rates, carbon fixation rates, cellular Chl a content and elemental
stoichiometry, suggesting that more attention should be paid to the overall effects of multiple
climate change variables rather than only to their independent impacts.
The second chapter examined how short-term thermal variability affected the growth and
physiology of Trichodesmium erythraeum GBRTRLI101 in two seasons (winter / summer), as
ix
well as the interaction between temperature variation and phosphate availability. P limitation
played a bigger role in stressing the growth of Trichodesmium than temperature variability. The
responses of Trichodesmium to thermal variability varied with the seasonal temperature regimes
and ambient nutrient availability. In particular, thermal variability significantly decreased the
growth and nitrogen and carbon fixation rates of Trichodesmium cells under P-replete conditions
in wintertime. Increases in cellular phosphorus use efficiencies of Trichodesmium were observed
at high temperature, suggested a lower P requirement in the future warming ocean. This chapter
incidentally illustrated that a nonlinear averaging model commonly used to predict growth rates
under fluctuating temperatures is not appropriate to apply to nutrient-limited cells. Future
warming and greater thermal variability could significantly interact with projected reduced P
supplies, and thus impact the growth and physiology of marine diazotrophic cyanobacteria.
The third chapter explored the short-term physiological responses and potential for
evolutionary adaptation of Trichodesmium erythraeum IMS101 and Crocosphaera WH0005
after long-term exposure to different temperatures. Long-term thermally-selected Trichodesmium
IMS101 showed a quick re-acclimation to temperature shifts from 20-34°C, while a true thermal
adaptation or possibly a much slower plastic acclimation occurred in Crocosphaera WH0005. A
remarkable finding was that 32°C-selected Crocosphaera cells can survive and fix nitrogen at a
normally lethal temperature of 34°C, suggesting that Crocosphaera can adaptively extend its
upper thermal limit if warming occurs gradually. These distinct responses of Trichodesmium and
Crocosphaera may stem from their different genetic backgrounds and lifestyles. These results
provide insights into evolutionary versus plastic responses, competitive interactions, niche
partitioning, and changes in new nitrogen supplies from these two keystone diazotrophs in the
future tropical and subtropical ocean.
The three studies in this dissertation help us to better understand the synergistic effects of
multiple climate change variables on marine diatom growth, elemental stoichiometry and the
silicification process. They also provide unique information on the interactions of thermal
variability and nutrient limitation on the growth and physiology of an important marine
diazotroph, and allow better predictions of short-term acclimation and possible evolutionary
adaptation to future warming conditions in two representative nitrogen-fixing cyanobacteria.
Together, this dissertation research provides novel insights into the potential trends of marine
primary productivity and biogeochemistry under future ocean global change scenarios.
x
Key words: diatom, diazotrophic cyanobacteria, climate change, nutrient availability,
temperature, carbon dioxide.

1
Chapter 1: Introduction of the Dissertation
The word “Anthropocene” was coined to describe the modern era of human domination of
the planet, in which human activities are reshaping global and regional ecosystems and giving
rise to climate changes (Crutzen 2002; Riebesell 2004). Consequently, all organisms in marine
ecosystems are faced with huge challenges caused by climate change variables, including
elevated CO2 concentration, global warming, reduced nutrient availability, and increased
environmental variability (IPCC 2007; 2012; 2013). In particular, marine phytoplankton
communities in the euphotic zone are anticipated to undergo strong disturbance in the future,
since changes in the surface layer will be more significant than those occurring in the ocean
interior (Rost et al. 2008).
Close attention has been paid to the acclimation and adaptation of marine phytoplankton to
these projected changes because of their contribution of nearly half of the global primary
productivity, their critical role in carbon export to the deep ocean through the biological pump,
and their importance in the biogeochemical cycles of important elements such as nitrogen,
phosphorous, silicon and trace metals (Falkowski 1994; Field et al. 1998). Moreover, the
capacity of phytoplankton to sequester carbon under global change scenarios may be a key factor
to relieve or intensify elevated CO2 levels and future climate change (Riebesell 2004; Sabine et
al. 2004).
Among the important phytoplankton functional groups, cyanobacteria are the main
prokaryotic group, while the three main eukaryotic groups are coccolithophores, diatoms and
dinoflagellates (Falkowski et al. 2004). This dissertation investigated the impacts of important
climate change variables on two of these keystone functional groups, diatoms and diazotrophic
cyanobacteria. Detailed information about the climate change variables studied and their effects
on marine phytoplankton groups is introduced in the following sections.
1.1 Climate Change Variables and Their Impacts on Marine Phytoplankton
1.1.1 Ocean Acidification
Rising atmospheric CO2 concentration due to fossil fuel burning and changes in land use
leads to an increase in seawater pCO2 and a decrease in the pH of seawater through air-seawater
exchange (IPCC 2007; Arnold et al. 2012). During the past several decades, ~25-30% of the CO2
released due to anthropogenic activities has entered the surface ocean (Field and Raupach 2004).
2
As a result, the acidity of the ocean increases, producing the phenomenon known as ocean
acidification.
Since the preindustrial period, the CO2 level in the atmosphere has increased from ~290
parts per million (ppm) to ~415 ppm currently (IPCC 2007; Bala 2013) and the pH value has
consequently dropped down from approximately 8.2 to 8.1 (Gattuso and Hansson 2011). By the
end of this century, atmospheric CO2 concentration is expected to approach 800-1150 ppm
(IPCC 2007; 2013), and the pH value of seawater is predicted to decrease by 0.2-0.4 units (Orr et
al. 2005), or even by as much as 0.7 units with unabated CO2 emissions (Caldeira and Wickett
2003). This projected huge perturbation of ocean carbonate chemistry is much faster compared to
the variations between glacial to interglacial periods, and than historical changes in ocean surface
waters (Haugan and Drange 1996; Caldeira and Wickett 2003).
With this major change in seawater acidity, the carbonate saturation state is expected to
decrease globally (Sabine et al. 2004; Schulz et al. 2009; Gao and Campbell 2014). By the end of
the 21st century, surface seawater dissolved inorganic carbon (DIC) and H
+
concentration are
predicted to increase by >12% and 100-150% respectively, and carbonate ion (CO3
2-
)
concentration is expected to decrease by almost 60% (Brewer 1997; Orr et al. 2005). These
changes are anticipated to impact the photosynthetic processes of marine photoautotrophs
utilizing DIC as an important C source (Maat et al. 2014). For instance, Biswas et al. (2014)
showed that the rates of DIC uptake and net photosynthetic oxygen evolution and the biomass of
diatom-dominated phytoplankton communities increased in response to increasing CO2 levels.
Similar positive effects of rising CO2 concentration on growth and photosynthesis have also been
observed in multiple phytoplankton species (Riebesell et al. 1993; Schippers et al. 2004;
Ainsworth and Long 2005; Riebesell et al. 2007; Johnson et al. 2013; Low-Décarie et al. 2014;
Matt et al. 2014). Growth stimulation by high CO2 levels can often be attributed to the
downregulation of carbon concentration mechanisms (CCMs) in phytoplankton cells with
increased inorganic carbon availability in the surrounding seawater (Reinfelder 2011;
Kupriyanova et al. 2013). Moreover, elevated CO2 level has also been found to increase nitrogen
fixation of marine diazotrophic cyanobacteria (Levitan et al. 2007; Fu et al. 2008; Hutchins et al.
2007, 2013, 2015), suggesting a potential influence of high CO2 on global nitrogen cycling.
Rising CO2 concentration can also shift the composition of phytoplankton communities
when species have different physiological responses (Niklaus et al. 2001; Kardol et al. 2010).
3
Johnson et al. (2013) and Xu et al. (2014) suggested that diatoms may be winners relative to
other functional groups of phytoplankton under future CO2 conditions. However, other studies
suggest that the community may shift away from diatoms under elevated CO2 (Riebesell 2004;
Reinfelder 2011). A possible reason for this observation could be that the possible advantage
conferred at ambient CO2 levels by the more efficient CCMs of diatoms compared to
coccolithophores and dinoflagellates may be weakened with higher environmental CO2 levels
(Riebesell 2004; Reinfelder 2011). These conflicting results indicate that the responses of
phytoplankton to climate change may be species-specific or group-specific, and thus worthy of
more investigation.
Simultaneously, ocean acidification-driven decreases in CO3
2-
concentration are changing
the saturation state with respect to calcium carbonate (CaCO3), including both aragonite and
calcite. As regions of under-saturated CaCO3 expand, the dissolution of marine carbonates will
increase (Sabine et al. 2004). As a result, it will become more difficult for marine calcifying
organisms such as coccolithophores and foraminiferans to form biogenic CaCO3 (Orr et al. 2005;
Brandenburg 2014). Calcification rates of the two bloom-forming coccolithophores Emiliania
huxleyi and Gephyrocapsa oceanica were found to decrease by 25% and 45%, respectively, at
pCO2 concentrations three times those of the preindustrial value (Riebesell et al. 2000;
Zondervan et al. 2001). Although not examined in this thesis, the responses of marine calcifying
phytoplankton to future ocean acidification are a research hotspot and are receiving close
attention (Hutchins and Fu 2017).
Notably, the capacity of oceans to take up atmospheric CO2 has also been found to be
weakened with higher levels of anthropogenic pCO2 and lower CO3
2-
concentration in the
surface seawater (Sarmiento et al. 1995, Sabine et al. 2004). Based on observations from 1959-
2012, the oceanic CO2 sink has declined by a factor of about 1/3, suggesting that CO2 uptake
increased more slowly than rising CO2 (Raupach et al. 2013). The CO3
2-
: CO2 (aq) ratio is also
predicted to decrease from 4:1 to 1:1 in the future Southern Ocean (Orr et al. 2005).
Consequently, future elevated CO2 may have even more severe consequences for global climate
and ecosystems as the buffering ability of oceans to sequestrate CO2 decreases.
1.1.2 Warming and Increased Thermal Variability
Elevated atmospheric CO2 along with other greenhouse gases such as methane, water
vapor and nitrous oxide, also bring about global warming (Riebesell 2004; IPCC 2007; Crosson
4
2008; Arnold et al. 2012). The concentrations of these greenhouse gases in the atmosphere
exceed their corresponding concentrations over the past thousands of years (IPCC 2007).
Consequently, average global temperature has risen by 0.76°C during the last century (Jones and
Mann 2004), and sea surface temperature has increased ~0.5°C from 1971-2010 (IPCC 2013;
Abram et al. 2016). By the end of the 21st century, global temperature is predicted to rise by 2.6-
4.7°C (IPCC 2013) and sea surface temperature is projected to increase by 1-3°C (Bopp et al.
2013; Roemmich et al. 2015). Moreover, short-term environmental variability is also projected to
be stronger (IPCC 2012). As a result, marine phytoplankton in the upper layer of the ocean will
need to deal with an environment that will be both warmer and more thermally variable in the
future.
It has been long acknowledged that temperature change has significant effects on the
growth, metabolism and physiology of marine phytoplankton (Eppley 1972; Goldman and
Carpenter 1974; Montagnes and Franklin 2001). Within the thermal limits of phytoplankton,
rising temperature generally stimulates growth by increasing metabolic rates. The Arrhenius
equation is widely applied to quantify this relationship between temperature and maximum
growth rates of phytoplankton (Eppley 1972; Goldman and Carpenter 1974; Goldman 1979).
The responses of phytoplankton functional groups have been extensively investigated
under global warming scenarios, and have been found to be diverse (Beardall et al. 2009;
Huertas et al. 2011; Chen et al. 2014; Lewandowska et al. 2014; Fu et al. 2014; Jiang et al.
2018). As suggested by Boyd et al. (2013), warming generally promotes the growth and biomass
of numerous phytoplankton species before their upper thermal threshold is reached, but will
dramatically decrease their growth if temperature continues rising beyond their upper thermal
limit. Particularly, tropical phytoplankton species are predicted to be the most vulnerable to
future warming conditions, since their maxima are close to current ambient temperatures
(Thomas et al. 2012). The biogeographic ranges of many tropical species may thus shift away
from their original habitats towards higher latitude regions (Breitbarth et al. 2007; Fu et al. 2014;
Boatman et al. 2017; Li et al. 2018). In other words, near future warming is likely to promote the
growth of marine phytoplankton in high-latitude regions, but will pose a severe challenge to the
persistence of tropical phytoplankton.
Current research on thermal variability consequences for marine ecosystems remains limited,
and the possible effects of variability have been overlooked in phytoplankton culture studies for a
5
long time. In the past decade, the diverse impacts of thermal variation have been mainly explored
on terrestrial ectothermic animals (Estay et al. 2011; Bozinovic et al. 2011; Paaijmans et al. 2013)
and soil bacterial communities (Langenheder et al. 2012). In marine ecosystems, distinct responses
have been documented for corals, intertidal polychaetes and other marine animals (Dong et al.
2006; Tian and Dong 2006; Putman and Edmunds 2011; Godbold and Solan 2013). To more
accurately estimate the growth and physiology of phytoplankton in the future ocean, thermal
variability needs to be recognized as an important environmental factor and addressed
experimentally.
1.1.3 Reduced nutrient availability
The growth of marine phytoplankton depends on macronutrients that are required in large
quantities, such as phosphorus (P), nitrogen (N) and silica (Si), and micronutrients such as iron
(Fe) and copper (Cu) which play critical roles but are required in only minor quantities (Sunda
2006). These nutrients have various biological functions in marine phytoplankton cells. For
instance, N is an important component in cellular proteins and chlorophyll (Geider and La Roche
2002; Arrigo 2004; Li et al. 2015) while P is involved in genetic information storage and
expression, cell membrane synthesis, and energy generation and signal pathways of cells (Sohm
et al. 2011; Toseland et al. 2013; Karl 2014; Lin et al. 2016). Si is required in the cellular
frustules of diatoms, and can sometimes limit the growth of diatoms (Anderson et al. 2002). The
micronutrient Fe plays a critical role in multiple key processes in phytoplankton cells, such as
photosynthesis, nitrogen fixation and nitrate assimilation (Kolber et al. 1994; Timmermans et al.
1994; Behrenfeld et al. 1996; Milligan and Harrison 2000; Franck et al. 2003).
Due to the importance of nutrients in the structure and function of phytoplankton cells,
nutrient deficiency often severely limits or colimits the growth of marine phytoplankton (Arrigo
2004). In the low-latitude surface ocean and many coastal areas, photosynthetic organisms are
primarily limited by N (Dugdale and Goering 1967; Glibert 1988; Moore et al. 2013). Fe-
limitation prevails in the High-Nitrate Low-Chlorophyll regimes, and also many other regions
(Wells 1999; Hutchins et al. 2002; Eldridge et al. 2004; King and Barbeau 2007). The growth of
diazotrophic cyanobacteria in the oligotrophic ocean is commonly limited by P and/or Fe
(Sañudo-Wilhelmy et al. 2001; Berman-Frank et al. 2001; Fu and Bell 2003; Mills et al. 2004;
Garcia et al. 2015). In turn, the biogeochemical cycles of these nutrients are also largely
controlled by marine phytoplankton and other microbes.
6
With shifting climate patterns, the ocean is predicted to be more stratified, and upwelling
will be weakened and could even virtually disappear in some areas (Sarmiento et al. 1998; IPCC
2013; Deutsch et al. 2014). Consequently, nutrient supplies from the deep ocean are projected to
be reduced, and thus particularly the low- and mid-latitude oceans will become more
oligotrophic (Behrenfeld et al. 2006; Taucher and Oschlies 2011; Garrison 2012). As a result,
marine primary production and biogeochemical cycles controlled by marine phytoplankton will
be profoundly affected (Gregg et al. 2003; Doney 2006; Boyd and Hutchins 2012; Hutchins and
Fu 2017).
Based on current observations and model predictions, these nutrient reductions will cause
global primary production to decline (Gregg et al. 2003; Schmittner 2005; Taucher and Oschlies
2011; Moore et al. 2018). In some cases, the importance of nutrient supplies can overwhelm or
offset the biological effects of temperature (Lewandowska et al. 2014; Marañón et al. 2014).
Moreover, altered nutrient availability shifts the elemental ratios of marine phytoplankton (Losh
et al. 2012; Reinfelder 2012; Brandenburg 2014; Verspagen et al. 2014). For instance, cellular C:
N, C: P and N: P ratio of the marine picoeukaryote Micromonas pusilla increased with
intensified P limitation (Maat et al. 2014). In addition, the species composition of marine
phytoplankton community also varies with different nutrient regimes (Yung et al. 1997; Heisler
et al., 2008). Riegman (1995) found that the prymnesiophyte Phaeocystis became a dominant
species under N-limitation during the North Sea summer bloom, and also in laboratory
competition experiments. Changing nutrient supplies and ratios in the Tolo Harbor of Hong
Kong also altered the abundance of diatoms and dinoflagellates (Yung et al. 1997).
Furthermore, changes in nutrient inputs and forms may also enhance the toxicity of
harmful algal blooms. For instance, the production of domoic acid (DA), the neurotoxin
produced by the bloom forming diatom Pseudo-nitzschia spp., varied with different forms of N,
including nitrate, urea and ammonium. In particular, Pseudo-nitzschia cuspidate was most toxic
when cultured with nitrate, while Pseudo-nitzschia australis produced more DA production
when urea was taken up (Cochlan et al., 2005; Auro, 2007; Howard et al., 2007).
In summary, nutrient availability and composition play a key role in the growth and
physiology of marine phytoplankton. The predicted changes in nutrient supplies under climate
change are thus likely to make a large difference in future marine primary productivity,
community structure, and biogeochemical cycling. In this context, research on marine
7
phytoplankton should clearly take nutrient availability into account along with other climate
change variables.
1.1.4 Other Climate Change Variables
Besides warming, ocean acidification and decreased nutrient availability, other climate
change variables such as more intense irradiance exposures in a shallower upper mixed layer,
including both photosynthetically active radiation and solar ultraviolet radiation, will also affect
the growth of phytoplankton in differential ways (Tremblay and Gagnon 2009; Behrenfeld 2011;
Williamson et al. 2014). For instance, increased irradiance has been found to lead to loss of
marine phytoplankton biomass because of its inhibitory effects on the growth and its interactions
with other environmental factors (Feng et al. 2008; Gao et al. 2012 a, b; Xu et al. 2014). The
future ocean is also widely predicted to experience a loss of dissolved O2, resulting in an
expansion of existing oxygen minimum zones and the establishment of new ones (Stramma et al.
2008; Long et al. 2016; Breitburg et al. 2018). Although these climate change variables were not
examined in this dissertation, their effects on the growth and survival of marine phytoplankton
also need further investigation.
1.1.5 The Interactive Effects of Multiple Climate Change Variables
The individual effects of climate change variables on marine phytoplankton have been
explored in numerous recent studies, as discussed above. Relatively fewer studies have tested
combinations of multiple environmental variables, however, and conflicting responses of
phytoplankton are often reported to the interactive effects of these variables (Riebesell 2004;
Feng et al. 2008; Reinfelder 2012; Gao et al. 2012b; Johnson et al. 2013; Brandenburg 2014;
Gao and Campbell 2014; Paul et al. 2016). For instance, the positive effects of warming in the
growth of marine phytoplankton could be completely offset by decreased nutrient supplies
(Doney 2006). Contradictory effects of combines temperature increases and elevated CO2 levels
on growth have also observed in the studies of diatoms (Low-Décarie et al. 2014; Sommer et al.
2015). However, warming appears to counteract to a large extent the negative effects of Fe
limitation on marine nitrogen fixers (Jiang et al. 2018).
The sometimes contradictory conclusions reached by previous studies indicate that the
responses of phytoplankton to a changing environment may be species- or group-specific. These
taxon-specific effects of multiple climate change variables highlight the need for more research
on synergistic or antagonistic interactions among them.
8
1.2 Key Phytoplankton Functional Groups: Diatoms and Diazotrophic Cyanobacteria
Diatoms and cyanobacteria are two of the most important functional groups in marine
phytoplankton communities (Riebesell 2004; Falkowski et al. 2004). The responses of diatoms
and diazotrophs to global change were investigated in this dissertation, as they are crucial to
predictions of future primary productivity, carbon acquisition and storage, and biogeochemical
cycles in marine ecosystems.
1.2.1 Diatoms
Diatoms contribute approximately 40% of marine primary productivity, and provide a major
food source for grazers in marine food webs (Field et al. 1998; Reinfelder 2011; Gao and
Campbell 2014). Covered by silicon frustules, diatoms are also important in the biogeochemistry
of silicon and export of carbon and multiple nutrient elements due to their fast sinking rates
(Miller and Wheeler 2012; Hutchins and Boyd 2016; Tréguer et al. 2018). Potential changes in
the persistence, abundance and species composition of diatoms under climate change scenarios
will therefore generate profound impacts on global ecology and biogeochemistry. Hence, the
diverse responses of diatom growth, physiology and cell size to climate change is a priority
research area.
Due to this ecological and biogeochemical significance of diatoms, their responses to
changing environmental factors have been extensively explored (Riebesell 2004; Gao et al.
2012b; Gao and Campbell 2014; Low-Decarie et al. 2014; Xu et al. 2014; Sommer et al. 2015;
Qu et al. 2018). The study of Tatters et al. (2013) examined the responses of an assemblage of
diatoms, including Coscinodiscus spp., Pseudo-nitzschia delicatissima, Chaetoceros criophilus
and other species, to future acidification and warming. Their results indicate that both warming
and elevated CO2 concentrations differentially affect the growth rates of these diatom species,
and therefore shift the community structure. As discussed in section 1.1 above, whether diatoms
are favored by future global changes still depends on the specific conditions and taxa being
examined (Johnson et al. 2013; Xu et al. 2014; Tatters et al. 2018).
Diatoms play a dominant role in the global silicon cycle due to their obligate requirement for
a silica frustule. Under ocean acidification, cellular Si/C ratios have been found to decrease in
multiple diatom species, indicating decreased silicification, especially under nutrient limiting
conditions (Tatters et al. 2012; Mejía et al. 2013). One possible explanation for this decrease is
associated with a possible pH-buffering function of the silica frustule that may contribute to the
9
efficiency of the enzyme carbonic anhydrase (CA), which helps the cell acquire CO2 for
photosynthesis. The silica frustule may thus help to maintain an optimal pH for the catalytic
activity of CA (Milligan et al., 2004). With elevated CO2 concentrations in seawater, the need
for efficient activity of CA decreases, and this possible function of the frustule becomes less
important. However, more research is needed to examine whether a decline in silica content
under climate change is common to all diatoms, and to reveal the mechanisms behind this
response.
Diatom cell size is another trait that is relevant to inter-species competition and carbon
export (Ploug et al. 1999; Smetacek 1999; Litchman and Klausmeier 2008), and that can be
shifted by environmental factors. Sommer et al. (2015) found that cell sizes may enlarge with
elevated CO2 levels, but shrink under warmer conditions. Differences in cell size may be also
make a contribution to determining success or failure in competitive interactions. Smaller species
are commonly thought to have an advantage in active and diffusive uptake of dissolved inorganic
carbon and nutrients because of their greater surface area: volume ratios (Miller and Wheeler
2012). However, with elevated CO2 concentrations the disadvantage of larger diatoms in carbon
acquisition may be reduced, and they become more competitive (Tortell et al. 2008; Johnson et
al. 2013; Gao and Campbell 2014; Low-Decarie et al. 2014). Another study indicates that
smaller diatom species seem to be more susceptible to photo-inactivation under ocean
acidification conditions (Gao et al. 2012a). Hence under future global change, larger diatom
species have been suggested to be the winners in competition with smaller ones (Gao and
Campbell 2014).
Diatoms are the most diverse marine phytoplankton group, and are composed of more than
200,000 species (Armbrust 2009; Falkowski and Knoll 2011). Diatoms can be morphologically
classified into two groups, pennates and centrics (Armbrust 2009). Pseudo-nitzschia is an
important globally-distributed genus of pennate diatoms that is capable of producing the
neurotoxin domoic acid and forming harmful algal blooms (Bates et al. 1998; Parsons et al.
2002). Due to the potential of this genus to bring about enormous ecological and economic
losses, previous research has mainly focused on the responses of its toxicity to nutrient
availability and other environmental factors. As mentioned in section 1.3 above, increasing
nutrient inputs and shifts in nutrient forms may enhance the abundance and toxicity of Pseudo-
nitzschia (Pan 2001; Cochlan et al., 2005; Auro 2007; Howard et al., 2007). Tatters et al. (2012)
10
found that high CO2 can increase the growth rate and toxicity of Pseudo-nitzschia fraudulenta,
while silicate limitation could intensify its toxicity.
With respect to centric diatoms, Thalassiosira and Coscinodiscus are two representative
genera. The growth, metabolism, nutrient utilization, and genomes of the cosmopolitan
Thalassiosira genus have been extensively examined (Paasche 1973; Berges et al. 2002; Stramsk
et al. 2002; Armbrust et al. 2004) while studies on the very large genus Coscinodiscus have
focused on cell size, reproduction and nutrient uptake (Holmes 1966; Burckle and McLaughlin
1977; Boyd and Gradmann 1999). Considering the important role of Coscinodiscus in marine
primary productivity, food-web interactions and carbon export, there is a need to investigate the
physiological responses of Coscinodiscus under climate change scenarios. The first chapter of
this dissertation investigates the growth, elemental stoichiometry and carbon fixation responses
of a representative Coscinodiscus isolate in relation an interacting set of three global change
factors (warming, elevated CO2 and nitrate availability).
1.2.2 Diazotrophic Cyanobacteria
Diazotrophic cyanobacteria play a critical ecological and biogeochemical role by
providing new nitrogen to the surface ocean through their nitrogen fixing activities, in turn
supporting carbon export and contributing to global carbon and nitrogen cycles (Sohm et al.
2011; Zehr 2011; Tang et al. 2019). One of the most important and globally distributed
diazotrophs in the tropical and subtropical ocean is the colonial and filamentous genus
Trichodesmium (Capone et al. 1997, 2005; Zehr 2011). The nitrogen fixed by Trichodemium
accounts for approximately one third to one half of marine biological new nitrogen (Capone et al.
2005; Westberry and Siegel 2006; Boatman et al. 2017). The sympatric unicellular diazotroph
Crocosphaera makes a contribution to total nitrogen fixation that is comparable or even bigger
than that of Trichodesmium in certain regions (Zehr et al 2001; Montoya et al 2004; Knapp et al.
2012). Interestingly, Berthelot et al. (2016) found that the filamentous diazotroph Trichodesmium
is more efficient in transferring fixed nitrogen to non-diazotrophic communities than
Crocosphaera.
The growth of Trichodesmium and Crocosphaera in the oligotrophic ocean is commonly
limited by phosphorus and iron (Sohm et al. 2011). Numerous studies have investigated the
effects of individual limitation by each element, or effects of co-limitation by both (Berman-
Frank et al. 2001; Sañudo-Wilhelmy et al. 2001; Fu and Bell 2003; Mills et al. 2004; Garcia et al.
11
2015; Walworth et al. 2016). Fu et al. (2005) compared P uptake and growth kinetics of two
Trichodesmium strains (IMS101 and GBRTRLI101), which were isolated from areas with
contrasting P concentrations.
The development of molecular biology, genomic, transcriptomic and proteomic studies of
diazotrophs has helped to reveal the mechanisms behind N2 fixation and nutrient uptake
processes (Dominic et al. 1998; Dyhrman et al. 2006; Hewson et al. 2009; Shi et al. 2010;
Walworth et al. 2015, 2016). Furthermore, genomic information on several strains of the two
diazotrophs is available (Dyhrman et al. 2006). In particular, the complete genome sequence of
Trichodesmium erythraeum IMS101 has been determined and published (Walworth et al. 2015).
Considering the significance of Trichodesmium and Crocosphaera in both the N and C
cycles, it is clearly a priority to investigate their responses to climate change. For instance,
Hutchins et al. (2007) revealed pronounced positive effects of increased CO2 on the growth rates
and nitrogen fixation of Trichodesmium. Similarly enhancing impacts of elevated CO2 were also
found on the growth and nitrogen fixation rates of Crocosphaera under Fe-replete conditions (Fu
et al. 2008). The effects of other environmental factors such as temperature and solar radiation
on these two diazotrophs have also been examined in previous studies (Garcia et al. 2014;
Gradoville et al. 2014; Boatman et al. 2017; Li et al. 2018; Jiang et al. 2018). In particular, the
temperature norms of a global collection of Trichodesmium and Crocosphaera isolates were
determined in the study of Fu et al. (2014) and found to be relatively invariant.
However, there are few studies looking at the effects of increased thermal variability on
diazotrophs, or on the interactions of dynamic temperatures with nutrient availability. Thermal
variability is relatively minor in the tropical and subtropical ocean compared to temperate or
coastal areas (Mantyla et al. 2008; De'ath et al. 2009; Nezlin et al. 2012). For instance, the strain
Trichodesmium erythraeum GBRTRLI101 was isolated from the Great Barrier Reef (Fu and Bell
2003) and thus is assumed to have experienced relatively little natural temperature fluctuation
during its evolutionary history (Lough 2007; De'ath et al. 2009), making it likely to be sensitive
to thermal variation in the future or in experimental simulations. Considering both this
expectation and the projected reduced nutrient supplies in the future ocean, the second chapter of
this dissertation explored the response of Trichodesmium erythraeum GBRTRLI101 to the
interactions between P-limitation and thermal variation.
12
Other than short-term acclimation to changing environment, it is intriguing and imperative to
investigate the long-term capacity of diazotrophs to evolve genetically under selection by climate
changes variables. As microbes, Trichodesmium and Crocosphaera cultures have relatively short
generation times and large population sizes, potentially facilitating their adaptation to the
changing ocean as well as making them tractable organisms for laboratory experimental
evolution studies. In the recent studies of Hutchins et al. (2015) and Walworth et al. (2016), up-
regulated N2 fixation rates of Trichodesmium IMS101 after long-term selection for hundreds of
generations by projected future CO2 concentration seem to indicate a constitutive or irreversible
adaptive response to high CO2 levels.
It is clear that long-term evolutionary experiments can provide more accurate and
potentially more realistic information to predict the responses of phytoplankton to future global
changes compared to short-term physiological acclimation studies. Besides elevated CO2 levels,
warming is another important climate change variable. It is still an enigma if and how tropical
phytoplankton species will respond evolutionarily to the major challenge posed by future
warming condition. It is clear though that these potential adaptive responses could have large
consequences for ocean biogeochemical cycles in a changing ocean. In this context, the third
chapter of this dissertation is aimed at investigating the possible long-term adaptation of the two
important subtropical-tropical diazotrophs Trichodesmium and Crocosphaera to changing
temperatures.
1.3 Summary
Key marine phytoplankton groups are facing huge challenges posed by climate change,
and their responses will have profound influences on global ecology, biogeochemistry and
climate. To better understand and predict the future trends of marine phytoplankton productivity
and community structure under global change scenarios, the interactive effects of multiple
climate change variables should be further investigated. In addition, natural variability has been
overlooked in most phytoplankton culture work, possibly leading to misleading results and
problematic conclusions. Although short-term responses of phytoplankton to climate changes
have been thoroughly investigated, research on their possible long-term adaptive responses is
still in its infancy (Hutchins et al. 2015, Hutchins and Fu 2017). Considering that climate change
will be a long-lasting process, research into the possible adaptation of phytoplankton under
13
extended global change scenarios is critical in order to accurately predict changes in biological
and geochemical processes in future marine ecosystems.
In order to achieve a deeper understanding and formulate better predictions of future
trends in the biology and biogeochemistry of marine phytoplankton under global change
scenarios, these three issues need to be taken into account and addressed with experimental
research methods. The three major conceptual components of my dissertation are intended to
help make progress in these neglected aspects of ocean global change biology, by providing
insights into the relationship between the marine environment and phytoplankton under climate
change scenarios.
The three projects that make up this dissertation are as follows:
1) Responses of the large centric diatom Coscinodiscus sp. to interactions between warming,
elevated CO2, and nitrate availability (published in Limnology and Oceanography, 2018).
2) Distinct responses of the nitrogen-fixing marine cyanobacterium Trichodesmium to a
thermally-variable environment as a function of phosphorus availability (published in Frontiers
in Microbiology, 2019).
3) The marine nitrogen-fixing cyanobacteria Trichodesmium and Crocosphaera employ
contrasting acclimation and adaptation strategies during long-term thermal selection (in
preparation for publication at the time that this dissertation was submitted).

14
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30
Chapter 2: Responses of the large centric diatom Coscinodiscus sp. to
interactions between warming, elevated CO2, and nitrate availability
Pingping Qu
1
, Feixue Fu
1
, David A. Hutchins
1

1. Marine and Environmental Biology, Department of Biological Sciences, University of
Southern California
Corresponding author: David Hutchins
1
(Email address: dahutch@usc.edu)
Running head: Diatom responses to climate changes
Key words: diatom, Coscinodiscus, climate change, nitrate, temperature, carbon dioxide

The manuscript was published in 2018 as:
Qu, P., Fu, F.-X. and Hutchins, D. A. (2018). Responses of the large centric diatom
Coscinodiscus sp. to interactions between warming, elevated CO2, and nitrate availability.
Limnology and Oceanography, 63(3), 1407-1424. doi:10.1002/lno.10781.

31
Abstract
Marine ecosystems are facing multiple anthropogenic global changes, including ocean
acidification, warming, and reductions in limiting nutrient supplies. Together, these will
challenge phytoplankton including large centric diatoms such as Coscinodiscus sp., a group that
is particularly important to many ocean food webs and to carbon export. We investigated the
interactive effects of warming, elevated CO2 levels, and nitrate availability on Coscinodiscus
growth, elemental stoichiometry and Fe and C uptake rates in a four-treatment triplicated
factorial experiment combining two CO2 levels (ambient CO2 and predicted future CO2
concentration) and two temperatures (16°C and 20°C) across a series of 7 nitrate concentrations
ranging from growth-limiting (1 μmol/L) to replete (100 μmol/L). Higher temperatures led to
higher maximum growth rates (μmax), but also decreased higher half saturation constants for
nitrate (K1/2), while elevated CO2 increased K1/2 only at the warmer temperature. Lowered
μmax/K1/2 ratio by warming and rising CO2 indicated a higher nitrate requirement at these
conditions. High temperature also decreased cellular P and Si contents, especially at ambient
CO2, leading to higher N: P and C: Si elemental ratios. Fe: C uptake ratios responded positively
to lower nitrate levels, lower CO2 concentrations, and warming. Furthermore, significant
interactions between nitrate availability and temperature or CO2 levels were observed in the
responses of specific growth rates, cellular Chl a and Si contents, Fe: C, PON: POP and BSi:
POC ratios, while interactions between temperature and CO2 levels were only found to be
significant for the μmax/K1/2 ratio and cellular phosphorus contents. The mutual interactions
among CO2 concentrations, temperature, and nitrate supply may all affect future growth,
physiology, and carbon export by Coscinodiscus sp., however in general warming and nitrate
availability appear to be more influential than CO2 level.

32
2.1 Introduction
Global changes caused by human activities have posed an enormous challenge to natural
ecosystems and human societies (Crutzen 2002; Riebesell 2004). In marine ecosystems, elevated
atmospheric CO2 concentrations bring about ocean acidification through air-seawater gas
exchange (Riebesell 2004; Solomon et al. 2007; Arnold et al. 2012). Since the preindustrial
period, atmospheric CO2 concentrations have increased from ~290 parts per million (ppm) to a
current value of ~400 ppm (Solomon et al. 2007; Bala 2013). As one of the most important
greenhouse gases, rising CO2 levels also give rise to atmospheric warming by trapping infrared
radiation, which in turn increases the sea surface temperature (Schneider 1989; Rodhe 1990).
Correspondingly, average global temperatures have risen 0.76°C during the last century (Jones
and Mann 2004). By the end of the 21st century, when the projected CO2 concentration will
approach 800-1150 ppm, global temperatures are predicted to rise 2.6-4.7°C (IPCC 2013). The
shift in climate patterns will also result in a series of other physical and chemical changes in the
ocean, such as shoaling of upper mixed layers and consequent increases in effective underwater
irradiance reaching phytoplankton, while intensified stratification will result in reduced nutrient
supplies from the deep ocean (Behrenfeld et al. 2006; Cermeño et al. 2008; Gao et al. 2012b). As
a result, marine ecosystems and global biogeochemical cycles will be profoundly affected (Boyd
and Hutchins 2012; Hutchins and Fu 2017).
Diatoms are one of the most important functional groups of marine phytoplankton (Riebesell
2004; Falkowski et al. 2004), contributing approximately 20% of global primary productivity
and supporting higher trophic levels in marine food webs (Field et al. 1998; Reinfelder 2011;
Gao and Campbell 2014). The production of siliceous frustules by diatoms dominates the marine
biogeochemical cycle of silicon, and this group also influences the cycles of other nutrients such
as nitrogen, phosphorus and iron (Miller and Wheeler 2012, Hutchins and Boyd 2016). Hence,
the response of diatoms to global change will be crucial to predictions of future primary
productivity, community structure shifts in marine ecosystems, and the role of the ocean as a
carbon sink.
To date, studies with cultured diatoms have revealed diverse responses of growth rates,
carbon fixation, elemental ratios, cell size and community composition to changing
environmental factors (Gao et al. 2012b; Gao and Campbell 2014; Xu et al. 2014). The carbon
fixation rates of natural diatom communities also exhibit a range of responses under elevated
33
CO2 levels (Tortell and Morel 2002; Tortell et al., 2008; Hare et al. 2007). Warming and rising
CO2 concentrations had contradictory impacts on the growth of diatoms in the studies of Low-
Décarie et al. (2014) and Sommer et al. (2015), and temperature seems to play a larger role than
CO2 levels in determining cell size and species composition (Tatters et al. 2013; Sommer et al.
2015). Nutrient levels that can vary from growth-limiting to replete complicate these interactions
even further, and can especially affect shifts in elemental ratios (Cermeño et al. 2008; Losh et al.
2012; Reinfelder 2012).
Several mechanisms and models have been developed to interpret these responses to climate
change variables. The effects of temperature on the growth of phytoplankton have long been
recognized (Eppley 1972; Goldman and Carpenter 1974; Montagnes and Franklin 2001). The
Arrhenius equation helps define the relationship between temperature and maximum growth
rates in many cases, and thermally-elevated metabolic rates explain the generally positive effects
of warming within the thermal limits of phytoplankton (Goldman and Carpenter 1974; Goldman
1979; Marañón et al. 2014). With respect to the effects of elevated CO2 levels, down-regulation
of carbon concentration mechanisms (CCMs) is one possible explanation for the increasing
growth rate and higher carbon: nutrient ratios of some species under higher CO2 levels
(Reinfelder 2011; Kupriyanova et al. 2013). CCMs are inducible and energy-consuming
mechanisms that boost supplies of CO2 and so compensate for the inefficiency of the carbon-
fixing enzyme Rubisco, thus allowing maintenance of photosynthetic rates under low CO2 levels
(Reinfelder 2011; Low-Décarie et al. 2014). At higher CO2 concentrations, more CO2 is provided
to cells through passive diffusion and therefore CCMs are predicted to be down-regulated,
allowing more energy to be reallocated to growth-supporting processes such as uptake of
nutrients and nitrogen assimilation (Reinfelder 2012; Johnson et al. 2013; Eberlein et al. 2016).
Although it seems the down-regulation of CCMs will benefit some diatom species through
energy reallocation, the actual effects on competition with other groups are debatable. Some
research suggests that diatoms may compete less successfully with other functional groups such
as coccolithophores and dinoflagellates at higher CO2, since the advantage of more efficient
CCMs which diatoms enjoy at current CO2 levels will be lessened (Riebesell 2004; Reinfelder
2011). Moreover, other changing factors may also stimulate shifts in community composition.
Cermeño et al. (2008) predicted that strengthened nutrient limitation in the future ocean would
reduce the advantage of diatoms compared to coccolithophorids. Alternately, several other
34
studies have suggested diatoms to be superior competitors under future conditions of CO2,
temperature, and light relative to currently co-existing groups such as prymnesiophytes (Xu et al.
2014; Zhu et al. 2016). According to the conflicting results obtained so far, the responses of
diatoms to integrated multiple variable global changes may be a function of distinct traits and
strategies as determined by local adaptation, and worthy of more investigation.
As the largest contributors to biogenic silica production in the ocean, diatoms have also
received attention concerning their silicification processes under ocean acidification (Martin-
Jézéquel et al. 2000). Declining Si/C ratios at higher CO2 concentrations and lower pH have been
reported in Pseudo-nitzschia fraudulenta, Thalassiosira pseudonana and Thalassiosira
weissflogii, especially under nutrient-limiting conditions (Tatters et al. 2012; Mejía et al. 2013).
However, more studies are needed to ascertain whether a decline in silicon content under ocean
acidification or other climate-change factors is a general response for diatoms, providing
information for further investigations into the mechanisms involved.
In addition to the major nutrients mentioned above, the micro-nutrient iron plays a critical
role in numerous key processes in phytoplankton cells, including photosynthesis (Kolber et al.
1994; Behrenfeld et al. 1996) and nitrate assimilation (Timmermans et al. 1994; Milligan and
Harrison 2000; Franck et al. 2003). Several studies have examined the interactions between iron
availability and climate change variables to predict future scenarios in marine ecosystems (Sunda
and Huntsman et al. 2011; Hoppe et al. 2013; Hutchins and Boyd 2016). Considering the
importance of iron in photosynthesis and nitrate assimilation, investigations into iron uptake and
use efficiency under varying nutrient, temperature, and carbonate system conditions may provide
new insights into the interactive effects of climate change variables on carbon and nitrogen
metabolism and biogeochemistry.
The widespread centric diatom genus Coscinodiscus plays an important role in marine
primary productivity, supports higher trophic levels, and contributes to global biogeochemistry.
It is particularly important to vertical carbon export, due to the high sinking rates of these very
large diatom cells, which range from several tens to hundreds of microns, depending on species
and conditions (Bienfang et al. 1982; Tada et al. 2000; Key et al. 2010). Moreover, although it
lacks toxins, it can still form harmful blooms due to high biomass (Nishikawa et al. 2010).
Recently, several studies on Coscinodiscus have focused on its morphology, elemental
composition and competitive interactions with other species (Ono et al. 2008; Nishikawa et al.
35
2010; Tatters et al. 2013). Due to its regular shape and relatively large size, Coscinodiscus has
also been chosen to investigate the interactions between cell size and multiple environmental
variables (Gao et al. 2012a; Nishikawa et al. 2008). However, few studies have focused on the
interactive effects of CO2 levels, warming and nitrate availability on Coscinodiscus.
Here, we investigate the responses of Coscinodiscus sp. growth, elemental stoichiometry,
carbon fixation, and iron uptake rates to a set of multiple factor global change scenarios. Based
on previous studies of this and related diatom species, we hypothesized that interactions between
warming and nitrate availability would have a greater impact on Coscinodiscus growth and
physiology than would CO2/nitrate interactions. We further hypothesized that due to the two- and
three-way interactions among warming, elevated CO2 and reduced nitrate availability, the overall
biological effects of these factors would in general be larger than the sum of their individual
effects.
2.2 Methods
2.2.1 Culturing and Incubation Methods
The large centric diatom Coscinodiscus sp. (cell volume 1397 ± 563 μm
3
) was isolated from
nearshore waters of the South Island of New Zealand (45, 45.09°S 170, 48.6°E) in January 2011
(Tatters et al. 2013). Ambient seawater temperature during the sample collection period was
~16°C. Experiments consisted of four triplicated factorial treatments combining two CO2 levels
(ambient CO2 and predicted future CO2 concentrations) and two temperatures (16°C and 20°C),
chosen to simulate current conditions and projected future condition in 2100, as predicted by
IPCC scenarios (Solomon et al. 2007; IPCC 2013). In view of the importance of nitrogen in the
growth of phytoplankton, its limiting nutrient status in many regimes (Dugdale and Goering
1967; Losh et al. 2012), and the fact that it is predicted to become scarcer in the future ocean
(Hutchins et al. 2009; Hutchins and Fu 2017), each of the four temperature/CO2 treatments was
tested across a series of seven nitrate concentrations ranging from growth-limiting to replete
(nitrate concentrations: 1, 2, 5, 10, 30, 50 and 100 μmol/L). The selection of nitrate
concentrations was based on previous experience with Coscinodiscus spp. cultures in our
laboratory (unpublished data).
All the Coscinodiscus cell lines were grown in 250 ml acid-washed polycarbonate bottles
using an artificial seawater medium, with replete levels of nutrients (except nitrogen), trace
metals and vitamin mixtures according to the AQUIL recipe (Sunda et al. 2005), with
36
modifications as in Garcia et al. (2011). Artificial seawater was autoclaved after adjusting
salinity to 35-36 by adding Milli-Q water. Cool white fluorescent bulbs were used to provide a
12h dark: 12h light cycle at 120 μmol photons m
-2
s
-1
in two laboratory incubators set to 16°C and
20°C. The two CO2 concentrations were maintained by gently bubbling the cultures with
ambient air and commercially prepared air/CO2 mixtures (800 ppm, Praxair) respectively, which
has proven to be a reliable way to control carbonate chemistry in past cultures of numerous
phytoplankton species (Hutchins et al. 2007, 2009; Fu et al. 2008).
Semi-continuous incubation methods were employed for all the cell lines in order to
maintain cells in the exponential growth phase. Cultures were diluted every two days, and
dilution volumes were determined based on Chl a in vivo fluorescence readings to obtain steady-
state specific growth rates. For one dilution cycle of two days, all the bottles on Day 0 were set
up to a fixed biomass based on Chl a in vivo fluorescence. After two days (48 hours), the final
biomass was measured at the same time of day to determine the volume of culture to remove and
fresh medium to be added, in order to dilute each replicate back to the fixed initial biomass for
the next cycle. The initial and final biomass were used to calculate growth rates for each bottle.
For cell lines under nitrogen-limiting conditions (nitrate concentrations from 1 to 10 μmol/L,
based on preliminary experiments with this isolate), the nitrate concentrations in the cultures
before dilution is extremely low and can be considered to be nearly zero, although care was
taken to make transfers before stationary growth phase could set in. Semi-continuous incubations
were maintained for more than six weeks. After three to six consecutive measurements in which
specific growth rates were not significantly different (< 15% variability between dilutions),
cultures were considered to be in steady state exponential growth, and sampling was conducted
for the physiological and biogeochemical analyses (King et al. 2011).
2.2.2 Physiological and Biogeochemical Analyses
Growth rates. During the semi-continuous incubation, real-time biomass was estimated with
in vivo fluorescence before and after dilution, and subsequently validated by microscopy using
preserved cell counts.
Specific growth rates (μ) were calculated based on the in vivo fluorescence readings as:
μ= ln(Nt2 / Nt1)/(t2-t1), where Nt1, and Nt2 refer to biomass (as Chl a in vivo fluorescence
readings) at time 1 (t1) and 2 (t2) (in days) respectively (Ihnken et al. 2011). Reported growth
37
rates for each treatment are the means of specific growth rates of triplicate cultures in each
treatment.
In the four CO2/temperature treatments, the maximal growth rates (μmax) and half saturation
constants (K1/2) relative to nitrate concentration were calculated with μ, based on the Monod
equation (Monod 1949): μ = μmax S / (K1/2 +S), where μ refers to the specific growth rate of
phytoplankton, μmax stands for the maximum specific growth rate, S is the concentration of
nutrients, and K1/2 is the half saturation constant, namely the nitrate concentration at which half
of μmax is reached (Monod 1949). In this study, the initial nitrate concentration was used in this
equation because after two days the cultures were still in exponential growth phase, hence
growth could still be assumed as a function of the original concentration of the limiting nutrient
nitrate. The only constraint was that the fit was forced through zero, due to the assumption that
with no nutrient, there is no growth of cultures.
Cell counts and size. Samples of 1 ml volume were collected from each incubation replicate
and preserved at 4°C in the dark with the addition of 25% glutaraldehyde. Cell numbers were
counted and recorded with a Spears-Levy Eosinophil counting chamber using an Olympus
BX51microscope. The diameter, length or width of 50-60 cells from each treatment was
precisely measured with a calibrated micrometer on an Accu-Scope 3032 inverted phase
microscope. Coscinodiscus sp. cells were treated as cylinders, therefore the volume was
calculated from the measurements of diameters of base planes and the heights.
Carbonate buffer system. Carbonate chemistry was monitored using dissolved inorganic
carbon (DIC) and pH measurements. The pH in each bottle was monitored daily using a pH
meter (Orinon 5 STAR, Thermo Fisher Scientific), calibrated with pH 4, 7 and 10 NBS buffer
solutions each time before use (King et al. 2011). Total DIC measurements were conducted using
coulometric protocols (King et al. 2011) before sampling was conducted. For each replicate,
samples for DIC measurements were fixed with 100 μl 5% HgCl2, in a 25-ml borosilicate
scintillation vial fitted tightly with Teflon coated caps. DIC samples were analyzed on a UIC
CM140 CO2 coulometer using DIC standards from Dickson (2010) and JGOFS protocols.
Seawater pCO2 was calculated from measured DIC and pH using CO2SYS software (Lewis et al.
1998). The measured pH, DIC and calculated pCO2 values are shown in Table 1.
Chlorophyll a (Chl a). A sample volume of 20 ml from each replicate bottle was filtered
through GF/F glass fiber filters for Chl a. After adding 6 ml of 90% acetone, Chl a was extracted
38
in the freezer at -20°C and measured using the non-acidification method with a Turner Designs
10-AU
TM
fluorometer after 24 h (Welschmeyer 1994).
Elemental stoichiometry. Elemental ratios were obtained by measuring particulate organic
carbon and nitrogen (POC and PON), particulate organic phosphorus (POP) and biogenic silica
(BSi). Using these measurements, cellular contents of C, N, P, Si were calculated based on cell
counts. For particulate organic carbon and nitrogen (POC and PON), 100 ml was filtered onto
pre-combusted (500 °C, 6-8 h) GF/F filters, which were then wrapped in aluminum foil and dried
at 55 °C. POC and PON were analyzed on a Costech Elemental Analyzer using methionine and
atropine as references to calibrate the system at the beginning and between measurements of
every twelve samples (Fu et al. 2007).
For particulate organic phosphorus (POP) samples, 30 ml was filtered onto pre-combusted
(500°C, 6-8 h) GF/F filters and rinsed twice with 2 ml 0.17 mol L
-1
Na2SO4 solution. The filters
were placed in 20 ml borosilicate scintillation vials (pre-combusted at 500°C, overnight) to
which was added 2ml 0.017 mol L
-1
MgSO4 solution. The vials were then covered with
aluminum foil and dried at 95 °C, followed by combustion at 450-500 °C for 2 h. After cooling
to room temperature, 5 ml of 0.2 mol L
-1
HCl solution was added to each vial, which were then
tightly capped and heated at 80°C for thirty minutes to digest POP into inorganic phosphate. The
standard molybdate colorimetric method was used to analyze the samples (Solorzano and Sharp
1980; Fu et al. 2005, 2007). Three GF/F filters were treated in the same way as the samples for
blank determinations.
For biogenic silica (BSi) analyses, a 30 ml sample was filtered through a 2.0-μm
polycarbonate membrane and dried at 95°C. Biogenic Si was dissolved with NaOH in a boiling
water bath, neutralized with HCl solution, and the resulting silicate concentration was measured
colorimetrically (Nelson et al. 1995).
C fixation and Fe uptake. To measure uptake rates of carbon and iron, 0.2 μCi
14
C-
NaH
14
CO3 or 0.2 μCi
55
Fe-
55
FeCl3 was added to 30 ml subsamples from each replicate (specific
activity for final solutions was roughly 0.25 kBq/ml; PerkinElmer). The background dissolved
inorganic carbon in the medium was determined by DIC measurements. In the final solution with
radioactive Fe, the non-radioactive background Fe was 450 nmol/L while radioactive Fe added
was ~70 nmol/L. Samples were then incubated for 24 h under their respective experimental
conditions, and filtered onto GF/F filters. To correct for filter adsorption, 30 ml of cultures from
39
each treatment (10ml from each replicate bottle) was filtered immediately after adding equal
amounts of NaH
14
CO3 or
55
FeCl3. All filters were rinsed with chelexed seawater. For Fe uptake
measurements, cell surface-adsorbed Fe was removed using 0.1 mol/L oxalate reagent (pH ~ 8.0)
for 5-10 min before rinsing (Tovar-Sanchez et al. 2003). Filters were then placed in 7 ml
scintillation vials in the dark overnight after adding 4 ml scintillation fluid. To determine the
total radioactivity (TA), 1 μCi
14
C-NaH
14
CO3 together with 100 μl Phenylalanine or 1 μCi
55
Fe-
55
FeCl3 was placed in identical scintillation vials with the addition of 4 ml scintillation solution.
14
C and
55
Fe radioactivity were measured using liquid scintillation counting (Perkin Elmer) for
TA, blanks and samples (Xu et al. 2014).
2.2.3 Statistics
SigmaPlot 12.5 software was used to calculate the maximum growth rates (μmax) and half
saturation constants (K1/2). To test the interactive effects among the nitrate, CO2 and temperature
treatments, Univariate Analysis of Variance (three-way ANOVA) was employed with SPSS
Statistics 23.0. Two-way ANOVA was applied when examining the interactions among CO2 and
temperature combinations while one-way ANOVA was used to analyze the different responses
of Coscinodiscus sp. to a series of nitrogen concentrations under each CO2 and temperature
treatment. The Tukey multiple comparison test was used for comparisons between individual
treatments. All significance testing was done at the p < 0.05 level. We also noted it when p <
0.01.
2.3 Results
2.3.1 Maximum growth rates (μmax) and half saturation constants (K1/2)
Temperature, CO2 levels, and nitrate concentrations all regulated the growth rates of
Coscinodiscus sp. (Fig. 1). The values of maximum growth rates (μmax) and half saturation
constants (K1/2) relative to nitrate concentrations for the four temperature/CO2 treatments are
presented in Table 2. No results are reported for nitrate concentrations of 1 and 2 μmol/L at
20°C, as after three trials Coscinodiscus sp. was found to be unable to grow in these two lowest
nitrogen treatments at the warmer temperature. Cell sizes of diatoms in the four temperature/CO2
treatments were also determined for three nitrate concentrations (5, 50, 100 μmol/L) and no
significant difference was found among treatments (one-way and two-way ANOVA, p > 0.05;
Supplementary Table S1). Cell count data that were used to verify the growth rates and the initial
and final cell numbers of the last dilution are presented in Supplementary Table S2.
40
As expected, nitrogen availability regulated the growth rates of Coscinodiscus cells under all
four combinations of temperatures and CO2 levels, and specific growth rates at nitrogen
concentrations ≤5 μmol/L) were always significantly lower than those at 50 and 100 μmol/L
(one-way ANOVA and multiple comparisons, p < 0.05). Specific growth rates increased along
with incremental increases of nitrate concentrations, and reached maximal growth rates (μmax) at
nitrate concentrations >50 μmol/L at 20°C and >30 μmol/L at 16°C (Fig. 1).
Both μmax and K1/2 positively correlated with rising temperatures and elevated CO2
concentrations (Table 2). At both CO2 levels, the μmax and K1/2 of Coscinodiscus at 20°C were
significantly higher than at 16°C (two-way ANOVA and multiple comparison, p < 0.01), while
CO2 concentrations did not have significant impacts on μmax at either temperature (p > 0.05). K1/2
values were more sensitive than μmax to CO2 levels under warmer conditions. At 20°C, the K1/2
value of Coscinodiscus at high CO2 was significantly higher than at ambient CO2 (p < 0.05). The
ratio of μmax to K1/2 was also calculated for each temperature/CO2 combination to estimate the
simultaneous effects of both of these kinetic parameters on growth, as either increases in μmax or
decreases in K1/2 can act to increase growth affinities for nitrate (Table 2). The μmax/K1/2 ratios in
the two CO2 levels at 20°C were significantly lower than at 16°C (two-way ANOVA and
multiple comparison, p < 0.01) while the ratio in the 16°C/high CO2 treatment was higher than in
the 16°C/ambient CO2 condition (p < 0.01). Both temperature and CO2 were found to play
significant roles in the μmax/K1/2 ratio (two-way ANOVA, p < 0.01).
2.3.2 Chl a contents
Similar to specific growth rates, cellular Chl a contents increased with nitrate concentrations
from limiting to replete status (Fig. 2). Specifically, under all combinations of temperatures and
CO2 levels, the cellular Chl a content under nitrogen-limiting conditions (5 μmol/L) was
significantly lower than under nitrogen-replete conditions (50 and 100 μmol/L) (One-way
ANOVA and multiple comparisons, p < 0.05).
At most nitrate concentrations, there were no significant differences in cellular Chl a among
the four temperature/CO2 treatments (Fig. 2). However, at a nitrate concentration of 100 μmol/L,
cellular Chl a content at 20°C/high CO2 level was significantly higher than at 20°C/ambient CO2
(p < 0.05) and the other two treatments at 16°C (p < 0.01, multiple comparisons), suggesting a
possible synergistic effect of high CO2, warming, and nitrogen availability on this photosynthetic
pigment.
41
2.3.3 Carbon fixation rates and iron uptake rates
The carbon fixation rates of Coscinodiscus sp. increased with incrementally increasing
nitrate concentrations under the four temperature/CO2 treatments (Fig. 3A). The carbon fixation
rates in nitrogen-replete treatments (at nitrate concentrations of 50 and 100 μmol/L) were
significantly higher than in nitrogen limiting treatments (one way ANOVA and multiple
comparisons, p < 0.01) under each temperature/CO2 combination.
At most nitrogen concentrations, the carbon fixation rates at the high CO2 level were higher
than at ambient CO2. However, the difference between two CO2 levels was not always
significant due to the relatively large error bars. From nitrogen limiting to replete status, the
influence of CO2 levels on carbon fixation rates also shifted. At a nitrate concentration of 5
μmol/L, there was no significant difference in carbon fixation rates among the four treatments.
However, at 50 μmol/L and 100 μmol/L, the rates under 20°C/high CO2 were significantly higher
than under 20°C/ambient CO2 (Two-way ANOVA and multiple comparisons, p < 0.05) and the
rates under 16°C/high CO2 were significantly (p < 0.05, at 50 μmol/L) or slightly (at 100
μmol/L) higher than at 16°C/ambient CO2, showing the contribution of elevated CO2 to
increasing carbon fixation rates under nitrogen-replete growth conditions.
Fe uptake rates appeared to negatively respond to rising CO2 levels (Fig. 3B). At a nitrate
concentration of 100 μmol/L Fe uptake rates under 20°C/ambient CO2 were significantly higher
relative to 16°C /high CO2 treatments (Two-way ANOVA and multiple comparisons, p < 0.05).
At the other nitrogen concentrations, the difference among different CO2 and temperature
combinations was not significant.
However, Fe: C uptake ratios responded positively to lower nitrate levels, lower CO2
concentrations and warming (Fig. 3C). With the increase of nitrogen concentrations, Fe: C ratios
showed a declining trend. Under each temperature/CO2 combination, the differences in Fe: C
ratios among nitrate concentrations were all highly significant (one-way ANOVA, p < 0.01). The
ratios in nitrogen-replete treatments (50 and 100 μmol/L) were significantly lower than in
nitrogen-limited treatments (5 μmol/L) (multiple comparisons, p < 0.01) while there was no
significant difference in Fe: C ratios between the two nitrate-replete concentrations (50 and 100
μmol/L) for the four temperature/CO2 combinations.
At nitrate concentration of 5 and 100 μmol/L, Fe: C uptake ratios at 20°C/ ambient CO2
were significantly higher than all the other three combinations of CO2 and temperature (two-way
42
ANOVA and multiple comparisons, p < 0.05). At 50 μmol/L, the ratios in the 20°C/ambient CO2
treatment were significantly higher than in the 16 °C/high CO2 treatment (p < 0.05).
Interestingly, at 50 μmol/L, only CO2 was responsible for the difference (two-way ANOVA, p <
0.05) while at 5 and 100 μmol/L, the contribution of either CO2 or temperature was extremely
significant (p < 0.01).
2.3.4 Elemental stoichiometry
Although there were no significant impacts of temperature and CO2 level on cellular carbon
and nitrogen contents (Supplementary Fig. S1A, B) in this study, higher temperature decreased
cellular P and Si contents (Fig. 4A, B). Consequently, elemental ratios involving these two
elements were shifted by warming. Moreover, C: N ratios declined due to the increased cellular
N content with increasing nitrate concentrations (Supplementary Fig. S1C).
Cellular P content at 16°C/ambient CO2 was always significantly higher than in the
20°C/ambient CO2 treatment (two-way ANOVA and multiple comparisons, p < 0.05) at nitrate
concentrations of 5, 50 and 100 μmol/L. At three nitrate concentrations, rising temperature
contributed to the decline of cellular P content (two-way ANOVA, p < 0.05 at 50 μmol/L and p <
0.01 at 5 and 100 μmol/L), but the interaction between temperature and CO2 levels was also
significant for the 100 μmol/L nitrate concentration (p < 0.05).
For cellular Si content, the situation was more complicated. The responses of Si content of
Coscinodiscus to rising temperatures and CO2 seemed to be weakened by increasing nitrate
concentrations. No significant differences were observed at 100 μmol/L, while at nitrate
concentrations of 5 and 50 μmol/L, the differences in cellular Si contents among the four CO2
and temperature combinations were significant (two-way ANOVA, p < 0.01). At a nitrate
concentration of 5 μmol/L, cellular Si contents at 16°C/ambient CO2 significantly exceeded all
the rest of the CO2 and temperature combinations, while significant differences were only found
between 20°C/ambient CO2 and 16°C/high CO2 at 50 μmol/L. These differences were caused by
both temperature (p < 0.01) and CO2 (p < 0.05) at the nitrate concentration of 5 μmol/L, and by
temperature at 50 μmol/L (p < 0.01).
Due to the changes in cellular P and Si contents, a series of elemental ratios was shifted (Fig.
4). At 16°C, N: P ratios ranged from 9.57 to 11.62, well below the Redfield ratio of 16 (Redfield
1958), while at 20°C the ratios were higher at all three nitrate levels (Fig. 4C). At 100 umol/L at
20°C, N: P ratios were 15.00 to 16.13, close to 16. At nitrate concentrations of 5 and 100, the
43
difference in N: P ratio was significant among the four CO2/temperature combinations, and was
brought about by temperature difference (two-way ANOVA, p < 0.01 at 5 μmol/L; p < 0.05 at
100 μmol/L). The results of one-way ANOVA demonstrated that nitrogen availability is not a
key factor for the N: P ratio in this study, since no significant difference was found among the
different nitrate concentrations under each temperature/CO2 combination.
Si: C ratios showed a pattern opposite to N: P ratios in response to temperature conditions
(Figure 4D). At 16°C, Si: C ratios ranged from 0.067 to 0.107, while at 20°C the ratios are from
0.040 to 0.069. At nitrate concentrations of 5, 50 and 100 μmol/L, the difference in Si: C was
significant among the four temperature/CO2 combinations (two-way ANOVA, p < 0.01 at 5
μmol/L; p < 0.05 at 50 and 100 μmol/L). Warming contributed to the decline of Si: C ratio in a
significant way (p < 0.01) at all the three concentrations, while the effects of CO2 were
significant at nitrate levels of 5 μmol/L (p < 0.01) and 50 μmol/L (p < 0.05).
2.3.5 Interactive effects
The interactions between temperature and CO2 at fixed nitrate concentrations are presented
above, together with the individual effects of the three factors. The interactive effects among
nitrate concentrations, temperature increase and CO2 levels on growth rates and physiological
parameters were also examined with Univariate Analysis of Variance analysis, and the
significance of the interactions is shown in Table 3. Since the calculations of μmax and K1/2 value
involved nitrate concentration, only the interaction between temperature and CO2 was tested with
two-way ANOVA. For other parameters, interactions of the three factors were analyzed with
three-way ANOVA.
For both μmax and K1/2 values, the interaction of warming and rising CO2 was not significant
(p > 0.05). However, there was a significant interaction of temperature and CO2 on the μmax/K1/2
ratio (p < 0.05). When comparing the specific growth rates of all treatments, temperature and
nitrate concentration had significant interactive effects (three-way ANOVA, p < 0.01).
Moreover, significant interactions between nitrate availability and temperature or CO2 levels
were also observed in the responses of cellular Chl a and Si contents, Fe: C, PON: POP and BSi:
POC ratios (p < 0.05 or p < 0.01), while interactions between temperature and CO2 levels were
only found to be significant for cellular phosphorus contents (p < 0.05).
2.4 Discussion
44
Our results suggest that warming, elevated CO2 concentrations, changing nitrate availability
and their mutual interactions can play critical roles in the growth and physiology of
Coscinodiscus sp., and their importance varies among different parameters. It is also important to
note that while we examined nitrate effects over a wide range of concentrations from severely
limiting to replete, we only investigated interactions using two environmentally-relevant levels
of temperature and CO2; results would undoubtedly vary if different levels of the two climate
change variables had been chosen. Although the growth of Coscinodiscus sp. was regulated by
all three factors, warming and nitrate availability were more influential than the impacts of
elevated CO2, supporting our first hypothesis. Warming shifted elemental ratios significantly,
while carbon fixation rates were only altered by CO2 and nitrate concentrations. Since nitrate
availability had an overwhelming role in determining most parameters, we discuss the effects of
nitrate first and then focus on the individual and interactive effects of temperature and CO2 on
each parameter.
The importance of the nutrient nitrogen (usually in the form of nitrate or ammonium) for
phytoplankton growth has been described by much previous research (Hecky and Kilham 1988;
Beardall and Giordano 2002; Capone et al. 2008). Our results are consistent with these studies, in
that C: N ratios decreased with increasing nitrate availability (Supplementary Fig. S1C). In some
cases, the importance of supplies of nutrients such as nitrogen can overwhelm the effects of
temperature. In the recent field studies of Marañón et al. (2014), growth rates of marine
phytoplankton in polar, temperate and tropical areas were largely influenced by resource supply
instead of temperature. In our studies, growth rates of Coscinodiscus from nitrate limited (5
μmol/L) to replete (50 and 100 μmol/L) status increased by ~200% and ~300% at 16 and 20°C,
respectively. In contrast, the largest increases in growth and carbon fixation rates that we
observed between different temperatures or CO2 treatments were roughly 30% - 40%. In this
sense, our results may provide another example supporting the conclusions of Marañón et al.
(2014).
2.4.1 Effects of temperature and CO2 on growth and half saturation constants
In this study, μmax and K1/2 were calculated with the Monod equation (Monod 1949),
using the initial nitrate concentration. We assume that the nutrient concentration underwent
minor serrated fluctuations due to the nutrient addition and uptake process, especially for nitrate-
limited treatments (1-10 μmol/L). To avoid nitrate depletion in nitrate-limited treatments, we
45
employed the semi-continuous incubation method and diluted cultures frequently (every two
days), as well as keeping biomass as low as practical while still meeting our sampling needs
(Supplementary material Table 2). In this sense, the growth rate in the dilution interval is
relatively stable and can be accepted as the growth rate at the initial nitrate concentration. Based
on this assumption, we discussed the results of μmax and K1/2 as follows.
Rising temperature, elevated CO2 and high nitrate concentrations all enhanced the growth of
Coscinodiscus sp.. Among the three factors in this case, temperature increase and nitrate
availability appeared to play significant roles in promoting growth, while rising CO2
concentrations contributed only slightly, again supporting our original hypothesis. The effects of
nitrate on phytoplankton growth are discussed above. It has long been recognized that warming
also stimulates phytoplankton, within thermal limits that vary according to species (Eppley 1972;
Goldman and Carpenter 1974; Montagnes and Franklin 2001). In the study of Montagnes and
Franklin (2001), the maximum growth rates of most diatom species examined showed a linear
increase in response to temperature increases until their upper temperature thresholds were
reached, and the growth rate of the Coscinodiscus sp. strain they examined was maximal at
16°C. Nishikawa et al. (2010) found that 20°C was the optimal temperature of Coscinodiscus
wailesii isolated from the eastern part of the Seto Inland Sea and maintained at 15°C. In our
study, we set the temperature at 16°C and 20°C based on the seawater temperature (16°C) of the
original isolation site of this strain. The results demonstrated the stimulatory effects of warming
across this range, indicating that the warming condition (20°C) we used in this study did not
exceed the upper thermal threshold of this isolate.
The effects of rising CO2 levels in phytoplankton growth have also been investigated by
numerous studies. Most of the studies suggested that elevated CO2 concentration might enhance
the growth of phytoplankton by reducing carbon limitation of photosynthesis, especially for
species with inefficient CCMs such as coccolithophores and chrysophytes (Riebesell et al 1993;
Schippers et al. 2004; Low-Décarie et al. 2014). However, research on the responses of diatoms
to rising CO2 has shown a host of contradictory results. In several studies, diatoms were thought
to have highly efficient CCMs, indicating possibly negligible effects of future elevated CO2 on
this group (Riebesell 2004; Reinfelder 2011). Other studies have proposed that large diatoms
such as Coscinodiscus might be exceptions, due to a greater potential for diffusion limitation of
CO2 uptake (Tortell et al. 2008). Trimborn et al. (2013) investigated the effects of different CO2
46
levels (160, 390 and 1000 ppm) on multiple phytoplankton species, and surprisingly found that
although the diatoms Chaetoceros debilis and Pseudo-nitzschia subcurvata both had efficient
CCMs, growth of the former species was significantly stimulated by increased CO2 levels while
the growth rates of the latter remained constant. These contradictions in previous studies imply
that diatoms respond to CO2 in various ways, and CCM changes may not be the only relevant
factor. In our study, the maximum growth rates of Coscinodiscus sp. at the higher CO2
concentration were slightly but not significantly higher than at the lower CO2 level, which seems
to support the prediction of Riebesell (2004).
The K1/2 value is an important indicator of the ability to grow under low nutrient conditions
(Eppley et al. 1969). A higher K1/2 value suggests a requirement for higher external nutrient
concentrations to maintain growth. Several previous studies reported the increase of K1/2 for
nutrients with increasing temperature. Tilman et al. (1981) showed that K1/2 of Asterionella
formosa for uptake of silicate increased sharply for temperatures above the optimal temperature.
Nishikawa et al. (2009; 2010) examined the affinity of Coscinodiscus wailesii and Eucampia
zodiacus for nitrate and phosphate uptake at two temperatures (9°C and 20°C). For both species,
the K1/2 for both nitrate and phosphate were higher at 20°C than at 9°C. Notably, the K1/2 in
Nishikawa et al. (2009, 2010) was calculated based on the uptake rates for nitrate. In their
research, 20°C was also the optimal temperature for the growth of Coscinodiscus wailesii. In our
study, K1/2 values relative to growth were significantly increased by the 4°C temperature increase
and by elevated CO2 concentration at 20°C, and also corresponded to higher growth rates. The
μmax/K1/2 ratio also indicated a higher requirement of Coscinodiscus sp. for nitrate under
warming conditions and high CO2 levels. This observation may explain the inability of
Coscinodiscus to survive at the lowest nitrate concentrations of 1 and 2 μmol/L at 20°C.
Although we did not determine the biochemical basis of this response, in general rapidly
growing phytoplankton cells need more nitrogen to synthesize proteins, nucleic acids,
chlorophyll and other biomolecules, resulting in elevated nitrogen requirements (Geider and La
Roche 2002).
2.4.2 Effects of nitrate availability on Chl a content
In our study, cellular Chl a did not show significant responses to warming and elevated
CO2 levels at most nitrate concentrations. However, it increased significantly at the highest
nitrate concentrations. Chlorophyll represents a considerable pool of N in phytoplankton cells
47
(Geider and La Roche 2002; Arrigo 2005), with the photosynthetic apparatus in diatoms
accounting for 15-25% of cellular N (Li et al. 2015), so it is reasonable to assume that cellular
Chl a benefits from higher nitrate availability. Under climate change scenarios, cellular Chl a
content of Coscinodiscus may decrease due to more nutrient-limiting conditions in the future.
2.4.3 Responses of carbon fixation rates and iron uptake rates to the three variables
Increased phytoplankton carbon fixation under elevated CO2 concentrations could intensify
the marine biological carbon pump in the future, thus mitigating climate changes by sequestering
more carbon from the atmosphere to the deep ocean (Tortell et al. 2008; Losh et al. 2012; Song
et al. 2014). According to previous studies, though, the responses of diatom carbon fixation to
climate change factors appear to be different among species and communities (Tortell and Morel
2002; Tortell et al., 2008; Hare et al. 2007). For some diatoms such as Phaeodactylum
tricornutum, Thalassiosira weissflogii (Burkhardt et al. 2001), and Skeletonema costatum (Rost
et al. 2003), carbon fixation rates were unresponsive to elevated CO2 levels, implying that carbon
assimilation of these species may be saturated at current CO2 concentrations (Riebesell 2004;
Beardall et al. 2009). Diatom-dominated assemblages from the California Upwelling showed a
lower carbon uptake rate at 750 ppm CO2 than at 150 ppm, due to the down-regulation of CCMs
(Tortell and Morel 2002). However, in another study Tortell et al. (2008) found stimulating
effects of increasing CO2 levels (from 100 ppm to 800 ppm) for carbon fixation rates of diatom-
dominated Southern Ocean assemblages, especially for large diatoms. In our study, the carbon
fixation rates were positively related to elevated CO2 levels under nitrate-replete conditions, but
were not significantly enhanced under nitrate-limitation.
Considering the important role of nitrogen in CCMs (Beardall and Giordano 2002), it is
plausible that the operation of CCMs was inhibited by the nitrate-limiting conditions, leading to
significantly lower carbon fixation rates and inconspicuous impacts of CO2 levels. Conversely,
with sufficient nitrogen supply for CCMs relatively large diatoms like the Coscinodiscus sp. in
our study are more likely to be limited by inorganic carbon availability (Riebesell et al. 1993;
Tortell et al. 2008). Hence a reasonable explanation for our results is that under nitrate-
limitation, carbon fixation rates of Coscinodiscus sp. were simultaneously limited by nitrate and
CO2, while increased CO2 levels enhanced carbon fixation with sufficient nitrate supply. Thus,
different responses of diatom carbon acquisition to climate change may be attributed to different
48
levels of nutrient supplies or other environmental factors, although the mechanisms involved still
require further investigation.
Unlike carbon fixation rates, Fe uptake rates did not respond consistently to nitrate
concentrations, CO2 concentrations and temperatures. The only significant difference in Fe
uptake rates was found between the 20°C/ambient CO2 and 16°C/high CO2 treatments at a nitrate
concentration of 100 μmol/L. This implies that Fe uptake rate was stimulated by the interaction
of warming and low CO2 under nitrate replete conditions.
As a result of shifting carbon and iron uptake rates between different treatments, Fe: C
uptake ratios showed an inverse correlation with high nitrate concentration, high CO2 and low
temperature. It is clear that the highly significant increase of Fe: C ratios at lower nitrate levels (5
μmol/L compared to 50 and 100 μmol/L) was mainly caused by the lowered carbon fixation rates
under nitrate-limiting conditions. Interestingly, at each of the three nitrate concentrations (5, 50
and 100 μmol/L), the Fe: C ratios showed the same treatment-related trends (from high to low):
20°C/ambient CO2, 16°C/ambient CO2, 20°C/high CO2, 16°C/high CO2. It appeared that higher
CO2 concentration decreased the Fe: C ratio, while warming elevated it. The effects of elevated
CO2 levels on the Fe: C ratio could be attributed to the corresponding increase in carbon fixation
rates and slight decrease of iron uptake, suggesting that higher diffusive CO2 availability reduced
the requirement for Fe to support carbon fixation processes such as carbon concentrating
mechanisms. On the other hand, increases in Fe: C ratios following warming may be caused by
an increased demand for Fe to support higher nitrogen requirements at more rapid growth rates,
considering the central role of Fe in nitrate uptake and assimilation (Milligan and Harrison 2000;
Franck et al. 2003).
Much previous research has demonstrated the important role of Fe in promoting the growth
and carbon sequestration of phytoplankton (Watson et al. 2000, Pollard et al. 2009), but Fe: C
ratios have been rarely examined relative to climate change variables (Hutchins and Boyd 2016).
Sunda and Huntsman (2011) found that intracellular Fe: C ratios of the diatom Thalassiosira
pseudonana (in contrast to the Fe: C uptake ratios measured in our study) decreased under
warmer conditions, suggesting a reduced requirement for Fe at high temperature. Xu et al. (2014)
found that the Fe: C uptake ratio of the Antarctic phytoplankton species Phaeocystis antarctica
(a prymnesiophyte) and Fragilariopsis cylindrus (a diatom) increased under a combination of
higher irradiance, temperature, and CO2 levels, suggesting a higher Fe requirement relative to C
49
requirements in the future. Our results do not fully agree with either study above, perhaps due to
taxonomic differences or to different experimental designs in the combinations of environmental
variables. More research is needed to understand the cellular mechanisms involved, and to
predict trends in C and Fe uptake and Fe use efficiencies of phytoplankton in the future ocean.
2.4.4 Cellular C, N and P contents and stoichiometry under three variables
Numerous studies have found that climate-change factors not only affect the growth rates
of phytoplankton, but also can potentially shift their cellular elemental contents and
stoichiometry (Burkhardt and Riebesell 1997; Hutchins et al. 2009; Spilling et al. 2015). The
effects of CO2 impacted elemental stoichiometry in diverse ways. Riebesell et al. (2007) found
that one natural phytoplankton community in a mesocosm experiment consumed up to 27% and
39% more dissolved inorganic carbon respectively at 700 and 1050 ppm compared to 350 ppm,
while nutrient uptake was relatively stable. Consequently, C: N ratios increased from 6.0 at 350
ppm CO2 to 7.1 at 700 ppm and 8.0 at 1050 ppm CO2 (Riebesell et al. 2007; Bellerby et al.
2008). Similarly, Losh et al. (2012) demonstrated that the C: N ratios of N-limited, diatom-
dominated California Current communities increased with elevated CO2 (150 - 900 ppm) due to
increased POC production but relatively unchanged PON content. In contrast, Reinfelder (2012)
tested three diatom species (Phaeodactylum tricornutum, Thalassiosira pseudonana and
Thalassiosira weissflogii) and found that C: N, C: P, and N: P ratios were all elevated with rising
CO2 levels (from 150 ppm to 1500 ppm), due to a decline in cellular nitrogen and phosphorus
content relative to carbon quotas. Li et al. (2015) found that higher growth rates of Thalassiosira
pseudonana at elevated CO2 were associated with a lower requirement for N in the
photosynthetic apparatus. Thus, previous research seems to suggest that the mechanisms behind
the altered phytoplankton elemental stoichiometry under elevated CO2 may vary with taxonomic
affiliation or community composition, as well as due to changing nutrient conditions.
Although the effects of varying CO2 levels on elemental stoichiometry of phytoplankton
have been investigated by a myriad of studies, less research has focused on the influence of
warming. Spilling et al. (2015) analyzed the interactive effects of temperature, light and nutrient
limitation on stoichiometry and found low temperature and light conditions lowered the C: P and
N: P ratio, although the sole role of temperature was not statistically significant. Tew et al.
(2014) also found that the N: P ratio of the diatom Amphora coffeaeformis was not significantly
different at high temperature (31°C compared to 28°C).
50
Unlike many of the studies cited above, cellular carbon and nitrogen contents did not vary
significantly between different CO2 and temperature treatments in our study, while C: N
decreased due to the increased N content under nitrate-replete conditions (Supplementary
Fig.S1). However, phosphorous content declined at higher temperature (20°C), and consequently
elemental ratios involving this element were altered by temperatures. We observed significantly
lowered C: P and N: P at 16°C compared to 20°C, implying an important effect of warming in
shifting elemental composition in our study. Similar results have been obtained in several
previous studies (Hutchins and Boyd 2016). Based on molecular physiology and biochemistry
experiments with the diatoms Fragilariopsis cylindrus and Thalassiosira pseudonana, Toseland
et al. (2013) built a phytoplankton cell model that revealed the potential effects of temperature on
resource allocation and elemental stoichiometry of phytoplankton. Their study suggested that at
low temperature, phosphorus-rich ribosomes and associated rRNAs are likely to increase while
cellular protein synthesis would decrease, resulting in lowered C: P and N: P ratios in
phytoplankton cells. This pattern has also been observed in the diatoms Amphora coffeaeformis
(Tew et al. 2014), Chaetoceros wighamii (Spilling et al. 2015), and Pseudo-nitzschia subcurvata
(Boyd et al. 2016), and would appear to be a general trend at least among many diatoms
(Hutchins and Boyd 2016).
2.4.5 Cellular Si contents and stoichiometry under three variables
Considering the central role of silicifying diatoms in the biogeochemical cycle of silicon, it
is requisite to investigate the response of their silicification processes to climate changes.
According to the Redfield-Brzezinski nutrient ratio, the classic Si: C is 15:106, or 0.141.
However, the Si: C ratios in this study were well below the classic ratio at 16°C, and even lower
at 20°C, suggesting a “limiting” status of Si relative to carbon in this study, especially at the
higher temperature (Redfield 1958; Brzezinski 1985). Only a few previous studies have
presented results for diatom Si quotas and Si: C ratios under warming (Paasche 1980) or ocean
acidification (Milligan et al. 2004; Tatters et al. 2012; Mejía et al. 2013). Paasche (1980)
examined the response of five diatom species to a temperature range from 8 to 23°C, and found
that the cellular silicon content of Rhixosolenia fragilissima and Chaetoceros affinis dropped
with increasing temperature, while that of the other three species (Cerataulina pelagica,
Thalassiosira pseudonana, Skeletonema costatum) did not show a clear trend with temperature.
With respect to ocean acidification, studies have showed declining Si quotas and Si: C ratios at
51
elevated CO2 levels or lower pH in Pseudo-nitzschia fraudulenta (800 ppm CO2 versus 200 ppm,
Tatters et al. 2012), Thalassiosira pseudonana (pH 8.63 compared to 7.41, Mejía et al. 2013),
and Thalassiosira weissflogii (pH 8.33 compared to 7.44, Milligan et al. 2004). Hervé et al.
(2012) found that frustule-bound Si content and Si quota of Thalassiosira weissflogii reached the
lowest point at pH 7.8 (corresponding to 730-770 ppm CO2), and increased at more acidic or
basic pH values.
An inverse relationship has been discovered between the growth rates and Si contents of
multiple diatom species, although exceptions exist (Martin-Jézéquel et al. 2000; Hervé et al.
2012; Mejía et al. 2013). Our results examining silicification in Coscinodiscus sp. are in good
agreement with this relationship. At higher temperature (20°C) and elevated CO2 levels, both
cellular Si quota and Si: C declined with increasing growth rates. One possible explanation for
this pattern is that more incorporation of silicon into the diatom frustule occurs at low growth
rates (Martin-Jézéquel et al. 2000). However, the responses of silicon content to multiple
simultaneous climate change variables have not been well established for diatoms, and this is an
area that needs further study.
2.4.6 Interactive effects of warming, CO2 and nitrate availability
As discussed above, warming, CO2 concentration and nitrate availability each could solely
regulate the growth and/or other physiological parameters of Coscinodiscus sp. in our study.
However, the interactions among the three factors seem to be far more complicated, and may be
far more relevant for the multi-factorial shifts occurring in a changing ocean (Hutchins and Fu
2017).
Since warming and rising CO2 are two important climate change variables, several previous
studies investigated their interactions along with their individual effects on marine
phytoplankton. The responses of phytoplankton groups and communities to their interactions
seem to vary (Fu et al. 2007; Hutchins et al. 2007; Feng et al. 2008), and no unambiguous
conclusions can be made about the interactive effects of temperature increase and CO2 addition
on biogeochemical cycles (Gao et al. 2012a). Recently, Tatters et al. (2013) found interactive
effects of warming with CO2 addition in shifting community structure during natural community
and artificial community competition experiments with diatoms. Coello-Camba et al. (2014)
found weak antagonistic interactions between the two variables on biomass, but synergistic
interactions on primary production of an Arctic phytoplankton community. In our study, no
52
interaction of warming and CO2 addition was found on μmax and K1/2 values, hence the promoting
effects of the two variables seemed to be additive. This, our second hypothesis was not supported
for these two parameters. However, significant interactions between temperature and CO2 levels
were observed for the μmax/K1/2 ratio and cellular phosphorus contents. This result on one hand
implies that in the future, the requirement of Coscinodiscus sp. for nitrate may be influenced by
both the independent and cumulative effects of warming and rising CO2 concentrations,
supporting our second hypothesis. On the other hand, under future climate change scenarios, the
interactive effects of temperature and rising CO2 could also shift cellular P contents and
corresponding elemental stoichiometry by reducing the strong impacts of warming, going against
our second hypothesis.
Furthermore, the interactions of nitrate with other variables were found to be significant for
the responses of multiple parameters, including growth rates, cellular Chl a and Si contents, Fe:
C, N: P, and Si: C ratios. These results further reveal the importance of nitrate availability on the
parameters above, and can help us to predict the specific responses of Coscinodiscus sp. in the
future ocean with expected reductions in nitrate supplies to the euphotic zone.
Both temperature and nitrate supply play important roles independently for the growth and
physiology of phytoplankton. However, there are surprisingly few studies on their interactions
(Thomas and Litchman 2016). In this sense, our results provide new evidence of the interactions
between these two major influences on diatom growth and stoichiometry. In this study, nitrate
concentration and warming had significant interactive effects on specific growth rates, cellular
Chl a content and N: P ratio where interaction between nitrate and CO2 was not found,
supporting our first hypothesis. With sufficient nitrate source both the growth and cellular Chl a
of Coscinodiscus sp. were strongly promoted by temperature increase, while with limiting nitrate
warming had negative effects, supporting the second hypothesis. Considering the prediction of
enhanced nutrient-stress in large areas of the future ocean, the actual effects of warming may
depend on the specific nitrate concentration in the ambient seawater. The incremental changes in
N: P ratio driven by warming are likely to be smaller under nitrate-limiting conditions than
nitrate-replete conditions. In other words, the interactive effects between reduced nitrate
availability and warming weakened the effect of warming, going against our second hypothesis.
Losh et al. (2012) reported that the interactive effects of CO2 with nitrate availability shifted
the C: N ratio of a natural phytoplankton community in the California Current. Verspagen et al.
53
(2014) proposed a hypothesis whereby the interactions of elevated CO2 with different nutrient
levels may result in contrasting effects on phytoplankton biomass and C: nutrient stoichiometry
and confirmed it with a model and experiments with the freshwater cyanobacterium Microcystis
aeruginosa at two CO2 levels and two nitrate concentrations. There are also studies examining
the interactions between CO2 and other nutrients (Feng et al. 2010; Tatters et al. 2012; Maat et al.
2014). However, no studies have examined changes in diatom Si: C ratios when elevated CO2
interacts with changing nitrate concentrations. In our study, Si: C ratios were impacted by the
interaction between CO2 and nitrate, as a result of a shift in cellular Si content. Under nitrate-
limited conditions in the future, the diatom silicification process may decline with rising CO2
levels. Together with the adverse effects of warming on silicification, the overall biological
effects of three climate change variables would be bigger than the sum of their individual effects,
consistent with our second hypothesis.
Another interesting finding in this study is that there was no interactive effect among the
three factors on carbon fixation and iron uptake rates. Instead, both temperature and nitrate and
CO2 and nitrate had strong two-way interactive effects on Fe: C uptake ratios. This result was not
supportive of our first hypothesis, and might be caused by the inherently large impacts of
elevated CO2 concentration as a C source for carbon fixation. Furthermore, under nitrate-limited
conditions, warming and the lower CO2 level had stronger effects by elevating the uptake of Fe
relative to C, supporting our second hypothesis. As discussed in Hutchins and Boyd (2016), there
are evidently complex interactions of warming, rising CO2 and nutrient co-limitation with Fe
biogeochemistry. To better understand these interactions, more research is needed to investigate
the feedbacks between acquisition pathways for Fe and C uptake and the synthesis and export of
molecules involving these two elements.
2.5 Conclusions
Our study revealed the individual and interactive effects of warming, elevated CO2 and
nitrate supply on the growth, carbon fixation rates and elemental stoichiometry of the diatom
Coscinodiscus sp. These results suggested that all three factors regulated the growth and
physiological responses of this species. Temperature increases and elevated nitrate availability
played significant roles in stimulating growth. High temperature also lowered the growth affinity
for nitrate, possibly indicating a more severely nitrate-limited status for Coscinodiscus sp. if
future warming is accompanied by reduced supplies of nitrate. Rising CO2 promoted carbon
54
fixation rates, implying that ocean acidification may provide a negative feedback to increasing
atmospheric CO2 as a carbon sink. Moreover, warming may alter the elemental composition of
phytoplankton. Cellular P and Si content decreased at warmer temperatures in our experiments,
resulting in increased C: P, N: P, and C: Si ratios. Specifically, the silicification processes of this
diatom may be substantially weakened by warming.
Besides the independent effects of these three variables, the interactions among them further
complicate the responses of phytoplankton. Intriguingly, neither of our two hypotheses was fully
supported, based on the results of multiple parameters. For most parameters such as growth,
cellular Chl a content and N: P ratio, the first hypothesis was supported and stronger interactive
effects of nitrate and warming were found compared to the effects between nitrate and CO2,
while for Fe: C ratio, the interactive effects between nitrate and CO2 are at least as important as
interactions of temperature and nitrate. The second hypothesis was supported by the trends in the
μmax/K1/2 ratio, specific growth rates, cellular Chl a contents, Fe: C uptake ratio and Si: C ratio,
indicating that the interactive effects synergistically enlarged the overall biological effects of the
three variables for these parameters. However, for the cellular P content and N: P ratio, the
interactions between warming and elevated CO2 or warming and reduced nitrate seemed to be
antagonistic to the effects of warming. The interactions of nitrate with global change variables
will be especially important to consider in the future ocean. Our research provides further
evidence for such multiple variable responses in the biogeochemically-important diatom
Coscinodiscus, but more investigations will be needed to reveal the full array of biochemical and
regulatory mechanisms behind our observations.

55
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iron availability. Limnology and Oceanography, 59(6), 1919-1931.
Zhu, Z., Xu, K., Fu, F., Spackeen, J. L., Bronk, D. A., & Hutchins, D. A. (2016). A comparative
study of iron and temperature interactive effects on diatoms and Phaeocystis antarctica
from the Ross Sea, Antarctica. Marine Ecology Progress Series, 550, 39-51.

65
Chapter 2-Tables and Figures
Table 1. Seawater carbonate buffer system values- pH, total dissolved inorganic carbon
(DIC) and pCO2 values calculated from the former two parameters. In the text pCO2 values
of 382 to 417 are referred to as “ambient CO2” and values of 792 to 832 are referred to as “high
CO2.”
Treatment pH (NBS) DIC (μmol/kg)
Calculated
pCO2(ppm)
16°C + ambient CO2 8.2 ± 0.04 2060 ± 7 417 ± 42
16°C + high CO2 7.9 ± 0.06 2198 ± 18 832 ±47
20°C + ambient CO2 8.2 ± 0.07 2140 ± 37 382 ± 31
20°C + high CO2 8.0 ± 0.05 2308 ± 11 792 ± 17

66
Table 2. Maximum specific growth rates μmax, half saturation constants K1/2 and μmax/K1/2
ratios of the four CO2/temperature treatments relative to nitrate concentration. The values
in the table are the average values of each triplicated treatment ± standard deviation.
16°C + ambient CO2 16°C + high CO2 20°C + ambient CO2 20°C + high CO2
μmax
(day
-1
)
0.61 ± 0.01 0.63 ± 0.02 0.88 ± 0.04 0.92 ± 0.05
K1/2
(μmol)
2.88 ± 0.35 3.96 ± 0.49 10.37 ± 1.59 12.81 ± 2.26
μmax /K1/2
(day
-1
/μmol)
0.20 ± 0.02 0.16 ± 0.02 0.08 ± 0 0.07 ± 0

67
Table 3. Significance of interactive effects of warming, CO2 and nitrate availability on 12
growth and physiological parameters of Coscinodiscus sp. The values in the table are the p
values of the results of Univariate Analysis of Variance. The symbols * and ** indicate that the
corresponding p value is smaller than 0.05 or 0.01, respectively.

Temperature&
CO2
Temperature &
Nitrate
CO2 &
Nitrate
Temperature &
CO2 & Nitrate
μmax > 0.05 n.a. n.a. n.a.
K1/2 value > 0.05 n.a. n.a. n.a.
μmax/K1/2 ratio < 0.05* n.a. n.a. n.a.
Growth rate > 0.05 < 0.01** > 0.05 > 0.05
Cellular Chl a > 0.05 < 0.01** > 0.05 > 0.05
Carbon
fixation rate
> 0.05 > 0.05 > 0.05 > 0.05
Iron uptake
rate
> 0.05 > 0.05 > 0.05 > 0.05
Fe: C uptake
ratio
> 0.05 < 0.05* < 0.01** > 0.05
Phosphorus
content
< 0.05* > 0.05 > 0.05 > 0.05
PON: POP > 0.05 <0.01** > 0.05 > 0.05
Silicon content > 0.05 > 0.05 < 0.05* > 0.05
BSi: POC > 0.05 > 0.05 < 0.05* > 0.05
n.a.: not applicable.

68
Figure Legends
Figure 1. Relationship between specific growth rates μ (day
-1
) and nitrate concentrations
(μmol/L) in the four temperature / CO2 treatments. Symbols represent values of all three
replicates in each treatment.
Figure 2. Relationships between cellular Chl a content (ng cell
-1
) and nitrate availability in
four temperature / CO2 treatments. Values and error bars respectively represent the means and
the standard deviations of triplicate cell cultures under each treatment. The Arabic numerals and
letters respectively show the significant differences among nitrate concentrations and the
significant differences among the four temperature/CO2 treatments at the 100 μmol/L nitrate
concentration, based on the results of multiple comparisons. No significant differences were
observed among temperature/CO2 treatments at other nitrate concentrations.
Figure 3. Carbon fixation rate (10
-13
mol C/h/cell) (A), Iron uptake rate (10
-17
mol Fe/h/cell)
(B) and Iron: Carbon uptake ratio (10
-3
mol: mol) (C) of Coscinodiscus sp. at three nitrate
concentrations (5, 50 and 100 μmol/L) under four CO2 / temperature treatments. Values
and error bars represent the means and the standard deviations of triplicate cell cultures under
each treatment. The Arabic numerals and letters respectively show the significant differences
among nitrate concentrations and the significant differences among the four temperature/CO2
treatments at each nitrate concentration, based on the results of multiple comparisons.
Figure 4. Cellular P contents (pmol cell
-1
)(A), cellular Si contents (pmol cell
-1
)(B), PON:
POP ratios (mol: mol) (C) and BSi: POC ratios (mol: mol) (D) at three nitrogen
concentrations (5, 50 and 100 μmol/L) in four temperature / CO2 treatments. Values and
error bars represent the means and the standard deviations of triplicate cell cultures under each
treatment. The Arabic numerals and letters respectively show the significant differences among
nitrate concentrations and the significant differences among the four temperature/CO2 treatments
at each nitrate concentration, based on the results of multiple comparisons.
69

Figure 1.

70

Figure 2.

1 2 5 10 30 50 100
16°C + ambient CO
2
16°C + high CO
2
20°C + ambient CO
2
20°C + high CO
2
nitrate concentration (µmol/L)
0
1
2
3
4
5
6
Chl a(pg cell
−1
)
4 4 4 1-2 3 a a a b 1 2-3
71

Figure 3.

16°C + ambient CO
2
16°C + high CO
2
20°C + ambient CO
2
20°C + high CO
2
0
5
10
15
Carbon fixation rate (10
−13
mol C/h/cell)
(A)
0.0
0.5
1.0
1.5
2.0
2.5
Iron uptake rate (10
−17
mol Fe/h/cell)
(B)
5 50 100
Nitrate concentration (µmol/L)
0
20
40
60
80
100
Iron: Carbon uptake ratio (10
−3
mol: mol)
(C)
1 2 2 1 2 2 1 c d b c ef ef e f a a a a b a c ab bc b c bc f f g f de d e de a a a a a a a a
72

Figure 4.

0.00
0.05
0.10
0.15
0.20
(A)
Cellular P contents (pmol cell
−1
)
16°C + ambient CO
2
16°C + high CO
2
20°C + ambient CO
2
20°C + high CO
2
0.0
0.5
1.0
1.5
2.0
Cellular Si contents (pmol cell
−1
)
(B)
5 50 100
0
5
10
15
20
PON: POP (mol: mol)
(C)
Nitrate concentration (µmol/L)
5 50 100
0.00
0.05
0.10
0.15
BSi: POC (mol: mol)
(D)
Nitrate concentration (µmol/L)
2 1 1 b ab a a d cd c cd f ef e ef 2 1 1-2 e e e e cd d c cd b a a a 1 1 1 f f f f de e d de c b ab a e ef ef f d d d d a ab c bc 1 1 1
73
Chapter 2-Supplementary Materials
Table S1. Coscinodiscus sp. cell volume (μm
3
) in the four CO2 / temperature treatments at
three nitrate concentrations (5, 50 and 100 μM). The values in the table are the average values
of each triplicated treatment ± standard deviation.

16°C + ambient
CO2
16°C + high
CO2
20°C + ambient
CO2
20°C + high
CO2
5 μM 1272 ± 682 1411 ± 653 1366 ± 484 1287 ± 447
50 μM
1373 ± 490
1595 ± 750 1369 ± 420 1310 ± 425
100 μM 1455 ± 551 1458 ± 629 1548 ± 651 1372 ± 454

74
Table S2. Coscinodiscus sp. cell density (ml
-1
) in the four CO2 / temperature treatments at
three nitrate concentrations (5, 50 and 100 μM) for the last dilution interval. The values in
the table are the average values of each triplicated treatment ± standard deviation. The values for
Day 0 represent the initial cell number after dilution and the values for Day 2 are the final cell
numbers after 48 hours.
16°C+ambient CO 2 16°C + high CO 2 20°C + ambient CO 2 20°C + high CO 2
5 μM
Day 0 5250 ± 507 5069 ± 543 6090 ± 714 6361 ± 937
Day 2 11431 ± 956 11810 ± 1108 11917 ± 741 14111 ± 994
50 μM
Day 0 6250 ± 507 5642 ± 600 8653 ± 1369 6306 ± 316
Day 2 20131 ± 4146 18139 ± 1385 32778 ± 1974 24708 ± 3068
100 μM
Day 0 4629 ± 1027 4807 ± 84 5789 ± 342 4646 ± 308
Day 2 21000 ± 3041 20428 ± 1060 39299 ± 3522 22972 ± 4882

75
Figure S1. Cellular C content (pmol cell
-1
) (A), N content (pmol cell
-1
)(B), and POC: PON
ratios (mol: mol)(C) at three nitrogen concentrations (5, 50 and 100μM) in four
temperature / CO2 treatments. Values and error bars represent the means and the standard
deviations of triplicate cell cultures under each treatment. The Arabic numerals and letters
respectively show the significant differences among nitrate concentrations and the significant
differences among the four temperature / CO2 treatments at each nitrate concentration, based on
results of multiple comparisons.

Figure S1.

0
5
10
15
20
5 50 100
Cellular C contents
(pmol cell
-1
) 16°C + ambient CO2
16°C + high CO2
20°C + ambient CO2
20°C + high CO2
0
0.5
1
1.5
2
2.5
5 50 100
Cellular N contents
(pmol cell
-1
) 0
2
4
6
8
10
12
14
5 50 100
POC: PON (mol: mol) Nitrate concentraDon (μmol/L) 16°+ ambient CO
2
16°+ high CO
2 20°+ ambient CO
2 16°+ high CO
2 (A) (C) (B) 1 2 2 a a a a a a a a a a a a a a a a a a a a a a a a 1 1 2 2 d d d d a a a a b bc c bc
76
Chapter 3: Distinct Responses of the Nitrogen-fixing Marine Cyanobacterium
Trichodesmium to a Thermally-variable Environment as a Function of
Phosphorus Availability
Pingping Qu
1
, Feixue Fu
1
, Joshua Kling
1
, Megan Huh
2
, Xinwei Wang
3
, David A. Hutchins
1*

1
Department of Biological Sciences, University of Southern California, Los Angeles, California,
USA
2
Department of Preventative Medicine, Keck School of Medicine, University of Southern
California, Los Angeles, California, USA
3
School of Life Sciences, Xiamen University, Xiamen, China
Corresponding author: Dr. David A. Hutchins (Email address: dahutch@usc.edu)
Key words: ocean warming, thermal variation, Trichodesmium, nitrogen fixation, phosphorus
limitation, phosphorus use efficiency, climate change

The manuscript was published in 2019 as:
Qu, P., Fu, F.-X., Kling, J., Megan, H., Wang, X., and Hutchins, D.A. (2019). Distinct responses of
the nitrogen-fixing marine cyanobacterium Trichodesmium to a thermally-variable environment as a
function of phosphorus availability. Frontiers in Microbiology, 10, 1282.
doi: 10.3389/fmicb.2019.01282.

77
Abstract
Surface temperature in the ocean is projected to be elevated and more variable in the future,
which will interact with other environmental changes like reduced nutrient supplies. To explore
these multiple stressor relationships, we tested the influence of thermal variation on the key
marine diazotrophic cyanobacterium Trichodesmium erythraeum GBRTRLI101 as a function of
the limiting nutrient phosphorus (P). Two constant temperature treatments represented current
winter (22°C) and summer (30°C) mean values. Three variable temperature treatments fluctuated
around the constant control values: Mean 22 °C, either ± 2 °C or ± 4°C; and mean 30°C ± 2°C.
Each thermal treatment was grown under both P-replete (10 μmol/L) and P-limiting conditions
(0.2 μmol/L).
Effects of thermal variability on Trichodesmium were mainly found in the two winter variable
temperature treatments (22°C ± 2°C or ± 4°C). P availability affected growth and physiology in
all treatments and had significant interactions with temperature. P-replete cultures had higher
growth and nitrogen and carbon fixation rates in the 22°C constant control, than in the
corresponding variable treatments. However, physiological rates were not different in the P-replete
constant and variable treatments at 30°C. In contrast, in P-limited cultures an advantage of constant
temperature over variable temperature was not apparent. Phosphorus use efficiencies (PUE, mol
N or C fixed
.
hour
-1

.
mol cellular P
-1
) for nitrogen and carbon fixation were significantly elevated
under P-limited conditions, and increased with temperature from 22°C to 30°C, implying a
potential advantage in a future warmer, P-limited environment. Taken together, these results imply
that future increasing temperature and greater thermal variability could have significant feedback
interactions with the projected intensification of P-limitation of marine N2-fixing cyanobacteria.

78
3.1 Introduction
All environmental factors that affect the growth and fitness of organisms vary over time, and
these biological impacts are projected to intensify as environmental variability increases under
global climate change scenarios (Burroughs, 2007; IPCC, 2012; Thornton et al., 2014; Boyd et al.,
2016). In particular, sea surface temperature fluctuations are mainly driven by air-sea heat
exchange and short-wave radiation penetration (Hazeleger and Haarsma 2005; Hosoda et al. 2016;
Schneider and Zhu 1998). In a more stratified warming ocean, an expected shallower upper mixed
layer will become more vulnerable to disturbance and more sensitive to varying air-sea heat fluxes
and solar radiation (Behrenfeld et al. 2006; Boyd et al. 2016; Häder et al. 2011). Consequently,
thermal variability is predicted to increase in the future surface ocean (IPCC 2012).
Global warming effects have been addressed in voluminous studies, but until recently the
influence of changing thermal variability on ecosystems has received relatively little attention.
Several recent studies in terrestrial ecosystems examined the diverse impacts of thermal variation
on ectothermic animals (Bozinovic et al., 2011; Estay et al., 2011; Paaijmans et al., 2013) or soil
bacterial communities (Langenheder et al., 2012).The biological effects of thermal variability have
also been examined in marine metazoans (Dong et al., 2006; Tian and Dong, 2006; Putnam and
Edmunds, 2011; Godbold and Solan, 2013). For phytoplankton, Bernhardt et al. (2018) proposed
a non-linear averaging model based on the principle of Jensen's inequality, suggesting that
temperature fluctuations exceeding the thermal optimum can depress temperature growth response
curves compared to the constant temperature condition.
Most investigations of thermal effects on marine phytoplankton, however, have used constant
experimental temperatures (Nishikawa and Yamaguchi, 2008; Fu et al., 2014; Sommer et al., 2016;
Jiang et al., 2018; Sett, 2018), thus neglecting all natural temperature fluctuations from diel to
seasonal timescales. This use of oversimplified thermal conditions in experimental design has the
potential to lead to biased conclusions and problematic predictions. Thus, there is a need to
investigate the impacts of thermal variability on important phytoplankton groups, especially under
climate change scenarios. In this study, we explored the influence of thermal variation on the
growth and physiology of the diazotrophic cyanobacterium Trichodesmium erythraeum
GBRTRLI101.
As a dominant, globally-distributed nitrogen-fixer in the tropical-subtropical oligotrophic
ocean (Capone et al., 1997; Karl et al., 1997; Bell et al., 1999), Trichodesmium contributes a
79
substantial fraction of marine biological nitrogen fixation and so in turn plays a key role in global
nitrogen and carbon cycling (Karl et al., 2002; Capone et al., 2005; Bergman et al., 2013). The
strain GBRTRLI101 used in this study was isolated in the Northern Great Barrier Reef (GBR)
Lagoon (Fu and Bell, 2003), where frequent Trichodesmium blooms have been observed (Bell et
al., 1999). The annual average sea surface temperature (SST) in this region is 26-27°C (De'ath et
al., 2009) and the annual temperature range is approximately 4°C (Lough, 2007), with a seasonal
SST range of 22-24°C in the winter and 28-32°C in the summer (Lough, 1998). This relatively
stable SST provides optimal conditions for the growth of GBRTRLI101. Fu et al. (2014)
determined that the optimal growth range of this strain is 24-28°C, coincidental with the average
ambient temperatures. This culture isolate is presumably well-adapted to the relatively stable
temperature of the tropical open ocean, and thus potentially sensitive to rapid thermal variation.
The growth of Trichodesmium in the northern GBR region also benefits from a relatively high
ambient inorganic phosphate concentration (0.08-0.12 μM) (Furnas, 1997). As a constitutive
element in nucleotides, phospholipids, certain coenzymes and other important molecules in
phytoplankton cells (Geider and Roche, 2002; Sterner, 2002; Sohm et al., 2011), P plays important
roles in cell structure, genetic information storage, gene expression, energy generation, protein
synthesis and regulation of metabolic processes and signal pathways of cells (Sohm et al., 2011;
Karl, 2014; Lin et al., 2016). The scarcity of P in the open ocean can lead to growth limitation of
diazotrophic cyanobacteria (Sañudo-Wilhelmy et al., 2001; Ammerman et al., 2003; Fu and Bell,
2003), or to co-limitation with a concurrent deficiency of iron (Mills et al., 2004; Garcia et al.,
2015; Walworth et al., 2016). Effects of P availability on the nitrogen fixation and growth of
Trichodesmium GBRTRLI101 were investigated in previous studies (Fu and Bell, 2003; Fu et al.,
2005).
The GBR region faces multiple climate change stressors, but ocean warming is notably
problematic and is predicted to escalate in the future. The average SSTs of the GBR area in 1976-
2005 were 0.4°C warmer than during the last three decades of the 19
th
century (Lough, 2007). By
the end of this century, a 1-3°C average SST increase is projected to occur in this region, with the
largest temperature increases in winter (Nakicenovic, 2000; Lough, 2007). More warm SST
extremes and fewer cold SST extremes are also predicted to occur in the future GBR area (Lough,
2007). Understanding the differing effects of changing temperature magnitudes and amplitudes
80
requires the use of experimental designs that can explore these aspects of intensive temperature
variability.
Moreover, the strengthened stratification of the water column caused by future warming is
predicted to sequentially reduce nutrient supplies from the deep ocean (Behrenfeld et al., 2006;
IPCC, 2013). Bioavailable phosphorous in the surface ocean is thus likely to decrease, since it is
mainly supplied to the sea surface by mixing and advective processes from the interior ocean (Karl,
2014). Consequently, the growth of Trichodesmium may be further stressed by P-limitation. The
net effects of potential interactions between increasing thermal variability and decreasing P
availability are however unknown.
We hypothesized that: 1) Thermal variability will have adverse impacts on the growth and
physiology of Trichodesmium GBRTRLI101, and increasing the intensity of thermal variation will
magnify these adverse influences, 2) P limitation will play a bigger role in stressing
Trichodesmium growth than temperature variability; and 3) P limited and P-replete cells will
interact with temperature variation differently, with distinct consequences for physiology and
growth. To test these hypotheses, we measured the growth rate, carbon fixation rate and nitrogen
fixation rate, elemental stoichiometry, and Chl a to C ratio as physiological proxies of
Trichodesmium GBRTRLI101 fitness. Finally, phosphorus use efficiency (PUE) for nitrogen
fixation was calculated to quantitatively explore the interactive relationship between cellular
phosphorus requirements and temperature.
3.2 Materials and Methods
3.2.1 Cultures and Incubation Methods
The Trichodesmium strain GBRTRLI101 used in this study was collected from waters close
to the Low Isles (16°23'S,145°34'E) in the Northern Great Barrier Reef Lagoon (Fu and Bell,
2003). Cultures were maintained with autoclaved artificial seawater with iron, EDTA, trace metals
and vitamins added as in the Aquil recipe (Sunda et al., 2005). All nutrients, metals and vitamins
were 0.2 μm syringe-filtered before being added to the medium. Based on the previous study of
Fu et al. (2005) on the relationship between inorganic P and growth of GBRTRLI101, 10 μmol/L
of phosphate was used to maintain laboratory stock cultures. This concentration was also selected
for the P-replete experimental condition in our study, while 0.2 μmol/L of phosphate was used for
the P-limited condition. Before starting experiments using P-limited treatments, the P-replete stock
cultures were acclimated to the phosphate concentration of 0.2 μmol/L for ~3 weeks, until they
81
achieved consistent P-limited growth rates. Cool white fluorescent bulbs were used to provide a
12h dark: 12h light cycle at 150 μmol photons m
-2
s
-1
. Cultures were grown in acid-washed 120-ml
plastic jars fitting into an aluminum thermal block that provided an even temperature gradient,
with uniform irradiance for all replicates. Constant temperature treatments were maintained at the
same location within the thermal gradient throughout the experiments, while variable temperature
treatments were achieved by manually switching the culture jars between two different
temperatures every two days (see below).
The thermal performance curves (TPC) of GBRTRLI101 strain were determined across a
temperature range from 16°C to 36°C at intervals of 2°C, under both P-replete and P-limited
conditions. Semi-continuous incubation methods were employed for 16 days, as described in Qu
et al. (2018) and below. Cultures were diluted every four days. The initial biomass of all treatments
for each cycle was carefully controlled to be low and invariant, at ~100 μmol/L of particulate
organic carbon (POC), and cultures were in early exponential growth stage, allowing the cultures
to maintain exponential growth during the whole dilution cycle. To achieve this objective, Chl a
in vivo fluorescence readings were measured with a Turner Designs 10-AU
TM
fluorometer at both
the beginning and end of the growth cycle to obtain the initial and final biomass of each cycle and
determine the volume of the next dilution. Growth rates and nitrogen fixation rates were measured
for all the temperature points in the thermal range, as described in Physiological and
Biogeochemical Analyses below.
Based on the thermal limits and optimal range of GBRTRLI101 we obtained from the TPC
experiment, and the SST records in the GBR area (Lough, 1998; 2007), we designed the constant
and variable temperature treatments as shown in Table S1. Two constant temperatures of 22°C and
30°C represented the mean values for “winter” (cool) and “summer” (warm) periods, respectively.
For each constant temperature, one or two variable treatments were used simultaneously, each with
an average temperature equal to the corresponding constant temperature. An “intense” 22 ± 4°C
and a “mild” 22 ± 2°C variable treatment were included for comparison to the constant 22°C
treatment. For 30°C, only one variable treatment 30 ± 2°C was used. The “intense” 30 ± 4°C
treatment was not performed because the 34°C maximum temperature would have exceeded the
maximum thermal tolerance of GBRTRLI101, especially under P-limited conditions. In each 4-
day cycle, the first 48 hours of variable treatments were at the lower temperature phase (LT phase,
respectively at 18, 20 and 28°C), and the second 48 hours were at the higher temperature phase
82
(HT phase, respectively at 26, 24 and 32°C). Triplicate cultures were grown at both P levels for
each constant and variable temperature treatment in order to investigate the interactions between
phosphate availability and thermal variation. For thermal variation experiments semi-continuous
incubation methods were employed in the same way as in the TPC determination experiment
described above, except that dilutions of all temperature treatments were conducted between 4-
day thermal cycles.
A steady state was reached after 15-16 cycles, at which point sampling began for specific
growth rates, nitrogen and carbon fixation rates, Chlorophyll a content, and elemental
stoichiometry. There were three sampling points in a growth cycle: the initial point (immediately
after the dilution and before transferring cultures to the LT phase), the midpoint (48 hours after
the dilution, at the end of LT phase) and the final point (96 hours after the dilution, the end of HT
phase). For variable temperature treatments, the data collected at the middle and final points were
averaged as the mean value to compare with the data from the constant treatments.
3.2.2 Physiological and Biogeochemical Analyses
Growth rates. Specific growth rates (μ) of each replicate were calculated as in Qu et al. (2018),
and the growth rate for each treatment was calculated as the mean growth rate of the triplicate
bottles in each treatment. In view of the possible influence of temperature on Chl a in vivo
fluorescence, only the in vivo readings at the initial and final points of each cycle, which were
measured at the same temperature, were used to calculate the growth rates of each cycle. The in
vivo readings collected at the middle point were only used to roughly estimate the growth of
cultures in real time, but were not employed in the subsequent analyses. In vivo-derived specific
growth rates were subsequently validated by particulate organic carbon/nitrogen (POC/PON)
measurements.
Nitrogen fixation measurements. N2-fixation rate was determined using the acetylene
reduction method (Capone, 1993) with a gas chromatograph GC-8a (Shimadzu Scientific
Instruments). For all treatments, 10 ml of culture from each replicate was transferred to a 27 ml
serum vial, which was immediately sealed. Afterward, 2 ml of air was extracted while 2 ml of
acetylene was injected with a syringe to the headspace of each serum vial. A theoretical 3:1 ratio
(mol acetylene to mol N2 reduced) was used to calculate the N2 fixation rates based on the rates of
ethylene production (Montoya et al., 1996). After 4 h incubation (from 11:00 am to 15:00 pm)
during the light period (Tuit et al., 2004), ethylene production was measured by injecting 200 μl
83
of headspace to the GC-8a device and comparing the reading to that of the same amount of a 100
ppm ethylene standard (GMT10325TC, Matheson Gas Products). The raw N2-fixation rates were
normalized to the PON concentration of each sample. In the calculation of PUE, raw N2-fixation
rates were normalized to cellular P content measured as particulate organic phosphorus (POP).
C fixation rates. Photosynthetic carbon fixation rates were measured using the
14
C-uptake
technique. 50 nCi
14
C-NaH
14
CO3 (PerkinElmer, Inc.) was added to 10 ml subsamples of each
replicate, resulting in a final specific activity of ~0.185 kBq/ml. Dissolved inorganic carbon in the
background culture was determined as in Qu et al. (2018). As with the nitrogen fixation
measurements, all subsamples were incubated for 4h (11:00 to 15:00) in the middle of the light
period, and then filtered onto GF/F filters. Each filter was then placed in 5 ml scintillation fluid in
a 7 ml scintillation vial overnight. Triplicated total radioactivity (TA) samples and blanks of each
treatment were prepared following the same protocol in Qu et al. (2018).
14
C radioactivity was
determined using liquid scintillation counting (PerkinElmer, Inc.) for TA, blanks and culture
samples.
Elemental stoichiometry. Elemental ratios were calculated based on the measurements of
POC, PON and POP. 30 ml of culture was filtered onto a pre-combusted (500 °C, 2-3 h) GF/F
filter and dried at 55-65°C for POC and PON analysis. POC and PON were determined with a
Costech Elemental Analyzer using methionine and acetanilide standard for standard curves. Blank
GF/F filters were used to measure the background carbon in the filter (Qu et al., 2018). Another
20 ml aliquot from each replicate was filtered through pre-combusted GF/F filters and prepared
for POP measurements following the protocol described in (Qu et al., 2018). The standard
molybdate colorimetric method was applied in the analysis of the POP samples (Solorzano and
Sharp, 1980). Blank GF/F filters were treated as samples to determine background phosphorous
contents.
Chlorophyll a (Chl a). As an important indicator of photo-acclimation and chlorophyll-
specific biomass in phytoplankton (Cloern et al., 1995; Geider et al., 1997), Chl a was extracted
from Trichodesmium cultures. 20 ml of culture from each replicate was filtered onto GF/F glass
fiber filters, which were then extracted in 6 ml of 90% acetone for 24 hours at -20°C. The non-
acidification method was employed to measure Chl a content with a Turner Designs 10-AU
TM

fluorometer (Welschmeyer, 1994). Chl a content was normalized to POC to test for changes in the
Chl a: carbon ratios in the treatments, which is a commonly used proxy of growth, productivity
84
and photosynthetic capacity of phytoplankton (Cloern et al., 1995; Geider et al., 1997; Jakobsen
and Markager 2016; Sathyendranath et al., 2009).
3.2.3 Statistics
Based on the actual growth rates and nitrogen fixation rates at constant temperature points
from 16-36°C, the thermal performance curves (TPC) were calculated from the model of
Norberg (2004) and Thomas et al. (2012). To make predictions about the effects of temperatures
fluctuating every two days on a magnitude of ± 2°C or ± 4°C, a non-linear averaging model that
takes the principle of Jensen's inequality into account was employed (Bernhardt et al., 2018).
This model is based on the time cells spend at each portion of their TPC during thermal
fluctuations and incorporates the observation that growth rates typically decline much more
precipitously at the warm end of the TPC compared to the cool end. Unlike previous models
applying an unrealistic linear relationship between temperature and population growth, this non-
linear averaging model provides quantitative and accurate estimates of the growth and
persistence of phytoplankton species in a thermally variable environment. It was tested with the
green alga Tetraselmis tetrahele under light and nutrient-saturated conditions, and provides
useful projections such as critical threshold temperatures for phytoplankton growth (Bernhardt et
al., 2018).
To test the interactive effects between thermal variation and P availability, Univariate
Analysis of Variance (two-way ANOVA) was carried out with R v3.3.1, while one-way ANOVA
was used to analyze the difference among temperature treatments for both P conditions. The Tukey
multiple comparison test was applied for multiple comparisons between individual treatments once
significant difference was observed by ANOVA. Student’s t-test was employed when comparing
the difference between constant 30°C and its variable 30 ± 2°C treatment at both P concentrations.
All significance testing was done at the p-value < 0.05 level.
3.3 Results
3.3.1 Thermal performance curves (TPC) for growth and nitrogen fixation rates
Incorporating the measured growth rates at constant temperatures from 16-36°C into the
thermal response model (Norberg, 2004; Thomas et al., 2012) yielded the TPCs under P-replete
(Fig. 1A) and P-limited (Fig. 1B) conditions, which showed an expected “increase-peak-decline”
pattern. As expected, P limitation substantially decreased both nitrogen fixation rates and growth
rates. The optimal growth temperature (T-opt) for both TPCs at constant temperatures under both
85
P conditions was ~27°C (Table 1). No growth was observed at either the coldest (16°C) or
warmest (36°C) temperatures tested, regardless of the P concentration. The maximum growth
rates were 0.32 d
-1
and 0.12 d
-1
respectively for P-replete and P-limited cultures in the TPCs. P-
limitation also narrowed the width of the thermal niche by ~2°C (minimum to maximum limits)
compared to the P-replete condition (Fig. 1A and 1B).
The TPCs based on nitrogen fixation rates at the same constant temperature range of 16-
36°C (Fig. 1C and 1D) were consistent with the TPCs of growth rates. The highest nitrogen
fixation rates across the TPC corresponded with the optimal temperature for growth (24-30°C).
Moreover, with sufficient P supply GBRTRLI101 could survive and fix nitrogen at 18°C, while
at the same temperature the growth and nitrogen fixation in P-limited cultures stopped. Although
both P-replete and P-limited cultures showed slight growth at 34°C, the measured nitrogen
fixation rates under P limitation were close to zero.
The non-linear averaging model-predicted TPCs under thermally-variable conditions of ± 2°C
and ± 4°C (Bernhardt et al., 2018) are shown in Fig. 2. In general, the model-predicted TPCs,
especially with the magnitude of ± 4°C, were narrowed and flattened compared to the
corresponding TPCs based on measured growth rates at constant temperatures (Table 1). The
comparisons between model-predicted TPCs and actually measured growth rates at constant and
variable temperature conditions are presented in the following section.
3.3.2 Growth rates of constant and variable temperature treatments
Two-way ANOVA testing of the effects of temperature treatments, P concentrations, and their
interactions on the measured growth rates of strain GBRTRLI101 in both seasons showed that
temperature variations played a minor role (p-value > 0.05). In contrast, the impacts of P conditions
and the interactions between temperature and P condition were significant (p-value < 0.05). The
effects of thermal fluctuation were further analyzed for two seasons and two P concentrations as
follows.
For “winter” treatments, the measured growth of strain GBRTRLI101 showed different
responses to thermal variation at the two P concentrations (Fig. 2). Under P-replete conditions, the
average growth rates in three temperature treatments were significantly different (one-way
ANOVA, p-value < 0.05), with a sequential declining trend from constant 22°C to “mildly” and
“intensely” variable temperature treatments. Specifically, Tukey multiple comparison showed that
the growth rate of the variable 22 ± 4°C treatment was significantly lower than that of the constant
86
22°C treatment (Fig. 2C, p-value < 0.05). At the growth-limiting P concentration, the declining
trend driven by thermal variation relative to the constant 22°C treatment disappeared (Fig. 2B and
2D, one-way ANOVA, p-value > 0.05).
Likewise, for constant 30°C and its variable treatment 30 ± 2°C, the adverse effect of thermal
variation on the actually measured growth rate showed a smaller but still significant difference
only in the P-replete condition (Fig. 2A, Student’s t-test, p-value < 0.05). Under the P-limited
condition, the measured growth rates at constant and variable temperature treatment showed no
significant difference (Fig. 2B, p-value > 0.05).
In the modeled TPC based on non-linear averaging (Bernhardt et al., 2018), temperature
fluctuation was predicted to decrease the growth rate of GBRTRLI101 compared to the constant
temperature condition, and more intense thermal variations were predicted to have a more
profound adverse effect on growth (Fig. 2). Under P-replete conditions, the measured growth
rates in the 22 ± 2°C, 22 ± 4°C, and 30 ± 2°C variable treatments all closely fit the predicted
curves (Fig. 2A and 2C). In P-limited cultures, however, the measured growth rates in the 22 ±
2°C, 22 ± 4°C and 30 ± 2°C variable treatments did not respond to the thermal variability of ±
2°C (Fig. 2B) and ± 4°C (Fig. 2D) as predicted by the model and showed similar growth rates at
constant and variable temperature treatments.
3.3.3 Nitrogen and carbon fixation rates in thermal variation experiments
Nitrogen and carbon fixation rates of GBRTRLI101 were also driven by the interactive effects
of temperature fluctuation and P concentration (Fig. 3A and 3B). For “winter” temperature
treatments, the average nitrogen fixation rates of GBRTRLI101 in both 22 ± 2°C and 22 ± 4°C
treatments were significantly lower than in the constant 22°C treatment (one-way ANOVA and
Tukey multiple comparison, p-value < 0.05) under P-replete conditions (Fig. 3A). The average
carbon fixation rates of P-replete cultures in variable treatments showed a slight decrease,
compared to the rate at constant 22°C (one-way ANOVA, p-value > 0.05) (Fig. 3B). In P-limited
cultures, however, the advantage of constant 22°C temperature treatment in both carbon and
nitrogen fixation over the corresponding variable temperature treatments disappeared (p-value >
0.05).
For the summertime treatment, the average nitrogen and carbon fixation rate showed no
significant difference between constant 30°C and variable 30 ± 2°C treatment at P-replete status
(Fig. 3A and 3B, Student’s t-test, p-value > 0.05). However, under the P-limiting condition, the
87
carbon fixation rate at variable 30 ± 2°C was significantly higher than at constant 30°C (Fig. 3B,
Student’s t-test, p-value < 0.05). No difference was found for the nitrogen fixation rate (p-value >
0.05).
3.3.4 Elemental stoichiometry and Chl a to carbon ratio
Cellular C: N, C: P and N: P ratios (mol to mol) based on POC, PON and POP measurements
changed with the two P levels in both “winter” and “summer” treatments (Table 2, two-way
ANOVA, p-value < 0.05). As expected, P limitation dramatically increased C: P and N: P ratios at
all temperature treatments. For all the three elemental ratios, though, the effects of temperature
fluctuation and the interaction between P conditions and thermal variation were minor (Table 2,
p-value > 0.05).
In the P-replete “winter” treatment groups, C: N, C: P and N: P ratios at constant 22°C were
consistent with the Redfield Ratio (Redfield, 1958). The C: N at variable treatments was slightly
higher than the ratio at constant 22°C for both P concentrations (one-way ANOVA, p-value >
0.05). The P-replete C: P and N: P ratios in the 22 ± 4°C treatment were significantly higher than
in the constant 22°C treatment (Tukey multiple comparison, p-value < 0.05), but none of the three
elemental ratios were significantly different between the constant 22°C and the two variable
treatments in the P-limited cultures (Table 3). In the P-replete “summer” treatment groups,
elemental ratios of all three thermal treatments were close to the Redfield Ratio, and no significant
differences were observed at either P level (Table 2, p-value > 0.05).
The P-replete Chl a to carbon ratios (g : mol) were significantly higher than ratios under P-
limitation for both “winter” and “summer” time (Fig. 4, two-way ANOVA, p-value < 0.05).
However, due to the relatively large error bars, neither temperature variation individually nor the
interactions between temperature and P conditions significantly changed the Chl a to C ratios (two-
way ANOVA, p-value > 0.05). Moreover, different responses to thermal variation were observed
for “winter” and “summer” treatments and the role of P concentrations varied for the two seasons
(Fig. 4). In the low temperature treatments, thermal variability slightly decreased the P-replete Chl
a to C ratios, while P-limited cultures did not respond to thermal variation (one-way ANOVA, p-
value > 0.05). In summertime, temperature variation slightly lifted the Chl a to C ratio for both P
conditions (Student’s t-test, p-value > 0.05). These patterns were similar to the responses of
nitrogen-fixation to temperature variation (Fig. 4).
88
3.3.5 Phosphorous use efficiency (PUE)
The PUE for nitrogen and carbon fixation at different temperatures was determined by the
amount of N or C fixed per unit time (mol ・hour
-1
) per unit cellular P (mol
-1
), respectively named
as PUEN and PUEC here. P availability, temperature treatment and their interactions all played
significant roles in determining the PUE of cultures (Fig. 5, two-way ANOVA, p-value < 0.05).
In both “summer” and “winter” treatments, PUEN values in P-limited cultures were
significantly higher than under P-replete conditions (two-way ANOVA, p < 0.05; Fig. 4A). In the
“winter” treatment group, PUEN values of the three thermal treatments were significantly different
under P-replete conditions (one-way ANOVA, p-value < 0.05). The P-replete PUEN of the constant
22°C treatment was the highest, followed by the 22 ± 4°C treatment and then the 22 ± 2°C
treatment. The difference between 22 ± 2°C and the other two treatments was significant (p-value
< 0.05). The higher PUEN at constant 22°C compared to the two variable temperature treatments
was consistent with the higher growth rate at constant temperature. However, this pattern was not
observed in P-limited “winter” treatments, or in the “summer” treatments with either of the two P
conditions (Fig. 5A, p-value > 0.05).
In most temperature treatments, PUEC in P-limited cultures was only slightly higher than in
the cultures with sufficient P, except at 30 ± 2°C where the difference in PUEC between the two P
concentrations was significant (Fig. 5B, p-value < 0.05). Moreover, under P limitation, the PUEC
at 30 ± 2°C greatly exceeded the value at constant 30°C (p-value < 0.05), consistent with the
pattern for carbon fixation rates (normalized to POC) in these treatments. In addition, under both
P-replete and P-limited conditions and both constant and variable temperatures, the PUE for both
nitrogen or carbon fixation increased when temperature rose from 22°C to 30°C. This increase of
PUE with rising temperature was especially significant for carbon fixation at both P concentrations
(Fig. 5B, Student’s t-test, p < 0.05).
3.4 Discussion
Our study examined the individual effects of temperature variability and P availability and
their interactions, using temperature treatments representative of two seasons. Temperature
variability affected growth, nitrogen and carbon fixation rates and elemental ratios differently
compared to constant temperatures at the same mean value. This was especially evident in the
“winter” treatments, suggesting that the responses of Trichodesmium to thermal variability may
89
vary in different seasons or in different parts of their latitudinal temperature range. These results
thus partly supported our first hypothesis that thermal variability would have adverse impacts on
Trichodesmium growth and physiology.
P limitation obviously decreased all the growth and physiological rates measured, and shifted
the elemental ratios, under both “winter” and “summer” temperature treatments. As expected in
our second hypothesis, these nutrient limitation effects were often larger individually than those
of variable thermal regimes. Interactions between P levels and thermal variation were widely
observed when comparing measured growth rates with the model-predicted TPCs. Interactions
were also manifested through differences in nitrogen and carbon fixation rates, particularly in the
three “winter” temperature treatments. This provided support for our third hypothesis, that P
availability would affect the responses of Trichodesmium to thermal variability due to interactive
effects.
3.4.1 P availability and phosphorus use efficiency (PUE)
P limitation played a large role in narrowing the temperature range of the TPC, decreasing
growth and fixation rates, and shifting the elemental ratios and Chl a content of Trichodesmium
GBRTRLI101. Generally, the effects of P limitation were larger than the impacts of temperature
variability, supporting our second hypothesis. In our study, growth and nitrogen fixation rates
under P-limitation dropped down significantly compared to the corresponding temperature
treatment in the P-replete condition. The importance of P in cellular structural components like
membranes and other key biomolecules such as nucleic acids accounts for the severely stressed
growth and metabolic rates of phytoplankton cells under P limitation (Toby, 1999; Ji and Sherrell,
2008; Liu et al., 2011). Specifically, the negative effects of P deficiency on nitrogen fixation in
Trichodesmium have been reported by numerous studies (Sañudo-Wilhelmy et al., 2001; Fu et al.,
2005; Hynes et al., 2009; Orchard et al., 2009).
It is not surprising that a large increase in the C: P and N: P ratios relative to the Redfield
Ratio (Redfield, 1958) was observed under P limitation, since the cellular P content decreased
relative to other elements. Moreover, P limitation also significantly decreased the Chl a to C ratio.
An increasing cellular C: P ratio was found to give rise to a decrease in the Chl a to C ratio in
marine phytoplankton under nutrient limitation (Tett et al., 1975), in accordance with our results.
Guildford and Hecky (2000) also extrapolated a universal positive correlation between Chl a
90
abundance and P availability. The decreased Chl a to C ratio and the concurrent reduced carbon
fixation rate under conditions of P limitation both implied a decline in photosynthetic capacity.
The positive correlation between PUE and nitrogen or carbon fixation rates at constant and
variable temperature in our study indicated that PUE is a useful parameter to evaluate the potential
capacity of Trichodesmium cells to survive and grow in a P-stressed environment. A major increase
in PUE under P-limited conditions was observed when compared to P-replete conditions. This may
be at least partly attributed to the luxury uptake of P under sufficient P availability, leading to
excess intracellular P storage and hence a lower PUE (Droop, 1973; Ducobu et al., 1998; Lin et
al., 2016). Thus, intracellular P used in our calculation of PUE in P replete cultures likely included
not only P actively involved in cell structure and functions, but also stored P, which may lead to a
minimum estimate of marginal PUE. However, within the same temperature range the pattern of
PUE increasing with temperature was observed under both P-replete and P-limited conditions,
suggesting that PUE could be still be used as a proxy in environmental surveys, although
interpretations will need to be made in the context of in situ P availability. It is also notable that
after several growth cycles under P limitation, the stored P in our cultures had been consumed to
support growth, nitrogen fixation and other cell activities. In this sense, our results imply
considerable flexibility in P storage and utilization in Trichodesmium cells.
Elevated iron and phosphorous use efficiencies with temperature increase have also been
observed in both Fe-replete and Fe-limited cultures of the Atlantic isolate Trichodesmium
erythraeum IMS101 (Jiang et al., 2018). This suggests a commonly occurring positive correlation
between Fe or P use efficiency and temperature in Trichodesmium. Elevated intracellular
metabolic rates at high temperature within the thermal limits of phytoplankton (Goldman and
Carpenter, 1974; Goldman, 1979) could thus allow for higher growth and nitrogen fixation rates
supported by less cellular P and Fe (Jiang et al. 2018).
Another possible interpretation is varying nutrient allocation at different temperatures.
Toseland et al. (2013) found that phytoplankton require less cellular P relative to N at high
temperatures, as faster protein synthesis rates decrease the numbers of phosphorus-rich ribosomes
and associated rRNAs required to support growth. Thus, the combination of up-regulated
metabolic rates and a reduced P requirement in ribosome synthesis may explain elevated PUE
values at warmer temperatures. However, the hypothesis of Toseland et al. (2013) needs to be
further tested for Trichodesmium by measuring the ribosomal content of cells grown at different
91
temperatures. Clearly though, in the future warming ocean increased PUE at higher temperature
may help Trichodesmium to persist under P-limited conditions.
3.4.2 Impacts of intensity of temperature variation
In the “winter” temperature treatments, we used “mildly” and “intensely” variable treatments
to investigate the effects of intensity of thermal variation. For some physiological proxies, the
intensity of thermal variation was found to modulate the responses of Trichodesmium to thermal
variability and P concentrations. For instance, the growth rate under P-replete conditions
sequentially decreased in 22 ± 2°C and 22 ± 4°C treatments, compared to the constant 22°C
treatment. The statistically significant difference was between 22°C and 22 ± 4°C, which indicated
an enhancement effect of intensified temperature variability. A similar pattern was also observed
in the N: P ratios of the three “winter” treatments under P replete conditions. Notably, under P
limited conditions, the growth rate and elemental ratios were similar at variable 22 ± 2°C, 22 ±
4°C and constant 22°C treatments. These results imply that the effects of thermal fluctuation
intensity are dependent on nutrient availability.
Marañón et al. (2014) indicated that in general the impact of nutrient availability on the growth
of phytoplankton exceeds that of temperature. However, our observation that the growth rates at
two P concentrations were close in variable 22 ± 4°C treatments suggests a comparable effect of
“intensely” variable temperature with nutrient limitation on stressing the growth. In other words,
the magnitude of the temperature fluctuation range matters when comparing effects of nutrient
availability and thermal variation.
3.4.3 Interactions between temperature variability and P availability in two seasons
Much previous research has indicated that until some upper threshold is reached, temperature
increases generally promote the growth of nutrient-replete phytoplankton by stimulating their
metabolic rates (Eppley, 1972; Goldman and Carpenter, 1974; Fu et al., 2014; Sherman et al.,
2016). This trend is in agreement with our results from two TPCs at both P concentrations, and
also with the growth rates of P-replete cultures at constant 22°C and 30°C. However, our study
suggests that this trend becomes much more complicated when temperature fluctuates instead of
simply rising, and also when nutrient availability is involved.
A comparison of our TPCs measured at two P concentrations shows that under P-limited
conditions growth rates declined, and the survival and nitrogen fixation temperature range
narrowed. In the marine diatom Thalassiosira pseudonana, the interaction between nutrient
92
limitation and temperature was found to intensify vulnerability to warming. This is because
growth-limiting conditions shifted the optimum temperature zone of the species towards the lower
temperature end of the nutrient-replete TPC (Thomas et al., 2017). Fe-limited Trichodesmium
however exhibit an opposite trend, in that their TPC is shifted to the right, towards warmer
temperatures (Jiang et al., 2018). Temperature-nutrient interactions also played a large role in
shaping the TPC and determining thermal limits in our Trichodesmium experiments. Specifically,
the strain GBRTRLI101 became more vulnerable to extreme temperatures, with constrictions of
both the upper and lower thermal limits under P limitation.
An evaluation of physiological responses to thermal variation under two distinct P conditions
clearly revealed interactions between the two variables as well. Under P-replete conditions,
temperature variation decreased the growth and nitrogen fixation rates of Trichodesmium, as
predicted in our first hypothesis. This observation was consistent with the projection of the non-
linear averaging model in Bernhardt et al. (2018). Under the P replete condition, the measured
growth rates in the variable temperature treatments fit the model-predicted TPC well, supporting
the validity of the model at least in the suboptimal and supraoptimal temperature range. Under P-
limitation, however, the growth advantage of constant temperature treatments over variable
temperature treatments became negligible. P-limited growth rates did not further decline at
variable temperatures, and so deviated from the predicted curves, especially in the ± 4°C treatment.
The reason for this observation could be that when the Bernhardt et al. (2018) model was
developed, experimental quantification in constant and fluctuating environments was only carried
out under nutrient-saturated conditions. This suggests a limitation of the model under low nutrient
availability.
Furthermore, as expected P-limited cultures showed consistently lower nitrogen and carbon
fixation rates and very elevated N: P and C: P ratios. Consequently, these proxies remained
statistically identical among the three “winter” temperature treatments. In contrast, significant
responses to thermal variation were observed among the same three treatments under P-replete
conditions.
Several recent studies focusing on the responses of phytoplankton to temperature under
nutrient limiting conditions may help to explain the inconsistent effects of temperature variation
at the distinct P conditions in our study. Marañón et al. (2018) discovered a lack of temperature
sensitivity in the metabolic rates of three widely-distributed and biogeochemically important
93
phytoplankton species (Synechococcus sp., Skeletonema costatum and Emiliania huxleyi) under
nitrogen-limitation, which was distinct from the results of nitrogen-replete treatments. They
interpreted this result based on classical Monod or Michaelis-Menten enzyme kinetics, and
attributed the minimal temperature dependence under N-limiting conditions to the similar
temperature sensitivity of maximum reaction rate (Vm) and half-saturation constant (Km)
(Marañón et al., 2018). Thingstad and Aksnes (2019) further extended this explanation and
concluded that the growth of nutrient-limited phytoplankton was limited by the molecular
diffusion of extracellular nutrients, which is less temperature dependent compared to intracellular
enzymatic processes. Jiang et al. (2018) however suggested that rate limitation by micronutrient
elements that are enzymatic co-factors, such as iron, may be much more responsive to temperature
than nutrients that are major cellular structural components, such as N. In short, there is evidence
that major nutrient limitation in particular may decrease the sensitivity of phytoplankton cells to
temperature changes. This could help explain some of the less obvious effects of thermal
variability on the physiology of Trichodesmium GBRTRLI101 under P limitation.
3.4.4 Implications for Trichodesmium growth and biogeochemistry under climate change
Under climate changes, our results suggest that the future trends of Trichodesmium
GBRTRLI101 growth in the winter GBR area will depend on the combination of temperature
increases and intensified thermal variability. Predictions of SST changes the GBR region during
the winter season include 1-3°C warming and less cold extremes (Lough, 2007), which would
seem to be beneficial to the growth of Trichodesmium. However, these climate changes are also
likely to bring about potentially intensified thermal variability (Burroughs, 2007), which would
adversely impact the growth and physiology of Trichodesmium cells especially in the winter, based
on the results of this study.
In our study, 30 ± 2°C did not profoundly change the growth and physiology of
Trichodesmium cells. However, according to the temperature norm of this strain obtained in this
study and in previous research (Fu et al., 2014), summertime temperatures above 33-34°C would
greatly decrease the growth rate. In the future, more warm extremes are predicted to occur in the
GBR area, similar to those that have already occurred in northeast Australia during 2015-2016
(Wolanski et al., 2017). In the future, a sea surface temperature increase of 1-3°C is expected to
occur in the GBR area (Nakicenovic, 2000; Lough, 2007), with the maximal seawater temperature
exceeding the upper growth threshold of Trichodesmium. Moreover, even with only 1°C of
94
warming, the number of summer days with a temperature higher than 33°C is projected to increase
by 3-4 fold compared to the years from 1961-1990 (Lough, 2007). Under this scenario,
Trichodesmium GBRTRLI101 may find it difficult to survive in future summers with more very
warm days and episodic heatwaves.
Other Trichodesmium species and strains are widely distributed in the tropical and subtropical
Pacific, North Atlantic and Indian Ocean (Capone et al. 1997, 2005; Sohm et al. 2011), where the
expected trends of future warming, reduced nutrient supplies and increased thermal variability are
similar to those of the GBR area (Basu and Mackey, 2018; Beardall et al. 2009; Häder et al. 2014;
IPCC 2012). In this context, the pattern we observed in this study could provide insights into the
potential responses of other Trichodesmium strains in different ocean regions to climate changes.
For instance, the current annual SST range of the tropical and subtropical Pacific Ocean is ~23-
30°C, with a diurnal thermal variability of 0.4-1.5°C observed in wintertime when the SST is
~23.5°C (Clayson and Weitlich, 2007; Dunstan et al. 2018). This provides a comparable
temperature background to our study examining the GBR area, and may result in similar growth
declines of Trichodesmium in the winter if thermal variability is intensified in the future. On the
other hand, Trichodesmium in the tropical Atlantic Ocean experience an annual temperature range
of ~26-29°C (Keenlyside and Latif, 2007; Muñoz et al. 2012) which resembles the summertime
situation in the GBR region, possibly leading to only minor responses of Trichodesmium to thermal
variability. However, more investigations will be needed to verify these extrapolations of our
results to other ocean basins dominated by Trichodesmium species and strains with potentially
different thermal adaptation histories.
Climate change is also predicted to decrease nutrient supplies to the euphotic zone of the
ocean, and in turn the export of organic particles to the deep ocean (Hutchins and Fu, 2017). Under
P-replete conditions, thermal variation had negative effects on most cellular parameters, while
under P-limitation, the adverse impacts became negligible. As a result, the possible implications
of temperature variability for future biogeochemistry should be considered to depend on P
availability.
Adverse effects on Trichodesmium growth and nitrogen fixation and increased N: P and C: P
ratios were especially evident under wintertime P-replete, thermally variable conditions. This trend
was even more marked when the intensity of the temperature fluctuations increased. Hence, if P
availability in the surface seawater of the GBR area is maintained at currently sufficient levels (Fu
95
and Bell, 2003; Fu et al., 2005) and more intensified temperature variability occurs in winter time,
the contribution of Trichodesmium GBRTRLI101 to primary production and new nitrogen supply
would be reduced. In the meanwhile, more C and N relative to P might be exported to the deep
ocean with ratios exceeding the Redfield Ratio. In summer, the growth of Trichodesmium
GBRTRLI101 and related biogeochemistry would not be obviously changed by thermal variation,
other than adverse effects of extreme heat wave events as discussed above.
If vertically advected P supplies are lower in the future as predicted, P deficiency in the GBR
region would affect the growth and physiology of Trichodesmium GBRTRLI101 in a similar
pattern in both winter and summer. All the physiological proxies of Trichodesmium GBRTRLI101
would be profoundly stressed, and C: P and N: P in sinking organic particles would dramatically
increase while the effects of thermal variation would be comparatively minor, as observed in our
study.
Today, P availability in the GBR area (0.08-0.12 μM) is higher than in the North Atlantic gyre
(0.2-1.0 nM), southwest Pacific Ocean (< 20 nM) and eastern Mediterranean (below 2.0 nM)
(Furnas 1997; Moutin et al., 2007; Thingstad et al., 2005; Wu et al., 2000). As a result,
Trichodesmium in the GBR area may suffer less than in the other ocean regions from intensified P
limitation in the future ocean. Based on our results, the growth of Trichodesmium in the GBR area
is more likely to be adversely impacted by thermal variability, while Trichodesmium in other more
P-limited regions may not respond as much to temperature variations. Our findings also suggest
that because Trichodesmium in the GBR area are relatively P-replete, it will be easier to use non-
linear averaging models such the one we employed from Bernhardt et al. (2018) to accurately
predict their responses to heightened future thermal variability, compared to Trichodesmium
populations in more P-limited areas.
Trichodesmium in the oligotrophic ocean is also often limited by iron (Fe), or is co-limited by
both P and Fe simultaneously (Berman-Frank et al., 2001; Fu and Bell, 2003; Mills et al., 2004;
Walworth et al. 2016). Although effects of Fe limitation on the growth of Trichodesmium are
beyond the scope of our study, investigations of interactions between this other primary limiting
nutrient and climate change variables such as warming are needed to provide better predictions of
the physiology and distribution of Trichodesmium in the future (Hutchins and Boyd 2016, Jiang et
al. 2018).
3.5 Conclusions
96
The distinct responses of Trichodesmium to thermal variation under two phosphate conditions
suggest that the physiological effects of thermal variability on GBRTRL101 strain would depend
on ambient nutrient availability. Our results also indicate that the responses of Trichodesmium to
thermal variability would vary with seasonal (winter / summer) temperature regimes and would be
impacted by different intensities (± 2°C / ± 4°C) of variation. In addition, this study implies that a
thermal performance curve obtained at constant temperatures can be used along with the non-linear
averaging model (Bernhardt et al., 2018) to provide accurate predictions of growth rates under
fluctuating temperatures when P is replete. However, application of this method under P-limited
conditions may require further refinement.
Moreover, the phosphorus use efficiency for nitrogen and carbon fixation was elevated by
rising temperature and P limited conditions in this study, suggesting a potential for this strain to
maintain fitness despite future warmer, more nutrient-limited conditions. In conclusion, the overall
effects of ambient temperature, intensity of thermal variation, nutrient supplies and their
interactions should be considered together to accurately predict how the growth and nitrogen
fixation of Trichodesmium may be altered in the future changing ocean.

Acknowledgments
We thank E. McParland, E. Mak and the E. Webb lab groups for help with culturing and
analyses.

Funding
This work was supported by a USC Provost’s Fellowship to PQ, and by NSF Biological
Oceanography grants OCE1538525 and OCE1638804 to F-XF and DH.

97
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105
Chapter 3-Tables and Figures
Table 1. Thermal performance curve (TPC) parameters at constant and two variable
temperature treatments (± 2°C and ± 4°C), all at two P concentrations (10 and 0.2 μmol/L).
Values in the table were calculated by incorporating the measured growth rates at constant
temperatures (16-36°C) and variable temperature treatments (22 ± 2°C, 22 ± 4°C, 30 ± 2°C) into
the thermal response model of Norberg (2004) and Thomas et al. (2012). T-max: upper
temperature limit; T-min: lower temperature limit; T-opt: optimal temperature; T-width:
temperature range; µmax: maximal growth rate; R
2
: the correlation factor, in the range of 0~1. The
closer R
2
is to 1, the better the model-predicted growth rates match the

actual measured ones.
Since only one ± 4°C temperature treatment was conducted, the R
2
for it is not available (N.A.).
Phosphate
status
TPC type T-max T-min T-opt T-width µ max R
2

P-replete
Constant temp 35.9 16.3 27.7 19.6 0.32 0.97
Variable ± 2°C 35.6 16.3 27.5 19.3 0.30 0.93
Variable ± 4°C 34.5 16.6 26.8 17.9 0.26 N.A.
P-limited
Constant temp 36.6 18.2 26.3 18.4 0.12 0.75
Variable ± 2°C 36.5 18.5 26.5 18 0.12 0.70
Variable ± 4°C 36 19.6 27.0 16.4 0.10 N.A.

106
Table 2. Elemental stoichiometry in five constant and variable temperature treatments at
two P concentrations (10 and 0.2 μmol/L) in summer and winter experiments. The values in
the table are the average values of triplicates± standard deviations.

P-replete P-limited
“Winter”
treatment
C: N C: P N: P C: N C: P N: P
Average
constant
22°C
6.70 ± 0.59 106.48 ± 1.98 15.86 ± 0.51 6.98 ± 0.81 350.91 ± 65.80 46.30 ± 13.85
Average
22 ± 2°C
7.40 ± 0.47 126.09 ± 10.91 17.04 ± 1.34 8.09 ± 0.57 342.08 ± 28.75 42.12 ± 4.15
Average
22 ± 4°C
7.18 ± 0.44 125.90 ± 4.91* 18.73 ± 0.55* 7.66 ± 0.36 342.09 ± 21.96 44.59 ± 1.53
“Summer”
treatment
C: N C: P N: P C: N C: P N: P
Average
constant
30°C
6.76 ± 0.66 116.99 ± 15.87 18.02 ± 1.20 7.35 ± 0.58 317.66 ± 15.73 42.94 ± 1.01
Average
30 ± 2°C
6.30 ± 0.55 115.94 ± 5.26 18.44 ± 0.71 7.10 ± 1.02 324.26 ± 21.96 45.15 ± 1.53
107
Figure Legends
Figure 1. Measured thermal response curves (TPC) based on growth rates of
Trichodesmium erythraeum strain GBRTRLI101 under (A) P-replete (solid symbols) and
(B) P-limited (open symbols) conditions. Nitrogen fixation rates normalized to PON are shown
in (C) P-replete and (D) P-limited cultures at nine temperatures. Values and error bars represent
the means and the standard deviations of triplicate cell cultures in each treatment. The arrows
labeled with temperature values show the temperatures where growth or N2 fixation was not
detectable.
Figure 2. Non-linear averaging model-predicted TPCs for the ± 2°C (dashed line) (A and B)
and ± 4°C (dotted line) (C and D) treatments. (A) and (C) are under P-replete conditions and
(B) and (D) under P-limited status. The actual measured growth rates of constant 22 and 30°C
(solid cycle: P-replete; open circle: P-limited), ± 2°C (solid triangle: P-replete; open triangle: P-
limited) and ± 4°C (solid rectangle: P-replete; open rectangle: P-limited) variable treatments are
shown.
Figure 3. Average (A) nitrogen-specific fixation rates and (B) carbon-specific fixation rates
normalized to PON and POC, respectively, of Trichodesmium GBRTRLI101 in five
constant and variable temperature treatments under two P conditions (10 and 0.2 μmol/L)
in summer and winter experiments. Values and error bars respectively represent the means and
the standard deviations of triplicate cell cultures under each treatment. The letters represent the
significant differences based on the grouping results of the Tukey multiple comparison among
ten temperature ´ P treatments.
Figure 4. Chl a to C ratios of Trichodesmium GBRTRLI101 at five constant and variable
temperature treatments and two P conditions (light grey symbols: P-limited culture, 0.2
μmol/L; dark grey symbols: P-replete culture, 10 μmol/L) in summer and winter
experiments. Values and error bars respectively represent the means and the standard deviations
of triplicate cell cultures under each treatment. The letters represent the significant differences
based on the grouping results of the Tukey multiple comparison among ten temperature ´ P
treatments.
Figure 5. Phosphate Use Efficiency (PUE) for nitrogen (A) and carbon (B) fixation under
P-replete and P-limited conditions (10 and 0.2 μmol/L) in five different constant and
108
variable temperature treatments in summer and winter experiments. Values and error bars
respectively represent the means and the standard deviations of triplicate cell cultures under each
treatment. The letters represent the significant differences based on the grouping results of the
Tukey multiple comparison among ten temperature ´ P treatments.

109

Figure 1.

1 8 ° C 1 6 ° C 3 6 ° C 3 6 ° C
1 8 ° C
0 . 0
0 . 1
0 . 2
0 . 3
0 . 4
G r ow t h r a t e s
( d a y
- 1
)
N f i x a t i on r a t e
(10
- 3
m o l · ( m ol P O N )
- 1
· h o u r
- 1
)
0
1 0
2 0
3 0
4 0
1 4 1 8 2 2 2 6 3 0 3 4 3 8
T e m p e r a t u r e ( C ° )
1 4 1 8 2 2 2 6 3 0 3 4 3 8
( A ) ( B )
( C ) ( D )
S p e c i f i c N f ixa t ion r a t e
( 10
-3
· h r
-1
)
S p e c i f i c g r o w t h r a t e
( d ay
-1
)
1 6 ° C 3 6 ° C 3 4 ° C
P-replete
P-limited
110

Figure 2.
●
●
0. 0
0. 1
0. 2
0. 3
0. 0
0. 1
0. 2
0. 3
0. 4
●
●
1 4 1 8 2 2 2 6 3 0 3 4
Tempe r at u r e (°C)
1 4 1 8 2 2 2 6 3 0 3 4 3 8
(C) P-replete, ± 4°C (D) P-limited, ± 4°C
Growth rates (day
-1
)
0. 4
(A) P-replete, ± 2°C (B) P-limited, ± 2°C
P-replete P-limite d
Constant temperatur e
V ariable ± 2° C
V ariable ± 4° C
Model-predicted ± 2°C TP C
Model-predicted ± 4°C TP C
22°C
30°C
22°C
30°C
22°C
30°C
22°C
30°C
3 8
111

Figure 3.

0
10
20
30
P-replete
P-limited
(A)
0
10
20
30
40
Temperature (℃)
a cd
22 22 2 22 4 30 30 2
“Winter”
treatments
“Summer”
treatments
(B)
Specific N fixation rate
(10
-3
hour
-1
)
Specific C fixation rate
(10
-3
hour
-1
)
bc e
ab b
cd e de e
cd d
ab d
ab bc
a cd
cd d
112

Figure 4.

0 .06
0 .08
0 . 1 0
0 . 12
22 22 ± 2 22 ± 4 30 30 ± 2
T e m p e r a t u r e ( ° C )
C h l a : C r ati o ( μ g C h l a · ( μ m o l P O C )
-1
)
“ W i n t e r ”
t r ea t m e n ts
“ S u mm e r ”
t r ea t m e n ts
P-r eplete P-limited
ab
c
bc
c c
bc
a
bc
ab
a
113

Figure 5.

0
3
6
9
12
0
0.3
0.6
0.9
1.2
P-replete
P-limited
“Winter”
treatments
“Summer”
treatments
22 22 2 22 4 30 30 2
Temperature (℃)
mol fixed N/mol POP/hr mol fixed C/mol POP/hr
(A)
(B)
cd cd d d cd cd
bc b
bcd a
cd bcd d bc cd bc
bcd a
bc ab
114
Chapter 3-Supplementary Materials
Table S1. Experimental temperature and phosphate availability treatments. LTP: low
temperature phase; HTP: high temperature phase. Constant temperatures of 22°C and 30°C
were used, along with variable treatments with these same two mean temperatures. For the mean
temperature of 22°C, the “intense” variable treatment consisted of 18-26°C fluctuations, and the
“mild” variable treatment was 20-24°C. For 30°C, only one variable treatment (28-32°C) was
used. All the thermal treatments were run under P-replete (10 μmol/L) and P-limited (0.2
μmol/L) conditions.

Temperature setting Phosphate status
LTP
(first 48 hrs)
HTP
(second 48 hrs)
Constant 22°C
P-limited
22°C
P-replete
“Intensely” variable 22 ±
4°C
P-limited
18°C 26°C
P-replete
“Mildly” variable 22 ± 2°C
P-limited
20°C 24°C
P-replete
Constant 30°C
P-limited
30°C
P-replete
“Mildly” variable 30 ± 2°C
P-limited
28°C 32°C
P-replete
115
Table S2. Elemental stoichiometry during the low temperature phase (LTP) and high
temperature phase (HTP) in three variable temperature treatments at two P concentrations
(10 and 0.2 μmol/L). The values in the table are the average values of each triplicated treatment
± standard deviation.

P-replete P-limited
“Winter”
treatment
C: N C: P N: P C: N C: P N: P
LTP
22 ± 2°C
7.73 ± 0.42 131.81 ± 19.52 17.04 ± 2.27 7.77 ± 0.38 253.33 ± 43.5 32.63 ± 5.79
LTP
22 ± 4°C
7.27 ± 0.36 122.71 ± 4.9 16.90 ± 0.54 7.51 ± 0.27 313.39 ± 16.89 41.72 ± 0.93
HTP
22 ± 2°C
7.07 ± 0.19 120.38 ± 4.38 17.03 ± 0.50 8.41 ± 0.59 430.83 ± 91.62 51.61 ± 12.75
HTP
22 ± 4°C
7.09 ± 0.57 146.19 ± 22.22 20.55 ± 1.45 7.81 ± 0.42 370.78 ± 28.45 47.45 ± 2.30
“Summer”
treatment
C: N C: P N: P C: N C: P N: P
LTP
30 ± 2°C
5.88 ± 0.14 107.71 ± 16.60 18.31 ± 2.78 6.32 ± 0.55 248.66 ± 14.18 39.40 ± 1.45
HTP
30 ± 2°C
6.72 ± 0.46 124.18 ± 7.92 18.57 ± 2.36 7.87 ± 0.71 399.86 ± 26.9 50.89 ± 1.52
116
Chapter 4: The Marine Nitrogen-Fixing Cyanobacteria Trichodesmium and
Crocosphaera Employ Contrasting Acclimation and Adaptation Strategies
during Long-Term Thermal Selection
Pingping Qu
1
, Feixue Fu
1
, Xinwei Wang
2
, Joshua D. Kling
1
, Mariam Elghazzawy
1
, Megan Huh
3
,
Esther Mak Wing Kwan
4
, Michael D. Lee
5
, David A. Hutchins
1

1. Department of Biological Sciences, University of Southern California, Los Angeles, California,
90089, USA
2. School of Life Sciences, Xiamen University, Xiamen, 361005, China
3. Department of Preventative Medicine, Keck School of Medicine, University of Southern
California, Los Angeles, California, USA
4. Department of Ocean Sciences and Institute of Marine Sciences, University of
California, Santa Cruz, 95064, USA
5. The National Aeronautics and Space Administration, Washington, D.C., 20546, USA

Corresponding author: David Hutchins
1
(Email address: dahutch@usc.edu)
Key words: Trichodesmium, Crocosphaera, nitrogen fixation, adaptation, plasticity, evolution,
climate changes.

The manuscript is to be submitted in 2019.

117
Abstract
Filamentous Trichodesmium and unicellular Crocosphaera are two of the most important
and widely distributed diazotrophic cyanobacteria in subtropical-tropical oceans. This study used
microbial experimental evolution methods to test their possible thermal adaptation during two
years of long-term selection at three temperatures (22, 28 and 32°C). Trichodesmium erythraeum
IMS101 and Crocosphaera WH0005 were thermally selected for hundreds of generations, and
thermal performance curves of temperature-selected cell lines were determined to test for
changes in temperature-related traits. The morphological and physiological differences in
nitrogen and carbon fixation rates, Chl a to carbon ratios, elemental stoichiometry, and cell size
of Crocosphaera were also measured for long-term cell lines of the two diazotrophs.
Our thermal response curve results suggest that Trichodesmium IMS101 did not adapt to
the three temperature regimes during the long-term selection, and instead showed a rapid
acclimation to temperature shifts from 20-34°C. In contrast, true thermal adaptation may have
occurred in Crocosphaera WH0005. Particularly in the warmest condition (34°C), 32°C-selected
Crocosphaera cells had an advantage in growth and nitrogen fixation over cell lines selected at
22°C and 28°C. One month reciprocal switch experiments between all three selection
temperatures using all three sets of temperature-selected Crocosphaera cell lines further
suggested apparent adaptive responses in this strain.
It appears that Trichodesmium IMS101 possesses a considerable amount of inherent
thermal plasticity, allowing it to quickly acclimate to new environments. Changes in thermal
characteristics of selected Crocosphaera cell lines may however represent an evolutionary
response, indicating a potential capacity of this diazotroph to adapt to global warming. These
contrasting responses of two crucial N2-fixers to long-term temperature change may help to
determine the shape of nitrogen and carbon global biogeochemical cycles in the future ocean.

118
4.1 Introduction
Diazotrophic cyanobacteria play crucial ecological and biogeochemical roles by fixing
new nitrogen that supports ocean food webs and carbon export (Sohm et al. 2011; Tang et al.
2019; Zehr 2011). Among the key groups of diazotrophs, Trichodesmium is a colonial,
filamentous cyanobacterium widely distributed in the subtropical and tropical ocean that
contributes a substantial fraction of marine new nitrogen inputs (Bergman et al. 2013; Capone et
al. 1997, 2005; Hood et al. 2004). Crocosphaera is a sympatric unicellular N2-fixer, with a
comparable nitrogen fixation rate to Trichodesmium, and an even bigger contribution to total
nitrogen fixation in oligotrophic ocean regions (Knapp et al. 2012; Moisander et al. 2010;
Montoya et al. 2004; Zehr et al. 2001).
The capabilities of these two diazotrophs to fix nitrogen and their new nitrogen transfer
efficiencies have been explored in several studies (Berthelot et al. 2016; Knapp et al. 2012).
Furthermore, genomic and transcriptomic studies of multiple strains of Trichodesmium and
Crocosphaera have revealed distinct genetic characteristics of the two diazotrophs, and
described molecular mechanisms behind their physiological responses (Bench et al. 2011, 2013;
Walworth et al. 2015, 2016; Webb et al. 2009; Zehr et al. 2007).
As a dominant N2-fixer in the marine ecosystem, Trichodesmium has received a lot of
attention relative to its responses to changing environmental factors such as CO2 concentrations,
temperature and light intensity (Boatman et al. 2017; Hutchins et al. 2007; Li et al. 2018; Qu et
al. 2019). In the past decade, the impacts of climate change variables on the representative
unicellular diazotroph Crocosphaera have also been investigated extensively (Fu et al. 2008;
Garcia et al. 2014; Gradoville et al. 2014). Comparative studies on these two important
diazotrophs have provided insights into controls on diazotroph community composition, and how
their influences on biogeochemistry may change in the future ocean (Fu et al. 2014; Hutchins et
al. 2013; Jiang et al. 2018).
In addition to short-term physiological responses, long-term evolutionary experiments
can provide valuable mechanistic information to understand the responses of phytoplankton to
future environmental changes (Jin and Agustí 2018; O'Donnell et al. 2018; Padfield et al. 2016).
The long-term evolutionary responses of Trichodesmium IMS101 to future CO2 concentration
have been investigated, and found to include apparently irreversible adaptation after selection
119
under projected future CO2 concentrations for ~ 4.5 years (Hutchins et al. 2015; Walworth et al.
2016).
It has been long recognized that warming is an important climate change variable
affecting marine ecosystems. The temperature of the surface ocean has increased ~0.44°C from
1971-2010, and is projected to continue rising by 0.7~2.7°C by the end of this century (Bopp et
al. 2013; IPCC 2013; Roemmich et al. 2015). The tropical and subtropical regions where
Trichodesmium and Crocosphaera thrive are predicted to be among the fastest warming parts of
the global ocean (Collins et al. 2010; Liu et al. 2005). Moreover, climate change gives rise to
intensified temperature variability and extreme temperature events (Boyd et al. 2016; IPCC
2012; Thornton et al. 2014), which may also challenge the survival of marine cyanobacteria (Qu
et al. 2019).
Currently, the annual SST ranges of the tropical Atlantic and Pacific Oceans can reach
26-28°C and 25-30°C respectively (Dunstan et al. 2018; Hurrell et al. 2008; Keenlyside and Latif
2007; Muñoz et al. 2012), while the upper threshold of the optimal temperature range for both
Trichodesmium and Crocosphaera is ~30°C (Breitbarth et al. 2007; Fu et al. 2014; Sohm et al.
2011). With projected mean temperature increases and potential for intensified extreme heat
wave events, the maximal SST of the tropical ocean is likely to exceed the upper thermal limit of
these two diazotrophs.
Thomas et al. (2012) suggested that tropical phytoplankton species are the most
vulnerable to future warming, since their optima are close to the current mean temperature of
their surroundings. In this context, it is imperative to investigate whether these two diazotrophs
have the capacity to acclimate or adapt to the future warming ocean, or whether they will be
excluded from the warmest parts of their present low latitude tropical habitat as predicted by
previous studies (Breitbarth et al. 2007; Fu et al. 2014; Boatman et al. 2017; Li et al. 2018).
Thus, the thermal responses of these key N2-fixers have the potential to influence
biogeochemical processes on a global scale.
Our study examined the potential for short-term acclimation and long-term adaptation of
the two important diazotrophs Trichodesmium erythraeum IMS101 and Crocosphaera WH0005
to optimal (28°C), supraoptimal (32°C) and suboptimal (22°C) temperatures. In particular, 32°C
is a representative temperature for the future warming condition. We hypothesized that: 1) The
physiology of Trichodesmium and Crocosphaera will respond to changing temperatures in the
120
short-term due to their intrinsic plasticity, while evolutionary adaptation will require a much
longer time to occur. 2) Divergent adaptation is predicted to first emerge between cell lines
selected at the two thermal extremes (suboptimal 22°C and supraoptimal 32°C) due to the large
temperature differential, so cell lines selected at these two temperatures will not be able to
effectively or quickly re-acclimate to the other temperature. 3) Trichodesmium and
Crocosphaera are likely to show different acclimation processes or adaptive responses after
long-term selection, due to their distinct genetic backgrounds and physiological characteristics.
To test our hypotheses, adaptive responses of both species were examined following two
years of experimental evolution under thermal selection, using reciprocal transfer experiments.
Acclimation capacity was tested using short-term temperature-response-curve determinations for
both diazotrophs. Results of these experiments reveal fundamental differences in the acclimation
and adaptation strategies that these two key N2-fixing cyanobacteria groups may employ to
respond to the challenges of living in a rapidly warming ocean.
4.2 Methods and Materials
4.2.1 Cultures and experimental evolution
Trichodesmium erythraeum IMS101 and Crocosphaera WH0005 were respectively
isolated from the Atlantic Ocean (Chen et al. 1996; Prufert-Bebout et al. 1993) and from the
North Pacific Ocean basin (Webb et al. 2009). All the cultures were grown in 250 ml sterile
flasks using autoclaved artificial seawater, with replete phosphorous, iron, trace metals and
vitamins added according to the AQUIL recipe (Sunda et al. 2005). An identical 12h dark: 12h
light cycle at 150 μmol photons•m
-2
•s
-1
was provided with cool white fluorescent bulbs for all
the cultures.
Ancestral cell lines of both strains were transferred from 26°C to the three selection
temperatures, which covered the optimal (28°C), suboptimal (22°C) and supraoptimal (32°C)
temperature for both strains (Fu et al. 2014). Initially, there were six replicates of both
Trichodesmium IMS101 and Crocosphaera WH0005. Unfortunately, several Crocosphaera cell
lines died or got contaminated, hence only three cell lines at each temperature remained at the
end of the two year selection period.
Semi-continuous incubation methods were used to maintain all the cell lines for ~2 years.
The dilution interval was 2-3 days, and the initial biomass of each growth cycle was set at ~150
μmol/L of particulate organic carbon (POC). The real-time initial and final biomass of each cycle
121
was determined by measuring Chl a in vivo fluorescence of all replicates with a Turner Designs
10-AUTM fluorometer. The in vivo reading at the end of the growth cycle was used to determine
the dilution volume for the next cycle.
4.2.2 Temperature performance curves (TPCs) Determination
After ~2 years’ selection at the three temperatures, temperature-related traits such as
temperature norm and thermal niche of selected Trichodesmium and Crocosphaera cell lines
were examined by determining their thermal performance curves (hereafter called TPC) across a
temperature range from 16°C to 36°C at intervals of 2°C. Specifically, The TPCs of
Crocosphaera WH0005 cells maintained in the three temperature treatments were measured in
the 18
th
-21
st
month after the initial transfer. The TPCs of Trichodesmium erythraeum IMS101
cell lines were determined after 24 months maintenance at the three selection temperatures. The
experimental conditions were identical to those used in the long-term incubations.
For the 16 day short-term incubations, semi-continuous incubations were conducted, as
described above and in Qu et al. (2018). Cultures were diluted every four days. The initial biomass
of each replicates for each cycle was carefully controlled at ~80-100 μmol/L of POC, to make sure
the cells remained within the exponential growth stage throughout the entire dilution cycle.
The specific growth rates were used as a proxy of relative fitness (Hutchins et al. 2015).
Nitrogen and carbon fixation rates were measured at three selection temperatures 22°C, 28°C,
32°C, as well as at an extension temperature of 34°C at the warm end. Since the temperature
range of Trichodesmium was found to be wider than that of Crocosphaera, physiological rates
were also determined at 20°C for Trichodesmium cell lines. Elemental stoichiometry were
measured, due to the significant implications of both groups of diazotrophs for global
biogeochemical processes. Chl a to carbon ratios were also examined as a proxy for
photosynthetic capacity. Cell sizes of Trichodesmium and Crocosphaera were also measured as
an important parameter related to their role in marine food webs, carbon export and competitive
abilities. Detailed descriptions of all these measurements are presented in the Physiological
measurements section below.
4.2.3 Switch experiments
Since the results of the TPC determination experiments showed potential adaptive
responses in long-term temperature-selected Crocosphaera cells, a reciprocal transfer or
“switch” experiments were conducted with these cell lines among the three selection
122
temperatures (Hutchins et al. 2015). The experimental conditions and dilution frequencies in
switch experiments were identical to those used in the long-term thermal selection incubations.
Following the switches, physiological sampling of short-term switch and long-term selected cell
lines at three temperatures took place after one month, to document the potential changing
phenotypes of each set of cell lines at the other two assay temperatures. In the switch experiment,
the parameters measured were identical with those examined in the TPC determination
experiments.
4.2.4 Physiological measurements
Growth rates. For the semi-continuous incubation, specific growth rates (μ) were
calculated based the following formula:
μ= ln(Ntf / Nti)/(tf-ti),
where Nti, and Ntf refer to biomass respectively at the initial (ti) and final (tf) time point
(in days) of each growth cycle (Ihnken et al. 2011; Qu et al. 2018). In our study, real-time
biomass was estimated with in vivo fluorescence measured before and after dilution, and
subsequently verified by microscopy using preserved cell counts and POC. The specific growth
rate of each treatment was the average value of the growth rate of all replicates.
Nitrogen fixation measurements. N2-fixation rates were measured over 6 h in the light
period (Trichodesmium) or the entire 12 h dark period (Crocosphaera), due to their different diel
strategies for N2 fixation (Shi et al. 2010; Tuit et al. 2004). For each replicate, 10 ml of culture
was transferred to a 27 ml air-tight vial. After replacing 2 ml of air in the headspace of each vial
with 2 ml of acetylene, the vials were incubated under the same conditions as the long-term cell
lines. The acetylene reduction assay (Capone 1993) was employed with a Shimadzu gas
chromatograph GC-8a (Shimadzu Scientific Instruments). The detailed protocol and calculations
were described in Montoya et al. (1996) and Fu et al. (2014). After raw N2-fixation readings
were obtained, particulate organic nitrogen (PON) measurements of each sample were used for
the normalization of N-specific N2-fixation rates.
C fixation rates. The photosynthetic efficiency of both diazotrophs was measured with
the
14
C uptake method (Nielsen 1952). 25 nCi
14
C-NaH
14
CO3 (PerkinElmer, Inc.) was added to
10 ml subsamples of each replicate. The specific activity in the subsample was roughly ~0.09
kBq/ml.
14
C radioactivity of each subsample was measured after 6 hours’ incubation in the light
cycle using liquid scintillation counting method (Perkin Elmer). Total radioactivity of
14
C added
123
and blank radioactivity of each treatment were measured as described in Qu et al. (2018). After
subtracting the blank radioactivity, the product of intercellular
14
C detected in the given volume
and the concentration of dissolved inorganic carbon was divided by total radioactivity and given
incubation time to calculate the raw C fixation rate, which was afterward normalized to POC to
attain the C-specific C fixation rate.
Elemental stoichiometry. Elemental ratios were determined by measurements of POC,
PON, and particulate organic phosphorus (POP). For POC and PON, 30 ml of each replicate was
filtered onto a GF/F filter (pre-combusted at 500°C for 3 h), which was afterward dried at 55 °C.
For POP samples, 20 ml of culture was filtered onto pre-combusted GF/F filters and rinsed twice
with 2ml of 0.17 mol/L Na2SO4 solution. Each filter was then placed in a 20 ml borosilicate
scintillation vial (pre-combusted at 500°C overnight) with the addition of 2ml of 0.017 mol/L
MgSO4 solution. Vials with POP filters were covered with foil cap and dried at 55°C. Three pre-
combusted GF/F filters were treated in the same way as samples, and used as blanks in the
following analysis.
After drying, POC and PON samples were pelleted and analyzed on a Costech Elemental
Analyzer, with pre-combusted GF/F filters analyzed as blanks, following the same protocol as
described in Fu et al. (2007) and Qu et al. (2018). The POP concentration of samples and in
blanks was measured following the standard molybdate colorimetric method (Fu et al. 2005;
Solorzano and Sharp 1980).
Chlorophyll a (Chl a). A 20 ml sample from each replicate bottle was filtered through
GF/F glass fiber filters. Chl a was extracted in 6 ml of 90% acetone at -20°C and measured using
the non-acidification method with a Turner Designs 10-AU
TM
fluorometer after 24 h (Jeffrey and
Humphrey 1975; Welschmeyer 1994).
Cell counts. For each replicate, 1 ml of culture was transferred to a 1.8 ml Eppendorf tube
and preserved by add 20 µl of 25% glutaraldehyde. The cell count samples were stored at 4°C in
the dark. Cell numbers were counted and recorded with a Spears-Levy Eosinophil counting
chamber using an Olympus BX51 microscope.
Trichome and cell size. Cell size of the long-term Trichodesmium trichomes and
Crocosphaera cells were measured at their selection temperatures. An Olympus BX51
microscope with a camera was used to capture photos of cells. The radius of Crocosphaera cells
124
and the length and width of trichomes were precisely measured with the calibrated micrometer
using the software Captavision Imaging.
4.2.5 Statistics
R 3.3.1 software was used for the statistical analysis. One-way ANOVA was used to
analyze the physiological differences among long-term cell lines, a series of temperature
treatments of TPC determination experiments for both diazotrophs, and the switch experiments
of Crocosphaera. When significance was found, the TukeyHSD method was applied for multiple
comparisons between treatments. All significance testing was done at the p < 0.05 level.
4.3 Results
4.3.1 Growth rates in temperature response curves (TPCs)
The growth rates of the ancestral lines of both isolates were ~0.30 day
-1
at 28°C, and
these decreased at 22°C and 32°C after the initial transfer for both Trichodesmium (Fig. 1A) and
Crocosphaera (Fig. 1B). After approximately 2 years’ maintenance at the three selection
temperatures, the specific growth rates of cell lines of Trichodesmium erythraeum IMS 101 were
~0.3 day
-1
at 28°C, ~0.26 at 32°C and ~

0.2 day
-1
at 22°C (Fig. 1A). For long-term thermally-
selected Crocosphaera WH0005, specific growth rates were ~0.3 day
-1
at 28°C and 32°C, and ~

0.18 day
-1
at 22°C (Fig. 1B). The generation time for both strains was thus 2-3 days at 28 and
32°C, and 3-4 days at 22 °C. Long-term Crocosphaera cell lines experienced temperature
selection for ~270 generations at 28°C and 32°C, and ~160 generations at 22°C. while
Trichodesmium erythraeum IMS101 cell lines underwent selection for ~180 generations at 22°C
and ~360 generations at 28 and 32°C.
The temperature response curves of Trichodesmium erythraeum IMS101 and
Crocosphaera WH0005 cell lines at the end of the long-term selection period are shown in
Figure 2. After short-term (16 day) TPC incubations, the “increase-peak-decline” pattern was
found in all the TPCs, but the temperature range of Trichodesmium was 3~4°C wider than for
Crocosphaera (Fig. 2). The optimal temperature range of both diazotrophs was 27-30°C, and the
maximal growth rates of all Trichodesmium and Crocosphaera cell lines were ~0.30 day
-1
and
comparable (Fig. 2).
As shown in Fig. 2A, the growth rates of Trichodesmium cell lines selected under all
three different temperatures were congruent at each temperature across the entire TPC (one-way
ANOVA for all temperatures from 16°C-36°C individually, p value > 0.05). However, long-term
125
thermally-selected Crocosphaera cell lines responded differently at the two ends of the
temperature range (Fig. 2B). Under temperatures ranging from 24°C to 30°C, the thermally-
selected Crocosphaera cells had similar growth rates (one-way ANOVA, p value > 0.05).
However, the 22°C-selected cell lines had significantly higher growth rates than the 28°C and
32°C-selected cell lines at 22°C (one-way ANOVA and TukeyHSD multiple comparison, p
value <0.05), while the growth rates of the 32°C-selected cell lines were also higher at 32°C than
those of the 22°C- and 28°C- selected Crocosphaera (Fig 2B).
Notably, Crocosphaera selected at 32°C maintained a stable level of biomass at 34°C for
16 days, and were still able to fix nitrogen at the end of the two-week incubation (see below). In
contrast, at 34°C long-term cell lines selected at 22 and 28°C died within one week. At the
lowest temperature tested (20°C), however, 32°C-selected cell lines grew significantly more
slowly than 22°C- and 28°C-selected cells (one-way ANOVA and TukeyHSD multiple
comparison, p value < 0.05).
4.3.2 Nitrogen and carbon fixation rates
After two years’ thermal selection, nitrogen fixation (Fig. 3A) and carbon fixation (Fig.
3B) rates at the respective selection temperatures were highest for long-term 28°C-selected
Trichodesmium cells, followed by those of 32°C-selected cells, while the rates of 22°C-selected
Trichodesmium were the lowest. The difference in the carbon and nitrogen fixation rates between
long-term 28°C-selected and 22°C-selected Trichodesmium cell lines was significant (Fig. 3,
one-way ANOVA and Tukey multiple comparison, p value < 0.05).
During the two weeks after being moved to novel temperatures in the final TPC
experiments, long-term Trichodesmium cells showed a rapid acclimation. The nitrogen and
carbon fixation rates of cell lines selected at three different temperatures were not significantly
different at each individual temperature from 20°C to 34°C (Fig. 3, one-way ANOVA, p value >
0.05). In the 2-week TPC determination experiments, the nitrogen and carbon fixation rates of
Trichodesmium at 28°C were significantly higher (TukeyHSD multiple comparison, p value <
0.05) than at either the cold-end temperatures (20°C, 22°C) or the warm-end temperature 34°C
(Fig. 3, TukeyHSD multiple comparison, p value < 0.05).
Like Trichodesmium, long-term 28°C and 32°C-selected Crocosphaera cell lines had
identical nitrogen and carbon fixation rates at their selection temperatures, while 22°C-selected
cells had significantly lower rates (Fig. 4, one-way ANOVA and TukeyHSD multiple
126
comparison, p value < 0.05). After the two-week incubations at 22°C and 28°C, all the
temperature-selected Crocosphaera cell lines matched the carbon and nitrogen fixation rates of
22°C-selected and 28°C-selected cells, respectively (Fig. 4, one-way ANOVA and TukeyHSD
multiple comparison, p value > 0.05). In contrast, at the assay temperature of 32°C, long-term
22°C and 28°C-selected cell lines had lower nitrogen and/or carbon fixation rates than 32°C-
selected cells, although the difference was not always significant. In particular, the carbon
fixation rates of 28°C-selected and the nitrogen fixation rates of 22°C-selected cell lines
transferred to 32°C were significantly lower than those of 32°C-selected cells at the end of the
two-week TPC determination experiments (Fig. 4, p value < 0.05). In the switch experiments,
22°C-selected Crocosphaera cells were observed to have a significantly lower carbon fixation
rate after one-month’s transfer to 32°C, compared to the original 32°C-selected cells (p value <
0.05).
Notably, only 32°C-selected Crocosphaera cells survived and had the ability to fix a
considerable amount of carbon and nitrogen at 34°C. At this temperature the 28°C and 22°C-
selected cell lines stopped growing and nitrogen or carbon fixation rates were mostly below
detection limits (Fig. 4, TukeyHSD multiple comparison, p value < 0.05), providing more direct
evidence that the 32°C-selected cell lines were more tolerant of elevated temperature than the
other two cell lines. The letters in Fig. 4 represent the overall grouping results among all
treatments at four temperatures, and hence fail to reflect the significant differences detected by
TukeyHSD methods between nitrogen fixation of 28°C- and 22°C-selected Crocosphaera cells
and 32°C-selected cells when assayed at 34°C.
4.3.3 Elemental stoichiometry and Chl a to POC ratio
The C: N: P ratios of Trichodesmium and Crocosphaera cell lines had different responses
to long-term thermal selection (Fig. 5 & Table S1). The C: N ratio was relatively stable among
all the long-term Trichodesmium cell lines, although 22°C-selected cell lines ratios were slightly
but not significantly higher than in the 28°C and 32°C-selected cells (Fig. 5A, p > 0.05). The N:
P and C: P ratios (Fig. 5B and 5C) were lower in long-term 22°C-selected Trichodesmium cells
compared to 28°C and 32°C-selected Trichodesmium cell lines (one-way ANOVA and
TukeyHSD multiple comparison, p value < 0.05) and also below the Redfield ratio.
The elemental ratios of long-term Crocosphaera cell lines and their corresponding
responses in short-term treatments showed both similarities and distinct differences, compared to
127
Trichodesmium. No significant difference was found in the C: N ratios of the three long-term
temperature-selected Crocosphaera cell lines (Fig. 5A). N: P and C: P ratios of long-term
thermally-selected Crocosphaera cell lines showed a consistent pattern (Figs 5B and 5C). The
28°C-selected Crocosphaera cells had lower N: P and C: P ratios than 22°C- or 32°C-selected
cells. This difference was particularly pronounced for the N: P ratio (one-way ANOVA and
TukeyHSD multiple comparison, p value < 0.05).
After two years’ temperature selection, all the long-term Trichodesmium cell lines had
nearly identical Chl a: POC ratios (one-way ANOVA, p value > 0.05) (Fig. 5D). In contrast to
the long-term Trichodesmium cell lines, the Chl a: POC ratio of long-term 28°C-selected
Crocosphaera was highest among all the three long-term Crocosphaera cell lines, followed by
the 32°C-selected cells and then the 22°C-selected cells (Fig. 5D). The difference in the Chl a:
POC ratio between 28°C-selected and 22°C-selected Crocosphaera cell lines was significant
(one-way ANOVA, Tukey multiple comparison, p value < 0.05).
4.3.4 Trichome and cell size
After ~2 years’ selection, Crocosphaera cell sizes and Trichodesmium trichome sizes
were significantly different among the three thermal selection treatments (one-way ANOVA, p
value < 0.05; Fig 6). The 22°C-selected Trichodesmium trichomes were significantly wider and
longer than 28°C- and 32°C-selected trichomes (TukeyHSD multiple comparison, p value <
0.05; Fig 6A). Similarly, the 22°C-selected Crocosphaera cells were significantly bigger than the
cells selected at the other two temperatures (TukeyHSD multiple comparison, p value < 0.05; Fig
6B). In addition, 28°C-selected Trichodesmium trichomes and Crocosphaera cells were slightly
but not significantly smaller than the 32°C-selected ones (p value > 0.05).
4.4 Discussion
4.4.1 Plasticity versus adaptation in thermally-selected Trichodesmium and Crocosphaera
In short-term TPC determination experiments, the optimal temperature range and thermal
limits of Trichodesmium IMS101 and Crocosphaera WH0005 cell lines previously maintained
for ~2 years at optimal temperature (28°C) were consistent with previous laboratory and field
studies (Breitbarth et al. 2007; Foster et al. 2009; Moisander et al. 2010; Webb et al. 2009).
However, cell lines of the two diazotrophs selected at suboptimal (22°C) and supra-optimal
(32°C) temperatures displayed quite different long-term responses.
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Our results suggested that Trichodesmium IMS101 did not adapt to the three temperature
regimes during two years of thermal selection. It appears to instead possess a considerable
amount of inherent thermal plasticity, allowing it to quickly acclimate to temperature shifts
within the range of 18-34°C. In contrast, Hutchins et al. (2015) and Walworth et al. (2016) found
an irreversible adaptation of the same strain Trichodesmium IMS101 to the projected future CO2
level after being selected under that CO2 concentration for > 4.5 years. Walworth et al. (2016)
also found that this adaptive response happened following an initial plastic acclimation period, so
the fact that the selection time in our thermal study was shorter (~44%) than that in the CO2
selection experiment is one possible explanation for these differing responses to selection by
these two different global change drivers in the same Trichodesmium strain .
Alternatively, CO2 enrichment and temperature increase may very well elicit distinct
responses in Trichodesmium. In acclimated experiments, Hutchins et al. (2013) showed that
Trichodesmium and Crocosphaera isolates exhibited considerable strain-level variability in
growth responses to CO2, while Fu et al. (2014) found that TPCs were nearly identical for all the
strains examined within each genus. Jerney et al. (2019) found that like Trichodesmium in our
study, the dinoflagellate Alexandria ostenfeldii did not adapt to warming, but instead
demonstrated significant plastic responses. Investigations targeting metabolic and molecular
mechanisms will be needed for mechanistic understanding of the biochemical pathways involved
in the rapid acclimation of Trichodesmium to warming.
In contrast to Trichodesmium, true thermal adaptation may have occurred in
Crocosphaera WH0005 cell after 18 months of temperature selection. Adaptation was indicated
by the apparently stable shifts in thermal response curve parameters in cell lines at each selection
temperature. Distinct responses of 32°C-selected and 22°C-selected cell lines to cold and warm
temperature extremes were observed, supporting our second hypothesis. Another piece of
evidence suggesting true adaptation is that only 32°C-selected Crocosphaera could survive and
fix nitrogen at the most intense warming temperature of 34°C, while the two cell lines selected at
lower temperatures died quickly there. This pattern in the thermal range width of these 32°C-
selected Crocosphaera cell lines seems to support the “hotter is broader” model (Jin and Agustí
2018; Knies et al. 2009).
Selection history helps determine the responses of CO2-selected cultures of the
picoplankter Ostreococcus when transferred to a new environment ( Schaum et al. 2015).
129
Ostreococcus lineages selected at high CO2 levels (~1000 μatm) achieved a higher growth rate
than lineages selected at ambient CO2 levels (~400 μatm) when both were moved to a further
elevated CO2 concentration of 2000 μatm. In this view, prior exposure to projected future
conditions such as mild warming or CO2 enrichment could help phytoplankton maintain fitness
under more intense subsequent environmental changes.
4.4.2 The link between physiological plasticity and evolutionary adaptation
Previous studies show that adaptation can protect phytoplankton from extinction in new,
stressful environments (Bell and Collins 2008; Collins et al. 2014), but plasticity can also move
the population closer to a new phenotypic optimum (Ghalambor et al. 2007; Hendry et al. 2008).
Not surprisingly, many studies have found that diatoms (Anning et al. 2001), cyanobacteria
(Stomp et al. 2008), green algae (Kremer et al. 2018) and dinoflagellates (Jerney et al. 2019), can
respond to changing environmental factors (temperature, light and salinity) using inherent
plasticity instead of evolutionary adaptation. In recent experimental evolution studies, adaptive
responses to temperature, CO2 and salinity changes have also been reported in phytoplankton
taxa including Trichodesmium (Hutchins et al. 2015, Walworth et al. 2016), diatoms (Jin and
Agustí 2018; O'Donnell et al. 2018), green algae (Schaum et al. 2015; Padfield et al. 2016),
coccolithophores (Schlüter et al. 2014) and dinoflagellates (Kremp et al. 2016).
Notably, distinguishing between plastic acclimation and rapid adaptation can be difficult,
especially after the concept of “gradual plasticity” was proposed by Kremer et al. (2018). Even
studies using the same species and identical environmental variables can reach different
conclusions. For instance, Jerney et al. (2019) suggested that the dinoflagellate A. ostenfeldii
responded to warming and salinity change only using phenotypic plasticity, as noted above.
However, a previous study by Kremp et al. (2016) pointed out that genotypic shifts and
adaptation were expected to occur in the cyst pool of A. ostenfeldii under selection by warming
and salinity changes.
Although it remains unclear why particular phytoplankton species exhibit responses to
environment changes involving acclimation, adaptation, or both, the relationship between
phenotypic plasticity and evolutionary adaptation has been extensively discussed (Chevin et al.
2010; Collins 2011; Ghalambor et al. 2007; Price et al. 2003). On one side, plasticity may
facilitate evolution by maintaining a large population size, with greater genetic and mutational
variance (Collins et al. 2014; Draghi and Whitlock 2012; Lande 2009). On the other hand, if
130
acclimation alone increases fitness without a large trade-off or cost, plastic genotypes may be
shielded from selection pressure. Thus, further evolutionary adaptation might not occur
(Ghalambor et al. 2007; Price et al. 2003).
The latter situation may be why no apparent adaptation occurred for our Trichodesmium
after two years of thermal selection. The considerable capacity of Trichodesmium to quickly
acclimate to new temperatures may alleviate thermal selection, thus retarding evolution. In
contrast, due to its lack of thermal plasticity Crocosphaera may have responded to strong
selective pressure with genotypic shifts, especially at the challenging temperatures of 32°C and
22°C. Moreover, it is unclear whether adaptation was halted for Trichodesmium, or might occur
later. Despite this open question, our study may help provide insights into modes of correlation
between plasticity and evolution, including positive, negative, and timescale-dependent
interactions.
4.4.3 Genetic backgrounds and lifestyles of the two diazotrophs
Although mechanistic information accounting for distinct plastic and evolutionary
trajectories of the two diazotrophs under long-term thermal selection is lacking, their genetic
backgrounds and lifestyles can perhaps provide some hints for us to interpret their differences,
and to predict their possible strategies to maximize fitness in the future ocean. Since evolutionary
potential has commonly been acknowledged to be positively associated with the genotypic
diversity or genetic variation of the population (Collins et al. 2014; Ghalambor et al. 2007), the
large effective population sizes and fast reproduction rates of marine phytoplankton are thought
to facilitate the adaptive process (Bell and Collins 2008; Hutchins and Fu 2017).
Building on previous research, Trichodesmium and Crocosphaera share similarity in their
highly conserved genomes, and the relatively low genetic diversity of populations leading to
small effective population sizes (Bench et al. 2011; Orcutt et al. 2002; Walworth et al. 2015;
Zehr et al. 2007). Both of these traits would seem to be disadvantageous for rapid evolution.
However, Trichodesmium and Crocosphaera do not exhibit genomic streamlining, in contrast to
sympatric non-diazotrophic cyanobacteria such as Prochlorococcus and Synechococcus (Swan et
al. 2013). Instead, the two diazotrophs have abundant transposons and pseudogenes in their
genomes (Bench et al. 2011; Walworth et al. 2015). The large number of mobile genetic
elements in their genomes has been associated with the maintenance of phenotypic and genetic
diversity in their populations (Bench et al. 2011; Webb et al. 2009; Zehr et al. 2007). Moreover,
131
positive selection has been detected in the transposase genes of Crocosphaera (Mes and
Doeleman 2006), indicating the importance and sensitivity of these mobile elements in the
evolutionary process.
The Crocosphaera strain WH0005 used in this study is characterized by a large-cell
phenotype, some genetic redundancy and potentially better adaptation to nutrient limitation than
the small-cell type (Webb et al. 2009). The Trichodesmium IMS101 genome contains
widespread, highly expressed non-coding spacers with unclear functions. As a result, the coding
percentage in Trichodesmium IMS101 is ~60%, while in Crocosphaera it is ~75-80% (Walworth
et al. 2015). Recently, epigenetic cytosine methylation has also been described in Trichodesmium
IMS101 (Walworth et al. 2017). There is little information about whether these genomic and
epigenomic differences might account for the observed rapid plastic responses in
Trichodesmium, but they may be a starting point for future work exploring the molecular
mechanisms behind the differing thermal responses of the two diazotrophs.
The different lifestyles of Trichodesmium and Crocosphaera may also be involved in
their differing responses. For instance, Trichodesmium performs vertical migrations in the water
column using gas vacuoles, allowing them to take advantage of vertical light and nutrient
gradients in their surroundings (Zehr 2011). This reflects a physiological and behavioral capacity
to rapidly respond to environmental changes. Furthermore, Trichodesmium can live as free
trichomes, or form colonies along with sister cells and a consortium of other microbes (Lee et al.
2017; Orcutt et al. 2013). This interaction between colony-forming Trichodesmium and a diverse
bacterial microbiome has been widely observed in both natural populations and laboratory
cultures, and is thought to facilitate Trichodemium in important metabolic processes such as
nutrient uptake and nitrogen fixation (Lee et al. 2017). In this context, the responses of
Trichodesmium to environmental change may be modulated to some extent by the other
members of this consortium, and thus differ from those of largely free-living unicellular cells
such as the Crocosphaera WH0005 isolate used in this study. However, some Crocosphaera-like
cells have also been found to aggregate in colonies, and even be involved in cellular interactions
with a diatom in situ (Foster et al. 2013; Thompson and Zehr 2013), suggesting that the evolution
of natural Crocosphaera populations may also be influenced by other marine microbes.
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4.4.4 Possible impacts of future warming on cellular stoichiometry and biogeochemistry
The C: N: P ratio of the two diazotrophs varied with temperature change, and thus
deviated from the canonical Redfield ratio of 106:16:1 (Redfield 1958). This occurred at non-
optimal temperatures in most cases, suggesting that thermally-stressed growth and physiology
shifted the elemental ratios. Considering expected future warming conditions, these altered
elemental ratios are likely to change global marine biogeochemical cycles.
An elevated C: N ratio was observed at extreme temperatures (at the cold end for
Trichodesmium, and at both ends for Crocosphaera), compared to the ratio at optimal
temperatures. This suggests that at extreme temperatures, carbon export to the deep ocean may
increase relative to nitrogen. The effects of temperature in driving shifts in N: P and C: P ratios
seemed to be greater than the influence on C: N ratios for both diazotrophs. The N: P and C: P
ratios of 22°C-selected Trichodesmium cells were observed to be significantly lower than the
ratios of 28°C or 32°C-selected cells, and also lower than the Redfield ratio (Redfield 1958),
indicating that Trichodesmium accumulated less P relative to N at higher temperatures. A
reduction of P requirements relative to N in phytoplankton cells at warm temperatures was
proposed by Toseland et al. (2013), providing possible mechanistic explanation for this ratio
shift. In further support of this trend, Trichodesmium has been shown to dramatically increase its
phosphorus use efficiency (mol N fixed per mol cellular P per hour) with warming (Jiang et al.
2017, Qu et al. 2019). The elevated N: P and C: P ratios of Crocosphaera at 32°C and 34°C
suggest that in the future warm ocean, more C and N will be sequestered by Crocosphaera
relative to P.
The Chl a: C ratio is a commonly used photo-acclimation proxy in phytoplankton
research (Geider et al. 1997; Jakobsen and Markager 2016; Sathyendranath et al. 2009), and
showed huge flexibility in the two diazotrophs. While the identical Chl a: C ratio in all the long-
term Trichodesmium cell lines is another evidence of the notable plasticity of this species, the
positive correlation between the Chl a: C ratios and growth rates in temperature-selected
Crocosphaera cell lines may imply a greater sensitivity of Crocosphaera to environmental
control. A non-optimal growth status in Crocosphaera was coupled with reduced Chl a: C, and
declines in photosynthetic efficiency.
The coincidence between Chl a: C and P: C in Crocosphaera is in line with previous
research on the correlation between Chl a abundance and P supply (Guildford and Hecky 2000).
133
Moreover, in the temperature range of 20-34°C, the Chl a: C of Crocosphaera decreased not
only at the cold temperature end like 28°C and 32°C-selected Trichodesmium, but also at the
warm end. This variation pattern is opposite to the increase of C: N ratios at these extreme
temperatures, and may be attributed to the fact that Chl a is a major nitrogen pool in marine
phytoplankton cells (Geider and Roche 2002; Arrigo 2004; Li et al. 2015).
4.4.5 Future N2 fixation and species competition between the two diazotrophs
The two sympatric diazotrophic cyanobacteria Trichodesmium and Crocosphaera have
different morphologies (Prufert-Bebout et al. 1993; Webb et al. 2009), abundance and vertical
distributions (Church et al. 2005; Langlois et al. 2005), diel patterns (Shi et al. 2010; Tuit et al.
2004; Wilson et al. 2017) and nutrient utilization abilities (Dyhrman and Haley 2006; Knapp et
al. 2012). With various but successful strategies, both diazotrophs have adapted to the
contemporary oligotrophic central gyres, and contribute substantially to new nitrogen inputs in
the tropical and subtropical ocean (Bonnet et al. 2009; Goebel et al. 2010; Langlois et al. 2005;
Moisander et al. 2010; Westberry and Siegel 2006). How future warming will influence the
survival and competitive success of these two crucial diazotrophs is hence of paramount interest.
In our study, changes in growth, nitrogen-, and carbon-fixation rates with temperature
had generally parallel trends for the two diazotrophs. Within the optimal temperature range, the
growth and nitrogen/carbon fixation rates of two diazotrophs were comparable. Under the
warming condition (32°C), all those rates decreased compared to optimal temperatures,
suggesting the possibility of reduced nitrogen fixation and carbon sequestration in the future
ocean. Nevertheless, the growth and nitrogen fixation rates of the two diazotrophs were still
relatively equally matched at this fairly moderate level of warming.
When temperature was further increased to 34°C, nitrogen fixation of Trichodesmium
and surviving Crocosphaera decreased substantially, to one third or less of the amount at optimal
temperatures. However, the relative competitive fitness of the two diazotrophs might change
with extreme warming. All our Trichodesmium cell lines acclimated rapidly to 34°C, while only
32°C-selected Crocosphaera cell lines could survive and fix nitrogen at this elevated
temperature.
More frequent and intense marine heat-waves have already been observed, and further
increases are predicted by numerous studies for the future ocean (Frölicher and Laufkötter 2018;
Oliver et al. 2018; Scannell et al. 2016; Smale et al. 2019). Under these conditions, our results
134
suggest that Crocosphaera may fail to survive, or be outcompeted by Trichodesmium. However,
if given the chance to adapt to the gradual temperature increases which actually constitute the
mean rate of climate change, Crocosphaera may be able to widen their upper thermal limit to
34°C. This prediction differs from those based on short-term acclimated experiments (weeks, Fu
et al. 2014). Our study suggests that the thermal acclimation of Trichodesmium and potential
adaptation of Crocosphaera may help sustain the biodiversity of diazotrophs, and thus help
mitigate any declines in new nitrogen inputs in the warming near-future tropical and subtropical
ocean.
4.4.6 Size change, growth and carbon export
Cell size of phytoplankton is an important parameter impacting on their nutrient uptake,
sinking rate and trophic role. Small cell size has been thought to be an ecological advantage,
because the increased surface area: volume ratio of cells could improve the uptake efficiency of
nutrients (Miller and Wheeler 2012). Moreover, phytoplankton cell size has often been found to
vary with environmental change (Atkinson et al. 2003; Daufresne et al. 2009; Schlüter et al.
2014; Sommer et al. 2016).
In our study, low temperature-selected Trichodesmium trichome size and Crocosphaera
cell size was significantly larger than in the optimal temperature- or warming-selected cell lines
after ~2 years of selection. These results revealed an inverse relationship between cell size and
the thermally-determined growth rate. This temperature-induced size change has also been
observed in another strain of Crocosphaera WH5801 (Webb et al. 2009), as well as in other
phytoplankton species (Schlüter et al. 2014; Sommer et al. 2016). Interestingly, the study of
Bach et al. (2012) proposed that warming will increase the sinking velocity of organic particles
to the deep ocean. According to our research, however, the trend towards decreasing cell sizes
with temperature increase may tend to offset the effects of warming on sinking rates. Along with
the observed shifts in elemental stoichiometry, such altered cell sizes of long-term selected
Trichodesmium and Crocosphaera suggest possible changes in export and trophic dynamics
driven by temperature.
4.5 Conclusion
Long-term thermally-selected Trichodesmium IMS101 showed a quick re-acclimation
ability within the temperature range of 20-34°C. This type of phenotypic plasticity is thought to
be a “double-edged sword” relative to evolutionary adaptation, but no evidence of an adaptive
135
response was seen over the time frame of our experiments. In contrast, a true adaptation or
possibly a much slower plastic acclimation occurred in selected Crocosphaera WH0005 cell
lines. The finding that 32°C-selected Crocosphaera cells can survive and fix nitrogen at 34°C
indicates that as long as temperature increases are gradual, Crocosphaera may be able to adapt
and continue contributing to marine nitrogen fixation, despite moderate future warming. The
distinct responses of these two diazotrophs may stem from their different genetic backgrounds
and lifestyles. These contrasting thermal acclimation and adaptation responses of Trichodesmium
and Crocosphaera can help to provide insights into competitive interactions, niche partitioning,
and changes in new nitrogen supplies from these two key diazotrophs in the future tropical and
subtropical ocean.

136
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147
Chapter 4-Figures
Figure legend
Figure 1. The growth rates of Trichodesmium (A) and Crocosphaera (B) at 22
o
C, 28
o
C and
32
o
C, showing rates of the ancestral strain just before the initial transfer to long-term
cultivation at the three temperatures (left), as well as the three thermally-selected cell lines
at the end of the ~2-year selection period (right). Values and error bars represent the means
and standard deviations of triplicate cell cultures in each treatment.
Figure 2. Measured thermal response curves (TPC) based on growth rates of
Trichodesmium erythraeum IMS101 (A), solid lines; and Crocosphaera WH0005 (B), dashed
lines; in the two weeks after transferring cell lines thermally selected at three temperatures
(22
o
C, 28
o
C and 32
o
C) for ~2 years to the TPC. Values and error bars represent the means and
standard deviations of triplicate cell cultures in each treatment. The lines and shading show the
smoothing results using the loess method and the 95% confidence intervals.
Figure 3. Nitrogen (A) and carbon (B) fixation rates of long-term thermally-selected
Trichodesmium erythraeum IMS101, respectively normalized to PON and POC at five
temperatures at the end of the two week TPC incubation. Values represent the means and
error bars are the standard deviations of three replicates. Bars marked with an asterisk * show the
rates of the long-term cell lines at their corresponding selection temperatures. Bars marked with
different letters are significantly different from each other (p value < 0.05). The letters represent
the grouping results after the Tukey multiple comparison among all 15 treatments at five
temperatures.
Figure 4. Nitrogen (A and B) and carbon (C and D) fixation rates of long-term thermally-
selected Crocosphaera WH0005, respectively normalized to PON and POC at five
temperatures after the two week TPC incubation (A and C) and one month’ switch
experiment (B and D). Values represent the means and error bars are the standard deviations of
three replicates. Bars marked with an asterisk * show the rates of the long-term cell lines at their
corresponding selection temperatures. Bars marked with different letters are significantly
different from each other (p value < 0.05). The letters represent the grouping results after the
Tukey multiple comparison among all 12 treatments at four temperatures.
148
Figure 5. POC: PON (A), PON: POP (B), POC: POP (C) and Chl a: POC (D) ratios of
long-term thermally-selected Trichodesmium erythraeum IMS101 (solid fill) and
Crocosphaera WH0005 (diagonal stripes) cell lines at their selection temperatures (22°C-
selected: light grey; 28°C-selected: grey; 32°C-selected: dark grey). Values represent the
means and error bars are the standard deviations of three replicates. The letters a-d represent the
grouping results after the Tukey multiple comparison on all temperature-selected cell lines of
two species for each parameter.
Figure 6. Trichome length and width of long-term Trichodesmium erythraeum IMS101 (A,
length: solid fill; width: dot fill) and cell radius of long-term Crocosphaera WH0005 (B,
radius: diagonal stripes) at their selection temperatures (22°C-selected: light grey; 28°C-
selected: grey; 32°C-selected: dark grey). Values represent the means and error bars are the
standard deviations of three replicates. The letters a-b represent the grouping results after the
Tukey multiple comparison on three temperature-selected cell lines of two species respectively
for each parameter.

149

Figure 1.
0
0.1
0.2
0.3
0.4
Ancestral strains after initial transfer Selected strains after 2 years
22°C 28°C 32°C
0
0.1
0.2
0.3
0.4
22°C 28°C 32°C
Growth rate (day
-1
)
(A) Trichodesmium
(B) Crocosphaera
150

Figure 2.

0.0
0.1
0.2
0.3
0.4
16 18 20 22 24 26 28 30 32 34 36
Temperature (°C)
Specific growth rate (day
-1
)
22°C-selected
28°C-selected
32°C-selected
0.0
0.1
0.2
0.3
0.4
16 18 20 22 24 26 28 30 32 34 36
Temperature (°C)
Specific growth rate (day
-1
)
22°C-selected
28°C-selected
32°C-selected
(A) Trichodesmium
(B) Crocosphaera
151

Figure 3.

Specific N fixation rate
(10
-3
*hr
-1
)
Specific C fixation rate
(10
-3
*hr
-1
)
-5
5
15
25
35
45
20°C 22°C 28°C 32°C 34°C
22°C-selected 28°C-selected 32°C-selected
0
10
20
30
40
50
20°C 22°C 28°C 32°C 34°C
Temperature (°C)
d d d bc cd bcd a a a abc a ab bcd bcd bcd
cd d d bc ab ab ab a ab abc abc ab d d d
(A)
(B)
*
*
*
*
*
*
152

Figure 4.

0
10
20
30
40
50
22°C 28°C 32°C
22°C-selected 28°C-selected 32°C-selected
abc bc c a a ab bc abc abc
0
10
20
30
40
50
22°C 28°C 32°C 34°C
22°C-selected 28°C-selected 32°C-selected
0
10
20
30
40
50
60
22°C 28°C 32°C 34°C
Temperature (°C)
Specific N fixation rate
(10
-3
*hr
-1
)
Specific C fixation rate
on POC (10
-3
*hr
-1
)
(A) After 2 weeks
(D)
(B) After 1 month
(C) After 2 weeks
0
10
20
30
40
50
60
22°C 28°C 32°C
Temperature (°C)
c abc bc abc ab abc c abc a
(D) After 1 month
cde bcde cde abc a ab de abcd abc f f ef
cd c bc a a a ab c a d d bc
*
*
*
*
*
*
*
*
*
*
*
*
153

Figure 5.

5. 0
6. 0
6. 6
7. 0
8. 0
9. 0
10. 0
1 1. 0
22°C-selecte d 28°C-selecte d 32°C-selecte d
POC : PON (mol : mol )
T richodesmiu m Crocosphaera
10
15
16
20
25
22°C-selecte d 28°C-selecte d 32°C-selecte d
PON : POP (mol : mol )
50
100
106
150
200
22°C-selecte d 28°C-selecte d 32°C-selecte d
POC : POP (mol : mol )
dab cd bc c d a
0.15
0.20
0.25
0.30
22°C-selecte d 28°C-selecte d 32°C-selecte d
Chl a to POC (ug/umol )
bc ab c abc c a
bc c bca
ca b ab b a (B)
(D) ab
Selection condition Selection condition
bc
(A)
(C)
154

Figure 6.

2
2.5
3
3.5
4
Cell radius (µm)
22°C 28°C 32°C
0
2
4
6
8
10
0
200
400
600
800
1000
Trichome length (µm)
length width
Trichome width (µm)
(A) Trichodesmium
(B) Crocosphaera
a
b
a
b
b
b
a
b b
155
Chapter 4-Supplementary Materials
Supplementary Results

Elemental Ratio and Chl a: POC Shift in TPC Experiments
In the TPC determination experiment, 22°C-selected Trichodesmium cell lines had a
significantly higher C: N ratio when transferred to 18°C than at 26°C, 28°C, or 30°C (Table S1,
Tukey multiple comparison among all 22°C-to-new temperature treatments, p value < 0.05).
Likewise, after transferring the 28°C-selected Trichodesmium cell lines to the TPC, the C: N
ratio in the 18°C treatment was significantly higher than at all temperatures from 22°C to 34°C
(Table S1, TukeyHSD multiple comparison among all 28°C-to-new temperature treatments, p
value < 0.05). Notably, the grouping results shown as different letters in Table S1 were based on
the statistical analysis of all cell lines of each species, which was less sensitive than the
TukeyHSD multiple comparison only carried out on the cell lines selected by each individual
temperature. Nevertheless, similar patterns were found in the multiple comparisons and overall
grouping, as also happened for the N: P and C: P ratios.
In the TPC determination experiment, the N: P and C: P ratios in 28°C- and 32°C-
selected Trichodesmium cell lines did not change in the temperature range of 18°C to 34°C
(Table S1, one-way ANOVA and TukeyHSD multiple comparison, p value > 0.05). However,
22°C-selected Trichodesmium retained their lower N: P and C: P ratios at the cold end (18°C to
22°C), while the two ratios increased within two weeks to levels similar to the 28°C- and 32°C-
selected cell lines at other temperatures (24°C to 34°C). In particular, the N: P ratios in the 22°C-
to-18°C, 20°C and 22°C treatments were significantly lower than in the 22°C-to-30°C, 32°C and
34°C treatments (Table S1, TukeyHSD multiple comparison, p value < 0.05). A significant
increase in the C: P ratios was also observed in the 22°C-to-30°C, 32°C and 34°C treatments
compared with the 22°C-to-20°C treatment (TukeyHSD multiple comparison, p value < 0.05)
(Table S1).
The C: N ratios of Crocosphaera cell lines were slightly higher at the two extremes of
the temperature range of 18°C to 34°C in the two-week TPC determination (Table S1). In some
cases, this increment was found to be significant. For instance, the C: N ratio of the 28°C-
selected to18°C assay treatment was significantly higher than the ratios of the 28°C-selected to-
22°C, 24°C, 26°C, 28°C, and 30°C assay treatments (Table S1, one-way ANOVA and
156
TukeyHSD multiple comparison, p value < 0.05), and also higher than the Redfield ratio.
Moreover, in the TPC determination experiment using the long-term 32°C cells, the C: N ratios
in the 32°C-selected to 30°C, 32°C, and 34°C assay treatments were significantly higher than
those of the 32°C-selected to 24°C assay treatment (Table S1, one-way ANOVA and TukeyHSD
multiple comparison, p value < 0.05).
In the temperature range of 20°C to 34°C in the TPC determination experiments, N: P
and C: P ratios of all the long-term Crocosphaera cell lines were generally higher at the warm
end than at the cold end (Table S1). The N: P ratio of the 28°C-selected was significantly lower
after transfer to the 18°C assay treatment, than in the 30°C, 32°C, and 34°C assay treatments
(TukeyHSD multiple comparison, p value < 0.05). The N: P ratio was also significant higher in
the 32°C-selected cells transferred to 34°C, relative to 22°C (Table S1, TukeyHSD multiple
comparison, p value < 0.05). Moreover, the C: P value in the 32°C-selected Crocosphaera
moved to 34°C was significantly higher compared to all temperatures from 18°C to 26°C (Table
S1, Tukey multiple comparison, p value < 0.05).
In the TPC determination experiment, the Chl a: POC ratio of 22°C-selected
Trichodesmium stayed relatively stable across the temperature range of 20°C to 34°C, although
the ratio was significantly decreased at 18°C compared to assay temperatures from 24°C to 32°C
(Tukey multiple comparison, p value < 0.05) (Fig. S1A). The Chl a: POC ratio of
Trichodesmium cultures transferred from the long-term 28°C and 32°C treatments decreased at
cold-end assay temperatures (18°C, 20°C and 22°C) after two weeks, compared to optimal or
warm-end temperatures (Fig. S1A). For instance, the Chl a: POC ratio in the 32°C-selected to
20°C assay treatment was significantly lower than for cells transferred to the 34°C assay
treatment (Tukey multiple comparison, p value < 0.05). When 28°C-selected Trichodesmium
were moved to 18°C and 20°C, significant decreases in Chl a: PO C ratios were found compared
to temperatures from 24°C to 34°C (Tukey multiple comparison, p value < 0.05).
In the TPC experiment, the Chl a: POC ratio in all three thermally selected Crocosphaera
cell lines was lower at both low and high temperature extremes, compared to the optimal
temperatures (26-30°C) (Fig. S1B). Significant decreases in the Chl a: POC ratios were observed
from 18°C-24°C and 34°C for 22°C-selected cells, from 18-20°C and at 34°C for 28°C-selected
cells, and from 18°C-22°C for 32°C-selected cells, compared to their corresponding ratios in the
optimal growth range of 26-30°C (Fig. S1B, one-way ANOVA, Tukey multiple comparison, p
157
value < 0.05). Especially, the Chl a: POC of the 32°C-selected Crocosphaera cell line was
higher at 34°C and lower at 22°C compared to 22°C- and 28°C-selected cell lines, although the
difference was not significant (one-way ANOVA, p value > 0.05).

158
Table S1. Elemental stoichiometry (C: N, N: P, and C: P) of long-term Trichodesmium
erythraeum IMS101 (A) and Crocosphaera WH0005 (B) two weeks after transfer to the TPC
from 18-34°C. The values in the table are the averages of triplicates ± standard deviations. The
letters under “Grouping” in the table show the grouping results among temperature treatments of
each species.
“NA”: not available. The 22°C- and 28°C-selected Crocosphaera cell lines could not survive at
34°C, and so did not produce enough biomass for elemental ratio analyses.

Selection-temp
TPC
treatment
Elemental ratios (mol : mol)
POC : PON Grouping PON : POP Grouping POC : POP Grouping
Trichodesmium
22°C-selected 18°C 6.59 ± 0.56 abc 13.12 ± 2.31 bc 86.24 ± 13.96 abc
22°C-selected 20°C 5.89 ± 0.61 abc 11.39 ± 1.91 c 67.48 ± 16.58 c
22°C-selected 22°C 5.89 ± 0.73 abc 13.08 ± 4.05 bc 75.26 ± 17.10 bc
22°C-selected 24°C 5.50 ± 0.15 bc 18.75 ± 2.20 abc 103.41 ± 14.83 abc
22°C-selected 26°C 5.22 ± 0.43 c 22.17 ± 2.35 abc 115.10 ± 5.37 abc
22°C-selected 28°C 5.23 ± 0.20 c 19.01 ± 2.66 abc 99.19 ± 10.87 abc
22°C-selected 30°C 5.20 ± 0.14 c 25.18 ± 3.20 a 130.99 ± 17.52 ab
22°C-selected 32°C 5.61 ± 0.46 bc 22.91 ± 6.37 abc 128.43 ± 35.17 abc
22°C-selected 34°C 5.58 ± 0.28 bc 23.99 ± 4.47 ab 134.64 ± 30.68 ab
28°C-selected 18°C 7.64 ± 0.24 a 15.06 ± 1.17 abc 114.84 ± 5.75 abc
28°C-selected 20°C 6.71 ± 0.58 abc 16.81 ± 2.03 abc 113.36 ± 23.30 abc
28°C-selected 22°C 6.35 ± 0.14 abc 17.40 ± 1.77 abc 110.70 ± 13.57 abc
28°C-selected 24°C 5.91 ± 0.15 abc 18.00 ± 5.99 abc 106.28 ± 34.42 abc
28°C-selected 26°C 6.01 ± 0.05 abc 23.52 ± 9.42 ab 110.75 ± 27.39 abc
28°C-selected 28°C 5.85 ± 0.44 abc 17.07 ± 1.24 abc 103.34 ± 8.45 abc
28°C-selected 30°C 5.78 ± 0.18 bc 19.24 ± 0.86 abc 109.67 ± 6.34 abc
28°C-selected 32°C 5.99 ± 0.27 abc 15.65 ± 3.57 abc 95.12 ± 20.89 abc
28°C-selected 34°C 6.49 ± 0.53 abc 21.32 ± 3.68 abc 140.36 ± 25.50 a
32°C-selected 18°C 6.36 ± 1.06 abc 17.26 ± 0.46 abc 116.87 ± 15.44 abc
32°C-selected 20°C 6.36 ± 1.75 abc 18.40 ± 4.26 abc 124.33 ± 6.34 abc
32°C-selected 22°C 5.81 ± 0.04 abc 17.15 ± 5.12 abc 100.60 ± 21.64 abc
32°C-selected 24°C 6.28 ± 0.69 abc 17.17 ± 3.29 abc 109.29 ± 33.34 abc
32°C-selected 26°C 6.23 ± 0.35 abc 19.30 ± 1.99 abc 120.64 ± 19.35 abc
32°C-selected 28°C 5.60 ± 0.33 bc 20.65 ± 1.14 abc 115.79 ± 12.89 abc
32°C-selected 30°C 6.54 ± 0.11 abc 17.42 ± 2.86 abc 114.03 ± 20.03 abc
32°C-selected 32°C 6.30 ± 0.16 abc 20.38 ± 2.49 abc 128.50 ± 17.53 abc
32°C-selected 34°C 7.17 ± 0.69 ab 16.58 ± 1.53 abc 118.22 ± 1.25 abc
Crocosphaera
159
22°C-selected 18°C 8.98 ± 0.33 ab 18.61 ± 3.56 ab 166.16 ± 26.42 abc
22°C-selected 20°C 8.39 ± 2.03 ab 21.05 ± 1.99 ab 174.14 ± 29.17 abc
22°C-selected 22°C 7.75 ± 1.43 ab 23.05 ± 2.07 a 178.16 ± 34.10 abc
22°C-selected 24°C 7.77 ± 1.73 ab 22.32 ± 6.60 a 166.02 ± 21.08 abc
22°C-selected 26°C 8.02 ± 0.71 ab 22.41 ± 2.91 a 178.35 ± 7.38 abc
22°C-selected 28°C 7.03 ± 0.98 ab 23.99 ± 4.90 a 165.87 ± 13.67 abc
22°C-selected 30°C 8.18 ± 0.45 ab 23.90 ± 1.05 a 195.46 ± 11.46 ab
22°C-selected 32°C 7.53 ± 1.36 ab 21.57 ± 0.70 ab 194.54 ± 29.39 ab
22°C-selected 34°C NA NA NA NA NA NA
28°C-selected 18°C 9.80 ± 0.69 a 11.28 ± 0.99 b 110.10 ± 17.19 c
28°C-selected 20°C 7.83 ± 0.76 ab 15.72 ± 0.25 ab 123.19 ± 13.89 bc
28°C-selected 22°C 6.77 ± 0.31 b 19.83 ± 1.42 ab 133.97 ± 4.56 bc
28°C-selected 24°C 6.61 ± 0.53 b 18.70 ± 1.62 ab 123.50 ± 13.58 bc
28°C-selected 26°C 6.96 ± 0.25 ab 18.51 ± 1.85 ab 128.71 ± 12.71 bc
28°C-selected 28°C 7.19 ± 0.29 ab 18.25 ± 3.63 ab 130.91 ± 23.72 bc
28°C-selected 30°C 7.47 ± 0.26 ab 20.62 ± 4.11 ab 154.00 ± 30.93 abc
28°C-selected 32°C 7.95 ± 1.17 ab 21.29 ± 5.36 ab 171.57 ± 57.73 abc
28°C-selected 34°C NA NA NA NA NA NA
32°C-selected 18°C 7.91 ± 0.76 ab 18.38 ± 4.67 ab 147.20 ± 47.45 abc
32°C-selected 20°C 8.53 ± 0.80 ab 17.07 ± 1.20 ab 145.81 ± 17.88 abc
32°C-selected 22°C 7.54 ± 0.35 ab 20.21 ± 1.04 ab 153.95 ± 2.32 abc
32°C-selected 24°C 6.47 ± 0.48 b 19.08 ± 2.05 ab 123.97 ± 19.57 bc
32°C-selected 26°C 6.96 ± 0.70 ab 21.35 ± 1.45 ab 149.17 ± 24.82 abc
32°C-selected 28°C 7.22 ± 0.66 ab 24.20 ± 2.51 a 174.02 ± 14.62 abc
32°C-selected 30°C 9.20 ± 0.79 ab 21.12 ± 2.86 ab 193.35 ± 19.27 ab
32°C-selected 32°C 8.90 ± 1.09 ab 22.22 ± 0.38 a 197.95 ± 27.63 ab
32°C-selected 34°C 8.83 ± 1.02 ab 25.52 ± 5.13 a 222.18 ± 23.89 a

160
Figure. S1 Chl a to POC ratio of long-term thermally-selected Trichodesmium erythraeum
IMS101 (solid line) (A) and Crocosphaera WH0005 (dashed line) (B) two weeks after
transfer to the thermal performance curve from 18-34°C (22°C-selected: light grey; 28°C-
selected: grey; 32°C- selected: dark grey). Values represent the means and error bars are the
standard deviations of three replicates. The letters a-i show the grouping results of all the 27
treatments for each species, based on the TukeyHSD multiple comparison.

Figure S1.

0.0
0.1
0.2
0.3
18°C 20°C 22°C 24°C 26°C 28°C 30°C 32°C 34°C
22°C-selected
28°C-selected
32°C-selected
0.0
0.1
0.2
0.3
18°C 20°C 22°C 24°C 26°C 28°C 30°C 32°C 34°C
Temperature (°C)
22°C-selected
28°C-selected
32°C-selected
Chl a :POC(ug/umol)
(A) Trichodesmium
(B) Crocosphaera
bcedf abcd abcde abc abcd abc a abcd abcdef
ff cdefabcdabcdefabcdefabcd ababcdef
ef f def abcdef abcdef abcdef abcde abcd abcd
ifghi cdefghdefghiabcde aba aefghi
hi defghi bcdefg abcdef abcd abcd abcde abcd fghi
ighi defghiabcdef abc abc abc abcd cdefgh
Chl a :POC(ug/umol)
161
Chapter 5: Dissertation Conclusion
5.1 Major Achievements and Implications for Future Studies
This dissertation included three data chapters with multiple objectives, all focused on
different aspects of how key phytoplankton groups may respond in the future changing ocean. As
a whole, this dissertation achieved number of major outcomes and pointed to several important
implications for current and future global change studies of marine phytoplankton. First, the
interactions of multiple stressors need to be taken into account. The results of the first data
chapter suggested that all three climate change variables (warming, elevated CO2 and nitrate
supply) regulated the growth and physiology of the diatom Coscinodiscus. Synergistic and
antagonistic effects among all the variables were observed in various physiological proxies. For
instance, the overall effect of warming and increased nitrate concentration exceeded the additive
impacts of the two variables. However, growth stimulation by warming was coupled with a
decreased growth affinity for nitrogen, implying that it may become more challenging for
Coscinodiscus to grow under the warmer and more severely nitrate-limited conditions expected
in the much of the future ocean.
Moreover, the significance of each variable differed for the different physiological
proxies measured. For instance, CO2 increase played a bigger role in enhancing carbon uptake
rate than it did in promoting the growth of Coscinodiscus. In the second chapter, the diazotrophic
cyanobacterium Trichodesmium responded distinctly to thermal variability under phosphate-
replete and phosphate-limited conditions, suggesting a strong interaction between phosphate
availability and thermal variation. This interaction also indicated limitations exist in applying the
current non-linear averaging model under nutrient-limited conditions, and suggested further
refinements may be needed to accurately predict growth in dynamic thermal environments when
they are paired with commonplace and widespread resource shortages (Bernhardt et al. 2018). In
short, the effects of multiple environmental factors on marine phytoplankton are not simply the
sum of their individual impacts. To more accurately predict the responses of marine
phytoplankton to climate changes, future studies should examine multiple changing variables.
Second, less-studied climate change variables such as environmental variability may play
a large but relatively underexplored role in the growth and physiology of marine phytoplankton.
Unlike ocean acidification, warming and changing nutrient supply, the impact of environmental
variability on marine phytoplankton has received little attention until recently. The second
162
chapter indicated that intensified thermal variability can significantly stress the growth and
nitrogen/carbon fixation of Trichodesmium, and is thus likely to profoundly change the global
carbon and nitrogen cycles. In future research, other previously neglected climate change
variables should be studied, along with those that have long been recognized as being important.
Third, in general the effects of major nutrient availability override those of temperature
change in the studies presented in this dissertation. For instance, the results of the first chapter
suggested that sufficient nitrate supply can promote the growth of Coscinodiscus sp. by 200-
300% relative to nitrate-limited samples, while realistically expected levels of warming can only
increase growth by 30-40%. According to the results of the second data chapter, Trichodesmium
largely lost the ability to respond to thermal variability under phosphate-limited conditions.
Although this is a consistent pattern that has also been observed in multiple previous studies
(Marañón et al. 2014, 2018; Taucher and Oschlies 2011), studies will still be needed to examine
the interactions of temperature and nutrients under other environmental conditions, for other
nutrients such as required trace elements, and using different phytoplankton species, to explore
whether the relative importance of these two climate change factors would be inverted in other
circumstances.
Fourth, the responses to climate change variables of phytoplankton are often group- or
species-specific. The first chapter examined the responses of the large centric diatom
Coscinodiscus to a combination of three climate change variables. Based on previous studies,
however, diatoms as a group show diverse responses to various climate change variables, as
discussed in the Introduction to the Dissertation. Other convincing evidence is presented in the
third data chapter, which showed that Trichodesmium IMS101 took advantage of its considerable
physiological plasticity to acclimate to temperature changes, while the other diazotrophic
cyanobacterium Crocosphaera WH0005 appears to deal with warming through true thermal
adaptation. In this context, there is a need to carry out more comparative studies on multiple
keystone phytoplankton species to better understand and predict the coming changes in marine
phytoplankton community structure, food-web dynamics and biogeochemical cycling in the
future ocean.
Fifth, long-term evolutionary studies can provide valuable information to reveal the
adaptive responses of phytoplankton to gradual climate changes. In addition to the physiological
methods used to determine phenotypic changes in the third data chapter, omics methods can help
163
to reveal the molecular mechanisms behind acclimation and adaptive responses of
phytoplankton. Global climate change is a long-term process, so studies that address only short-
term physiological responses of marine phytoplankton to environmental changes could be biased
or misleading. For instance, in the long-term study presented here, Crocosphaera was able to
broaden its thermal limit to 34°C after long-term thermal selection at 32°C, whereas previous
short-term studies suggested this diazotroph species could not survive at this temperature (Fu et
al. 2014). Moreover, other long-term studies by Hutchins et al. (2015) and Walworth et al.
(2016) have revealed an irreversible upregulation of nitrogen fixation in long-term high CO2
selected Trichodesmium IMS101, even after being transferred back to ambient CO2 level. This
remarkable finding could not have been acquired in short term experiments. Thus, long-term
experimental evolutionary research is a requisite for future research. Genomics, transcriptomics
and proteomics samples were collected and will be used provide in-depth mechanistic insights
into the physiological evolutionary responses described here. This will be the next step for the
study presented in the third data chapter, although these omics results are not included as part of
this dissertation.
In short, the results in this dissertation revealed the short-term and long-term responses of
keystone marine phytoplankton species to the interactions of multiple climate change variables.
More work is needed to involve more environmental factors and phytoplankton species on longer
timescales, and to further investigate the molecular mechanisms behind the observed
physiological responses. Besides these major recommendations for additional studies, there are
other minor implications for future research. For instance, additional physiological parameters
such as respiratory rate and photosynthetic capacity could be useful when examining the
responses of phytoplankton to environmental changes. Moreover, field work involving natural
communities with multiple trophic levels and competition experiments utilizing multiple
phytoplankton species would be useful ways to provide more information for making predictions
of changes in species composition in the future ocean.
5.2 Contributions and Broader Impacts
The scientific results from my dissertation have been published in part, and the remainder
will continue to be disseminated internationally through publications and presentations, thereby
shedding light on outstanding questions in the ocean global change field. The work in this
dissertation helped us to better understand the interactions of multiple global changes on the
164
growth, elemental stoichiometry and silicification processes of a representative marine diatom,
as well as leading to a better prediction of the physiological responses and possible evolutionary
adaptation of two keystone diazotrophic cyanobacteria to future warming conditions. This
project also provided insights into the potential trends of marine primary productivity and
biogeochemistry under future climate change scenarios and shed light on possible directions for
future research in environmental management and policy of marine ecosystems. The scientific
data produced during this project could also be used help to inform non-governmental
organizations concerned with environmental protection, or local fishery and tourism associations.
This project also provided training opportunities in biological oceanography and
physiological methods for two undergraduate students at USC, Mariam Elghazzawy of Human
Biology, and Megan Huh of the Department of Preventative Medicine. Through hands-on
experiments in the laboratory under my supervision, they have acquired practical experimental
skills and been inspired by natural science while carrying out their own research projects in our
lab. Thus, my dissertation work made a contribution to both undergraduate and graduate
education at USC. Perhaps the most important impact of my dissertation, though, is to provide
critically needed scientific information about how key phytoplankton functional groups and
related ocean biogeochemical cycles may respond to a rapidly changing ocean environment.

165
References
Bernhardt, J.R., Sunday, J.M., Thompson, P.L., & O'connor, M.I. (2018). Nonlinear averaging of
thermal experience predicts population growth rates in a thermally variable environment.
Proceedings of the Royal Society B: Biological Sciences, 285(1886), 20181076.
Fu, F. X., Yu, E., Garcia, N. S., Gale, J., Luo, Y., Webb, E. A., & Hutchins, D. A. (2014).
Differing responses of marine N2 fixers to warming and consequences for future
diazotroph community structure. Aquatic Microbial Ecology, 72, 33-46.
Hutchins, D. A., Walworth, N. G., Webb, E. A., Saito, M. A., Moran, D., McIlvin, M. R., ... &
Fu, F. X. (2015). Irreversibly increased nitrogen fixation in Trichodesmium
experimentally adapted to elevated carbon dioxide. Nature Communications, 6, 8155.
Marañón, E., Cermeño, P., Huete-Ortega, M., López-Sandoval, D. C., Mouriño-Carballido, B., &
Rodríguez-Ramos, T. (2014). Resource supply overrides temperature as a controlling
factor of marine phytoplankton growth. PloS One, 9(6), e99312.
Marañón, E., Lorenzo, M.P., Cermeño, P., & Mouriño-Carballido, B. (2018). Nutrient limitation
suppresses the temperature dependence of phytoplankton metabolic rates. The ISME
Journal, 12, 1836-1845.
Taucher, J., & Oschlies, A. (2011). Can we predict the direction of marine primary production
change under global warming? Geophysical Research Letters, 38(2), L02603.
Walworth, N. G., Lee, M. D., Fu, F., Hutchins, D. A., & Webb, E. A. (2016). Molecular and
physiological evidence of genetic assimilation to high CO2 in the marine nitrogen fixer
Trichodesmium. Proceedings of the National Academy of Sciences, 113(47), E7367-
E7374.

Abstract (if available)

Abstract Several functional groups of marine phytoplankton groups play especially critical roles in global primary productivity, carbon export and biogeochemistry. In particular, diatoms substantially contribute to primary production, carbon sequestration and the food web. Diazotrophic cyanobacteria are equally important as a source of new nitrogen (N) through nitrogen fixation. The relationship between environmental forcing and these key marine phytoplankton groups needs more attention, especially in the context of future global change. ❧ Elevated atmospheric concentrations of the greenhouse gas carbon dioxide (CO₂) are bringing about ocean acidification and giving rise to sea surface warming. By the end of this century, atmospheric CO₂ concentration is predicted to rise from current levels of ∼415 parts per million (ppm) to 800-1150 ppm, while average surface seawater temperatures will increase by 2-4℃. Global warming will also result in indirect changes, such as intensified stratification of seawater, increased temperature variability and decreased nutrient inputs from the interior of oceans. Phytoplankton communities in the euphotic zone will be forced to accommodate to all of these concurrent climate change trends. ❧ The three research chapters of this dissertation aim to increase our understanding of the possible responses of marine diatoms and diazotrophic cyanobacteria to this changing ocean environment. The first project investigated the physiological responses of the widespread centric diatom Coscinodiscus sp. to interactions between elevated CO₂, warming, and nitrogen availability. The growth and physiology of Coscinodiscus were found to be regulated by both individual and interactive effects of these three variables. Growth stimulation by warming was coupled with a decreased growth affinity for nitrate, indicating increased vulnerability to nitrate-limitation. Rising CO₂ promoted carbon fixation rates, which may provide a negative feedback to increasing atmospheric CO₂. Cellular phosphorus (P) and silicon (Si) contents declined and consequently N: P and C: Si ratios were elevated at high temperature, especially at ambient CO₂ level. Moreover, numerous mutual interactions among the three climate change variables were observed for growth rates, carbon fixation rates, cellular Chl a content and elemental stoichiometry, suggesting that more attention should be paid to the overall effects of multiple climate change variables rather than only to their independent impacts. ❧ The second chapter examined how short-term thermal variability affected the growth and physiology of Trichodesmium erythraeum GBRTRLI101 in two seasons (winter / summer), as well as the interaction between temperature variation and phosphate availability. P limitation played a bigger role in stressing the growth of Trichodesmium than temperature variability. The responses of Trichodesmium to thermal variability varied with the seasonal temperature regimes and ambient nutrient availability. In particular, thermal variability significantly decreased the growth and nitrogen and carbon fixation rates of Trichodesmium cells under P-replete conditions in wintertime. Increases in cellular phosphorus use efficiencies of Trichodesmium were observed at high temperature, suggested a lower P requirement in the future warming ocean. This chapter incidentally illustrated that a nonlinear averaging model commonly used to predict growth rates under fluctuating temperatures is not appropriate to apply to nutrient-limited cells. Future warming and greater thermal variability could significantly interact with projected reduced P supplies, and thus impact the growth and physiology of marine diazotrophic cyanobacteria. ❧ The third chapter explored the short-term physiological responses and potential for evolutionary adaptation of Trichodesmium erythraeum IMS101 and Crocosphaera WH0005 after long-term exposure to different temperatures. Long-term thermally-selected Trichodesmium IMS101 showed a quick re-acclimation to temperature shifts from 20-34℃, while a true thermal adaptation or possibly a much slower plastic acclimation occurred in Crocosphaera WH0005. A remarkable finding was that 32℃-selected Crocosphaera cells can survive and fix nitrogen at a normally lethal temperature of 34℃, suggesting that Crocosphaera can adaptively extend its upper thermal limit if warming occurs gradually. These distinct responses of Trichodesmium and Crocosphaera may stem from their different genetic backgrounds and lifestyles. These results provide insights into evolutionary versus plastic responses, competitive interactions, niche partitioning, and changes in new nitrogen supplies from these two keystone diazotrophs in the future tropical and subtropical ocean. ❧ The three studies in this dissertation help us to better understand the synergistic effects of multiple climate change variables on marine diatom growth, elemental stoichiometry and the silicification process. They also provide unique information on the interactions of thermal variability and nutrient limitation on the growth and physiology of an important marine diazotroph, and allow better predictions of short-term acclimation and possible evolutionary adaptation to future warming conditions in two representative nitrogen-fixing cyanobacteria. Together, this dissertation research provides novel insights into the potential trends of marine primary productivity and biogeochemistry under future ocean global change scenarios.

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Creator Qu, Pingping (author)

Core Title Thermal acclimation and adaptation of key phytoplankton groups and interactions with other global change variables

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Biology (Marine Biology and Biological Oceanography)

Publication Date 07/18/2019

Defense Date 05/01/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag carbon dioxide,climate change,diatom,diazotrophic cyanobacteria,nutrient availability,OAI-PMH Harvest,temperature

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Advisor Hutchins, David Allen (committee chair), Capone, Douglas George (committee member), Feakins, Sarah J. (committee member), Sanudo-Wilhelmy, Sergio (committee member)

Creator Email ppqu2012@gmail.com,pqu@usc.edu

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