University of Southern California Dissertations and Theses

Future impacts of warming and other global change variables on phytoplankton communities of coastal Antarctica and California

(USC Thesis Other)

Future impacts of warming and other global change variables on phytoplankton communities of coastal Antarctica and California

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FUTURE IMPACTS OF WARMING AND OTHER GLOBAL CHANGE
VARIABLES ON PHYTOPLANKTON COMMUNITIES OF
COASTAL ANTARCTICA AND CALIFORNIA

Zhi Zhu

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOLOGY)

May 2017

Approved by Advisory Committee:
Professor David Hutchins (Chair)
Professor Feixue Fu dddd
Professor Douglas Hammond ddd

ii
This document was formatted in accordance to the author guidelines specified for the Journal of
Harmful Algae.

iii
Table of Contents
Acknowledgments .......................................................................................................................... iv
List of Tables .................................................................................................................................. v
List of Figures ................................................................................................................................ vi
Introduction ..................................................................................................................................... 1
Chapter 1: A comparative study of iron and temperature interactive effects on diatoms and
Phaeocystis antarctica from the Ross Sea, Antarctica ................................................................. 22
Chapter 2: Individual and interactive effects of warming and CO
2
on Pseudo-nitzschia
subcurvata and Phaeocystis antarctica, two dominant phytoplankton from the Ross Sea,
Antarctica ...................................................................................................................................... 61
Chapter 3: Understanding the blob bloom: warming increases toxicity and abundance of the
harmful bloom diatom Pseudo-nitzschia in California coastal waters. ........................................ 99
Conclusions ................................................................................................................................. 129
Comprehensive list of references ................................................................................................ 136

iv
Acknowledgments
I would like to thank my advisor, Dr. David A. Hutchins, and Dr. Feixue Fu for being
encouraging and supporting from the beginning of the journey. I appreciate all the support, help,
patience, and advice you have given me. To Dr. Doug Hammond for serving as my qualification
committee and dissertation committee and giving me valuable suggestions. To all the other
faculties, lab mates, and friends who helped in the five years. I also would like to thank
California Sea Grant, University of Southern California, and the National Sciences Foundation
for funding this research. Finally, I would like to thank my parents, my wife Ling, and baby
Derek, I couldn’t finish this journey without your support.

v
List of Tables
Chapter 1 Table 1: Elemental ratios of Pseudo-nitzschia subcurvata, Chaetoceros sp.,
Fragilariopsis cylindrus, and Phaeocystis antarctica cultures ..................................................... 52
Chapter 2 Table 1: The measured pH and dissolved inorganic carbon (DIC), and calculated pCO
2

of Pseudo-nitzschia subcurvata and Phaeocystis antarctica at 2°C and 8°C ............................... 86
Chapter 2 Table 2: Optimum temperature, maximum growth rate and temperature niche width of
Pseudo-nitzschia subcurvata and Phaeocystis antarctica ............................................................ 87
Chapter 2 Table 3: The C quota, N quota, P quota, Si quota, and chl a per cell of Pseudo-
nitzschia subcurvata and Phaeocystis antarctica over a series of CO
2
levels .............................. 88
Chapter 2 Table 4: The maximum growth rate and half saturation constants (K
m
) of Pseudo-
nitzschia subcurvata and Phaeocystis antarctica ......................................................................... 89
Chapter 2 Table 5: The elemental ratios and C: Chl a ratios of P. subcurvata and P. antarctica at
2°C and 8°C .................................................................................................................................. 90
Chapter 2 Table 6: The C quota, N quota, P quota, Si quota, and chl a per cell of P. subcurvata
and P. antarctica at 2°C and 8°C .................................................................................................. 91
Chapter 3 Table 1: The elemental ratios of of cultured Southern California Pseudo-nitzschia
australis isolate S7 and phytoplankton assemblages from SB1 ................................................. 120

vi
List of Figures
Chapter 1 Fig. 1: Specific growth rates and Q10 of Pseudo-nitzschia subcurvata, Chaetoceros
sp., Fragilariopsis cylindrus, and Phaeocystis antarctica cultures .............................................. 55
Chapter 1 Fig. 2: Carbon fixation rates, iron uptake rates, and Fe:C uptake ratios of Pseudo-
nitzschia subcurvata, Chaetoceros sp., Fragilariopsis cylindrus, and Phaeocystis antarctica
cultures .......................................................................................................................................... 56
Chapter 1 Fig. 3: Particulate organic carbon (POC), nitrogen (PON), and phosphorus (POP), and
biogenic silica (BSi) content of Pseudo-nitzschia subcurvata, Chaetoceros sp., Fragilariopsis
cylindrus, and Phaeocystis antarctica cultures ............................................................................. 57
Chapter 1 Fig. 4: Cell volume, surface area to cell volume ratios, and relationship of particulate
organic carbon and volume of Pseudo-nitzschia subcurvata, Chaetoceros sp., Fragilariopsis
cylindrus, and Phaeocystis antarctica cultures ............................................................................. 58
Chapter 1 Fig. 5: POC to chl a ratio and Chl a per cell of of Pseudo-nitzschia subcurvata,
Chaetoceros sp., Fragilariopsis cylindrus, and Phaeocystis antarctica cultures ......................... 59
Chapter 1 Fig. 6: F
v
/F
m
of Pseudo-nitzschia subcurvata LN and Chaetoceros sp. LN ................ 60
Chapter 2 Fig. 1: Thermal functional response curves showing specific growth rates (and fitted
curves) of Pseudo-nitzschia subcurvata and Phaeocystis antarctica cultures ............................. 94
Chapter 2 Fig. 2: The elemental ratios of Pseudo-nitzschia subcurvata and Phaeocystis
antarctica cultures over a range of temperatures from 0
o
C to 10
o
C. ............................................ 95
Chapter 2 Fig. 3: The C: Chl a ratios of Pseudo-nitzschia subcurvata and Phaeocystis antarctica
cultures over a range of temperatures from 0
o
C to 10
o
C .............................................................. 96
Chapter 2 Fig. 4: The relative abundance of Pseudo-nitzschia subcurvata in a 6 day competition
experiment with Phaeocystis antarctica at 0°C and 6°C .............................................................. 97
Chapter 2 Fig. 5: CO
2
functional response curves of Pseudo-nitzschia subcurvata and
Phaeocystis antarctica showing specific growth rates (and fitted curves) across a range of CO
2

concentrations from ~100 ppm to ~1730 ppm at 2°C and at 8°C ................................................. 98
Chapter 3 Fig. 1: Cell-specific growth rates, Chl a: C ratios and photosystem II efficiency
(Fv/Fm) of cultured Southern California Pseudo-nitzschia australis isolate S7 grown across a
range of temperatures from 12-30
o
C ........................................................................................... 123
Chapter 3 Fig. 2: Cellular domoic acid concentrations, and DA production rates of cultured
Southern California Pseudo-nitzschia australis isolate S7 grown across a range of temperatures
from 12-30
o
C .............................................................................................................................. 124
Chapter 3 Fig. 3: The percentage of Chl a > 10 µm of the phytoplankton assemblages in SB1 and
SB2 experiment across a range of temperatures ......................................................................... 125
vii
Chapter 3 Fig. 4: The relative abundance of different phytoplankton groups of the phytoplankton
assemblage at the end of experiments in the SB1 and SB2 experiments across a range of
temperatures ................................................................................................................................ 126
Chapter 3 Fig. 5: The relative abundance of Pseudo-nitzschia delicatissima in the phytoplankton
assemblage incubations initially and as function of temperature at the end of experiments SB1
and SB2 ....................................................................................................................................... 127
Chapter 3 Fig. 6: Microscopic image of final phytoplankton communities in the SB2 experiment
at 15
o
C and 28
o
C ......................................................................................................................... 128

1
Introduction
Anthropogenic emissions have increased the atmospheric CO
2
concentration from the
pre-Industrial Revolution level of 280 ppm to the current level of approximately 400 ppm, and
concentrations are predicted to reach 750–1,000 ppm by 2100. Meanwhile, the ‘greenhouse’
effect of CO
2
is projected to lead to a global temperature elevation of 2.6°C to 4.8 (6)°C by the
end of this century (IPCC, 2014). The oceans have absorbed around one-third of the CO
2

released into the atmosphere over the past 200 years, which has caused the pH of surface
seawater to drop by ~0.1 units (Caldeira and Wickett, 2003; Raven et al., 2005; Boyd, 2011). If
current global CO
2
emissions trends continue, the average pH of the oceans could decrease by a
further ~0.5 units by the end of this century (Raven et al., 2005). Together, warming and ocean
acidification may dramatically affect phytoplankton productivity, biogeochemical cycles, and
harmful algal blooms around the world (Boyd et al., 2010; Doney, 2010; Fu et al., 2012; Capone
and Hutchins, 2013). The research presented in this dissertation focuses on the effects of these
two global change variables on key phytoplankton groups from two important areas of the ocean,
the coastal upwelling region of California, and the Southern Ocean High-Nutrient Low-
Chlorophyll (HNLC) region.
Global change and the Ross Sea, Antarctica phytoplankton community
The Southern Ocean is designated as an HNLC region because biological utilization of
the abundant nitrate and phosphate in surface waters is limited by the availability of the trace
metal iron. Nevertheless, Southern Ocean phytoplankton are still major drivers of global carbon
cycling, contributing ~ 2 Pg C of annual primary production (Martin et al., 1990; Arrigo et al.,
2008). The coastal polynya of the Ross Sea, Antarctica is one of the most productive regions in
the Southern Ocean, as the annual algal blooms in this region contribute as much as 28% of total
2
oceanic primary production in the Southern Ocean (Arrigo et al., 1999, 2008; Smith et al., 2000).
The Southern Ocean is also a relatively large anthropogenic CO
2
sink, and so contributes to
moderating the anthropogenic CO
2
level of the atmosphere (Caldeira and Duffy, 2000; Arrigo et
al., 2008).
The austral spring and summer blooms in the Ross Sea are usually dominated by the
colonial prymnesiophyte Phaeocystis antarctica and multiple medium sized diatom species
(Arrigo et al., 1999, 2000; DiTullio and Smith, 1996). Representative species of diatoms often
observed in the Ross Sea include Pseudo-nitzschia subcurvata, Fragilariopsis spp.,
Thalassiosira spp., and Chaetoceros spp. (Arrigo et al., 1999; Leventer and Dunbar, 1996;
Goffart et al., 2000; Rose et al., 2009). However, the spatial and temporal distribution of P.
antarctica and diatoms varies. P. antarctica blooms earlier than diatoms, and typically
dominates in the southeast polynya of the Ross Sea from austral spring to mid-summer when the
mixed layer is deep. In contrast, diatoms tend to bloom after P. antarctica and dominate in the
northwest Ross Sea (Arrigo et al., 1999; DiTullio and Smith, 1996).
The cellular N: P and C: P ratios of P. antarctica are higher than those of diatoms, so the
prymnesiophyte can export more nitrogen and carbon per unit P assimilated (Arrigo et al., 1999,
2000). In addition, P. antarctica produces large amounts of the potentially climatically active
compound dimethylsulfoniopropionate (DMSP), and so plays a significant role in the global
sulfur cycle. Diatoms are dominant in the global biogenic silicon cycle because of their silica
frustule (Arrigo et al., 1999; Tréguer et al., 1995; Schoemann et al., 2005). Furthermore, diatoms
and P. antarctica may contribute differently to the food webs in Southern Ocean. Diatoms are
the major food of krill, and so support the highly productive food webs of the Southern Ocean,
3
whereas the colonial morphology and possibly chemical defenses of P. antarctica may deter
predation by both macro- and microzooplankton (Knox, 1994; Caron et al., 2000).
The temperature of the surface Southern Ocean has been observed to be increasing faster
than that of the global ocean since the 1950s, and is predicted to keep increasing with rising
atmospheric CO
2
(Manabe and Stouffer, 1993; Sarmiento et al., 1998; Gille, 2002). The marine
biological community along the western Antarctic Peninsula has been affected by the diminished
ice cover and strengthened stratification caused by ongoing climate change (Montes-Hugo et al.,
2009). Although sea ice duration and extent increases with cooling in the Ross Sea have been
observed in recent years, this trend is predicted to reverse and warming and sea ice losses seem
inevitable by the end of this century (Ainley et al., 2010; Comiso et al., 2011; Pritchard et al.,
2012). The projected warming in Southern Ocean caused by rising anthropogenic CO
2
may lead
to intensified stratification and thus influence the mixed layer depth, the light regime, and the
nutrient upwelling dynamics of the Ross Sea (Boyd and Doney, 2002; Montes-Hugo et al.,
2009). It is important to understand how these changes may affect the phytoplankton community
and the whole ecosystems of the Southern Ocean in the future.
Multiple iron (Fe) addition experiments conducted in Southern Ocean have proven Fe is
the limiting nutrient for the growth of phytoplankton in the HNLC regime of the Southern Ocean
(de Baar et al., 1990; Martin et al., 1990; Takeda, 1998; Boyd et al., 2000; Sedwick et al., 2000;
Hutchins et al., 2001; Coale et al., 2004). The Fe input pathways into the Southern Ocean include
aeolian deposition, deep-water upwelling, continental shelf sources, and sea ice melting
(Sedwick and DiTullio, 1997; Elrod et al., 2004; Jickells et al., 2005; Hutchins and Boyd, 2016).
However, how Fe inputs into the Southern Ocean will be changed due to future global change is
still unclear (Hutchins and Boyd, 2016). The predictions from two models show opposite trends
4
for global dust fluxes coming into the Southern Ocean by the end of this century (Mahowald and
Luo, 2003; Tegen et al., 2004). A warming climate may reduce terrestrial ice and snow cover on
the Antarctic continent and result in an increase in local dust inputs into the Antarctic coastal
ocean (Cook et al., 2005; Overpeck et al., 2006; Raiswell et al., 2006). Additionally, melting of
icebergs and glacial ice may also contribute to the future Fe inputs into coastal Southern Ocean
(Overpeck et al., 2006; Raiswell et al., 2006, 2008; Hutchins and Boyd, 2016). At the same time,
strengthened stratification in the future may decrease Fe inputs from upwelling, and thus
exacerbate Fe limitation of the HNLC region (Boyd and Doney, 2002; Montes-Hugo et al.,
2009). Alternately, increased wind speeds in a warmer climate may increase Fe inputs to the
mixed layer by intensifying upwelling (Anderson et al., 2009). Clearly, much work remains to
be done to accurately forecast the net impacts of all these inter-connected global change
processes on Fe supply to phytoplankton in the Southern Ocean (Hutchins and Boyd, 2016).
Research is still scarce on how the interactions between diatoms and P. antarctica will
change due to the effects of temperature increase and other environmental factors such as Fe
availability and acidification in the Ross Sea (Rose et al., 2009; Xu et al., 2014). A shipboard
experiment showed that Fe addition and temperature increases synergistically increased the
growth of a natural phytoplankton assemblage, and the final phytoplankton community was
dominated by diatoms instead of P. antarctica (Rose et al., 2009). The diatom Fragilariopsis
cylindrus had a competitive advantage over P. antarctica under a “cluster” of factors simulating
future conditions, which included warming, ocean acidification, and increases in light intensity
and Fe availability (Xu et al., 2014). The interactive affects of temperature increase and Fe
availability on phytoplankton may be due to several factors. Temperature increase can elevate
diffusion rates and enzyme efficiency, and reduce the cellular Fe requirement for respiration and
5
nitrogen assimilation (Sunda and Huntsman, 2011). Under warming, ribosome numbers per algal
cell decrease along with increasing protein translation rates, thus decreasing the phosphorus
requirement of phytoplankton (Toseland et al., 2013). Thus, predicted temperature increases may
alleviate Fe-limitation of phytoplankton in Southern Ocean HNLC waters in the future, allowing
them to draw down nutrients below current levels (Hutchins and Boyd, 2016).
Ocean acidification caused by rising anthropogenic CO
2
emission may affect the growth
of different groups of phytoplankton differently (Boyd et al., 2010; King et al., 2015; Boyd and
Hutchins 2012; Gao and Campbell 2014; Hutchins 2013; Trimborn et al., 2013). Species-specific
metabolic pathways may determine the responses of phytoplankton to CO
2
increase and pH
decrease, and these may in turn interact with other environmental factors such as temperature,
light and nutrients (Gao et al., 2012). Trimborn et al. (2013) examined the effects of CO
2

increase on several Southern Ocean phytoplankton and observed that the growth of P.
subcurvata and P. antarctica remained the same at different CO
2
levels, while the growth of
Chaetoceros debilis significantly increased with a CO
2
increase from 160ppm to 1000ppm.
Hoogstraten et al. (2012) found that the specific growth rates of Phaeocystis globosa decreased
from 1.4 d
-1
to 1.1 d
-1
with a CO
2
increase from 190ppm to 750ppm at a light level of 240 µmol
photons m
-2

s
-1
, whereas growth rates were unaffected by changing CO
2
at 80µmol photons m
-2

s
-
1
.
Studies show that rising CO
2
and warming may also shift the phytoplankton community
in high latitude environments (Feng et al., 2009, 2010; Hinder et al., 2012). An unchanged
abundance of diatoms coupled with decreased dinoflagellate abundance was revealed by a 50
year time series survey in the north-east Atlantic and North Sea, and attributed to warming and
windier conditions (Hinder et al., 2012). An increase of diatom abundance (largely the pennate
6
diatom Pseudo-nitzschia sp.) with CO
2
increase was observed in a shipboard continuous culture
experiment during the spring bloom in the North Atlantic, and temperature increase also changed
phytoplankton community structure (Feng et al., 2009). Another shipboard continuous cultures
experiment in the Ross Sea in summer showed that high CO
2
stimulated the growth of the large
centric diatom Chaetoceros lineola over the small pennate diatom Cylindrotheca closterium, and
high light increased the abundance of P. antarctica colonies relative to diatoms (Feng et al.,
2010).
One major goal of this dissertation is to further explore the potential effects of warming,
Fe availability, and ocean acidification on P. antarctica and ecologically important diatom
species from the Southern Ocean. These results are intended to help us to predict the potential
effects of global change on the phytoplankton community, food webs, and biogeochemical
cycles of carbon, iron, and nutrients in the tremendously productive coastal polynyas of
Antarctica.
Global change and toxic diatoms in the coastal California upwelling region
In addition to Antarctica, phytoplankton communities in temperate regions can also be
profoundly affected by global change. Wang et al. (2010) observed that the growth rates of the
temperate prymnesiophyte species Phaeocystis globosa (which is closely related to P.
antarctica) increased 30% with a CO
2
increase from 380ppm to 750ppm at 20
o
C. King et al.
(2015) studied seven temperate marine phytoplankton species and found the specific growth
rates of four species increased and one species decreased with CO
2
increase, while no significant
change was observed in two species. Wu et al. (2014) found that phytoplankton size affected
responses to CO
2
increases, and observed that changing CO
2
concentration from 190ppm to
750ppm enhanced the growth of small diatoms by ~ 5% and that of larger diatoms by ~30%. In
7
contrast, Gao et al. (2012) observed that the growth of a temperate diatom slowed in high CO
2

(1000ppm) relative to current CO
2
level (390ppm) at light intensities representative of the upper
surface layer. Hervé et al. (2012) observed that silicon biomineralization of the diatom
Thalassiosira weissflogii is pH-dependent, and thus ocean acidification may decrease the growth
of diatoms.
Harmful bloom forming algae in coastal regions constitute another group of important
ocean phytoplankton that needs further research under global change scenarios. For instance,
harmful blooms of the pennate diatom Pseudo-nitzschia recur every year during spring and
summer along the California coast, and domoic acid produced by some members of this genus
has been identified as the potent neurotoxin that causes sea lion mortalities and seabird deaths
(Scholin et al., 2000; Schnetzer et al., 2007; Bejarano et al., 2008; Trainer et al., 2000, 2009,
2012; Bargu et al., 2012). Domoic acid accumulates throughout marine food webs, and has been
detected in mussels (Mytilus spp.), Dungeness crabs (Cancer magister), razor clams (Siliqua
patula), anchovies (Engraulis mordax), Pacific sardines (Sardinops sagax), brown pelicans
(Pelecanus occidentalis), and blue whales (Work et al., 1993; Lefebvre et al., 2002; Schnetzer et
al., 2007; Bargu et al., 2008; Bejarano et al., 2008). Frequent shellfish harvesting closures have
occurred due to the expansion of blooms of Pseudo-nitzschia, causing economic losses by
impacting both commercial and recreational activities (Bill et al., 2006; Trainer et al., 2007).
Various factors including temperature, irradiance, nutrient availability, CO
2

concentration, trace metal availability, cellular elemental ratios of nutrients, and bacterial
interactions can all affect the domoic acid production of Pseudo-nitzschia spp. (Bates et al.,
1998; Maldonado et al., 2002; Sun et al., 2011; Lelong et al., 2012; Tatters et al., 2012; Sison-
Mangus et al., 2014). However, temperature has been addressed in only the studies of Lundholm
8
et al. (1994) and Lewis et al. (1993). These two studies found contradictory results, with the
former demonstrating decreased domoic acid and the latter increased domoic acid at warmer
temperatures. In addition, Sun et al. (2011) found that CO
2
increase stimulated the domoic acid
production of Pseudo-nitzschia multiseries, especially when the cultures were phosphorus-
limited. Similarly, Tatters et al. (2012) observed elevated domoic acid production in P.
fraudulenta with CO
2
increase and silicate-limitation.
Other than pure cultures of Pseudo-nitzschia spp., the effects of both high CO
2
and
warming on domoic acid production in a natural P. multiseries bloom collected at Fish Harbor,
in the Los Angeles/Long Beach harbor area were examined by Tatters et al. (submitted). The
results show that both warming and acidification promoted significantly higher domoic acid
production per Pseudo-nitzschia cell. Domoic acid quota approximately tripled when CO
2

increased from 380 to 800 ppm at 19
o
C, and also increased 50% (not significantly, due to high
standard deviation) with rising CO
2
at 23
o
C. Considering the effect of temperature increases, the
domoic acid cellular quota doubled when temperature increased from 19
o
C to 23
o
C at 380 ppm,
while at 800 ppm, domoic acid production was not significantly promoted by temperature
increases (Tatters et al., 2013).
The frequency and severity of harmful algal bloom may increase with global warming
(Peperzak, 2005; Moore et al., 2008; Paerl and Huisman, 2008; Fu et al., 2012). A large patch of
warm-water anomaly (dubbed “the Blob”) started in the winter of 2013-2014 and persisted
through the end of 2015 in the north-eastern Pacific, stretching from Alaska to Baja California
(Bond et al., 2015; Cavole et al., 2016; McCabe et al., 2016). Concurrently, an extraordinarily
widespread harmful algal bloom dominated by Pseudo-nitzschia stretched from the Aleutian
Islands down to southern California. The toxic bloom formed during the course of this
9
extraordinary regional warming event spanned early spring 2014 until the summer of 2015, a
period during which sea surface temperatures were up to 4
o
C above long-term averages from
Alaska to Southern California (Bond et al 2015; Cavole et al., 2016). Unprecedented high
concentrations of domoic acid were present throughout the marine food webs in this area,
creating widespread mortality of marine mammals and birds (Cavole et al., 2016; McCabe et al.,
2016). Economic losses from this massive bloom due to closure of the Dungeness crab fishery
were estimated at >$50 million (McCabe et al., 2016; Leising et al.; 2015, Cavole et al., 2016).
Research on the impact of warming on the toxin production of Southern California
coastal Pseudo-nitzschia species, and on the succession of natural phytoplankton communities
containing Pseudo-nitzschia remains scarce. The second focus of this dissertation research aims
to explore the effects of temperature on the toxin production of Pseudo-nitzschia isolated from
the Southern California Coast, and the effects of temperature increase on the succession and
dominance of Pseudo-nitzschia spp. in the natural phytoplankton community of this region.
Summary
My work aims to expand our understanding of how several major climate change factors
may affect key phytoplankton groups from two oceanic regions. The first part of this work
focuses on two distinct phytoplankton groups in the Southern Ocean, diatoms and the
prymnesiophyte Phaeocystis antarctica, which contribute considerably differently to nutrient
cycles. In this study, the interactive effects of Fe availability and warming on the physiology and
community structure of several species of diatom and P. antarctica is investigated.
Subsequently, the individual and interactive responses of P. subcurvata and P. antarctica across
wide ranges of temperature and CO
2
are also examined. This study addresses how these two
10
factors influence the physiology of and competition between these two dominant phytoplankton
from the Southern Ocean.
The final part of this work focuses on the effects of impending increasing temperature on
harmful algal bloom (HAB) forming algae Pseudo-nitzschia spp. in the Southern California
Bight. These experiments address the effects of warming on domoic acid production of Pseudo-
nitzschia, and on the phytoplankton composition of Pseudo-nitzschia-containing coastal
phytoplankton communities from Southern California. This study aims to understand the
relationship between temperature and the extent and toxicity of HABs formed by Pseudo-
nitzschia. Collectively, these dissertation research projects are targeted at providing needed
information to understand how phytoplankton physiology, community structure change, and
nutrient cycles may be altered in both the coastal Southern Ocean and during HAB events in the
Southern California Bight under future global change scenarios.

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22
Chapter 1: A comparative study of iron and temperature interactive effects on
diatoms and Phaeocystis antarctica from the Ross Sea, Antarctica

Zhi Zhu
1
, Kai Xu
1
, Feixue Fu
1
, Jenna L. Spackeen
2
, Deborah A. Bronk
2
, David A. Hutchins
1*
1. Department of Biological Science, University of Southern California, Los Angeles, CA
90089, USA.
2. Department of Physical Sciences, Virginia Institute of Marine Science, College of
William & Mary, P.O. Box 1346, Gloucester Point, VA 23062, USA.
* Corresponding author: dahutch@usc.edu

Abstract: Future temperature and iron availability is predicted to change in the most biologically
productive regions of the Southern Ocean, the coastal polynyas of Antarctica. We examined the
individual and combined effects of iron addition (+500 nM) and temperature increase (4°C) on
Phaeocystis antarctica and several dominant diatom species isolated from the McMurdo Sound
sector of the Ross Sea. Iron addition increased growth, carbon fixation, and iron uptake rates,
cellular carbon quota, and cell size of almost all tested species, while temperature increase only
affected certain species. Concurrent increases in temperature and iron synergistically stimulated
the growth rates of some tested species, particularly Pseudo-nitzschia subcurvata. The
diversified responses of these phytoplankton to iron and temperature may help to explain the
current spatial and temporal distributions of diatoms and prymnesiophytes in the Ross Sea. In
the future, potential temperature and iron increases may promote the growth of the diatoms
Chaetoceros sp., Fragilariopsis cylindrus, and especially P. subcurvata. In contrast, growth rates
of P. antarctica did not increase at higher temperature, suggesting a shift in community
23
composition toward diatoms may occur under warmer conditions in this biologically and
biogeochemically important Southern Ocean polynya region.
Key words: Antarctic, global warming, Fe input, phytoplankton community, diatom,
Phaeocystis, Pseudo-nitzschia

1 Introduction
The annual austral spring and summer algal blooms in the Ross Sea, Antarctica
contribute as much as 28% of total oceanic primary production in the Southern Ocean (Arrigo et
al., 1998). These primary producers support a thriving food web, and make this polynya one of
the most biologically productive areas in the Southern Ocean (Arrigo et al., 1999, 2008; Smith et
al., 2000). Carbon fixation and export by these algal blooms also make the coastal Southern
Ocean an important anthropogenic CO
2
sink (Arrigo et al., 2008).
The colonial prymnesiophyte Phaeocystis antarctica and multiple species of diatoms are
the two dominant phytoplankton taxa in the Ross Sea (Arrigo et al., 1999, 2000; DiTullio and
Smith 1996). Diatom assemblages are usually composed of Pseudo-nitzschia subcurvata,
Fragilariopsis spp., Thalassiosira spp., and Chaetoceros spp., as well as various other species
(Arrigo et al., 1999; Leventer and Dunbar, 1996; Goffart et al., 2000; Rose et al., 2009). P.
antarctica typically initiates the bloom in austral spring when the mixed layer depth is deep and
continues to dominate until mid-summer, especially in the southeast polynya of the Ross Sea
(Arrigo et al., 1999; DiTullio and Smith, 1996). In contrast, diatoms normally bloom after P.
antarctica during the late austral summer, and are particularly dominant in the northwest Ross
Sea, along the coast of Victoria Land and in Terra Nova Bay (Arrigo et al., 1999; DiTullio and
Smith 1996). Mixed layer depth and water column temperature have been suggested as the main
24
factors that determine the spatial and temporal distributions of P. antarctica and diatoms in the
Ross Sea (Arrigo et al., 1999; Liu and Smith 2012).
In addition to being important contributors to the global carbon cycle and anthropogenic
CO
2
drawdown, P. antarctica plays a large role in global sulfur biogeochemistry and diatoms
have a major influence on the global silicon cycle (Arrigo et al., 1999; Tréguer et al., 1995;
Schoemann et al., 2005). P. antarctica has higher N: P and C: P ratios, so it can export more
nitrogen and carbon per mol of PO
4
3-
removed relative to diatoms (Arrigo et al., 1999, 2000).
The colonial morphology of P. antarctica may deter grazing by microzooplankton (Caron et al.,
2000), and diatoms are important in supporting the krill-based food webs of the Southern Ocean
(Knox, 1994). Thus, any shift in Ross Sea phytoplankton community structure has the potential
to affect the global biogeochemical cycles of carbon, sulfur, silicon, and other major nutrients
(Arrigo et al., 1999; DiTullio et al., 2000).
The Southern Ocean is predicted to experience significant warming caused by increasing
atmospheric CO
2
(Manabe and Stouffer 1993; Sarmiento et al., 1998), and the West Antarctic
Peninsula (WAP) has been notable as one of the most rapidly warming areas on the planet
(Vaughan et al., 2003). Decreased ice cover and increased stratification caused by ongoing
climate change has already influenced the marine biological community along the WAP
(Montes-Hugo et al., 2009). In contrast, the Ross Sea has experienced cooling and increases in
sea ice duration and extent in recent years (Comiso et al., 2011; Stammerjohn et al., 2012; Smith
et al., 2012). Nevertheless, given current climate trends, the Ross Sea is expected to also
experience significant warming and loss of sea ice by the end of this century (Ainley et al.,
2010). The intensified stratification caused by this projected warming may profoundly influence
25
the nutrient upwelling, mixed layer depth, and light regime of the Ross Sea (Boyd and Doney
2002; Montes-Hugo et al., 2009).
Iron (Fe) has been conclusively proven to be the primary limiting nutrient for the HNLC
regime in the Southern Ocean. Many experiments have shown that Fe addition stimulates the
growth of phytoplankton, especially the proliferation of diatoms (de Baar et al., 1990; Martin et
al., 1990; Takeda 1998; Boyd et al., 2000; Sedwick et al., 2000; Hutchins et al., 2002; Coale et
al., 2004). Aeolian deposition, deep water upwelling, and sea ice melting are the major iron input
pathways into the Southern Ocean (Sedwick and DiTullio 1997; Elrod et al., 2004; Jickells et al.,
2005). The effects of global change on these iron inputs into the Southern Ocean are currently
unresolved. For instance, predictions from two models show very different trends of global dust
fluxes (range from a 60% decrease to a 12% increase) in the next 100 years (Mahowald and Luo
2003; Tegen et al., 2004). Local dust inputs could also become a significant source of iron to the
coastal Southern Ocean, due to reduced terrestrial ice and snow cover as a result of a warming
climate (Cook et al., 2005; Overpeck et al., 2006; Raiswell et al., 2006). Melting of glacial ice
and icebergs may increase future iron inputs into the Antarctic coastal ocean (Overpeck et al.,
2006; Raiswell et al., 2006, 2008). Changes to stratification and upwelling in the future may also
impact Fe concentrations in the upper water column. Fe inputs from upwelling may decrease
with increased stratification, and thus intensify Fe limitation of the HNLC region (Boyd and
Doney 2002; Montes-Hugo et al., 2009); alternately, Fe inputs from upwelling may instead
increase due to increased wind speeds in a warmer climate (Anderson et al., 2009). Although it is
currently challenging to predict the net trends in iron inputs to the Ross Sea, it seems quite likely
that both future iron availability and sea surface temperature may deviate substantially from
current conditions.
26
The biological consequences of potential interactions between iron supply changes and
temperature increase are poorly understood. Rose et al. (2009) found that iron addition and
temperature increase synergistically promoted the growth of a natural phytoplankton community
from the Ross Sea in a shipboard experiment, and diatoms dominated the final assemblages
rather than P. antarctica. Xu et al. (2014) examined the effects of “clustered” global change
factors including warming, light, ocean acidification, and iron availability on P. antarctica and
the diatom Fragilariopsis cylindrus. Their results suggested that diatoms may outcompete P.
antarctica under a combined suite of simulated future conditions.
Research on the interactive effects of temperature and iron on representative individual
phytoplankton species from Ross Sea is scarce. This study aimed to explore the effects of
temperature increase and Fe addition on P. antarctica and several ecologically important diatom
species. The results are intended to help us predict the potential effects of global change on the
phytoplankton community, food webs, and biogeochemical cycles of carbon, iron, and nutrients
in the coastal polynyas of Antarctica.
2 Materials and Methods
2.1 Strains and growth conditions
Unialgal cultures of Pseudo-nitzschia subcurvata, Chaetoceros sp., Fragilariopsis
cylindrus, and Phaeocystis antarctica were isolated from the ice edge in McMurdo Sound
(77.62°S, 165.47°E) in the Ross Sea, Antarctica during January and February 2013. P. antarctica
grew in the non-colonial form, as single flagellated cells. All stock cultures were maintained in
0.2 µM-filtered seawater that was collected using trace metal clean techniques from the same
locale as the culture isolates (Hare et al., 2007; King et al., 2012). Cultures were grown at 0°C in
a walk-in incubator under 24 hour (h) cold white fluorescence light (80 µmol photons m
-2
s
-1
).
27
Experiments examined interactions between temperature and iron availability under four
conditions: 0°C, Fe limited (+1nM Fe, abbreviated as 0C-Fe), 0°C, Fe replete (+500nM Fe, or
0C+Fe), 4°C, Fe limited (+1nM Fe, or 4C-Fe), and 4°C, Fe replete (+500nM Fe, or 4C+Fe). Fe
concentration was amended by adding EDTA chelated FeCl
3
(100: 1) into 0.2 µM-filtered trace
metal-clean Ross Sea seawater. The seawater was collected late in the Antarctic summer, and so
the concentrations of NO
3
-
, PO
4
3-
were relatively low for this region at 6.95 µmol L
-1
and 0.66
µmol L
-1
respectively. Si(OH)
4
and dissolved Fe concentrations were 52.91 µmol L
-1
, and 0.2
nmol L
-1
respectively (Feng et al., 2010). Chaetoceros sp. and one strain of Pseudo-nitzschia
subcurvata (P. subcurvata) were grown in this seawater medium without any added nutrients.
Another isolate of P. subcurvata, Fragilariopsis cylindrus, and Phaeocystis antarctica were
maintained in the same four conditions, but the seawater was enriched with chelexed nutrient
stocks to 50 µmol L
-1
NO
3
-
and 10 µmol L
-1
PO
4
3-
to examine growth effects of these two
variables at higher nutrient levels. Cultures grown at high and low major nutrient levels will be
identified as HN and LN treatments, respectively.
Experimental cultures were grown in triplicate 500 ml acid washed polycarbonate bottles
under the same light condition as stock cultures. Semi-continuous culturing methods were used,
whereby the cultures were diluted every 2 days with medium pre-acclimated to their respective
temperatures. Dilution rates were based on the individually calculated growth rate of each
replicate bottle (see ‘Growth rates’ section below), allowing each bottle to reach its own steady-
state exponential growth rate. All of the cultures were acclimated to their respective
environmental conditions for 8 weeks before the commencement of the experiment. After the
growth rates remained stable for at least three to five consecutive transfers, indicating steady-
state growth had been attained, the cultures were sampled 48 h after dilution.
28
2.2 Growth rates
Ten ml aliquot of culture samples were taken for visual cell counts directly before and
after each treatment was diluted. Cell count samples were preserved with 0.5% glutaraldehyde
(final concentration) and stored at 4°C for subsequent counting on a hemocytometer or Sedgwick
Rafter Grid using an Olympus BX51 microscope (Olympus, Japan). Due to poor preservation,
cell count samples of P. antarctica at 4°C for phosphorus cell quotas and Chl a per cell
calculations were lost. Specific growth rates, expressed as d
-1
, were calculated as: µ = (ln N
1
– ln
N
0
)/t, where N
0
and N
1
are the cell density at the beginning and end of a dilution period,
respectively, and t is the duration of the dilution period. Q
10
for growth rates of all the
phytoplankton was calculated as: Q
10
= (µ
2
/ µ
1
)
10/(T1-T2)
(Chaui-Berlinck et al., 2002), where µ
1

and µ
2
are the specific growth rates of the phytoplankton at temperature T
1
(Celsius) and T
2
,
respectively.
2.3 Elemental analysis
50 ml and 20 ml culture samples of each treatment were filtered onto pre-combusted GF/F
filters (500°C for 2h) and dried in a 60°C oven overnight for particulate organic carbon/nitrogen
(POC/PON) and particulate organic phosphorus (POP) analyses, respectively. POC/PON samples
were analyzed using a 440 Elemental Analyzer (Costech Inc, Valencia, CA) following Fu et al.
(2007) and Garcia et al. (2014). POP was analyzed using a molybdate colorimetric method
according to Fu et al. (2007). A 20 ml aliquot of Chaetoceros sp. and P. subcurvata LN sample
from each treatment was filtered onto 2 µm polycarbonate filters and dried in a 60°C oven
overnight for biogenic silica (BSi) analysis (Pasche et al., 1973).
2.4 Chlorophyll a analysis
29
20 to 50 ml culture samples were filtered onto GF/F filters (Whatman) and extracted with
90% aqueous acetone for 24 h at -20°C, and measured using the non-acidification method on a
10-AU
TM
fluorometer (Turner Designs, CA) (Fu et al., 2007).
2.5 Cell volume and surface area
A minimum of 50 cells (fresh samples for P. antarctica and preserved samples for
diatoms) from each treatment were measured using an Olympus BX51 microscope (Olympus,
Japan) with a coupled Excelis HD camera (ACCU-SCOPE, NY). The length, height, or diameter
of all cells were measured using ImageJ (NIH), and the volume and surface area of each cell was
calculated following Hillebrand et al. (1999).
2.6 Active fluorescence characteristics
A 6 ml aliquot of culture sample of P. subcurvata LN and Chaetoceros sp. LN from each
treatment was dark-adapted for ~15 min, and minimum fluorescence (F
0
) was measured using a
10-AU
TM
fluorometer (Turner Designs, CA). Next, maximum fluorescence (F
m
) was recorded by
adding 6 µl DCMU (dichloromethylurea) to each sample followed by shaking for 30 seconds.
The quantum efficiency of photosystem II F
v
/F
m
was calculated according to the equation F
v
/F
m

= (F
m
– F
0
)/F
m
(Schreiber, 2004).
2.7 C fixation and Fe uptake rates:
To measure carbon fixation rates and iron uptake rates, a 30 ml aliquot of culture sample
from each treatment was incubated with 37 kBq
14
C

–bicarbonate (MP Biomedicals), or ~2 kBq
55
FeCl
3
(PerkinElmer, 0.33 nM
55
FeCl
3
complexed to 120 µmol L
-1
EDTA) under their
respective treatment conditions. Samples were filtered onto GF/F filters after 24 h incubation.
For Fe uptake rate samples, the filters were washed in oxalate reagent for 5 min followed by a
trace-metal clean seawater rinse to remove surface-adsorbed Fe (Tovar-Sanchez et al., 2003,
30
Tang and Morel, 2006). To correct for filter absorption of both radiotracers, the same amount of
stock solution was added to a 30 ml aliquot of sample and filtered immediately; these filter
absorption count values were subtracted from reported activities. The radioactivities of
14
C and
55
Fe in each sample were counted in a Tri-Carb 2500TR (Packard, now Perkin Elmer). Carbon
fixation rates and Fe uptake rates were calculated using the initial dissolved inorganic carbon
(DIC) concentrations and initial total Fe concentrations of each bottle (1nM Fe and 500 nM for
iron limited and iron replete cultures, respectively), and were normalized to cell density (Garcia
et al., 2014). Because
55
Fe additions were a large fraction of the total Fe present in the Fe-
limited samples and were calculated using initial concentrations, these uptake values represent an
upper rate estimate for this treatment.
2.8 Statistical analysis
All statistical analyses, including student t-tests, ANOVA, Tukey’s HSD test, and two-
way ANOVA were conducted using the open source statistical software R version 3.1.2 (R
Foundation).
3 Results
3.1 Growth rates and Q10 values
Fe addition significantly increased the growth rates of all the phytoplankton tested (p <
0.05) (Fig. 1A) at both 0°C and 4°C, confirming that Fe limitation had been successfully
achieved for all low-Fe treatments. While the effect of temperature varied between species, the
growth rates of both Fe-limited and Fe-replete cultures of P. subcurvata HN and F. cylindrus HN
significantly increased at 4°C (p< 0.05), but only the Fe-replete cultures of P. subcurvata LN and
Chaetoceros sp. LN were stimulated by temperature increase (p < 0.05) (Fig. 1A). The 4°C
temperature increase did not influence the growth rates of the prymnesiophyte P. antarctica HN
31
(Fig. 1A). Additionally, the growth rates of P. subcurvata LN and P. subcurvata HN were
significantly stimulated by the interactive effects of concurrent temperature and Fe increase;
these responses were significantly greater than the additive effects of individual temperature
increase and Fe addition (p < 0.05). The growth rates of Fe-limited P. subcurvata HN
significantly increased relative to Fe-limited P. subcurvata LN by 63% at 0°C and by 81% at
4°C (p < 0.05), while the growth rates of Fe-replete P. subcurvata HN significantly increased by
30% relative to Fe-replete P. subcurvata LN at 4°C (p < 0.05) (Fig. 1A).
The Q
10
values of Fe-limited P. subcurvata LN, P. subcurvata HN, and Chaetoceros sp.
LN were in the range of 2.0 ~ 2.8. The Q
10
value of Fe-replete P. subcurvata LN was 3.07,
which was lower than Fe-replete P. subcurvata HN (4.24) (Fig. 1B). The Q
10
values of F.
cylindrus HN (6.56 and 5.06 for Fe-limited and Fe-replete cultures, respectively) were highest
amongst all the phytoplankton tested (Fig. 1B). In contrast, the Q
10
values of P. antarctica HN
(0.63 and 0.80 for Fe-limited and Fe-replete cultures, respectively) were much lower than those
of all the diatoms (Fig. 1B).
3.2 Carbon fixation and Fe uptake responses
The effects of Fe addition on carbon fixation rates were similar to growth rates. Carbon
fixation rates of all Fe-replete diatom cultures significantly increased relative to Fe-limited
cultures at both 0°C and 4°C (p < 0.05) (Fig. 2A), while the carbon fixation rates of Fe-replete P.
antarctica HN significantly increased relative to Fe-limited rates only at 0°C (p < 0.05) (Fig.
2A). Temperature increase led to significantly higher carbon fixation rates of Fe-replete P.
subcurvata HN, F. cylindrus HN, and Chaetoceros sp. LN (p < 0.05), but significantly decreased
the carbon fixation rates of P. antarctica HN in Fe-replete cultures by 72% (p < 0.05) (Fig. 2A).
In addition, temperature increase and Fe addition interactively stimulated a 6.6 fold increase of
32
carbon fixation rates of P. subcurvata HN and a 1.9 fold increase of Chaetoceros sp. LN (p <
0.05). The carbon fixation rates of Fe-replete P. subcurvata HN significantly increased 1.7 fold
relative to P. subcurvata LN at 4°C (p < 0.05) (Fig. 2A).
Similar to growth and carbon uptake rates, Fe fertilization significantly increased the Fe
uptake rates of all five phytoplankton species at both 0°C and 4°C (p < 0.05) (Fig. 2B).
Temperature increase significantly elevated the Fe uptake rates of Fe-replete cultures of P.
subcurvata LN, P. subcurvata HN, and F. cylindrus HN (p < 0.05) (Fig. 2B). Additionally,
temperature increase and Fe fertilization interactively increased the Fe uptake rates of these same
three strains (p < 0.05). The Fe uptake rates of Fe-replete P. antarctica HN cultures were
significantly decreased by 86% at 4°C relative to 0°C (p < 0.05) (Fig. 2B). The Fe uptake rates of
P. subcurvata LN were 41% higher than P. subcurvata HN in 4°C Fe-replete cultures (p < 0.05)
(Fig. 2B), suggesting that nutrient concentrations may affect Fe uptake rates of this species.
The molar Fe: C uptake ratios of all four diatoms were significantly increased with Fe
addition at both 0°C and 4°C (p < 0.05) (Fig. 2C). Fe addition significantly elevated the Fe: C
uptake ratio of P. antarctica HN (2.4 fold) at 0°C (p < 0.05), but not at 4
o
C. Furthermore,
temperature increase significantly increased the Fe: C uptake ratios of Fe-replete P. subcurvata
LN by 1.8 fold, and Fe addition and warming interactively affected the Fe: C uptake ratio of P.
antarctica HN (p < 0.05) (Fig. 2C).
3.3 Cellular elemental quotas and stoichiometry
The effects of Fe addition and temperature increase on cellular carbon quotas varied
among the five strains tested. Fe addition significantly increased the carbon quota of P.
subcurvata LN and P. subcurvata HN at both temperatures (p < 0.05) (Fig. 3A). P. antarctica
HN carbon quotas increased 42% with Fe addition at 0°C (p < 0.05) (Fig. 3A). Warming
33
decreased the carbon quota of F. cylindrus HN by 30%, regardless of Fe concentration (p < 0.05)
(Fig. 3A). There was no significant effect of either warming or Fe availability on the carbon
quota of Chaetoceros sp. LN (p > 0.05) (Fig. 2C).
The phosphorus quota of P. subcurvata LN was significantly lower than that of P.
subcurvata HN in all the four treatments (p < 0.05) (Fig. 3C), but the low nutrient condition did
not affect the cellular carbon quota (Fig. 3A). Nitrogen quotas of P. subcurvata LN and P.
subcurvata HN increased significantly with Fe addition at both 0°C and 4°C (p < 0.05) (Fig. 3B).
Temperature increase significantly decreased the nitrogen quota of Fe-limited (37%) and Fe-
replete (32%) F. cylindrus HN (p < 0.05) (Fig. 3B). Iron addition significantly increased
phosphorus quota of P. subcurvata HN at 0°C and 4°C, but temperature increase significantly
decreased phosphorus quota of Fe-replete P. subcurvata HN by 32% and Fe-replete F. cylindrus
HN by 39% (p < 0.05) (Fig. 3C). Temperature increase significantly decreased the phosphorus
quota of both Fe-limited (58%) and Fe-replete (30%) P. subcurvata LN (p < 0.05) (Fig. 3C). Fe
addition significantly increased the phosphorus quota of Chaetoceros sp. LN at 0°C (p < 0.05),
and temperature increase significantly increased the phosphorus quota of Fe-limited Chaetoceros
sp. LN (p < 0.05) (Fig. 3C). Fe addition significantly increased the cellular Si quota of P.
subcurvata LN and Chaetoceros sp. LN at both 0°C and 4°C (p < 0.05) (Fig. 3D). Temperature
increase decreased the Si quota of Fe-replete P. subcurvata LN by 19%(p < 0.05) (Fig. 3D).
Elemental ratios of the phytoplankton in all experimental treatments are shown in Table
1. Fe addition significantly increased the C: N ratio of P. subcurvata LN and Chaetoceros sp.
LN at both temperatures (p < 0.05) and the C: N ratio of F. cylindrus HN at 0°C (p < 0.05).
Temperature increase significantly elevated the C: N ratio of Fe-limited F. cylindrus HN (p <
0.05) (Table 1). The C: N ratios of Fe-limited P. subcurvata LN at 0°C and Fe-replete P.
34
subcurvata LN at 4°C were significantly lower than those of the same species in the HN
treatment (p < 0.05) (Table 1).
The N: P ratio of F. cylindrus HN was significantly increased by Fe addition at both 0°C
and 4°C (p < 0.05), as was the N: P ratio of P. subcurvata LN at 4°C (p < 0.05) (Table 1). In
contrast, Fe addition significantly decreased the N: P ratio of P. subcurvata HN and Chaetoceros
sp. LN at 4°C (p < 0.05) (Table 1). In addition, temperature increase significantly increased the
N: P ratio of both Fe-limited and Fe-replete P. subcurvata LN (p < 0.05) and N: P ratio of Fe-
replete P. antarctica HN (p < 0.05) (Table 1). The N: P ratios of both Fe-limited and Fe-replete
P. subcurvata LN at 4°C were significantly higher than those of P. subcurvata HN (p < 0.05)
(Table 1).
Fe addition significantly increased the C: P ratio of P. subcurvata LN at both 0°C and
4°C (p < 0.05), and the C: P ratio of F. cylindrus HN at 4°C (p < 0.05) (Table 1). Warmer
temperatures significantly increased the C: P ratio of both Fe-limited and Fe-replete P.
subcurvata LN (p < 0.05), and the C: P ratio of Fe-replete P. antarctica HN (p < 0.05) (Table 1).
Temperature increase and Fe addition had no significant effect on the stoichiometry of P.
subcurvata HN or Chaetoceros sp. LN (Table 1). However, the C: P ratio of Fe-replete P.
subcurvata LN at 0°C was significantly higher than P. subcurvata HN (p < 0.05) (Table 1).
3.4 Cell morphology
Fe addition significantly enlarged the cell volume of P. subcurvata HN, P. subcurvata
LN and Chaetoceros sp. LN at both temperatures (p < 0.05) (Fig. 4A), and cell volume of F.
cylindrus HN was significantly enlarged with Fe addition at 4°C (p < 0.05) (Fig. 4A). Warming
significantly enlarged the cell volume of Fe-replete P. subcurvata LN by 17% (p < 0.05) (Fig.
4A). Corresponding to the effects on cell volume, Fe addition significantly decreased the surface
35
area to volume ratios of P. subcurvata HN, P. subcurvata LN and Chaetoceros sp. LN at both
temperatures (p < 0.05) (Fig. 4B), but only at 4°C for F. cylindrus HN (p < 0.05) (Fig. 4B).
Temperature increase diminished the surface area to volume ratio of Fe-replete P. subcurvata LN
by 5 % (p < 0.05) (Fig. 4B). The cell volume of P. subcurvata LN was significantly smaller than
P. subcurvata HN at all the four treatments (p < 0.05, Fig. 4A), and the surface area to volume
ratio of Fe-limited P. subcurvata LN was significantly higher than P. subcurvata HN at both
temperatures (p < 0.05, Fig. 4B). Cell volume increased in parallel with carbon quota increases
for all the five strains tested (Fig 4C).
3.5 Chlorophyll a and photosynthetic characteristics
The carbon to Chl a ratio of Fe-replete Chaetoceros sp. LN and F. cylindrus HN
significantly decreased relative to Fe-limited cultures at both 0°C and 4°C (p < 0.05) (Fig. 5A).
In addition, warmer temperature decreased the carbon to Chl a ratio of Fe-limited Chaetoceros
sp. LN by 30% and F. cylindrus HN by 49% (p < 0.05) (Fig. 5A). The Chl a per cell was
significantly elevated in Fe-replete cultures of all the four strains of diatom relative to Fe-limited
cultures at both 0°C and 4°C (p < 0.05) (Fig. 5B). The Chl a per cell of Fe-replete P. antarctica
HN significantly increased 50% relative to Fe-limited cultures at 0°C (p < 0.05) (Fig. 5B).
Temperature increase significantly decreased the Chl a per cell of Fe-replete F. cylindrus HN by
36% (p < 0.05) (Fig. 5B). The carbon to Chl a ratio of Fe-replete P. subcurvata LN was
significantly higher than Fe-replete P. subcurvata HN at both 0°C and 4°C, and the carbon to
Chl a ratio of Fe-limited P. subcurvata LN was significantly higher than Fe-limited P.
subcurvata HN at 0°C (p < 0.05) (Fig. 5A). In addition, the Chl a per cell of Fe-replete P.
subcurvata LN was significantly lower than Fe-replete P. subcurvata HN at both 0°C and 4°C,
36
and the Chl a per cell of Fe-limited P. subcurvata LN was significantly smaller than Fe-limited
P. subcurvata HN at 0°C (p < 0.05) (Fig. 5B).
Higher Fe availability significantly increased the F
v
/F
m
of P. subcurvata LN and
Chaetoceros sp. LN (p < 0.05) (Fig. 6). With Fe addition, F
v
/F
m
of both P. subcurvata LN and
Chaetoceros sp. LN increased significantly from 0.18 ± 0.05 and 0.15 ± 0.04 to 0.58 ± 0.03 and
0.56 ± 0.01 (p < 0.05) at 0
o
C, respectively; and from 0.22 ± 0.04 and 0.19 ± 0.02 to 0.49 ± 0.03
and 0.50 ± 0.03 (p < 0.05) at 4
o
C, respectively. The F
v
/F
m
of both species slightly decreased in
response to warming, but this was only significant for P. subcurvata LN (p < 0.05) (Fig. 6).

4 Discussion
Studies have shown that individually iron addition or temperature increase can promote
the growth of Antarctic phytoplankton (Neori and Holm-Hansen 1982; Martin et al., 1990;
Hoffman et al., 2006; Boyd et al., 2007); however, the interactive effects of iron and temperature
increase on representative phytoplankton species from the Ross Sea have seldom been reported.
In our study, as expected, iron-addition alone significantly promoted the growth rates of all the
diatoms and of P. antarctica HN. However, while higher temperature also increased the growth
rates of Fe-limited diatoms and Fe-replete diatoms by 60% and 61%, respectively, it didn’t
significantly affect the growth rate of the prymnesiophyte. The Q
10
for growth rates of P.
antarctica HN is 0.62-0.79, which is lower than 2.0, the typical Q
10 v
alue for phytoplankton as
suggested by Eppley (1992), and also much lower than the Q
10
values for the diatom species
tested in our study (2.02-6.56). This indicates that Ross Sea diatoms may be better adapted to
higher temperatures than P. antarctica.
The growth rates of P. antarctica HN (0.17 d
-1
at 0°C and 0.14 d
-1
at 4°C in Fe-limited
cultures, and 0.40 d
-1
at 0°C and 0.37 d
-1
at 4°C in Fe-replete cultures) in our study are similar to
37
the growth rates observed by Alderkamp et al. (2012). In their study, P. antarctica (strain
CCMP1871) was incubated with a 2h dynamic light cycle at 2°C. They observed that the
average growth rate was 0.2 d
-1
and 0.38 d
-1
in in Fe-limited cultures and Fe-replete cultures,
respectively. Either lack of growth stimulation or decreased growth has also been reported for
other cultured P. antarctica isolates under higher temperatures. Wang et al. (2010) incubated
their strain of P. antarctica (CCMP1871) at 0°C, 2°C, 4°C, and 6°C, and found that the growth
rates of P. antarctica increased from 0.16 d
-1
at 0°C to a maximum of 0.35 d
-1
at 4°C, but then
sharply decreased to 0.12 d
-1
at 6°C. Xu et al. (2014) incubated P. antarctica (CCMP3314) in
three clustered matrices of environment factors, and found that growth rates were similar (~0.6 d
-
1
) under “current conditions” (2°C, 39 Pa CO
2
, and 50 µmol photons m
-2
s
-1
light) and “2060
conditions” (4°C , 61 Pa CO
2
, and 100 µmol photons m
-2
s
-1
light), but significantly decreased to
~0.2 d
-1
under simulated year “2100 conditions” (6°C, 81 Pa CO
2
, 150 µmol photons m
-2
s
-1

light). The slight differences between maximum growth rates and optimum temperatures in these
studies and ours may be due to different strains, and/or different incubation conditions.
There is also a limited amount of research on cultured diatom isolates from the Ross Sea.
Alderkamp et al. (2012) observed that the growth rates of F. cylindrus (strain CCMP1102) were
0.05 d
-1
in Fe-limited and 0.16 d
-1
in Fe-replete media at 2°C. Xu et al. (2014) found the growth
rates of Fe-replete F. cylindrus (CCMP3323) increased from ~0.3 d
-1
at “current conditions” to ~
0.6 d
-1
at “2060 conditions” (see above), and those of Fe-limited F. cylindrus (CCMP3323)
increased from ~0.16 d
-1
at “current conditions” to ~ 0.22 d
-1
at “2060 conditions”. The growth
rates of F. cylindrus we observed are similar to Xu et al. (2014), and higher than those of
Alderkamp et al. (2012), perhaps because our light cycle differed from that used by Alderkamp
et al. (2012).
38
Carbon fixation and iron uptake rates of Fe-replete P. antarctica HN both responded to a
4°C temperature rise with decreasing trends. Warming-mediated decreases in carbon fixation
rates may lead to a reduced demand for Fe to support synthesis of Chl a and associated
photosystem and electron carrier components. In contrast, like growth and carbon fixation rates,
in our Antarctic diatoms Fe uptake rates generally increased with temperature. Xu et al. (2104)
found that the Fe uptake rates of Fe-limited P. antarctica (CCMP3314) decreased from the
“current conditions” to the “year 2100 conditions” clusters, which included warming as well as
rising CO
2
concentration and light intensity. However, they found that Fe uptake rates of Fe-
replete P. antarctica actually increased slightly under clustered future conditions. The interactive
effects of all these environmental factors on Fe uptake rates of P. antarctica and diatoms need
further research.
Our results show that Ross Sea diatoms may be better adapted to higher temperatures
than P. antarctica, which corresponds to Ross Sea field surveys suggesting that the temporal and
spatial distributions of this species are negatively correlated to elevated temperature (Liu and
Smith, 2012). Phytoplankton community dominance shifts caused by experimental warming
have also been observed in other high altitude regions. Feng et al. (2009) observed the dominant
algal groups of the North Atlantic Spring Bloom changed from diatoms to coccolithophores
following incubation under high temperature and high CO
2
conditions for 14 days. Likewise, in
experiments in the subarctic Pacific Noiri et al. (2005) documented that unidentified
prymnesiophytes became dominant at 18 °C, while diatoms dominated the phytoplankton
community at temperatures below 13°C. Interestingly, both of these other high latitude studies
showed that warming shifted the community towards prymnesiophytes, not away from them as
our results suggest for the prymnesiophyte P. antarctica.
39
Iron-addition and temperature increase interactively promoted the growth rates of P.
subcurvata LN and P. subcurvata HN in a synergistic manner; that is, the cumulative effects of
iron-addition and temperature increase far exceeded the magnitude of the additive effects of
these two factors. Rose et al. (2009) documented synergistic effects of temperature increase and
iron addition on total phytoplankton biomass and on the abundance of specific groups including
diatoms and nanoplankton in a Ross Sea shipboard experiment. In addition, temperature
increase in combination with iron addition in the Rose et al. (2009) study shifted diatom
community structure towards the centric Chaetoceros sp., and away from pennate diatoms.
Such a shift would not be predicted by the results of our laboratory culture study, in which both a
centric diatom (Chaetoceros) and a pennate diatom (Pseudo-nitzschia) benefited from a
synergistic interaction between Fe and warming. In multifactor (temperature, light, CO
2
and Fe)
cluster competition experiments between F. cylindrus and P. antarctica, Xu et al. (2014) showed
that the diatom outcompeted P. antarctica under simulated future conditions.
The effects of temperature increase and changes in iron input on phytoplankton
community succession and species composition may also affect the biogeochemical cycle of
carbon and nitrogen in the Southern Ocean (DiTullio et al., 2000; Smetacek et al., 2012). P.
antarctica has higher C: N and C: P ratios than diatoms (Arrigo et al., 1999; Xu et al., 2014).
Indeed, our results indicate that the N: P ratio of Fe-replete P. antarctica HN was higher than Fe-
replete P. subcurvata HN and Fe-replete F. cylindrus HN at 4°C, and the N: P ratio of Fe-limited
P. antarctica HN was higher than Fe-limited F. cylindrus HN at 4°C (p < 0.05) (Table 1). In
addition, the C: P ratios of Fe-replete P. antarctica HN were higher than Fe-replete P.
subcurvata HN at 4°C and Fe-replete F. cylindrus HN at both temperatures, and the C: P ratio of
Fe-limited P. antarctica HN was higher than Fe-limited F. cylindrus HN at 4°C (p < 0.05) (Table
40
1). Thus, if warming or interactions between changing iron and temperature cause shifts away
from P. antarctica and towards diatoms, carbon export per mole of phosphorus utilized and the
N: P ratio of exported organic matter may also decrease in the future Ross Sea (Arrigo et al.,
1999; DiTullio et al., 2000; Smetacek et al., 2012).
Our results suggest that decreasing cell sizes and consequent increases in surface area to
cell volume quotients is a common response of Ross Sea phytoplankton to iron limitation. The
cell size of all the diatoms and P. antarctica decreased in iron-limited culture relative to iron-
replete cultures. This was particularly evident for P. subcurvata grown at both nutrient levels;
for this species, cell size decreased by half in iron-limited cultures. Increased surface area to cell
volume quotients may facilitate iron-uptake under iron-limited conditions (Sunda and Huntsman,
1997; Sunda and Hardison 2010). Decreased cell size also means less iron, carbon and nutrients
needs to be accumulated before cell division can occur, allowing the maintenance of higher cell-
specific growth rates (Garcia et al., 2015). Decreases in cell size under iron limitation have been
recorded in marine cyanobacteria, diatoms, and dinoflagellates, suggesting that this is common
response to lack of this essential micronutrient (Sunda and Huntsman 1997; Hutchins et al.,
1998; Timmermans et al., 2001; Garcia et al., 2015).
Our results indicate that iron addition increased the efficiency of photosystem II in P.
subcurvata LN and Chaetoceros sp. LN at both 0°C and 4°C, consistent with many studies in the
Southern Ocean, including mesoscale Fe enrichment and shipboard incubation experiments
(Boyd et al., 2000; Coale et al., 2004; Rose et al., 2009). As F
v
/F
m
measures electron transfer
efficiency rather than enzyme-based biochemical reactions, it is usually assumed to be relatively
insensitive to temperature. In our experiments P. subcurvata LN exhibited a small but significant
decrease in F
v
/F
m
under Fe-replete conditions with increasing temperature. Negative effects of
41
temperature increase on photosystem II electron flow have occasionally also been reported in
other phytoplankton and plants (Geel et al., 1997; Warner et al., 1996; Zobayed and Kozai,
2005), but the reason for this warming effect is unclear.
Our results also suggest that P. subcurvata may have the potential become Fe and
nutrient co-limited at the end of the austral summer in the future, especially in nearshore areas
such as McMurdo Sound where nutrients are often largely drawn down by the end of the
growing season. The growth rates of Fe-limited P. subcurvata LN were lower than those of Fe-
limited P. subcurvata HN at both temperatures, and the growth rates of Fe-replete P. subcurvata
LN were lower than those of replete HN cultures at 4°C. Furthermore, in both the Fe-limited and
Fe-replete warmed treatments, the C: P ratio of P. subcurvata LN was higher than that of P.
subcurvata HN, and higher than the Redfield ratio (Falkowski, 2000; Geider and LaRoche 2002).
The C: P ratio of Fe-replete P. subcurvata LN was also significantly higher than that of the HN
treatment and the Redfield ratio at 0°C. In addition, the cell size and phosphorus quota of P.
subcurvata LN were significantly smaller than P. subcurvata HN in all the four treatments.
Smaller cell size and thus lower cellular nutrient quotas may be a potential strategy for P.
subcurvata to maintain a competitive advantage during periods of lower seasonal nutrient
availability, as seen in some phytoplankton from other environments (Garcia et al., 2015). We
did not test our other three isolates (Chaetoceros, Fragilariopsis, and Phaeocystis) under both
nutrient conditions. However, future investigations may find that there are significant Fe and
major nutrient co-limitation effects for them as well, interactions that could be important in
coastal portions of the Southern Ocean where nutrients can be seasonally drawn down to
relatively low levels.
Our results may support suggestions that future global warming and potential changes in
42
iron supplies may alter phytoplankton community structure in the future Ross Sea. In general,
our study suggests that warming, either with or without concurrent iron fertilization, may cause
the spatial and temporal distributions of diatoms to expand while the distribution of P. antarctica
may shrink. It is important to consider, however, that the competitive balance between diatoms
and P. antarctica will likely also be affected by other factors that are changing along with global
warming, such as ocean acidification, irradiance and salinity changes, shifts in the grazing
community (Boyd and Hutchins 2012), and the availability of other micronutrients and organic
cofactors such as vitamins to the phytoplankton community in the coastal Southern Ocean
(Bertrand et al., 2015). Additionally, the responses of diatoms and P. antarctica to a wider
temperature range will be important to determine, as response curves that include a range of
temperatures may provide deeper insight into the competitive interplay between these two groups
of phytoplankton in a rapidly changing Southern Ocean.

Acknowledgements
This work was supported by National Science Foundation grants ANT 1043748 to DAH and
1043635 to DAB. We thank Avery Tatters for isolating these strains.
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52
Table 1. Effects of temperature and Fe addition on the C:N, N:P, and C:P ratios of P. subcurvata LN, P.
subcurvata HN, Chaetoceros sp. LN, F. cylindrus HN, and P. antarctica HN Values are means and
errors are standard deviations of triplicate bottles, different letters indicate significant different at p < 0.05
level, * indicates interactive effects of temperature and iron.

P. subcurvata
LN
P. subcurvata
HN
Chaetoceros sp.
LN
F. cylindrus
HN
P. antarctica
HN
C: N
0C-Fe 5.1±0.5
a
6.5±0.5
a
6.5±0.5
a
6.3±0.3
a
6.5±0.4
a

0C+Fe 6.9±0.4
b
6.9±0.2
a
7.9±0.6
b
7.0±0.0
b
7.2±0.8
a

4C-Fe 4.9±0.8
a
6.6±0.3
a
6.6±0.0
a
7.0±0.4
ab
6.6±0.5
a

4C+Fe 6.3±0.2
b
6.8±0.1
a
7.3±0.3
b
6.9±0.1
ab
6.6±0.5
a

N: P
0C-Fe 18.1±2.7
a
15.3±0.5
a
23.0±5.0
a
11.6±0.6
a
18.5±3.8
a

0C+Fe 21.3±1.4
a
15.7±3.7
abc
18.9±1.6
ab
15.4±0.8
b
16.7±0.5
a

4C-Fe 28.6±5.4
b
13.5±0.4
b
19.6±0.5
a
11.3±1.4
a
25.6±5.2
a

4C+Fe 38.1±1.5
c
12.5±0.4
c
16.3±0.3
b
15.1±0.3
b
32.5±2.0
b

C: P
0C-Fe 92.4±17.9
a
90.6±6.5
a
151.5±43.9
abc
75.9±2.7
ab
134.5±27.5
a

0C+Fe 147.0±6.4
b
80.1±17.6
a
147.5±6.0
c
85.0±6.7
a
100.4±5.5
a

4C-Fe 136.4±4.3
b
102.1±4.5
a
128.4±3.5
a
63.2±6.5
b
187.8±37.7
a

4C+Fe 241.6±13.7
c
80.9±2.5
a
118.3±6.8
b
82.0±3.5
a
214.9±20.4
b

53
Figure legends
Fig. 1. Specific growth rates (A) and Q
10
(B) of Pseudo-nitzschia subcurvata LN, Pseudo-
nitzschia subcurvata HN, Chaetoceros sp. LN, Fragilariopsis cylindrus HN, and Phaeocystis
antarctica HN at 0°C–Fe limited (0C-Fe), 0°C–Fe replete (0C+Fe), 4°C–Fe limited (4C-Fe),
4°C–Fe replete (4C+Fe)

Fig. 2. Carbon fixation rates (A), Fe uptake rates (B), and Fe: C uptake ratios (C) of Pseudo-
nitzschia subcurvata LN, Pseudo-nitzschia subcurvata HN, Chaetoceros sp. LN, Fragilariopsis
cylindrus HN, and Phaeocystis antarctica HN at 0°C–Fe limited (0C-Fe), 0°C–Fe replete
(0C+Fe), 4°C–Fe limited (4C-Fe), 4°C–Fe replete (4C+Fe)

Fig. 3 POC (A), PON (B), POP (C), and BSi (D) of Pseudo-nitzschia subcurvata LN, Pseudo-
nitzschia subcurvata HN, Chaetoceros sp. LN, Fragilariopsis cylindrus HN, and Phaeocystis
antarctica HN at 0°C–Fe limited (0C-Fe), 0°C–Fe replete (0C+Fe), 4°C–Fe limited (4C-Fe),
4°C–Fe replete (4C+Fe), BSi was measured only for Pseudo-nitzschia subcurvata LN and
Chaetoceros sp. LN

Fig. 4. Cell volume (A), surface area to cell volume ratios (B), and relationship of POC and
volume of Pseudo-nitzschia subcurvata LN, Pseudo-nitzschia subcurvata HN, Chaetoceros sp.
LN, Fragilariopsis cylindrus HN, and Phaeocystis antarctica HN at 0°C–Fe limited (0C-Fe),
0°C–Fe replete (0C+Fe), 4°C–Fe limited (4C-Fe), 4°C–Fe replete (4C+Fe)

54
Fig. 5. POC to chl a ratio (g g
-1
) (A) and Chl a per cell (pg cell
-1
) (B) of Pseudo-nitzschia
subcurvata LN, Pseudo-nitzschia subcurvata HN, Chaetoceros sp. LN, Fragilariopsis cylindrus
HN, and Phaeocystis antarctica HN at 0°C–Fe limited (0C-Fe), 0°C–Fe replete (0C+Fe), 4°C–
Fe limited (4C-Fe), 4°C–Fe replete (4C+Fe)

Fig. 6. F
v
/F
m
of Pseudo-nitzschia subcurvata LN and Chaetoceros sp. LN at 0°C–Fe limited (0C-
Fe), 0°C–Fe replete (0C+Fe), 4°C–Fe limited (4C-Fe), 4°C–Fe replete (4C+Fe)

55
Fig. 1

B
A
56

Fig. 2

C
B
A
57
Fig. 3

A
C
B
D
58
Fig. 4

C
A
B
59
Fig. 5

B
A
A
60
Fig. 6

61
Chapter 2: Individual and interactive effects of warming and CO
2
on Pseudo-nitzschia
subcurvata and Phaeocystis antarctica, two dominant phytoplankton from the Ross Sea,
Antarctica
Zhi Zhu
1
, Pingping Qu
1
, Jasmine Gale
1
, Feixue Fu
1
, David A. Hutchins
1
1. Department of Biological Science, University of Southern California, Los Angeles, CA 90089,
USA.
Correspondence to: David A. Hutchins (dahutch@usc.edu)

Abstract: We investigated the effects of temperature and CO
2
variation on the growth and
elemental composition of cultures of the diatom Pseudo-nitzschia subcurvata and the
prymnesiophyte Phaeocystis antarctica, two ecologically dominant phytoplankton species
isolated from the Ross Sea, Antarctica. To obtain thermal functional response curves, cultures
were grown across a range of temperatures from 0
o
C to 14
o
C. In addition, a competition
experiment examined the relative abundance of both species at 0
o
C and 6
o
C. CO
2
functional
response curves were conducted from 100 to 1730 ppm at 2
o
C and 8
o
C to test for interactive
effects between the two variables. The growth of both phytoplankton was significantly affected
by temperature increase, but with different trends. Growth rates of P. subcurvata increased with
temperature from 0°C to maximum levels at 8°C, while the growth rates of P. antarctica only
increased from 0°C to 2°C. The maximum thermal limits of P. subcurvata and P. antarctica
where growth stopped completely were 14°C and 10°C, respectively. Although P. subcurvata
outcompeted P. antarctica at both temperatures in the competition experiment, this happened
much faster at 6°C than at 0°C. For P. subcurvata, there was a significant interactive effect in
which the warmer temperature decreased the CO
2
half saturation constant for growth, but this
62
was not the case for P. antarctica. The growth rates of both species increased with CO
2
increases
up 425 ppm, and in contrast to significant effects of temperature, the effects of CO
2
increase on
their elemental composition were minimal. Our results suggest that future warming may be more
favorable to the diatom than to the prymnesiophyte, while CO
2
increases may not be a major
factor in future competitive interactions between Pseudo-nitzschia subcurvata and Phaeocystis
antarctica in the Ross Sea.

1 Introduction
Global temperature is predicted to increase 2.6°C to 4.8°C by 2100 with increasing
anthropogenic CO
2
emissions (IPCC, 2014). The temperature of the Southern Ocean has
increased even faster than global average temperature (Gille, 2002), and predicted future climate
warming may profoundly change the ocean carbon cycle in this region (Sarmiento et al., 1998).
The Ross Sea, Antarctica, is one of the most productive area in the ocean, and features annual
austral spring and summer algal blooms dominated by Phaeocystis and diatoms that contribute as
much as 30% of total primary production in the Southern Ocean (Arrigo et al., 1999, 2008;
Smith et al., 2000). The response of phytoplankton in the Southern Ocean to future temperature
change may offset the decrease of carbon export caused by intensified stratification (Sarmiento
et al., 1998), and the physiological effects of warming may partially compensate for a lack of
iron throughout much of this region (Hutchins and Boyd, 2016).
In the Ross Sea, the colonial prymnesiophyte Phaeocystis antarctica typically blooms in
austral spring and early summer, and diatoms including Pseudo-nitzschia subcurvata and
Chaetoceros spp. bloom later in the austral summer (Arrigo et al., 1999, 2000; DiTullio and
Smith, 1996; Goffart et al., 2000; Rose et al., 2009). Both diatoms and P. antarctica play an
important role in anthropogenic CO
2
drawdown and the global carbon cycle; additionally, they
63
contribute significantly to the global silicon and sulfur cycles, respectively (Arrigo et al., 1999;
Tréguer et al., 1995; Schoemann et al., 2005). Furthermore, the elemental ratios of P. antarctica
and diatoms are different and thus they contribute unequally to the carbon, nitrogen, and
phosphorus cycles (Arrigo et al., 1999, 2000). Diatoms are preferred by zooplankton grazers
over P. antarctica, and so the two groups also differentially influence the food webs of the
Southern Ocean (Knox, 1994; Caron et al., 2000).
Arrigo et al. (1999) suggested that the spatial and temporal distributions of P. antarctica
and diatoms in the Ross Sea are determined by the mixed layer depth, while Liu and Smith
(2012) indicated that temperature is more important in shaping the distribution of these two
dominant groups of phytoplankton. Zhu et al. (2016) observed that a 4ºC temperature increase
promoted the growth rates of several dominant diatoms isolated from Ross Sea, including P.
subcurvata, Chaetoceros sp., and Fragilariopsis cylindrus, but not the growth rates of P.
antarctica. In addition, both field and laboratory research has suggested that temperature
increase and iron addition can synergistically promote the growth of Ross Sea diatoms (Rose et
al., 2009; Zhu et al., 2016; Hutchins and Boyd, 2016). Thus, it is possible that phytoplankton
community structure in the Southern Ocean may change in the future under a global warming
scenario.
In addition to temperature increases, ocean uptake of 30% of total emitted anthropogenic
CO
2
has led to a 0.1 pH unit decrease in surface water, corresponding to a 26% increase in
acidity (IPCC, 2014). The global CO
2
concentration is predicted to increase to around 800 ppm
by 2100, which will lead to a further decrease in surface seawater pH of 0.3–0.4 units (Orr et al.,
2005; IPCC, 2014). CO
2
increases have been found to promote the growth and affect the
64
physiology of many but not all phytoplankton species tested (Fu et al., 2007, 2008; King et al.,
2011; Xu et al., 2014).
Research on the effects of CO
2
increases on Phaeocystis antarctica and Antarctic diatoms
is still scarce. Xu et al. (2014) suggested that future conditions (higher temperature, CO
2
, and
light intensity) may shift phytoplankton community structure towards diatoms and away from P.
antarctica in the Ross Sea. Trimborn et al. (2013) discovered that the growth rates of P.
antarctica and P. subcurvata were not significantly promoted by high CO
2
relative to ambient
CO
2
at 3°C. In contrast, Wang et al. (2010) observed that the growth rates of the closely related
temperate colonial species Phaeocystis globosa increased significantly at 750 ppm CO
2
relative
to 380 ppm CO
2
.
Thus, an important goal of phytoplankton research is to understand how global warming
together with ocean acidification may shift the phytoplankton community in the Southern Ocean
(Arrigo et al., 1999; DiTullio et al., 2000). This study aimed to explore the effects of increases in
temperature and CO
2
availability, both individually and in combination, on P. antarctica and P.
subcurvata isolated from the Ross Sea, Antarctica. These results may shed light on the potential
effects of global change on the marine ecosystem and the cycles of carbon and nutrients in the
highly productive coastal polynyas of Antarctica.

2 Materials and Methods
2.1 Strains and growth conditions
P. subcurvata and P. antarctica were isolated from the ice edge in McMurdo Sound
(77.62° S, 165.47° E) in the Ross Sea, Antarctica during January 2015. All stock cultures were
grown in Aquil* medium (100 µmol L
-1
NO
3
-
, 100 µmol L
-1
SiO
4
4-
,10 µmol L
-1
PO
4
3-
) made
with 0.2 µM-filtered seawater that was collected from the same Ross Sea locale as the culture
65
isolates (Sunda et al., 2005). Because stock and experimental cultures were grown in Fe-replete
Aquil medium (0.5 µM), culture conditions most closely resembled the McMurdo Sound ice
edge environment in the early spring when Fe is not limiting, prior to being drawn down over the
course of the seasonal algal bloom (Bertrand et al., 2015). Cultures were maintained at 0°C in a
walk-in incubator under 24 h cold white fluorescence light (80 µmol photons m
-2
s
-1
).
2.2 Experimental design
For thermal functional response curves, experimental cultures of both phytoplankton
were grown in triplicate 500 ml acid washed polycarbonate bottles and gradually acclimated by a
series of step-wise transfers to a range of temperatures, including 0°C, 2°C, 4°C, 6°C, 8°C, and
10°C (P. antarctica died at 10°C) under the same light cycle as stock cultures. Cultures were
diluted semi-continuously following Zhu et al. (2016). All of the cultures were acclimated to
their respective temperatures for 8 weeks before the commencement of the experiment. At this
point, after the growth rates were verified to be stable for at least three to five consecutive
transfers, the cultures were sampled 48 h after dilution (Zhu et al., 2016).
For CO
2
functional response curves, P. antarctica and P. subcurvata were also grown in
triplicate in a series of six CO
2
concentrations from ~100 ppm to ~1730 ppm in triplicate 500 ml
acid washed polycarbonate bottles at both 2°C and 8°C using same dilution technique as above.
The CO
2
concentration was achieved by gently bubbling with 0.2 µm filtered air/CO
2
mixture
(Gilmore, CA) and carbonate system equilibration was ensured by pH and dissolved inorganic
carbon (DIC) measurements (King et al., 2015, see below).
To examine thermal effects on competition between the two species, P. antarctica and P.
subcurvata (pre-acclimated to respective temperatures) were mixed at equal Chl a (chlorophyll
a) concentrations and grown together for 6 days in triplicate bottles at both 0°C and 6°C. The
66
relative abundance of each phytoplankton was then calculated based on cell counts taken on days
0, 3 and 6.
2.3 Growth rates
Cell count samples were counted on a Sedgewick Rafter Grid using an Olympus BX51
microscope before and after dilution for each treatment. Samples that couldn’t be counted
immediately were preserved with Lugol’s (final concentration 2%) and stored at 4°C until
counting. Specific growth rates (d
-1
) were calculated following Eq. (1):
µ = (ln N
1
– ln N
0
)/t, (1)
where N
0
and N
1
are the cell density at the beginning and end of a dilution period, respectively,
and t is the duration of the dilution period (Zhu et al. 2016). The Q
10
of growth rates was
calculated following Chaui-Berlinck et al. (2002) as Eq. (2):
Q
10
= (µ
2
/ µ
1
)
10/(T
2
-T
1
)
, (2)
where µ
1
and µ
2
are the specific growth rates of the phytoplankton at temperatures T
1
and T
2
,
respectively. The growth rates were fitted to Eq. (3) to estimate the thermal reaction norms of
each species:
f(T) = ae
bT
(1- ((T-z)/(w/2))
2
), (3)
where specific growth rate f depends on temperature (T), temperature niche width (w), and other
empirical parameters z, a, and b were estimated by maximum likelihood (Thomas et al., 2012;
Boyd et al., 2013). Afterwards, the optimum temperature for growth and maximum growth rate
were estimated by numerically maximizing the equation (Boyd et al., 2013). The growth rates of
all the species at all the CO
2
levels were fitted to Michaelis-Menten equation as Eq. (4):
µ = µ
max
S/(K
m
+ S), (4)
67
to estimate maximum growth rates (µ
max
) and half saturation constants (K
m
) for CO
2

concentration (S).
2.4 Elemental and Chl a analysis
Culture samples for particulate organic carbon/nitrogen (POC/PON) and particulate
organic phosphorus (POP) analyses were filtered onto pre-combusted (500°C for 2 h) GF/F
filters and dried at 60°C overnight. A 30 ml aliquot of P. subcurvata culture samples for each
treatment were filtered onto 2 µm polycarbonate filters (GE Healthcare, CA) and dried in a 60°C
oven overnight for biogenic silica (BSi) analysis. The analysis method of POC/PON and POP
followed Fu et al. (2007), and BSi analysis followed Paasche et al. (1973). An aliquot of 30 to 50
ml from each treatment replicate was filtered onto GF/F filters and extracted with 90% acetone at
-20°C for 24 h for Chl a analysis. The Chl a concentration was then determined using the non-
acidification method on a 10-AU
TM
fluorometer (Turner Design, CA) (Fu et al., 2007).
2.5 pH and dissolved inorganic carbon (DIC) measurements
pH was measured using a pH meter (Thermo Scientific, MA), calibrated with pH 7 and
10 buffer solutions. For DIC analyses, an aliquot of 25 mL was preserved with 200 µL 5% HgCl
2

and stored in the dark at 4
o
C until analysis. Total DIC was measured using CM140 Total
Inorganic Carbon Analyzer (UIC Inc., IL). An aliquot of 5 mL sample was injected into the
sparging column of Acidification Unit CM5230 (UIC Inc., IL) followed by 2 ml 10% phosphoric
acid. By using flow rates controlled pure nitrogen as carrier gas, the CO
2
released from the DIC
pool in the sample was quantified by CM5015 CO
2
Coulometer (UIC Inc., IL) using absolute
coulometric titration. The carbonate buffer system was sampled for each of the triplicate bottles
in each treatment at the beginning and end of the experiments; reported values are final ones.
The pCO
2
in growth media was calculated using CO2SYS (Pierrot et al., 2006). These carbonate
68
system measurements are shown in Table 1, along with the corresponding calculated pCO
2

values calculated. Kinetic parameters were calculated using the individual calculated pCO
2

values for each replicate (see above), but for convenience, the CO
2
treatments are referred to in
the text using the mean value of all experimental bottles, rounded to the nearest 5 ppm: these
values are 100 ppm, 205 ppm, 260 ppm, 425 ppm, 755 ppm, and 1730 ppm.
2.6 Statistical analysis
All statistical analyses and model fitting, including student t-tests, ANOVA, Tukey’s
HSD test, two-way ANOVA, and thermal reaction norms estimation were conducted using the
open source statistical software R version 3.1.2 (R Foundation).
3 Results
3.1 Temperature effects on growth rates
Temperature increase significantly affected the growth rates of both P. antarctica and P.
subcurvata, but with different trends (p < 0.05) (Fig. 1). The specific growth rates of P.
subcurvata increased from 0°C to 8°C (p < 0.05), and then significantly decreased at 10°C (p <
0.05) (Fig. 1). The growth rates of P. antarctica significantly increased from 0°C to 2°C, and
plateaued at 4°C and 6°C, and then significantly decreased from 6°C to 8°C (p < 0.05) (Fig. 1).
P. antarctica and P. subcurvata stopped growing at 10°C and 14°C, respectively (Fig. 1A). The
specific growth rates of P. subcurvata were not significantly different from those of P. antarctica
at 0°C, 2°C and 4°C, but became significantly higher than P. antarctica at 6°C, and remained
significantly higher than P. antarctica through 8°C and 10°C (p < 0.05) (Fig. 1A). The optimum
temperatures for growth of P. antarctica and P. subcurvata were 4.9°C and 7.4°C, respectively
(Table 2). In addition, the estimated temperature niche width of P. subcurvata
69
(-2.0°C – 12.2°C) is wider than that of P. antarctica (-2.0°C to 9.5°C) (Table 2); calculated
minimum temperatures estimated from the thermal niche width equation were less than -2.0°, the
freezing point of seawater, and so growth is assumed to terminate at -2.0°. The Q10 value of the
growth rate of P. antarctica from 0°C to 4°C is 2.1, which is lower than the Q10 values 3.2 for
P. subcurvata over the same temperature interval (p < 0.05) (Table 2).
3.2 Temperature effects on elemental composition
The C: N and N: P ratios of P. subcurvata were unaffected by changing temperature (Fig.
2A, B), but the C: P, C: Si, and C: Chl a ratios of this species were significantly affected (p <
0.05) (Fig. 2C, D, Fig. 3). The C: P ratios of P. subcurvata were slightly but significantly lower
in the middle of the tested temperature range. They were higher at 8°C and 10°C than at 2°C,
4°C, and 6°C (p < 0.05) (Fig. 2C), and also significantly higher at 10°C than at 0°C (Fig. 2C).
The C: Si ratios of P. subcurvata showed a similar pattern of slightly lower values at mid-range
temperatures; at 0°C and 2°C they were significantly higher than at 6°C and 8°C (p < 0.05) (Fig.
2D), and significantly higher at 2°C and 10°C than at 4°C and 8°C, respectively (Fig. 2D). The
C: Chl a ratios of P. subcurvata also showed this trend of somewhat lower values in the middle
of the thermal gradient. At 0°C, 8°C and 10°C, C: Chl a ratios were significantly higher than at
2°C, 4°C, and 6°C (p < 0.05), and also significantly higher at 10°C than at 0°C and 8°C (Fig. 3).
The C: N, N: P, C: P, and C: Chl a ratios of P. antarctica were not significantly different
across the temperature range (Fig. 2A, B, C, Fig. 3). The N: P ratios of P. antarctica were
significantly higher than those of P. subcurvata at 2°C, 6°C, and 8°C (p < 0.05) (Fig. 2B).
Additionally, the C: P ratios of P. antarctica were significantly higher than those of P.
subcurvata at 6°C and 8°C (p < 0.05) (Fig. 2C), and the C: Chl a ratios of P. antarctica were
significantly higher than values of P. subcurvata at all the temperatures tested (p < 0.05) (Fig. 3).
70
Temperature change significantly affected the cellular carbon (C) quotas, cellular
nitrogen (N) quotas, cellular phosphorus (P) quotas, cellular silica (Si) quotas, and cellular Chl a
quotas of P. subcurvata (p < 0.05) (Table 3). The cellular C and N quotas of P. subcurvata were
significantly higher at 8°C than at 0°C (p < 0.05) (Table 3), the cellular P quotas of P.
subcurvata were significantly higher at 4°C than at 0°C, 2°C, and 10°C (p < 0.05) (Table 3), and
the cellular Si quotas of P. subcurvata were significantly higher at 8°C than at 0°C and 2°C. Si
quotas were also significantly higher at 4°C and 6°C than at 0°C (p < 0.05) (Table 3). The
extreme temperatures significantly decreased the cellular Chl a quotas of P. subcurvata, as the
cellular Chl a quotas of this species were significantly higher at 4°C, 6°C, and 8°C than at 0°C
and 10°C (p < 0.05) (Table 3).
Temperature change significantly affected the cellular P quotas and cellular Chl a quotas
of P. antarctica (p < 0.05), but not the cellular C and N quotas (p > 0.05) (Table 3). The cellular
P quotas of P. antarctica were significantly higher at 0°C than at 8°C (p < 0.05) (Table 3), and
the Chl a quotas of the prymnesiophyte were significantly lower at 8°C than at 0°C, 2°C, and
6°C (p < 0.05) (Table 3).
3.3 Competition at two temperatures
A warmer temperature favored the dominance of P. ubcurvata over P. antarctica in the
competition experiment. Although P. subcurvata increased its abundance relative to the
prymnesiophyte at both temperatures by day 6, this increase was larger and happened much
faster at 6°C (from 31% to 72%) relative to 0°C (from 31% to 38%) (p < 0.05) (Fig. 4).
3.4 CO
2
effects on specific growth rates at two temperatures
The carbonate system was relatively stable across the range of CO
2
levels during the
course of the experiment (Table 1). CO
2
concentration significantly affected the growth rates of
71
P. subcurvata at both temperatures. The growth rates of the diatom at 2°C increased steadily
with CO
2
concentration increase from 205 ppm to 425 ppm (p < 0.05), but were saturated at at
755 ppm and 1730 ppm (Fig. 5A). Similarly, the growth rates of P. subcurvata at 8°C increased
with CO
2
concentration increase from 205 ppm to 260 ppm (p < 0.05), and were saturated at 425
ppm, 755 ppm and 1730 ppm (Fig. 5B). The growth rates of the diatom at all CO
2
concentrations
tested at 8°C were significantly higher than at 2°C (p < 0.05); for instance, the maximum growth
rate of P. subcurvata at 8°C was 0.88 d
-1
, significantly higher than the value of 0.60 d
-1
at 2°C (p
< 0.0.5) (Table 4). In addition, the pCO
2
half saturation constant (K
m
) of P. subcurvata at 8°C
was 10.7 ppm, significantly lower than 66.0 ppm at 2°C (p < 0.0.5) (Table 4). Thus,
temperature and CO
2
concentration increase interactively increased the growth rates of P.
subcurvata (p < 0.05).
CO
2
concentration also significantly affected the growth rates of P. antarctica
at both 2°C and 8°C. The growth rates of the prymnesiophyte at both 2°C and 8°C increased with
CO
2
concentration increase from 100 ppm to 260 ppm (p < 0.05), and were saturated at 425 ppm
and 755 ppm (Fig. 5C, D). The growth rates of P. antarctica at 2°C decreased slightly at 1730
ppm relative to 425 ppm and 755 ppm (p < 0.05) (Fig. 5C). The maximum growth rate of P.
antarctica at 8°C was 0.43 d
-1
, significantly lower than the value of 0.61 d
-1
at 2°C (p < 0.05) (
Table 4). The pCO
2
half saturation constants of P. antarctica at 2°C and 8°C were not
significantly different (Table 4), and thus no interactive effect of temperature and CO
2
was
observed on the growth rate of the prymnesiophyte (p > 0.05).
3.5 CO
2
effects on elemental composition at two temperatures
CO
2
concentration variation didn’t affect the C: N, N: P, or C: P ratios of P. subcurvata at
either 2°C or 8°C. The C: Si ratios of P. subcurvata were significantly higher at 1730 ppm
72
relative to lower pCO
2
levels, except at 755 ppm at 8°C (p < 0.05) (Table 5). The N: P ratios of
P. subcurvata at 8°C were significantly higher than at 2°C at all the CO
2
levels tested except 100
ppm (p < 0.05) (Table 5). The C: P ratios of P. subcurvata at 8°C were significantly higher than
at 2°C at all the CO
2
levels tested (p < 0.05) (Table 5). The C: Si ratios of P. subcurvata at CO
2

levels lower than 755 ppm at 8°C were significantly lower than at 2°C (p < 0.05) (Table 5). The
higher temperature also significantly increased the C: Chl a ratios of P. subcurvata at all the CO
2
levels tested (p < 0.05) (Table 5). Additionally, the temperature increase and CO
2
concentration
increase interactively decreased the C: Chl a ratios of P. subcurvata (p < 0.05) (Table 5).
The CO
2
concentration increase did not affect the C: N, N: P, and C: P ratios of P.
antarctica at either 2°C or 8°C. The carbon to Chl a ratios of P. antarctica were significantly
higher at 1730 ppm than at all lower CO
2
concentrations at 2°C. Similarly, at 8°C the carbon to
Chl a ratios of this species also were significantly higher at 425 ppm, 755 ppm, and 1730 ppm
than at lower CO
2
concentrations (p < 0.05) (Table 5), and significantly higher at 1730 ppm than
at 425 ppm and 755 ppm (p < 0.05) (Table 5).
The warmer temperature significantly decreased the C: N ratios of P. antarctica at 260
ppm and 755 ppm CO
2
(p < 0.05) (Table 5), and C: P ratios also decreased at 100 ppm and 205
ppm (p < 0.05) (Table 5). The C: Chl a ratios of P. antarctica at CO
2
levels higher than 205 ppm
were significantly higher at 8°C relative to 2°C (p < 0.05) (Table 5). Temperature and CO
2

concentration increase interactively increased the C: Chl a ratios of P. antarctica (p < 0.05)
(Table 5).
The CO
2
concentration increase didn’t affect the cellular C, N, P, or Si quotas of P.
subcurvata at 2°C, or the C quotas and N quotas at 8°C. The Si quotas of P. subcurvata were
significantly lower at 1730 ppm CO
2
than at 100 ppm and 205 ppm at 8°C (p < 0.05) (Table 6).
73
The cellular Chl a quotas of P. subcurvata were significantly lower at 8°C relative to 2°C at CO
2
higher than 205 ppm (p < 0.05) (Table 6). The temperature increase significantly increased the
cellular Si quota of P. subcurvata at all the CO
2
levels tested except 1730 ppm (p < 0.05) (Table
6). Additionally, warming and CO
2
concentration interactively decreased the cellular Si quotas of
P. subcurvata (p < 0.05) (Table 6).
The C, N, and P quotas of P. antarctica were not affected by CO
2
increase at 2°C, and N
and P quotas were not affected by CO
2
increase at 8°C, either. However, the C quota of P.
antarctica at 1730 ppm CO
2
was significantly higher than CO
2
levels lower than 755 ppm at 8°C
(p < 0.05) (Table 6). The Chl a per cell of P. antarctica at 1730 ppm CO
2
was significantly less
than at lower CO
2
levels at both 2°C and 8°C (p < 0.05) (Table 6). For P. antarctica, the Chl a
per cell values at 100 ppm, 205 ppm, and 755 ppm CO
2
at 8°C were significantly lower relative
to 2°C (p < 0.05) (Table 6). Temperature increase and CO
2
concentration increase interactively
increased the C and N quotas of P. antarctica (p < 0.05) (Table 6).
4 Discussion
As has been documented in previous work, the diatom P. subcurvata and the
prymnesiophyte P. antarctica responded differently to warming (Xu et al., 2014; Zhu et al.
2016). In the Southern Ocean as elsewhere, temperature determines both phytoplankton
maximum growth rates (Bissinger et al., 2008) and the upper limit of growth (Smith, 1990) in a
species-specific manner. Thermal functional responses curves of phytoplankton typically
increase in a normally distributed pattern, with growth rates increasing up to the optimum
temperature range, and then declining when temperature reaches inhibitory levels (Boyd et al.,
2013; Fu et al., 2014; Xu et al., 2014). Specific growth rates of P. subcurvata reached optimal
levels at 8°C, while those of P. antarctica saturated at 2°C. Zhu et al. (2016) found that 4°C
74
warming significantly promoted the growth rates of P. subcurvata but not P. antarctica. Xu et al.
(2014) found that the growth rates of another strain of P. antarctica (CCMP3314) decreased in a
multi-variable “year 2100 cluster” condition (6°C, 81 Pa CO
2
, 150 µmol photons m
−2
s
−1
)
relative to the “current condition” (2°C, 39 Pa CO
2
, and 50 µmol photons m
−2
s
−1
) and the “year
2060 condition” (4°C, 61 Pa CO
2
, and 100 µmol photons m
−2
s
−1
). In our study, the Q10 value of
P. subcurvata from 0°C to 4°C was 3.11, nearly 50% higher than the Q10 value of P. antarctica
across the same temperature range (2.17), and similar to the Q10 values observed for different
strains of these two species in Zhu et al. (2016). Our results showed that the maximal thermal
limit of P. antarctica was reached at 10°C, as was also observed by Buma et al. (1991), while P.
subcurvata did not cease to grow until 14°C. Clearly, P. subcurvata has a superior tolerance to
higher temperature compared to P. antarctica.
The competition experiment between P. subcurvata and P. antarctica at 0°C and 6°C
confirmed that the diatom had an additional competitive advantage over P. antarctica at the
higher temperature. Xu el al. (2014) observed that the diatom Fragilariopsis cylindrus also
outcompeted P. antarctica under “year 2060 conditions” (4°C, 61 Pa CO
2
, and 100 µmol
photons m
−2
s
−1
). These competition experiments support the results of a Ross Sea field survey
which suggested that water temperature structured the phytoplankton assemblage (Liu and
Smith, 2012), and may shed light on why P. antarctica is often dominant in cooler waters in the
springtime, while diatoms often dominate in summer (DiTullio and Smith, 1996; Arrigo et al.,
1999; DiTullio et al., 2000; Liu and Smith, 2012).
Besides temperature, mixed layer depth and light intensity also likely play a role in the
competition between diatoms and P. antarctica (Arrigo et al., 1999; Arrigo et al., 2010). Arrigo
et al. (1999) observed that P. antarctica dominated the southern Ross Sea region with deeper
75
mixed layers, while diatom dominated the regions with shallower mixed layer depths. To some
extent temperature and irradiance can often be considered co-variables, as shallow surface
stratification promotes both solar heating and high irradiance, while deep mixing lowers both
light and temperatures. Thus, rather than being segregated by either light or by temperature, it is
worth considering whether these two phytoplankton groups are each best adapted to a different
environmental matrix of both variables. This concept of different light/temperature niches for
Ross Sea diatoms and P. antarctica is worthy of further investigation.
Temperature change affected the C: P, N: P and C: Si ratios of P. subcurvata, due to the
combined effects of the different responses of cellular C, P, and Si quotas. The C: P and N:P
ratios of P. subcurvata increased at the two highest temperatures tested. This might be due to an
increase in protein translation efficiency and a corresponding decrease in phosphate-rich
ribosomes with warming, which can result in a decreased cellular P requirement per unit of
carbon in marine phytoplankton (Toseland et al., 2013). Similarly lowered P quotas at higher
temperatures have been documented in other studies as well (Xu et al., 2014; Boyd et al., 2015;
Hutchins and Boyd, 2016). This result suggests that the amount of carbon exported per unit
phosphorus by P. subcurvata (and perhaps other diatoms) in the Southern Ocean may increase as
temperature increases in the future (Toseland et al., 2013).
In contrast, the decreasing trend of C: Si ratios in P. subcurvata appears to be largely due
to higher cellular Si quotas at temperatures at and above 4°C. Although the physiological
reason(s) for increased silicification with warming are currently not understood, this trend also
may have significant biogeochemical consequences. These results suggest that Si export by
diatoms in the Southern Ocean could be enhanced under future global warming.
76
Previous studies have shown that nutrient drawdown by diatoms and P. antarctica are
different, due to differing elemental ratios of these two groups (Arrigo et al., 1999; Xu et al.,
2014). Our results generally corresponded to this trend, as the N: P ratios of P. antarctica were
higher than P. subcurvata at 2°C, 6°C and 8°C and C: P ratios of P. antarctica were higher than
P. subcurvata at 6°C and 8°C (p < 0.05) (Fig. 2). Although elemental ratios of the
prymnesiophyte were largely unaffected by temperature. phytoplankton relative abundance shifts
caused by warming (such as those observed in our competition experiment) will likely change
nutrient export ratios. Thus, N and C export per unit P may decrease with a phytoplankton
community shift from P. antarctica dominance to diatom dominance (Arrigo et al., 1999; Xu et
al., 2014).
Our results showed that the growth rates of both P. subcurvata and P. antarctica
exhibited moderate limitation by CO
2
levels lower than ~425 ppm at both 2°C and 8°C; this
observation is significant, since pCO
2
during the intense Ross Sea summertime phytoplankton
bloom can sometimes drop to very low levels (Tagliabue and Arrigo, 2016). However, at CO
2

concentrations beyond current atmospheric levels of ~400 ppm, growth rates of P. subcurvata or
P. antarctica were CO
2
-saturated. Although a general model prediction suggests that an
atmospheric CO
2
increase from current levels to 700 ppm could increase the growth of marine
phytoplankton by 40% (Schippers et al., 2004), our results instead correspond to several other
studies which showed negligible effects of elevated CO
2
on different groups of phytoplankton,
including P. subcurvata and P. antarctica (Goldman, 1999; Fu et al., 2007, Trimborn et al.,
2013). The minimal effects of CO
2
levels higher than 400 ppm on these phytoplankton has been
suggested to be due to efficient carbon concentrating mechanisms (CCMs) (Burkhardt et al.,
2001; Fu et al., 2007; Tortell et al., 2008; Trimborn et al., 2013), although clearly for our two
77
species their CCM activity was not sufficient to completely compensate for carbon limitation at
low pCO
2
levels. Our results also showed that very high CO
2
(1730 ppm) significantly reduced
the growth rate of P. antarctica relative to 425 ppm and 755 ppm at 2°C; negative effects of high
CO
2
on an Antarctic microbial community were also observed by Davidson et al. (2016). This
inhibitory effect might be due to the significantly lower pH at 1730 ppm (~7.4), which could
entail expenditures of additional energy to maintain pH homeostasis within cells.
Warming from 2°C to 8°C had a significant interactive effect with CO
2
concentration in
P. subcurvata, as maximum growth rates were higher and the half saturation constant (K
1/2
) for
growth was much lower at the warmer temperature. In contrast, warming decreased the maximal
growth rates of P. antarctica over the range of CO
2
concentrations tested, and failed to change its
K
1/2
for growth. The decreased CO
2
K
1/2
of P. subcurvata at high temperature might confer a
future additional competitive advantage over P. antarctica in the late growing season when pCO
2

can be low (Tagliabue and Arrigo, 2016) and temperatures higher, although temperatures are
generally never as high as 8°C in the current Ross Sea (Liu and Smith, 2012). The CO
2
K
1/2
of P.
antarctica at 2°C was however significantly lower than that of P. subcurvata at this temperature,
which may be advantageous to the prymnesiophyte when water temperatures are low in the
spring.
The effects of pCO
2
variation on the elemental ratios of P. subcurvata and P. antarctica
were minimal relative to those of temperature increase. Previous research on the effects of CO
2

on the elemental ratios of phytoplankton has shown that the elemental composition of
phytoplankton may change with CO
2
availability (Burkhardt et al., 1999; Fu et al., 2007, 2008;
Tew et al., 2014; reviewed in Hutchins et al., 2009). Hoogstraten et al. (2012) found that CO
2

concentration change didn’t change the cellular POC, PON, C: N ratios, or POC to Chl a ratios
78
of the temperate species Phaeocystis globosa. In contrast, Reinfelder (2014) observed that the N
and P quotas of several diatoms decreased with increasing CO
2
and led to increased C: N, N: P,
and C: P ratios. King et al. (2015) found that high CO
2
could increase, decrease or not affect the
C: P and N: P ratios of several different phytoplankton species. Our results resemble those of
studies with other phytoplankton that found that the effects of CO
2
concentration can be
negligible on C: N, N: P, or C: P ratios (Fu et al., 2007; Hutchins et al., 2009; Hoogstraten et al.,
2012; King et al., 2015).
In contrast to C:N:P ratios, we observed that the C: Si ratios of P. subcurvata were
significantly higher at 1730 ppm compared to almost all of the lower CO
2
levels. This increase in
C: Si ratios was due to a decrease in cellular Si quotas at 1730 ppm CO
2
. Milligan et al. (2004)
observed that the silica dissolution rates of a temperate diatom increased significantly in high
CO
2
relative to in low CO
2
cultures. Tatters et al. (2012) found a similar trend in the temperate
toxic diatom Pseudo-nitzschia fraudulenta, in which cellular C: Si ratios were higher at 765 ppm
than at 200 ppm CO
2
. This suggests that future increases in diatom silicification at elevated
pCO
2
could partially or wholly offset the decreased silicification observed at warmer
temperatures (above); to fully predict net trends, further interactive experiments focusing on
silicification as a function across a range of both temperature and pCO
2
are needed.
In conclusion, our results indicate that P. subcurvata from the Ross Sea are better adapted
to higher temperature than is P. antarctica, suggesting that the relative dominance of P.
antarctica in this region may wane in under future global warming scenarios. Such an ecological
shift may significantly change the biogeochemical cycles of carbon, nitrogen, phosphorus,
silicon, and sulfur. This conclusion must be qualified as it was obtained using Fe-replete culture
conditions; which often prevail early in the growing season in McMurdo Sound. However, Fe
79
limitation generally prevails later in the season here, and elsewhere in the offshore Ross Sea.
Irradiance is another key environmental factor to consider in both the present and future in this
region. Thus, in addition to warming and CO
2
increases, the interactive effects of light and Fe
with these two factors should also be considered (Xu et al., 2014; Boyd et al., 2015). Considering
the differences between the responses of the diatom and P. antarctica to warming and ocean
acidification seen here, as well to warming and Fe in previous work (Zhu et al., 2016), models
attempting to predict future changes in community structure and primary production in Southern
Ocean coastal polynyas may need to realistically incorporate a complex network of interacting
global change variables.
Author contribution
Z. Zhu, F. X. Fu, D. A. Hutchins designed the experiments, Z. Zhu, P. Qu, and J. Gale carried
them out, and Z. Zhu and D. A. Hutchins wrote the manuscripts.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgments
We want to thank Kai Xu for isolating all these phytoplankton strains. Support for this research
was provided by National Science Foundation grant ANT 1043748 to D. A. Hutchins
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87
Table 1 The measured pH and dissolved inorganic carbon (DIC), and calculated pCO
2
of P. subcurvata
and P. antarctica at 2°C and 8°C in each treatment. Values represent the means and errors are the
standard deviations of triplicate bottles.

P. subcurvata P. antarctica
2°C 8°C 2°C 8°C
pH
8.36±0.04 8.51±0.04 8.40±0.03 8.45±0.03
8.25±0.04 8.36±0.01 8.22±0.04 8.29±0.01
8.07±0.01 8.17±0.01 8.09±0.02 8.14±0.00
7.86±0.02 7.99±0.01 7.85±0.01 7.94±0.00
7.68±0.01 7.79±0.02 7.65±0.01 7.75±0.00
7.35±0.01 7.46±0.02 7.34±0.01 7.45±0.00
DIC (µmol/kg)
1890±27 1847±16 1847±30 1831±23
2049±11 1986±2 2034±15 2014±20
2131±9 2068±5 2137±6 2085±15
2190±3 2156±14 2168±12 2167±22
2260±22 2235±10 2252±12 2239±12
2340±19 2335±19 2338±12 2324±12
pCO
2
( ppm)
109±9 94±10 97±10 109±9
159±16 150±4 171±14 184±4
263±6 254±10 246±10 280±1
450±17 415±12 462±12 481±5
741±11 709±24 787±10 784±5
1751±36 1675±49 1770±60 1720±18

88
Table 2 Statistical comparison of the results for each of the three thermal traits: Optimum temperature
(ºC), Maximum growth rate (d
-1
) and temperature niche width (W)* of P. subcurvata and P. antarctica.

Species
Optimum
temperature
(ºC)
Maximum
growth rates (d
-1
)
W upper CI W lower CI Q
10

P. subcurvata 7.36 0.86 12.19 < -2.0 3.17
P. antarctica 4.85 0.66 9.52 < -2.0 2.11

* The statistical results for the lower bound of temperate niche width in both species were lower
than -2.0ºC, the freezing point of seawater

89
Table 3 The effects of temperature on the C quota (pmol cell
-1
), N quota (pmol cell
-1
), P quota (pmol cell
-
1
), Si quota (pmol cell
-1
), and chl a per cell (pg cell
-1
) of P. subcurvata and P. antarctica. Values represent
the means and errors are the standard deviations of triplicate bottles.

P. subcurvata P. antarctica
C quota
0°C 1.91±0.14 2.64±0.34
2°C 2.11±0.19 2.49±0.41
4°C 2.15±0.12 2.50±0.23
6°C 2.07±0.13 2.26±0.18
8°C 2.33±0.14 2.17±0.22
10°C 2.17±0.13
N quota
0°C 0.27±0.03 0.39±0.03
2°C 0.29±0.03 0.36±0.02
4°C 0.33±0.02 0.40±0.01
6°C 0.31±0.01 0.35±0.02
8°C 0.36±0.05 0.34±0.03
10°C 0.33±0.04
P quota
0°C 0.02±0.00 0.03±0.00
2°C 0.02±0.00 0.02±0.00
4°C 0.03±0.00 0.03±0.01
6°C 0.03±0.00 0.02±0.00
8°C 0.03±0.00 0.02±0.00
10°C 0.02±0.00
Si quota
0°C 0.23±0.02
2°C 0.23±0.06
4°C 0.30±0.01
6°C 0.30±0.03
8°C 0.34±0.01
10°C 0.28±0.04
Chl a per cell (pg/cell)
0°C 0.48±0.01 0.23±0.03
2°C 0.57±0.07 0.22±0.02
4°C 0.64±0.01 0.20±0.01
6°C 0.68±0.05 0.21±0.00
8°C 0.58±0.03 0.17±0.02
10°C 0.46±0.03

90
Table 4 Comparison of the curve fitting results for maximum growth rate (d
-1
) and half saturation
constants (K
m
), calculated from the CO
2
functional response curves of P. subcurvata and P. antarctica at
2°C and 8°C. Values represent the means and errors are the standard errors from fitting.
.
Species
Maximum
growth rates (d
-1
)
K
m

P. subcurvata
2°C 0.60±0.18 66.40±10.39
8°C 0.88±0.02 9.80±5.34
P. antarctica
2°C 0.61±0.02 26.40±8.23
8°C 0.41±0.02 22.10±11.15

91
Table 5 The effects of CO
2
on the C: N, N: P, C: P, C: Si, and C: Chl a ratios of P. subcurvata and P.
antarctica at 2°C and 8°C. Values represent the means and errors are the standard deviations of triplicate
bottles.
P. subcurvata P. antarctica
2°C 8°C 2°C 8°C
C: N
100 ppm 6.6±0.3 7.1±0.7 7.2±0.5 7.0±0.4
205 ppm 6.7±0.2 7.5±0.3 7.7±0.2 6.6±1.2
260 ppm 6.7±0.3 7.3±0.2 8.1±0.5 7.0±0.3
425 ppm 6.7±0.1 6.6±0.1 7.2±0.8 6.2±0.1
755 ppm 6.8±0.2 7.1±0.7 8.0±0.4 6.8±0.2
1730 ppm 7.1±0.8 7.4±1.1 8.2±0.5 7.1±0.9
N: P
100 ppm 10.4±0.9 14.5±2.3 16.4±1.2 13.9±0.2
205 ppm 10.8±1.0 13.3±0.4 16.6±1.1 15.7±2.8
260 ppm 10.3±1.3 14.0±0.6 14.3±1.2 14.5±2.4
425 ppm 11.3±0.8 16.5±0.3 17.1±1.8 17.2±2.0
755 ppm 9.9±0.3 14.3±1.3 14.2±2.6 11.6±4.1
1730 ppm 10.4±1.0 15.5±1.8 15.5±0.6 15.1±1.9
C: P
100 ppm 68.6±3.1 101.0±6.4 117.7±4.1 96.7±4.9
205 ppm 72.7±4.8 99.3±7.1 128.2±6.0 101.0±1.9
260 ppm 69.1±7.7 103.0±4.9 115.5±7.3 101.0±13.0
425 ppm 76.3±5.2 109.0±2.2 122.3±4.9 106.0±11.1
755 ppm 67.2±1.4 101.0±5.8 113.5±22.5 78.6±27.1
1730 ppm 73.4±1.2 114.0±6.0 126.2±12.1 105.0±6.3
C: Si
100 ppm 7.8±0.8 5.6±0.3
205 ppm 7.4±0.3 5.6±0.2
260 ppm 7.3±0.2 6.1±0.4
425 ppm 7.5±0.2 6.1±0.1
755 ppm 7.4±0.7 6.3±0.4
1730 ppm 8.0±0.9 7.1±0.5
C: Chl a (µg/µg)
100 ppm 43.6±1.1 70.7±5.0 160.4±6.7 197.4±29.4
205 ppm 45.2±2.9 67.3±4.4 157.5±5.0 194.0±17.1
260 ppm 41.6±3.3 60.1±9.5 138.3±15.2 169.8±9.2
425 ppm 37.2±2.6 72.5±2.4 180.2±20.1 232.4±20.5
755 ppm 42.2±3.6 68.7±6.3 167.5±5.1 282.5±15.3
1730 ppm 46.3±2.2 85.3±15.7 276.5±36.6 460.3±15.2

92
Table 6 The effects of CO
2
on the C quota (pmol cell
-1
), N quota (pmol cell
-1
), P quota (pmol cell
-1
), Si
quota (pmol cell
-1
), and chl a per cell (pg cell
-1
) of P. subcurvata and P. antarctica at 2°C and 8°C.
Values represent the means and errors are the standard deviations of triplicate bottles.
P. subcurvata P. antarctica
2°C 8°C 2°C 8°C
C quota
100 ppm 2.0±0.2 2.6±0.1 2.6±0.0 2.2±0.2
205 ppm 2.1±0.1 2.7±0.3 2.7±0.3 2.4±0.2
260 ppm 1.9±0.0 2.3±0.2 2.5±0.4 2.2±0.0
425 ppm 1.8±0.0 2.4±0.2 2.3±0.1 2.3±0.5
755 ppm 2.1±0.1 2.3±0.1 2.5±0.2 2.8±0.2
1730 ppm 2.1±0.3 2.5±0.2 2.4±0.1 3.0±0.3
N quota
100 ppm 0.30±0.03 0.38±0.04 0.36±0.03 0.31±0.03
205 ppm 0.30±0.03 0.36±0.03 0.35±0.03 0.36±0.06
260 ppm 0.29±0.01 0.31±0.02 0.31±0.06 0.32±0.02
425 ppm 0.27±0.01 0.37±0.06 0.32±0.03 0.37±0.05
755 ppm 0.30±0.02 0.32±0.03 0.31±0.03 0.41±0.01
1730 ppm 0.29±0.05 0.34±0.06 0.30±0.03 0.43±0.10
P quota
100 ppm 0.03±0.00 0.03±0.00 0.02±0.00 0.02±0.00
205 ppm 0.03±0.00 0.03±0.00 0.02±0.00 0.02±0.00
260 ppm 0.03±0.00 0.02±0.00 0.02±0.00 0.02±0.00
425 ppm 0.02±0.00 0.02±0.00 0.02±0.00 0.02±0.01
755 ppm 0.03±0.00 0.02±0.00 0.02±0.00 0.04±0.02
1730 ppm 0.03±0.00 0.02±0.00 0.02±0.00 0.03±0.00
Si quota
100 ppm 0.26±0.02 0.47±0.04
205 ppm 0.28±0.02 0.48±0.07
260 ppm 0.27±0.01 0.37±0.03
425 ppm 0.25±0.01 0.40±0.04
755 ppm 0.28±0.03 0.36±0.03
1730 ppm 0.26±0.01 0.35±0.05
Chl a per cell (pg/cell)
100 ppm 0.54±0.05 0.45±0.04 0.19±0.01 0.13±0.02
205 ppm 0.54±0.04 0.48±0.05 0.21±0.02 0.15±0.02
260 ppm 0.56±0.03 0.46±0.04 0.22±0.04 0.16±0.01
425 ppm 0.60±0.04 0.40±0.04 0.16±0.02 0.12±0.01
755 ppm 0.59±0.06 0.40±0.03 0.18±0.01 0.12±0.00
1730 ppm 0.53±0.06 0.35±0.05 0.11±0.02 0.08±0.01

93

Figure legends
Fig. 1. Thermal functional response curves showing specific growth rates (and fitted curves) of
Pseudo-nitzschia subcurvata and Phaeocystis antarctica across a range of temperatures from 0
o
C
to 14
o
C. Values represent the means and error bars represents the standard deviations of triplicate
samples.
Fig. 2. The C: N ratios (A), N: P ratios (B), and C: P ratios (C) of Pseudo-nitzschia subcurvata
and Phaeocystis antarctica and (D) the C: Si ratios of Pseudo-nitzschia subcurvata from the
thermal response curves shown in Fig. 1 for a range of temperatures from 0
o
C to 10
o
C. Values
represent the means and error bars represents the standard deviations of triplicate samples.
Fig. 3. The C: Chl a ratios of Pseudo-nitzschia subcurvata and Phaeocystis antarctica from the
thermal response curves shown in Fig. 1 for a range of temperatures from 0
o
C to 10
o
C. Values
represent the means and error bars represents the standard deviations of triplicate samples.
Fig. 4. The relative abundance of Pseudo-nitzschia subcurvata in a 6 day competition
experiment with Phaeocystis antarctica at 0°C and 6°C. The competition experiments were
started with equal Chl a concentrations for both species, and the relative abundance was
calculated based on cell counts. Values represent the means and error bars represents the
standard deviations of triplicate samples.
Fig. 5. CO
2
functional response curves showing specific growth rates (and fitted curves) across a
range of CO
2
concentrations from ~100 ppm to ~1730 ppm at 2°C and at 8°C. Pseudo-nitzschia
subcurvata at 2°C (A) and 8°C (B) and Phaeocystis antarctica at 2°C (C) and 8°C (D). Values
represent the means and error bars represents the standard deviations of triplicate samples.

94

Fig. 1

95
Fig. 2

A B
C D
96
Fig. 3
97
Fig. 4

98
Fig. 5

A B
C
D
99
Chapter 3: Understanding the blob bloom: warming increases toxicity and abundance of
the harmful bloom diatom Pseudo-nitzschia in California coastal waters

Zhi Zhu
1
, Pingping Qu
1
, Feixue Fu
1
, Nancy Tennenbaum
1
, Avery O. Tatters
1
, David A.
Hutchins
1*
1. Marine and Environmental Biology, University of Southern California, Los Angeles, CA
90089, USA.

* Corresponding author: dahutch@usc.edu

Abstract: The toxic diatom genus Pseudo-nitzschia produces environmentally damaging
harmful algal blooms (HABs) along the U.S. west coast and elsewhere, and a recent ocean
warming event coincided with toxic blooms of record extent. We examined the effects of
temperature on growth, domoic acid toxin production, and competitive dominance of Pseudo-
nitzschia spp. from Southern California. Growth rates of cultured P. australis were maximal at
23°C (~0.8 d
-1
), similar to maximum temperature recorded during the 2014-2015 warming
anomaly, and decreased to ~0.1 d
-1
by 30°C. In contrast, cellular domoic acid concentrations
only became detectable at 23°C and increased to maximum levels at 30°C. In two incubation
experiments using natural Southern California phytoplankton communities, warming also
increased the relative abundance of another potentially toxic local species, P. delicatissima.
These results suggest that both the toxicity and the competitive success of Pseudo-nitzschia spp.
100
can be positively correlated with temperature, and therefore harmful blooms of this diatom genus
may be increasingly problematic in a warmer future coastal ocean.

Key words: Domoic acid, Pseudo-nitzschia, warming, temperature, HAB, the Blob

1 Introduction
With increasing anthropogenic CO
2
emissions, global temperatures are predicted to
increase 2.6-4.8°C by 2100 (IPCC, 2014). Increases in the frequency and severity of harmful
algal blooms (HABs) may be among the many impacts of this warming (Hallegraeff, 2010; Fu et
al., 2012). One such potentially toxic algal group is the diatom genus Pseudo-nitzschia spp.
Many Pseudo-nitzschia species produce the neurotoxin domoic acid (DA), the causal agent of
amnesic shellfish poisoning. Pseudo-nitzschia blooms recur nearly every year along the
California coast, where they cause mass mortalities of sea lions and seabirds (Trainer et al.,
2012). DA is capable of dispersing widely in coastal food webs, where it has been detected in
organisms ranging from mussels and sardines up to blue whales (Lefebvre et al., 2002; Schnetzer
et al., 2013). The occurrence of toxic harmful algal blooms dominated by Pseudo-nitzschia spp.
is often difficult to predict, because the same species can be toxic or non-toxic (Lundholm et al.,
1994; Lelong et al., 2012). Various factors including temperature, irradiance, nutrient
availability, and bacterial interactions can also affect DA production (Bates et al., 1998; Sun et
al., 2011; Tatters et al., 2012; Lelong et al., 2012; Sison-Mangus et al., 2014).
In 2014-2015, an extraordinarily widespread and sustained harmful Pseudo-nitzschia
bloom was observed along nearly the entire west coast of the United States, coinciding with a
persistent warm-water anomaly that was nicknamed ‘the Blob’ (Bond et al., 2015). The toxic
101
bloom formed during the course of this unprecedented regional warming event from early spring
2014 until the summer of 2015, a period during which sea surface temperatures were up to 4
o
C
above long-term averages from Alaska to Southern California (Bond et al., 2015; McCabe et al.,
2016). The bloom was promoted by upwelled nutrients after the spring transition (McCabe et al.,
2016). Historically high concentrations of domoic acid were present throughout the marine food
web in this area, creating widespread mortality of marine mammals and birds (McCabe et al.,
2016). Economic losses from this massive bloom due to closure of the Dungeness crab fishery
were estimated at >$50 million (McCabe et al., 2016; Leising et al., 2015; Cavole et al., 2016).
Research on the combined effects of upwelling and short-term warming events on the
occurrence of harmful algal bloom is still scarce. However, a recent study found correlations
between historical outbreaks of elevated domoic acid levels, and warm water transitions due to
regional Pacific Decadal Oscillation trends and El Niño occurrences (McKibben et al., 2017).
The linkage of the most devastating harmful Pseudo-nitzschia bloom ever observed along the
west coast to the 2014/2015 regional warming event underscores the need for research on the
impacts of warming on DA production and the composition of natural phytoplankton
communities containing Pseudo-nitzschia spp. Although many factors have been shown to
stimulate DA production (Lelong et al., 2012), temperature has been addressed in only a few
studies (Lundholm et al., 1994; Lewis et al., 1993). These studies have found contradictory
results, with the former demonstrating decreased DA, and the latter increased DA at warmer
temperatures.
Here, we address this knowledge gap by presenting a study that explored the effects of
warming on domoic acid production in a Pseudo-nitzschia australis strain isolated from the
Southern California Bight near the beginning of the Blob event. In addition, we examined the
102
effects of temperature increases on the species composition of two Pseudo-nitzschia
delicatissima-containing natural phytoplankton communities from this region. Our aim was to
gain a better understanding of how anomalous warming events such as the Blob, or future
sustained climate warming, may affect the occurrence and toxicity of harmful Pseudo-nitzschia
blooms in the California Current region.
2 Materials and Methods
2.1 Culture thermal response curves
P. australis S7 was micropipette isolated from public nearshore water collected at 33.73
N, 118.35 W in Los Angeles County, California in March, 2014. This strain was maintained at
16°C on a 12h light: 12h dark cycle in Aquil* media (Sunda et al., 2005) under 150 µmol photons
m
−2
s
−1
of cool white fluorescent light.
For thermal response curve experiments, the P. australis S7 culture was transferred to
temperatures of 12°C, 15°C, 18°C, 20°C, 23°C, 26°C, 28°C, and 30°C step by step in triplicate
500 ml acid washed polycarbonate bottles, under the same light conditions as stock cultures.
Cultures were diluted every 2 days to keep the culture at exponential growth stage with Aquil*
medium pre-acclimated to the appropriate temperature. After steady-state growth was attained, as
indicated by stable growth rates for 3-5 consecutive transfers, the cultures were sampled 48 h after
dilution.
2.2 Natural community temperature manipulations
We conducted two experiments with Pseudo-nitzschia-containing assemblages in which
we examined changes in community structure over a range of temperatures after adding nutrients
to simulate an upwelling event. Whole seawater was collected at Stearns Wharf (34.41N,
119.69W), Santa Barbara, California in February and March 2016 at ambient temperatures of 13°C
103
and 15°C, respectively. The seawater was filtered with 80 µm mesh to remove large zooplankton.
The February experiment (SB1) used 800ml of seawater in triplicate 1L polycarbonate bottles with
additions of 20 µM nitrate (NaNO
3
), 20 µM silicate (Na
2
SiO
3
) and 2 µM phosphate (NaH
2
PO
4
)
and vitamin and trace metal additions following the Aquil* recipe to simulate nutrient
supplementation from upwelling (Sunda et al., 2005). Bottles were incubated at 13°C, 18°C, and
23°C. For seawater collected in March (SB2), 800ml aliquots of seawater were added to triplicate
1L polycarbonate bottles with 100 µM nitrate (NaNO
3
), 100 µM silicate (Na
2
SiO
3
) and 10 µM
phosphate (NaH
2
PO
4
) and vitamins and trace metals following the Aquil* recipe. In SB2, the
bottles were incubated at 15°C, 20°C, 25°C, and 28°C. Both incubation experiments were
incubated on a 12h light: 12h dark cycle under 150 photons m
−2
s
−1
of cool white fluorescent light.
2.3 Growth rates
In vivo fluorescence of each culture was measured using a 10-AU
TM
fluorometer (Turner
Designs, CA) to calculate growth rates. Specific growth rates, expressed as d
-1
, were calculated
as: µ = (ln N
1
– ln N
0
)/t, where N
0
and N
1
are in vivo fluorescence at the beginning and end of a
dilution period, respectively, and t is the duration of the dilution period. Q
10
of growth rates of P.
australis was calculated as: Q
10
= (µ
2
/ µ
1
)
10/(T2-T1)
(Chaui-Berlinck et al., 2002), where µ
1
and µ
2
are the specific growth rates of the phytoplankton at temperature T
1
(Celsius) and T
2
,
respectively.
2.4 Elemental analysis
In addition to potential temperature effects, nutrient limitation has also been shown to
affect the toxicity of Pseudo-nitzschia spp. (Bates et al., 1998; Sun et al., 2011; Tatters et al., 2012;
Lelong et al., 2012). As an indicator of potential thermal effects on nutrient stress, we measured
the C:N:P ratios of the cultures and communities in our experiments. 20-100 ml culture samples
104
of each treatment were filtered onto pre-combusted GF/F filters (500°C for 2h) and dried in a 60°C
oven overnight for particulate organic carbon/nitrogen (POC/PON) and particulate organic
phosphorus (POP) analyses, respectively. POC/PON samples were analyzed using a 440 Elemental
Analyzer (Costech Inc, CA) following Fu et al. (2007). POP was analyzed using a molybdate
colorimetric method according to Fu et al. (2007). 20 to 100 ml of culture samples of each
treatment was filtered onto 2 µm polycarbonate filters (GE Healthcare, CA) and dried in a 60°C
oven overnight for biogenic silica (BSi) analysis following Paasche et al. (1980).
2.5 Chlorophyll a analysis
20 to 100 ml culture samples were filtered onto GF/F filters and 10 µm polycarbonate
filters (Whatman) at low vacuum, frozen overnight, and extracted with 90% aqueous acetone for
24 h at -20°C, followed by measurements using the non-acidification method on a 10-AU
TM

fluorometer (Turner Designs, CA), as in Fu et al. (2007).
2.6 Photosynthetic competency (F
v
/F
m
)
Chlorophyll fluorescence was measured using PHYTO-PAM (Walz, Germany) following
Fu et al. (2007). An aliquot of 5 ml cultures were dark-adapted for 30 minutes, then dark-adapted
minimal fluorescence yield (F
0
) was measured at light intensity < 1µmol photon m
-2
s
-1
and dark-
adapted maximal fluorescence yield (F
m
) was measured using saturation pulses (400 ms, 3000
µmol photon m
-2
s
-1
), and variable fluorescence of dark-adapted sample (F
v
) was calculated
follows: F
v
= F
m
– F
0
.
2.7 Domoic acid analysis
100 ml culture samples were filtered onto 2 µm polycarbonate filters and frozen at -20°C
until analysis. DA was quantified by high performance liquid chromatography with ultraviolet
detection (242 nm) (HPLC-UV) using an Agilent 1200 HPLC (Agilent, CA) according to Mafra
105
et al. (2009) and Tatters et al. (2012) with modification. The cells on filters were lysed in 10%
MeOH by Vibra-Cell VC50 ultrasonic processor (Sonics & Materials, CT) for 60 seconds at
50% duty cycle in ice-water bath, and filtered by 0.2 µm Acrodisc® syringe filters (Pall, NY)
into 2.0 ml Target DP
TM
vials (National Scientific, CA). Trifluoroacetic acid (TFA) was added to
all the filtrate to a final concentration of 0.15% prior to analysis. The chromatographic separation
and DA concentration calculation followed Tatters et al. (2012). The DA production rates (y),
expressed as pg cell
-1
d
-1
, were calculated as: y = e
µ
* c, where µ and c are growth rates and DA
concentration per cell, respectively.
2.8 Cell counts and classification
Cell count samples were preserved with 2% Lugol’s solution (final concentration) and stored at
4°C for subsequent counting on a Sedgwick Rafter Grid using an Olympus BX51 microscope
(Olympus, Japan). Phytoplankton identification and classification was based on Tomas (1997).
2.9 Statistical analysis
All statistical analyses, including student t-tests, ANOVA, and Tukey’s HSD test were
conducted using the open source statistical software R version 3.1.2 (R Foundation).
3 Results
3.1 The responses of cultured P. australis S7 to warming
3.1.1 Growth Rates
Growth rates of P. australis S7 increased from 0.21 d
-1
at 12°C to a maximum of 0.81 d
-1

at 23°C (p < 0.05) (Fig. 1A). This represents an increase of 275% over this temperature range, or
a Q10 value of 3.3. Above the optimal temperature of 23°C, growth rates of P. australis S7
decreased rapidly down to <0.1 d
-1
at 30°C (p < 0.001) (Fig. 1A).
3.1.2 Photosynthetic characteristics
106
The Chl a to carbon (Chl a : C) ratio (mg: g) of P. australis S7 was positively correlated
to thermally-determined growth rates (R
2
= 0.63, Fig. 1B). Chl a : C steadily increased by ~97%
from 12°C (13.8 ± 1.19) to 26°C (27.3 ± 2.72), and then decreased by ~34% from 26°C to 30°C
(17.1 ± 1.87) (p <0.05) (Fig. 1B). Photosynthetic competency (F
v
/F
m
) of P. australis S7 showed
a similar pattern to Chl a : C, and was also positively correlated to growth rates (R
2
= 0.84) (Fig.
1C), increasing from 12°C to 23°C and then decreasing at temperatures >26°C (p < 0.05) (Fig.
1C).
3.1.3 Domoic Acid
The concentrations of cellular DA in P. australis S7 cells at temperatures lower than
23°C were below the analytical detection limit (0.1 ng ml
-1
), but increased exponentially with
rising temperatures from 23°C to 30°C (R
2
= 0.96, p < 0.01). Over this range, cellular DA quotas
increased from 0.08 pg cell
-1
to 5.9 pg cell
-1
, or by ~70-fold (p < 0.001) (Fig. 2A). The DA
production rates of P. australis S7 also increased exponentially with temperature increase from
23°C to 30°C (R
2
= 0.92, p < 0.001)(Fig. 2B).
3.1.4 Elemental stoichiometry
C: N ratios of P. australis S7 were significantly lower at optimum growth temperatures
of 23°C and 26°C than at 12°C, 28°C, and 30°C (p < 0.05) (Table 1), but N: P ratios were lowest
at the extreme temperatures tested, 12°C and 30°C (Table 1). The C: N ratios of P. australis S7
were inversely correlated to thermally-determined growth rates (R
2
= 0.71, Table 1). C: P ratios
of P. australis S7 ranged from 104.7 ± 7.9 (23°C) to 137.5 ± 5.2 (28°C), and were not
significantly different across the tested temperature range (Table 1). C: Si ratios of P. australis
S7 were significantly reduced at higher temperatures (23°C to 30°C) compared to lower
temperatures (12°C to 20°C) (p < 0.05) (Table 1).
107
3.2 Natural Southern California phytoplankton community responses to warming
3.2.1 Phytoplankton community composition
In experiment SB1, the relative abundance of phytoplankton > 10 µm significantly
increased from 38% to 80-88% during the incubation (p < 0.05, Fig. 3A). The original collected
phytoplankton community in experiment SB1 was dominated by dinoflagellates (64% of all cells
counted), but diatoms increased to make up 80-87% of the community at the end of the
experiments, and there was no significant difference among the relative abundances of total
diatoms of all species at the three temperatures (Fig. 4A). However, in this experiment, the
relative abundance of the diatom Pseudo-nitzschia delicatissima increased linearly with
temperature (R
2
= 0.61) from the lowest temperature tested (16% at 13°C) to the highest
temperature (42% at 23°C) (Fig. 5A).
Similarly, in experiment SB2 the relative abundance of phytoplankton > 10 µm increased
from 47% initially to 82-90% at 25°C and 28°C at the end of the incubation (p < 0.05, Fig. 3B).
Dinoflagellates were again dominant in the originally collected water (55%) but were largely
replaced by a mixed diatom community in all treatments in experiment SB2 (p < 0.05, Fig. 4B).
In this experiment, the relative abundance of P. delicatissima only increased significantly at the
warmest temperature tested (22% at 28°C, p < 0.05), but remained at ~4% in the three lower
temperature treatments (Fig. 5B). These large increases in P. delicatissima abundance at the
warmest temperature were clearly evident from microscopic observation of water samples from
the experiment (Fig. 6).
3.2.2 Elemental stoichiometry
C: N ratios in the SB1 experiment significantly increased with temperature increase from
13°C to 18°C, and were significantly higher than the initial C:N ratio of the phytoplankton
108
community at 18°C (p < 0.05) (Table 2). The C: P, N: P and C: Si ratios of the SB1 plankton
community were significantly higher at all the three temperatures relative to the initial ratios
after incubation in the nutrient-enriched seawater medium (p < 0.05) (Table 1). N: P ratios of the
SB1 community at 18°C and 23°C were significantly lower than at 13°C (p < 0.05), and the C: P
ratios at 18°C were significantly higher than 13°C (p < 0.05) (Table 1).
4 Discussion
Our culture results suggest that temperature increase alone could significantly increase
the severity of HABs caused by P. australis, by elevating its growth rates and toxin production.
The particulate DA concentration of our P. australis S7 strain was below the detection limit at
temperatures < 20°C, but increased exponentially from 23°C to 30°C. The current spring and
summer average sea surface temperatures (SST) of Southern California coastal waters are in the
range of 14°C-22°C (http://www.nodc.noaa.gov/dsdt/cwtg/all_meanT.html), levels at which the
tested strain of P. australis was not toxic. However, the future summertime SST of Southern
California may increase to 26°C or more, considering predicted global temperature increases
(IPCC, 2014).
This future scenario closely resembles the expansive Pseudo-nitzschia HAB with record-
breaking DA concentrations that occurred in spring 2015 along the West Coast of the U.S.,
coinciding with the persistent warm water anomaly dubbed “the Blob” (McCabe et al., 2016;
Bond et al., 2015). During this event, the SST in the northeast Pacific basin increased 4-5°C
above normal (Bond et al., 2015; Zeba and Rudnick, 2015), with temperatures as high as 24°C
recorded in some parts of the Southern California Bight (Leising et al., 2015). Our Southern
California P. australis S7 culture, isolated in 2014 towards the beginning of this event, had
maximal growth rates between 23°C and 26°C. The highest cellular Chl a contents (Chl a: C)
109
and photosynthetic efficiencies (Fv/Fm) were also measured within this thermal range,
supporting the suggestion that these temperature conditions were optimal for the growth and
photosynthesis of our isolate. At lower temperatures growth was not only slower, but DA
production was undetectable. It is also noteworthy that for our P. australis culture, DA
concentration and DA production rates increased much more in concert with rising temperatures
up to 30°C, even as the optimal thermal range for growth was exceeded and growth rates
declined. Clearly, our P. australis isolate has the potential for considerable additional cell-
normalized toxicity if future temperatures were to exceed those seen during the Blob, although
growth and so perhaps relative abundance of this strain might be reduced.
There are many other toxic species and strains of Pseudo-nitzschia, some of which may
share similar physiological and toxin-production characteristics with P. australis S7, while
others may not. McCabe et al. (2016) presented growth curves for three P. australis isolates
from Monterey Bay, California that had higher maximum growth rates (up to ~1.5 d
-1
) and lower
optimum growth temperatures (15-17
o
C) than our Southern California isolate. Maximum growth
rate differences could be due to intrinsic strain variability, or to differing culture medium and
growth conditions. Differences in optimum temperatures may reflect regional variations, as sea
surface temperatures are significantly lower in Monterey Bay than in the Southern California
Bight. McCabe et al. (2016) did not however measure domoic acid production by their isolates.
Bates et al. (1998) and Lelong et al. (2012) reviewed the effects of a number of environmental
factors on growth and DA production of different species of Pseudo-nitzschia, and reported that
the effects of temperature increase on DA content of Pseudo-nitzschia spp. were species specific.
Lewis et al. (1993) reported Nitzschia pungens f. multiseries (now P. multiseries) isolated from
Nova Scotia, Canada produced significantly more DA at 15°C than 4°C, while Lundholm et al.
110
(1994) discovered P. seriata collected in Europe produced significantly more DA at 4°C than
15°C. It is not clear how comparable such studies of strains from colder northern environments
may be to our results using a strain from a Mediterranean-type climatic zone. We found that the
threshold temperature for DA production of P. australis S7 was around 23°C, and it is possible
that the thresholds of some other strains are lower. Clearly, more research is needed on the
thermal responses of various Pseudo-nitzschia species to reliably make general, global
predictions about the impacts of warming on DA production and harmful blooms.
In addition to promoting DA production by our cultured P. australis S7, our results also
show that warming increases the competitive dominance of another potentially toxic local
species, P. delicatissima. In our bottle incubation experiments, the mixed natural phytoplankton
community shifted to diatom-dominated assemblages, with similar final relative abundance of
diatoms at all the temperatures tested. Although the relative abundances of all diatoms were not
significantly different among temperatures at the end of both natural community incubations, the
relative abundance of Pseudo-nitzschia delicatissima significantly increased stepwise with
temperature elevation in SB1, and was highest at 28°C in SB2. In addition, the relative
abundance of Pseudo-nitzschia delicatissima significantly increased after 6 days incubation at
15°C and 20°C. Our results indicate that warming may increase the relative abundance of this
and perhaps other Pseudo-nitzschia species in upwelling regions of the California coast, which
corresponds to previous suggestions that there is a linkage between increasing temperatures and
this genus of diatoms (Hinder et al., 2012; McCabe et al., 2016; McKibben et al., 2017)
Although we did not detect DA in our natural community samples, P. delicatissima has
been reported to be capable of producing this toxin, and toxic and non-toxic strains of Pseudo-
nitzschia spp. often occur within the same species (Lundholm et al., 1994; Lelong et al., 2012).
111
Furthermore, the species of Pseudo-nitzschia that dominates during harmful algal blooms often
varies along the California coast (Anderson et al., 2006; Busse et al., 2006; Schnetzer et al.,
2007, 2013; McCabe et al., 2016). Many factors other than temperature can affect DA production
(Bates et al., 1998; Lelong et al., 2012). Such uncertainties about the taxonomic and
environmental factors that control DA production make it very difficult to reliably predict the
toxicity, location, timing, and duration of HABs dominated by Pseudo-nitzschia spp. (Caron et
al., 2010).
Our results also showed that the relative abundance of larger diatoms in general increased
with warming and nutrient additions. Lehman and Smith (1991) found that phytoplankton
community composition correlated to water column temperature and nutrients along the
California Coast. Anderson et al. (2008) also observed that mixed-phytoplankton assemblages
shifted to diatom-dominated phytoplankton assemblages following spring upwelling in the Santa
Barbara Channel, California. The nutrients we added to simulate such upwelling events might be
the main factor that drove the shift of the phytoplankton communities to large diatom-dominated
assemblages in our experiment, since this group can grow faster and outcompete other groups of
phytoplankton when nutrients are replete (Anderson et al., 2008).
Reay et al. (1999) found that phytoplankton-specific affinity for nitrate is temperature-
dependent below optimal temperatures, and Lomas and Glibert (1999) showed that diatom
nitrate reductase activity declines rapidly as temperatures exceed optimal growth levels. The
significantly higher C: N ratios of P. australis S7 at 12°C, 28°C and 30°C compared to optimal
temperatures suggest that sub-optimal and supra-optimal temperature could have limited nitrate
uptake relative to carbon fixation (Fig. 2A). Increased C:N ratios at the lower and upper
extremes of the thermal functional response curve of P. australis suggest that it may be growing
112
closer to nitrogen limitation outside of its optimal temperature range, with possible implications
for competitive success and bloom formation.
In addition, our results showed that C: Si ratios of P. australis S7 were significantly
decreased at higher temperatures, suggesting an increased Si requirement with warming and
potentially making the diatom more susceptible to Si limitation. This effect may exacerbate the
secondary environmental silicate limitation resulting from the highly silicified diatom growth
that occurs in parts of the California upwelling region due to iron limitation (Hutchins and
Bruland, 1998). The excess drawdown of silicate in Fe-limited regions such as along the Big Sur
coast may increase the toxicity of P. australis, as Si limitation is known to greatly increase
cellular DA production (Tatters et al., 2012).
Pseudo-nitzschia species have been recorded in the Southern California Bight since the
1920s (Fryxell et al., 1997), and toxic events caused by Pseudo-nitzschia spp. along the
California coast have been increasing since the 1990s, including in the Santa Barbara Channel
(Trainer et al., 2000), the San Pedro Channel (Busse et al., 2006; Schnetzer et al., 2013), and off
San Diego (Busse et al., 2006). Upwelling and nutrient availability certainly play an important
role in the initiation of HABs along the California coast, and our results suggest warming may
need to be considered as well. Notably, the West Coast of North America experiences regular
warm water anomalies caused by El Niño and the Pacific Decadal Oscillation, and the frequency
of extreme warming events is predicted to increase (Fisher et al., 2005; Cai et al., 2013; McCabe
et al., 2016; McKibben et al., 2017). Further research on the interactive effects of short-term
warming events and nutrient inputs on natural phytoplankton communities along in this region
are needed for better predictions of the occurrence of harmful algal blooms. For instance,
whether more frequent warming events will drive natural selection for Pseudo-nitzschia strains
113
adapted to higher temperatures is currently unknown (Boyd and Hutchins, 2012). Other global
change factors may also come into play. Sun et al. (2011) and Tatters et al. (2012) reported that
increased CO
2
concentrations can promote DA production by two different Pseudo-nitzschia
species, including a P. fraudulenta isolate from Southern California waters. In the future, this
region will likely experience a combination of simultaneous ocean acidification (CO
2
increase)
and warming (Feely et al., 2008), suggesting the interactive effects of these two global change
variables on the growth and DA production of Pseudo-nitzschia spp. should also be examined
(Fu et al., 2012).
5 Conclusion
In conclusion, our results show that the toxin production of P. australis S7 increased
exponentially with temperature increase higher than 23°C, and the relative abundance of P.
delicatissima increased with temperature increase. These observations suggest that HABs
dominated by toxic Pseudo-nitzschia species have the potential to increase in frequency and
severity along the California coast under projected future global warming scenarios. These
experiments demonstrate that temperature increase alone can stimulate growth, toxin production,
and ecological dominance in Pseudo-nitzschia spp., regardless of other possible trigger factors. It
seems likely that future warm water intrusions like the Blob, or just predicted general long-term
warming trends, could greatly aggravate the already serious threat that Pseudo-nitzschia blooms
pose to the California Current ecosystem. Regional sea surface temperature observations may
need to be an integral part of efforts to build effective monitoring systems and forecasting
models aimed at evaluating the health of the coastal environment and local economies based on
predicting future, more toxic Pseudo-nitzschia blooms.

114
Funding
This project was supported by USC Urban Ocean Sea Grant funding to DAH and FFU, and a Sea
Grant Graduate Student Traineeship to ZZ.
Author Contributions
ZZ, FF, and DAH designed the study, ZZ, PQ, AOT, and NT conducted the experiments, ZZ
conducted the statistical analyses, and ZZ and DAH wrote the paper.

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120
Table 1 The elemental ratios of of cultured Southern California Pseudo-nitzschia australis isolate
S7 grown across a range of temperatures from 12-30
o
C, and the elemental ratios of
phytoplankton assemblages from SB1 at the beginning and end of incubation at a series of
temperatures from 13-23
o
C.
Temperature
(°C)
C: N N: P C: P C: Si
P. multiseries S7
12 10.2±0.8 11.2±1.2 113.6±6.8 7.6±0.6
15 9.1±0.5 15.1±0.3 137.2±5.6 7.3±0.3
18 8.9±0.3 14.1±2.2 125.1±16.2 7.2±0.8
20 8.7±0.8 17.2±0.5 140.7±2.7 7.4±0.5
23 7.5±0.3 13.9±0.4 104.7±7.9 5.6±0.3
26 8.2±0.4 12.9±1.5 105.4±12.1 6.0±0.2
28 9.8±0.4 14.4±0.0 137.5±5.2 5.7±0.4
30 11.0±0.3 9.8±1.6 108.7±19.9 5.6±0.1
SB1
T0 9.9±0.9 7.0±0.9 68.3±5.3 3.7±0.1
13 7.1±0.2 19.1±0.9 136.3±6.5 10.9±0.4
18 14.8±0.5 14.9±0.9 220.0±5.7 11.4±0.2
23 12.6±2.6 14.9±0.5 188.9±41.5 9.7±1.9

121
Figure Legends
Fig. 1 A) Cell-specific growth rates (d
-1
), B) Chl a: C ratios (mg: g) and C) photosystem II
efficiency (Fv/Fm) of cultured Southern California Pseudo-nitzschia australis isolate S7 grown
across a range of temperatures from 12-30
o
C. Values are the means and error bars are the
standard deviations of triplicate samples.
Fig. 2 A) Cellular domoic acid concentrations (pg cell
-1
), and B) DA production rates (pg cell
-1
d
-
1
) of cultured Southern California Pseudo-nitzschia australis isolate S7 grown across a range of
temperatures from 12-30
o
C. Values are the means and error bars are the standard deviations of
triplicate samples.
Fig. 3 The percentage of Chl a > 10 µm of the phytoplankton assemblages in SB1 (A) and SB2
(B) experiment across a range of temperatures. Values are the means and error bars are the
standard deviations of triplicate samples.
Fig. 4 The relative abundance of different phytoplankton groups of the phytoplankton
assemblage at the end of experiments in the (A) SB1 and (B) SB2 experiments across a range of
temperatures. Values are the means and error bars are the standard deviations of triplicate
samples.
Fig. 5 The relative abundance of Pseudo-nitzschia delicatissima in the phytoplankton assemblage
incubations initially (T0) and as function of temperature at the end of experiments (A) SB1 and
(B) SB2. Values are the means and error bars are the standard deviations of triplicate samples.

122
Fig. 6 Microscopic image of final phytoplankton communities in the SB2 experiment at 15
o
C
and 28
o
C, showing the increase in relative abundance of Pseudo-nitzschia delicatissima (species
denoted by arrows).

123
Fig. 1

A
C
B
124
Fig. 2

A
B
125
Fig. 3

A
B
126

Fig. 4
A
B
127
Fig. 5

A
B
128
Fig. 6

15
o
C 28
o
C
129
Conclusions
We are gradually expanding our knowledge about the potential effects of climate change
on phytoplankton in the ocean, and the consequences for the biogeochemical cycles of carbon
and nutrients. However, due to the complexity of all the individual chemical, physical, and
biological factors involved in changing marine ecosystems, as well as their many possible
interactions, it remains extremely difficult to accurately predict shifts in phytoplankton
physiology and community structure in different regions of the ocean. The research presented in
this dissertation explored several key aspects of climate change effects on dominant
phytoplankton of two significant and representative regions, the Ross Sea, Antarctica, and the
Southern California coast. This Conclusion to the dissertation will explore the implications of
the findings from each of the major data chapters individually, followed by a discussion of the
overall significance of the contributions this research has made to our knowledge of ocean global
change biology. Finally, this Conclusion will offer some suggestions for promising directions of
future studies in this field, based on the results of the research presented here.
Effects of warming and Fe availability on Ross Sea diatoms and Phaeocystis antarctica
The goal of this part of my dissertation was to understand how several diatom species and
the prymnesiophyte Phaeocystis antarctica from an ice edge community in the Ross Sea,
Antarctica, would respond to warming and changes in Fe availability. My results showed that as
expected, when any of these phytoplankton were Fe-limited, their growth was significantly
promoted by Fe addition. However, the responses of these two groups of phytoplankton to
warming were quite distinct. Warming increased the growth rates of the diatoms, but not P.
antarctica. Additionally, warming and Fe addition synergistically affected the growth of the
pennate diatom Pseudo-nitzschia subcurvata. These results suggest that Ross Sea phytoplankton
130
community composition may shift towards dominance by diatoms in general, and P. subcurvata
in particular, under predicted future scenarios that include both higher temperatures and
increased Fe inputs (Zhu et al., 2016; Hutchins and Boyd, 2016). The elemental composition (C:
P and N: P) of diatoms and P. antarctica are significantly different, and the respective roles of
diatoms and P. antarctica in the silicon and sulfur cycles. Therefore, such changes in
phytoplankton community structure may alter the biogeochemical cycle of not only C, but also
N, P, Si, and S in the future Southern Ocean (Arrigo et al., 1999, 2000). Furthermore, these
climate change-driven phytoplankton community changes may affect the broader food web in the
Southern Ocean, since diatoms and P. antarctica contribute very differently to trophic
interactions in the Southern Ocean (Knox, 1994; Caron et al., 2000). Specifically, shifts away
from P. antarctica and towards diatoms may allow more efficient transfer of materials and
energy to higher trophic levels, since the latter group is typically considered to be a preferred
food for herbivorous zooplankton grazers. Taken together, these experiments would seem to
suggest that simultaneous changes in temperature and Fe inputs could have far-reaching effects
for both the biogeochemistry and ecology of the Ross Sea.
Effects of warming and ocean acidification on Ross Sea Pseudo-nitzschia subcurvata and
Phaeocystis antarctica
The second results chapter of this dissertation continued and expanded on the theme of
multiple variable global change impacts on dominant phytoplankton species from the Ross Sea,
Antarctica. This study focused on the individual and interactive effects of warming and CO
2

variations on the diatom Pseudo-nitzschia subcurvata and the prymnesiophyte Phaeocystis
antarctica. Thermal functional response curves revealed that both optimum and maximum
growth temperatures of P. subcurvata were significantly higher than those of P. antarctica (Zhu
131
et al., in review). This conclusion, which supported the findings of the experiments presented in
the preceding chapter, was also reinforced by the results of a competition experiment conducted
at two temperatures. CO
2
functional response curves at two temperatures revealed a significant
interactive effect between warming and CO
2
that decreased the CO
2
half saturation constant for
growth for P. subcurvata, while P. antarctica showed no interactive response to these two
variables. In contrast to significant effects of temperature, the effects of CO
2
increase on the
elemental composition of both species were minimal. While the finding that higher temperatures
are more favorable to the diatom than to the prymnesiophyte was robustly replicated, CO
2

increases beyond current levels may not affect either of these two phytoplankton profoundly,
suggesting it may not be an important factor in future competitive interactions between Pseudo-
nitzschia subcurvata and Phaeocystis antarctica in the Ross Sea (Zhu et al., in review b).
Warming and the toxicity and abundance of the harmful bloom diatom Pseudo-nitzschia in
California coastal waters
This study examined the effects of temperature on production of the neurotoxin domoic
acid by a strain of the harmful algal bloom forming diatom Pseudo-nitzschia australis isolated
from the Southern California coast. The results from a thermal response curve from 12°C to
30°C showed that toxin production exponentially increased at temperatures above 23°C, but was
undetectable at lower temperatures. Two additional studies of thermal effects on natural
phytoplankton communities from the California coast indicated that warming increased the
relative abundance of diatoms, and decreased the relative abundance of dinoflagellates. Notably,
the relative abundance of the diatom Pseudo-nitzschia delicatissima increased with temperature
increase in both incubation experiments. Although domoic acid was not detected in the natural
community incubation experiments, these experiments showed that temperature increase may
132
benefit Pseudo-nitzschia spp. (Zhu et al., in review b). This work sheds light on the causes of the
historical harmful algal bloom formed by Pseudo-nitzschia observed in 2015 along the West
Coast of the United States, which coincided with anomalous ocean conditions that were warmer
than usual by 1°C to 4°C (McCabe et al., 2016). Together, our results suggested that warming
may not only promote the growth rates and competitive advantage of toxin producing Pseudo-
nitzschia spp., it may also increase toxin production by this diatom genus (Zhu et al., in review
b). This set of experiments offers a cautionary note about future trends in harmful bloom
impacts along the California coast, in which warmer average temperatures driven by climate
change could also be accompanied by increased frequency and intensity of warm anomaly events
like the one in 2015.
Significance and future research direction
Many studies have shown that predicted warming and ocean acidification trends are
likely to affect phytoplankton assemblages in general, although impacts vary considerably
between taxa, regions, and with different combinations of global change variables. Considering
the diversity of phytoplankton community composition, food web structure, and chemical and
physical conditions, it is impossible to accurately predict how climate change will affect every
part of the world ocean. My aim with this dissertation work was to provide some insight into the
potential effects of climate change in two important coastal regions, one in the Southern Ocean,
and one closer to home in California.
My research confirmed the interactive effects of warming and Fe addition on the growth
of Antarctic isolates of the diatom Pseudo-nitzschia, as well as the compensatory effects of
warming on the growth of Fe-limited diatoms in this region. Also, I discovered that the
prymnesiophyte Phaeocystis antarctica has a different response to warming compared to
133
diatoms, which has the potential to cause a dramatic phytoplankton community composition shift
in the Southern Ocean in the future. In particular, I found that the thermal tolerance of P.
antarctica is narrower and the optimum temperature is lower than that of the diatom P.
subcurvata. The effects of CO
2
increase beyond present day atmospheric concentrations on both
species were minimal, since both are CO
2
-saturated at current CO
2
levels. This work suggested
that temperature and Fe availability play a more important role than CO
2
in controlling the
growth of diatoms and P. antarctica in the Southern Ocean. My results indicate that further
research should perhaps invest more effort into understanding the interactive effects of these two
climate change factors, rather than on the narrower focus on acidification alone seen in many
previous studies.
Besides temperature, Fe availability and CO
2
concentration, other factors including light
intensity and nutrients may also be altered by climate change. To accurately predict how climate
change will affect the ecosystems of the Southern Ocean, more complex experimental designs
similar to Xu et l. (2014) and Boyd et al. (2016) that include a larger suite of the important
factors are needed. Such matrix experimental designs can detect interactive effects between
different factors that cannot be determined by single factor experiments. Furthermore, other
dominant diatom species in addition to P. subcurvata should also be studied to provide a broader
body of knowledge about the responses of diatoms as a group to climate change. In situ
metagenomic and metatranscriptomic analyses of the Southern Ocean algal bloom time series
relative to physical and chemical parameters may also provide deeper insights into the
competition between the two dominant groups of phytoplankton in this region, diatoms and the
prymnesiophyte Phaeocystis antarctica.
134
The other aspect of my work discovered that temperature increase promotes toxin
production by Pseudo-nitzschia australis, and also increased the competitive advantage of
Pseudo-nitzschia delicatissima in natural phytoplankton communities. This research indicates the
urgency of understanding how climate change may affect coastal harmful algal blooms,
especially considering the extensive public health risk and potential economic losses and
environmental damage due to harmful algal blooms. More strains of P. australis and other
domoic acid producing Pseudo-nitzschia spp. should be similarly investigated in the future.
Furthermore, the individual and interactive effects of other factors, such as nutrient limitation,
light, and ocean acidification on these species should also be examined to help provide more
accurate information to predict the occurrences and toxicity of harmful algal blooms. In addition,
the genetic mechanism of domoic acid production is still not clearly understood, and more
investment on this aspect is needed in order to better understand and predict the occurrence of
toxic harmful algal blooms of Pseudo-nitzschia.
Collectively, my work suggests climate change may profoundly affect phytoplankton
community in both the Southern Ocean and the California coast. Among these factors that would
change under climate change, temperature is the most dominant factor that may significantly
change the phytoplankton community composition in both study regions. Warming may expand
the distribution of diatoms at the expense of Phaeocystis antarctica abundance in the Southern
Ocean, consequently affecting regional biogeochemistry and ecology. There was however no
significant difference between the effects of Fe addition on Fe-limited diatoms and Phaeocystis
antarctica, so climate-related Fe additions may promote the growth of both Fe-limited diatoms
and Phaeocystis antarctica. Temperature increase can also promote domoic acid production and
competition advantage of Pseudo-nitzschia spp. in the California coast.
135
Besides temperature, Fe availability, and CO
2
investigated in my work, many other
factors will also be altered by climate change. Further research including both in situ
investigation and laboratory experiments should examine the interactive effects of temperature
and other factors on phytoplankton communities in the Southern Ocean,, especially considering
that polar regions are warming much faster than other regions on the earth. Furthermore, the
California coast experiences regular warming events, and more temperature extremes are
predicted to happen with global warming. A final take-home message from my dissertation
work is that more research is needed into the interactive effects of warming with other
environmental factors on the biota of both temperate and polar marine regimes, in order to fully
understand and predict the impacts of future anthropogenic change on all of the unique and
fragile ocean ecosystems of the planet.

136
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Creator Zhu, Zhi (author)

Core Title Future impacts of warming and other global change variables on phytoplankton communities of coastal Antarctica and California

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Marine Biology and Biological Oceanography

Publication Date 04/11/2017

Defense Date 03/07/2017

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

Tag climate change,CO₂,diatom,Fe,harmful algal bloom,OAI-PMH Harvest,Phaeocystis,phytoplankton,Ross Sea,Southern Ocean,temperature

Language English

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Advisor Hutchins, David (committee chair), Fu, Feixue (committee member), Hammond, Douglas (committee member)

Creator Email zhizhu@usc.edu,zhuzhi33@gmail.com

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