Quantitative biogeography of distinct clades of picoplanktonic marine green algae establishes that

87 Pages·2016·1.92 MB·English

by  Alexander Limardo

UC Santa Cruz
UC Santa Cruz Electronic Theses and Dissertations
Title
Quantitative biogeography of distinct clades of picoplanktonic marine green algae
establishes that Bathycoccus ecotypes coexist more frequently than Ostreococcus ecotypes
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https://escholarship.org/uc/item/09c977pb
Author
Limardo, Alexander Joseph
Publication Date
2016
Supplemental Material
https://escholarship.org/uc/item/09c977pb#supplemental
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UNIVERSITY OF CALIFORNIA
SANTA CRUZ
Quantitative biogeography of distinct clades of picoplanktonic marine green
algae establishes that Bathycoccus ecotypes coexist more frequently than
Ostreococcus ecotypes

A thesis submitted in partial satisfaction
of the requirements for the degree of

MASTER OF SCIENCE
in
OCEAN SCIENCES
by
Alexander Joseph Limardo
December 2016

The Thesis of Alexander Joseph Limardo
is approved:

Professor Marilou Sison-Mangus

Professor Alexandra Z. Worden

Professor Jonathan Zehr

Tyrus Miller
Vice Provost and Dean of Graduate Studies
Copyright © by
Alexander Joseph Limardo
2016
Table of Contents
Title Page i
Copyright ii
Table of Contents iii
List of Tables and Figures iv
Acknowledgements vi
Abstract vii
Chapter 1: Background and Introduction 1
Chapter 2: Quantitative biogeography of distinct clades of picoplanktonic marine
green algae establishes that Bathycoccus ecotypes coexist more frequently than
Ostreococcus ecotypes 14
Summary 14
Introduction 17
Materials and Methods 22
Results 32
Discussion 55
Chapter 3: Conclusions and Future Directions 73

iii
List of Figures & Tables
Figure 1: Alignment of partial ITS1 region showing regions targeted by Bathycoccus
BI and BII qPCR primers and probes. 28
Figure 2: Bathycoccus BI and BII qPCR assays testing different mixtures of BI and
BII genomic DNA. 29
Figure 3: Map showing locations of environmental samples and phylogenetic tree of
Mamiellophyceae ITS sequences. 34
Figure 4: Results from Station M1 of Monterey Bay Time-Series (MBTS). 40
Figure 5: Results from Station ALOHA of the Hawaii Ocean Time-series Study
(HOT). 41
Figure 6: Results from GOC12 transect. 47
Figure 7: Results from WFAD09 transect. 48
Figure 8: Clade ratios (BI/BII or OI/OII) from GOC12 and WFAD09 cruises plotted
against depth. 49
Figure 9: QPCR abundance for all samples where Bathycoccus or Ostreococcus were
detected plotted against temperature and salinity. 53
Figure 10: Non-metric multidimensional scaling with k-means clustering analysis. 54
Table 1: Metadata for cruises and time-series sampled in this study. 23
Table 2: Nucleotide sequences for Bathycoccus BI and BII qPCR assays. 29
Table 3: Cross reactivity of Bathycoccus BI and BII qPCR assays. 30
Table 4: Results from environmental clone libraries targeting Mamiellophyceae. 33
iv
Table 5: Environmental variables at Station M1 (MBTS) where BII or OII were
detected and where absent. 42
Table 6: Environmental variables at Station ALOHA (HOT) where BII and OII were
detected and where absent. 42
Table 7: Spearman’s rank correlation coefficient (ρ) between ecotype qPCR
abundances and environmental variables. 52

v
Acknowledgements
First, I want to thank my parents, Lynn and Ken Limardo. Obviously I wouldn’t
exist without them, but they also helped instill in me the value of education, and as a
child they nurtured my scientific curiosity. Also, none of this would have been
possible without the love and support of my significant other, Amy Klebesadel, who
has stood by my side throughout the good times as well as the bad.
I wanted to say a big THANK YOU to all Worden Lab members (past and
present) who have helped me tremendously in so many ways these last 4 years (5
years including my time in the lab prior to becoming a grad student) as well as many
MBARI staff members. A special thanks to those of you that sacrificed your time to
give constructive feedback on my thesis presentation and on the thesis text itself. I
feel fortunate to be a part of such a talented lab group with people who genuinely care
about each other, and who are willing to help fellow lab-mates succeed in achieving
their goals.
Additional thanks to the amazing UCSC Ocean Sciences Department, my thesis
reading committee (Marilou Sison-Mangus, Alex Worden and Jon Zehr), and all of
my Santa Cruz and MBARI friends. Last, but certainly not least, I wanted to give a
special thanks to my advisor Alex Worden for all the support and guidance she has
provided these last few years. During my time with Alex, she has provided me with
invaluable experiences, both in my professional as well as personal life, and these
experiences will continue to influence my life for many years into the future.

vi
Abstract
Quantitative biogeography of distinct clades of picoplanktonic marine green
algae establishes that Bathycoccus ecotypes coexist more frequently than
Ostreococcus ecotypes
by
Alexander Joseph Limardo

We describe the biogeography of two clades of Bathycoccus, a picoplanktonic group
of marine green algae, that are phylogenetically distinct based on analysis of the
ITS1/5.8S/ITS2 region of the nuclear rRNA operon, but identical across the 18S
rRNA gene. QPCR assays were developed against the ITS1 to allow quantification of
these taxa (clades BI and BII). These were used alongside prior 18S rRNA primer-
probes for two Ostreococcus clades that have previously been classified as different
ecotypes (clades OI and OII) to analyze 266 photic zone samples from the tropical
Atlantic and North Pacific Oceans. Clades BI and OI were observed in mesotrophic
locations, whereas BII and OII were found in oligotrophic regions. Bathycoccus
ecotypes co-occurred more often than Ostreococcus ecotypes. In contrast to
Ostreococcus ecotypes, the adaptive differences between Bathycoccus clades have yet
to be resolved and both Bathycoccus ecotypes appear to be more flexible in their
niche(s) than Ostreococcus.
vii
CHAPTER 1: Background & Introduction

Marine phytoplankton are responsible for approximately half of global net primary
production, and comprise the base of the marine food web (Field et al 1998). By
utilizing energy from sunlight to fix CO into organic carbon molecules, the growth
2
of phytoplankton biomass has a strong impact on the entire marine ecosystem as well
as the physico-chemical conditions of the surrounding environment (Redfield 1958).
Phytoplankton growth and mortality rates are controlled by numerous physical,
chemical and biological factors. Understanding how these complex environmental
parameters interact to determine phytoplankton community structure and biomass
production is crucial to improving models of biogeochemical cycling and trophic
interactions, and thus our ability to predict future changes to those systems.
Oceanographers conventionally classify plankton based on size fractions retrieved
from sequential filtration through different pore sizes. The smallest size class, the
picophytoplankton (<3 µm cell diameter) are widely distributed in all aquatic
habitats, and comprise a diverse group of organisms, including cyanobacteria and the
‘picoeukaryotes’ (Johnson and Sieburth 1982, Waterbury et al 1979). Due to their
small cell size which has a high surface area to volume ratio, picophytoplankton are
effective competitors for nutrients, therefore this group dominates primary production
in marine habitats where nutrients are scarce (Chisholm 1992, Raven 1998).
However, the small size of picophytoplankton and lack of distinguishable
morphological features presents major obstacles for researchers attempting to study
1
these organisms, therefore they were largely overlooked until advances such as flow
cytometry, epifluorescence microcopy (combined with in-situ hybridization methods)
and nucleic acid sequencing enabled more detailed investigations.
Flow cytometry, a tool utilized frequently to study picophytoplankton, allows
oceanographers to discriminate, sort and quantify cyanobacteria (Synechococcus and
Prochlorococcus) and photosynthetic picoeukaryotes (PPE) based on optical
properties such as fluorescence of photosynthetic pigments and cell size-dependent
light scattering (Olson et al 1991). Using flow cytometry to measure cell abundances,
researchers began to recognize that picophytoplankton account for a significant
proportion of photosynthetic biomass in many regions of the world’s oceans
(Campbell et al 1994, Li 1994). While cyanobacteria were often numerically more
abundant than PPE, studies incorporating biomass and growth estimates found that
PPE can dominate primary productivity due to their larger cell volumes and relatively
high growth rates (Li 1994, Worden et al 2004). However, a significant drawback of
flow cytometry is limited taxonomic resolution of picoeukaryotes, where cell
abundances of many different species are often summed together and reported as a
single group. When DNA sequencing techniques were applied to ocean samples, the
immense genetic diversity and complexity of the PPE component was revealed, and
several novel branches were added to the eukaryotic tree of life (Diez et al 2001,
Lopez-Garcia et al 2001, Moon-van der Staay et al 2001). Efforts to understand the
ecological roles played by different PPE taxa were confounded by massive
biodiversity and a lack of quantitative observations, although certain PPE taxa
2