A GENETIC MARKER FOR COASTAL DIATOMS BASED ON PSBA
Meghan E Chafee
A Thesis Submitted to the
University of North Carolina Wilmington in Partial Fulfillment
of the Requirements for the Degree of
Master of Science
Center for Marine Science
University of North Carolina Wilmington
2008
Approved by
Advisory Committee
Dr. Bongkeun Song
Dr. Lynn Leonard
Dr. Lawrence B. Cahoon
Dr. J. Craig Bailey
Chair
Accepted by
Robert D. Roer
Digitally signed by Robert D. Roer
DN: cn=Robert D. Roer, o=UNCW, ou=Dean of the
Graduate School & Research, email=roer@uncw.edu,
c=US
Date: 2008.05.20 11:42:39 -04'00'
___________________________
Dean, Graduate School
TABLE OF CONTENTS
ABSTRACT ..........................................................................................................iv
ACKNOWLEDGEMENTS..................................................................................... v
LIST OF TABLES .................................................................................................vi
LIST OF FIGURES .............................................................................................. vii
CHAPTER 1: LITERATURE REVIEW .................................................................. 1
Environmental Sampling ............................................................................ 1
Diatoms...................................................................................................... 4
The psbA Gene.......................................................................................... 5
Biodiversity ................................................................................................ 8
Objective.................................................................................................. 11
CHAPTER 2: A GENETIC MARKER FOR MARINE DIATOMS BASED ON
PsbA ............................................................................................ 12
INTRODUCTION ..................................................................................... 12
MATERIALS AND METHODS ................................................................. 14
Cultures......................................................................................... 14
Diatom Isolation ............................................................................ 15
DNA Extraction from Cultures ....................................................... 15
Environmental Samples ................................................................ 16
18S Sequence Generation............................................................ 16
Primer Design ............................................................................... 17
psbA Sequence Generation .......................................................... 18
Cloning.......................................................................................... 19
ii
Allelic Diversity .............................................................................. 19
Distance Analysis.......................................................................... 20
Phylogenetic Analysis......................................................................................... 20
DOTUR Analysis ........................................................................... 21
RESULTS ................................................................................................. 22
Isolation and Analysis of Pure Cultures......................................... 22
18S rRNA and psbA Gene Comparisons ...................................... 24
Environmental Sampling ............................................................... 25
DISCUSSION ........................................................................................... 26
Primer Specificity .......................................................................... 28
psbA Diversity ............................................................................... 24
CONCLUSIONS ...................................................................................... 30
LITERATURE CITED............................................................................... 32
APPENDIX 1............................................................................................ 55
APPENDIX 2............................................................................................ 63
iii
ABSTRACT
Molecular ecological studies of marine diatoms have been hindered by the
lack of taxon-specific eukaryote genetic markers. Diatoms are among the most
abundant and diverse of all photosynthetic eukaryotes and account for nearly
25% of all global primary productivity. The ability of algae, and of diatoms in
particular, to quickly respond to a wide range of environmental parameters and
their position as the base of food webs make them prime sentinels of
environmental change. Recently a ‘universal’ psbA gene marker was developed
for environmental studies of photosynthetic prokaryotes and eukaryotes. The
objective of this study was to develop a diatom-specific eukaryotic gene marker
based on this ‘universal’ psbA marker to enhance ecological studies of marine
diatoms. Using nucleotide alignments of diatom psbA sequences, an
oligonucleotide primer was designed that amplifies diatoms along with some
other photosynthetic eukaryotes without the inclusion of homologous
cyanobacterial psbA genes. Comparison of psbA and 18S diatom sequences
from unialgal, non-axenic cultures confirms the use of psbA as an accurate
marker for resolving relationships within Phylum Bacillariophyta (diatoms). The
subsequent application of this modified psbA gene marker to environmental DNA
samples from the Cape Fear River Plume showed that primarily diatoms were
amplified from the environment. This marker proves useful in the detection of
highly resolved levels of diversity within Bacillariophyta.
iv
ACKNOWLEDGEMENTS
Special thanks goes to Kristine Sommer, one of the greatest resources at
the Center for Marine Science. I could not have completed this project without
her and I would not have learned all that I did if not for her patience and
dedication to taking care of her girls. I would also like to thank my lab mates,
Lyndsay Bell for always being so thoughtful, Cory Dashiell for keeping things light
and Erika Schwartz for keeping my cultures alive.
I would also like to thank my Wilmington and Charleston friends for their
encouragement and opportunities to blow off a little steam every now and then. I
must of course thank my family who still has no idea what it is that I do with DNA
but who nevertheless remain proud and supportive.
The National Science Foundation provided funding for this project. The
Coastal Ocean Research and Monitoring Program (CORMP) collected water
samples for this project from the Cape Fear River Plume.
Finally, I would like to thank my advisor Dr. Craig Bailey and my
committee members Drs. Lawrence Cahoon, Bongkeun Song and Lynn Leonard
for their dedication to teaching and for guidance and support throughout this
project.
v
LIST OF TABLES
Table
Page
1.
Forward and reverse primer matrix.............................................................. 41
2.
Putative species identifications for CMS diatom cultures ............................ 42
3.
psbA allelic diversity for CMS01 (Cylindrotheca cloisterium) ....................... 43
4.
psbA allelic diversity for CMS09 (Thalassiosira eccentrica)......................... 44
5.
Diversity and predicted richness of the 18S rRNA gene and psbA ............. 45
vi
LIST OF FIGURES
Figure
Page
1.
Map of southeastern North Carolina ........................................................... 46
2.
Sequence chromatograms from cloned isolate CMS01 (Cylindrotheca
cloisterium) ................................................................................................. 47
3.
Sequence chromatograms from cloned isolate CMS09 (Thalassiosira
eccentrica) .................................................................................................. 48
4.
18S rRNA gene parsimony phylogeny........................................................ 49
5.
psbA parsimony phylogeny......................................................................... 50
6.
18S rRNA and psbA neighbor-joining phylogenies..................................... 51
7.
Rarefaction analyses of 18S rRNA and psbA genes .................................. 52
8.
Haplotype diversity of psbA sequences ...................................................... 53
vii
CHAPTER 1: LITERATURE REVIEW
Environmental sampling
Ecological studies of microorganisms have resorted to a variety of
techniques to characterize natural assemblages. In the past, delineation of
community structure was based primarily on morphological identification of pure
cultures isolated from the environment. Identification of some microorganisms
requires that live specimens be examined (Butler and Rogerson 1995); however,
cultivation requirements of species widely vary and for many may not be feasible.
The lack of phenotypic traits available for reliable identification also hinders
comprehensive ecologically based studies of microorganisms. Microscopic
analysis at the bright field level is possible for some taxa (Flöder and Burns
2004), but often proves insufficient, and electron microscopy is subsequently
required for classification (Hoepffner and Haas 1990; Johnson and Sieburth
1982). Such techniques are feasible for larger protists that often have distinctive
characters (Andersen et al. 1996) but cannot be used to identify prokaryotes and
small eukaryotes, many of which share a nondescript round shape (Potter et al.
1997). Epifluorescence microscopy or flow cytometry is useful for estimates of
picoplankton abundance but cannot discriminate lower taxonomic levels (Lange
et al. 1996; Li 1994; Li et al. 1983; Simon et al. 1994). Pigment analysis by highperformance liquid chromatography (HPLC) is widely used to estimate
abundance and diversity (Bidigare and Ondrusek 1996). Although this technique
is useful for generating estimations of biomass for major taxonomic groups based
1
on pigment concentrations and their profiles, it does not have a sufficient
resolution for classifications below class levels (Mackey and Holdsworth 1998;
Zapata et al. 2000). Running blind HPLC analyses with no species level
taxonomic information from microscopic identification can result in significant
error where a major bloom species can be misidentified based on pigments alone
(Irigoien et al. 2004). Additionally, pigment concentrations vary among species
(Stolte et al. 2000) and environmental conditions (van Leeuwe and Stefels 1998)
resulting in inaccurate measures of biodiversity and abundance.
As a result of the above technological limitations, it was long suspected
that the biodiversity of microorganisms was inadequately characterized. Cultureindependent DNA sequencing and cloning methods made it technologically and
economically feasible to examine large numbers of sequences that make
ecological studies of microorganisms more meaningful. Giovannoni et al. (1990)
first sequenced 16S ribosomal RNA genes directly from environmental DNA
samples rather than from pure cultures, revealing many prokaryotic species
previously unknown. Since then numerous studies have discovered the
presence of many uncultured prokaryotes (DeLong 1992; Field et al. 1997;
Fuhrman et al. 1992; Moyer et al. 1995; Rooney-Varga et al. 1997), furthering
our understanding of prokaryotic abundance and distribution and their roles in
ecosystems (Fuhrman 2002; Pace 1997).
Ribosomal rRNA was the pioneering gene used extensively in the
detection of microbial diversity from natural communities. Prior to large-scale
eukaryote biodiversity studies, the 16S rRNA gene, designed to be specific for
2
bacteria, was applied to environmental nucleic acid samples and resulted in the
amplification of eukaryotic plastid genes (Rappé et al. 1998), which share a
common phylogenetic origin with cyanobacterial (Wolfe et al. 1994). Several of
these plastid genes appeared to be novel phytoplankton lineages and the
molecular tools developed for prokaryotic diversity studies were finally applied to
eukaryotes. This lag can be attributed to the fact that for most protozoa and
microalgae, the defining species-specific morphological characters required for
identification are more obvious than those of prokaryotes, generating a sizeable
group of protistan morphological experts. Although many eukaryotic plankton are
large enough in size to examine with bright field microscopy, the discovery of
phototrophic picoeukaryotes led to a taxonomic challenge similar to that posed
by prokaryotes in which few conspicuous morphological characters are available
for identification (Potter et al. 1997). Since the application of the same
techniques used in prokaryotic studies, many novel eukaryotic lineages have
been uncovered using genetic markers, some of which represent new
phylogenetic lineages (Dawson and Pace 2002; Dièz et al. 2001; Edgecomb et
al. 2002; Guillou et al. 1999; Lopez-Garcia et al. 2001; Massana et al. 2002;
Moon-van der Staay et al. 2000; Moon-van der Staay et al. 2001; Rappé et al.
1998; Stoeck and Epstein 2003; Stoeck et al. 2003).
The expansion of molecular ecological studies has made it abundantly
clear that there exists a much larger amount of prokaryotic and eukaryotic
diversity than has been observed through the conventional techniques described
above. While morphological experts are becoming less common, large-scale
3
studies of microorganism ecology are increasing in number, providing the public
databases needed to improve our understanding of interactions between
prokaryotic and eukaryotic communities and their bottom-up effects on higher
trophic levels.
Diatoms
Diatoms are among the most abundant and diverse group of all
photosynthetic eukaryotes. Found in all aquatic environments, these unicellular
chromophyte algae account for nearly half of total marine primary production
(Falkowski et al. 1998) and 25% of all global primary production (Field et al.
1998, Mann 1999). Diatoms (Phylum Bacillariophyta) are a unique group of
heterokont microalgae with a distinctive siliceous cell wall known as a frustule.
Generally diatoms are classified into two groups based on frustule symmetry:
centric and pennate forms. Centric diatoms are radially symmetrical and are
inclined to exhibit a planktonic lifestyle and pennate forms display bilateral
symmetry and tend to inhabit benthic microalgal assemblages. Raphid pennates
possess a raphe or slit through which mucilage is secreted as a substrate to glide
upon. Araphid pennates are bilateral but do not have a raphe (Falciatore and
Bowler 2002; Round et al. 1990).
The importance of diatoms cannot be overemphasized; they have
traditionally been used in many different scientific sub-disciplines. For example,
high-resolution stratigraphic analyses and species abundances for fossil diatoms
are key tools for developing paleolimnological-based reconstructions of past
4
biodiversity, trophic status and broad-scale climate change (Bigler and Hall 2002,
2003; Finkel et al. 2005; Smol et al. 2005; Wolfe 2003). Assemblages of extant,
attached diatoms are increasingly being used as biological reporters for
assessing trophic status and water quality in freshwater and coastal marine
systems (de la Rey et al. 2004, Poulíčková et al. 2004; Reavie and Smol 1998;.
Previous studies have demonstrated that diatoms are sensitive and respond
rapidly to changes in nutrient availability and are accurate predictors of other
water quality variables (e.g., chlorophyll a content: Cunningham et al. 2005;
Reavie and Smol 1998; Vaultonburg and Pederson 1994). For these reasons,
diatom indexes are useful monitors of eutrophication and other environmental
change (McCormick and Cairns 1994, Weckström and Juggins 2005, de la Cruz
et al. 2006). The development of more specific eukaryotic gene markers has
potential to improve molecular ecology studies of diatoms that are often
employed in water quality assessment and monitoring programs where the
presence or absence of particular diatom species have been correlated to
specific environmental parameters.
The psbA gene
The lag in development of species-specific eukaryotic genetic markers has
led to a current deficit in eukaryotic environmental sampling studies, thereby
inhibiting the development of large-scale ecologically-based comparisons.
Ribosomal RNA (rRNA) genes are found universally in all organisms and have
widely been used in the past to characterize evolutionary relationships (Woese
5
1987). The rRNA molecules show a high degree of functional constraint among
lineages, which allows for the comparison of evolutionary relationships across
phyla. Although much of the rRNA sequence is conserved, many regions of this
gene vary at different positions and at very different rates among species
providing a more resolved phylogeny of closely related organisms (Woese 1987).
The rRNA gene has proven highly versatile in the types of phylogenetic and
ecological analyses that can be performed. However, rRNA sequence
divergence may not be capable of distinguishing between two closely related
species that are only subtly different from one another. Alternative genetic
markers such as the chloroplast-encoded genes could prove useful in revealing
eukaryotic genetic diversity not detected by 18S rRNA, specifically in
photoautotrophic plankton.
In addition to ribosomal RNA, the ribulose bisphosphate
carboxylase/oxygenase enzyme (RUBISCO), encoded by the rbcL plastid gene,
has been employed in studies of microbial ecology as part of a quantification of
gene expression in the water column (Paul et al. 1999; Pichard and Paul 1991;
Pichard et al. 1993; Pichard et al 1996; Pichard et al. 1997a; Wawrik et al. 2002;
Xu and Tabita 1996). Studies involved in the detection of rbcL diversity in natural
phytoplankton communities, however, are limited in number as well as scope
(Pichard et al. 1997b; Xu and Tabita 1996).
The psbA gene used in this study encodes the D1 protein of photosystem II in all
oxygenic photosynthesizers. Literature regarding psbA in algae is scarce,
however, the nature of this gene could prove useful in a higher resolution
6
analysis of more cryptic genetic diversity of photosynthetic organisms. D1 and
D2 proteins form the reaction center heterodimer of PSII (Nanba and Satoh
1986) where oxygen evolution takes place (reviewed in Debus 1992). The D1
protein of photosystem II is continually damaged by light (photoinhibition)
(Osmond et al. 1993). The rapid, light-dependent turnover of D1 replaces
damaged proteins (Kyle 1987), serving as an important response to light stress in
plants and algae.
Zeidner et al. (2003) recently developed a “universal” genetic marker for
psbA that amplifies the D1 protein from prokaryotic cyanobacteria and eukaryotic
algae within environmental DNA samples. Comparison of psbA genes with
nuclear small subunit rRNA genes through use of environmental bacterial
artificial chromosome (BAC) libraries affirmed the phylogenic association
(Zeidner 2003). With support of psbA accuracy, Zeidner et al. (2003) applied this
photosynthetic marker to marine environmental DNA and subsequently amplified
psbA fragments from photosynthetic eukaryotes and cyanobacteria. The
advantage to using psbA instead of rRNA genes is that it eliminates heterotrophs
from analysis and focuses instead on the photoautotrophs. Such a marker could
be particularly beneficial in the ecological study of eukaryotic algae whose
nutritional requirements and role as primary producers make them unique
sentinels of environmental change and overall ecosystem health (McCormick and
Cairns 1994).
The primary practical impediment to employing psbA in a photosynthetic
eukaryote biodiversity study is excluding homologous sequence data from
7
prokaryotic cyanobacteria. Cyanobacteria are ubiquitous and numerous in
marine and freshwater environments. Because photosynthetic eukaryotes
derived their chloroplast genome endosymbiotically from a photosynthetic
prokaryote (Wolfe et al. 1994), obtaining a sufficient number of eukaryotic algal
genes through direct sequencings methods from environmental DNA requires
many rounds of cloning. An ideal photosynthetic eukaryote psbA marker would
also eliminate the amplification of cyanobacterial DNA, allowing for a more direct
assessment of the biodiversity of algal assemblages.
Biodiversity
An ongoing debate within scientific communities is the question regarding
the extent of microorganism dispersal and ubiquity. Because of their high
dispersal capabilities and lack of formidable boundaries, microorganisms in
general exhibit an overall low species richness compared to larger organisms
that are more confined to spatial niches (Finlay and Clarke 1999; Finlay 2002).
It was in the nineteenth century that Baas Becking first formulated the hypothesis
that with respect to microorganisms, ‘everything is everywhere, but, the
environment selects’ (Baas Becking 1934). In other words, there is a large
‘seedbank’ of organisms subjected to random global dispersal that subsequently
proliferate wherever conditions are suitable for growth (Finlay 2002). Under this
hypothesis, species samples taken from opposite corners of the world, but with
similar environmental conditions, would be identical. Perhaps microorganism
8
genetic differentiation must be examined at a more resolved level in order to
determine the extent of their ubiquity and ecological plasticity (Sarno et al. 2005).
Although microorganisms tend to exhibit wide-ranging dispersal, genetic
and ecological differentiation is becoming more apparent with the application of
appropriate genetic markers and a reexamination of cryptic morphological
characteristics. It has recently been suggested by Sarno et al. (2005) that the
easily identified diatom Skeletonema costatum actually consists of more than one
distinctive morphological species as revealed by light microscopy and scanning
and transmission electron micrograph. Out of eight specimens examined, four
main morphological groups were identified (Sarno et al. 2005). Specimens
belonging to the same morphological species shared identical large subunit
(LSU) rRNA sequences with similar or identical small subunit (SSU) rRNA
sequences. Even though SSU and LSU maximum likelihood phylogenies were
in agreement, their similar topologies may be a result of their close proximity
within rDNA cistrons. Sarno et al. (2005) recommends that phylogenies from
unrelated DNA regions, such as mitochondria or plastid genes, are needed to
confirm patterns revealed by these ribosomal genes. In addition to
morphological and phylogenetic differentiation, variations in seasonal
distributions were found among the species examined, indicating that this
“cosmopolitan” diatom is actually composed of many species that are
differentially selected upon by the environment, confining them to spatial niches.
Samples were collected from a small portion of the Skeletonema costatum
9
spatial distribution; there likely remains a higher level of diversity within this
species yet to be uncovered.
Sarno et al. (2005) found genetic and morphological differentiation among
geographically distant populations, however, a study by Rynearson and Armbrust
(2004) found substantial genetic differentiation among populations of the diatom
Ditylum brightwellii within the connected estuaries of the Strait of Juan de Fuca
and Puget Sound (WA, USA). The three genetically distinct populations were
identified by different microsatellite allele distributions and unique alleles.
Isolates from the two most genetically diverged species had identical nuclear 18S
rRNA sequences, indicating a close evolutionary relationship; however, isolates
from two of the populations examined in this study exhibited different
physiological capabilities (i.e., growth rates), suggesting differential selection. In
this particular region, the recirculation of waters within Puget Sound created the
‘geographical barrier’ required for genetic and physiological divergence between
these two populations of diatoms despite their close proximity (Rynearson and
Armbrust 2004). In this study microsatellites were able to detect a level of
diversity not found by 18S rRNA. Although microsatellites are extremely useful in
resolving relationships within species, development can be time consuming. The
direct use of a gene marker is likely to be more efficient and can be more broadly
applied.
Although morphology remains important to the study and classification of
microorganisms, DNA sequencing methods have provided the ultimate
examination of interspecific genetic variation at the level of nucleotide base pair.
10
Furthermore, the manipulation of bacteria to separate and clone DNA fragments
has exposed genetic variation within single isolates, potentially revealing multiple
gene haplotypes within a single species. Skeletonema costatum is broadly
distributed and has been extensively studied for more than a century (Sarno et
al. 2005), yet in addition to newfound genetic differentiation, new morphological
characters are still presenting themselves after many microscope hours. With
the use of more appropriate biodiversity gene markers such as psbA,
environmental sampling could prove to be a powerful tool to expose extant levels
of microorganism ubiquity and diversity on a global scale.
Objective
The primary objective of this project is to develop a diatom-specific
eukaryotic genetic marker based on the membrane-encoded psbA gene that can
be applied to ecological studies of coastal diatoms.
11
CHAPTER 2: A GENETIC MARKER FOR MARINE DIATOMS BASED ON psbA
INTRODUCTION
Development of taxon-specific prokaryotic genetic markers has preceded
that of the eukaryotes by nearly a decade. In general, prokaryotes are very small
(0.2-2 μm) in size with a nondescript round shape (Potter et al. 1997), many of
which cannot be cultured in the laboratory. For most protozoa and microalgae,
the defining species-specific morphological characters required for identification
are more obvious than those of prokaryotes. The discovery of picoeukaryotes
(0.2-2 μm), however, posed a similar taxonomic challenge where convergent
evolution has resulted in similar phenotypes among distantly related eukaryotic
taxa (Potter et al. 1997).
The recent application of 18S rRNA gene markers to environmental DNA
has revealed many novel eukaryotic lineages (Dawson and Pace 2002; Dièz et
al. 2001; Edgecomb et al. 2002; Guillou et al. 1999; Lopez-Garcia et al. 2001;
Massana et al. 2002; Moon-van der Staay et al. 2000; Moon-van der Staay et al.
2001; Rappé et al. 1998; Stoeck and Epstein 2003; Stoeck et al. 2003). Nearly
every environmental eukaryotic study to date has employed the 18S rRNA gene
marker for assemblage characterization. The 18S rRNA gene is useful in
determining community structure of both heterotrophic and autotrophic
eukaryotes but a genetic marker specific to photoautotrophs would enhance
ecological studies of marine microalgae.
The rbcL gene, which codes for the ribulose bisphosphate
carboxylase/oxygenase enzyme (RUBISCO) responsible for carbon fixation, has
12
also been applied to environmental DNA samples. Studies employing RUBISCO
focus largely on the quantification of gene expression of photosynthetic plankton
within the water column (Paul et al. 1999; Pichard and Paul 1991; Pichard et al.
1993; Pichard et al 1996; Pichard et al. 1997a; Wawrik et al. 2002; Xu and Tabita
1996), although some have examined diversity of the rbcL gene in the natural
phytoplankton community (Pichard et al. 1997b; Xu and Tabita 1996). The rbcL
gene, however, has not been employed is any large-scale investigations of
diversity.
Molecular ecological studies of marine diatoms have been hindered by the
lack of taxon-specific eukaryote genetic markers. Diatoms are among the most
diverse of all photosynthetic eukaryotes and account for nearly 25% of all global
primary productivity (Field et al. 1998, Mann 1999). Their characteristic siliceous
frustules preserve well in sediments providing indispensable tools for developing
paleolimnological-based reconstructions of past biodiversity, trophic status and
broad-scale climate change (Bigler and Hall 2002, 2003; Wolfe 2003; Finkel et al.
2005; Smol et al. 2005). The ability of diatoms to quickly respond to a wide
range of environmental parameters and their position at the base of food webs
make them prime sentinels of environmental change (McCormick and Cairns
1994; Weckström and Juggins 2005; de la Cruz et al. 2006). Diatoms have been
employed in environmental monitoring and assessment programs in freshwater
and coastal marine systems (Reavie and Smol 1998; de la Rey et al. 2004,
Poulíckovã et al. 2004). The development of genetic markers that specifically
target this important and diverse group has potential to reveal ecological
13
information that is central to their use as monitors of environmental change (e.g.,
eutrophication), particularly in coastal regions where nutrients are delivered in
pulses by rivers.
Zeidner et al. (2003) developed a “universal” genetic marker for the
membrane-embedded psbA gene that amplifies all oxygenic photosynthesizers,
including phototrophic cyanobacteria and eukaryotic algae. The ubiquity of
cyanobacteria in the marine environment complicates biodiversity studies of
eukaryotic algae. Direct application of psbA to environmental samples results in
a clonal library composed primarily of cyanobacteria.
In this study, I developed a new set of psbA gene primers based on those
developed by Zeidner et al. (2003) such that eukaryotic algae are amplified from
pure culture and the environment but cyanobacteria are now eliminated from
clone libraries. When applied to study site CFP6 in the Cape Fear River Plume
off southeastern coastal North Carolina, this modified psbA gene primarily
amplified diatoms from river plume waters. This genetic marker, based on the
psbA gene, is a useful for estimations of biodiversity and proves itself as a
suitable marker for phylogenetic studies of diatoms (Phylum Bacillariophyta).
MATERIALS AND METHODS
Cultures
A total of 30 pure cultures were examined in this study. Six diatom
cultures were obtained from The Provasoli-Guillard National Center for Culture of
Marine Phytoplankton (CCMP, Bigelow, ME, USA) (Appendix 1). Twenty-four
14
cultures from other photosynthetic eukaryotes were obtained from CCMP, UTEX
(University of Texas at Austin, TX, USA), SCCAP (The Scandinavian Culture
Centre for Algae and Protozoa, Copenhagen, Denmark), SAG (Culture Collection
of Algae at the University of Göttingen, Göttingen, Germany), MBIC (Marine
Biotechnology Institute Culture Collection, Japan), PCC (Pasteur Culture
Collection of Cyanobacteria, France), NIEHS (The National Institute of
Environmental Health Sciences, NC, USA) and CCAP (Culture Collection of
Algae and Protozoa, Scotland, UK) (Appendix 2).
Diatom isolation
Additional diatoms were isolated from the Intracoastal Waterway at the
University of North Carolina’s Center for Marine Science (CMS) (Fig. 1). In total
46 single diatom cells were isolated under an Olympus Bx60 microscope with a
capillary pipette and grown into unialgal, non-axenic cultures in f/2 media at room
temperature (~25°C) (Appendix 1).
DNA extraction from cultures
Diatom cells from CMS/ICW cultures and CCMP culture collections were
harvested by centrifuging at 14,000 rpm and total cellular DNA was extracted as
described by Bailey et al. (1998).
15
Environmental samples
Five liters of benthic (~10 m depth) and surface water samples were
collected on 13 June 2007 from the Cape Fear River Plume site CFP6 (Fig. 1).
Water was filtered through 0.45-μm non-sterile Nalgene membrane filters to
collect planktonic biomass and stored at -80°C. DNA was then extracted from
filters as described above.
18S sequence generation
The 18S rRNA gene was amplified from the six diatom cultures from CCMP and
for 34 of the 46 unialgal, non-axenic CMS cultures for comparison with diatom
psbA sequences. PCR reactions were performed in 50 μL volumes with 10 μL of
5X Green GoTaq Reaction Buffer (Promega, Madison, WI), 1 μL 10mM dNTP
(Promega), 0.5 μL 10 μM of the forward and reverse primers, 1.25 units of
GoTaq (Promega), 36.75 μL sterile distilled water and 1 μL DNA template. The
PCR thermocycling program included an initial denaturing step at 94 °C for 4
min, followed by 40 cycles of DNA denaturation at 94 °C for 30 s, primer
annealing at 50 °C for 1 min and fragment extension at 72 °C for 1.5 min. Final
extension ran for an additional 7 min at 72 °C. PCR products were then purified
using StrataPrep PCR Purification Kit (Stratagene, La Jolla, CA) according to the
manufacturer’s instructions.
Partial 18S RNA gene sequences were generated from cleaned PCR
products using BigDye v3.1 Terminator Sequencing Kits (ABI, Foster City, CA)
determined on a 3130 xl Genetic Analyzer (ABI). All sequences were edited in
16
Sequencher 4.7 (Gene Codes Corp., Ann Arbor, MI) automatically aligned using
ClustalX (Thompson et al. 1994) and edited by eye in MacClade 4.0 (Maddison
and Maddison 2000). Hypervariable regions were excluded from analysis.
Primer design
Diatom specific primer design based on psbA was initiated by comparing
the psbA gene sequences from the complete chloroplast genomes of diatoms
Odontella sinensis (GenBank Acc. No. Z67753), Phaeodactylum tricornutum
(GenBank Acc. No. EF067920) and Thalassiosira pseudonana (GenBank Acc.
No. EF067921) and from the six diatom CCMP cultures. Eight additional diatom
psbA sequences from Onslow Bay were added and aligned from a previous
study (unpublished data) to aid in primer development. Based on a visual
comparison of conserved regions, a matrix of six forward and six reverse
degenerative primers (Table 1) were tested using diatom cultures from CCMP.
Application of primer combination dF11 (5’- GGT ATT CGT GAG CCT
GTT GC -3’) and dR12 (5’- CCT CCA TAC CTA AAT CAG CAC -3’) to diatom
cultures resulted in positive PCR bands for all cultures. Cloned fragments from
diatom cultures were PCR amplified and sequenced in the forward and reverse
direction. Contigs were BLASTed in GenBank and those closely resembling
diatom sequences were maintained and aligned with previously determined
diatom psbA sequences. The dF11/dR12 primer set was then applied to
environmental DNA from CFP6 and a clonal library was generated.
17
With a suite of diatom psbA sequences available, primer specificity was
further streamlined. A second primer combination dsF (5’-GAT TGG TTR AAG
TTG AAA CC-3’) and dsR (5’-AGG TTC TTT ATT ATA YGG TAA-3’)] was
designed that directly amplified CCMP diatom cultures and CMS/ICW diatom
isolates in culture with cyanobacteria without cloning. dsF/dsR primers were
then applied CFP6 environmental samples and a second clonal library was
generated.
psbA sequence generation
The psbA gene was amplified from the six diatom cultures from CCMP, for
all 46 CMS cultures and for the 24 other photosynthetic eukaryote cultures. PCR
amplification and purification of psbA by the dF11/dR12 primer combination was
performed the same as described above for the 18S rRNA gene. A gradient
PCR amplification of the dsF/dsR primer combination showed an annealing
temperature at 45 °C for 1 min to be optimal for this set of primers but all other
thermocycle conditions were the same as described above.
Partial psbA gene sequences were generated from cleaned PCR products
using BigDye v3.1 Terminator Sequencing Kits (ABI, Foster City, CA) determined
on a 3130 xl Genetic Analyzer (ABI). All sequences were edited in Sequencher
4.7 and manually aligned in MacClade 4.0.
18
Cloning
PCR products of the psbA gene were cloned using pGEM-T Easy Vector
Systems Kit (Promega) according to manufacturer’s instructions. Positive
colonies were picked, boiled in 25 μL of distilled water at 99 ºC for 5 min, and
amplified as follows for the dF11/dR12 primer set: initial denaturing step at 95 °C
for 15 min, followed by 35 cycles of DNA denaturation at 94 °C for 1 min, primer
annealing at 50 °C for 1 min and fragment extension at 72 °C for 2 min. Final
extension ran for an additional 7 min at 72 °C. Clones generated from the
dsF/dsR primer set were amplified under the same conditions as above with the
exception of a lower annealing temperature at 45 °C for 1 min. All clonal PCR
products were cleaned using StrataPrep PCR Purification Kit.
Allelic diversity
Two diatom cultures, pennate CMS01 (Cylindrotheca closterium) and
centric CMS09 (Thalassiosira eccentrica), were chosen for analysis of psbA
allelic diversity. A brightline hemacytometer (Sigma, St. Louis, MO, USA) was
used to determine cell counts of each culture. DNA was extracted from ~335,000
cells of each culture for comparison of allelic diversity between species with
different numbers of chloroplasts. PCR amplification, cloning and sequencing
was carried out as described above for the dsF/dsR primer set. Partial psbA
sequences were edited and manually aligned. Segregating sites found in psbA
alignments for the two species were visually confirmed by sequence
chromatograms. Because the sequences were generated in the forward and
19
reverse direction, the changes observed in the alignment could be seen in the
raw sequence data in both directions.
Distance analysis
All psbA sequences used in analyses were generated with the dsF/dsR
primer. Partial 18S rRNA gene and psbA diatom sequences from CMS and
CCMP diatom cultures and GenBank sequences were edited and maintained in
separate alignments. Neighbor-joining trees were generated for each gene using
an uncorrected-p distance and 10,000 neighbor-joining bootstrap replicates.
Partial psbA sequences from CFP6 clonal library, CMS and CCMP diatom
cultures, GenBank diatom sequences and other photosynthetic eukaryote
reference sequences were edited and manually aligned. All analyses in this study
were performed in PAUP* 4.0b10 (Swofford 2002). A neighbor-joining (Saitou
and Nei 1987) psbA haplotype diversity was generated using an uncorrected-p
distance and 1000 neighbor-joining bootstrap replicates.
Phylogenetic analyses
Partial 18S rRNA gene and psbA sequences from CMS diatom isolates
were edited and aligned. Parsimony, maximum likelihood and neighbor-joining
analyses of the 18S rRNA gene and psbA alignments were performed.
Maximum parsimony analyses were generated using a full heuristic search with
10 random sequence additions and TBR branch swapping. ModelTest (Posada
and Crandall 1998) was used to generate the best-fit model of molecular
20
evolution for the maximum likelihood analyses. The model used for the 18S
rRNA gene was TrN (Tamura-Nei: Tamura and Nei 1993) with a proportion of
invariable sites (=0.4982) and with a gamma distribution (α=0.5145). The model
used for psbA was GTR (General Time Reversible: Lanave et al. 1984) with a
proportion of invariable sites (=0.6384) and with a gamma distribution
(α=1.1035). Maximum likelihood bootstrap values were generated using 100 fast
stepwise additions.
DOTUR analysis of 18S rRNA gene and psbA genes
The sequences of the 18S rRNA and psbA genes from CMS and CCMP
diatom cultures and reference diatom sequences from GenBank were used for
distance-based operational taxonomic unit and richness (DOTUR: Scholss and
Handelsman 2005) analysis. DOTUR calculated various diversity indices and
richness estimators. The sequences were aligned with ClustalW (Thompson et
al. 1994). The DNA distance program in the PHYLIP package (Felsenstein
1993) was used to calculate genetic distances with Kimura-2-parameter methods
(Kimura 1980). The distance matrix was then used for the DOTUR analysis.
The determination of operational taxonomic units (OTUs) was based on 1% and
3% genetic differences. Rarefaction curves were generated to compare the
sequence variation between 18S rRNA and psbA genes. The bias-corrected
Chao1 richness estimator (Chao 1984) was used to assess species richness and
the Shannon-Weiner diversity index (Shannon and Weaver 1963) was used to
assess species richness and evenness for the two genes.
21
RESULTS
Isolation and analysis of pure cultures
In total 46 diatoms were isolated from the CMS/ICW and have been
maintained in the laboratory. 18S rRNA gene sequences were obtained for 34 of
these cultures. BLASTn results provided putative identifications (Table 2) for the
previously unknown CMS diatom isolates, and species names were added to
trees to provide reference sequences for anonymous clonal haplotypes in the
psbA haplotype diversity tree. 18S rRNA gene IDs were also used in
phylogenetic comparisons of 18S rRNA and psbA gene sequences from CMS
and CCMP diatom cultures and diatom sequences from GenBank.
The psbA gene was amplified from each CMS diatom isolate using the
first set of primers developed in this study, dF11/dR12. PCR products were
sequenced and upon analysis of direct sequence chromatograms, it was
apparent that multiple psbA sequences were present and that cloning was
necessary to obtain separate sequences. BLASTn search results of cloned psbA
sequences indicated that the diatom cultures were contaminated with
photosynthetic cyanobacteria indicating that the CMS diatom cultures were
unialgal but not axenic. dF11/dR12 was then applied to the environment.
BLASTn results from this clone library indicated that primarily cyanobacteria were
amplified from CFP6 (results not shown).
The second primer set designed, dsF/dsR, directly amplified diatoms from
the CMS cultures contaminated with cyanobacteria without cloning. dsF/dsR
22
was then used to amplify all CMS and CCMP diatom cultures as well as the 24
other photosynthetic eukaryote cultures used as indicators of primer specificity.
Upon application of this primer set to environmental samples from CFP6 it was
found that cyanobacteria had been eliminated from the clone library.
PCR amplification and cloning of CMS cultures with the first primer set
dF11/dR12 resulted in the separation of diatom and cyanobacteria sequences
provided sequence alignments of multiple psbA copies from each diatom culture.
As a result, a significant number of nucleotide substitutions were revealed within
each isolate. Subsequently, two CMS cultures were chosen to determine the
extent of psbA variation within a culture derived from a single diatom cell: CMS01
with 18S BLASTn ID Cylindrotheca closterium and CMS09 with Thalassiosira
eccentrica BLASTn ID. A total of 57 psbA gene fragments were obtained from
the Cylindrotheca closterium clone library and 39 psbA gene fragments were
obtained from the Thalassiosira eccentrica clone library using the second primer
set developed dsF/dsR. Only those sequences containing segregating sites are
shown (Table 3 and Table 4). Sequences were aligned and compared to raw
sequence chromatograms to ensure that the observed segregating sites were
sound and not due to sequencing error (Fig. 2 and 3). Cylindrotheca sp. clones
had 22 segregating sites out of a total of 655 nucleotide positions (with an
estimated 96.6% sequence similarity) with a total of 11 non-synonymous
substitutions (Table 3). Thalassiosira sp. clones had 10 segregating sites out of
a total of 663 nucleotide positions (with an estimated 98.5% sequence similarity)
with 8 non-synonymous substitutions (Table 4).
23
Thalassiosira sp. has many more chloroplasts than Cylindrotheca sp.
Despite this obvious difference, however, Cylindrotheca sp. and Thalassiosira sp.
had nearly equal substitution rates at about 7.3 x 10-4 substitutions/ total sites.
Substitution rates for Thalassiosira sp. were extrapolated to account for the
unequal number of sequences available for the two isolates.
18S rRNA and psbA gene comparisons
The 18S rRNA gene and psbA maximum parsimony and maximum
likelihood phylogenies with supporting maximum parsimony and maximum
likelihood bootstrap values (Fig. 4 and 5) were generated for sequences from
CMS and CCMP diatom cultures and for GenBank diatom sequences.
Topologies for phylogenies of both the 18S rRNA gene and psbA show that the
raphid pennate diatoms are monophyletic, but that the centric and araphid
pennate diatoms are not. A similar topology is observed for each gene in a
neighbor-joining comparison of genetic distance using uncorrected p-distance
(Fig.6) providing confidence in psbA as an appropriate marker for biodiversity
that is also accurate in resolving phylogenetic relationships among the diatoms.
Rarefaction curves were generated based on 41 18S rRNA genes and 50
psbA genes. Rarefaction analysis calculated the same number of OTUs for the
18S rRNA and psbA genes (Fig. 7). Twenty-four OTUs were observed at the 1%
cutoff and 16 were observed at the 3% cutoff for both genes. Richness of both
sequences did not reach saturation based on rarefaction analysis. The
nonparametric Chao1 richness estimator calculated 41 and 75 OTUs based on
24
1% cutoff for 18S rRNA and psbA genes, respectively. However, richness of the
psbA genes was lower than the 18S rRNA genes based on the 3% cutoff. The
Shannon-Weiner richness and evenness index values for psbA genes are lower
than those observed for 18S rRNA gene at both cutoff levels (Table 5).
Environmental sampling
In total, direct sequencing methods were used to determine partial psbA
sequences for 140 diatoms obtained from clone libraries constructed with the
dsF/dsR marker using environmental DNA extracted from surface and bottom
samples collected at CFP6 (Appendix 1, Fig. 8). The average length of the psbA
fragment sequenced in this study was 665 bp.
Diatom psbA sequences from the CFP6 clone library were compiled with
sequences from CMS and CCMP diatom cultures and diatom sequences from
GenBank and Onslow Bay (Appendix 1). Twenty-four other eukaryotic taxa
comprising a phylogenetically diverse assemblage of photoautotrophs were
combined with 13 psbA sequences from GenBank for a total of 37 psbA
sequences from other photosynthetic eukaryotes (Appendix 2) added to analysis
for assessment of primer specificity of photosynthetic groups outside Phylum
Bacillariophyta (diatoms). The neighbor-joining tree of all psbA sequences
analyzed in this study with supporting bootstrap values (Fig. 8) reflects haplotype
diversity at site CFP6. Although the dsF/dsR primers are not specific to diatoms,
a diatom psbA sequence can be distinguished from that of other photosynthetic
eukaryotes; anonymous environmental sequences can be identified as a diatom
25
if it groups within the monophyletic clade of Phylum Bacillariophyta (diatoms).
CMS cultures shown in bold have been putatively identified according to the 18S
rRNA gene sequence BLASTn search. The presence of a branch with a species
name indicates a positively identified reference haplotype from a culture
collection or a GenBank sequence that can be used to infer relationships among
anonymous psbA haplotypes. The application of the photosynthetic eukaryote
specific psbA marker dsF/dsR to site CFP6 on 13 June 2007 revealed that
primarily diatoms are amplified from river plume waters with only three
sequences out of 140 that grouped with other photosynthetic eukaryotes.
DISCUSSION
Primer specificity
The primary goal of this study was to design a diatom-specific maker for
genetic and ecological studies. When tested on pure cultures, the psbA-based
genetic marker developed amplifies some other photosynthetic eukaryotes
including other stramenopiles, chlorophytes, haptophytes, cryptophytes and
rhodophytes; it is not strictly, by definition, diatom-specific. Nevertheless, our
field studies clearly demonstrate that application of these oligonucleotide primers
to environmental samples proves sufficient for ecological studies of marine
diatoms. A diatom sequence can easily be distinguished from other members of
the photosynthetic eukaryotic assemblage based on an analysis of genetic
distance and the grouping of a diatom sequence within the monophyletic
Bacillariophyta clade.
26
Phylogenetic and genetic distance analyses of the 18S rRNA gene
sequences from pure diatom cultures indicates that centric and araphid pennate
diatoms do not form monophyletic groups but that raphid pennates do share a
common ancestor. This pattern has been observed in previous studies that
employed the 18S rRNA gene in determining diatom phylogeny (Kooistra and
Medlin 1996; Medlin et al. 1996; Sorhannus 2007). The same analyses carried
out for diatom psbA sequences show that the two genes share a similar topology,
providing support for the use of psbA as reliable phylogenetic marker to resolve
relationships among diatoms.
Upon neighbor-joining analysis (Fig. 8), it is apparent that diatoms are
primarily amplified from the eukaryotic microalgal assemblage at CFP6 (Fig. 1).
Such a result is not surprising in this high nutrient region. CFP6 is located 7 km
west of the estuary and has depressed nutrient concentrations, indicative of high
biological activity at this site. CFP6 contains high concentrations of nitrate (NO3-)
at 1.36+1.56 μM (Mallin et al. 2005), the form of inorganic nitrogen that often
supports the growth of larger-celled organisms such as diatoms (Franck et al.
2005). Silica, which is vital to diatom growth, shows a strong riverine signal and
while silicate concentrations are higher within the estuary, levels are high enough
to support the growth of diatoms in the surrounding coastal sites with
concentrations at CFP6 at ~5 μM (Mallin et al. 2005).
Because the primer set developed in this study was designed based on
diatom psbA sequences, there likely exists a bias for the preferential
amplification of diatoms from the environment. However, this bias does not
27
appear to preferentially amplify any particular group of diatoms from
environmental samples; all three major morphological groups are represented
from the sample site CFP6. The genetic marker dsF/dsR proves highly useful in
the extraction of diatom sequences from the environment without the inclusion of
homologous cyanobacterial DNA, while also proving accurate for the
establishment of phylogenetic relationships among diatoms for use in studies of
ecology and biodiversity of this globally important group of heterokont algae.
psbA diversity
Substantial allelic diversity of psbA was found in two morphologically
distinct diatom genera. The pennate CMS01 (Cylindrotheca closterium) and
centric CMS09 (Thalassiosira eccentrica) were amplified with psbA and
subsequently cloned to separate alleles. Separate sequence alignments of the
two species show multiple segregating sites within the psbA gene within each
diatom isolate. Sequence chromatograms from the forward and reverse direction
confirm the nucleotide changes; the observed segregating sites are not due to
sequencing error but may arise as a result of PCR bias where Taq polymerase
errors may result in an overestimate in the amount of nucleotide variation.
However, these errors in DNA replication during PCR amplification can be
sufficiently accounted for by the clustering of sequences based on 99% similarity,
as Taq polymerase errors account for about 1% of nucleotide variation (Acinas et
al. 2005). The Cylindrotheca closterium and Thalassiosira eccentrica clones
showed approximately 96.6% and 98.5% sequence similarity, respectively, both
28
outside the range of PCR bias due to Taq polymerase errors. Consequently,
these changes are likely due to mutations that accumulate in the chloroplast
genomes within each diatom cell.
Rarefaction analysis using DOTUR indicates that at 99% and 97%
sequence similarity, the 18S rRNA and psbA genes show the same number of
OTUs. This finding suggests that the variability within these two very different
genes is nearly equal with respect to defining OTUs. However, the Chao1 value
for psbA at the 1% cutoff was three-fold higher than seen for the 3% cutoff. This
high value of gene richness correlates to the high level of allelic diversity found in
the Cylindrotheca and Thalassiosira clone libraries, but does not result it a larger
number of OTUs determined by rarefaction analysis indicating that many of the
sequences are very nearly identical and are thus treated as one taxonomic unit.
Because psbA genes show this high level of variation at the 1% level, either
because of mutational change or Taq polymerase errors, it is advisable to only
include taxa at 99% sequence similarity in phylogenetic and distance analyses
when employing psbA in environmental studies.
Despite these singleton changes in psbA, the alignment analyzed in this
study shows many regions to be highly conserved. Presumably, primary
structure must be maintained to confer full function of the D1 protein. A
substitution that affects primary structure may cause that D1 protein to lose
function. The presence of many more chloroplast genomes and thus psbA copies
within the cell ensures that photosystem II will be provided a functional D1
protein. The nature of chloroplast replication may explain the presence of
29
segregating sites within a single diatom isolate. Unlike the nuclear genome,
chloroplasts divide independently of cellular binary fission (reviewed in
Osteryoung and Nunnari 2003). Multiple chloroplasts can be found within a
single cell. With the separate division of each chloroplast, there likely exists a
higher probability of mutation than for nuclear genomes that are replicated only
upon binary division of the cell.
This finding brings up an interesting question regarding differential rates of
chloroplast genome evolution. Does the presence of more chloroplasts within a
particular diatom species result in faster rates of evolution due to an increase in
mutation pressure? Answering this question would require the measurement of
growth rates in order to determine mutation rates in species with differing
numbers of chloroplasts. Results of such a study may shed light on gene
evolution within the chloroplast genomes of algae, which to date have been
largely underrepresented in the literature.
CONCLUSIONS
The discovery that multiple psbA alleles may be present within diatom
isolates reveals an additional level of genetic diversity that to my knowledge has
not yet been addressed. Such a finding underlies the importance of analyzing
DNA variability at the most resolvable level at which cloning and subsequent
sequencing provides an analysis of distinct alleles of the same gene within a
single isolate. The psbA gene is an excellent molecular marker for the
30
characterization of biodiversity and if applied more broadly to environmental
samples will likely provide a more accurate estimate of microalgal genetic
variation that may also correlate to physical, morphological and ecological
differentiation resulting from natural selection. Key water quality parameters (eg.,
light, nutrients, chlorophyll) can be measured with great precision and accuracy.
These water mass characteristics may very well correlate to specific microalgal
assemblages. In other words, microorganisms may be more confined to spatial
niches than suggested by previous genetic studies. The supplement of a genetic
marker such as psbA that is sensitive to the more cryptic genetic diversity of
planktonic algae may result in a higher resolution analysis of these water masses
with respect to species biodiversity and changes in community structure that
likely correlate to specific environmental parameters.
31
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40
Table 1. Forward and reverse primer matrix tested on CCMP diatom cultures during initial primer development.
dF10
dF11
dF12
dF13
dF14
dF15
dR10
df10/dR10
dF11/dR10
dF12/dR10
dF13/dR10
dF14/dR10
dF15/dR10
dR11
df10/dR11
dF11/dR11
dF12/dR11
dF13/dR11
dF14/dR11
dF15/dR11
dR12
df10/dR12
dF11/dR12
dF12/dR12
dF13/dR12
dF14/dR12
dF15/dR12
41
dR13
df10/dR13
dF11/dR13
dF12/dR13
dF13/dR13
dF14/dR13
dF15/dR13
dR14
df10/dR14
dF11/dR14
dF12/dR14
dF13/dR14
dF14/dR14
dF15/dR14
dR15
df10/dR15
dF11/dR15
dF12/dR15
dF13/dR15
dF14/dR15
dF15/dR15
Table 2. Putative species identifications for CMS diatom cultures based on 18S rRNA gene sequences.
Culture No.
18S rRNA BLASTn ID
Culture No.
18S rRNA BLASTn ID
CMS01
CMS02
CMS03
CMS07
CMS08
CMS09
CMS11
CMS13
CMS14
CMS18
CMS19
CMS20
CMS21
CMS22
CMS24
CMS25
CMS27
Cylindrotheca closterium
Licmopha abreviata
Planktoniella sol
Navicula sp.
Skeletonema grethae
Thalassiosira eccentrica
Navicula sp.
Nitzschia communis
Navicula cryptocephala
Cylindrotheca closterium
Thalassiosira anguste-lineata
Leptocylindrus sp.
Chaetoceros negracile
Thalassiosira nordenskioeldii
Navicula phyllepta
Skeletonema menzelii
Thalassiosira antarctica
CMS29
CMS31
CMS32
CMS33
CMS38
CMS39
CMS40
CMS42
CMS46
CMS47
CMS48
CMS49
CMS50
CMS51
CMS52
CMS54
CMS55
Papiliocellulus elegans
Thalassiosira nordenskioeldii
Papiliocellulus elegans
Skeletonema costatum
Skeletonema tropicum
Navicula sp.
Skeletonema concavivscula
Pleurosigma planktonicum
Skeletonema tropicum
Skeletonema tropicum
Skeletonema tropicum
Skeletonema tropicum
Skeletonema tropicum
Thalassiosira frauenfeldii
Skeletonema tropicum
Cylindrotheca closterium
Skeletonema tropicum
42
Table 3. psbA allelic diversity for CMS01 (Cylindrotheca sp.). Each unique psbA sequence was treated as a unique allele.
Base pair position of the segregating site is included along with the change in nucleotide. Nucleotide changes are
classified as transitions or transversions and changes in amino acid are noted.
Clone No.
Segregating
Site
Nucleotide
Change
Ts/Tr
Amino Acid
Change
CMS01C10
CMS01C02
CMS01C25
CMS01C12
CMS01C18
CMS01C47
CMS01C17
CMS01C14
CMS01C06
CMS01C23
CMS01C19
CMS01C33
CMS01C24
CMS01C10
CMS01C19
CMS01C34
CMS01C13
CMS01C19
CMS01C45
CMS01C02
CMS01C28
CMS01C15
43
69
152
195
219
260
293
302
313
324
347
371
416
431
431
443
465
497
497
504
553
572
T-->C
A-->G
T-->G
T-->C
G-->A
A-->G
C-->T
T-->C
T-->C
C-->T
G-->A
T-->C
A-->G
Deletion of T
T-->C
A-->G
A-->G
A-->G
A-->G
A-->G
C-->T
T-->C
Ts
Ts
Tv
Ts
Ts
Ts
Ts
Ts
Ts
Ts
Ts
Ts
Ts
N/A
Ts
Ts
Ts
Ts
Ts
Ts
Ts
Ts
F-->S
I-->V
None
F-->L
A-->T
None
None
None
M-->T
Q-->Stop
M-->T
None
S-->P
N/A
None
None
N-->D
None
None
N-->D
A-->V
None
43
Table 4. psbA allelic diversity for CMS09 (Thalassiosira sp.). Each unique psbA sequence was treated as a unique allele.
Base pair position of the segregating site is included along with the change in nucleotide. Nucleotide changes are
classified as transitions or transversions and changes in amino acid are noted.
Clone No.
Segregating
Site
Nucleotide
Change
Ts/Tr
Amino Acid
Change
CMS09C10/C11
CMS09C10/C11
CMS09C25
CMS09C37
CMS09C04
CMS09C22
CMS09C37
CMS09C31
CMS09C33
CMS09C05
46
78
79
191
198
224
272
297
340
438
G-->A
T-->C
T-->A
C-->T
G-->A
G-->A
T-->C
G-->A
A-->G
G-->A
Ts
Ts
Tv
Ts
Ts
Ts
Ts
Ts
Ts
Ts
M-->I
V-->A
None
R-->C
W-->Stop
V-->I
S-->P
G-->D
None
S-->N
44
Table 5. Diversity and predicted richness of the 18S rRNA gene and psbA gene fragments from CMS and CCMP diatom
cultures and diatom GenBank sequences as estimated by the Shannon-Weiner diversity index and Chao1 richness
estimator computed using DOTUR for 1% and 3 % differences in nucleic acid sequence alignments. Numbers in
parentheses are lower and upper 95% confidence intervals for the Choa1 estimator.
18S rRNA gene
psbA
No. OTUs
1% cutoff
3% cutoff
24
16
24
16
Chao1
1% cutoff
3% cutoff
41 (29, 80)
31 (19, 80)
75 (39, 198) 25 (18, 56)
45
Shannon-Weiner
1% cutoff
3% cutoff
2.869
2.32
2.573
2.166
?
Wi l m i n g t o n , N C
CMS/ICW
0 1.5 3
Atlantic Ocean
6
9
Miles
12
OB27
CFP6
Fig. 1. Map of southeastern North Carolina. Diatoms were collected from
CMS/ICW (Center for Marine Science/ Intercoastal Waterway). Field primer tests
were conducted at site CFP6 (Cape Fear River Plume). Diatom psbA sequences
obtained from a previous study at OB27 (Onslow Bay) were used in primer
development.
46
A)
C2
C3
C4
C6
C7
C9
C10
C11
C12
(B)
(C)
Fig. 2. Alignment and sequence chromatograms from cloned isolate CMS01
(Cylindrotheca closterium). (A) Alignment of psbA sequences. (B) Clone 4
showing the wildtype psbA allele. (C) Clone 10 showing a deletion of T.
47
(A)
C1
C2
C3
C4
C5
C6
C7
C8
C9
(B)
(C)
Fig. 3. Alignment and sequence chromatograms from cloned isolate CMS09
(Thalassiosira eccentrica). (A) Alignment of psbA sequences. (B) Clone 5
showing the wildtype psbA allele. (C) Clone 4 showing a substitution of A for G.
48
100
100
100
100
100
99
57
69
98
99
100
99
100
99
57
64
100
100
50
51
64
63
72
71
92
87
76
100
100
100
54
raphid
pennate
97
97
Skeletonema tropicum
Skeletonema tropicum
Skeletonema tropicum
Skeletonema tropicum
Skeletonema tropicum
Skeletonema tropicum
Skeletonema tropicum
Skeletonema tropicum
Skeletonema grethae
Skeletonema menzelii
Skeletonema costatum
Thalassiosira nordenskioeldii
Thalassiosira nordenskioeldii
Thalassiosira pseudonana
Thalassiosira concavivscula
Thalassiosira eccentrica
Thalassiosira anguste-lineata
Thalassiosira antarctica
Planktoniella sol
Helicotheca tamesis
Papiliocellulus elegans
Papiliocellulus elegans
Biddulphia sp.
Odontella sinensis
Navicula sp.
Navicula sp.
Navicula sp.
Navicula cyrptocephala
Navicula phyllepta
Pleurosigma planktonicum
Nitzschia communis
Phaeodactylum tricornutum
Cylindrotheca closterium
Cylindrotheca closterium
Cylindrotheca closterium
Licmopha abbreviata
Thalassiosira frauenfeldii
Asterionellopsis gracialis (Black Sea)
Asterionellopsis gracialis (CA, USA)
Leptocylindrus sp.
Melosira octogana
Chaetoceros neogracile
araphid
pennate
87
93
CMS48
CMS52
CMS50
CMS49
CMS38
CMS55
CMS47
CMS46
CMS08
CMS25
CMS33
CMS22
CMS31
Thalassiosira pseudonana
CMS40
CMS09
CMS19
CMS27
CMS03
Helicotheca tamesis
CMS32
CMS29
Biddulphia sp.
Odontella sinensis
CMS11
CMS39
CMS07
CMS14
CMS24
CMS42
CMS13
Phaeodactylum tricornutum
CMS54
CMS01
CMS18
CMS02
CMS51
Asterionellopsis gracialis (Black Sea)
Asterionellopsis gracialis (CA, USA)
CMS20
Melosira octogana
CMS21
Bolidomonas mediterranea
Bolidomonas pacifica
centric
BLASTn ID
centric
18S rRNA
Fig. 4. 18S rRNA maximum parsimony phylogeny of CMS and CCMP diatom cultures and GenBank diatom sequences.
Maximum parsimony bootstrap values are given above the branches and maximum likelihood bootstrap values are given
below. CMS diatom cultures are putatively identified and labeled centric, raphid pennate or araphid pennate according to
18S rRNA BLASTn results (right).
49
18S rRNA BLASTn ID
psbA
61
77
74
62
75
65
85
86
100
100
95
97
86
92
86
94
84
75
98
89
100
100
76
80
66
61
54
71
86
84
Navicula sp.
CMS11
Navicula sp.
CMS39+CMS44
Navicula sp.
CMS07
Navicula phyllepta
CMS24
Navicula cytptocephala
CMS14
CMS06
CMS43
CMS04
Pleurosigma planktonicum
CMS42
Navicula salinicola
Cylindrotheca closterium
CMS01
Cylindrotheca closterium
CMS18
CMS41
Phaeodactylum tricornutum
CMS13
Nitzschia communis
CMS30
Chaetoceros neogracile
CMS21
Skeletonema tropicum
CMS52
CMS35
CMS02
Licmorpha abbreviata
CMS45
Asterionellopsis glacialis
(Black Sea)
Asterionellopsis glacialis
(CA, USA)
Leptocylindrus
sp.
CMS20
Papiliocellulus elegans
CMS32
Papiliocellulus elegans
CMS29
CMS34
Thalassiosira nordenskioeldii
CMS31
Odontella sinensis
Skeletonema tropicum
CMS49
Skeletonema tropicum
CMS55
CMS26
Skeletonema tropicum
CMS38
Thalassiosira frauenfeldii
CMS51
CMS50
Skeletonema tropicum
Skeletonema tropicum
CMS48
CMS10,08,47,46 *
CMS23
Thalassiosira pseudonana
Thalassiosira antarctica
CMS27
Skeletonema menzelii
CMS25
CMS22
Thalassiosira nordenskioeldii
Skeletonema costatum
CMS33
CMS09
Thalassiosira eccentrica
CMS19
Thalassiosira anguste-lineata
CMS53
CMS40
Thalassiosira concavivscula
Biddulphia sp.
Melosira octogona
Helicotheca tamensis
Bolidomonas mediterranea
Bolidomonas pacifica
*CMS 08 Skeletonema grethae
CMS 46,47 Skeletonema tropicum
Fig. 5. psbA maximum parsimony phylogeny of CMS and CCMP diatom cultures
and diatom GenBank sequences. Maximum parsimony bootstrap values are
given above the branches and maximum likelihood bootstrap values are given
below. CMS diatom cultures are putatively identified by 18S rRNA BLASTn
results (right).
50
63
58
88
0.005 substitutions/site
araphid
pennate
raphid pennate
100
raphid pennate
87
centric
araphid
pennate
93
centric
67
CMS11
CMS39+CMS44
CMS07
100 CMS24
67
CMS14
CMS06
100
CMS43
96
CMS01
CMS18
CMS41
Navicula salinicola
60
61
CMS04
CMS42
CMS13
Phaeodactylum tricornutum
CMS30
CMS52
97
61
CMS35
60
CMS02
CMS45
Asterionellopsis
glacialis
(CA,
USA)
98
Asterionellopsis glacialis (Black Sea)
CMS21
CMS20
CMS49
CMS55
CMS48
62 CMS51
CMS26
CMS38
CMS10+CMS08+CMS47+CMS46
81
CMS50
CMS23
Thalassiosira pseudonana
CMS27
CMS53
CMS40
CMS19
64
CMS09
99 CMS25
CMS22
57
CMS33
100 CMS32
81
100
CMS29
100
CMS34
CMS31
Odontella sinensis
Biddulphia sp.
Helicotheca tamesis
Melosira octogona
Bolidomonas mediterranea
Bolidomonas pacifica
0.005 substitutions/site
psbA
centric
18S rRNA
CMS48
CMS52
gene
CMS50
95 CMS46
CMS49
CMS55
CMS38
100
CMS47
96
CMS08
100
CMS33
CMS25
CMS40
100
CMS19
CMS03
53
CMS27
CMS09
97
CMS22
100
67
CMS31
Thalassiosira pseudonana
100 CMS32
CMS29
Biddulphia sp.
Odontella sinensis
Helicotheca tamesis
CMS20
Melosira octogana
CMS21
CMS39
100
CMS07
CMS11
100
100 CMS14
67
CMS24
CMS42
99 CMS54
92
CMS01
CMS18
CMS51
CMS13
Phaeodactylum tricornutum
100 Asterionellopsis gracialis (CA, USA)
Asterionellopsis gracialis (Black Sea)
CMS02
Bolidomonas mediterranea
Bolidomonas pacifica
Fig. 6. 18S rRNA and psbA neighbor-joining phylogenies of CMS and CCMP diatom culture sequences and GenBank
diatom sequences using uncorrected p-distance with supporting bootstrap values above the branches. Classification of
CMS diatom cultures as centric, raphid pennate and araphid pennate diatoms are based on 18S rRNA BLASTn results.
51
Fig. 7. Rarefaction curves for the 41 18S rRNA gene sequences and 50 psbA gene sequences. Twenty-four OTUs were
observed for each gene at the 1% cutoff and 16 were observed at the 3% cutoff.
52
psbA
haplotype
diversity
61
58
99
85
77
84
87
56
100
70
100 CFP6B.60,CFP6B.63
CFP6B.62
CFP6S.8
CFP6S.48
CFP6S.52
CFP6S.47
CFP6S.24
CFP6S.6
CMS51
CFP6B.96
CFP6B.8
CMS26
CMS38
CMS08,CMS10,CMS46,CMS47
CMS48
CMS49
CMS23
CMS50
CMS55
Thalassiosirapseudonana
EF460704
CMS27
CMS33
CFP6S.18
99 CFP6B.42
CFP6B.79
CMS22
CMS25
CFP6B.95
CMS09
CFP6B.43
EF460761
EF460757
EF460759
EF460760
EF460755
EF460762
CMS40
CFP6B.85
85 CFP6S.22
CFP6S.30
100 CFP6S.21
79
CFP6S.4
CFP6S.3
CFP6S.17
CFP6B.86
CFP6S.5
CFP6S.2
CFP6S.32
CMS19
CFP6B.19
CFP6B.36
CFP6B.16
CFP6B.34
83 CMS29
70 CMS32
CFP6B.39
66
CFP6S.20
CMS34
CFP6S.29
CFP6B.110
CFP6B.7
CFP6B.18
96
CMS31
OB27B34
CFP6B.64
Odontellasinensis
CFP6B.51
Helicothecatamensis
CFP6B.44
Biddulphia
sp.
CFP6B.90
CFP6B.93
Melosiraoctogona
CMS53
CFP6B.87
OB27BB22
CMS03
CFP6B.15
CFP6B.46
CFP6B.97
CFP6B.48
CFP6B.105
Bolidomonaspacifica
99
83
Bolidomonas
CFP6B.45
OB27SS51
CFP6B.5
CFP6B.82
CFP6B.59
CFP6S.33
78 CFP6S.34
CFP6S.35
CFP6S.39
CFP6S.43
CFP6S.31
CFP6S.37
CFP6B.56,CFP6S.44,CFP6S.45
CFP6B.89
CFP6B.81
CFP6S.19
CFP6B.78
CFP6S.25
CFP6B.103
CFP6S.49
CFP6B.107
CFP6S.50
CFP6B.106
CFP6S.12
CFP6S.10
CFP6B.88,CFP6B.94,CFP6B.101,CFP6B.108,CFP6B.109
Pterocladiafiliformis
CFP6B.98
CFP6B.99
CFP6B.91
CFP6S.42
CFP6S.36
CFP6B.92
Ahnfeltia plicata
CMS21
CFP6B.37
18S rRNA BLASTn ID
Thalassiosirafrauenfeldi
Skeletonematropicum
CMS08 Skeletonema
Skeletonematropicum
Skeletonematropicum
Skeletonematropicum
Skeletonematropicum
grethae
CMS46,47
Skeletonematropicum
Thalassiosiraantarctica
Skeletonemacostatum
Thalassiosiranordenskioeldi
Skeletonemamenzeli
Thalassiosiraeccentrica
Thalassiosiraconcavivscula
Thalassiosiraanguste-lineata
Papiliocelluluselegans
Papiliocelluluselegans
Thalassiosiranordenskioeldi
Planktoniela
sol
mediterranea
Chaetocerosneogracile
53
centrics
72
89
Cylindrothecaclosterium
Cylindrothecaclosterium
raphid pennate
Navicula sp.
Navicula sp.
Navicula sp.
Naviculacyrptpcephala
Naviculaphyllepta
Pleurosigmaplanktonicum
Nitzschiacommunis
sp.
centric
Leptocylindrus
Skeletonematropicum
Licmophaabbreviata
Stramenopiles
araphid
pennate
araphid
pennate
CFP6B.71
CFP6B.73
CFP6B.74
CFP6B.75
100 CFP6S.1
CFP6S.41
98
CFP6S.40
CFP6S.11
100 CFP6B.100
62 90
CFP6B.104
CFP6B.102
CMS41
96 CMS18
95
CFP6B.22
73
CFP6B.6
CFP6B.47
52
CMS01
CFP6S.51
AY176629
85 CMS11
87
CMS39,CMS44
92
CMS07
100 CMS14
CMS24
CMS06
94
CMS43
100 CFP6B.24
CFP6B.80
100 CFP6B.76
100
CFP6B.77
CFPB6.61
CFP6B.49,CFP6.53
98 CFP6B.54
93
CFP6B.13
CFP6B.14
65
CFP6B.69
93
CMS42
67
100 CFP6B.65
CFP6B.67
CFP6B.58
Naviculasalinicola
79
CMS04
CMS13
95
CFP6B.52
Phaeodactylumtricornutum
CMS30
CFP6B.38
100 CFP6B.41
CFP6B.40
CFP6S.9
81
CFP6S.14
CMS20
63
68 CFP6S.13
CFP6B.9
CFP6S.46
CFP6S.15
OB27SS59
CFP6B.84
OB27B26
Asterionellopsisglacialis
(CA, USA)
98
Asterionellopsisglacialis
(BlackSea)
CMS45
CFP6B.83
99
CMS52
92
CFP6S.38
CMS35
56
CMS02
OB27BB43
Bumileriopsisfiliformis
100
Tribonema aequale
51
Gonyostomum
semen
Fibrocapsa japonica
OB27SS28
Aureococcus
anophagefferens
CFP6B.33
OB27SS62
100 CFP6B.72
83
CFP6S.16
Tetraselmis
sp.
99
Chlorosarcinopsis
gelatinosa
Geminella
terricola
CFP6B.68
Chrysochromulina
polylepis
Pleurochrysiscarterae
65
Chrysotilalamellosa
100 67
Emiliana huxleyi
Phaeocystis
antarctica
Pavlova gyrans
Pyramonas obovata
100
Rhodomonas
sal
ina
74
Protomonas
sulcata
Hemiselmis
andersonii
Porphyridium
aerugineium
89
Cumagloia andersonii
Chlorophyta
Haptophyta
Chlorophyta
Cryptophyta
Rhodophyta
0.005substitutions/site
Fig. 8. Haplotype diversity of all psbA sequences obtained from cultures,
environmental clones and GenBank. Evolutionary distances determined using
neighbor-joining analysis with an uncorrected p-distance. psbA sequences
amplified from unialgal diatom cultures are indicated by bold letters and
putatively identified (right) based on 18S rRNA BLASTn results. Diatoms are
classified as raphid pennate, araphid pennate and centric. Environmental
sequences were processed from the Cape Fear River Plume (CFP) surface (S)
and bottom (B) water samples. The black circle indicates the monophyletic
Phylum Bacillariophyta (diatoms) clade.
54
Appendix 1. Diatom psbA sequences generated with the dsF/dsR primer and analyzed in this study from CMS and CCMP
diatom cultures, CFP6 clone library and GenBank diatom sequences.
Sequence
Clone/
I.D.
Centric/Pennate Origin
Direct
psbA GenBank Acc. No.
Cultured Diatoms
Thalassiosira pseudonana
Odontella sinensis
Phaeodactylum tricornutum
Asterionellopsis glacialis (CA)
A. glacialis (Black Sea)
Biddulphia sp.
Melosira octogana
Helicotheca tamensis
Navicula salinicola
CMS01
CMS02
CMS03
CMS04
CMS06
CMS07
CMS08
CMS09
CMS10
CMS11
CMS13
CMS14
Centric
Centric
Raphid Pennate
Araphid Pennate
Araphid Pennate
Centric
Centric
Centric
Raphid Pennate
GenBank
GenBank
GenBank
CCMP134
CCMP135
CCMP147
CCMP483
CCMP824
CCMP1730
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
55
N/A
N/A
N/A
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
EF067921
Z67753
EF067920
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
CMS18
CMS19
CMS20
CMS21
CMS22
CMS23
CMS24
CMS25
CMS26
CMS27
CMS29
CMS30
CMS31
CMS32
CMS33
CMS34
CMS35
CMS38
CMS39
CMS40
CMS41
CMS42
CMS43
CMS44
CMS45
CMS46
CMS47
CMS48
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
CMS
56
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
CMS49
CMS50
CMS51
CMS52
CMS53
CMS55
Environmental DNA Sequences
OB27B26
OB27B34
OBO27BB22
OB27BB43
OB27SS28
OB27SS51
OB27SS59
OB27SS62
CFP6B.5
CFP6B.6
CFP6B.7
CFP6B.8
CFP6B.9
CFP6B.13
CFP6B.14
CFP6B.15
CFP6B.16
CFP6B.18
CFP6B.19
CFP6B.22
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
CMS
CMS
CMS
CMS
CMS
CMS
D
D
D
D
D
D
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
OB27
OB27
OB27
OB27
OB27
OB27
OB27
OB27
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
57
CFP6B.24
CFP6B.33
CFP6B.34
CFP6B.36
CFP6B.37
CFP6B.38
CFP6B.39
CFP6B.40
CFP6B.41
CFP6B.42
CFP6B.43
CFP6B.44
CFP6B.45
CFP6B.46
CFP6B.47
CFP6B.48
CFP6B.49
CFP6B.51
CFP6B.52
CFP6B.53
CFP6B.54
CFP6B.56
CFP6B.58
CFP6B.59
CFP6B.60
CFP6B.61
CFP6B.62
CFP6B.63
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
58
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
CFP6B.64
CFP6B.65
CFP6B.67
CFP6B.68
CFP6B.69
CFP6B.71
CFP6B.72
CFP6B.73
CFP6B.74
CFP6B.75
CFP6B.76
CFP6B.77
CFP6B.78
CFP6B.79
CFP6B.80
CFP6B.81
CFP6B.82
CFP6B.83
CFP6B.84
CFP6B.85
CFP6B.86
CFP6B.87
CFP6B.88
CFP6B.89
CFP6B.90
CFP6B.91
CFP6B.92
CFP6B.93
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
59
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
CFP6B.94
CFP6B.95
CFP6B.96
CFP6B.97
CFP6B.98
CFP6B.99
CFP6B.100
CFP6B.101
CFP6B.102
CFP6B.103
CFP6B.104
CFP6B.105
CFP6B.106
CFP6B.107
CFP6B.108
CFP6B.109
CFP6B.110
CFP6S.1
CFP6S.2
CFP6S.3
CFP6S.4
CFP6S.5
CFP6S.6
CFP6S.8
CFP6S.9
CFP6S.10
CFP6S.11
CFP6S.12
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
60
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
CFP6S.13
CFP6S.14
CFP6S.15
CFP6S.16
CFP6S.17
CFP6S.18
CFP6S.19
CFP6S.20
CFP6S.21
CFP6S.22
CFP6S.24
CFP6S.25
CFP6S.29
CFP6S.30
CFP6S.31
CFP6S.32
CFP6S.33
CFP6S.34
CFP6S.35
CFP6S.36
CFP6S.37
CFP6S.38
CFP6S.39
CFP6S.40
CFP6S.41
CFP6S.42
CFP6S.43
CFP6S.44
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
61
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
CFP6S.45
CFP6S.46
CFP6S.47
CFP6S.48
CFP6S.49
CFP6S.50
CFP6S.51
CFP6S.52
Uncultured algal clone RED122-4-8
Uncultured isolate JC306
Uncultured isolate JD819
Uncultured isolate JA126
Uncultured isolate JD710
Uncultured isolate JD810
Uncultured isolate JD828
Uncultured isolate JD815
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
CFP6
Zeidner et al. 2003
Jiaozhou Bay, N. China
Jiaozhou Bay, N. China
Jiaozhou Bay, N. China
Jiaozhou Bay, N. China
Jiaozhou Bay, N. China
Jiaozhou Bay, N. China
Jiaozhou Bay, N. China
62
C
C
C
C
C
C
C
C
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
XXXXXXXXXXX
AY176629
EF460704
EF460755
EF460757
EF460759
EF460760
EF460761
EF460762
Appendix 2. Other photosynthetic eukaryote psbA sequences obtained from
GenBank and pure cultures from culture collections analyzed in this study for
analysis of primer specificity outside Phylum Bacillariophyta (diatoms).
Taxon
Strain
psbA GenBank No.
Bolidophyceae
Bolidomonas mediterranea
Bolidomonas pacifica
CCMP1867
CCMP1866
XXXXXXXX
XXXXXXXX
Chlorophyta
Chlorosarcinopsis gelatinosa
Geminella terricola
CCMP1511
UTEX2540
XXXXXXXX
XXXXXXXX
Chrysomerophyceae
Chrysowaernella hieroglyphica
SCCAP K-0368
XXXXXXXX
Chrysophyceae
Krematstochrysopsis sp.
Ochromonas sphaerocystis
CCMP260
CCMP586
XXXXXXXX
XXXXXXXX
Cryptophyceae
Hemiselmis andersenii
Proteomonas sulcata
Rhodomonas salina
CCMP644
CCMP704
CCMP1319
XXXXXXXX
XXXXXXXX
XXXXXXXX
Eustigmatophyceae
Goniochloris sculpta
Unidentified coccoid sp.
SAG29.96
N/A
XXXXXXXX
XXXXXXXX
Dinophyceae
Amphidinium gibbosum
Symbiodinum sp.1
CCMP120
N/A
XXXXXXXX
XXXXXXXX
Pelagophyceae
Aureococcus anophagefferens
CCMP1707
XXXXXXXX
Phaeophyceae
Laminaria sp.
N/A
XXXXXXXX
Pinguiophyceae
Pinguiochrysis pyriformis
MBIC 10872
XXXXXXXX
Prasinophyceae
Pyramimonas obovata
CCMP722
XXXXXXXX
63
Tetraselmis sp.
CCMP908
XXXXXXXX
Prymnesiophyceae
Chrysochromulina polylepis
Chrysotila lamellosa
Emiliania huxleyi
Pavlova gyrans
Phaeocystis antarctica
Pluerochrysis carterae
N/A
CCMP1307
PCC 1516
N/A
N/A
N/A
AJ575572
XXXXXXX
XXXXXXXX
AJ575575
AY119756
AY119757
Rhodophyta
Ahnfeltia plicata
Cumagloia andersonii
Porphyridium aerugineum
Pterocladia lucida
N/A
UTEX1694
CCMP674
N/A
XXXXXXXX
XXXXXXXX
XXXXXXXX
XXXXXXXX
Xanthophyceae
Bumilleriopsis filiformis
Chloridella miniata
Chlorocloster engadinensis
Sphaerosorus compositus
Tribonema aequale
Tribonema vulgare
N/A
UTEX491
UTEX307
CCAP876/1
N/A
SCCAPK-0339
X79223
XXXXXXXX
XXXXXXXX
XXXXXXXX
AY528860
XXXXXXXX
Rhaphidopyceae
Fibrocapsa japonica
Gonyostomum semen
1
ex. Siderastrea sp.
64