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. Lawrence B. Cahoon

Dr. Lynn Leonard

Dr. J. Craig Bailey

Chair

Accepted by

___________________________ 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 high-

performance 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. Culture-

independent 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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39

40

Zeidner, G, Preston, CM, Delong, EF, Massana, R, Post, AF, Scanlan, DJ & Béjà, O. 2003. Molecular diversity among marine picophytoplankton as revealed by psbA analysis. Env. Microbiol. 5: 212-216.

Table 1. Forward and reverse primer matrix tested on CCMP diatom cultures during initial primer development.

dR10 dR11 dR12 dR13 dR14 dR15 dF10 df10/dR10 df10/dR11 df10/dR12 df10/dR13 df10/dR14 df10/dR15 dF11 dF11/dR10 dF11/dR11 dF11/dR12 dF11/dR13 dF11/dR14 dF11/dR15 dF12 dF12/dR10 dF12/dR11 dF12/dR12 dF12/dR13 dF12/dR14 dF12/dR15 dF13 dF13/dR10 dF13/dR11 dF13/dR12 dF13/dR13 dF13/dR14 dF13/dR15 dF14 dF14/dR10 dF14/dR11 dF14/dR12 dF14/dR13 dF14/dR14 dF14/dR15 dF15 dF15/dR10 dF15/dR11 dF15/dR12 dF15/dR13 dF15/dR14 dF15/dR15

41

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 Cylindrotheca closterium CMS29 Papiliocellulus elegans CMS02 Licmopha abreviata CMS31 Thalassiosira nordenskioeldii CMS03 Planktoniella sol CMS32 Papiliocellulus elegans CMS07 Navicula sp. CMS33 Skeletonema costatum CMS08 Skeletonema grethae CMS38 Skeletonema tropicum CMS09 Thalassiosira eccentrica CMS39 Navicula sp. CMS11 Navicula sp. CMS40 Skeletonema concavivscula CMS13 Nitzschia communis CMS42 Pleurosigma planktonicum CMS14 Navicula cryptocephala CMS46 Skeletonema tropicum CMS18 Cylindrotheca closterium CMS47 Skeletonema tropicum CMS19 Thalassiosira anguste-lineata CMS48 Skeletonema tropicum CMS20 Leptocylindrus sp. CMS49 Skeletonema tropicum CMS21 Chaetoceros negracile CMS50 Skeletonema tropicum CMS22 Thalassiosira nordenskioeldii CMS51 Thalassiosira frauenfeldii CMS24 Navicula phyllepta CMS52 Skeletonema tropicum CMS25 Skeletonema menzelii CMS54 Cylindrotheca closterium CMS27 Thalassiosira antarctica CMS55 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 43 T-->C Ts F-->S CMS01C02 69 A-->G Ts I-->V CMS01C25 152 T-->G Tv None CMS01C12 195 T-->C Ts F-->L CMS01C18 219 G-->A Ts A-->T CMS01C47 260 A-->G Ts None CMS01C17 293 C-->T Ts None CMS01C14 302 T-->C Ts None CMS01C06 313 T-->C Ts M-->T CMS01C23 324 C-->T Ts Q-->Stop CMS01C19 347 G-->A Ts M-->T CMS01C33 371 T-->C Ts None CMS01C24 416 A-->G Ts S-->P CMS01C10 431 Deletion of T N/A N/A CMS01C19 431 T-->C Ts None CMS01C34 443 A-->G Ts None CMS01C13 465 A-->G Ts N-->D CMS01C19 497 A-->G Ts None CMS01C45 497 A-->G Ts None CMS01C02 504 A-->G Ts N-->D CMS01C28 553 C-->T Ts A-->V CMS01C15 572 T-->C Ts 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 46 G-->A Ts M-->I CMS09C10/C11 78 T-->C Ts V-->A

CMS09C25 79 T-->A Tv None CMS09C37 191 C-->T Ts R-->C CMS09C04 198 G-->A Ts W-->Stop CMS09C22 224 G-->A Ts V-->I CMS09C37 272 T-->C Ts S-->P CMS09C31 297 G-->A Ts G-->D CMS09C33 340 A-->G Ts None CMS09C05 438 G-->A Ts S-->N

44

45

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.

No. OTUs Chao1 Shannon-Weiner1% cutoff 3% cutoff 1% cutoff 3% cutoff 1% cutoff 3% cutoff

18S rRNA gene 24 16 41 (29, 80) 31 (19, 80) 2.869 2.32psbA 24 16 75 (39, 198) 25 (18, 56) 2.573 2.166

Wi l m i n g t o n , N C

CFP6

OB27

CMS/ICW

A t l a n t i c O c e a n

?0 3 6 9 121.5

Miles

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

48

C1 C2 C3 C4 C5 C6 C7 C8 C9

(A) (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.

49

CMS48CMS52CMS50CMS49CMS38CMS55CMS47CMS46CMS08CMS25CMS33CMS22CMS31Thalassiosira pseudonanaCMS40CMS09CMS19CMS27CMS03Helicotheca tamesisCMS32CMS29Biddulphia sp.Odontella sinensisCMS11CMS39CMS07CMS14CMS24CMS42CMS13Phaeodactylum tricornutumCMS54CMS01CMS18CMS02CMS51Asterionellopsis gracialis (Black Sea)Asterionellopsis gracialis (CA, USA)CMS20Melosira octoganaCMS21Bolidomonas mediterraneaBolidomonas pacifica

18S rRNA BLASTn ID

centr

icra

phid

penn

atear

aphi

dpe

nnate

centr

ic

87

97

100

100100

57

100

50

64 100

100 98

92 72

100 100

76

54

100

93

97

100

69

99

51

100

99

63 99

99

87 71

57

100

64

Skeletonema tropicum

Skeletonema tropicumSkeletonema grethae

Thalassiosira nordenskioeldiiThalassiosira pseudonana

Thalassiosira anguste-lineataThalassiosira antarcticaPlanktoniella solHelicotheca tamesisPapiliocellulus elegansPapiliocellulus elegansBiddulphia sp.Odontella sinensis

Skeletonema tropicumSkeletonema tropicumSkeletonema tropicumSkeletonema tropicumSkeletonema tropicum

Skeletonema tropicum

Skeletonema menzeliiSkeletonema costatumThalassiosira nordenskioeldii

Thalassiosira concavivsculaThalassiosira eccentrica

Navicula sp.Navicula sp.Navicula sp.Navicula cyrptocephalaNavicula phylleptaPleurosigma planktonicumNitzschia communisPhaeodactylum tricornutumCylindrotheca closteriumCylindrotheca closteriumCylindrotheca closteriumLicmopha abbreviataThalassiosira frauenfeldiiAsterionellopsis gracialis (Black Sea)Asterionellopsis gracialis (CA, USA)Leptocylindrus sp.Melosira octoganaChaetoceros neogracile

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).

50

858675

65746261

77

1001009597

86 92

86 94

84 75

76 80 98

89

66 61

86 84 54

71

CMS11CMS39+CMS44CMS07CMS24CMS14CMS06CMS43CMS04CMS42Navicula salinicolaCMS01CMS18CMS41Phaeodactylum tricornutumCMS13CMS30CMS21CMS52CMS35CMS02CMS45

100100

Asterionellopsis glacialis (Black Sea)Asterionellopsis glacialis (CA, USA)CMS20CMS32CMS29CMS34CMS31Odontella sinensisCMS49CMS55CMS26CMS38CMS51CMS50CMS48CMS10,08,47,46 *CMS23Thalassiosira pseudonanaCMS27CMS25CMS22CMS33CMS09CMS19CMS53CMS40Biddulphia sp. Melosira octogonaHelicotheca tamensisBolidomonas mediterraneaBolidomonas pacifica

psbA 18S rRNA BLASTn ID

Navicula sp. Navicula sp.Navicula sp.Navicula phylleptaNavicula cytptocephala

Pleurosigma planktonicum

Cylindrotheca closteriumCylindrotheca closterium

Nitzschia communis

Chaetoceros neogracileSkeletonema tropicum

Licmorpha abbreviata

*CMS 08 Skeletonema grethae CMS 46,47 Skeletonema tropicum

Leptocylindrus sp.Papiliocellulus elegansPapiliocellulus elegans

Thalassiosira nordenskioeldii

Skeletonema tropicumSkeletonema tropicum

Skeletonema tropicumThalassiosira frauenfeldiiSkeletonema tropicumSkeletonema tropicum

Thalassiosira antarcticaSkeletonema menzeliiThalassiosira nordenskioeldiiSkeletonema costatumThalassiosira eccentricaThalassiosira anguste-lineata

Thalassiosira concavivscula

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).

CMS48CMS52CMS50CMS46CMS49CMS55CMS38CMS47

CMS08CMS33

CMS25CMS40

CMS19CMS03CMS27

CMS09CMS22

CMS31Thalassiosira pseudonana

CMS32CMS29

Biddulphia sp.Odontella sinensis

Helicotheca tamesisCMS20

Melosira octoganaCMS21

CMS39CMS07

CMS11CMS14CMS24

CMS42CMS54

CMS01CMS18

CMS51CMS13

Phaeodactylum tricornutumAsterionellopsis gracialis (CA, USA)Asterionellopsis gracialis (Black Sea)

CMS02Bolidomonas mediterranea

Bolidomonas pacifica0.005 substitutions/site

psbA

raph

id p

enna

tear

aphid

pe

nnate

centr

ic

centr

ic

raph

id p

enna

tear

aphid

pe

nnate

centric

CMS11CMS39+CMS44

CMS07CMS24CMS14

CMS06CMS43

CMS01CMS18

CMS41Navicula salinicola

CMS04CMS42

CMS13Phaeodactylum tricornutum

CMS30CMS52

CMS35CMS02

CMS45Asterionellopsis glacialis (CA, USA)

Asterionellopsis glacialis (Black Sea)CMS21CMS20

CMS49CMS55CMS48CMS51CMS26CMS38CMS10+CMS08+CMS47+CMS46

CMS50CMS23

Thalassiosira pseudonanaCMS27

CMS53CMS40CMS19

CMS09CMS25CMS22

CMS33CMS32CMS29

CMS34CMS31

Odontella sinensisBiddulphia sp.

Helicotheca tamesisMelosira octogona

Bolidomonas mediterraneaBolidomonas pacifica

0.005 substitutions/site

18S rRNA gene95

10096

100

53

10067

100

63

58

100

67

100

100100

67

9992

10088

100

97

62

81

64

99

5710081

100100

98

9761

60

8793

100

100

96

67

6061

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

52

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.

CFP6B.60, CFP6B.63CFP6B.62

CFP6S.8CFP6S.48CFP6S.52

CFP6S.47CFP6S.24CFP6S.6CMS51CFP6B.96

CFP6B.8CMS26CMS38CMS 08, CMS10, CMS46, CMS47CMS48CMS49

CMS23CMS50

CMS55Thalassiosira pseudonanaEF460704CMS27CMS33CFP6S.18

CFP6B.42CFP6B.79

CMS22CMS25CFP6B.95

CMS09CFP6B.43

EF460761EF460757

EF460759EF460760EF460755

EF460762CMS40CFP6B.85

CFP6S.22CFP6S.30CFP6S.21

CFP6S.4CFP6S.3

CFP6S.17CFP6B.86

CFP6S.5CFP6S.2

CFP6S.32CMS19CFP6B.19

CFP6B.36CFP6B.16CFP6B.34

CMS29CMS32

CFP6B.39CFP6S.20

CMS34CFP6S.29

CFP6B.110CFP6B.7CFP6B.18CMS31

OB27B34CFP6B.64

Odontella sinensisCFP6B.51Helicotheca tamensis

CFP6B.44Biddulphia sp.

CFP6B.90CFP6B.93

Melosira octogonaCMS53

CFP6B.87OB27BB22

CMS03CFP6B.15CFP6B.46

CFP6B.97CFP6B.48

CFP6B.105Bolidomonas pacifica

Bolidomonas mediterraneaCFP6B.45

OB27SS51CFP6B.5

CFP6B.82CFP6B.59

CFP6S.33CFP6S.34CFP6S.35

CFP6S.39CFP6S.43

CFP6S.31CFP6S.37CFP6B.56, CFP6S.44, CFP6S.45CFP6B.89CFP6B.81

CFP6S.19CFP6B.78CFP6S.25CFP6B.103CFP6S.49CFP6B.107CFP6S.50CFP6B.106

CFP6S.12CFP6S.10CFP6B.88, CFP6B.94, CFP6B.101, CFP6B.108, CFP6B.109Pterocladia filiformisCFP6B.98CFP6B.99CFP6B.91CFP6S.42CFP6S.36CFP6B.92

Ahnfeltia plicataCMS21

CFP6B.37

Skeletonema tropicumSkeletonema tropicum

Skeletonema tropicum

Thalassiosira antarctica

Skeletonema menzelii

CMS 08 Skeletonema grethae CMS 46,47 Skeletonema tropicum

Skeletonema tropicum

Thalassiosira frauenfeldii

Skeletonema costatum

Thalassiosira eccentrica

Thalassiosira nordenskioeldii

Thalassiosira concavivscula

Thalassiosira anguste-lineata

Papilliocellulus elegansPapilliocellulus elegans

Thalassiosira nordenskioeldii

Planktoniella sol

Chaetoceros neogracile

Skeletonema tropicum

18S rRNA BLASTn IDpsbA haplotype diversity

centr

ics

8370

66

96

99

9958

85100

79

77

70

87

61

9983

78

56

85

84

100

100

72

53

54

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.

CFP6B.71CFP6B.73CFP6B.74CFP6B.75

CFP6S.1CFP6S.41CFP6S.40

CFP6S.11CFP6B.100CFP6B.104

CFP6B.102CMS41

CMS18CFP6B.22

CFP6B.6CFP6B.47

CMS01CFP6S.51AY176629

CMS11CMS 39, CMS 44CMS07

CMS14CMS24

CMS06CMS43

CFP6B.24CFP6B.80

CFP6B.76CFP6B.77

CFPB6.61CFP6B.49, CFP6.53CFP6B.54CFP6B.13

CFP6B.14CFP6B.69

CMS42CFP6B.65CFP6B.67

Navicula sp.

Navicula sp.

Navicula phyllepta

Cylindrotheca closterium

Cylindrotheca closterium

Navicula sp.

Navicula cyrptpcephala

Pleurosigma planktonicum

CFP6B.58Navicula salinicola

CMS04CMS13

CFP6B.52Phaeodactylum tricornutum

CMS30CFP6B.38CFP6B.41CFP6B.40

CFP6S.9CFP6S.14CMS20CFP6S.13CFP6B.9CFP6S.46

CFP6S.15OB27SS59

CFP6B.84OB27B26

Asterionellopsis glacialis (CA, USA)Asterionellopsis glacialis (Black Sea)

CMS45CFP6B.83

CMS52CFP6S.38

CMS35CMS02

OB27BB43Bumilleriopsis filiformis

Tribonema aequaleGonyostomum semen

Fibrocapsa japonicaOB27SS28

Aureococcus anophagefferensCFP6B.33

OB27SS62CFP6B.72CFP6S.16

Tetraselmis sp.Chlorosarcinopsis gelatinosa

Geminella terricolaCFP6B.68

Chrysochromulina polylepisPleurochrysis carterae

Chrysotila lamellosaEmiliana huxleyi

Phaeocystis antarcticaPavlova gyrans

Pyramonas obovataRhodomonas salina

Protomonas sulcataHemiselmis andersonii

Porphyridium aerugineiumCumagloia andersonii

0.005 substitutions/site

Nitzschia communis

Leptocylindrus sp.

Skeletonema tropicum

Licmopha abbreviata

raph

id p

enna

tece

ntric

arap

hid

penn

ate

arap

hid

penn

ate

Rhodophyta

Cryptophyta

Haptophyta

Chlorophyta

Stramenopiles

Chlorophyta

81

6368

10083

99

6765

100

10074

8989

9992

56

100

98

7995

10051

10098

1009062

9893

65

10093

67

969573

52

8587

10092

100100

94100

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

I.D. Centric/Pennate Origin Clone/ Direct psbA GenBank Acc. No.

Cultured Diatoms Thalassiosira pseudonana Centric GenBank N/A EF067921 Odontella sinensis Centric GenBank N/A Z67753 Phaeodactylum tricornutum Raphid Pennate GenBank N/A EF067920 Asterionellopsis glacialis (CA) Araphid Pennate CCMP134 D XXXXXXXXXXX A. glacialis (Black Sea) Araphid Pennate CCMP135 D XXXXXXXXXXX Biddulphia sp. Centric CCMP147 D XXXXXXXXXXX Melosira octogana Centric CCMP483 D XXXXXXXXXXX Helicotheca tamensis Centric CCMP824 D XXXXXXXXXXX Navicula salinicola Raphid Pennate CCMP1730 D XXXXXXXXXXX CMS01 CMS D XXXXXXXXXXX CMS02 CMS D XXXXXXXXXXX CMS03 CMS D XXXXXXXXXXX CMS04 CMS D XXXXXXXXXXX CMS06 CMS D XXXXXXXXXXX CMS07 CMS D XXXXXXXXXXX CMS08 CMS D XXXXXXXXXXX CMS09 CMS D XXXXXXXXXXX CMS10 CMS D XXXXXXXXXXX CMS11 CMS D XXXXXXXXXXX CMS13 CMS D XXXXXXXXXXX CMS14 CMS D XXXXXXXXXXX

55

CMS18 CMS D XXXXXXXXXXX CMS19 CMS D XXXXXXXXXXX CMS20 CMS D XXXXXXXXXXX CMS21 CMS D XXXXXXXXXXX CMS22 CMS D XXXXXXXXXXX CMS23 CMS D XXXXXXXXXXX CMS24 CMS D XXXXXXXXXXX CMS25 CMS D XXXXXXXXXXX CMS26 CMS D XXXXXXXXXXX CMS27 CMS D XXXXXXXXXXX CMS29 CMS D XXXXXXXXXXX CMS30 CMS D XXXXXXXXXXX CMS31 CMS D XXXXXXXXXXX CMS32 CMS D XXXXXXXXXXX CMS33 CMS D XXXXXXXXXXX CMS34 CMS D XXXXXXXXXXX CMS35 CMS D XXXXXXXXXXX CMS38 CMS D XXXXXXXXXXX CMS39 CMS D XXXXXXXXXXX CMS40 CMS D XXXXXXXXXXX CMS41 CMS D XXXXXXXXXXX CMS42 CMS D XXXXXXXXXXX CMS43 CMS D XXXXXXXXXXX CMS44 CMS D XXXXXXXXXXX CMS45 CMS D XXXXXXXXXXX CMS46 CMS D XXXXXXXXXXX CMS47 CMS D XXXXXXXXXXX CMS48 CMS D XXXXXXXXXXX

56

CMS49 CMS D XXXXXXXXXXX CMS50 CMS D XXXXXXXXXXX CMS51 CMS D XXXXXXXXXXX CMS52 CMS D XXXXXXXXXXX CMS53 CMS D XXXXXXXXXXX CMS55 CMS D XXXXXXXXXXX Environmental DNA Sequences OB27B26 N/A OB27 C XXXXXXXXXXX OB27B34 N/A OB27 C XXXXXXXXXXX OBO27BB22 N/A OB27 C XXXXXXXXXXX OB27BB43 N/A OB27 C XXXXXXXXXXX OB27SS28 N/A OB27 C XXXXXXXXXXX OB27SS51 N/A OB27 C XXXXXXXXXXX OB27SS59 N/A OB27 C XXXXXXXXXXX OB27SS62 N/A OB27 C XXXXXXXXXXX CFP6B.5 N/A CFP6 C XXXXXXXXXXX CFP6B.6 N/A CFP6 C XXXXXXXXXXX CFP6B.7 N/A CFP6 C XXXXXXXXXXX CFP6B.8 N/A CFP6 C XXXXXXXXXXX CFP6B.9 N/A CFP6 C XXXXXXXXXXX CFP6B.13 N/A CFP6 C XXXXXXXXXXX CFP6B.14 N/A CFP6 C XXXXXXXXXXX CFP6B.15 N/A CFP6 C XXXXXXXXXXX CFP6B.16 N/A CFP6 C XXXXXXXXXXX CFP6B.18 N/A CFP6 C XXXXXXXXXXX CFP6B.19 N/A CFP6 C XXXXXXXXXXX CFP6B.22 N/A CFP6 C XXXXXXXXXXX

57

CFP6B.24 N/A CFP6 C XXXXXXXXXXX CFP6B.33 N/A CFP6 C XXXXXXXXXXX CFP6B.34 N/A CFP6 C XXXXXXXXXXX CFP6B.36 N/A CFP6 C XXXXXXXXXXX CFP6B.37 N/A CFP6 C XXXXXXXXXXX CFP6B.38 N/A CFP6 C XXXXXXXXXXX CFP6B.39 N/A CFP6 C XXXXXXXXXXX CFP6B.40 N/A CFP6 C XXXXXXXXXXX CFP6B.41 N/A CFP6 C XXXXXXXXXXX CFP6B.42 N/A CFP6 C XXXXXXXXXXX CFP6B.43 N/A CFP6 C XXXXXXXXXXX CFP6B.44 N/A CFP6 C XXXXXXXXXXX CFP6B.45 N/A CFP6 C XXXXXXXXXXX CFP6B.46 N/A CFP6 C XXXXXXXXXXX CFP6B.47 N/A CFP6 C XXXXXXXXXXX CFP6B.48 N/A CFP6 C XXXXXXXXXXX CFP6B.49 N/A CFP6 C XXXXXXXXXXX CFP6B.51 N/A CFP6 C XXXXXXXXXXX CFP6B.52 N/A CFP6 C XXXXXXXXXXX CFP6B.53 N/A CFP6 C XXXXXXXXXXX CFP6B.54 N/A CFP6 C XXXXXXXXXXX CFP6B.56 N/A CFP6 C XXXXXXXXXXX CFP6B.58 N/A CFP6 C XXXXXXXXXXX CFP6B.59 N/A CFP6 C XXXXXXXXXXX CFP6B.60 N/A CFP6 C XXXXXXXXXXX CFP6B.61 N/A CFP6 C XXXXXXXXXXX CFP6B.62 N/A CFP6 C XXXXXXXXXXX CFP6B.63 N/A CFP6 C XXXXXXXXXXX

58

CFP6B.64 N/A CFP6 C XXXXXXXXXXX CFP6B.65 N/A CFP6 C XXXXXXXXXXX CFP6B.67 N/A CFP6 C XXXXXXXXXXX CFP6B.68 N/A CFP6 C XXXXXXXXXXX CFP6B.69 N/A CFP6 C XXXXXXXXXXX CFP6B.71 N/A CFP6 C XXXXXXXXXXX CFP6B.72 N/A CFP6 C XXXXXXXXXXX CFP6B.73 N/A CFP6 C XXXXXXXXXXX CFP6B.74 N/A CFP6 C XXXXXXXXXXX CFP6B.75 N/A CFP6 C XXXXXXXXXXX CFP6B.76 N/A CFP6 C XXXXXXXXXXX CFP6B.77 N/A CFP6 C XXXXXXXXXXX CFP6B.78 N/A CFP6 C XXXXXXXXXXX CFP6B.79 N/A CFP6 C XXXXXXXXXXX CFP6B.80 N/A CFP6 C XXXXXXXXXXX CFP6B.81 N/A CFP6 C XXXXXXXXXXX CFP6B.82 N/A CFP6 C XXXXXXXXXXX CFP6B.83 N/A CFP6 C XXXXXXXXXXX CFP6B.84 N/A CFP6 C XXXXXXXXXXX CFP6B.85 N/A CFP6 C XXXXXXXXXXX CFP6B.86 N/A CFP6 C XXXXXXXXXXX CFP6B.87 N/A CFP6 C XXXXXXXXXXX CFP6B.88 N/A CFP6 C XXXXXXXXXXX CFP6B.89 N/A CFP6 C XXXXXXXXXXX CFP6B.90 N/A CFP6 C XXXXXXXXXXX CFP6B.91 N/A CFP6 C XXXXXXXXXXX CFP6B.92 N/A CFP6 C XXXXXXXXXXX CFP6B.93 N/A CFP6 C XXXXXXXXXXX

59

CFP6B.94 N/A CFP6 C XXXXXXXXXXX CFP6B.95 N/A CFP6 C XXXXXXXXXXX CFP6B.96 N/A CFP6 C XXXXXXXXXXX CFP6B.97 N/A CFP6 C XXXXXXXXXXX CFP6B.98 N/A CFP6 C XXXXXXXXXXX CFP6B.99 N/A CFP6 C XXXXXXXXXXX CFP6B.100 N/A CFP6 C XXXXXXXXXXX CFP6B.101 N/A CFP6 C XXXXXXXXXXX CFP6B.102 N/A CFP6 C XXXXXXXXXXX CFP6B.103 N/A CFP6 C XXXXXXXXXXX CFP6B.104 N/A CFP6 C XXXXXXXXXXX CFP6B.105 N/A CFP6 C XXXXXXXXXXX CFP6B.106 N/A CFP6 C XXXXXXXXXXX CFP6B.107 N/A CFP6 C XXXXXXXXXXX CFP6B.108 N/A CFP6 C XXXXXXXXXXX CFP6B.109 N/A CFP6 C XXXXXXXXXXX CFP6B.110 N/A CFP6 C XXXXXXXXXXX CFP6S.1 N/A CFP6 C XXXXXXXXXXX CFP6S.2 N/A CFP6 C XXXXXXXXXXX CFP6S.3 N/A CFP6 C XXXXXXXXXXX CFP6S.4 N/A CFP6 C XXXXXXXXXXX CFP6S.5 N/A CFP6 C XXXXXXXXXXX CFP6S.6 N/A CFP6 C XXXXXXXXXXX CFP6S.8 N/A CFP6 C XXXXXXXXXXX CFP6S.9 N/A CFP6 C XXXXXXXXXXX CFP6S.10 N/A CFP6 C XXXXXXXXXXX CFP6S.11 N/A CFP6 C XXXXXXXXXXX CFP6S.12 N/A CFP6 C XXXXXXXXXXX

60

CFP6S.13 N/A CFP6 C XXXXXXXXXXX CFP6S.14 N/A CFP6 C XXXXXXXXXXX CFP6S.15 N/A CFP6 C XXXXXXXXXXX CFP6S.16 N/A CFP6 C XXXXXXXXXXX CFP6S.17 N/A CFP6 C XXXXXXXXXXX CFP6S.18 N/A CFP6 C XXXXXXXXXXX CFP6S.19 N/A CFP6 C XXXXXXXXXXX CFP6S.20 N/A CFP6 C XXXXXXXXXXX CFP6S.21 N/A CFP6 C XXXXXXXXXXX CFP6S.22 N/A CFP6 C XXXXXXXXXXX CFP6S.24 N/A CFP6 C XXXXXXXXXXX CFP6S.25 N/A CFP6 C XXXXXXXXXXX CFP6S.29 N/A CFP6 C XXXXXXXXXXX CFP6S.30 N/A CFP6 C XXXXXXXXXXX CFP6S.31 N/A CFP6 C XXXXXXXXXXX CFP6S.32 N/A CFP6 C XXXXXXXXXXX CFP6S.33 N/A CFP6 C XXXXXXXXXXX CFP6S.34 N/A CFP6 C XXXXXXXXXXX CFP6S.35 N/A CFP6 C XXXXXXXXXXX CFP6S.36 N/A CFP6 C XXXXXXXXXXX CFP6S.37 N/A CFP6 C XXXXXXXXXXX CFP6S.38 N/A CFP6 C XXXXXXXXXXX CFP6S.39 N/A CFP6 C XXXXXXXXXXX CFP6S.40 N/A CFP6 C XXXXXXXXXXX CFP6S.41 N/A CFP6 C XXXXXXXXXXX CFP6S.42 N/A CFP6 C XXXXXXXXXXX CFP6S.43 N/A CFP6 C XXXXXXXXXXX CFP6S.44 N/A CFP6 C XXXXXXXXXXX

61

62

CFP6S.45 N/A CFP6 C XXXXXXXXXXX CFP6S.46 N/A CFP6 C XXXXXXXXXXX CFP6S.47 N/A CFP6 C XXXXXXXXXXX CFP6S.48 N/A CFP6 C XXXXXXXXXXX CFP6S.49 N/A CFP6 C XXXXXXXXXXX CFP6S.50 N/A CFP6 C XXXXXXXXXXX CFP6S.51 N/A CFP6 C XXXXXXXXXXX CFP6S.52 N/A CFP6 C XXXXXXXXXXX Uncultured algal clone RED122-4-8 N/A Zeidner et al. 2003 N/A AY176629 Uncultured isolate JC306 N/A Jiaozhou Bay, N. China N/A EF460704 Uncultured isolate JD819 N/A Jiaozhou Bay, N. China N/A EF460755 Uncultured isolate JA126 N/A Jiaozhou Bay, N. China N/A EF460757 Uncultured isolate JD710 N/A Jiaozhou Bay, N. China N/A EF460759 Uncultured isolate JD810 N/A Jiaozhou Bay, N. China N/A EF460760 Uncultured isolate JD828 N/A Jiaozhou Bay, N. China N/A EF460761 Uncultured isolate JD815 N/A Jiaozhou Bay, N. China N/A 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 CCMP1867 XXXXXXXX Bolidomonas pacifica CCMP1866 XXXXXXXX

Chlorophyta Chlorosarcinopsis gelatinosa CCMP1511 XXXXXXXX Geminella terricola UTEX2540 XXXXXXXX

Chrysomerophyceae Chrysowaernella hieroglyphica SCCAP K-0368 XXXXXXXX

Chrysophyceae Krematstochrysopsis sp. CCMP260 XXXXXXXX Ochromonas sphaerocystis CCMP586 XXXXXXXX

Cryptophyceae Hemiselmis andersenii CCMP644 XXXXXXXX Proteomonas sulcata CCMP704 XXXXXXXX Rhodomonas salina CCMP1319 XXXXXXXX

Eustigmatophyceae Goniochloris sculpta SAG29.96 XXXXXXXX Unidentified coccoid sp. N/A XXXXXXXX

Dinophyceae Amphidinium gibbosum CCMP120 XXXXXXXX Symbiodinum sp.1 N/A 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 N/A AJ575572 Chrysotila lamellosa CCMP1307 XXXXXXX Emiliania huxleyi PCC 1516 XXXXXXXX Pavlova gyrans N/A AJ575575 Phaeocystis antarctica N/A AY119756 Pluerochrysis carterae N/A AY119757 Rhaphidopyceae Fibrocapsa japonica Gonyostomum semen Rhodophyta Ahnfeltia plicata N/A XXXXXXXX Cumagloia andersonii UTEX1694 XXXXXXXX Porphyridium aerugineum CCMP674 XXXXXXXX Pterocladia lucida N/A XXXXXXXX

Xanthophyceae Bumilleriopsis filiformis N/A X79223 Chloridella miniata UTEX491 XXXXXXXX Chlorocloster engadinensis UTEX307 XXXXXXXX Sphaerosorus compositus CCAP876/1 XXXXXXXX Tribonema aequale N/A AY528860 Tribonema vulgare SCCAPK-0339 XXXXXXXX 1 ex. Siderastrea sp.

64