J. Phycol.

43, 992–1009 (2007)

 2007 Phycological Society of America
DOI: 10.1111/j.1529-8817.2007.00384.x

GENETIC AND PHENOTYPIC CHARACTERIZATION OF PHAEODACTYLUM

TRICORNUTUM (BACILLARIOPHYCEAE) ACCESSIONS 1

Alessandra De Martino
Laboratory of Molecular Plant Biology, CNRS UMR 8186, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France
Laboratory of Cell Signalling, Stazione Zoologica Anton Dohrn, Villa Comunale, I-80121 Naples, Italy

Agne`s Meichenin
Laboratory of Molecular Plant Biology, CNRS UMR 8186, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France

Juan Shi, Kehou Pan

College of Fisheries, Ocean University of China, Qingdao, Shangdong Province 266003, P. R. China

and Chris Bowler2

Laboratory of Molecular Plant Biology, CNRS UMR 8186, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France
Laboratory of Cell Signalling, Stazione Zoologica Anton Dohrn, Villa Comunale, I-80121 Naples, Italy

In the last few years, genome-based studies in
diatoms have received a major boost following the
genome sequencing of the centric species Thalassi-
osira pseudonana Hasle et Heimdal and the pleio-
morphic raphid pennate diatom Phaeodactylum
tricornutum Bohlin. In addition, molecular tools,
such as genetic transformation, have been devel-
oped for both species. Despite these molecular
advances, relatively little is known regarding the
genetic diversity of the available strains of these
diatoms. In this study, we have compiled a histori-
cal summary of the known P. tricornutum species
resources and have provided a genetic and pheno-
typic overview of 10 different axenic strains.
Examination of intraspecies genetic diversity based
on internal transcribed spacer 2 (ITS2) sequence
and amplified fragment length polymorphism
(AFLP) analyses indicate four different genotypes.
Seven strains are predominantly fusiform, whereas
one strain is predominantly oval, and another is
predominantly triradiate. Another is defined as a
tropical strain because it appears better acclimated
to growth at higher temperatures. Observations in
the natural environment indicate that P. tricornutum
is a coastal marine diatom that is able to adapt to
unstable environments, such as estuaries and rock
pools. Because it has rarely been noted in nature,
we have developed specific primers to amplify
ITS2 sequences and have successfully identified it
in environmental samples. These resources should
become useful tools for the diatom community
when combined with the whole genome sequence
and will open up a range of new possibilities
1
Received 3 November 2006. Accepted 17 April 2007.
2
Author for correspondence: e-mail cbowler@biologie.ens.fr.
for experimental investigations that can exploit
the genotypic and phenotypic characteristics
described.
Key index words: AFLP; ecophenotype; genotype;
ITS probe; model diatom; pleiomorphism
Abbreviations: AFLP, amplified fragment length
polymorphism; CAPS, cleaved amplified polymor-
phic sequence; CCAP, Culture Collection of Algae
and Protozoa; CCCM, Canadian Center for the
Culture of Microorganisms; CCMP, Provasoli-
Guillard National Center for Culture of Marine
Phytoplankton; EST, expressed sequence tag;
ITS2, internal transcribed spacer 2
Over the last decade, diatoms have become a rep-
resentative algal group to explore the biology of
marine phytoplankton and the phylogeny of hetero-
konts (Falciatore and Bowler 2002, Kooistra et al.
2003). Genetic markers have been developed to
study the diversity of planktonic diatoms, revealing
surprisingly high genetic diversity even at the popu-
lation level (Rynearson and Armbrust 2000, 2004,
Evans et al. 2004, 2005). In parallel, over the last
few years, genomic data from diatoms have
increased enormously, following the genome
sequencing of the centric diatom Thalassiosira
pseudonana (Armbrust et al. 2004) and the pennate
diatom Phaeodactylum tricornutum (http://genome.
jgi-psf.org/Phatr2/Phatr2.home.html), both of
which have been sequenced by the Joint Genome
Institute (Walnut Creek, CA, USA). In addition,
>10,000 expressed sequence tags (ESTs) have been
generated from both species and have been
organized in a searchable diatom EST database
(http://www.biologie.ens.fr/diatomics/EST/; Mahes-

992
 G E N O T Y P E S AN D P H E N O T Y P E S O F A MO D E L D I A T O M
wari et al. 2005, Montsant et al. 2005). Another
100,000 ESTs have been generated from P. tricornu-
tum by Genoscope (Evry, France) from a range of
cDNA libraries prepared from cells grown in
different conditions (http://www.biologie.ens.fr/
diatomics/EST/). These data have already revealed
several surprising peculiarities of diatom biology
that likely reflect specialized evolutionary
adaptations to the marine environment (Armbrust
et al. 2004, Montsant et al. 2005). For example,
transgenic P. tricornutum lines have been used to
study the response of diatoms to nutrients
(Falciatore et al. 2000) and, more recently, to study
calcium signaling and cell–cell communication in
response to allelochemicals (Vardi et al. 2006). The
species has also been used for studies of protein
targeting to diatom plastids (Kilian and Kroth
2005).
Phaeodactylum tricornutum has the unusual prop-
erty of being pleiomorphic, and this plasticity is
related to the atypical nature of the cell wall,
which is only poorly silicified compared with other
diatoms (Lewin et al. 1958, Borowitzka and
Volcani 1978). The species appears to be unique
in that it does not have an obligate require-
ment for silicic acid. Nothing is known about
the physiological significance of these characteris-
tics. Furthermore, despite the abundance of
genome information, the basic biological features
of P. tricornutum are relatively poorly studied, in
particular its life cycle and its ecological relevance.
These are serious shortcomings, given the likely
convergence of several areas of diatom research
toward this species now that a whole genome
sequence is available.
Our aim in the present study was to better define
the P. tricornutum accessions that are available in
stock centers around the world. The first objective
was to trace the species in a historical context,
which we considered important to provide a single
reference source that documents the recorded
observations of P. tricornutum and describes the ori-
gins of different strains available in different stock
centers. This information should permit each labo-
ratory using P. tricornutum to know the origins of
the strains being used. Subsequently, we have
sought to characterize the phenotypic traits and
genetic diversity of 10 different accessions. This has
revealed that certain accessions have specific charac-
teristics that can be exploited experimentally
to explore particular aspects of diatom biology.
Furthermore, the availability of 10 characterized
P. tricornutum accessions can permit the search for
polymorphisms of functional significance, as has
been championed in Arabidopsis (Mitchell-Olds and
Schmitt 2006). Finally, we have developed a PCR
primer set that can be used for the detection of the
species in the marine environment, which should
be useful to clarify the ecology and biogeography of
P. tricornutum.
993
MATERIALS AND METHODS
Accessions and culture conditions. Nine different accessions of
P. tricornutum were obtained from the culture collections of the
Provasoli-Guillard National Center for Culture of Marine
Phytoplankton (CCMP, http://ccmp.bigelow.org), the Culture
Collection of Algae and Protozoa (CCAP, http://www.ccap.
ac.uk), and the Canadian Center for the Culture of Micro-
organisms (CCCM, http://www3.botany.ubc.ca/cccm/index.
html). An additional pennate diatom described as Nitzschia
closterium forma minutissima, harvested from coastal waters near
Dalian, Yellow Sea, China, by Xuecun Qu was obtained from
the Microalgae Culture Collection of Qingdao University
(MACC).
All of the accessions were axenic except two (NEPCC 640,
MACC B228), which were made axenic by treatment of the cells
with 50 lg Æ mL)1 streptomycin (Sigma Inc., St. Louis, MO,
USA) +50 lg Æ mL)1 ampicillin (Sigma). Cells were grown in
batch in flask cultures with a photon fluence rate of 60–
80 lmol Æ m)2 Æ s)1 provided by cool-white fluorescent tubes
(F 36 W ⁄ 33 640 4000 K; Claude, Blanc Industry, Franco Belge
d’électricité, Malakoff, France) in a 12:12 light:dark (L:D)
photoperiod at 19C. Cells were subcultured in f ⁄ 2 medium
without additional silicic acid for routine growth of the cells
(Guillard 1975) or in artificial seawater medium (ASW;
Falciatore et al. 2000) for physiological analyses. Cells were
counted in a Malassez chamber. Clonal cultures were gener-
ated from single cells originally isolated using a pipette in an
Axiovert inverted light microscope (Zeiss, Jena, Germany). The
clone Pt1 8.6 used for genome sequencing has been deposited
at CCAP, with the accession number CCAP 1055/1, and at
CCMP, with the accession number CCMP2561.
Marine samples were collected by surface pump into carboys
and filtered onto Sterivex (0.2 lm pore size; Millipore Corp.,
Billerica, MA, USA). After filtration, filters were stored at )80C
until DNA extraction. The DNA was extracted from the filters
using the Gentra PureGene (Gentra Systems Inc., Minneapolis,
MN, USA) tissue protocol (information provided by Bess Ward,
Department of Geosciences, Princeton University, NJ, USA).
Ribotype analysis. Genetic analysis of the nine P. tricornutum
accessions was performed by sequencing the fast-evolving
internal transcribed spacer region, ITS2, and the beginning
of the 28S rDNA, including the highly variable D1 region
(Coleman 2003). The rDNA regions comprising the 3¢ end of
the 5.8S, the hypervariable ITS2 and the 5¢ end of the 28S,
including the variable D1 region, were amplified by PCR with
the universal forward primer ITS3 (5¢-gcatcgatgaagaacgcagc-3¢)
and reverse primer TW13 (5¢-ggtccgtgtttcaagacg-3¢). The PCR
amplification was performed on lysates of P. tricornutum cells as
described in Falciatore et al. (1999), using 2.5 U of Pfx high-
fidelity enzyme (Invitrogen, Cergy Pontoise, France) following
the manufacturer’s instructions. The PCR fragments were
purified with the QIAEX II kit (Qiagen Inc., Valencia, CA,
USA) and directly sequenced by MWG-Biotech (Ebersberg,
Germany). The PCR fragments were sequenced in both
directions and checked with three different forward and
reverse primer pairs specific to the sequences (F1: ITS3 primer,
F2: 5¢-acccgctgaatttaagcatataat-3¢, F3: 5¢-ggtggtaaattccatctaaagc-
ta-3¢; R1: TW13 primer, R2: 5¢-gttagtttcttttcctccgcttaa-3¢, R3:
5¢-aactctcttttcaaagttctgttgcatc-3¢). Complete sequences of 5.8S
rDNA and ITS2 from the diatom from the Yellow Sea were
obtained after PCR amplification with a forward primer
that was specific to the ITS1 sequence (F4: 5¢-acccaaac-
ccaaccacgacgg-3¢) and the reverse primer R2, cloning into
pBluescript II KS ()), and sequencing with T7 primers.
Sequence alignments were made using the BioEdit program
(version 6.0.5; Hall 1999).
Cleaved amplified polymorphic sequence (CAPS; Scott and
Amasino 1998) analysis of the region denoted ITS2-D1 was
994 A L E S S A N D R A D E MA R T I N O E T A L .
performed with TaaI or HincII ⁄ SnaI (Fermentas). ITS2
sequences have been deposited in GenBank with the following
accession numbers: DQ085801 (Pt1), DQ085802 (Pt2),
DQ085803 (Pt3), DQ085804 (Pt4), DQ085805 (Pt5), DQ085806
(Pt6), DQ085807 (Pt7), DQ085808 (Pt8), DQ085809 (Pt9), and
DQ655656 (Pt10).
Amplified fragment length polymorphism (AFLP). AFLP analysis
was carried out as in Vos et al. (1995) according to the
manufacturer’s instructions (AFLP Analysis system II; Invitro-
gen). The first step of amplification was performed with 300 ng
of genomic DNA extracted from exponentially growing cells
according to the procedure previously described (Falciatore
et al. 1999). MseI primers with three selective nucleotides and
EcoR1 primers having two selective nucleotides were used for
PCR. Primer labeling was performed by phosphorylating the 5¢
end of the EcoR1 primers with c33P-ATP and T4 kinase
(Invitrogen). Following PCR amplification, reaction products
were checked on a 1% agarose electrophoresis gel and then
analyzed on a 6% denaturing polyacrylamide gel. After a
prerun of 30 min at 50 W, samples were run at 65 W in a sequi-
gen GT Nucleic Acid Electrophoresis Cell, 38 · 50 cm (Bio-
Rad, Division Bio-Recherche, Marnes la Coquette, France) for
2 h. After separation of the bands, the gel was dried and
exposed to an X-ray film for 1–3 d. All AFLP analyses were
performed at the same time on the different accessions with
different pairs of primers, and the polymorphic bands were
confirmed by analysis of two different genomic DNA samples.
The presence or absence of polymorphic bands was visually
recorded and compiled in a binary data table.
Light and epifluorescence microscopy. Microscopy was
performed with an Axioskop-2 epifluorescence microscope
(Zeiss), with differential interference contrast or with epifluo-
rescence using filter sets for YFP (XF104-2; Omega Optical,
Brattleboro, VT, USA). Images were captured with an Axiocam
MRc camera (Zeiss), and cell size and shape parameters were
measured with the Axiovision 4.1 software (Carl Zeiss S.A.S, Le
Pecq, France).
TEM. Frustules from the different accessions were exam-
ined by TEM. Exponentially growing cells were fixed with
formaldehyde (final concentration 0.8%) for 1 h at room
temperature and washed four times with distilled water by
centrifuging at 6000g for 10 min. Organic material was elimi-
nated by treating the cells with 16% HNO3 (v ⁄ v) and 48%
H2SO4 (2:1 v ⁄ v) for 2–3 min, and cells were then carefully
washed several times with distilled water until the solution
reached neutral pH. Monoclonal cultures corresponding to
each morphotype isolated from single cells of the accession Pt8
were also examined after different acid treatments with varia-
tions in time and ⁄ or acid concentration. A drop of the cleaned
material was placed on a Formvar Ni-coated 200 Mesh grid
(Agar Scientific Ltd., Essex, UK) and observed with a Philips
TEM 400 microscope (Philips, Eindhoven, the Netherlands).
SEM. Whole cells were examined by SEM. Cells were fixed
with glutaraldehyde (1% final concentration), rinsed with
distilled water to remove salts and fixatives, dehydrated in a
graded ethanol series (ethanol: 10% for 15 min, 25% for
15 min, 50% for 15 min, 75% for 15 min, 95% for 15 min,
100% for 60 min), and critical-point-dried. Preparations were
coated with a complete layer of gold-palladium and observed
with a scanning electron microscope (Philips 505 SEM;
Eindhoven, the Netherlands).
Diatom transformation. Diatom cells were cotransformed by
the biolistic method with the vector for resistance to phleomy-
cin pAF6 (Falciatore et al. 1999) and an expression vector for a
cytosolic eYFP (kindly provided by Anton Montsant). The
sequence encoding eYFP was obtained from the vector pEYFP-
N1 (Clontech, Mountain View, CA, USA) and cloned between
the FcpB promoter and the FcpA terminator of P. tricornutum
(accession number Z24768). The hepta-biolistic system from
Biorad was used to shoot cells on petri dishes. Several
parameters were tested to shoot the oval and the triradiate
accessions, with a range of delivery pressures between 1100 and
2000 psi and two shooting distances: the top level (6 cm) and
the intermediate level (9 cm). M17 tungsten microcarriers
(Bio-Rad) were used for transformation of all accessions and
morphotypes.
RESULTS
Historical overview. Now that a whole genome
sequence is available, P. tricornutum is likely to
become even more utilized than at present, and so
we believe it to be important to clarify the available
P. tricornutum species resources in a historical,
genetic, and phenotypic context, both to avoid erro-
neous classifications in the future and to permit
resources to be utilized to the maximum for physio-
logical studies that can also exploit intraspecific
polymorphisms for functional studies of diatom
genes.
Phaeodactylum tricornutum was first described as a
new genus with a single unique species by Bohlin in
1897. He found it in samples collected off Plymouth
(UK) as well as in Baltic rock pools. He described
only triradiate morphotype cells with three arms, a
yellow–brown chloroplast, and a weakly silicified
exterior. His description does not reveal whether he
also found fusiform or oval cells, but the star shape
was probably more noticeable and distinguished the
species from other phytoplankton cells. Since this
first description, the species has been isolated and
described on several occasions in a range of loca-
tions worldwide and is now available in several algal
culture collections. Below and in the supplementary
material, we present what we believe is a compre-
hensive list of P. tricornutum accessions that are avail-
able worldwide, with a focus on the 10 strains that
have been extensively analyzed in our laboratory.
The Blackpool strain and derived accessions (Pt1): This
strain was harvested off Blackpool, UK, ca. 1956 and
identified by S. Coughlan. We received this strain as
accession CCMP632 from CCMP in March 2001 and
denoted it as Pt1 (Table 1). Nine monoclonal cul-
tures of Pt1 were subsequently generated, and one
with a fusiform morphotype, denoted Pt1 8.6, was
selected for genome sequencing (http://genome.
jgi-psf.org/Phatr2/Phatr2.home.html). It was then
sent to CCAP (Oban, UK) in December 2004 and
given the accession number CCAP 1055/1. It was
subsequently sent from CCAP to CCMP, which gave
it the accession number CCMP2561.
The Plymouth strain and derived accessions (Pt2,
Pt3): E. J. Allen collected this strain off Plymouth
sometime before 1910, probably in 1908 (informa-
tion from M. Jutson, manager Plymouth Algal Cul-
ture Collection, Plymouth, UK). In a subsequent
publication (Allen and Nelson 1910), the authors
did not provide any description or illustrations, and
they identified it wrongly as Nitzschia closterium
Table 1. Origin and morphotype characteristics of the 10 accessions of P. tricornutum.

Accession Strain reference in Date of Accession numbers of

name permanent collectionsa Location of sampling collection Characteristics of collection site Major morphotype characterized strainsb

Pt1 CCMP632 Off Blackpool, UK ca. 1956 Coastal water, close Fusiform 95%–100% CCAP 1055 ⁄ 3,
54.0000N–04.0000W to estuaries CCAP 1055 ⁄ 1c
Pt2 CCAP 1052 ⁄ 1A English Channel, Prior 1910 Coastal water, close Fusiform 95%–100% CCMP2557
(PCC 100, SAG off Plymouth, UK to estuaries
1090-1a, UTCC 162) 50.3620N–04.1700W
Pt3 CCAP 1052 ⁄ 1B Brackish clonal culture 1930s Clonal bacteria-free isolation. Oval 60%–75% CCMP2558
(SAG 1090-1b) isolated from Plymouth Growth on freshwater, Clonal
strain (Pt2), UK soil extract
Pt4 CCAP 1052 ⁄ 6 (UTEX 646, Island of Segelskår, near 1951 Brackish water, isolated from Fusiform 95%–100% CCMP2559
SAG 1090-6) Tvarminne, Finland a supralittoral rock pool Clonal
63.0000N–27.0000E
Pt5 CCMP630 (NEPCC 738) Gulf of Maine, Cape 1972, Shallow tidal creek with wide Fusiform 95%–100% CCAP 1055 ⁄ 2
Cod Bay, West Dennis, winter ranging salinity Clonal
Chase Garden Creek,
MA, USA
41.7600N–70.2000W
Pt6 CCMP631 (NEPCC 31) Nantucket Bay, off Woods ca. 1956 Seawater tank with wide Fusiform 95%–100% CCAP 1055 ⁄ 4
Hole, MA, USA ranging salinity
41.5250N–70.6736W
Pt7 CCMP1327 Great South Bay, off Long 1952 Polluted seawater (duck industry). Fusiform 95%–100% CCAP 1055 ⁄ 6
Island, MA, USA Enclosed bay with low salinity.
40.6600N–73.2500W Isolated during bloom of
Nannochloropsis ⁄ Nannochloris
Pt8 NEPCC 640 Jericho Beach, Vancouver, 1987, Coastal water. Sample collected Triradiate 80%–85% CCAP 1055 ⁄ 7,
BC, Canada March with a plankton net 2 feet CCMP2560
40.6600N–73.2500W from surface
Pt9 CCMP633 Territory of Guam, 1981, From the shore, water Oval 60%–75% at CCAP 1055 ⁄ 5
Northern Mariana Feb. temperature 25C 15C–19C; but
Islands, Micronesia fusiform 80%–95%
13.8333N–144.7500E at 25C–28C
G E N O T Y P E S AN D P H E N O T Y P E S O F A MO D E L D I A T O M

Clonal
Pt10 MACC B228 Dalian, Yellow Sea 2000 Polluted seawater Fusiform 95%–100% CCAP 1055 ⁄ 8,
(industrial area and CCMP2928
seaside resort)
All the information indicated was provided by the person who isolated the strain or by people from the different culture collections.
CCMP, Provasoli-Guillard National Center for Culture of Marine Phytoplankton; CCAP, Culture Collection of Algae and Protozoa; SAG, Sammlung von Algenkulturen der
Universität Gottingen, Germany; NEPCC, North East Pacific Culture Collection; UTEX, Culture Collection of Algae at the University of Texas at Austin; MACC, Microalgae
Culture Collection of Qingdao University; UTCC, University of Toronto Culture Collection of Algae.
a
Bold indicates our source of each isolate.
b
Accession numbers of our strains deposited in culture collection.
c
Clonal isolate used for genome sequencing (http://genome.jgi-psf.org/Phatr2/Phatr2.home.html).
995
996 A L E S S A N D R A D E MA R T I N O E T A L .
W. Sm. forma minutissima. This Plymouth isolate has
been the basis for a large amount of physiological
and biochemical research, but during the first half
of the last century, it was referred to as N. closterium
W. Sm. forma minutissima (for references see Lewin
1958). It was only in the 1950s that cells from these
cultures were recognized as resembling P. tricornu-
tum, and the taxonomic position of the species was
subsequently revised by J. C. Lewin in 1958 (Woods
Hole Oceanographic Institution, MA, USA) to P. tri-
cornutum Bohlin emend. Since 1958, this clone and
others have been officially renamed as P. tricornu-
tum. Lewin assigned it to the class of the Bacillario-
phyceae. She also described P. tricornutum as only
one known genus and species of a new suborder,
Phaeodactylineae, and a unique and new family,
Phaeodactylaceae. The official designation is there-
fore as follows:
P. tricornutum Bohlin emend (Lewin 1958)
Class: Bacillariophyceae
Order: Bacillariales
Suborder: Phaeodactylineae (nov. suborder)
Family: Phaeodactylaceae (nov. family)
Genus: Phaeodactylum (Bohlin 1897)
Species: P. tricornutum
Care of the Plymouth strain was passed to D. P.
Wilson (Plymouth Marine Laboratory) in 1939 who
published the first detailed account of the organism
with illustrations (Wilson 1946). He described the
pleiomorphic character of P. tricornutum and
described each of the different morphotypes (albeit
under the name of N. closterium W. Sm. forma minu-
tissima). He states, on the authority of M. Parke
(Marine Biological Association, Plymouth), that
both the triradiate and fusiform phases were fre-
quent in water samples from the Irish Sea off Port
Erin. For our studies, we acquired the Plymouth
strain from the CCAP (Oban) in November 2001,
and we denoted it as Pt2 (Table 1). There are
several permanent stocks of the Plymouth strain
around the world (see supplementary material), and
all are described as displaying the fusiform morpho-
type.
The strain that we denote Pt3 was initially derived
from Pt2 in Plymouth in the 1930s as a subclonal
culture of Pt2 that was able to grow in freshwater
media (information kindly provided by T. Henning,
Culture Collection of Algae at the University of
Texas at Austin [UTEX]). We received this strain as
CCAP 1052 ⁄ 1B from CCAP in November 2001, and
it contained principally oval morphotype cells.
The Finnish strain (Pt4): The strain that we denote
Pt4 was obtained as CCAP 1052 ⁄ 6 from CCAP and
is described as a brackish strain of P. tricornutum
that was collected by M. R. Droop (Scottish Marine
Biological Association, Millport, UK) in 1951 from a
supralittoral rock pool on the Island of Segelskår,
near Tvarminne, Finland. In his notebook, he stated
that P. tricornutum often occurs in rock pools on
skerries, and he also found it within a few miles of
Runmarö (information from Droop’s notebook and
other records kindly provided by C. Campbell,
CCAP). He also stated that naturally occurring
material was always triradiate.
The Cape Cod Bay strain (Pt5): The Cape Cod Bay
strain was collected during the winter of 1972 by
Petrovitch and Guillard (CCMP) in a shallow tidal
creek with fluctuating salinity in the Gulf of Maine
(West Dennis, MA, USA). It was subsequently puri-
fied by R. Guillard and was cryopreserved by the
CCMP as accession CCMP630. We obtained the
strain from CCMP in November 2001.
The Woods Hole strain (Pt6): J. C. Lewin and R. A.
Lewin originally isolated this strain in September
1956 from a seawater tank behind the Woods Hole
Oceanographic Institution (MA, USA) that con-
tained seawater enriched with a commercial fertil-
izer (information kindly provided by R. Guillard).
They subsequently provided the first morphological
microscopic illustration of P. tricornutum using this
strain in 1958 (Lewin et al. 1958). In their report,
they describe it as being morphologically identical
to the Plymouth strain of N. closterium W. Sm.
forma minutissima. They reported two typical cell
forms, fusiform and oval, and noted that triradiate
cells were rare. The article also contains a descrip-
tion of the life cycle for the first time and reveals
that oval cells possess a siliceous valve. From
an ecological viewpoint, they considered that
P. tricornutum was not a normal component of mar-
ine phytoplankton, and that it occurs generally
only in intertidal rock pools or seawater tanks.
This Woods Hole strain was deposited at CCMP in
December 1986 and given the accession number
CCMP631. We obtained CCMP631 from CCMP in
November 2001, and we now denote the accession
as Pt6.
The Long Island strain (Pt7): This isolate of
P. tricornutum was collected in 1952 by J. Ryther
(Woods Hole Oceanographic Institution) and
deposited at CCMP in 1952 with the accession
CCMP1327. The site of collection is described as an
enclosed bay with low salinity (teens to twenties)
that was polluted from a local duck farm at that
time (information kindly provided from R. Guil-
lard). We obtained CCMP1327 from CCMP in
November 2001, and in our study, it is denoted Pt7.
The Vancouver strain (Pt8): The isolate that we
denote Pt8 was collected off Jericho Beach, Vancou-
ver, Canada, in March 1987 and deposited at CCCM
as accession NEPCC 640. Accession NEPCC 640 is
described as a fusiform morphotype, although the
subculture we received in November 2001 from
CCCM was predominantly triradiate and has
remained so since then.
The Micronesian strain (Pt9): A Micronesian strain
was collected in February 1981 in the territory of
Guam, Northern Mariana Islands, Micronesia, by
R. A. Lewin. At CCMP, it has the accession number
CCMP633, and it is typically grown at 20C–26C.
 G E N O T Y P E S AN D P H E N O T Y P E S O F A MO D E L D I A T O M
We received this accession in June 2003, and we
denote it Pt9.
The Chinese strain (Pt10): A strain described as
N. closterium W. Sm. forma minutissima was isolated
from Yellow Sea, Dalian, Liaoning Province, China,
by X. Qu in the spring of 2000 and maintained at
MACC (Qingdao, China) with accession number
B228. It was provided to us by Prof. Kehou Pan,
Ocean University of China, Qingdao, in May 2005,
and it was denoted Pt10.
Other Phaeodactylum strains: A range of other
strains are classified as P. tricornutum in different
stock centers around the world. We have not charac-
terized these strains because they are not axenic,
but they are described in the supplementary mate-
rial.
In addition to what has already been mentioned,
records of P. tricornutum from the field have been
reported off Port Erin in the Irish Sea; off
St. Andrews in the North Sea; and from the Canan-
éia Lagoon Estuarine System, Sao Paulo, Brazil
(Schaffer-Novelli and Cintron-Molero 1990), a large
mangrove-dominated estuary. Schaffer-Novelli and
Cintron-Molero reported that P. tricornutum became
a predominant component of the phytoplankton
following changes in salinity. It has also been
reported in rock cavities off the French coast (at
Dinard; Bourrelly and Dragesco 1955). In the 2001
World Ocean Database from the Ocean Biogeo-
graphic Information System (OBIS, NODC Plankton
Database, http://www.iobis.org), it is reported that
P. tricornutum has been collected in the Gulf of
Mexico. Several isolates of P. tricornutum have been
recorded in the Base de Dados Tropical (BDT), Bra-
zil, seven of which have been collected in Aguas
Costeiras Ubatuba (designated Phaeo-Ub0–Phaeo-
Ub7). More information about these reports is pro-
vided in the supplementary material.
Intraspecies genetic variability of Phaeodactylum
tricornutum. Genetic analysis of the 10 P. tricornu-
tum accessions Pt1–Pt10 was first performed by
sequencing the fast-evolving ITS2 and the beginning
of the 28S rDNA, including the highly variable D1
region (Coleman 2003). Universal forward and
reverse primers that anneal to highly conserved
regions of the 5.8S rDNA and 28S rDNA regions
were used for PCR amplification. From all acces-
sions, we obtained a unique band of 1.1 kb, and
PCR products were sequenced directly. A major
sequence of 1016 bp was obtained from all acces-
sions, although minor peaks were observed in the
ITS2 region (see below).
28S rDNA and 5.8S rDNA sequence analysis: No
differences in sequence were observed in the partial
28S rDNA sequences (494 bp) among the 10 acces-
sions of P. tricornutum (data not shown). blastn
analysis (http://www.ncbi.nih.gov/BLAST/) showed
a high percentage of sequence similarity with the
28S rDNA sequences of other diatoms and brown
algae. Recently, an rDNA sequence (AY574376),
997
including the 5.8S rDNA region and a partial ITS2
sequence, from a strain of P. tricornutum of unde-
scribed biogeographic origin has been deposited in
GenBank by Y. Joubert et al. (Institut des Sub-
stances et Organismes de la Mer [ISOMER] Nantes,
France). The 5.8S rDNA sequences of the 10 acces-
sions were identical to this 5.8S rDNA sequence
(data not shown).
Description of genotypes based on ITS2 and CAPS
markers: blastn analysis of the ITS2 regions did
not reveal any significant similarity with other dia-
tom ITS2 sequences available in GenBank (based
on 146 ITS2 sequences from six different diatom
species; data not shown). The ITS2 of P. tricornutum
was also significantly longer (439–446 nucleotides)
than these other diatom ITS2 sequences (which
range from 263 to 380 bp).
Sequence alignments of the ITS2 region showed
that the 10 accessions have a high percentage of simi-
larity throughout the sequence (97%–100% identity,
Fig. S1 in the supplementary material). However, five
nucleotide positions were phylogenetically informa-
tive and showed the presence of four different geno-
types, which we denoted A, B, C, and D (Fig. 1a).
Two polymorphic sites (denoted I and II) generated
differences in restriction enzyme sites, allowing the
possibility to distinguish the genotypes by CAPS
markers (Fig. 1, b–f). Heterogeneity of sequences was
observed for genotype A, because polymorphic site II
was not completely digested by SnaI in these acces-
sions (Pt1, Pt2, Pt3, and Pt9; Fig. 1d). This heteroge-
neity, subsequently confirmed by sequencing, could
imply either heterogeneity of these populations or
heterogeneity within different copies of ITS2 within
the genomes of these accessions.
To examine further this genetic variability, we
analyzed nine different monoclonal cultures derived
from Pt1 using CAPS markers. The results indicated
that the populations were genetically homogeneous
(Fig. 1, e and f), which was further confirmed by
sequencing the ITS2 and D1 regions (data not
shown), although the profile of restriction again
indicated heterogeneity at locus II (Fig. 1f). The
identical restriction patterns observed from each of
the subcultures of Pt1 indicate that this heterogene-
ity may be due to inherent heterogeneity within dif-
ferent copies of ITS2 or to heterozygosity.
Phylogenetic trees based on ITS2 sequences and
generated by the neighbor-joining or parsimony
methods showed the 10 accessions as four major
clades that confirm the presence of four different
genotypes of P. tricornutum (Fig. 2a and data not
shown), with genotype B (Pt4) and C (Pt5 and
Pt10) being the most distinct genetically.
High intraspecies genetic variability among genotypes
revealed by AFLP: We used AFLP fingerprinting to
further investigate the genetic variability among the
four genotypes of P. tricornutum. Pt1, Pt4, Pt5, and
Pt8 were chosen as representatives of each
genotype. AFLP is a powerful method of DNA
998 A L E S S A N D R A D E MA R T I N O E T A L .

c e

d f

Fig. 1. Genotypes of Phaeodactylum tricornutum. (a) Genetic differences in the ITS2 sequences (the numbers correspond to the nucleo-
tide on the rDNA regions [995 bp] submitted to GenBank). (b) Summary of genotypes identified by cleaved amplified polymorphic
sequence (CAPS) analysis. (c–f) Differences in profiles of restriction of internal transcribed spacer 2 (ITS2) region when cut with TaaI
and SnaI ⁄ HincII in polymorphic sites I (c and e) and II (d and f). (c and d) Profiles of restriction obtained from the four genotypes
(lanes 1–4). In genotype A (Pt1, Pt2, Pt3, Pt9), there was always a fraction of DNA that was not cut by SnaI (*), even after purification of
the band and redigestion (d). (e and f) Profile of restriction obtained from nine clones of Pt1 (lanes 5–13) that were each isolated from
a single fusiform cell. The identical profiles indicate that the populations were genetically homogeneous. Again, in all the clones, half of
the amount of DNA was not cut by SnaI (*) in polymorphic site II (f).

fingerprinting because, unlike single gene
sequences or microsatellites, it is based on the poly-
morphism of the entire genome (Vos et al. 1995).
Nonetheless, it is not routinely employed in marine
genetic studies. A high degree of polymorphism was
observed, confirming that the four accessions were
significantly different genetically (Table S1 in the
supplementary material). The 12 AFLP primer com-
binations generated a total of 330 bands (27 ± 8 for
each primer combination), of which 128 loci
(38.8%) were polymorphic across all P. tricornutum
strains. Pt5 showed the highest level of genetic poly-
morphism with Pt4 and Pt8 (70% and 66%, respec-
tively, of the polymorphic loci, observed with all
primer combinations tested; Table S1). The
distance trees generated from these AFLP analyses
 G E N O T Y P E S AN D P H E N O T Y P E S O F A MO D E L D I A T O M
a to a region of the ITS2 of P. tricornutum that highly
b could be detected in environmental samples isolated
Fig. 2. Intraspecies genetic and morphological variability
among the 10 Phaeodactylum tricornutum accessions. (a) Phyloge-
netic tree based on the neighbor-joining (NJ) method, obtained
from analysis of the internal transcribed spacer 2 (ITS2) region
(unrooted tree). (b) Phylogenetic tree of four accessions repre-
senting each genotype (Pt1, Pt4, Pt5, Pt8) based on amplified
fragment length polymorphism (AFLP) analysis. NJ trees (Saitou
and Nei 1987) were obtained with Treecon software (Van de Peer
and Dewachter 1994) according to the algorithm of Tajima and
Nei (1984), and node statistics were supported with 1000 boot-
strap replicates.
confirmed that accession Pt5 (genotype C) was the
most distant genetically from Pt4 and Pt8 (Fig. 2b),
in agreement with the ITS2 analysis (Fig. 2a).
ITS2 primer set to detect Phaeodactylum tricornutum
in natural environments: Because the ITS2 sequence
was highly conserved within P. tricornutum accessions
and was highly divergent from ITS2 sequences from
other diatom species, we tested whether it can be
used to detect P. tricornutum in the natural environ-
ment. For this we decided to examine isolates col-
lected off Plymouth (UK), because P. tricornutum
has been reported at this location on several
occasions in the past (see above). Two planktonic
DNA samples (ST30 and ST82) from the surface of
the English Channel (50.15¢N, 4.13¢W) were col-
lected on April 13, 2004, and on July 6, 2006, and
kindly provided by Bess Ward (Department of Geo-
sciences, Princeton University, NJ, USA). Specific
primers (ITS2Pt-fwd: 5¢-tctggctgctgttcaagtgt-3¢ and
ITS2Pt-rev: 5¢-tcggtttccgtctccagt-3¢), corresponding
999
differs from other diatoms and does not match any
sequences by blastn analysis, were used to amplify
a short sequence of 139 bp. Fragments with the
expected size were obtained from both environmen-
tal samples, and sequencing confirmed their identity
as being from P. tricornutum (Fig. S1). This specific
region of ITS2 was identical among genotypes A, B,
and D, but contained one polymorphic site in geno-
type C that was absent in the ST30 and ST82
sequences. One of the ST82 derived sequences was
100% identical to genotypes A, B, and D, while
another showed one polymorphic site located in a
distinct position of the sequence (Fig. S1). No PCR
amplification was obtained from genomic DNA from
the centric diatom T. pseudonana or the raphid pen-
nate diatom Amphora sp. (data not shown). These
results therefore demonstrated that P. tricornutum
during the spring and summer periods in the Eng-
lish Channel.
Morphotypes of Phaeodactylum tricornutum. Morpho-
logical variability among the accessions: The morpho-
logical characteristics of the 10 P. tricornutum
accessions are summarized in Tables 1 and 2. High
morphological variability was observed among the 10
accessions. These features have been stable for
5 years in our standard culture conditions. All cul-
tures displayed the three different shapes
(morphotypes) that have been described for
P. tricornutum, known as fusiform, oval, and triradiate
morphotypes (Borowitzka et al. 1977, Borowitzka
and Volcani 1978; Fig. 3, a–c). However, fusiform was
usually the most abundant morphotype, representing
>95% of the cells in seven accessions (Table 1). We
therefore denoted these as ‘‘fusiform accessions.’’
Three accessions contained only a minority of fusi-
form cells. The Canadian accession Pt8 was distinct
from the others by displaying a majority of triradiate
morphotype cells, and oval cells were usually absent
in this accession. We therefore denoted Pt8 a ‘‘trira-
diate accession.’’ Cultures of the English accession
Pt3 and the Micronesian accession Pt9 exhibited
60%–75% oval morphotype cells. However, cultures
of Pt3 were initially characterized as containing
mainly oval cells when we received the strain from
the culture collection, whereas cultures of Pt9 were
initially close to 100% fusiform and became oval after
a few weeks in culture at 19C. When transferred to
25C–28C, Pt9 cultures became fusiform, suggesting
that the appearance of the oval morphotype in acces-
sion Pt9 was related to culture conditions rather than
being an intrinsic characteristic of this strain, per-
haps being induced in response to cold-temperature
stress. Furthermore, Pt3 was originally isolated from
the fusiform accession Pt2 by its ability to grow at low
salinity (see supplementary material). This suggests
that the oval morphotype may be able to acclimate to
low salinity. We therefore denoted Pt3 an ‘‘oval
accession,’’ and Pt9 a ‘‘tropical accession.’’
1000 A L E S S A N D R A D E MA R T I N O E T A L .

Table 2. Intraspecific morphological variability of the 10 accessions of P. tricornutum, based on the characteristics of fusi-
form, triradiate, and oval cells.

Length size = a
Accession name Width size = b (lm) a⁄b Morphology

Fusiform morphotype
Pt1 a = 19–24 6.4 ± 0.7
b = 2.9–3.9
Pt2 a = 19–24 7.4 ± 0.6
b = 2.8–3.3
Pt3 a = 19–24 6.9 ± 0.3
b = 3.1–3.4
Pt4 a = 19–24 7.5 ± 0.6
b = 2.5–3.6
Pt5 a = 25–30 9.0 ± 1.1
b = 2.5–3.4
Pt6 a = 15–19 5.5 ± 0.3
b = 2.8–4.1
Pt7 a = 15–19 4.8 ± 0.6
b = 3.2–4.8
Pt8 a = 14–22 5.8 ± 1.0
b = 2.4–3.5
Pt9 a = 16–20 5.0 ± 0.8
b = 3.1–5.2
Pt10 a = 15–20 5.6 ± 0.9
b = 2.6–4.6

Oval morphotype
Pt3 a = 7.7–9.3 2.5 ± 0.4
b = 2.5–3.9
Pt9 a = 5.9–8.2 1.9 ± 0.3
b = 2.8–5.1
Accession name Length of each arm: a, a¢, a¢¢ (lm) a ⁄ a¢ and a ⁄ a¢¢ Morphology

Triradiate morphotype
Pt8 in batch culture a = a¢ = a¢¢ = 6.6–8.4 1.0 ± 0.1 1.0 ± 0.1

Pt8 in shaken culture a = a¢ = 5.3–8.2 1.0 ± 0.1 0.8 ± 0.1

a¢¢ = 8.1–11.0

a, a¢, a¢¢, and b, the minimum and maximum values of length or width of cells; a ⁄ b, the average values of length ⁄ width in fusi-
form and oval cells; a ⁄ a¢¢ and a ⁄ a¢, the average length of two arms in triradiate cells (number of cells examined = 60). Growth
conditions are described in Materials and Methods.

Concerning the fusiform morphotypes, when cells
were grown in the same laboratory conditions, three
major fusiform morphologies were observed (Fig 3,
d–m; Table 2). In addition to differences in the size
and shape of fusiform cells, the oval cells of Pt3
differed from those of Pt9 in that they were longer
and thinner (Fig. 3, n and o). Moreover, Pt9 oval cells
grown at 19C for several months tended to aggregate
in clusters of hundreds to thousands of cells within a
gelatinous matrix (Fig. 3, p and q), with round cells
typically present in the center of the aggregates
(Fig. 3r). We also observed plasticity of shape within
triradiate cells in cultures of Pt8. In particular, con-
stant shaking of cultures resulted in the appearance
of cells with nonsymmetrical and unequal arm
lengths (cf. Fig. 3, s and t). The observed morpho-
logical plasticity indicated that cell architecture was
able to vary in response to changing culture condi-
tions and was not strictly genetically based.
Siliceous frustules: The micromorphology and
nanostructure of diatom frustules is usually well
conserved within species and is used for descriptive
systematics. Phaeodactylum tricornutum cell walls have
the peculiarity of being only poorly silicified. After
acid treatment of cells derived from monoclonal
cultures of each morphotype, we observed that only
oval cells grown in the presence of silicic acid pos-
sessed a silicified cell wall organized into a typical
frustule (Fig. 4, a–f). Siliceous frustules were never
observed in fusiform or triradiate cells (Fig. 4, g
and h), nor in oval cells grown in the absence of
silicic acid (Fig. 4i). The silicified cell wall of oval
cells displayed typical features of silicified valves
from raphid pennate diatoms, with a feather-like
structure, although this frustule was rather reduced,
in agreement with previous observations (Lewin
et al. 1958). It was characterized by a longitudinal
and median slit, known as the raphe, and a large
 G E N O T Y P E S A N D P H E N O TY P E S O F A M O D E L D I A T O M 1001

central nodule with uniseriate striae containing sim-
ple areolae, like the raphid diatoms Sellaphora sp.
and Navicula sp. (Behnke et al. 2004). The apical
valve was slightly curved, producing a shape charac-
teristic of the genus Cymbella. We observed hooked
external polar raphe endings (Fig. 4c).
The number of costae and areolae per microme-
ter were strictly conserved in all 10 accessions, there-
fore representing a specific micromorphological,
taxonomic characteristic that could be useful for
the identification of P. tricornutum (Table 3). Never-
theless, the size of the frustule was highly variable
a c
pl b pl
d e f along two arms (Fig. S3 in the supplementary mate-
Pt1 Pt2 Pt3
g h
among the accessions. The frustule covered the
length of the oval cell wall, although the expansion
of silicified material on the lateral sides was highly
variable (Fig. S2 in the supplementary material;
Table 3). Valve morphology also varied significantly
within one monoclonal population (data not
shown). It is therefore not easy to identify any sig-
nificant differences among the accessions. This is
probably a consequence of the ability of oval cells to
be only facultatively silicified, and it probably also
reflects varying degrees of silicification during the
life cycle.
Scanning electron micrographs of Pt3 oval cells
indicated that only one valve of the oval cells pos-
sessed a raphe (Fig. 5). In general, no raphe was
pl observed in triradiate and fusiform cells (Fig. 5).
However, in spite of the absence of an organized
siliceous frustule, we nonetheless found a raphe in
triradiate cell walls that was oriented longitudinally
rial), an observation that had also been mentioned
previously (Wilson 1946) and that could correspond
to the differentiation of an oval cell into a triradiate
cell.
General physiological features of Phaeodactylum tri-
cornutum. Cell growth: Because certain physiological
features can be characteristic of particular ecotypes
or genotypes, we examined the growth characteris-
tics in a range of conditions. When cells of different
i accessions were grown in ASW without silicic acid in
acclimated culture conditions, we did not observe
significant differences in growth among fusiform
accessions (Table 4). They all displayed a maxi-
mum average growth rate (d)1) of 0.97 ± 0.06

Pt4 Pt5 Pt6

Fig. 3. Pleiomorphic character of Phaeodactylum tricornutum
j k l m and intraspecies morphological variability. (a–c) The three mor-
photypes of P. tricornutum cells. (a) Fusiform cells typically con-
sisted of a central body extended at both ends with two thinner
arms. The central body contained the main organelles, in particu-
lar one nucleus in a central position and a single, large golden-
brown parietal plastid (pl). (b) Two oval cells. Compared to the
morphology of the fusiform morphotype, oval morphotype cells
Pt7 Pt8 Pt9 Pt10 appeared like the central body without the arms. (c) Triradiate
cells consisted of a central body and three divergent arms of usu-
n p r ally equal length with radial symmetry. The three arms were not
arranged in the same plane but were derived from the central
body like a pyramid. Their cytology was similar to fusiform cells,
with the plastid often localized at the base of two arms and usu-
Pt9
ally spread in one of these arms. (d–m) Fusiform cells from the
s 10 different accessions. Three major fusiform morphologies were
observed. The three English (d–f) and the Finnish (g) accessions
were similar to each other in being long and thin. Cells from
Pt3 Pt9 Great South Bay (i), Nantucket Bay (j), Vancouver (k), Microne-
sia (l), and the Yellow Sea (m) were all characterized as short
o q and fat fusiforms. In contrast, fusiform cells of the Cape Cod Bay
Pt8 (h) were, on average, significantly longer and thinner than all
t the other accessions. (n and o) Morphology of oval cells from
Pt3 (n) and tropical accession Pt9 (o). (p and q) Aggregate of
oval cells from tropical accession Pt9 grown at 19C. (r) round
cells observed in the center of Pt9 cell aggregates. (s and t) Trira-
diate cells from the triradiate accession Pt8 grown in batch cul-
ture (s) and under agitation (t). Photographs were taken with
Pt9 Pt9 Pt8 differential interference contrast microscopy. Scale bars, 3 lm.
1002 A L E S S A N D R A D E MA R T I N O E T A L .

a b c a

pm
cm

d e f

b c

g h i

Fig. 4. TEM of the three morphotypes after acid treatment.
(a–f) Siliceous frustules of oval cells. The costae are arranged
roughly perpendicularly to the raphe, except in the region of the
central nodule where they sometimes diverge with radial orienta-
tion (e.g., [b], indicated by white arrows). In many frustules,
there was a Y ramification of one or two central costae, which
were always positioned immediately adjacent to the most central
costae (e.g., [b], indicated by black arrows). (a) Valve view of a
longitudinal raphe. (b) Central nodule region (cn). (c) Polar
nodule region (pn). (d–f) Micromorphological variability of the
central region. (g–i) Valves from fusiform (g) and triradiate (h)
morphotypes after very gentle acid treatment. Fusiform and trira-
diate morphotypes could be recognized, but without siliceous
frustules. In oval cells of accession Pt8 grown in the absence of
silicic acid, no frustule was observed (i). Black scale bar, 0.5 lm;
white scale bar, 3 lm.
corresponding to a doubling time (d) of
1.08 ± 0.06. However, both the oval (Pt3) and trira-
diate (Pt8) accessions grew significantly slower
(Table 4).
Despite these findings, the proportion of the dif-
ferent morphotypes in each culture was remarkably
well maintained. For example, morphotype stability
was observed over many generations for the oval
morphotype of accession Pt3 and for the triradiate
morphotype of accession Pt8 (Fig. 6). Conversely,
Fig. 5. SEM of the three morphotypes. (a) Fusiform cell from
Pt1. (b) Two oval cells, on valve top view from Pt3. The longitudi-
nal raphe is visible on one cell. (c) Triradiate cell from Pt8. No
raphe is visible in (a) and (c), but the junction between the two
valves can be seen. Scale bar, 1 lm.
addition of silicic acid (53 lM) did not modify the
growth rate of fusiform accessions, nor did it alter
the proportion of fusiform or triradiate cells in
these cultures, and no significant increase of oval
morphotype cells was observed (Fig. 7a, and data
not shown). However, we did observe an increase in
the number of oval but not fusiform cells in Pt3
and Pt9 cultures when silicic acid was added to the
medium (Fig. 7, b–e). The sensitivity of oval cells to
the presence of silicic acid was in agreement with
the fact that only oval cells contain organized silici-
fied frustules.
Morphotype plasticity: Strain Pt3, originally selected
for its ability to survive at low salinity (see above), is
characterized by an oval morphology. Conversely,
the tropical strain Pt9 is characterized by a change
from fusiform to oval morphotype at low tempera-
tures (Fig. 3, l and o). These observations indicate
that the oval morphotype is induced in suboptimal

Table 3. Frustule characteristics of oval cells isolated from different accessions: oval accession (Pt3), fusiform accession
(Pt2), and two monoclonal cultures of triradiate accession (Pt8, clone a and b).

Accession name Length (lm) Width (lm) Length ⁄ width Costaea number Areolaeb number Costae number in 1 lm Areolae number in 1 lm

Pt3 5.2–6.9 1.5–2.0 2.7–5.5 36–50 7–10 10 14

Pt2 6.1–7.6 1.4–2.0 3.3–4.2 44–48 7–8 10 14
Pt8 (clone a) 5.3–6.1 1.2–1.4 4.1–9 40–50 5–7 10 14
Pt8 (clone b) 5.8–6.9 0.9–2.0 3.8–5.7 46–58 6–9 10 14
a
Number of costae on each side of the raphe.
b
Number of areolae between the two subcentral costae. The two values correspond to the minimum and maximum values (aver-
age number of cells observed: n = 30).
 G E N O T Y P E S A N D P H E N O TY P E S O F A M O D E L D I A T O M
Table 4. Differences in maximum growth rates and doubling times among fusiform, oval, and triradiate accessions.
Fusiform
Accessions Pt1 Pt2 Pt4 Pt5
Growth 0.97 ± 0.04 1.08 ± 0.02 0.91 ± 0.04 0.99 ± 0.05
rate (d)1)
Doubling 1.03 ± 0.04 0.93 ± 0.02 1.10 ± 0.04 1.01 ± 0.05
time (d)
The data are based on three independent experiments. Growth conditions are described in Materials and Methods.
a
b sent different models for forward and reverse genet-
Fig. 6. Stability of the proportion of oval morphotype cells in
the oval accession Pt3 (a) and of triradiate morphotype cells in
the triradiate accession Pt8 (b). Each point represents an average
from three experiments. Cells were grown in artificial seawater
medium (ASW) without silicic acid.
conditions and suggest that such cells may have a
better capacity for acclimation compared to fusi-
form or triradiate cells. Moreover, in all cultures, we
observed that only oval cells could survive in low-
light conditions (5 lmol Æ m)2 Æ s)1) and without
enrichment of nutrients in the media (data not
shown). We also noted that the proportion of oval
cells increases significantly in stationary phase cul-
tures compared to fusiform or triradiate cells. These
observations were seen in all accessions, even when
oval cells were rare or absent in the starting cul-
tures, as was the case in the triradiate accession Pt8
(data not shown). Furthermore, the oval cells
tended to aggregate in clusters, and we sometimes
observed rounded cells in the center of the aggre-
gates (Fig. 3, p and q). These aggregates of hun-
dreds to thousands of mixed oval and round cells
were typically surrounded by mucilage and strongly
1003
Oval Triradiate
Pt6 Pt7 Average Pt3 Pt8
0.98 ± 0.03 0.91 ± 0.02 0.97 ± 0.06 0.71 ± 0.03 0.73 ± 0.04
1.02 ± 0.03 1.10 ± 0.02 1.08 ± 0.06 1.41 ± 0.03 1.37 ± 0.04
adhered to the culture flasks (data not shown). Fur-
thermore, triradiate cells were never observed in
these conditions, and a large number of ghost cells
were observed in stationary-phase cultures of Pt8,
indicating that the triradiate cells died in conditions
of nutrient starvation. Cell aggregation and polysac-
charide secretion could represent mechanisms of
physiological acclimation to protect cells during
nutrient starvation, and the round cells could repre-
sent a previously unknown resting stage in the life
cycle of P. tricornutum.
Genetic transformation of different morphotypes: In the
future, the different morphotypes and accessions of
P. tricornutum identified in this work might repre-
ics, as well as for genomic approaches in this
species. For example, the oval morphotype provides
a unique opportunity to study the silicification pro-
cess of diatom frustules in an inducible system.
Genetic transformation of P. tricornutum has only
been reported previously for the fusiform morpho-
type using the English accession Pt1 (Falciatore
et al. 1999, Siaut et al. 2007) and the Finnish acces-
sion Pt4 (Apt et al. 1996, Zaslavskaia et al. 2000, Kil-
ian and Kroth 2005). It is therefore important to
extend genetic transformation to all morphotypes
and accessions. In the current study, we have been
able to transform all three morphotypes and all
genotypes at highly similar efficiencies (data not
shown), although it is more difficult to obtain trans-
formants with the triradiate morphology. Typically,
after transformation of a monoclonal culture of
100% triradiate cells, only 25% of the transformants
were triradiate, whereas the rest were fusiform.
Moreover, the morphology of most of the triradiate
clones was not stable, and they became fusiform or
oval during successive subculturing (Fig. 8). This
finding demonstrates that one clonal triradiate cell
can change shape to fusiform or oval cells. In the
cultures, we observed new transient morphotypes, in
particular a ‘‘boomerang morphotype’’ and a fusi-
form cell containing two oval cells inside (Fig. 8),
which we describe as ‘‘transitory morphotypes’’ dur-
ing morphotype differentiation. This morphotype
instability was only rarely observed in the seven fusi-
form accessions and in the oval accession Pt3. These
observations suggest that the triradiate morphotype
may be less amenable to transformation and to
heterologous gene expression as compared to
1004 A L E S S A N D R A D E MA R T I N O E T A L .

b c

d e

Fig. 7. Effect of silicic acid on growth of Phaeodactylum tricornutum. (a) Growth rates and doubling times of Pt1 (fusiform cells), Pt3
(oval cells), and Pt9 (oval cells) in the presence and absence of silicic acid. (b–e) Differences in growth of oval and fusiform cells from
oval accession Pt3 (b and c, respectively) and from the tropical accession Pt9 (d and e, respectively) when cells were grown in the absence
(d) or presence (s) of silicic acid. Each point represents an average of two samples. Cells were grown in artificial seawater medium
(ASW) minus (–Si) or plus silica (+Si) at 53 lM.

fusiform and oval morphotypes. This phenomenon
may be because transformation imposes a stress on
triradiate cells that triggers a change to the fusiform
or oval morphotype. These results again suggest that
the different morphotypes correspond to different
phenotypes.
DISCUSSION
Although P. tricornutum is generally not consid-
ered to be widely distributed in nature, it has been
extensively used for physiological and biochemical
research in diatoms for several decades, and as a
food source for the aquaculture industry because of
its ease of cultivation and its rich oil content. As is
clear from the historical summary reported here, sev-
eral isolates have been collected around the world
over the last century and maintained as permanent
stocks in algal culture collections. In more recent
years, P. tricornutum has entered a new era following
the development of molecular tools for functional
genomics (Maheswari et al. 2005, Montsant et al.
2005) and its subsequent choice as the second dia-
tom for whole genome sequencing (http://genome.
jgi-psf.org/Phatr2/Phatr2.home.html). We therefore
consider it important to compile a summary of the
species resources currently available to researchers
and to provide a genetic and phenotypic overview of
their characteristics. This compilation should (i)
allow laboratories working with P. tricornutum to
know the origin of their strains, (ii) permit scientists
interested in studying specific characteristics to
choose the appropriate accession, and (iii) open up
possibilities to use P. tricornutum accession resources
to search for polymorphisms of functional signifi-
cance in genes of interest.
We decided to focus principally on stock center
accessions that were unialgal and axenic, and that
had a known geographical origin and date of collec-
tion. In total, we have characterized 10 different
accessions, which constitute a species complex in
that they represent four different genotypes that dis-
play different phenotypic characteristics. For exam-
ple, two accessions are characterized by differences
 G E N O T Y P E S A N D P H E N O TY P E S O F A M O D E L D I A T O M 1005

in the dominant morphotype: an oval accession
(Pt3), and a triradiate accession (Pt8). Another can
be defined as a tropical accession (Pt9) because it
appears better acclimated to growth at higher tem-
peratures. Our analyses indicate that changes in
a used for genome sequencing, because this is likely
b logeny (Table 1; Figs. 2 and 3). For example, even
c
d together, in spite of being characterized by very dis-
e morphotype and genotype. The fusiform cells from
f strains (Pt6, Pt7).
g
growth conditions are associated with morphotype
transformations, and so this strain represents useful
material for studying how pleiomorphy is related to
culture conditions.
In addition, we have documented the isolation of
the clonal population (denoted Pt1 8.6) that was
to become the strain of reference for future molecu-
lar studies, and we have developed a PCR primer
that can be used to detect P. tricornutum in field
samples.
Our analyses have revealed only limited correla-
tions between morphology, biogeography, and phy-
though the tropical accession (Pt9) has a morphology
different from the English strains (Pt1, Pt2, Pt3) and
is derived from a very different geographical location,
it clustered with them in the same clade; whereas the
Finnish strain (Pt4) did not, in spite of its similar fusi-
form cell morphology and closer geographical loca-
tion (Fig. 2a; Table 1). The three American strains
were collected in close proximity, but the Cape Cod
Bay strain (Pt5) did not cluster with the others, con-
sistent with its distinct morphology characterized by
long fusiform cells. Conversely, the Cape Cod Bay
strain (Pt5) and the Chinese strain (Pt10) clustered
tinct biogeographies and morphologies. Although
Cape Cod Bay, Great South Bay, and Nantucket Bay
are found in the same geographical area, the Cape
Cod Bay strain had been collected 20 years after the
other two accessions.
Only limited correlations were found between
the three English strains (Pt1, Pt2, Pt3) had similar
morphologies and clustered within the same clade,
although Pt3 cultures contain mainly the oval mor-
photype (Figs. 2a and 3). Conversely, although Pt8
cultures were characterized by a majority of triradi-
ate cells, the strain clustered with the two American
Phaeodactylum tricornutum is therefore character-
ized by a high intraspecies morphological variability,
related to the high level of pleiomorphy that charac-
terizes the species. Furthermore, the abundance of
oval and triradiate cells in the Pt3 and Pt8 strains,
respectively, might not be genetically determined
but could correspond to distinct ecophenotypes
(see below).

Fig. 8. Morphotype diversity and differentiation in a clonal

population of transformed cells from Pt8, expressing cytosolic
eYFP. In this clone, transformed cells were 100% triradiate after
transformation (a). During subcultures, we observed an increase
h of triradiate cells with short arm and ‘‘boomerang morphotype’’
(c and d), then a majority of fusiform cells (e) and oval cells (f).
We observed the differentiation of ovals cells within a fusiform
cell (g and h), showing that the cytosolic-YFP is located exclu-
sively in the oval cells and not in the arms of the mother fusiform
cells. Left panel: bright-field views; central panel: chl-derived red
autofluorescence; right panel: eYFP fluorescence. Scale bars,
3 lm.
1006 A L E S S A N D R A D E MA R T I N O E T A L .
The limited correlations observed between mor-
phology, genotype, and site of collection may imply
that heritable changes have occurred during culture
of the different strains in the laboratory. This
phenomenon has been very poorly studied in phyto-
plankton species. For P. tricornutum, the fact that
many of the strains were isolated a long time ago
and have been subsequently distributed to different
stock centers around the world could allow such
divergence to be examined.
In spite of the significant morphological variabil-
ity that was observed among different strains, the
high level of ITS2 sequence similarity that we
observed between them is similar to what has been
reported in other diatoms (Lundholm et al. 2003,
Behnke et al. 2004, Orsini et al. 2004) and implies
that all 10 accessions belong to the same species.
Our data from AFLP analysis is generally consistent
with the ITS2 analyses and also revealed levels of
polymorphism similar to what has been seen in
other diatoms (de Bruin et al. 2004). Four distinct
genotypes could be detected, and Pt5 appears to be
most different from the others, based on genetic
polymorphism (Figs. 1 and 2) and morphology
(longer fusiform cells; Fig. 3). However, evidence of
mating incompatibility will be needed to confirm
whether the four identified genotypes correspond
to cryptic species. Lewin et al. (1958) occasionally
observed spherical auxospores attached to an empty
wall of a fusiform cell, but her attempts to follow
the fate of individual auxospores on agar were
unsuccessful. We have also noted the appearance of
such structures, particularly in aging or stressed cul-
tures (data not shown). If sexual reproduction can
be controlled in the laboratory, it will be infor-
mative to study sexual capability and mating
ompatibility between the different genotypes and to
follow ITS2 genetic variability in F1 populations. For
this purpose, sexual reproduction in P. tricornutum
needs to be more carefully investigated.
The EM analysis showed that fusiform and triradi-
ate cells were devoid of a silicified valve, in agree-
ment with previous observations (Lewin et al. 1958,
Borowitzka and Volcani 1978). On the other hand,
oval cells do possess a siliceous valve with character-
istics typical of raphid pennate diatoms, resembling,
for example, those of the genus Cymbella (Lewin
et al. 1958). Most of our EM observations of oval
cells were made with Pt3, for which only one silici-
fied valve was present, in agreement with previous
observations on the Woods Hole strain (Pt6; Lewin
et al. 1958). Nonetheless, this finding contrasts with
observations of the Long Island strain (Pt7; Boro-
witzka and Volcani 1978), which indicated that both
valves of oval cells were sometimes silicified.
Although identical ITS2 sequences were found
among clonal populations of the Blackpool strain
(Pt1), variability in valve size was nonetheless
observed (Fig. S2; Table 3). It is therefore not possi-
ble to use these micromorphological characteristics
to draw conclusions about phylogenetic relation-
ships, and so intrapopulation variability among cells
derived from a single clone could be related either
to the variability in size of different daughter cells
or to the variation of silicification of their cell walls.
However, all the accessions of P. tricornutum have
identical striation patterns and densities (Table 3)
that do represent good criteria for the identification
of the species.
The limited information about P. tricornutum
distribution in nature indicates it to be a coastal
species that most often occurs in unstable environ-
ments, such as estuaries or rock pools. The ability of
the species to adapt to changing environmental con-
ditions could be related to the pleiomorphic charac-
ter of the cells, and our results indicate that the
three morphotypes are indeed physiologically differ-
ent. The oval morphotype appears to be induced as
an acclimation response to suboptimal growth con-
ditions, as evidenced by the fact that Pt3 was
selected by growth in low salinity and is oval, that
the tropical strain Pt9 is oval at low temperatures,
and that oval cells accumulate in all cultures when
left in stationary phase for extended periods
(1 month or more) and on agar plates (Wilson
1946). Clear differences in growth rate were also
observed between oval and triradiate cultures com-
pared to fusiform cultures; maximal growth rates of
fusiform accessions were 1.4 times higher
(Table 4). It was also observed that the absence of
silicic acid resulted in a 50% decrease in the growth
rate of oval cells in the oval strain Pt3, whereas the
availability of silicic acid does not appear to impact
growth of fusiform cultures (Fig. 7).
We also found evidence that the life cycle of
P. tricornutum includes a ‘‘round’’ morphotype
(Fig. 3r) that appears better enabled to survive long
periods of stress. The oval and round morphotypes
both secrete mucilage and grow as clumps attached
to the bottom of culture flasks, whereas fusiform
cells remain in suspension. The chemical structure
of the walls of oval cells compared with fusiform
cells has been described previously (Gutenbrunner
et al. 1994), and it was shown that the mucilaginous
capsular material of oval cells can represent 16% of
the dry weight. In contrast to our study, these
authors did not observe any effect of salinity
changes on the morphotypes. It should be noted,
however, that this study was based on accession
MUR-138, which is likely to be distinct from Pt3
(see above and supplementary material).
In contrast to oval cells, triradiate cells appear to
be much more sensitive to stress and rapidly disap-
pear (or convert to oval ⁄ fusiform cells) when
growth conditions are suboptimal (A. De Martino,
A. Meichenin, and C. Bowler, unpublished data). It
has also been noted that fusiform and triradiate
cells are more buoyant than oval cells (Lewin et al.
1958) and so would be expected to be better
adapted to a planktonic lifestyle than oval cells.
 G E N O T Y P E S A N D P H E N O TY P E S O F A M O D E L D I A T O M
These observations therefore infer the impor-
tance of pleiomorphic responses to changing envi-
ronmental conditions and suggest that the oval and
triradiate morphotypes may represent distinct ec-
ophenotypes, each one specifically adapted to
growth in particular conditions. Borowitzka and Vol-
cani (1978) have suggested that differences in vacu-
olar structure may be an important determinant of
this adaptive capacity, and Dickson and Kirst (1987)
reported that glycine betaine and proline levels
increase in P. tricornutum cells in response to high
salinity. The impressive adaptive capacity of P. tricor-
nutum is currently being explored in our laboratory
(A. De Martino, A. Meichenin, and C. Bowler,
unpublished data), and the contribution of genetic
and epigenetic factors in the regulation of pleio-
morphy will be addressed.
To further investigate the ecological distribution
and genetic diversity of P. tricornutum in natural
environments, it is now possible to detect it in
natural samples using the specific ITS2 primers
that we have developed in this study. Because
coastal waters are unstable environments, and
because our results indicate that oval and round
cells are the most abundant morphotypes in stress
conditions, it is possible that P. tricornutum typi-
cally occurs as these rather nondescript morpho-
types in natural environments, and so the
ecological distribution of the species may have
been underestimated. Moreover, our results indi-
cate that P. tricornutum should predominantly
occur in benthic communities because oval and
round cells tend to aggregate and sink and can
adhere strongly to surfaces (data not shown). This
hypothesis is reinforced by a recent ribotype analy-
sis of a biofilm collected from the hardwater creek
‘‘Westerhoefer Bach’’ in Lower Saxony (near
Braunschweig), Germany, based on 18S rDNA
sequencing, which revealed the presence of P. tri-
cornutum (T. Friedl, University of Goettingen, Ger-
many, personal communication). However, from
the information available, it appears that all iso-
lates (except the oval accession Pt3) are derived
from planktonic samples, so it is not surprising
that the majority have fusiform or triradiate mor-
photypes. The P. tricornutum–specific primers
reported in this manuscript should therefore be
useful for clarifying the ecological and geographi-
cal distribution of the species.
The presence of distinct genotypes and ecophe-
notypes of P. tricornutum has important implications
for future studies of this species as a model for for-
ward and reverse genetics, as well as genomics
approaches in diatoms. Studies with P. tricornutum
should take the accession number into account
and consider possible gene polymorphisms of func-
tional significance and differential gene expression
among accessions. It will be important to focus
studies on one model accession for comparative
studies. We consider the Pt1 accession to be the
1007
optimal reference model for genetic and genomic
studies because we have already generated
>100,000 ESTs from this strain (U. Maheswari and
C. Bowler, unpublished data), and the whole gen-
ome sequence has been generated from a mono-
clonal culture derived from Pt1 (named Pt1 8.6,
CCAP1055 ⁄ 1). Nonetheless, it will be interesting to
expand analyses to the other accessions that exhi-
bit particular physiological properties and that
offer advantages for specific biological processes.
For example, the oval strain Pt3 is more appropri-
ate to study silicic acid metabolism, gliding, and
adherence. This accession is also euryhaline, so it
will be interesting to study the response of oval
morphotypes to salinity stress or nutrient limitation
at the molecular level. The tropical accession is
interesting because it is acclimated to high temper-
atures and shows cold sensitivity as manifested by a
change of shape to the oval morphotype.
The availability of different accessions of P. tricor-
nutum that display different phenotypic variations
can also allow an exploration of polymorphism pat-
terns in particular genes of interest. By comparing
these patterns with those expected under standard
neutral models, it may be possible to identify func-
tionally significant polymorphisms related to partic-
ular traits. As such, links may be identified between
molecular analyses of gene function and evolution-
ary investigations of adaptation and natural selec-
tion. Such an approach has been used with great
success in Arabidopsis ecotypes to identify genes
underlying ecologically important complex traits,
such as responses to day length variation (reviewed
in Mitchell-Olds and Schmitt 2006), and its utility
will be further potentiated as new methods of high-
throughput DNA sequencing and polymorphism
analysis become adopted.
Finally, although P. tricornutum is an unusual dia-
tom in that it is pleiomorphic and uses silicic acid
only facultatively, these features can be exploited to
explore the molecular basis of cell-shape control
and to study silicon-based nanofabrication in an
inducible experimental system. We have been able
to transform genetically all the accessions as well as
the three morphotypes (Fig. 8), so genome-enabled
reverse genetic approaches to address fundamental
questions such as these are realistic future objec-
tives.
We thank members of CCAP, CCMP, CCCM, UTEX, SAG,
and CSIRO culture collection centers for their help in recov-
ering information on the history of their accessions of P. tri-
cornutum, in particular Tracey Riggens from CCMP, Christine
Campbell from CCAP, and Elaine Simons from CCCM. We
are grateful to Bess Ward who kindly provided the marine
samples. We thank people from the laboratories of Marina
Montresor and Adriana Zingone at the Stazione Zoologica
(Naples, Italy) for their valuable discussions and recommen-
dations, especially Mario De Stefano and Federica Cerino for
their technical help in the preparation of the samples for
electronic microscopy. We are grateful to Andrew Allen and
Chantal Guidi-Rontani for critical reading of the manuscript.
1008 A L E S S A N D R A D E MA R T I N O E T A L .
Partial funding for this work was obtained from the EU-
funded FP5 MarGenes project (QLRT-2001-01226), the EU-
FP6 Diatomics project (LSHG-CT-2004-512035), the EU-FP6
Marine Genomics Network of Excellence (GOCE-CT-2004-
505403), an ATIP ‘‘Blanche’’ grant (CNRS), and the Agence
Nationale de la Recherche (France).
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 G E N O T Y P E S A N D P H E N O TY P E S O F A M O D E L D I A T O M 1009

Supplementary Material
The following supplementary material is avail-
able for this article:
Table S1. Comparative amplified fragment
length polymorphism (AFLP) analysis of Pt1, Pt4,
Pt5, and Pt8 accessions.
Figure S1. Alignment of internal transcribed
spacer 2 (ITS2) sequences from the 10 accessions
of Phaeodactylum tricornutum (Pt1–Pt10) and from
the environmental samples (ST30–ST82). The
differences in nucleotides or gaps are marked
(*).
Figure S2. Interspecific and intraspecific
micromorphological variability of the frustules of
Phaeodactylum tricornutum. (a and b) Frustules of
oval cells from Pt1 (a) and Pt6 (b). (c, d, and e)
Frustules from monoclonal culture of Pt3 oval
cells. (f, g, and h) Frustules from monoclonal
cultures of Pt8 oval cells. Scale bars, 0.5 lm.
Figure S3. Scanning electron micrographs of a
triradiate cell from Pt8, on which a raphe is visi-
ble extending along two arms. Scale bar, 1 lm.
Appendix S1. Historical overview of P. tricornu-
tum.
This material is available as part of the online
article from: http://www.blackwell-synergy.com/
doi/abs/10.1111/j.1529-8817.2007.00384.x.
(This link will take you to the article abstract).
Please note: Blackwell Publishing is not
responsible for the content or functionality of
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