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J. Phycol. 42, 464–481 (2006)
r 2006 Phycological Society of America
DOI: 10.1111/j.1529-8817.2006.00211.x

INTER- AND INTRASPECIFIC VARIATION OF THE PSEUDO-NITZSCHIA

DELICATISSIMA COMPLEX (BACILLARIOPHYCEAE) ILLUSTRATED
BY rRNA PROBES, MORPHOLOGICAL DATA AND
PHYLOGENETIC ANALYSES1

Nina Lundholm, Øjvind Moestrup2

Department of Phycology, Biological Institute, University of Copenhagen, Øster Farimagsgade 2D, DK-1353
Copenhagen K, Denmark

Yuichi Kotaki
School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0101, Japan

Kerstin Hoef-Emden
Gyrhofstrasse 15, Botanical Institute, University of Cologne, 50931 Cologne, Germany

Chris Scholin
Monterey Bay Aquarium Research Institute, PO Box 628, Moss Landing, California 95039, USA

and
Peter Miller
Ocean Sciences, University of California, 1156 High Street, Santa Cruz, California 95039, USA

A study of 25 cultures tentatively identified as
Pseudo-nitzschia delicatissima (Cleve) Heiden, and
originating from geographically widely distributed
locations, showed both morphological and genetic
variation among strains. Use of rRNA-targeted DNA
probes on 17 different strains showed large varia-
tion in the hybridization patterns. Detailed morpho-
logical studies placed the isolates into three groups.
The sample on which the neotype of P. delicatissima
is based was also examined, and used to establish
the morphological identity of P. delicatissima.
Phylogenetic analyses of 16 strains, based on se-
quences of internal transcriber spacer 1 (ITS1), 5.8S
and ITS2 of the nuclear-encoded rDNA, supported
the morphological observations and the hybridiza-
tion studies, and revealed large genetic variation
among strains. A combination of the morphological
and molecular findings resulted in the description
of two new species, P. decipiens sp. nov. and P. do-
lorosa sp. nov. P. dolorosa has a mixture of one or two
rows of poroids in the striae whereas P. delicatissima
always has two rows. In addition, P. dolorosa has
wider valves and a lower density of poroids. P. deci-
piens differs from P. delicatissima by a higher density
of striae on the valve face as well as a higher density
of poroids on the girdle bands. Among the strains
referred to P. delicatissima, an epitype was selected.
Large genetic variation was found among the P. del-
1
Received 28 May 2003. Accepted 26 December 2005.
2
Author for correspondence: e-mail moestrup@bi.ku.dk.
icatissima strains and a subdivision into two major
clades represent cryptic species.
Key index words: Bacillariaceae; cryptic diversity;
diatoms; intraspecific; ITS rDNA; morphology; P.
decipiens; P. dolorosa; phylogeny; probes; taxonomy;
Pseudo-nitzschia delicatissima
Abbreviations: AU, approximately unbiased; DA, do-
moic acid; DIC, differential interference contrast;
ITS, internal transcriber spacer; LM, light micro-
scopy; ME, minimum evolution; ML, maximum
likelihood; MP, maximum parsimony; NJ, neigh-
bor-joining; SSCP, single stranded conformation po-
lymorphism; TVM, transversion model
The most detailed description of Pseudo-nitzschia del-
icatissima (Cleve) Heiden and its geographic distribu-
tion is by Hasle (1965; as Nitzschia actydrophila).
P. delicatissima has been reported worldwide (Hasle
1965, 2002, Kaczmarska et al. 1986, Hasle et al.
1996, Hasle and Syvertsen 1997, Skov et al. 1999),
sometimes as the dominant diatom species in the ma-
rine plankton, for example in the North Atlantic (Has-
le et al. 1996). In addition to being very common,
P. delicatissima has been shown to produce domoic acid
(DA) in a preliminary report by Smith et al. (1990).
Subsequently, Rhodes et al. (1996) detected small
amounts of DA in cultures, whereas Villac et al.
(1993) and Lundholm et al. (1994) failed to find DA
in their cultures. However, both toxic and non-toxic

464
 GENETIC VARIATION OF P. DELICATISSIMA
isolates of the same species have been reported for
several other Pseudo-nitzschia species (Villac et al. 1993,
Lundholm et al. 1994, Bates et al. 1998).
Large morphological variation among specimens
assigned to P. delicatissima was previously reported by
Rivera (1985) who expanded the described measure-
ments for P. delicatissima (Hasle 1965) to include speci-
mens with a wider valve, a lower density of fibulae and
striae, and a lower density of the poroids. Rhodes et al.
(1998) found isolates from New Zealand, which mor-
phologically represented a mixture of characters from
P. delicatissima and P. turgidula (Hustedt) Hasle, espe-
cially with respect to valve width and density of po-
roids. Similarly, Hallegraeff (1994) described variants
of P. turgidula from Australia that differed from the
description in Hasle (1965) by having a higher density
of fibulae and striae. And finally, large genetic variation
has been found among strains of P. delicatissima from
the Gulf of Naples (Orsini et al. 2004).
We have established a number of cultures tentative-
ly identified as P. delicatissima from geographically
widespread locations. Morphological variation was ob-
served between the isolates and 25 cultures were there-
fore selected and examined in detail. Additionally, 10
different rRNA-targeted probes developed by Miller
and Scholin (1996, 1998, 2000) were tested on 17 of
the cultures.
Following the results of the probe hybridization and
the morphological studies, 16 cultures were selected
and used for a phylogenetic study of sequences of in-
ternal transcriber spacer 1 (ITS1), 5.8S and ITS2 of
the nuclear-encoded rDNA. The two spacer regions
ITS1 and ITS2 show greater genetic variation than the
coding regions and have been used for phylogenetic
analyses at the intraspecific, interspecific, and interge-
neric level (Coleman and Mai 1997, Mai and Coleman
1997, Coleman 1999, Lundholm et al. 2003, Orsini
et al. 2004). Despite the presence of a high copy
number of the rDNA operon in general, the different
copies have usually been found to be homogeneous
(Baldwin et al. 1995) and this also applies to Pseudo-
nitzschia (Cangelosi et al. 1997, Orsini et al. 2004).
Finally, some of the cultures were tested for the
production of DA. The cultures chosen represent dif-
ferent morphological groups and different phylo-
genetic clades.
MATERIALS AND METHODS
Cultures and field material. All cultures were clonal and
non-axenic (Table 1). They were isolated by us or acquired
from Eun Cho, Francisco Rodrigues, Jette Skov, Lars
Holtegaard, and Yang Zhenbo. Cultures were grown at 241 C
or 151 C under a 12:12 or 16:8 light:dark (L:D), respectively,
and a photon flux rate of 20–60 mmol photons
m  2
s  1
in 32 psu L1-medium (Guillard and Hargraves 1993).
Microscopy. A subsample was taken soon after the culture
was established or acquired, and fixed in 2% glutaraldehyde.
Organic material was removed as described in Lundholm
et al. (2002a). Live cultures and Naphrax slides were studied
using a Zeiss Axiophot (Oberkochen, Germany) or an Olym-
pus BH-2 microscope (Tokyo, Japan).
465
For TEM, drops of cleaned material were placed on Form-
var-coated copper grids, dried, and studied in JEM-100SX or
JEM-1010 electron microscopes (JEOL, Tokyo, Japan) operat-
ing at 80 kV. The cells were examined for transapical axis
(width of valve) and apical axis (length of valve), density of
striae, fibulae and poroids on valves, structure of girdle bands,
and for the pattern of perforations in poroid hymenes. Meas-
urements of apical length were mainly performed using light
microscopy (LM). Fixed material of several cultures has been
deposited at the Botanical Museum in Copenhagen as num-
bers Phyc886 (Mex13, GranCan1), Phyc887 (300, Calif 1, Ca-
lif3, BP2, BP3), and Phyc888 (Læs5, ØM2, OFPd972, MexA,
Catsell2, Castell3, Castell4 and BP1). Terminology follows
Anonymous (1975), Ross et al. (1979), Mann (1981), and
Round et al. (1990).
Molecular probes. Ribosomal RNA-targeted probes devel-
oped for Pseudo-nitzschia (Miller and Scholin 1996, Scholin
et al. 1996) were applied to 17 different cultures (Table 2). All
probes were based on sequences of large subunit (LSU)
rDNA from cultures isolated from Monterey Bay, California
(Scholin et al. 1994). Application of the probes was by the
whole cell hybridization technique according to Miller and
Scholin (1998, 2000). In addition to a ‘‘no probe’’ control
reaction, 10 different probes were utilized for all isolates: the
negative control probe (uni-r), the positive control probe
(uni-c) and eight probes developed for different Pseudo-nit-
zschia species: auD1 (P. australis Frenguelli), muD1 (P. multi-
series (Hasle) Hasle), muD2 (P. multiseries and a species
identified as P. pseudodelicatissima (Hasle) Hasle), puD1 (P.
pungens (Grunow ex Cleve) Hasle), frD1 (P. fraudulenta
(Cleve) Hasle), deD1 (P. delicatissima), heD2-2 (P. heimii Man-
guin) and amD1 (P. americana (Hasle) Fryxell) (Miller and
Scholin 1996). The reactions were examined in a Zeiss
Axioskop with a fluorescein band-pass filter set (Miller and
Scholin 2000).
DNA extraction, amplification, and sequencing. Subsamples
of the cultures were concentrated by centrifugation and fro-
zen. Extraction followed the CTAB (N-Cetyl-N,N,N-tri-
methyl-ammoniumbromide) method (Doyle and Doyle
1987) with the modifications reported by Lundholm et al.
(2002a). The ITS1, 5.8S and ITS2 of the nuclear-encoded
rDNA (hereafter ‘‘ITS’’) were amplified using the primers
listed in Lundholm et al. (2003). The procedure was as fol-
lows: one initial denaturation at 941 C for 2 min followed by
30–32 cycles of 941 C for 30 s, 601 C for 25 s and 721 C for
25 s; and finally 721 C for 2 min. The PCR products were
visualized on a 2% Nusieve gel and subsequently purified
using QIAquick PCR Purification Kit (Qiagen GmbH, Hil-
den, Germany) as recommended by the manufacturer. Twen-
ty to forty nanograms PCR product were used in each 20 mL
sequencing reaction using the sequencing primers of Lund-
holm et al. (2003). Nucleotide sequences were determined
using the Dye Terminator Cycle Sequencing Ready Reaction
Kit (Perkin Elmer, Foster City, CA, USA) as recommended by
the manufacturer. Sequencing was performed using a ABI
Prism 377 DNA sequencer (Perkin Elmer). The accession
numbers of sequences obtained from the strains sequenced
in the present study or from Lundholm et al. (2003) are list-
ed in Table 1.
Alignment and phylogenetic analyses. The ITS sequences
were aligned by eye using the multiple alignment sequence
editor SeaView; non-alignable regions were excluded before
the phylogenetic analyses (Galtier et al. 1996). The final data
set comprised 38 taxa and 622 positions (for taxon sampling
and accession numbers, see Table 1). Modeltest 3.6 was used
to determine the evolutionary model that best fitted the data
according to the Akaike Information Criterion; a transver-
sion model (TVM) with invariable sites estimation and ap-
plication of a gamma distribution with four rate categories
466 NINA LUNDHOLM ET AL.

TABLE 1. Pseudo-nitzschia cultures studied morphologically, used in phylogenetic studies, and applied to rRNA probes.

Taxon Strain designation Origin Isolator Accession number

P. dolorosa BP2 Boca Piccola, Italy N. Lundholm DQ336155

BP3 Boca Piccola, Italy N. Lundholm DQ336151
Calif1 Monterey Bay, California N. Lundholm DQ336152
Calif3 Monterey Bay, California N. Lundholm DQ336154
300 Ria de Aveiro, Portugal F. Rodrigues DQ336153
P. delicatissima MexA Gulf of Mexico, off Tuxpam, Mexico Ø. Moestrup/N. Lundholm DQ329211
Zhenbo6 Port Shelter, HongKong Y. Zhenbo
OFPd972 Ofunato Bay, Japan Y. Kotaki DQ329208
OFPd973 Ofunato Bay, Japan Y. Kotaki
Tasm10 Hobart, Tasmania N. Lundholm AY257848
ØM2 Aveiro, Portugal Ø. Moestrup DQ329207
Læs1 Læs, Kattegat, Denmark N. Lundholm
Læs2 Læs, Kattegat, Denmark N. Lundholm DQ329206
Læs5 Læs, Kattegat, Denmark N. Lundholm AY257849
MexB Gulf of Mexico, off Tuxpam, Mexico Ø. Moestrup
BP1 Boca Piccola, Italy N. Lundholm DQ336150
Castell1 Castellamare, Italy N. Lundholm DQ329210
Castell2 Castellamare, Italy N. Lundholm DQ319212
Castell3 Castellamare, Italy N. Lundholm
Castell4 Castellamare, Italy N. Lundholm
P. decipiens Mex13 Gulf of Mexico, off Tuxpam, Mexico Ø. Moestrup/N. Lundholm DQ336156
Mex14 Gulf of Mexico, off Tuxpam, Mexico Ø. Moestrup
Gran Can1 Arguineguin, Gran Canaria, Canary Islands N. Lundholm
GranCan4-1 Arguineguin, Gran Canaria, Canary Islands N. Lundholm DQ336157
Tenerife1 Tenerife, Canary Islands N. Lundholm
P. australis ØM1 Aveiro, Portugal Ø. Moestrup AY257842
P. calliantha DS2 Do Son, North Vietnam J. Skov AY257856
TA-1 Thuan An, Central Vietnam J. Skov AY257855
P. cuspidate Mex12 Near Tuxpam, Mexico N. Lundholm/Ø. Moestrup AY257852
Sydney1 Bondi Beach, Sydney, Australia N. Lundholm AY257862
Tenerife8 Tenerife, Canary Islands N. Lundholm AY257853
P. fraudulenta Limens1 Limens, Spain K. Grostl/N. Lundholm AY257840
P. galaxiae Mex23 Near Tuxpam, Mexico N. Lundholm/Ø. Moestrup AY257850
Sydney4 Bondi Beach, Sydney, Australia N. Lundholm DQ336158
P. inflatula No7 Phuket, Thailand K. Priisholm DQ329204
P. micropora VPB-B3 Van Phong Bay, Vietnam J. Skov AY257847
No16 Phuket, Thailand K. Priisholm DQ329209
P. multiseries mu3 Monterey Bay, CA, USA P. Miller, C. Scholin AY257844
P. multistriata Korea A Chinhae Bay, Korea E. Cho AY257843
P. pseudodelicatissima P-11 Gafahna, Portugal N. Lundholm AY257854
P. pungens P-24 Costa Nova, Portugal N. Lundholm AY257845
Mex18 Near Tuxpam, Mexico Ø. Moestrup AY257846
P. seriata Nissum3 Nissum Bredning, Denmark N. Lundholm AY257841
P. sp. Hobart5 Hobart, Tasmania, Australia L. Holtegaard AY257851
P. subcurvata 1-F Ross Sea, 74102 0 S, 140176 0 W N. Lundholm DQ329205
P. cf. subpacifica RdA8 Ria de Arousa, Spain N. Lundholm AY257860
P. turgiduloides 3–19 Ross Sea, 74159 0 S, 145119 0 W N. Lundholm AY257839

(Posada and Crandall 1998). The proposed likelihood set-
tings were used for maximum likelihood (ML) analysis and
for calculating the distance matrix in neighbor-joining (NJ)
analysis. The NJ (set to minimum evolution), ML (three ran-
dom-addition replicates) and unweighted maximum parsi-
mony (MP) analyses (10 random-addition replicates) were
performed with the PAUP* 4.0b10 portable version under
Debian GNU/Linux 3.0 or Solaris (Swofford 2002). For 50%
majority-rule consensus trees, 1000 bootstrap subsamples
(NJ and MP) or 500 bootstrap subsamples (ML) were sum-
marized. Bayesian analyses were performed using MrBayes 3
(5 million generations, sampling every 100th generation,
burn-in 4000, likelihood settings: nst 5 6 rates 5 invgamma
ngammacat 5 4 covarion 5 yes) (Ronquist and Huelsenbeck
2003). To compare several user-defined trees versus the op-
timal tree obtained by ML analysis, the approximately un-
biased test (AU test) was performed using Consel 1.0f
(Shimodaira and Hasegawa 2001) (Table 3).
Toxin analyses. One hundred and-fifty milliliter cultures
(Mex13, GranCan1, Tenerife1, Zhenbo6, ØM2, Læs2, BP1,
300, and Castell2) were grown in 250 mL flasks at 241 C in a
12:12 L:D regime or at 151 C in a 16:18 L:D regime in 33 psu
L1-medium at a photon flux of 100 mmol photons
m  2
s  1.
Two or three subsamples of each culture in the stationary
growth phase were freeze dried, re-suspended in redistilled
water and tested using UV-DAD HPLC at the University of
Copenhagen (Lundholm et al. 2002a) and fluorescence-de-
tection HPLC with precolumn FMOC derivatization at Kita-
sato University; detection limit was 0.3 ng/mL whole culture
(Kotaki et al. 2000).
RESULTS
Morphological studies. Because of the morphologi-
cal variation among the strains, examination of the
 GENETIC VARIATION OF P. DELICATISSIMA 467

TABLE 2. Application of fluorescent rRNA probes developed for Pseudo-nitzschia species from Monterey Bay, California.

Reactions between different isolates and probes developed

for different Pseudo-nitzschia species

Cultures auD1 muD1 muD2 puD1 frD1 deD1 heD2-2 amD1

P. dolorosa (Calif 1,Calif3, BP2)   þ  þþ þ   

P. delicatissima (MexB) þ    þþ þ   
P. delicatissima (1001 1G1 5, Læs1, Læs2)    þþ þ   
P. delicatissima (OFPd972, OFPd973, Zhenbo6, MexA) þ  þ  þþ þ þþþ  
P. delicatissima (Castell1, Castell2, BP1) þþþ  þþ  þ   
P. decipiens (Mex13, Mex14, GranCan4-1) ?    þþ þ  þþ 

The probes were applied to 17 isolates, which morphologically are more or less similar to P. delicatissima. þ þ þ means bright
fluorescent reaction, þ þ and þ means less fluorescence, ? indicates a doubtful reaction and — means no fluorescence.

type material of P. delicatissima was essential. P. deli- Examination of the published light micrographs of
catissima was described by Cleve (1897; as Nitzschia the neotype material (Hasle 1976) demonstrated the
delicatissima) as having a narrow linear acute valve presence of 21 fibulae in 10 mm, casting doubt on the
with an apical axis of 55 mm, a transapical axis of magnification given for the electron micrograph. Frus-
1.5 mm, 24 fibulae in 10 mm, and a central raphe tules from the sample on which the neotype was based
(‘‘keel’’). Cleve did not observe the striae. The local- were kindly provided by Grethe R. Hasle and revealed
ities were given as the Atlantic (63110 0 N, 0136 0 E), 21–24 fibulae and 36–40 striae in 10 mm (TEM obser-
Spitzbergen and Sweden. Hasle (1965) examined vations, Table 4). This agrees with the measurements
samples from the North Atlantic and Scandinavian and the light micrographs in Hasle (1965).
coastal waters, and she described as N. actydrophila In the following, P. delicatissima sensu stricto will be
Hasle a similar species with an eccentric raphe. The described in detail. As the neotype material comprises
type material of P. delicatissima is missing and studies cleaned frustules only, an epitype comprising fixed
by Hasle (1976) revealed that in some of Cleve’s ma- material and associated molecular data (ITS1, 5.8S,
terial, the two valves of P. delicatissima were often ITS2 of the rDNA) have been selected for possible fu-
joined, giving the appearance of the central raphe ture taxonomic comparisons (according to article 9.7 in
described by Cleve. Hasle (1976) selected a neotype the International Code of Botanical Nomenclature, St
from Cleve’s collection (slides labelled ‘‘Nitzschia Louis Code (ICBN 2000)). The epitype is morpholog-
delicatissima, Helder 7/5-97’’ in Cleve’s handwriting), ically similar to the neotype, and both are from Scan-
which she examined with LM and TEM (Hasle 1976). dinavian coastal waters. Furthermore, both are similar
The neotype material agreed with the diagnosis of N. to specimens of P. delicatissima from field samples col-
actydrophila Hasle (1965) and may serve as an emend- lected in Scandinavian coastal waters (Table 4).
ed diagnosis for P. delicatissima. Two species differing morphologically and phyloge-
netically from P. delicatissima are described as P. decipiens
TABLE 3. Tests of congruence using approximately un- sp. nov. and P. dolorosa sp. nov.
biased test.
Pseudo-nitzschia delicatissima (Cleve) Heiden in
AU test of constraint trees
Heiden & Kolbe 1928
Constraint topologies D(  ln L)a P Fig. 1, A–G, Table 4
Best tree (maximum likelihood) (3475.6) b Basionym: Nitzschia delicatissima Cleve
P. dolorosa, P. delicatissima and P. 58.2 o0.001c Synonym: Nitzschia actydrophila Hasle
decipiens monophyletic Neotype: Slide labeled ‘‘Nitzschia delicatissima, Helder
P. delicatissima and P. decipiens 57.4 o0.001c 7/5-97’’ in P.T.Cleve’s handwriting in the Swedish Mu-
monophyletic
P. dolorosa and P. delicatissima 19.4 0.010c seum of Natural History (S), section of Botany.
monophyletic Epitype: Fixed material of the culture ‘‘Læs 5’’ has
P. decipiens and P. dolorosa 3.8 0.180 been deposited at the Botanical Museum, University of
monophyletic Copenhagen as number A2380. ITS1, 5.8S and ITS2
P. delicatissima monophyleticd 1.6 0.340
of the nuclear-encoded rDNA of the epitype have been
The best tree (the maximum likelihood tree; Fig. 4) is com- sequenced and deposited at GenBank with accession
pared with several user-defined trees. number AY257849.
a
Difference in  log likelihood between the best tree and the Morphology. The cells form stepped colonies and
constrained tree.
b are linear to lanceolate in valve view, tapering from
 log likelihood of the best tree (ML).
c
Indicates that the constrained tree is significantly different the middle toward the tips of the valve, 1.5–2.0 mm
from the best tree. wide, and 19–76 mm long (Fig. 1, D and E). The two
d
Does not include P. micropora. ends of the valve are similar. In girdle view, cells are
P., Pseudo-nitzschia; AU, approximately unbiased. lanceolate with cut-off ends. A larger interspace be-
TABLE 4. Morphological characters of strains studied morphologically.
468

Fibulae/ Striae / Row of Poroids/ Length Width Number of bandc Band striae: Number of
Taxa Valve shape 10 mm 10 mm poroids mm (m) (mm) Mantleb striae/10 mm poroids width  height

Pseudo-nitzschia delicatissima
MexAa Lanceolate 19–22 35–38 2 10–11 33–34 1.7–1.9 43
Zhenbo6 Lanceolate 20–24 35–37 2 10–12 38–41 1.5–1.7 1 split poroid
a
OFPd972 Lanceolate 19–23 35–38 2 9–10 26–35 1.4–1.8 1 split poroid 45–46
OFPd973 Lanceolate 19–20 36–37 2 10–11 23 1.8–2.0 1 split poroid
a
Tasm10 Lanceolate 19–22 35–37 2 9–12 49–51 1.6–2.0 1 split poroid 44–46 1 poroid divided into several parts
ØM2a Lanceolate 20–25 38–39 2 10 32 1.5–1.7 1 split poroid 48
Læs1 Lanceolate 20–22 35–38 2 9–12 68–70 1.5–1.9 1 split poroid 44–48 1 poroid divided into several parts
Læs2a Lanceolate 20–25 36–39 2 9–12 70–72 1.7–1.9 1 split poroid 47–48 1 poroid divided into several parts
a
Læs5 Lanceolate 20–22 36–39 2 12 76 1.5–1.9 1 split poroid
MexB Lanceolate 22–23 39–40 (41) 2 9–11 19–37 1.5–1.7 1 split poroid 43
BP1a Lanceolate 20–22 35–37 2 8–11 22–61 1.6–1.9 1 split poroid 44 1 poroid divided into several parts
Castell1a Lanceolate 20–22 34–37 2 8–11 35–60 1.7–2.1 1 split poroid 43
Castell2a Lanceolate 18–23 36–38 2 8–9 57–58 1.7–2.1 1 split poroid 43–44 1 poroid divided into several parts
Castell3 Lanceolate 18–22 36 2 8–9 2.0 43–45 1 poroid divided into several parts
Castell4 Lanceolate 20–22 35–37 2 8–11 43–46 1.8–1.9 1 split poroid 43
P. delicatissima Lanceolate 21–24 36–40 2 10–12 1.6–1.7
neotype material
Field samples from Lanceolate 20–26 37–40 2 9–12 1.3–1.6
Vejle fjord Aug. 1998
Field samples from Lanceolate 22–25 39–40 (41) 2 10–12 1.2–1.8 44
Kattegat Nov. 1986
Field sample from Lanceolate 21–24 36–39 2 9–11 52–67 1.3–1.7 43
Oslofjord 22nd March
2001
P. delicatissima Linear-lanceolate 19–25 36–40 2 10–12 40–76 c.2 Two to three different types
(Hasle 1965)
Pseudo-nitzschia decipens
NINA LUNDHOLM ET AL.

Mex13a Lanceolate 22–26 42–45 2 9–12 54 1.4–1.8 1 split poroid or as valve 48–53
Mex14 Lanceolate 23–26 42–45 2 9–12 1.8–1.9 1 split poroid or as valve 50–55 1 poroid divided into several parts
GranCan1 Lanceolate 22–25 41–45 2 10–13 32–34 1.8–2.2 1 split poroid or as valve 47–53 1 poroid divided into several parts
a
GranCan4-1 Lanceolate 20–25 41–46 2 9–12 29–31 2.0–2.4 (2.5) 1 split poroid or as valve 48–55
Tenerife1 Lanceolate 21–26 41–45 2 9–11 61–64 1.7–2.0 1 split poroid or as valve 48–53 1 poroid divided into several parts
Pseudo-nitzschia dolorosa
Calif 1a Lanceolate, 19–20 30–35 2 or 1 6–7 44–47 2.5–3.0 5 valve, 1–2 poroids high 41–42 2  2–3
asymmetrical
Calif 3a Lanceolate, 19–21 32–36 2 or 1 6–8 44–46 2.5–2.8 5 valve, 1–2 poroids high 40–43 23
asymmetrical
a
BP2 Lanceolate 20–21 33–36 2 or 1 5–8 56–59 2.5–2.8 42–43 23
BP3a Lanceolate, 18–21 33–35 2 or 1 5–8 55–56 2.5–2.8 (2.4) 5 valve, 1–2 poroids high
asymmetrical
300a Lanceolate, 18–22 33–36 2 or 1 6–8 29–33 2.5–2.9 5 valve, 1–2 poroids high 40–44 I. 2  2–3, II. 2  1, III. No
asymmetrical poroids
a
Indicates that ITS1, 5.8S and ITS2 was sequenced and used in the phylogenetic analyses.
b
See species descriptions for further details.
c
Girdle band.
 GENETIC VARIATION OF P. DELICATISSIMA 469

FIG. 1. TEM of Pseudo-nitzschia delicatissima. (A) Part of valve showing proximal and distal mantles; strain Castell4. (B, C) Details of
cingular bands in strain Tasm10 (B) and strain Læs1 (C). (D,E) Valves: strain Læs5 (D) and strain Tasm10 (E). (F, G) Details of cingular
bands in strain Tasm10 (F) and strain Læs2 (G). A–C and G scale bar, 0.2 mm. D,E scale bar, 5 mm. F scale bar, 0.5 mm.

tween the two central fibulae includes a central nod- fibulae are visible by LM. Each stria is biseriate, with
ule (Fig. 1, A and D). The fibulae and striae number eight to 12 poroids in 1 mm (Fig. 1A). The perfora-
19–26 and 35–40 in 10 mm, respectively. Only the tions of the hymen in both the valve and the girdle
470 NINA LUNDHOLM ET AL.
bands are arranged in a hexagonal pattern (Fig. 1,
A and B). Both the proximal and distal mantle are
one poroid high, but the poroid is often split into a
few sectors (Fig. 1A).
The cingulum comprises open bands, all bordered
by an often unperforated margin (Fig. 1, B, C, F, and
G). The bands contain 43–48 striae. Each stria in the
valvocopula consists of one poroid, which may be split
into a few sectors (Fig. 1, F and G). The second band
contains one longitudinal row of small poroids (Fig. 1,
B and C). In this band, the abvalvar part sometimes
contains scattered poroids (Fig. 1C). Unperforated
bands were also observed (Fig. 1F).
Distribution. The species appears to be cosmo-
politan in its distribution. It has been observed near
Svalbard, in Norwegian coastal waters (off Drbak,
Espegrend and Grnsfjord), in Skagerrak and Katte-
gat, in Danish coastal waters, off Plymouth, off
Den Helder (Netherlands), in the Dutch Wadden
Sea, the Mediterranean Sea (off Boca Piccola, Cas-
tellamare, Gulf of Naples), off Accra (Ghana) and
Port Shelter (Hong Kong), in Ofunato Bay (Japan),
near Hobart (Tasmania), in the Gulf of California,
and in the Gulf of Mexico (off Tuxpam) (Orsini et al.
2004, Grethe Hasle personal communication and
present study).
Toxicity. Toxin analyses of the isolates Zhenbo6,
ØM2, Læs2, BP1 and Castell2 did not show pres-
ence of DA.
Taxonomic remarks. The girdle band structure re-
sembles that illustrated in P. delicatissima by Hasle
et al. (1996), and the density of the striae is identical
to that published by Hasle (1965) (as P. actydrophila).
Accepting the diagnosis of N. actydrophila as an
emended diagnosis of P. delicatissima thus implies
that P. delicatissima as described above is identical to
P. delicatissima sensu stricto.
Pseudo-nitzschia dolorosa Lundholm et Moestrup
sp. nov.
Fig. 2, A–I, Table 4
Cellulae in catena longa imbricatae. Cellulae as-
pectu valvari lanceolatae, axe transapicali 2.5–3.0 mm,
axe apicali 30–59 mm. Raphe excentrica, nodulo cen-
trali divisa, fibulis aequidistantibus, 18–22 per 10 mm.
Striae 30–36 in 10 mm. Series pororum 1–2 per striam;
pori 5–8 per 1 mm. Limbus altitudine diametrum 1-(2)
pororum aequans, structura fronte valvae similis. Cin-
gulum ex copulis tribus constans. Copula proxima
(valvocopula) striis 40–44 per 10 mm, unaquaeque
biseriatae et altitudine diametros pororum 2–3 aequ-
ante. Copula secunda biseriatae et altitudine diamet-
rum pori 1–2 aequante. Copula tertia sine poris.
Holotype. Material of culture ‘‘300,’’ deposited at
the Botanical Museum, University of Copenhagen as
permanent slide A2187.
Isotype. Deposited at Department of Biology, Uni-
versity of Oslo as IMBB slide no. 111. Fixed material
of the culture ‘‘300’’ deposited at the Botanical Mu-
seum, University of Copenhagen as number A2378.
Type locality: The mouth of Ria de Aveiro, Portu-
gal.
Etymology: (Latin) dolorosa, painful; this species
gave us a lot of trouble before its taxonomic position
was clarified.
Morphology. The cells form stepped colonies and
are lanceolate in valve view with a tendency to being
slightly asymmetrical laterally (Fig. 2, A–C). The
valves taper from the middle toward the apices,
with a width of 2.5–3.0 mm at the center and a length
of 30–59 mm. The two ends of the valves are similar
(isopolar). In girdle view, the cells appear lanceolate
with cut-off ends (not shown). A central nodule is
present in the larger interspace between the two cen-
tral fibulae (Fig. 2, D–F). The fibulae are otherwise
more or less regularly spaced, 18–22 in 10 mm. They
are visible under the light microscope, using phase
contrast or differential interference contrast (DIC).
The striae number 30–36 in 10 mm and were not vis-
ible under the light miroscope. Each stria is biseriate,
sometimes uniseriate (Fig. 2, D–F). Most parts of the
valves have two opposite rows, but in some parts of
the same valve the two rows merge more or less into a
single row. The proportion of each valve with biseri-
ate striae varies. When two rows are present they of-
ten form pairs (Fig. 2F). The poroids number 5–8 in
1 mm. The perforations of the hymen in both the
valve and the girdle bands are arranged in a hexa-
gonal pattern (Fig. 2G). The proximal and distal
mantles are structured similar to the valve face.
Both are one (seldom two) poroid(s) high (Fig. 2F).
The cingulum comprises three open bands, all bor-
dered by an unperforated margin (Fig. 2, H and I).
The bands possess 40–44 striae in 10 mm, which in the
two most advalvar bands are two poroids wide. In the
valvocopula, the striae are two to three poroids high,
but in the second band only one to two poroids high
(Fig. 2, G–I). The third band lacks poroids (Fig. 2H).
Distribution. This species has been recorded from
the mouth of Ria de Aveiro, Portugal; Gulf of Naples
and Boca Piccola, Italy (present study, Orsini et al.
2004); off the south coast of Ireland (C. Cusack, per-
sonal communication); Monterey Bay, California (pre-
sent study); and the Drake passage, south of South
America (M. Ferrario, personal communication).
Toxicity. Toxin analyses of culture 300 did not show
production of DA.
Taxonomic remarks. In addition to the type materi-
al, the present description includes the cultures
Calif1, Calif3, BP2, BP3, all of which are morphologi-
cally identical (Table 4). The strain 20-02 from Gulf
of Naples, Italy was identical to P. dolorosa with respect
to both morphology and sequence data of ITS1, 5.8S
and ITS of rDNA (accession number AY519275)
(Orsini et al. 2004).
Comparison with other Pseudo-nitzschia species. P. do-
lorosa somewhat resembles P. turgidula, which has
more rounded valve ends and a smaller number of
fibulae and striae (Table 5, Hasle 1965). It also re-
sembles P. cf. subpacifica (Hasle) Hasle as described in
 GENETIC VARIATION OF P. DELICATISSIMA 471

FIG. 2. TEM of Pseudo-nitzschia dolorosa (A) Strain Calif 3. (B–D) Strain 300. (E) Strain BP2. (F–I) Strain 300. (A–C) Whole valves.
Valves may be symmetrical or slightly asymmetrical. (D) Part of valve showing uni- or biseriate striae. (E) Part of valve showing uni- or
biseriate striae. (F) Central part of valve showing distal and proximal mantles. (G) Detail of valvocopulae, showing hexagonal pattern of
perforation in the poroids. (H) Detail of cingular bands showing both perforated and unperforated bands. (I) Detail of the two ‘‘legs’’ of
second cingular band. A–C scale bars, 5 mm. D–F, H, I scale bars, 0.5 mm. G scale bar, 0.2 mm.

Lundholm et al. (2002a). The valves of this species as in P. dolorosa. P. dolorosa differs in the width of its
are lanceolate and asymmetrical with two rows of po- valves (2.4–3.0 vs. 4.1–4.8 mm in P. cf. subpacifica), a
roids and with the same number of fibulae and striae higher number of band striae in 10 mm (40–44 vs.
472 NINA LUNDHOLM ET AL.

29–32), the tendency of the two rows of poroids to

wide  poroids high

2  3–4, 2  1–2
Band striae: Poroids

1 divided poroid

1 divided poroid

1 divided poroid

1 divided poroid
2  3–4, 2  2
fuse and by being less asymmetrical.

2–3  several
P. lineola (Cleve) Hasle resembles P. dolorosa in having
one or two rows of poroids in the valve striae. It differs
in a lower density of fibulae (11–16 in 10 mm) and striae

22
ND

ND
(22–28 in 10 mm), a linear valve shape, and narrower
valves (1.8–2.7 mm) (Table 5, Hasle 1965). Other spe-
cies with uniseriate striae such as P. caciantha, P. pseudo-
44.2  1.6

51.8  1.7

42.0  1.4
Band striae/

Based on data from Hasle (1965), Priisholm et al. (2002), Lundholm et al. (2003). Numbers in italic show mean  SD. ND means no data available.
delicatissima, P. calliantha, and P. cuspidata, also slightly
43–48

48–55

40–44

33–38
42–45
48–51

48–54
48–53
10 mm

ND

ND
resemble P. dolorosa. They differ in a lower density of
poroids, and a higher (P. pseudodelicatissima, P. cuspidata)
or lower (P. caciantha) density of girdle band striae (Ta-
ble 5). P. pseudodelicatissima, P. calliantha, and P. cuspidata
(1.4)1.5–2.0(2.1)

are also narrower than P. dolorosa (Table 5).

1.8  0.2

1.9  0.3

2.6  0.2
1.4–2.4

2.5–3.0

2.7–3.5
1.3–1.8
1.4–2.2
1.8–2.7
1.3–2.0
1.1–1.4

2.5–3.5
Width
(mm)

Pseudo-nitzschia decipiens Lundholm et Moestrup

sp. nov.
Fig. 3, A–G, Table 4
Cellulae in catena longa imbricatae. Cellulae aspectu
TABLE 5. Comparison of Pseudo-nitzschia delicatissima, P. decipiens and P. dolorosa with other Pseudo-nitzschia speciesa.

valvari lanceolatae, axe transapicali 1.4–2.5 mm, axe

apicali 28–64 mm. Raphe excentrica, nodulo centrali di-
56–112
19–76

29–64

30–59

53–75
41–98
30–72

31–57
54–87

30–80
Length
(mm)

visa, fibulis aequidistantibus, 20–28 per 10 mm. Striae

40–46 in 10 mm. Series pororum 2 per striam; pori
9–13 per 1 mm. Cingulum ex copulis tribus constans.
Copulae saltem typis duobus. Typus unus cum series
Central
nodule

þ
þ
þ
þ

þ


pororum transversum, 48–55 in 10 mm, unaquaeque in

sectiones multis divisus. Typus secundus sine poris.
Holotype. Material of culture ‘‘Mex13’’ deposited
10.1  1.2

11.4  1.2

at the Botanical Museum, University of Copenhagen

6.6  0.8

3–6;4–7
Poroids/

3.5–5
8–12

9–13

9–12

as permanent slide number A2382.

1 mm

5–8

4–6
4–6

5–6

7–9

Isotype. Fixed material of the culture ‘‘Mex13’’ de-

posited at the Botanical Museum, University of
Copenhagen as number A2379.
Type locality. Tuxpam, Caribbean Coast of Mexico.
Rows of
poroids

1–2

1;2

Etymology: Decipiens: deceiving (used of a species

2

1
1
1

2
1

closely resembling another: Stearn 1966).

Morphology. The valves are linear to lanceolate in
valve view, tapering from the middle toward the ap-
(34)35–40(41)
36.8  1.5

43.2  1.2

34.5  1.4

ices of the valve, 1.4–2.4 mm wide and 29–64 mm long

41–46

30–36

28–31
34–39
35–40
22–28
41–46
36–42

23–28
Striae/
10 mm

(Fig. 3, A and B). In girdle view, the cells are lanceo-

late with cut-off ends. The fibulae number 20–26 in
10 mm and the striae 41–46 in 10 mm (Fig. 3, C–E).
The fibulae are visible under the light microscope
using phase contrast or DIC. The striae could not be
20–25 (28)
21.4  1.6

24.0  1.4

20.0  1.0
(18)19–26

seen in the LM. The biseriate striae contain nine to 13

Fibulae/

20–26

18–22

15–19
15–21
19–24
11–16
21–29

13–18
10 mm

poroids in 1 mm (Fig. 3C). The perforations of the

hymen in both the valve and the girdle bands are ar-
ranged in a hexagonal pattern (Fig. 3G). The prox-
imal and distal mantle are one to two poroids high
asymmetrical

(Fig. 3, C and E).

Valve shape

Lanceolate

Lanceolate

Lanceolate

Lanceolate

Lanceolate

Lanceolate

Lanceolate

The cingulum comprises open bands bordered by

an unperforated margin (Fig. 3, F and G). Two types of
Linear

Linear

Linear

bands were observed. One type contains a large poroid

separated into several perforated sectors, 48–55 po-
roids in 10 mm (Fig. 3, F and G). The second type lacks
poroids (not shown). The precise number and position
P. delicatissima

delicatissima
micropora
calliantha
caciantha

cuspidata

P. turgidula

of the bands relative to one another is uncertain.

P. decipiens

P. dolorosa

pseudo-
lineola

Distribution. P. decipiens has been recorded from

the Gulf of Mexico (off Tuxpam), the Canary Islands
Taxa

and the Black Sea (near Karadag).

P.
P.
P.
P.
P.
P.
 GENETIC VARIATION OF P. DELICATISSIMA 473

FIG. 3. TEM of Pseudo-nitzschia decipiens A and G. Strain Tenerife1. (B–F). Strain GranCan1. (A and B) Whole valves (C) Part of valve
showing two rows of poroids. (D) Tip of valve. (E) Central part of valve showing central nodule and distal and proximal mantles. (F)
Detail of copulae. (G) Detail of valvocopula showing hexagonal pattern of perforations in the poroids. A and B scale bars, 5 mm. D–F scale
bars, 0.5 mm. C, G Scale bar, 0.2 mm.
474 NINA LUNDHOLM ET AL.
Toxicity. Toxin analyses of the isolates Mex13,
GranCan1, and GranCan4-1 did not show presence
of DA.
Taxonomic remarks. In addition to the type materi-
al, the present description includes the cultures listed
in Table 4, all of which are morphologically similar.
Comparison with other Pseudo-nitzschia species.
P. decipiens and P. delicatissima both resemble P. turgid-
ula, which has a lower density of fibulae (13–18),
striae (23–28), and poroids (7–9), wider valves
(2.5–3.5 mm) and more rounded valve ends (Hasle
1965, Hasle et al. 1996). P. decipiens differ from
P. micropora in possessing a central nodule, a differ-
ent structure of the bands and a lower density of stri-
ae (Table 5, Priisholm et al. 2002). For further
differences, see Table 5.
Hybridization studies. Application of rRNA probes
revealed very large variation in the hybridization pat-
tern and grouped the isolates tested into six groups
FIG. 4. Maximum likelihood (ML) tree
based on ITS1, 5.8S and ITS2 of the nu-
clear encoded rDNA (583 positions includ-
ed) illustrating a phylogeny of the genus
Pseudo-nitzschia. The tree is unrooted and
four well-supported clades have been labe-
led I, II, III, and IV. Two clades of P. del-
icatissima have been named clade A and B.
The bootstrap values indicated on the
branches represent bootstrap values from
ML (500 replicates, before slash), neigh-
bor-joining (1000 replicates, between
slashes) and parsimony (1000 replicates,
after slash). Only values above 50% are
shown.
(Table 2). Three groups of isolates hybridized mainly
with the frD1 probe, but two of these groups also re-
acted weakly with the auD1 and the muD2 probes. A
fourth group hybridized strongly with both the frD1
and the deD1 probe, and weakly with two other
probes. A fifth group reacted with the auD1 and
the muD2 probes and weakly with the frD1 probe,
and the last group hybridized both with the frD1 and
the heD2-2 probes.
Phylogenetic inferences. The phylogenetic studies of
the ITS1, 5.8S and ITS2 regions of the nuclear-
encoded rDNA revealed similar tree topologies
using ML, NJ, Bayesian and parsimony analyses
(MP) of the data set (ML tree in Fig. 4, the other
trees not shown). The data set was not very homopla-
sic, only one island was found in both ML and MP
analyses. All analyses supported the existence of four
different clades (labeled I–IV in Fig. 4; I–III as in
Lundholm et al. (2003)). The only topological differ-
ences found were minor and occurred in clade II and
 GENETIC VARIATION OF P. DELICATISSIMA
clade I and were not related to taxa in the del-
icatissima-complex.
All analyses showed clade I to be very well support-
ed (bootstrap values 498%) similar to the results in
Lundholm et al. (2003). Within this clade, P. pungens
and P. multiseries always appeared as highly supported
sister taxa (bootstrap values 100%) and, similarly, P. se-
riata (Cleve) H. Peragallo and P. australis constituted
sister taxa (bootstrap values 491%). In all analyses ex-
cept the parsimony analyses, P. multistriata (Takano)
Takano formed a sister taxon to P. pungens and P. mul-
tiseries, whereas in the parsimony analyses it constituted
the basal taxon in the clade. In all analyses, P. fraudu-
lenta constituted a sister taxon to clade I (bootstrap
values above 79%).
A second clade (clade II) comprising a P. pseudo-
delicatissima isolate, three P. cuspidata isolates and strain
Hobart5 was well supported (bootstrap values 1000%),
but the branching within the clade was not resolved or
it was supported by low bootstrap values, and differ-
ences in branching order were found among the dif-
ferent types of analyses (Lundholm et al. 2003). Clade
III in this study comprised only P. calliantha Lund-
holm, Moestrup et Hasle and P. cf. subpacifica (Hasle)
475
FIG. 5. Box plots of morphometric data of
Pseudo-nitzschia delicatissima, P. decipiens and P. do-
lorosa. The morphometric data comprise density
of fibulae, striae and poroids of valve face, valve
width, and density of band striae. The grey ar-
eas indicate 25% and 75% percentiles, the line
within the fields is the median. The error bars
indicate the 10% and 90% percentiles and the
circles the 5% and 95% percentiles.
Hasle, and was supported by moderate to high boot-
strap values (75% in ML, 90% in NJ, and 95% in MP).
Large genetic variation was found among the strains
that had tentatively been termed P. delicatissima (Fig. 4).
The ITS regions were sequenced in all five isolates of
P. dolorosa and revealed similar sequences. The five
isolates formed a cluster supported by very high boot-
strap values (100%) in all the phylogenetic analyses. In
clade IV, isolates Mex13 and GranCan4-1, represent-
ing P. decipiens, clustered together, supported by high
bootstrap values (489%), and with P. galaxiae Lund-
holm et Moestrup as sister taxon in a well-supported
branch (498%). The clade comprising P. galaxiae and
P. decipiens made up the closest sister group to the clade
comprising the P. dolorosa strains, and was supported
by moderate to high bootstrap values (80, 94, and 73%
using ML, NJ, and MP, respectively).
In all the phylogenetic analyses P. delicatissima and
P. micropora formed a monophyletic clade (bootstrap
values between 50% and 90%). Branching within this
monophyletic clade was, however, either not support-
ed or poorly supported, and it is therefore unknown
whether the P. delicatissima strains form a monophyletic
cluster within the clade, with P. micropora as sister tax-
476 NINA LUNDHOLM ET AL.
on. The clade comprised two clades of P. delicatissima
(clade A and B; Fig. 4) and one clade of P. micropora.
Analyses of a reduced data set comprising all the
P. delicatissima and the P. micropora strains together
with an outgroup of three taxa (Sydney1, Sydney4,
and Mex13) did not result in any major changes in the
support for the branches. The AU test showed that
forcing the strains of P. delicatissima into a monophyletic
group did not result in a phylogenetic tree significantly
different from the best tree (Table 3), so one cannot
reject that the P. delicatissima strains constitute a mon-
ophyletic group. Constraint analyses with P. deli-
catissima strains forced to form a monophyletic clade
resulted in higher or similar bootstrap support for
clade A (94/100/95), clade B (100/100/100), P. micropora
(98/100/98) and for the clade comprising the three lat-
ter clades (94/99/98), given as (ML, NJ, MP).
Among the P. delicatissima strains, all the phyloge-
netic analyses (bootstrap values 497%) supported a
grouping of the Mediterranean isolates (Castell1, Cas-
tell2, BP1). These isolates clustered in clade B with a
Mexican isolate as sister taxon (MexA, Fig. 4), sup-
ported by high bootstrap values (497%). The Pacific
isolates (OFPd972 and Tasm10; from Japan and Tas-
mania) grouped together (bootstrap values 496%) as
did the European Atlantic isolates (Læs2, Læs5,
ØM2) (80%–85% bootstrap values, Fig. 4). The two
latter clades constituted clade A. Sequences of P. deli-
catissima from Orsini et al. (2004) (accession numbers
AY519334–AY519337) were similar to Castell 1, 2, and
BP1, and a sequence of P. dolorosa (accession number
AY519275) was identical to BP3 and was therefore
not included in the phylogenetic analyses. Analyses
including the only strain from Orsini et al. (2004)
(21-02; accession number AY519281) that was not sim-
ilar to any of our own strains did not affect the tree
topology or result in any obvious change in the sup-
port for the branches. The only effect was a slightly
lower support for the clade (clade A) to which the
strain belonged.
Following the results of the AU test (Table 3), the
constrained analyses forcing the strains of P. dolorosa, P.
delicatissima, and P. decipiens into a monophyletic clade
resulted in a tree significantly different from the best
(the ML tree topology), thus supporting separation
into different species. Similarly, the trees resulting
from the constrained analyses forcing either P. deli-
catissima and P. decipiens or P. delicatissima and P. dolorosa
into monophyletic groups were rejected as significantly
different from the best tree (Table 3). User defined
tree topologies forcing either the P. decipiens and P. do-
lorosa or the P. delicatissima strains (excluding P. micro-
pora) were, however, not rejected as significantly worse
than the best tree. Based on the ITS-data set, the mo-
lecular distances between P. decipiens and P. dolorosa was
0.105–0.108, between P. delicatissima and P. decipiens
0.075–0.090, and between P. dolorosa and P. delicatissima
0.129–151. The intraspecific molecular distances of the
three taxa was 0.000–0.002 in P. dolorosa, 0.000–0.005
in P. decipiens and 0.000–0.049 in P. delicatissima.
DISCUSSION
P. decipiens, P. dolorosa, and P. delicatissima. Erec-
tion of P. decipiens and P. dolorosa is supported by both
morphological and molecular data. Morphologically,
P. dolorosa is distinguished from P. delicatissima and
P. decipiens by a larger width of the valves, fewer
poroids in 1 mm, a different structure of the girdle
band striae. Also by the tendency to form only one
row of poroids in the striae in certain parts of the
valve, or opposite poroids in the biseriate parts.
P. decipiens and P. dolorosa can also be separated by
differences in the density of striae and the density of
band striae.
The distinction between P. dolorosa and the two oth-
er species is supported by the phylogenetic analyses of
the ITS regions, where P. dolorosa in all four analyses
constitutes a well-supported sister taxon to a clade
comprising P. galaxiae and P. decipiens (Fig. 4). This
clade (clade IV) branches out separately from the
P. delicatissima strains. In addition, the molecular dis-
tances between P. dolorosa and P. decipiens (0.105–0.108)
and P. dolorosa and P. delicatissima (0.129–0.151) are
within the distances between other species: P. decipiens
and P. delicatissima: 0.075–0.090, P. seriata and P. fraudu-
lenta: 0.116, P. pungens and P. multiseries: 0.096, and P.
multistriata and P. australis: 0.080. Furthermore, the AU
test supports the distinction of P. delicatissima and P. do-
lorosa as well as the delineation between P. delicatissima
and P. decipiens, but cannot reject that P. dolorosa and
P. decipiens may constitute a monophyletic cluster.
Application of the rRNA probes shows that the
P. dolorosa strains never hybridize with exactly the
same probes as P. decipiens or P. delicatissima and hence
supports the existence of three species. The differenc-
es between the P. dolorosa strains and some of the
P. delicatissima strains, however, result in only weak hy-
bridization reactions (Table 2).
Based on the morphological and phylogenetic sup-
port, as well the hybridization experiments, it appears
well justified to consider P. dolorosa as a separate spe-
cies. This is also supported by the phylogenetic studies
of Orsini et al. (2004) which demonstrated that strains
morphologically and genetically identical to P. dolorosa
(e.g. strain 20-02; ITS clade 2) cluster separately from
the remaining P. delicatissima strains. After presentation
of the new species at international meetings (Lund-
holm et al. 2000, 2002b), findings of unidentified Pseu-
do-nitzschia specimens from the Celtic Sea and the
southern Atlantic (M. Ferrario, personal communica-
tion, Cusack 2002) were readily identified as P. dolorosa.
P. decipiens is morphologically similar to P. del-
icatissima, but differs in two features. It has a higher
density of striae (41–46 in 10 mm in P. decipiens com-
pared with 35–40 in P. delicatissima) and a higher
number of band striae (48–55, compared with 43–48
in P. delicatissima). The difference in stria density does
not seem to constitute a continuum; rather the meas-
urements indicate a morphological disjunction, al-
though the gap is very narrow (t test: Po0.001).
 GENETIC VARIATION OF P. DELICATISSIMA
Similarly, the difference in density of band striae is dif-
ferent, the 95% percentiles do not overlap but was
close (Fig. 5, Table 5) (t test: Po0.001).
The phylogenetic analyses of the ITS regions sup-
port recognition of one more species, different from
P. delicatissima; P. decipiens. The isolates of P. decipiens
appear in a well-supported cluster with P. galaxiae as
sister group, which again clusters within a larger well-
supported clade that includes P. dolorosa. P. decipiens
and P. galaxiae resemble each other in being narrow
with a high number of striae, but they are otherwise
very different morphologically (Lundholm and Mo-
estrup 2002). The results of the AU test support that
P. delicatissima and P. decipiens are phylogenetically dif-
ferent. The molecular distances (based on the ITS-data
set) between the isolates of P. delicatissima and P. decipi-
ens (0.075–0.090) are similar to the distances between
other species.
The difference between P. delicatissima and P. decipi-
ens is also supported by single-stranded conformation
polymorphism (SSCP) results of ITS2 sequences of
several isolates (SSCP, a method by which nucleotide
differences between homologous sequence strands are
detected by electrophoresis of single-stranded DNA
under denaturing condition; Orita et al. 1989, Kjller
and Rosendahl 2000). Using this method, the banding
patterns of the isolates GranCan1, GranCan4-1, Tene-
rife1, and Mex14 (all P. decipiens; Table 4) are identical
and different from the isolates Læs2, Læs5, ØM2
and Castell1, Castell2, Castell3, and Castell4 (all P. deli-
catissima).
The above-mentioned molecular and morphologi-
cal data show that both morphological and phyloge-
netic species concepts support recognition of the three
species, P. dolorosa, P. delicatissima, and P. decipiens. We
would have liked to test the biological species concept
as well. However, Pseudo-nitzschia strains are relatively
short-lived in culture, and the strains died before mat-
ing studies could be performed.
Intraspecific variation of P. delicatissima. The intra-
specific distances among the isolates of P. delicatissima
(based on the ITS-data set) are relatively large,
0-0.049, compared with intraspecific differences in
P. seriata, P. australis, P. pungens, and P. galaxiae (0-0.017)
(Hasle and Lundholm 2005, present paper).
The distances among clade A and B isolates are
0-0.012 and 0-0.021, respectively. Hence, particularly
the distances between the strains from the two clades as
well as distances among the clade B isolates are large.
Application of the DNA probes also indicate large ge-
netic variation among the P. delicatissima strains. This
variation is, however, not reflected in any apparent
morphological variation. The AU-test does not reject
that the P. delicatissima strains make up a phylogenetic
monophyletic unit. When the P. delicatissima strains are
forced together, the P. micropora strains simply consti-
tute a sister clade to the P. delicatissima clade and the
support for the P. micropora and the two P. delicatissima
clades are either similar or increased. P. delicatissima
must therefore be considered a species whose genetic
477
variation is slightly larger than in other Pseudo-nitzschia
species. The variations indicate the existence of cryptic
species, represented by clade A and B. This supports
Orsini et al. (2004) who found different clades of
P. delicatissima, putative cryptic species, in the pre-
bloom phase of a bloom of P. delicatissima. In fact, the
two clades of P. delicatissima (clade 3 and 1) found in the
study by Orsini et al. (2004) correspond to clade A and
B in the present study.
A recent study has reported on mating between
strains of P. delicatissima from Gulf of Naples (Amato
et al. 2005). All four strains belonged to clade B of P.
delicatissima, the ITS sequences being similar to Castell
1, Castell2 and BP1, and the study therefore did not
contribute to the question regarding the difference be-
tween clade A and B. Apart from minor morphological
differences, the F1, the F2 and back cross-generations
were similar to the parental generations (Amato et al.
2005, Table 2) and identical to P. delicatissima (Table 5).
Differences in the shape of the tips on one or both
valve ends were observed in some strains, giving the
cells some similarity to P. pseudodelicatissima. Differences
in the arrangement of poroids gave a resemblance to
P. dolorosa in one of the backcross-generations. How-
ever, the strain with the aberrant poroids always had
narrow valves (o2 mm), a character differentiating it
from P. dolorosa (Amato et al. 2005). Such a strain has
never been found in the field. In general, the mating
experiments showed that the features characterizing
P. delicatissima are morphologically stable (Amato et al.
2005).
Large genetic variation within morphologically sim-
ilar strains has previously been reported among dia-
toms, including P. calliantha (identified as P. pseudo-
delicatissima) from the same bloom area (Skov et al.
1997). Using microsatellites, Evans et al. (2004) found
a high degree of genetic diversity among strains of
P. pungens isolated from the North Sea. However, no
support for cryptic diversity was found in P. pungens,
and the strains seem to constitute a single population
(Evans et al. 2005). Among strains of P. galaxiae from
the Gulf of Naples, Cerino et al. (2005) found low ge-
netic diversity using LSU rDNA, but in the field, dif-
ferent size populations occurred at different seasons.
In other diatoms like Thalassiosira weissflogii (Grunow)
G. Fryxell et Hasle, Murphy and Guillard (1976) found
genetic difference between neritic and oceanic clones.
Recently, Rynearson and Armbrust (2000, 2004) dis-
covered very large genetic diversity among strains of
Ditylum brightwellii (West) Grunow isolated from the
same locality, and proved the presence of genetically
distinct populations. Hence, genetic variation among
morphological identical strains i.e. cryptic diversity is
probably a frequent phenomenon among marine dia-
toms. Recent reports have shown cryptic diversity also
in the dinoflagellate Scrippsiella trochoidea (Montresor
et al. 2003) and within the Alexandrium tamarense com-
plex (Lily et al. 2003).
Previously, large intraspecific variation was reported
in Skeletonema costatum (Grev.) Cleve and some of the
478 NINA LUNDHOLM ET AL.
variation was related to seasonal changes (Gallagher
1980, 1982, Stabile et al. 1992). It was recently shown,
however, that at least some of the variation represent
interspecific variation, and ‘‘S. costatum’’ has now been
split into several species (Sarno et al. 2005). The new
observations on Skeletonema and those described in
P. delicatissima above indicate that the diversity within
the marine planktonic diatoms is much larger than
previously thought, and that our knowledge on intra-
and interspecific diversity is still very restricted (Mann
1997, 1999).
Reports of P. delicatissima in the literature. The de-
scription of the new species P. decipiens and P. dolorosa
enhances our knowledge of the variation in the
P. delicatissima complex. Morphological variation of
P. delicatissima has been reported several times (Rivera
1985, Rhodes et al. 1998) but it was only after studies
on a relatively large number of isolates from different
locations that the species-characteristic features be-
came apparent. It also required studies of cultures to
evaluate the morphological and molecular data. Sep-
aration of P. pungens and P. multiseries, as well as P. se-
riata and P. australis, are also based on differences in a
few morphological characters, which became ques-
tioned by other authors but subsequently received
strong molecular support (Douglas et al. 1994, Man-
hart et al. 1995, present paper). The same applies to
the P. pseudodelicatissima complex (Lundholm et al.
2003). Hence, the species delineation within the ge-
nus is getting based on both morphological and mo-
lecular data, representing both the morphological
and a phylogenetic species concept.
Some of the reports of morphological variation in
the ‘‘delicatissima’’ complex can now perhaps be ex-
plained. Hallegraeff (1994) found cells that differed
from P. turgidula in being shorter (30–40 mm) and with
a more delicate structure (32 striae and 21 fibulae in
10 mm). The isolates were lanceolate with a slight ten-
dency for being asymmetrical, and possessed seven to
nine poroids in 1 mm (Hallegraeff 1994, Fig. 5A). Meas-
urements on light micrographs in Hallegraeff (1994,
Fig. 5, A, B, and C) give a valve width of 2.3–2.7 mm. All
these features agree with P. dolorosa.
The large variation of P. delicatissima reported by
Rivera (1985) may be explained by Rivera’s material
being a mixture of P. delicatissima, P. decipiens, and P.
dolorosa. Only combined measurements are listed, but
Fig. 80 in Rivera (1985) shows a 2.5 mm-wide specimen
with 34 striae and 20 fibulae in 10 mm, and with a ten-
dency for the two rows of poroids to merge. This spec-
imen agrees with P. dolorosa. Similarly, the observations
of cells from New Zealand by Rhodes et al. (1998) with
characteristics of both P. delicatissima and P. turgidula
may be explained by the material being a mixture
of P. delicatissima, P. decipens, and P. dolorosa.
Hybridization pattern using rRNA probes. Some, but
not all, the variation observed when applying rRNA
probes to the various strains of presumed P. del-
icatissima can be explained by the presence of sever-
al species. Only the P. decipiens strains hybridized with
heD2-2. One group of P. delicatissima (Castell1, Cas-
tell2, and BP1) reacted as the only group with auD1
and muD2 and weakly with frD1, and another group
(OFPd972, OFPd973, Zhenbo6, and MexA) reacted
as the only group strongly with deD1. Only weak hy-
bridization reactions differentiated the two remain-
ing P. delicatissima groups and the P. dolorosa group
(Table 2).
Variation in the hybridization pattern of rRNA
probes applied to strains identified as P. delicatissima
has previously been reported by Rhodes et al. (1998),
Parsons et al. (1999), Scholin et al. (1999), Orsini et al.
(2002) and C. Cusack (personal communication). The
strains on which the probes are based were isolated
from Monterey Bay, California. They reacted with the
probe deD1 only, but, as in our studies, cross-reactions
with probe frD1 were also found in strains isolated
from Monterey Bay (California), Louisiana coastal wa-
ters and Irish waters (Miller and Scholin 1996, Parsons
et al. 1999, C. Cusack, personal communication). Sub-
sequently, cross-reactions of a P. delicatissima-like spe-
cies with the auD1 probe were reported in field
samples from Monterey Bay (Scholin et al. 1999).
The presence of species cross-reacting with auD1 was
believed to be typical of waters offshore Monterey Bay.
It was also considered to be more abundant during El
Niño years, indicating the presence of several geneti-
cally divergent strains of P. delicatissima in Monterey
Bay and possibly a physiological difference between
the strains (Scholin et al. 1999). Owing to the hybrid-
ization reactions, these strains may represent the two
clades (A and B) of P. delicatissima. Rhodes et al. (1998)
noted that some P. delicatissima-like strains did not react
with any of the probes they applied and, similarly, P.
delicatissima strains from Gulf of Naples did not react
with any probes (Orsini et al. 2002). In summary, the
above-mentioned data expand the type of cross-reac-
tions found in the present study and indicate the pres-
ence of a very large genetic diversity within the
P. delicatissima-complex.
Molecular probes may serve as a means of studying
intraspecific genetic variation. In addition, they may be
used to study intraspecific population dynamics in the
field (Parsons et al. 1999). In our experience, molec-
ular probes must always be used with caution and test-
ed on local strains, as inconsistent probing results may
be caused by intraspecific genetic variation as well as by
the presence of other, possibly unknown, species. It is
also a concern that weak hybridization may take place
also in case of base pair mismatches. In the present
study the strongest positive reactions ( þ þ or þ þ þ )
took place when there were none or only a single base
pair of mismatch. A weak reaction ( þ ) occurred in
cases of up to 3 base pair mismatches. With additional
modifications to the hybridization protocol such cross
reactivity should be eliminated.
Toxin data. The toxin analyses of isolates of P. del-
icatissima, P. decipiens, and P. dolorosa did not show the
presence of DA in any of the tested isolates. These
results do not exclude that the cultures may produce
 GENETIC VARIATION OF P. DELICATISSIMA
DA in minor concentrations or under different con-
ditions; only two or three samples from the stationary
growth phase were tested. The results agree with
analyses of P. delicatissima by Villac et al. (1993),
Lundholm et al. (1994), and Orsini et al. (2002). Oth-
er studies have reported toxin-producing P. del-
icatissima (Smith et al. 1990, Rhodes et al. 1996),
and the existence of both toxic and non-toxic strains
has also been found in several other Pseudo-nitzschia
species. In field samples, P. delicatissima-like cells
cross-reacted with the auD1 probe when low levels
of DA were detected (Scholin et al. 1999). When the
strains were brought into culture, however, they did
not show production of DA. Either a different source
of DA was present in the field samples or production
of DA differed between cells in culture and in the
field (Scholin et al. 1999). The latter scenario may be
comparable with an experiment in which P. calliantha
(as P. pseudodelicatissima) produced DA only under
some culture conditions (Lundholm et al. 1997). Fac-
tors triggering production of DA have still not been
identified. On the whole, present knowledge of toxin
production in the P. delicatissima-complex is very
sparse.
CONCLUSION
The present study aimed to solve some of the tax-
onomic confusion regarding species resembling P. del-
icatissima. Two new species (P. decipiens and P. dolorosa)
were found. The results may explain some of the pre-
vious morphological variation reported in P. del-
icatissima and P. turgidula. The detection of large
genetic variation in P. delicatissima indicate cryptic spe-
cies and/or show a species in the process of speciation.
Among phytoplankton species, and in diatoms in par-
ticular, large intraspecific variation, presence of cryptic
species and presence of morphologically similar spe-
cies is likely to be encountered more frequently as the
number of studies increases.
We thank Grethe Hasle for valuable discussions concerning
taxonomy and for use of photographic material, and David
Mann and Luisa Orsini for useful comments on the manu-
script. Lene Christiansen, Charlotte Hansen, Lisbeth Ha-
ukrogh, Kenn Kristiansen, and Niels Henry Larsen are
thanked for technical assistance. We also acknowledge the
persons providing subsamples of their cultures (Eun Cho,
Francisco Rodrigues, Jette Skov, Lars Hotegaard, Yang
Zhenbo and Nickolai Davidovich), giving us access to their
observations (Caroline Cusack, Martha Ferrario, Nicolai Da-
vidovich) or collecting water samples for us (Lisbeth Ha-
ukrogh, Jette Buch Østergård and Lis Hjlund). This study
was funded by Danish Science Research Council project
9601668 and by a grant from the Carlsberg Foundation to
Nina Lundholm (grant 0656/20).
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