Journal of Sea Research 46 (2001) 213±222
www.elsevier.com/locate/seares
Variation along ITS markers across strains of
Fibrocapsa japonica (Raphidophyceae) suggests hybridisation
events and recent range expansion
Wiebe H.C.F. Kooistra a,*, M. Karin de Boer b, Engel G. Vrieling b,
Laurie B. Connell c, Winfried W.C. Gieskes b
b
a
Stazione Zoologica ªA. Dohrnº, Villa Comunale, 80121 Naples, Italy
Department of Marine Biology, Center for Ecological and Evolutionary Studies, University of Groningen, Biological Center,
P.O. Box 14, 9750 AA Haren, The Netherlands
c
National Marine Fisheries Service, Northwest Fisheries Science Center, Environmental Conservation Division,
2725 Montlake Blvd E, Seattle, WA 98112, USA
Received 31 January 2001; accepted 29 May 2001
Abstract
The ¯agellate micro-alga Fibrocapsa japonica can form harmful algal blooms along all temperate coastal regions of the
world. The species was ®rst observed in coastal waters of Japan and the western US in the 1970s; it has been reported regularly
worldwide since. To unravel whether this apparent range expansion can be tracked, we assessed genetic variation among
nuclear ribosomal DNA ITS sequences, obtained from sixteen global strains collected over the course of three decades. Ten
sequence positions showed polymorphism across the strains. Nine out of these revealed ambiguities in several or most
sequences sampled. The oldest strain collected (LB-2161) was the only one without such intra-individual polymorphism. In
the others, the proportion of ambiguities at variable sites increased with more recent collection date. The pattern does not result
from loss of variation due to sexual reproduction and random drift in culture because sister cultures CS-332 and NIES-136
showed virtually the same ITS-pattern after seven years of separation. Neither are the patterns explained by recent range
expansion of a single genotype, because in that case one would expect lowest genetic diversity in the recently invaded North
Sea; instead, polymorphism is highest there. Recent ballast-water-mediated mixing of formerly isolated populations and
subsequent ongoing sexual reproduction among them can explain the increase in ambiguities. The species' capacity to form
harmful blooms may well have been enhanced through increased genetic diversity of regional populations. q 2001 Elsevier
Science Ltd All rights reserved.
Keywords: Dispersal; Fibrocapsa japonica; Harmful algal blooms (HAB); Hybridisation; ITS; Phylogeography; Raphidophyceae
1. Introduction
The marine algal species Fibrocapsa japonica
Toriumi et Takano is a common ¯agellate of the
* Corresponding author.
E-mail address: [email protected] (W.H.C.F. Kooistra).
class Raphidophyceae (Hara and Chihara, 1985),
which occasionally forms harmful blooms in coastal
waters (Taylor, 1990; Hilmer and Bate, 1991; Imai et
al., 1998; Smayda, 1998). Cells can produce neurotoxins (Khan et al., 1996) potentially threatening
marine wildlife (Anderson et al., 1998) and are occasionally held responsible for mass mortality events of
1385-1101/01/$ - see front matter q 2001 Elsevier Science Ltd All rights reserved.
PII: S 1385-110 1(01)00086-7
214
W.H.C.F. Kooistra et al. / Journal of Sea Research 46 (2001) 213±222
economically important ®sh stocks (Okaichi, 1972;
Toriumi and Takano, 1973; Ono, 1989). At unfavourable conditions, inconspicuous resting cysts are formed
(Yoshimatsu, 1987) and such resting stages have been
associated with sexual reproduction in numerous
phytoplankton species (Ono, 1988; Yamaguchi and
Imai, 1994; Matsuoka and Fukuyo, 1995). Meiotic
division, however, has yet never been observed in F.
japonica.
The species was reported ®rst in Japan (Okaichi,
1972) where it now abounds in coastal areas and
inland seas (Toriumi and Takano, 1973; Yoshimatsu,
1987; Iwasaki, 1989). It was also collected in southern
California in the 1970s (Loeblich and Fine, 1977),
although that strain was identi®ed only afterwards as
F. japonica by Gibbs et al. (1980). More recently the
species has been detected in coastal regions of the
northern Atlantic and the Mediterranean Sea (Smayda
and Villareal, 1989; Billard, 1992; Vrieling et al.,
1995; Tomas, 1998), the southern Atlantic (Odebrecht
and Abreu, 1995), and in southern Australia and New
Zealand (Chang et al., 1995). So far, the species has
never been reported from the tropical belt or from
Arctic or Antarctic regions. Its apparent con®nement
to disjunct temperate ocean regions and the chronology of the reports suggest a northern Paci®c donor
population and recent range expansion by means of
jump dispersal events, possibly resulting from discharge of ship ballast water loaded elsewhere or
with introduced mariculture species (Hallegraeff,
1993, 1998; Scholin et al., 1995; Hayes, 1998;
Nehring, 1998).
We used nuclear ribosomal DNA internal transcribed spacer sequences (ITS-1 and ITS-2) to detect
possible genealogical lineages (distinct ITS-types)
and phylogeographic structure within F. japonica,
and to track possible dispersal routes. The spacers
are grouped together with other ribosomal DNA
regions (18S, 5.8S and 28S genes) in cistrons, which
are repeated several hundred times in each genome
(Jorgensen and Cluster, 1988). Mutations occur
along these strings, but such resulting polymorphisms
among the various copies are eliminated rapidly
through a series of homogenising mechanisms
referred to as concerted evolution (Dover, 1982;
Arnheim, 1983). In rare cases, however, concerted
evolution substitutes the new type throughout the
chain of cistrons and throughout the population
changing the cistrons over time. The spacers are the
most rapidly changing sequences in the cistron
because of their relative lack of functional constraints.
Therefore, they have been applied to infer phylogenies and track dispersal routes at or below the
species level (Bakker et al., 1992; Coleman et al.,
1994; Zechman et al., 1994; Van Oppen et al., 1995;
Peters et al., 1997; Connell, 2000).
Here, we reveal high ITS polymorphism among and
within strains of F. japonica isolated throughout the
world's known distribution range and evaluate the
biological and phylogeographic signi®cance of this
polymorphism. Some intra-population and intraindividual polymorphism can be maintained as a
result of equilibrium between mutation rates and
homogenising forces of concerted evolution (Suh
et al., 1993; Sang et al., 1995; Wendel et al., 1995;
Pillmann et al., 1997; SerraÄo et al., 1999; FamaÁ et al.,
2000). Polymorphism will be encountered most likely
in the ITS-regions because of these regions' low functional constraits. Different levels of polymorphism
among species could result from differences in ploidy
levels of the vegetative life stage, frequency of sexual
reproduction and distribution of the rDNA cistrons in
the genome since these factors all affect the homogenisation rate (Hillis and Davis, 1988; Sang et al.,
1995; Wendel et al., 1995). Polymorphism can also
result from ecophysiological differentiation (Gallagher,
1980) or from hybridisation between genetically
distinct populations, either by infrequent attenuated
gene ¯ow or by mixing of formerly isolated populations (Hillis and Davis, 1988; Scholin et al., 1995).
2. Materials and methods
Fifteen strains of F. japonica and one of Fibrocapsa ªsp.º were obtained from culture collections
(Table 1). Of the North Sea strains, the three from
the Marsdiep in the south-eastern North Sea
(Md0913i, ii, iii) originated from individual cells
isolated from the same dm 3 of seawater taken in
September 2000. The DNA was processed only a
few weeks after establishment of the clonal strains.
The original BuÈsum harbour (eastern North Sea)
culture commenced in 1995 from a sample of a
mono-speci®c bloom (U. Tillmann, pers. comm.,
2000). Clones 3, 4, 5 and 6 originated each from a
W.H.C.F. Kooistra et al. / Journal of Sea Research 46 (2001) 213±222
215
Table 1
Origin and isolation dates of Fibrocapsa japonica strains
Strain
Locality or mother strain
Date of isolation and
culture coll.
Genbank reference
LB-2162 a
NIES-136
CS-220
CCMP-1661
Cawr-02
CS-332
K-0542
BuÈsum OR b
Clone 3 c
Clone 4 c
Clone 5 c
Clone 6 c
Cawr-19
Md0913i
Md0913ii
Md0913iii
Point Loma, California
Tsuda Bay, Japan
Hobson Bay, Victoria, Aus
Hobsons Bay, Australia
Leigh, New Zealand
NIES-136
Sea off BuÈsum, Germany
BuÈsum Harbour, Germany
BuÈsum OR
BuÈsum OR
BuÈsum OR
BuÈsum OR
Wellington Harbour, NZ
Marsdiep, Netherlands
Marsdiep, Netherlands
Marsdiep, Netherlands
1970
1978
1988
1988
1992
1993
1995
1995
1997
1997
1997
1997
1999
2000
2000
2000
AF112991
AF152603
UTEX
NIES
SCIRO
CCMP
Cawthron
CSIRO
SCCAP
BuÈsum
CCRUG d
CCRUG
CCRUG
CCRUG
Cawthron
CCRUG
CCRUG
CCRUG
a
In UTEX referred to as Chattonella japonica.
Original BuÈsum culture; this culture was established from a monospeci®c bloom (U. Tillmann, pers. comm., 2000).
c
The clonal strains were established in 1997 by taking a single cell from the original BuÈsum culture (Vrieling and Tjallingii, unpublished
data).
d
Culture collection of the University of Groningen.
b
single cell taken from the original BuÈsum harbour
culture after it had been established in our laboratory
at the University of Groningen in 1997 (Table 1).
Strain K-0542 also originated from the BuÈsum region
but was collected offshore. Strain CS-332 was
sampled from its parental strain NIES-136 in 1993
and was maintained since then as a distinct culture.
All cultures were grown in 75 cm 3 ¦/2-enriched
seawater medium (Guillard, 1975) with a salinity of
25 practical salinity units at 168C, at a light intensity
of 25 mMol photons m 22 s 21 provided by ¯uorescent
tubes (Osram 36W/19 daylight, spectral range:
^300±700 nm), with a 16/8 h light±dark cycle.
Samples of 2 cm 3 of medium containing growing
cells were ®ltered through 0.2 mm pore-diameter
polycarbonate membrane ®lters (Osmonics, Livermore,
CA). Filters were immersed in 500 mm 3 DNAextraction buffer containing 2% (w/v) CTAB, 1.4 M
NaCl, 20 mM EDTA, 100 mM TRIS-HCl, pH8, 0.2%
(w/v) PVP, 0.01% (w/v) SDS and 0.2% b -mercaptoethanol. Immersed ®lters were incubated at 658C for
5±10 min, vortexed for a few seconds and then cooled
brie¯y on ice. The DNA was extracted with an equal
volume of chloroform-isoamylalcohol (CIA; 24/1
[v/v]) and centrifuged in a table-top Eppendorff
microfuge at maximum speed (14 000 rpm) for
10 min. The aqueous phase was collected, reextracted with CIA and centrifuged as above. Next,
the aqueous phase was mixed thoroughly with 0.8
volumes ice-cold 100% isopropanol, then left on ice
for 5 min and subsequently centrifuged in a precooled Eppendorff microfuge under maximum speed
for 15 min. The DNA pellets were washed in
500 mm 3 70% (v/v) ethanol, centrifuged 6 min, and
after decanting of the ethanol, allowed to dry on air.
The obtained DNA pellets were dissolved overnight
in 100 mm 3 0.1X TE buffer. The quantity and quality
of the DNA was examined by agarose gel electrophoresis against known standards.
The targeted marker sequence comprises the internal
transcribed spacers (ITS-1 and -2) and the 5.8S rDNA
within the nuclear ribosomal DNA cistron. The marker
was PCR-ampli®ed in 25-mm 3 volumes containing 10±
25 ng DNA, 2.5 mM MgCl2, 10 mM Tris-HCl pH9.0,
50 mM KCl, 0.1% Triton X-100, 1 mM dNTPs, 0.5 mM
of forward primer AFP2-F (5 0 -AGC TCT TTC TTG
ATT CTA TG-3 0 ; Peters et al., 1997), 0.5 mM of
reverse primer 26B (5 0 -GGT CCG TGT TTC AAG
216
W.H.C.F. Kooistra et al. / Journal of Sea Research 46 (2001) 213±222
Table 2
Variable positions in the ITS region of the nuclear rDNA of Fibrocapsa japonica strains. Positions are numbered relative to the ®rst base at the
5 0 -end of ITS-1 from strain LB-2162. An ambiguity (e.g. CT) is depicted as Ct if C dominates, as cT if T dominates and as CT if both are
represented more or less equally (Y would be proper coding but we prefer CT for clarity). We de®ne Ct and cT as weak ambiguities and CT as a
strong one
Isolate
Position
ITS-1
LB-2162 a
NIES-136 a
CS-220
CCMP-1661
Cawr-02
CS-332
K-0542
BuÈsum OR
Clone 3
Clone 4
Clone 5
Clone 6
Cawr-19
Md0913i
Md0913ii
Md0913iii
a
ITS-2
32
50
58
213
553
712
719
758
785
786
T
T
cT
T
CT
cT
cT
CT
Ct
cT
CT
CT
cT
CT
CT
cT
A
Ac
aC
AC
AC
AC
AC
aC
aC
aC
aC
aC
aC
AC
AC
aC
C
C
C
C
C
C
Ct
Ct
CT
Ct
Ct
CT
C
Ct
Ct
Ct
C
C
CT
C
CT
C
CT
CT
CT
CT
CT
CT
C
CT
CT
CT
C
CT
CT
C
CT
Ct
CT
Ct
Ct
CT
CT
C
CT
Ct
CT
CT
C
C
C
C
C
C
C
CT
CT
Ct
CT
CT
C
C
Ct
Ct
G
GT
gT
GT
GT
GT
GT
gT
gT
GT
GT
gT
GT
GT
GT
GT
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
cT
cT
CT
cT
cT
cT
cT
T
cT
cT
cT
cT
cT
CT
cT
A
AC
AC
AC
aC
AC
aC
C
C
C
C
C
AC
aC
aC
AC
Sequences originally generated by L.C., but rechecked and corrected following comparison with sequences generated by W.H.C.F.K.
ACG GG-3 0 ), and 1 unit Taq DNA Polymerase
(Promega, Madison, WI, USA). The PCR cycling
comprised an initial 1-min heating step at 948C
followed by 30 cycles of 948C for 30 s, 558C for
30 s, 728C for 90 s and ®nal extension at 728C for
5 min. Quantity and length of products were examined by agarose gel electrophoresis against known
standards. Products were puri®ed using the QIAquick
PCR Puri®cation Kit (Qiagen GmBH, Hilden,
Germany) following manufacturer's instructions.
Cycle sequencing reactions were performed in 20mm 3 volumes containing 50 mM Tris-HCl pH 9,
1.25 mM MgCl2, 0.16 mM of primer, 90 ng PCRproduct and 3 mm 3 Big-dye terminator reaction mix
(Applied Biosystems, Perkin-Elmer, Foster City, CA,
USA) following manufacturer's instructions. The
internal forward primer was oBTG 005B (5 0 -CTG
CGG AAG GAT CAT TACC C-3 0 ) and internal
reverse primer ITS4-Fj (5 0 -TCC TCT GCT TAG
TTA TAT GC-3 0 ; this study, after comparing available sequences in GENBANK). Products were
cleaned using Sephadex G-50 in Centri-Sep Spin
columns (Princeton Separations, Adelphia NJ, USA)
and sequenced on an ABI model 377XL automated
sequencer (Applied Biosystems) according to manufacturer's instructions. Forward and reverse sequences
were combined and aligned with Sequence navigator
(Applied Biosystems) or Sequencher (Gene Codes
Corp, Ann Arbor, MI, USA).
3. Results
All sequences obtained from the Fibrocapsa strains
used in this study showed identical length; ITS-1
contains 283 base pairs, the 5.8S rDNA 142, and
ITS-2 405. A condensed alignment is presented in
Table 2; only variable positions are shown. Four positions in the alignment of ITS-1 and six in that of ITS-2
showed polymorphism. Of these, nine showed intraindividual variation in the form of ambiguities in
several strains, either between A and C, between C
and T, or between G and T. If a variable position
revealed ambiguity in certain strains, then it was
W.H.C.F. Kooistra et al. / Journal of Sea Research 46 (2001) 213±222
Fig. 1. The relationship between the number of ambiguous sites at
ten variable positions in the ITS regions of Fibrocapsa japonica
strains and year of collection. Clones 3±6 and daughter strain CS332 have been excluded. Symbols used for origin of strains: `X'
Paci®c USA, `f' Western Europe, `W' Japan, and `P' Southern
Australia and New Zealand. Linear regression revealed signi®cant
correlation with r 2 ˆ 0.77 (P , 0.001). Strains presented in the
graph are: above the regression line from left to right NIES-136,
CS-220, Cawr-02, BuÈsum OR 1 K-0542, and Md0913ii 1
Md0913iii; below the regression line from left to right LB-2162,
CCMP-1661, Cawr-19, Md0913i.
always of a single type; either AC, CG, or CT, with
the remaining strains showing one or the other base
present in that ambiguity. The proportion of two bases
in the ambiguities differed among the strains (see
Table 2). Observed polymorphism did not result
from technical problems, because sequenced PCR
products generated from different DNA extracts of
the same strains showed similar chromatograms.
The explanation of the ambiguities is straightforward
if one considers the multi-copy nature of the
sequences. Relative heights of two or more peaks at
the same sequence position in a chromatogram ideally
re¯ect approximate proportions of distinct ITS types
in the string of cistrons.
The oldest strain collected (LB-2126, collected
in 1970) did not reveal any ambiguities whereas
all others possessed several. Ambiguity numbers
increased with more recent collection dates (Fig. 1)
and the recently collected North Sea samples showed
the highest number (Table 2). The fact that there are
differences among the Marsdiep clones and between
the BuÈsum harbour and open sea clones implies that
variation exists among individual cells in the ®eld
population (Table 2). Three years after establishment
217
of the BuÈsum harbour strains, the derived clones and
the original culture showed the same ambiguities in
their ITS-sequences but the proportion of each base
within ambiguities varied among them (Table 2).
Small but persistent differences between the Marsdiep
and BuÈsum strains indicate spatial and/or annual
genetic differentiation within the North Sea. At the
time of our comparison, ITS-regions of strains
NIES-136 and its daughter strain CS-332 were almost
identical (Table 2). Strain Fibrocapsa sp. (CS-220)
did not differ phenotypically from the other strains
in our morphological examination and its sequence
showed ambiguities comparable to those in sequences
of remaining strains (Table 2).
4. Discussion
Our ®nding of intra-individual and intra-speci®c
ITS-polymorphism corroborates ®ndings by Pillmann
et al. (1997) and FamaÁ et al. (2000) in macroalgae and
Sgrosso (2001) in phytoplankton. Others (Gallagher,
1980; Medlin et al., 1996; Rynearson and Armbrust,
2000) also encountered genotypic polymorphism
although in these studies markers were used that are
believed to evolve different from ITS regions. Notably, Medlin et al. (1996) revealed genetic variation
independent of geographic scale.
One explanation for high intra-individual ITSpolymorphism in F. japonica is that it emerged from
sexual reproduction between parents with different
ITS haplotypes (combinations of alleles of closely
linked loci). The parents may have originated from
formerly separated populations and been brought
together, possibly through ballast water exchange
(Scholin et al., 1993, 1995). Hybrids continue to
reproduce sexually given observed small-scale intrapopulation differences and differences in proportion
among the alleles of each polymorphic locus. Mixing
must be of historic times because otherwise concerted
evolution would eliminate most intra-speci®c ITS
diversity. Although appealing, the explanation is
quite complex. Of course, more simple ones could
explain the observed genetic patterns in F. japonica
were it not that they all have their weaknesses.
A provocative aspect of the hypothesis is sexual
reproduction. Raphidophyceae are believed to be
diplontic (Guillou et al., 1999). Although meiosis
218
W.H.C.F. Kooistra et al. / Journal of Sea Research 46 (2001) 213±222
and gamete fusion has been recorded in several
genera, e.g. Chattonella spp. (Yamaguchi and Imai,
1994; Imai et al., 1998), it has yet never been observed
in F. japonica. Cyst formation in F. japonica and
Heterosigma akashiwo (Hada) Hada ex Hara et
Chihara seems to be alike (Yoshimatsu, 1987; Imai,
1989; Imai et al., 1996; Imai and Itakura 1999); this
life cycle stage, however, is more easily observed in
Chattonella spp. (Imai, 1989; Imai et al., 1998). We
suspect that sex is yet unknown for F. japonica as
nobody has ever really studied that phenomenon of
this species.
Several clues suggest that F. japonica also reproduces sexually. Exclusive mitosis of hybrids would
essentially freeze intra-individual polymorphism;
because only two parental ITS-haplotypes would be
maintained in these clonal diploid individuals. The
resulting chromatograms would show two nucleotides
in about equal proportions at each polymorphic site.
Random drift would then raise one or a few of those
hybrids to dominance; an unfavourable state of affairs
in a changing environment (Lynch et al., 1991). Anyway, this is not what our data show. If sex takes place
frequently, then various possible re-combinations
of the original parental ITS-haplotypes can be
encountered. Weak and strong ambiguities (Table 2)
at the polymorphic loci and inter-individual variation
among the Marsdiep clones as well as among those
from BuÈsum are readily explained by such recurrent
re-assorting of the original parental ITS-haplotypes.
The observation could be veri®ed through cloning of
PCR-products and subsequent sequencing of many
individual ITS regions. Alternatively, various ITShaplotypes in the PCR-products could be separated
with DGGE or comparable techniques (Guldberg et
al., 1993; Shef®eld et al., 1993; Moyret et al., 1994)
and then sequenced separately. The expectation is
that crossing over has recombined the haplotypes of
the original parents into a whole array of daughter
haplotypes.
Ongoing sexual reproduction among hybrids and
between hybrids and original parental populations
can thus explain the apparent increase of the number
of polymorphic sites and the unequal proportions of
the alleles among these ambiguities. Strictly clonal
reproduction of initial hybrids cannot. This very argument, however, can back®re on the hypothesis that
before the 1970s the species consisted of a series of
geographically isolated populations each essentially
containing a single haplotype. The reason is that
sexual reproduction could proceed in culture. Concerted evolution due to unequal crossing-over, and
genetic erosion caused by random drift could then
reduce the number of ambiguities with time, thereby
explaining the observed trend (Fig. 1) simply as a
culture artefact. Then, the oldest strain (LB-2162)
would have eliminated all ambiguities. Likewise, the
ITS-differences among the BuÈsum clones could re¯ect
similar post-isolation differential genetic erosion.
Still, the strains CS-332 and NIES-136 share virtually
identical ITS-patterns (Table 2), irrespective of the
fact that both have been separated since 1993. Consequently, we can safely exclude post-isolation or
culture artefacts because the probability of identically
changing ambiguities is very small.
Similar ambiguities across all Fibrocapsa strains
collected throughout the known distribution range
suggest that F. japonica now forms a cosmopolitan
population. Even strain CS-220 (known as Fibrocapsa sp.) belongs to F. japonica. Most likely, subtle
morphological differences in the ®eld material at the
time of collection have been induced by environmental conditions (Khan et al., 1998). In a comparable
phylogeographic study of H. akashiwo, Connell
(2000) could assign all the used specimens in the
genus to this species based on identical ITS patterns
and identical phenotype under the same culture
conditions.
The apparent lack of genotypic differentiation
among our global strains of F. japonica strains corroborates their similar eco-physiological responses and
identical phenotype in culture (De Boer et al., unpublished results). This ®nding is remarkable since they
originate from three disjunct temperate regions: the
northern Paci®c, the northern Atlantic and the southern temperate Ocean (southern Australia and New
Zealand). Although the species blooms in eutrophic
coastal habitats (Taylor, 1990; Hilmer and Bate, 1991;
Imai et al., 1998), it does occur at low densities (,10 3
cells dm 23) in the central North Sea (Vrieling et al.,
1995; Koeman, pers. comm., 2000). Distribution
patterns inferred from sightings match temperature
requirements for growth ranging from about 10 to
268C (Yoshimatsu and Ono, 1986; Khan et al. 1996;
De Boer, unpublished results). The species can
survive temperatures below 108C and above 268C as
W.H.C.F. Kooistra et al. / Journal of Sea Research 46 (2001) 213±222
long as temperatures for growth are met during at least
part of the year (Yoshimatsu and Ono, 1986). So far, it
has never been observed in strictly tropical or polar
regions simply because surface water temperatures
are either too warm or too cold throughout the year
(McIntyre et al., 1981).
Shared ITS-polymorphisms among disjunct regions
could result from attenuated gene ¯ow through tropical upwelling zones (Bowen and Grant, 1997). Ecophysiological constraints permit occurrence in such
relatively cool regions along western shores of the
Americas and western Africa (McIntyre et al., 1981;
Weaver, 1990; Vermeij, 1992). Their apparent
absence from these upwelling regions could well be
due to a paucity of surveys there or extremely low
concentrations. However, the lack of sightings in
Atlantic temperate regions before the 1990s remains
dif®cult to explain since these areas have always been
under close scrutiny. Fibrocapsa japonica is quite
characteristic and easily recognised in live samples,
but it should be noted that raphidophytes are delicate
species, which cannot be preserved well to allow
proper taxonomic identi®cation in stored samples
(Billard, 1992; Vrieling et al., 1995).
Alternatively, the observed patterns may result
from recent range expansion of a single population.
Donor population must then formerly have been
con®ned to the Northern Paci®c, the latter suggested
by the chronology of ®rst sightings (see Introduction).
The species may have dispersed into the southern
temperate ocean (southern Brazil, southern Australia,
New Zealand) and the Northern Atlantic. Heterosigma
akashiwo has similar eco-physiological requirements,
a comparable disjunct distribution pattern, and a similar history of ®rst sightings (Connell, 2000). Absence
of ambiguities in H. akashiwo suggests that it originates from such a single donor population, spreading
around the world only recently (Connell, 2000). The
pattern is comparable to that of a strain of Caulerpa
taxifolia (M. Vahl) C. Agardh, which also appeared
all over the world only recently (Jousson et al., 1998,
2000). However, in contrast to F. japonica, these
examples of invasive taxa lack any ITS-polymorphism. In that case, one would expect highest genotypic
variation in the North Paci®c donor and distinct subsamples thereof in the Australian and North Sea
founders. Yet, in F. japonica, the increase of ambiguities with more recent collection date and the high
219
number of ambiguities in the apparently recently
invaded North Sea remain dif®cult to explain if one
assumes a single donor population.
Apparently, there must have been more than one
parental population, each with its own ITS-genotype.
The oldest strain, LB-2162, may have been collected
from such a population since it lacks ambiguities.
Intra-individual polymorphism in the Japanese strain
NIES-136 (and its daughter CS-332) can then result
from sexual reproduction between indigenous and
introduced parents. The emergence of such genetically heterozigous offspring could be responsible for
the sudden appearance of F. japonica blooms in the
Seto Inland Sea of Japan in the early 1970s (Okaichi,
1972; Toriumi and Takano, 1973). High cell densities
in the water imply intake of large cell numbers in
ballast water tanks, increasing the survival probability
of at least a few individuals during transport and
subsequent discharge (Hallegraeff and Bolch, 1992;
Hallegraeff, 1998; Gollasch et al., 2000). These hitchhikers can then initiate blooms in other coastal
regions. There, further hybridisation with other
formerly isolated populations will then provide additional ITS-polymorphism, thus conveniently explaining the increase with time shown in Fig. 1.
Where the formerly isolated populations abounded
remains unclear. It is possible that they were con®ned
to the eastern and western sides of the northern Paci®c
because that is where the species were reported in the
1970s; twenty years before they were ®rst recorded in
the northern Atlantic. Their much more recent sightings in the northern Atlantic probably result from
jump dispersal of already highly polymorphic hybrids
explaining high polymorphism among North Sea
strains. This hypothesis does not need a formerly
endemic northern Atlantic population.
Scholin et al. (1993) uncovered similar intraindividual polymorphism in the SSU rDNA of a
North American strain belonging to the Alexandrium
tamarense (Lebour) Balech species complex (Dinophyceae). They tentatively suggested that the pattern
resulted from hybridisation between local and
imported genotypes. FamaÁ et al. (2000) encountered
extremely high levels of ITS-polymorphism in
Caulerpa racemosa (ForsskaÊl) J. Agardh, a species
that, like C. taxifolia, recently invaded the Mediterranean Sea. Distinct ITS-haplotypes were found
together in single individuals, thereby obliterating
220
W.H.C.F. Kooistra et al. / Journal of Sea Research 46 (2001) 213±222
phylogeographic patterns. Here, hybridisation also
forms a likely explanation, but it remains unclear if
the hybrids continue to reproduce sexually.
Such models of diverging and merging populations
have been put forward to explain the bewildering
population-genetic diversity in green plants (Riseberg
and Wendel, 1993) and corals (Veron, 1995). We
believe hybridisation should be taken seriously in
phytoplankton biodiversity as well. Exchange of
ballast water and of aquaculture species among
regions does not only transport existing HAB-forming
phytoplankton species; it also brings together genealogical lineages within such species (Scholin et al.,
1993, 1995). Hybridisation may then give rise to
vigorously blooming, potentially extremely toxic
varieties, apparently appearing out of the blue sky.
Assessments of ribosomal DNA sequences isolated
from seawater samples uncovered considerable
microbial and phytoplankton diversity, unknown to
morphological taxonomists (Rappe et al., 1995;
Medlin and Simon, 1998) and it may be this cryptic
diversity that could be a potent source of future HAB
forming hybrids. Geographically separated lineages
belonging to still unknown toxic species may just
await the opportunity to be introduced to one another.
Acknowledgements
We are thankful to the Dutch National Institute of
Coastal and Marine Management (RIKZ) and Dr
R.P.T. Koeman (Koeman & Bijkerk B.V., Ecological
Research and Advice, Haren, NL) for assistance in
providing samples, isolation, and taxonomic crossidenti®cation of F. japonica strains and taxonomic
identi®cation. Drs A. Zingone, W.T. Stam and J.L.
Olsen are thanked for critical discussion of the manuscript. WHCFK was supported by a grant from the
Priority Programme Theme 3: Sustainable Use and
Conservation of Marine Living Resources of the
Dutch National Science Foundation (NWO) MKDB
was supported by Earth and Life Science (ALW)
running under the auspices of NWO.
References
Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), 1998.
Physiological Ecology of Harmful Algal Blooms. NATO-ASI
series, Vol. G41, 662 pp.
Arnheim, N., 1983. In: Nei, M., Koehn, M. (Eds.). Evolution of
Genes and Proteins. Sinauer, Sunderland, MA, USA, pp. 38±61.
Bakker, F.T., Olsen, J.L., Stam, W.T., Van den Hoek, C., 1992.
Nuclear ribosomal DNA internal transcribed spacers (ITS1
and ITS2) de®ne discrete biogeographic groups in Cladophora
albida (Chlorophyta). J. Phycol. 28, 839±845.
Billard, C., 1992. Fibrocapsa japonica (Raphidophyceae), algue
planctonique nouvelle pour les coÃtes de France. Crypt. Algol.
13, 225±231.
Bowen, B.W., Grant, W.S., 1997. Phylogeography of the sardines
(Sardinops spp.): assessing biogeographic models and population histories in temperate upwelling zones. Evolution 51,
1601±1610.
Chang, F.H., Mackenzie, L., Till, K., Hannah, D., Rhodes, L., 1995.
The ®rst toxic shell®sh outbreaks and the associated phytoplankton blooms in early 1993 in New Zealand. In: Lassus, P.,
Arzul, G., Erard, E., Gentien, P., Marcaillou-Le Baut, C. (Eds.).
Harmful Marine Algal Blooms. Lavoisier Publishing, Paris,
pp. 145±150.
Coleman, A.W., Suarez, A., Goff, L.J., 1994. Molecular delineation
of species and syngens in volvocalean green algae (Chlorophyta). J. Phycol. 30, 80±90.
Connell, L.B., 2000. Nuclear ITS region of the alga Heterosigma
akashiwo (Chromophyta: Raphidophyceae) is identical in
isolates from Atlantic and Paci®c basins. Mar. Biol. 136,
953±960.
Dover, G., 1982. Molecular drive: a cohesive mode of species
evolution. Nature 299, 111±117.
FamaÁ, P., Olsen, J.L., Stam, W.T., Procaccini, G., 2000. High levels
of intra- and inter-individual polymorphism in the rDNA ITS1
of Caulerpa racemosa (Chlorophyta). Eur. J. Phycol. 35, 349±
356.
Gallagher, J.C., 1980. Population genetics of Skeletonema costatum
(Bacillariophyceae) in Narragansett Bay. J. Phycol. 16, 464±
474.
Gibbs, S.P., Chu, L.L., Magnussen, C., 1980. Evidence that Olisthodiscus luteus is a member of the Chrysophyceae. Phycologia 19,
173±177.
Gollasch, S., Lenz, J., Dammer, M., Andres, H.-G., 2000. Survival
of tropical ballast water organisms during a cruise from the
Indian Ocean to the North Sea. J. Plankton Res. 22, 923±937.
Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine
invertebrates. In: Smith, W.L., Chanley, M.H. (Eds.). Culture of
Marine Invertebrate Animals. Plenum Press, New York, pp. 29±
60.
Guillou, L., CreÂtiennot-Dinet, M.-J., Medlin, L.K., Claustre, H.,
Loiseaux-De GoÈer, S., Vaulot, D., 1999. Bolidomonas: a new
genus with two species belonging to a new algal class, the
Bolidophyceae (Heterokonta). J. Phycol. 35, 368±381.
Guldberg, P., Henriksen, K.F., Guttler, F., 1993. Molecular analysis
of phenylketonia in Denmark: 99% of the mutations detected by
denaturing gradient gel electrophoresis. Genomics 17, 141±146.
Hallegraeff, G.M., 1993. A review of harmful algal blooms and their
apparent global increase. Phycologia 32, 79±99.
Hallegraeff, G.M., 1998. Transport of toxic dino¯agellates via
W.H.C.F. Kooistra et al. / Journal of Sea Research 46 (2001) 213±222
ships' ballast water; bioeconomic risk assessment and ef®cacy
of possible ballast water management strategies. Mar. Ecol.
Prog. Ser. 168, 297±309.
Hallegraeff, G.M., Bolch, C.J., 1992. Transport of diatom and dino¯agellate resting spores in ships' ballast water: implications for
plankton biogeography and aquaculture. J. Plankton Res. 14,
1067±1084.
Hara, Y., Chihara, M., 1985. Ultrastructure and taxonomy of Fibrocapsa japonica (Class Raphidophyceae). Arch. Protistenk. 130,
133±141.
Hayes, K.R., 1998. Ecological risk assessment for ballast water introductions: a suggested approach. ICES J. Mar. Sci. 55, 201±212.
Hillis, D.M., Davis, S.K., 1988. Ribosomal DNA: intraspeci®c
polymorphism, concerted evolution, and phylogeny reconstruction. Syst. Zool. 37, 63±66.
Hilmer, T., Bate, G.C., 1991. Vertical migration of a ¯agellatedominated bloom in a shallow South African estuary. Bot.
Mar. 34, 113±121.
Imai, I., 1989. Cyst formation of the noxious red tide ¯agellate
Chattonella marina (Raphidophyceae) in culture. Mar. Biol.
103, 235±239.
Imai, I., Itakura, S., 1999. Importance of cysts in the population
dynamics of the red tide ¯agellate Heterosigma akashiwo
(Raphidopyceae). Mar. Biol. 133, 755±762.
Imai, I., Itakura, S., Yamaguchi,, M., Honjo, T., 1996. Selective
germination of Heterosigma akashiwo (Raphidophyceae) cysts
in bottom sediments under low light conditions: a possible
mechanism for red tide initiation. In: Yasumoto, Y., Oshima,
Y., Fukuyo, Y. (Eds.). Harmful and Toxic Algal Blooms. Int.
Oceanogr. Comm. UNESCO, Paris, pp. 197±200.
Imai, I., Yamaguchi, M., Watanabe, M., 1998. Ecophysiology, life
cycle and bloom dynamics of Chattonella in the Seto Inland
Sea, Japan. In: Anderson, D.M., Cembella, A.D., Hallegraeff,
G.M. (Eds.). Physiological Ecology of Harmful Algal Blooms.
NATO ASI series, G41, pp. 45±112.
Iwasaki, H., 1989. Recent progress of red-tide studies in Japan: an
overview. In: Okaichi, T., Anderson, D.M., Nemoto, T. (Eds.).
Red Tides: Biology, Environmental Science and Toxicology.
Elsevier, New York, pp. 3±9.
Jorgensen, R.A., Cluster, P.D., 1988. Modes and tempos in the
evolution of nuclear ribosomal DNA: new characters for evolutionary studies and new markers for genetic and population
studies. Ann. Missouri Bot. Gard. 75, 1238±1247.
Jousson, O., Pawlowski, J., Zaninetti, L., Meinesz, A., Boudouresque,
C.F., 1998. Molecular evidence for the aquarium origin of the
green alga Caulerpa taxifolia introduced to the Mediterranean
Sea. Mar. Ecol. Prog. Ser. 172, 275±280.
Jousson, O., Pawlowski, J., Zaninetti, L., Zechman, F.W., Dini, F.,
Di Guiseppe, G., Wood®eld, R., Millar, A., Meinesz, A., 2000.
Invasive alga reaches California. Nature 408, 157±158.
Khan, S., Arakawa, O., Onoue, Y., 1996. Growth characteristics of a
neurotoxin-producing Cloromonad Fibrocapsa japonica
(Raphidophyceae). J. World Aquacult. Soc. 27, 247±253.
Khan, S., Haque, M.M., Arakawa, O., Onoue, Y., 1998. In¯uence of
environmental factors on the morphology of red-tide producing
phyto¯agellate Fibrocapsa japonica. J. Aqua. Trop. 13, 119±
132.
221
Loeblich III, A.R., Fine, K., 1977. Marine Chloromonads: more
widely distributed in neritic environments that previously
thought. Proc. Biol. Soc. Wash. 90, 388±399.
Lynch, M., Gabriel, W., Wood, A.M., 1991. Adaptive and demographic responses of plankton populations to environmental
change. Limnol. Oceanogr. 36, 1301±1312.
McIntyre, C.D., and other CLIMAP project members, 1981. Seasonal
reconstructions of the earth's surface at the last glacial maximum. Geol. Soc. Am. Map Chart Ser. MC-36, Boulder, CO.
Matsuoka, K., Fukuyo, Y., 1995. Taxonomy of cycts. In:
Hallegraeff, G.M., Anderson, D.M., Cembella, A.D. (Eds.).
Manual on Harmful Marine Microalgae. Manuals and Guides.
Intergov. Oceanogr. Comm. UNESCO, Santiago de Compostela, Spain, pp. 381±401.
Medlin, L.K., Barker, G.L.A., Campbell, L., Green, J.C., Hayes,
P.K., Marie, D., Wrieden, S., Vaulot, D., 1996. Genetic characterisation of Emiliania huxleyi (Haptophyta). J. Mar. Syst. 9,
13±31.
Medlin, L.K., Simon, N., 1998. Phylogenetic analysis of marine
phytoplankton. In: Cooksey, K.E. (Ed.). Molecular Approaches
to the Study of the Ocean. Chapman and Hall, London, pp. 161±
186.
Moyret, C., Theillet, C., Puig, P.L., Moles, J.-P., Thomas, G.,
Hamelin, R., 1994. Relative ef®ciency of denaturing gradient
gel electrophoresis and single strand conformation polymorphism in the detection of mutations in exons 5 to 8 of the p53 gene.
Oncogene 9, 1739±1743.
Nehring, S., 1998. Non-indigenous phytoplankton species in the
North Sea: supposed region of origin and possible transport
vector. Arch. Fish. Mar. Res. 46, 181±194.
Odebrecht, C., Abreu, P.C., 1995. Raphidophyceans in southern
Brazil. Harmful Algae News 12/13, 4.
Okaichi, T., 1972. Occurrence of red-tides related to neritic water
pollution. In: Anonymous (Ed.). The Cause of Red-Tide in
Neritic Waters. Japan. Ass. Protec. Fish. Resour, Tokyo,
pp. 58±76.
Ono, C., 1988. Cell cycles and growth rates of red tide organisms in
Harima±Nada area, eastern part of Seto Inland Sea, Japan. Bull.
Akashiwo Res. Inst. Kagawa Prefecture 3, 1±67.
Ono, C., 1989. Red-tide problems in the Seto Inland Sea, Japan. In:
Okaichi, T., Anderson, D.M., Nemoto, T. (Eds.). Red Tides:
Biology, Environmental Science and Toxicology. Elsevier,
New York, pp. 137±142.
Peters, A.F., Van Oppen, M.J.H., Wiencke, C., Stam, W.T., Olsen,
J.L., 1997. Phylogeny and historical ecology of the Desmarestiaceae (Phaeophyta) support a southern hemisphere origin.
J. Phycol. 33, 294±309.
Pillmann, A., Woolcott, G.W., Olsen, J.L., Stam, W.T., King, R.J.,
1997. Inter- and intraspeci®c genetic variation in Caulerpa
(Chlorophyta) based on nuclear rDNA ITS sequences. Eur. J.
Phycol. 32, 379±386.
RappeÂ, M.S., Kemp, P.F., Giovannoni, S.J., 1995. Abundant
chromophyte plastid 16S ribosomal DNA genes found in a clone
library from Atlantic Ocean seawater. J. Phycol. 31, 979±988.
Riseberg, L.H., Wendel, J.F., 1993. Introgression and its consequences. In: Harrison, R.G. (Ed.). Hybrid Zones and the Evolutionary Process. Oxford Univ. Press, New York, pp. 70±109.
222
W.H.C.F. Kooistra et al. / Journal of Sea Research 46 (2001) 213±222
Rynearson, T.A., Armbrust, E.V., 2000. DNA ®ngerprinting reveals
extensive genetic diversity in a ®eld population of the centric
diatom Ditylum brightwellii. Limnol. Oceanogr. 45, 1329±
1340.
Sang, T., Crawford, D.J., Stuessy, T.F., 1995. Documentation of
reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution. Proc. Natl.
Acad. Sci. USA 92, 6813±6817.
Scholin, C.A., Anderson, D.M., Sogin, M., 1993. The existence of
two distinct small-subunit rRNA genes in the toxic dino¯agellate Alexandrium funduense (Dinophyceae). J. Phycol. 29, 209±
216.
Scholin, C.A., Hallegraeff, G.M., Anderson, D.M., 1995. Molecular
evolution of the Alexandrium tamarense ªspecies complexº
(Dinophyceae): dispersal in the North American and West
Paci®c regions. Phycologia 34, 472±485.
SerraÄo, E., Alice, L.A., Brawley, S.H., 1999. Evolution of the
Fucaceae (Phaeophyta) inferred from nrDNA-ITS. J. Phycol.
35, 382±394.
Sgrosso, S., 2001. Dino¯agellati produttori di cisti calcaree: fattori
che inducono l'incistamento e variabilitaÁ intraspeci®ca in
Scrippsiella trochoidea. Doctoral thesis UniversitaÁ degli studi
di Messina, Italy, 165 pp.
Shef®eld, V.C., Beck, J.S., Kwitek, A.E., Sandstrom, D.W., Stone,
E.M., 1993. The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions.
Genomics 16, 325±332.
Smayda, T.J., 1998. Ecophysiology and bloom dynamics of Heterosigma akashiwo (Raphidophyceae). In: Anderson, D.M.,
Cembella, A.D., Hallegraeff, G.M. (Eds.). Physiology of Harmful Algal Blooms. NATO-ASI series, G41, pp. 113±131.
Smayda, T.J., Villareal, T., 1989. The 1985 ªbrown tideº and the
open phytoplankton niche in Narragansett Bay during summer.
In: Cosper, E.M., Bricelj, V.M., Carpenter, E.J. (Eds.). Novel
Phytoplankton Blooms: Causes and Impacts of Recurrent
Brown Tides and Other Unusual Blooms. Coastal and Estuarine
Studies, 35. Springer Verlag, Berlin, pp. 165±187.
Suh, Y., Thien, L.B., Reeve, H.E., Zimmer, E.A., 1993. Molecular
evolution and phylogenetic implications of internal transcribed
spacer sequences of ribosomal DNA in Winteraceae. Am. J.
Bot. 80, 1042±1055.
Taylor, F.J.R., 1990. Redtides, brown tides and other harmful algal
blooms: the view in the 1990's. In: Graneli, E., Sundstroem, B.,
Edler, L., Anderson, D.M. (Eds.). Toxic Marine Phytoplankton.
Elsevier, New York, pp. 527±533.
Tomas, C.R., 1998. Blooms of potentially harmful Raphidophycean
¯agellates in Florida coastal waters. In: Reguera, B., Blanco, J.,
FernaÂndez, M.L. (Eds.). Harmful Algae. Xunta de Galicia &
Intergov. Oceanogr. Comm. UNESCO, Santiago de Compostela, Spain, pp. 101±103.
Toriumi, S., Takano, H., 1973. Fibrocapsa, a new genus in
Chloromodadophyceae from Atsumi Bay, Japan. Bull. Tokai
Regional Fish. Res. Lab. 76, 25±35.
Van Oppen, M.J.H., Draisma, S.G.A., Olsen, J.L., Stam, W.T.,
1995. Multiple trans-Arctic passages in the red alga Phycodrys
rubens: evidence from nuclear rDNA ITS sequences. Mar. Biol.
123, 179±188.
Vermeij, G.J., 1992. Trans-equatorial connections between biotas in
the temperate eastern Atlantic. Mar. Biol. 112, 343±348.
Veron, J.E.N., 1995. Corals in Space and Time: the Biogeography
and Evolution of the Scleractinia. Cornell, NY, 321 pp.
Vrieling, E.G., Koeman, R.P.T., Nagasaki, K., Ishida, Y., Peperzak,
L., Gieskes, W.W.C., Veenhuis, M., 1995. Chattonella and
Fibrocapsa (Raphidophyceae): ®rst observation of, potentially
harmful, red tide organisms in Dutch coastal waters. Neth. J. Sea
Res. 33, 183±189.
Weaver, A.J., 1990. Ocean currents and climate. Nature 47, 432.
Wendel, J.F., Schnabel, A., Seelanan, T., 1995. Bidirectional interlocus concerted evolution following allopolyploid speciation
in cotton (Gossypium). Proc. Natl. Acad. Sci. USA 92, 280±
284.
Yamaguchi, M., Imai, I., 1994. A micro¯uorometric analysis of
nuclear DNA at differents stages in the life history of Chattonella antiqua and Chattonella marina (Raphidophyceae).
Phycologia 33, 163±170.
Yoshimatsu, S., 1987. The cyst of Fibrocapsa japonica (Raphidophyceae) found in bottom sediment in Harima±Nada, eastern
Inland Sea of Japan. Bull. Plankton Soc. Japan 34, 25±31.
Yoshimatsu, S., Ono, C., 1986. The seasonal appearance of red tide
organisms and ¯agellates in southern Harima±Nada. Seto
Inland Sea. Bull. Akashiwo Res. Inst. Kagawa Prefecture 2,
1±42.
Zechman, F.W., Zimmer, E.A., Therit, E.C., 1994. Use of ribosomal
DNA internal transcribed spacers for phylogenetic studies in
diatoms. J. Phycol. 30, 507±512.