Morphology and genetic affinities of a novel Chattonella isolate (Raphidophyceae) isolated from Iran's south coast (Oman Sea)

Abstract

The morphology and genetic affinity of a novel raphidophyte belonging to the genus Chattonella Biecheler is described for the first time from the Oman Sea along the south-east coast of Iran. While morphologically very similar to Chattonella subsalsa Biecheler, the Iranian isolates contain a distinct red eyespot. A comparison of LSU-rDNA and rDNA-ITS show that the Iranian isolate is genetically distinct from other Chattonella subsalsa strains isolated from across a wide global range and indicates that the Iranian isolates represent a distinct species related to Chattonella subsalsa.

Full text

156
http://journals.tubitak.gov.tr/botany/
Turkish Journal of Botany
Turk J Bot
(2014) 38: 156-168
© TÜBİTAK
doi:10.3906/bot-1210-33
Morphology and genetic anities of a novel Chattonella isolate (Raphidophyceae)
isolated from Iran’s south coast (Oman Sea)
Gilan ATTARAN-FARIMAN1,*, Christopher John Stanley BOLCH2
1Chabahar Maritime University, Marine Science Faculty, Chabahar, Iran
2National Centres for Marine Conservation and Resource Sustainability, Australian Maritime College, University of Tasmania,
Launceston, Australia
* Correspondence: g.attaran@cmu.ac.ir
1. Introduction
e genus Chattonella Biecheler, belonging to the
class Raphidophyceae, consists of small golden-brown
agellates. Taxonomy of the class is based mainly on cell
shape and size and the ultrastructure of the chloroplasts,
mucocysts, trichocysts, and ejectosomes (Marshall
et al., 2002). However, the existing taxonomy is still
controversial (Hosini-Tanabe et al., 2006). Previously,
7 species in the genus Chattonella have been described:
C. subsalsa Biecheler, C. antiqua (Hada) Ono, C. marina
(Subrahmanyan) Hara & Chihara, C. minima Hara &
Chihara, C. ovata Hara & Chihara, C. globosa Hara &
Chihara, and C. verruculosa Hara & Chihara (Hara et al.,
1994; Hallegrae and Hara, 1995). Recently, 2 Chattonella
species (C. globosa and C. verruculosa) have been separated
from the class Raphidophyceae based on molecular
analysis. ey now belong to the class Dictyochophyceae
and are currently regarded as a taxonomic synonym
of Vicicitus globosus (Hara & Chihara) Chang and
Pseudochattonella verruculosa (Hara & Chihara) Hosoi-
Tanabe, Honda, Fukaya, Inagaki & Sako (Edvardsen et al.,
2007; Hosoi-Tanabe et al., 2007; Takano et al., 2007; Cheng
et al., 2012). erefore, the genus Chattonella has 5 species.
Demura et al. (2009) suggested a taxonomic revision of C.
marina, C. ovate, and C. antiqua based on morphological
characteristics and genetic diversity and considered these
species a variety of C. marina and oered a new status: C.
marina var. ovata (Hara & Chihara) Demura & Kawachi
and C. marina var. antiqua (Hada) Demura & Kawachi.
Wild resting cysts from sediment have been reported in
some species of the genus including C. marina var. antiqua,
C. marina (Yamaguchi and Imai, 1994), C. marina var.
ovata (Yamaguchi et al., 2008), and C. subsalsa (Steidinger
and Penta, 1999). ese cysts may have a role in bloom
initiation in coastal areas (Peperzak, 2001; Blanco et al.,
2009; Cucchiari et al., 2010; Imai and Yamaguchi, 2012).
Noxious blooms of C. marina var. antiqua, C. marina,
C. subsalsa, Fibrocapsa japonica Toriumi & Takano, and
Heterosigma akashiwo (Hada) Hada ex Hara & Chihara
have oen been associated with mortalities of both
cultured and wild sh and shellsh (Oda et al., 1997; Hard
et al., 2000; Imai et al., 2001; Landsberg, 2002; Hiroishi
et al., 2005; Matsubara et al., 2007; Shen et al., 2011).
C. marina var. ovata has also been reported to form a
harmful bloom (Imai and Yamaguchi, 2012). In the north
part of the Oman and Arabian seas phytoplankton blooms
occur during and aer the north-east monsoon every year
(Attaran-Fariman and Javid, 2013; Latif et al., 2013). In
autumn 2010 a massive bloom of Chattonella sp. occurred
along the south-east coast of Iran (Pasabandar, Bris) in
the north part of the Oman sea, causing massive mortality
of sh and shellsh and 4 green turtle species (Nabavi,
2010). Identication and characterisation of Iranian
Chattonella is an important step towards understanding
Abstract: e morphology and genetic anity of a novel raphidophyte belonging to the genus Chattonella Biecheler is described for the
rst time from the Oman Sea along the south-east coast of Iran. While morphologically very similar to Chattonella subsalsa Biecheler,
the Iranian isolates contain a distinct red eyespot. A comparison of LSU-rDNA and rDNA-ITS show that the Iranian isolate is genetically
distinct from other Chattonella subsalsa strains isolated from across a wide global range and indicates that the Iranian isolates represent
a distinct species related to Chattonella subsalsa.
Key words: Chattonella, phylogeny, morphology, raphidophyte, cyst, LSU-rDNA, rDNA-ITS
Received: 18.10.2012 Accepted: 04.09.2013 Published Online: 02.01.2014 Printed: 15.01.2014
Research Article

ATTARAN-FARIMAN and BOLCH / Turk J Bot
157
the potential harmful consequences of future blooms in
the area. Identication of raphidophytes species based
solely on morphology is problematic due to their fragile
nature and the pleomorphic morphology of some species
(Aizdaicher, 1993; Tyrrell et al., 2001; Demura et al.,
2009), leading to diculties distinguishing one species
from another, especially within the varieties of C. marina
(Bowers et al., 2006; Demura et al., 2009). e fragile cells
of this group are also dicult to identify by transmission
electron microscopy aer xation due to a range of xation
artefacts (rondsen, 1993; Marshall et al., 2002).
Utilisation of internal transcribed spacer sequence
comparison has been well established in plants and marine
algae (Hosoi-Tanabe et al., 2007; Terzioğlu et al., 2012;
Dündar et al., 2013). rDNA sequencing has been used to
examine relationships among marine microalgae including
Raphidophyceae taxa, both at the population and species
level (Connell, 2000, 2002; Hosoi-Tanabe et al., 2007).
Several studies of raphidophytes have successfully used the
rDNA-ITS region to examine raphidophyte relationships,
and there is relatively broad coverage of rDNA-ITS
sequences available in public databases. ese studies
have shown that, while there is a variation among dierent
species, there is little or no rDNA-ITS sequence variation
within species, even among isolates from across the globe
(Kooistra et al., 2001; Bowers et al., 2006; Edvardsen et al.,
2007). erefore, this region is a potentially useful means to
distinguish distinct species of raphidophytes. In this study
we describe motile cells of a novel Chattonella isolate from
the south coast of Iran (Oman Sea) by light and scanning
electron microscopy and phylogenetic analyses carried out
based on LSU-rDNA and rDNA-ITS sequences.
2. Materials and methods
2.1. Cell culture and microscopy
Single agellated raphidophyte cells were isolated from
incubated mixed sediment collected from the south-
east coast of Iran using a micropipette under a Leica
stereomicroscope. Isolated cells were placed into 55-
mm polystyrene petri dishes containing 15 mL of GSe
medium and incubated at 26 °C ± 0.5 °C under cool white
uorescent light (70–90 µmol photon m–1s–1) with a 12
h light/12 h dark cycle. Successfully established cultures
were subsequently transferred to 100-mL Erlenmeyer
asks containing 50 mL of GSe and sub-cultured every 3
weeks under the conditions described above.
Encystment in cultures was examined by transfer to
nitrate/phosphate-decient GSe medium and incubation
under the conditions above (Yoshimatsu, 1987; Imai and
Itakura, 1999). Cells were photographed with an Olympus
BH-2 microscope equipped with a Leica DC300F
digital imaging system and a Zeiss Axioplan 2-Plus
microscope (Zeiss, Gottingen, Germany) equipped with
a Zeiss AxioCam HR digital camera using bright eld
and dierential interference contrast illumination. For
SEM, 10 mL of mid-logarithmic growth-phase cultures
were concentrated by centrifuge, xed with 4% osmium
tetroxide (OsO4), and adhered to polylysine-coated
coverslips (Marchant and omas, 1983). Coverslips were
then critical-point dried via liquid CO2, mounted on SEM
stubs, sputter coated with gold, and examined with a JEOL
JSM-840 scanning electron microscope.
2.2. DNA extraction, PCR, and DNA sequencing
DNA was extracted by a phenol:chloroform:isoamyl
alcohol gentle-lysis method (Bolch et al., 1998), and the
internal transcribed spacer 1 (ITS1), 5.8S rRNA gene, and
internal transcribed spacer2 (ITS2) were amplied. For
amplication of the rDNA-ITS, primers ITSA and ITSB
(Adachi et al., 1994) were used, and for partial large subunit
(LSU) rRNA gene, D1R-F and 1483-R primers (Daugbjerg
et al., 2000) were used. Amplied PCR products were
puried using Montage PCR clean-up columns (Millipore,
USA), and 60 ng of puried product was used as template
in DNA sequencing reactions. PCR products were
sequenced using a Beckman-Coulter Dye Terminator
Sequencing Kit, according to standard protocols. Sequence
base-calling errors were corrected by manual inspection
of electropherograms using the soware program BioEdit
(Hall, 1999). DNA sequence data from Iranian Chattonella
sp. isolate CHPI36 was aligned to comparable nucleotide
sequences of other raphidophytes available from GenBank
using Clustal-X soware v.1.83 (Jeanmougin et al., 1998),
and alignments were improved by manual inspection.
Details of the taxa included in the analyses are summarised
in Tables 1 and 2.
2.3. Alignment and phylogenetic analyses
Two sequence alignments were used to infer relationships
among Chattonella spp. and the phylogenetic position
of the Chattonella sp. CHPI36. e rDNA-ITS dataset
contained 27 taxa and 730 characters in the sequence
alignment. Olisthodiscus luteus Carter, 1937 was used as
outgroup for the analysis. e large subunit rDNA-LSU
rRNA gene dataset contained 16 taxa and 1361 characters.
e diatom Cylindrotheca closterium (Ehrenberg) Reimann
& Lewin was used as an outgroup for the analysis. e
LSU-rDNA sequences C. subsalsa CCMP217 (AF409129)
and Chattonella sp. (CHPI36) were approximately 1360
bp in length, whereas all other sequences corresponded
to approximately the rst 680–700 bp of the alignment.
Analyses were repeated with all sequences truncated
to the rst 700 bp of the alignment, and any changes
in branching order were noted. PAUP* version 4.0b10
for Macintosh (PPC) was used (Swoord, 2002) for all
phylogenetic analysis of rDNA-ITS region and partial
LSU-rDNA. Phylogenetic structure was examined and
tested by the randomisation tests and probability tables

ATTARAN-FARIMAN and BOLCH / Turk J Bot
158
Table 1. Details of species used in the phylogenetic analysis of partial LSU-rDNA sequences.
Species GenBank accession no. Strain code Geographical locations
Chattonella sp. JF896100 CHPI36 Iran
Chattonella subsalsa AF409126 CCMP217 Gulf of Mexico
Chattonella subsalsa AF210736 CCMP217 Gulf of Mexico
Chattonella marina AY704162 Hong Kong
Chattonella marina AF210739 CCMP-217 Gulf of Mexico
Heterosigma akashiwo AY704161 Hong Kong
Heterosigma akashiwo AF086948 CCMP-452 Long Island Sound, USA
Heterosigma akashiwo AF042820 Masan Bay, Korea
Heterosigma akashiwo AF210741 CAWR05
Vacuolaria virescens AF210742 LB2236
Vacuolaria virescens AF409125 SAG1195.1 Wirral, Cheshire, England
Chattonella ovata AF210738 NIES-603 Seto Inland Sea, Japan
Chattonella ovata AY704163 Hong Kong
Chattonella antiqua AF210737 NIES-1 Seto Inland Sea, Japan
Olisthodiscus luteus AF210743 NIES-15 Seto Inland Sea, Japan
Cylindrotheca closterium AF417666 K-520
Table 2. List of species included in the phylogenetic analysis of ITS region of rDNA.
Species GenBank accession no. Strain code Geographical locations
Chattonella sp. JF896101 CHPI36 Iran
Chattonella subsalsa AF409126 CCMP217 Gulf of Mexico
Chattonella subsalsa AY858871 C. Tomas Texas
Chattonella subsalsa AY858870 C. Tomas Singapore
Chattonella subsalsa AY858869 C. Tomas Sardinia
Chattonella subsalsa AY858867 C. Tomas Delaware
Chattonella subsalsa AY858866 C. Tomas California
Chattonella subsalsa AY858864 C. Tomas Japan
Chattonella subsalsa AY858868 C. Tomas North Carolina
Chattonella marina AY858862 C. Tomas North Carolina
Chattonella marina AY858861 C. Tomas Maryland
Chattonella marina AY858860 C. Tomas Japan
Chattonella marina AY865604 CCMP 2049 Kagoshima Bay, Japan
Chattonella marina AY704165 Hong Kong
Chattonella marina AF137074 NIES 3 Osaka Bay, Japan
Heterosigma akashiwo AY858874 CCMP 1680 Sandy Hook Bay, USA
Heterosigma akashiwo AY858875 CCMP 1912 Kalaloch, USA
Vacuolaria virescens AF409125 SAG1195.1 Wirral, Cheshire, England
Chattonella antiqua AY858858 C. Tomas Japan
Chattonella antiqua AY858857 CCMP 2052 Mikawa Bay, Japan
Chattonella antiqua AY858856 CCMP 2050 Seto Inland Sea, Japan
Chattonella antiqua AF136761 NIES 1 Seto Inland Sea, Japan
Chattonella ovata AY858872 CCMP 216 Japan
Chattonella ovata AY858863 C. Tomas Japan
Chattonella ovata AY704166 Hong Kong
Fibrocapsa japonica AF112991 LB 2162
Olisthodiscus luteus AF112992 NIES-15 Seto Inland Sea, Japan

ATTARAN-FARIMAN and BOLCH / Turk J Bot
159
of critical values of g1 (Hillis and Huelsenbeck, 1992).
Neighbour-joining (NJ) trees were constructed with
the minimum evolutionary (ME) model using logdet
distances (ME-LgD) and the mean distance metric (Bolch
and Campbell, 2004). Maximum parsimony (MP) analyses
used the branch and bound search algorithm to nd the
most parsimonious trees. All characters were equally
weighted and gaps were treated as missing data; multistate
characters were interpreted as uncertainty. To estimate
the reliability of the MP trees and the NJ tree, bootstrap
analyses were carried out utilising 1000 replicates of the
full heuristic search algorithm.
3. Results
3.1. Morphology
Cells of Chattonella sp. are 24–43 µm long and 17–23 µm
wide, slightly compressed, and tear-shaped to lanceolate
in lateral view (Figure 1). e large oval-shaped nucleus
A B C
D E F
G I
n
n n
H
Figure 1. Motile cells of Chattonella sp. CHPI36 germinated from mixed incubated sediment. A- cell with 2 dark brown eyespots; B-
same cell as Figure A (note the eyespot colour changes to a lighter colour during observation); C- cell in lateral view, showing 1 visible
eyespot (bottom arrow). Note anterior depression (top arrow); D- cell showing a large oval nucleus; E- mucocysts on the surface of the
cell (arrow); F- tear-shaped cell showing the densely-packed chloroplasts (arrow); G- cell showing posterior tail (bottom arrow). Note
the nucleus; H- SEM. Anterior depression of the cell and 2 agella grooves (arrows); I- polar view of cell showing the nucleus (n) and
eyespot. All scale bars = 10 µm, except Figure G = 5 µm.

ATTARAN-FARIMAN and BOLCH / Turk J Bot
160
extends from beneath the anterior depression of the cell
toward the cell centre (Figure 1). Numerous mucocysts
are present on the cell surface. e numerous, densely
packed chloroplasts are green, peripherally placed, and
ellipsoid-to-ribbon shaped (Figure 1). Some cells show a
posterior projection. Two sub-equal agella project from
2 agella grooves that arise from a clearly dened anterior
depression (Figure 1). e cells are golden yellow in colour
under bright eld illumination. Two dark brown-red
eyespots are present in the posterior part of the cell (Figure
1). Examination of live cells under the light microscope
causes cells to quickly lose motility, and the eyespot colour
fades to a lighter brown. e eyespot is not visible in lateral
view (Figure 1).
Presumed encysted cells were observed only in
senescent, late stationary-phase cultures approximately 6
months aer transfer into nutrient replete medium. Cysts
were not produced in cultures transferred into nutrient-
decient media. When grown to senescence in nutrient-
replete medium, non-motile vegetative cells ranged from
9 to 12 µm in diameter (Figure 2). Putative resting cysts
were pale-brown, spherical, and ranged from 17–21 µm
in diameter and contained a brown accumulation body
(Figure 2).
3.2. Phylogenetic analyses
Based on partial LSU-rDNA data, both NJ (gures
not shown) and MP analyses generated a tree with the
same primary branching order (Figure 3). All analyses,
including raphidophyte taxa, formed a monophyletic
group with 100% bootstrap support. In both trees, O. luteus
branched rst followed by Vacuolaria Cienkowski, 1870
and Chattonella. e genus Chattonella was monophyletic
in both trees. Within Chattonella, 2 monophyletic groups
were formed, 1 comprising all strains of C. marina, C.
ovata, and C. antiqua with 100% bootstrap support
and near identical sequences (referred to as C. marina
group hereaer) and diering by only 1–2 nucleotide
substitutions. e second group included all C. subsalsa
strains. Chattonella sp. CHPI36 clustered with C. subsalsa
but was clearly distinct, diering by 17 base-pairs over the
1372 bp of LSU-rDNA compared.
e trees derived from analysis of rDNA-ITS sequences
supported the analyses of the LSU-rDNA data. Both NJ and
MP analyses of the rDNA-ITS resulted in trees with similar
branch order (Figures 4 and 5). e MP analysis resulted
in 2 most parsimonious trees with identical branching
order; therefore, only 1 is presented in Figure 5. In this tree
the branch orders are F. japonica, V. virescens (freshwater
species), and H. akashiwo, followed by Chattonella spp.,
respectively. Within Chattonella, 2 groups were evident.
e rst group comprised the C. marina group, diering
by 1–4 nucleotides across the rDNA-ITS. e second
group contained all C. subsalsa strains and Chattonella
sp. CHPI36, which was clearly distinct from C. subsalsa,
diering by 12 base pairs over the ITS region.
4. Discussion
4.1. Morphology of Chattonella sp. CHPI36
Due to the morphological similarity between C. subsalsa
and C. marina, identication based on light microscopy is
oen dicult (Figure 6). C. subsalsa is the type species for
the genus and morphologically related to C. marina. Hara
and Chihara (1982) separated these 2 species based on 2
ultrastructure characteristics: the presence of oboe-shaped
mucocysts in C. subsalsa and the relationship between the
thylakoid membranes and chloroplast pyrenoid matrix. In
C. subsalsa, the thylakoids do not penetrate the pyrenoid,
but in C. marina the thylakoids are in the pyrenoid matrix,
and the cells have distinctive mucocysts. However, there
are a number of unresolved questions regarding the type
AB C
Figure 2. Chattonella sp. putative resting cysts and non-motile cells. A- spherical
cyst showing large accumulation body (arrow). Note non-motile cells produced in
old cultures; B- cysts of Chattonella sp. surrounded with a mucilaginous layer; C-
non-motile spherical cells produced in nutrient-depleted medium. All scale bars =
10 µm.

ATTARAN-FARIMAN and BOLCH / Turk J Bot
161
material of C. subsalsa, and it has been suggested that re-
examination of cells from the type locality (from India)
is necessary to clarify the identity and circumscription
of both C. subsalsa and C. marina (Imai and Yamaguchi,
2012).
In the present study, the cell shape of Chattonella
sp. CHPI36 resembles C. marina more than C. subsalsa,
sometimes possessing a posterior tail similar to C. marina.
Comparing the Iranian isolate with C. subsalsa CCMP217
(Figure 7), the 2 strains have quite dierent cell outlines;
however, cell shape is known to be pleomorphic in most
Chattonella Raphidophytes and varies with the age of the
cells (Hara and Chihara, 1987; Aizdaicher, 1993; Tomas,
1998; Demura et al., 2009). In older cultures, the posterior
tail of C. marina cells become narrower and longer, similar
to those of mature cells of C. antiqua (Band-Schmidt et
al., 2004; Hosoi-Tanabe et al., 2006), indicating that cell
morphology alone is an unreliable taxonomic character
Chattonella subsalsa
Chattonella subsalsa AF409126
Chattonella sp JF896100
Chattonella antiqua AF210737
Chattonella marina AF210739
Chattonella ovata AF210738
Chattonella marina AY704162
Chattonella ovata AY704163
Vacuolaria virescens AF210742
Vacuolaria virescens AF409125
H
eterosigma akashiwo AF210741
H
eterosigma akashiwo AF086948
H
eterosigma akashiwo AY704161
H
eterosigma akashiwo AF042820
Olisthodiscus luteus AF210743
Cylindrotheca closterium
50 changes
100
100
100
84
59
100
100
100
Figure 3. Phylogenetic relationship among Chattonella-like species inferred from phylogenetic of partial LSU of rDNA gene. Most
parsimonious tree obtained using branch and bound search. Bootstrap values from 100 replicates are shown above the nodes.
Cylindrotheca closterium is the outgroup taxon.

ATTARAN-FARIMAN and BOLCH / Turk J Bot
162
for eld samples containing multiple species with cells of
dierent ages.
Chattonella sp. CHPI36 clearly diers from C. antiqua
due to its smaller size (C. antiqua, 70–130 µm long), lack of
a long posterior tail, and presence of mucocysts in the cell
surface (Table 3). Strain CHPI36 more closely resembles
C. subsalsa in many features. Both possess a tear-shaped
nucleus that is centrally positioned, both have oval-shaped
chloroplasts that are peripherally placed, both possess
many mucocysts on the cell surface, and neither possesses
contractile vacuoles. e anterior depression of strain
CHPI36 where agella arise is deep and clear; however,
this feature is not clearly documented for C. subsalsa
(Hallegrae and Hara, 2003).
ere is little published information describing the
resting cysts of C. subsalsa, and those that refer to the
Vacuolaria virescens
Heterosigma akashiwo
Chattonella subsalsa AF409126
Chattonella subsalsa AY858869
Chattonella subsalsa AY858870
Chattonella subsalsa AY858864
Chattonella subsalsa AY858866
Chattonella subsalsa AY858868
Chattonella subsalsa AY858871
Chattonella sp. JF896101
Fibrocapsa japonica
Olisthodiscus luteus
50 changes
Chattonella marina AY704165
Chattonella ovata AY704166
Chattonella antiqua AF 136761
Chattonella marina AF137074
Chattonella ovata AF858863
100
99
98
69
100
100
Figure 4. Molecular analysis of ITS regions of rDNA gene of Chattonella sp. CHPI36. Most parsimonious tree obtained using branch
and bound search. Values above nodes represent bootstrap values (100 replicates). O. luteus is the outgroup taxon.

ATTARAN-FARIMAN and BOLCH / Turk J Bot
163
production of a resting stage do not give morphological
descriptions (Steidinger and Penta, 1999). e putative
cyst stages described here for strain CHPI36 are smaller
than the yellow-greenish-to-brownish, hemispherical cysts
described for C. marina (20–30 µm diameter; Imai, 1989)
and C. ovata (30 µm diameter; Yamaguchi et al., 2008). e
large dark brown accumulation body in cysts of Iranian
isolate CHPI36 also diers from the several dark brown
spots or black material in the cysts of C. marina (Imai,
1989). e appearance of small cells (before encystment)
in N-limited medium has been reported for C. marina
(Imai et al., 1998), and Band-Schmidt et al. (2004) noticed
that the morphology of C. marina is aected by the age of
the culture. In older and N-limited cultures, cells become
Vacuola
r
ia
vi
r
escens
AF4
09
12
5
H
eterosigma akashiwo AY858875
Chattonella marina AY704165
Chattonella antiqua AF136761
Chattonella marina AF137074
Chattonella ovata AY858863
Chattonella marina AY858862
Chattonella ovata AY858872
Chattonella marina AY858861
Chattonella marina AY865604
Chattonella
ovata
AY
70
41
66
Chattonella subsalsa AF409126
Chattonella subsalsa AY858869
ChattonellasubsalsaAY858870
Chattonella subsalsa AY858864
Chattonella subsalsa AY858866
Chattonella subsalsa AY858868
Chattonella subsalsa AY858871
Chattonella sp. JF896101
Fibrocapsa japonica
AF112991
Olisthodiscus luteus
0.05
93
100
100
86
71
100
Figure 5. Phylogenetic analysis of the ITS-rDNA of Iranian Chattonella sp. isolate CHPI36 with other Chattonella-like species. e tree
was constructed by neighbour-joining from logdet genetic distance (ME-LgD analysis). Numbers above branches represent bootstrap
support values (100 replicates). O. luteus was used as an outgroup taxon.

ATTARAN-FARIMAN and BOLCH / Turk J Bot
164
smaller, more ovoid or spherical and non-motile. Similar
changes were noted in nutrient-limited cultures of Iranian
strain CHPI36.
Strain CHPI36 is similar in general morphology to
Chattonella subsalsa CCMP217, as both have similar
chloroplast arrangement, cell shapes, and mucocysts.
Despite these similarities, strain CHPI36 and C. subsalsa
show distinct dierences. e cell shape/outline of strain
CHPI36 is dierent (Figure 7), and it is slightly smaller
than C. subsalsa, although there is considerable overlap
in the size ranges (Hallegrae and Hara, 1995) (Table
3). CHPI36 isolate also has an obvious eyespot, whereas
C. subsalsa, and most other members of Chattonella, do
not possess an eyespot (e.g., C. ovata, C. antiqua, and C.
marina; Hara and Chihara, 1982; Yamaguchi et al., 2008;
Demura et al., 2009). Colour is also considered one of the
A B C
D E
F G
H
IJ
Figure 6. Comparison of dierent Raphidophyceae species (A–H), Dictyochophyceae (I–J; previously class Raphidophyceae). A-
Heterosigma carterae; B- Olisthodiscus luteus; C- Fibrocapsa japonica; D- Chattonella. antiqua; E- Chattonella ovata; F- Chattonella
marina; G- Chattonella subsalsa; H- Chattonella. minima; I- Pseudochattonella verruculosa; J- Dictyocha bula var. stapedia (aer Hara
and Chihara, 1987).

ATTARAN-FARIMAN and BOLCH / Turk J Bot
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A B
C
F
D
E
n n
Table 3. Comparison of Chattonella sp. CHPI36 with similar Chattonella spp.
Vegetative cell Chattonella sp. CHPI36 aC. subsalsa bC. marina cC. antiqua
Size: length (µm)
width (µm)
24–43
17–23
30–50
15–25
30–70
20–30
70–130
30–70
Chloroplast: colour Green-brown Green-brown Yellowish-green-brown Green-brown
Shape Ellipsoid Ellipsoid Ellipsoid Ellipsoid-to- tear-shaped
Eyespot Present Absent Absent Absent
(a,b,c): described in Hara and Chihara (1987) and Hallegrae and Hara (1995).
Figure 7. Comparing the morphology of vegetative cells of Chattonella sp. CHPI36 with C. subsalsa CCMP217. A- strain CHPI36 in
deep focus, showing 2 eyespots; B- CCMP217 in deep focus, note the lack of eyespot; C- and D- strains CHPI36 (le) and CCMP217
(right) in surface focus showing chloroplasts shape and arrangement; E- and F- strains CHPI36 (le) and CCMP217 (right). Note the
presence of mucocysts (arrow). All scale bars = 10 µm.

ATTARAN-FARIMAN and BOLCH / Turk J Bot
166
important features for distinguishing C. subsalsa from C.
marina (Hallegrae and Hara, 2003). C. subsalsa possesses
a green-brown colour, whereas C. marina possesses a
green yellowish-brown colour. Although colour can be
a somewhat subjective character, under identical culture
conditions isolate CHPI36 diers from C. subsalsa by
having a greener colour.
4.2. Molecular analyses
e analyses presented here show that the raphidophytes
included form a monophyletic group with high bootstrap
support that includes both the marine and freshwater
Vacuolaria genera. is agrees with previous studies
(Potter et al., 1997; Ben Ali et al., 2001; Ben Ali et al., 2002;
Hosoi-Tanabe et al., 2006). Members of the 4 dierent
genera are quite distinct from each other. Connell (2000)
suggested that the H. akashiwo ITS sequence was quite
divergent from both C. antiqua and C. subsalsa (20%
and 18% divergence, respectively), and the present study
supports this result. e 2 strains of H. akashiwo were
distinct from all members of other genera, with sequence
divergences of 22% and 19% in ITS sequence from the 2
later species. In addition, F. japonica and O. luteus showed
high ITS sequence divergence by pairwise comparison
(47%, Connell, 2000; 50%, present study). e amount of
either ITS sequence or partial LSU sequence divergence
and nucleotide base dierence between Chattonella species
and other species of dierent genera is high.
Many algal species show intra-specic sequence
variation in the rDNA-ITS and LSU-rDNA genes
between dierent geographical isolates (Chopin et
al., 1996; Bolch et al., 1998; Hirashita et al., 2000). For
example, Atlantic and Pacic isolates of Cladophora
albida (Nees) Kutzing, 1843 showed up to 1% sequence
divergence across the ITS region within each oceanic
basin and as much as 21% between the 2 oceanic
basins (Bakker et al., 1992). In contrast, past studies on
raphidophytes have demonstrated little or no variation
in LSU-rDNA and rDNA-ITS sequences within each
species (Connell, 2000; Hirashita et al., 2000; Connell,
2002). For example, 20 strains of H. akashiwo from
across the globe have almost 100% ITS sequence identity,
indicating that populations of this species represent only
1 worldwide species (Connell, 2000). Similar results have
been reported for 16 isolates of F. japonica (Kooistra et
al., 2001). e sequences from the 4 H. akashiwo used in
the present study were also virtually identical across the
700 bp of LSU examined, with a sequence divergence of
<0.6% between strains.
Within the genus Chattonella, strains of C. marina, C.
ovata, and C. antiqua show remarkable similarity across
the rDNA-ITS regions with <1.2% sequence divergence
between C. marina and C. ovata and only a few base
(maximum 7 nucleotides) dierences in nucleotide
sequence. Past studies on C. marina and C. antiqua
have considered these to be one species (Connell, 2000;
Hirashita et al., 2000; Sako et al., 2000; Connell, 2002).
Hosini-Tanabe et al. (2006) also documented high genetic
homogeneity of C. marina, C. antiqua, and C. ovata in the
5.8S rDNA D1/D2 region of the LSU-rDNA and rDNA-
ITS1 and ITS2 regions.
From this study and previous work, it is clear that
global geographical variation in both the LSU-rDNA and
rDNA-ITS is very low within raphidophyte species. Strain
CHPI36 from the Oman Sea is clearly distinct from C.
subsalsa and exhibits small but consistent morphological
dierences from C. subsalsa, indicating that isolate
CHPI36 is a distinct species related to C. subsalsa.
Acknowledgement
e authors thank Sharareh Khodami from the Fisheries
Research Institute in Iran for her help in sediment
sampling.
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