Blackwell Publishing AsiaMelbourne, AustraliaPREPhycological Research1322-08292006 Japanese Society of PhycologyDecember 20065512541Original
Article
Ultrastructure and LSU rDNA of Lepidodinium virideG. Hansen et al.
Phycological Research 2007; 55: 25–41
Ultrastructure and large subunit rDNA sequences of
Lepidodinium viride reveal a close relationship to
Lepidodinium chlorophorum comb. nov.
(= Gymnodinium chlorophorum)
Gert Hansen,1* Lizeth Botes2 and Miguel De Salas3
1
Department of Phycology, Institute of Biology, University of Copenhagen, Øster Farimagsgade 2D, 1353 Copenhagen K,
Denmark, 2Aquaculture Institute of South Africa, c/o MCM Research Aquarium, Private Bag X2, Rogge Bay, 8012, South
Africa, and 3School of Plant Science, University of Tasmania, Private Bag 55, Hobart, TAS 7001, Australia
SUMMARY
The ultrastructure of the green dinoflagellate Lepididodinium viride M. M. Watanabe, S. Suda, I. Inouye
Sawaguchi et Chihara was studied in detail. The
nuclear envelope possessed numerous chambers each
furnished with a nuclear pore, a similar arrangement
to that found in other gymnodinioids. The flagellar
apparatus was essentially identical to Gymnodinium
chlorophorum Elbrächter et Schnepf, a species also
containing chloroplasts of chlorophyte origin. Of particular interest was the connection of the flagellar apparatus to the nuclear envelope by means of both a fiber
and a microtubular extension of the R3 flagellar root.
This feature has not been found in other dinoflagellates
and suggests a close relationship between these two
species. This was confirmed by phylogenetic analysis
based on partial sequences of the large subunit (LSU)
rDNA gene of L. viride, G. chlorophorum and 16
other unarmoured dinoflagellates, including both the
‘type’ culture and a new Tasmanian isolate of
G. chlorophorum. These two isolates had identical
sequences and differed from L. viride by only 3.75%
of their partial LSU sequences, considerably less than
the difference between other Gymnodinium species.
Therefore, based on ultrastructure, pigments and partial LSU rDNA sequences, the genus Lepidodinium was
emended to encompass L. chlorophorum comb. nov.
Key words: dinoflagellates, endosymbiosis, Lepidodinium viride, nuclear pores, phylogeny, ultrastructure.
INTRODUCTION
The chloroplasts of dinoflagellates display an unusually
high diversity. Therefore, besides the typical peridinincontaining chloroplast probably of red algal origin
(Ishida & Green 2002), some dinoflagellate chloroplasts have originated from a cryptomonad, diatom,
haptophyte or chlorophyte (Watanabe et al. 1987,
1990; Elbrächter & Schnepf 1996; Chesnick et al.
1997; Schnepf & Elbrächter 1999; Takishita et al.
1999; Hackett et al. 2003). It has recently been
hypothesized that the seeming readiness of dinoflagellates to take up and incorporate foreign chloroplasts
might rely on the exceptionally high number of chloroplast genes transferred to the dinoflagellate nucleus
from the original chloroplast (Green 2004). As acquisition of chloroplast-containing endosymbionts seems
to be a relatively frequent event in apparently diverse
groups of dinoflagellates, the chloroplast type and color
have not been considered to be useful taxonomic criteria above species level (Elbrächter & Schnepf 1996).
However, recent investigations based on molecular
sequences have quite surprisingly shown that the diatom-containing dinoflagellates form a monophyletic
group (Inagaki et al. 2000; Horiguchi 2003; Tamura
et al. 2005; Horiguchi & Takano 2006), irrespective of
the fact that this group consists of morphologically very
diverse species (e.g. athecate, thecate, flagellate, coccoid and filamentous forms). This indicates that the
acquisition of endosymbionts might not be so frequent
a phenomenon as hitherto anticipated and that chloroplast type might be a taxonomic criterion to be used
also at higher than species level.
Several dinoflagellate species have been described
as having green chloroplasts (see Sournia et al. 1992;
Elbrächter & Schnepf 1996), but only two species have
been examined in detail; namely, Lepidodinium viride
M. M. Watanabe, S. Suda, I. Inouye Sawaguchi et Chihara and Gymnodinium chlorophorum Elbrächter et
Schnepf. Both species contain an endosymbiont, probably of prasinophyte origin, and are morphologically
very similar, but L. viride is distinguished by the pres*To whom correspondence should be addressed.
E-mail: [email protected]
Communicating editor: K. Okuda.
Received 23 February 2006; accepted 26 May 2006.
doi: 10.1111/j.1440-1835.2006.00442.x
26
Table 1.
G. Hansen et al.
Origin and GenBank accession numbers of sequences used in phylogenetic analyses
Species
Origin
Accession number
Gymnodinium aureolum (Hulburt) Gert Hansen
Gymnodinium catenatum L. W. Graham
Gymnodinium fuscum (Ehrenberg) F. Stein
Gymnodinium impudicum (Fraga et Bravo) Gert Hansen et Moestrup
Gymnodinium microreticulatum Bolch et Hallegraeff
Gymnodinium nolleri Ellegaard et Moestrup
Gymnodinium palustre A. J. Schilling
Gymnodinium cf. placidum Herdman
Karenia brevis (Davis) Gert Hansen et Moestrup
Karenia brevisulcata (Chang) Gert Hansen et Moestrup
Karenia mikimotoi (Miyake et Kominami ex Oda) Gert Hansen et Moestrup
Lepidodinium chlorophorum (Elbrächter et Schnepf) Gert Hansen, Botes
et de Salas
Lepidodinium chlorophorum
Lepidodinium viride M. Watanabe, S. Suda, I. Inouye Sawaguchi et Chihara
Karlodinium australe de Salas, Bolch et Hallegraeff
Karlodinium veneficum (Ballantine) J. Larsen
Takayama helix de Salas, Bolch, Botes et Hallegraeff
Takayama tasmanica de Salas, Bolch et Hallegraeff
Woloszynskia pseudopalustris (Schiller) Kisselev
USA
Spain
Australia
—
New South Wales, Australia
Denmark
Denmark
Denmark
Florida, USA
New Zealand
English Channel, UK
Tasmania, Australia
AF200671
AF200672
AF200676
AF200674
AY036078
AF200673
AF260382
AF260383
AF200677
AY243032
AF200678
EF010974
Germany
South Africa
New South Wales, Australia
Norway
Tasmania, Australia
Tasmania, Australia
Denmark
AF200669
AY464689
DQ151560
AF200675
AY284950
AY284948
AF260402
ence of an outer layer of body scales (Watanabe et al.
1987, 1990; Elbrächter & Schnepf 1996). The presence of a horseshoe-shaped apical groove suggests an
affiliation with the Gymnodinium group sensu Daugbjerg et al. (2000). Molecular phylogenies based on
small subunit (SSU) and large subunit (LSU) rDNA
confirm this affiliation (Saunders et al. 1997; Daugbjerg et al. 2000). In addition, new detailed analyses of
the flagellar apparatus and nuclear envelope have
shown that G. chlorophorum possesses nuclear chambers and a fibrous connection between the flagellar
apparatus and the nucleus (Hansen & Moestrup 2005),
both features considered to be key characters of the
Gymnodinium group. However, significant differences
compared to other Gymnodinium species were noted in
G. chlorophorum. The nuclear connective was much
reduced and, more importantly, the transverse microtubular root extension (TMRE) of the R3 flagellar root
was attached to the nucleus, a novel arrangement in
dinoflagellates (Hansen & Moestrup 2005). As flagellar
apparatus detail in protists are considered phylogenetically important (e.g. Moestrup 1982), we speculated
whether a similar arrangement was present in L. viride,
which would suggest that these two species form a
natural group. Previous preliminary analyses of the LSU
rDNA gene have shown that this might indeed be the
case (Botes 2003).
In the present paper, we provide novel ultrastructural
details, notably on the flagellar apparatus of L. viride,
and compare these findings with the present knowledge
of G. chlorophorum. In addition, new LSU rDNA
sequences of the South African strain of L. viride and
a Tasmanian strain of G. chlorophorum are used in
phylogenetic analyses comprising 16 other dinoflagellates available from GenBank, including the original
strain of G. chlorophorum from Germany (Table 1).
MATERIALS AND METHODS
Culturing
Lepidodinium viride (strain number CTCC 17) was collected in 1997 by Lizeth Botes in False Bay, South
Africa and grown at 18°C or 15°C in either F/2, Keller
or TL medium (Guillard & Ryther 1962; Keller & Guillard 1985; Larsen et al. 1994) at a salinity of 30 PSU
and a 12:12 h LD cycle. Light intensity was 50 or
200 µmol/m2/s.
Gymnodinium chlorophorum (strain no. LCDE01)
from the River Derwent, Tasmania was collected and
isolated by Miguel de Salas and grown in the University
of Tasmania’s collection of harmful microalgae. Culture
conditions are detailed in de Salas et al. (2003).
Light microscopy
Live cells were examined using an Olympus BHS microscope equipped with an Olympus Camedia 5060 digital
camera.
Scanning electron microscopy
Two different schedules were used. In schedule 1, a
culture was fixed for 25 min in 2% OsO4 made up in
sterile filtered seawater, while settling on a poly L-lysine
coated coverslip. The coverslip was subsequently
Ultrastructure and LSU rDNA of Lepidodinium viride
washed for approximately 1 h in distilled water, dehydrated in an ethanol series, and critical-point dried
through liquid CO2 in a BAL-TEC CPD 030 critical point
drying apparatus. The coverslip was then glued to a
scanning electron microscope (SEM) stub by doubleadhesive carbon-discs, sputter coated with platinumpalladium, and examined in a JEOL 6335F field emission SEM (Fig. 3). Schedule 2 was carried out according to the technique developed by Botes et al. (2002)
(Fig. 4).
Transmission electron microscopy
Whole mount preparations were made by pipetting
droplets of a culture onto formvar/carbon coated grids
and fixing for approximately 30 s in OsO4 vapour. The
grids were washed in distilled water, stained in 1%
aqueous uranyl acetate for approximately 15 min and
thoroughly washed in distilled water.
For thin sectioning, the culture was fixed in 2%
glutaraldehyde made up in 0.05 M Na-cacodylate
buffer with 0.5 M sucrose (final concentrations) for
25 min, pelleted by centrifugation and washed in four
changes of buffer with decreasing sucrose concentration: 0.4, 0.25, 0.125 M and pure buffer, 10 min in
each change.
Postfixation was for 2 h in 2% OsO4 made up in
0.1 M Na-cacodylate buffer, and the material was dehydrated in an ethanol series and embedded in Spurr’s
resin via propylene oxide. The material was sectioned
on an LKB 2088 Ultrotome V ultramicrotome (LKB,
Bromma, Sweden) using a diamond knife, and the
sections were collected on slot grids and placed on
formvar film. After staining in uranyl acetate and lead
citrate, sections were examined in a JEOL JEM-1010
electron microscope operated at 80 kV. Micrographs
were taken using a GATAN 792 digital camera.
DNA isolation, polymerase chain reaction
amplification and sequencing
The culture of G. chlorophorum was grown to midlogarithmic phase and 5–10 mL pelleted by gentle centrifugation. Total DNA was extracted using the gentle
lysis method (Bolch et al. 1998) or using a Qiagen
DNeasy plant tissue mini kit (Qiagen). Extracted DNA
was used as a template to amplify a fragment of the
large subunit ribosomal gene approximately 900 bp
Figs 1–4. Light and scanning electron microscopy (SEM) of Lepidodinium viride. 1,2. Live cells, high and low focus, respectively. The apical groove is barely visible (arrowheads). nucleus,
n. 3,4. SEM revealing the apical groove (arrowheads) and the
body scales (arrows). tf, transverse flagellum, lf, longitudinal
flagellum.
27
28
long, using the primers D1R and D3Ca (Scholin et al.
1994). Polymerase chain reaction (PCR) amplifications
were performed in volumes of 50–100 µL, as described
in de Salas et al. (2003). Amplification products were
checked by electrophoresis through 1% agarose gels
stained with ethidium bromide and visualized under
ultraviolet light. Successful reactions were purified
using QIAquick PCR purification columns (Qiagen),
according to the manufacturer’s instructions. PCR products were quantified with a Bio-Rad SmartSpec 3000
(Biorad, Hercules, California, USA), diluted to the
appropriate concentration and sequenced in both
directions using a Beckman-Coulter Dye Terminator
Sequencing Kit (Beckman-Coulter, Fullerton, California, USA), according to the manufacturer’s instructions. Sequencing reactions were electrophoresed on a
Beckman-Coulter CEQ8000 capillary electrophoresis
sequencer. Primers D1R and D3Ca (Scholin et al.
1994) were used to determine the nucleotide sequence
of approximately 900 bp of the amplified fragment.
Sequence chromatograms were examined visually and
base-calling errors corrected manually. Both forward
and reverse sequences were aligned and conflicts
resolved by manual inspection.
Individual live cells of L. viride were isolated directly
into PCR tubes containing the reaction ingredients following the protocol of Bolch (2001). DNA fragments
were checked on 1% agarose gels containing ethidium
bromide, cut out with a sterile surgical blade and purified using the QIAquick Gel Extraction Kit (Qiagen). The
PCR product was sequenced as described above, except
for using ABI PRISM BigDye terminator Cycle-Sequencing Ready Reaction Kit (v. 2), and sequence reactions
were run on an ABI PRISM 3100 Genetic Analyzer,
following the recommendations of the manufacturer.
Sequence alignment and
phylogenetic analyses
Corrected sequences were aligned using ClustalX
(Thompson et al. 1997), and alignments were refined
manually. Phylogenetic analyses using maximum parsimony, minimum evolution, and maximum likelihood
criteria were carried out using PAUP version 4.0* (Swofford 2003). Wolosyznskia pseudopalustris (Schiller)
Kisselev was used as an outgroup in all analyses. A
Bayesian phylogeny was inferred using MrBayes version
3.11 (Huelsenbeck & Ronquist 2001). Unlike PAUP*,
Bayesian methods provide likelihood trees that display
branch lengths, but also carry a measure of support for
branches in the form of posterior probability values. The
evolutionary model that best fits the data matrix was
chosen with the program MrModeltest version 2.2
(Nylander 2004), which is an adaptation of Modeltest
(Posada & Crandall 1998), and only the evolutionary
models supported by both MrBayes and PAUP* were
G. Hansen et al.
tested. The chosen model was a general-time reversible
evolutionary model, with a gamma-shaped among-site
rate variation and a proportion of invariable sites
(GTR + I + G). The Bayesian analysis was carried out
with two simultaneous runs for 106 generations, and
the trees were sampled every 1000 generations. Of the
1000 trees saved for each run, the last 500 were used
to construct a 50% majority-rule consensus tree.
Finally, bootstrap support values (1000 replicates) from
PAUP, maximum parsimony and minimum evolution
analyses were added to the Bayesian tree containing
posterior probabilities.
OBSERVATIONS AND DISCUSSION
Identity of the South African material
Cells of L. viride from South Africa are more or less
ovoid in shape and measure approximately 30 µm in
length and approximately 26 µm in width. The episome
is slightly smaller than the hyposome, but both are
hemispherical. The cingulum is displaced approximately one cingular width and is descending. The sulcus extends onto the episome and continues in a
delicate horseshoe-shaped apical groove running in a
counter-clockwise direction around the cell’s apex
(Fig. 1). The apical groove is barely visible in the light
microscope but quite distinct in the SEM (Figs 3,4).
The cells are bright green in color and the chloroplast(s)
is usually reticulated (not shown). The nucleus is
located in the central part of the cell (Fig. 2). The cell
surface is covered with box-shaped scales that measure
260–300 nm across. They have a somewhat complicated substructure consisting of two interconnected
arches and a square base subdivided into four larger
squares and a smaller centrally located rhomboid one
(Figs 5–8). In sectioned material the scale base was
seen to be furnished with delicate dense material that
was not visible in whole mount preparations or SEM
(Fig. 6). In SEM many cells displayed a protruded
peduncle located between the exit points of the transverse and longitudinal flagellum (Fig. 3).
The South African material is in agreement with the
original description of L. viride from Japan, both in
terms of cell size, morphology, chloroplast color and
structure and the micromorphology of the body scales.
A peduncle was not included in the original description,
but a small club-shaped protrusion with dense contents
was located at the same position. It was suggested to
be a peduncle homolog used to attach the cell to the
substratum rather than for food uptake (Watanabe et al.
1990). Similar protrusions have also been found in
Gymnodinium aureolum (Hulburt) Gert Hansen and the
gonyalacoid Protoceratium reticulatum (Claparède et
J. Lachmann) Bütschli (Hansen et al. 1997; Hansen
2001). However, they are not consistent features and
Ultrastructure and LSU rDNA of Lepidodinium viride
Figs 5–8. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of body scales. 5. SEM, revealing the two interconnected arches. 6. Thin section of the base
plate (TEM). 7,8. Wholemount preparations (TEM).
have been suggested to be a part of the peduncle,
which at certain stages or conditions takes up this
particular appearance (Hansen & Moestrup 2005). The
same probably applies to L. viride.
Ultrastructure
Generally the ultrastructure of L. viride from South
Africa is in agreement with previously published information, including the original description (Watanabe
et al. 1987, 1990). Therefore, we only present new
observations here.
The chloroplast has an interlamellar pyrenoid and is
separated from the dinoflagellate host by four membranes (Fig. 9). The two innermost membranes represent the chloroplast envelope, whereas the two
outermost membranes might represent the food vacuole
membrane of the host and the plasma membrane of
the symbiont, respectively. The space between the inner
and outer set of membranes is filled with ribosomes.
This arrangement is exactly as published before, and
similar to that found in G. chlorophorum (Watanabe
et al. 1987; Elbrächter & Schnepf 1996; Schnepf &
Elbrächter 1999). However, in fortuitous sections we
observed an eyespot-like structure (Fig. 9). We have
never observed this in G. chlorophorum, and it might
either have been overlooked, or the endosymbiont is
more reduced in this species. The eyespot-like structure
was not located in the sulcal area, as in other
dinoflagellates (Dodge 1984), and might, therefore, be
non-functional.
29
Fig. 9. The chloroplast. The inner pair of chloroplast membranes
marked with an arrowhead, the outer pair with an arrow. Notice
the putative eyespot and the ribosome-filled space between the
two membrane systems.
The nuclear envelope contains numerous chambers
that open towards the nucleoplasm (Figs 10,11). These
openings, measuring approximately 70 nm, are the
nuclear pores and each pore has a central plug-like
structure. The chambers are continuous with the endoplasmic reticulum (ER), but there appears to be no
direct opening from the pore to the lumen of the ER
and it remains a mystery how macromolecules are
transported in and out of the nucleus. Nuclear chambers are a characteristic of the Gymnodinium group
(Daugbjerg et al. 2000), but they are readily overlooked, in particular if the nuclear envelope is not well
fixed. In the present material, this was not a problem
and nuclear chambers were very abundant. The presence of nuclear chambers was not mentioned in the
original descriptions of L. viride (Watanabe et al. 1990)
and G. chlorophorum (Elbrächter & Schnepf 1996). In
the latter species they were actually suggested to be
absent (Honsell & Talarico 2004), but careful examination of serial sections revealed their presence also in
this species (Hansen & Moestrup 2005). The nuclear
pore arrangement of L. viride is exactly as in
G. chlorophorum, and G. aureolum and Gymnodinium
nolleri Ellegaard et Moestrup, all having one pore per
chamber. This is slightly different from the type species, Gymnodinium fuscum (Ehrenberg) F. Stein, where
numerous pores are present per chamber (Hansen et al.
2000). This has been suggested to be of phylogenetic
importance (Hansen & Moestrup 2005).
The peduncle of L. viride is located slightly above
and to the right of the exit point of the transverse
30
G. Hansen et al.
Figs 10 and 11. The nucleus (n). The nuclear envelope with nuclear chambers and nuclear pores (arrowheads). The outer envelope
membrane, close to a chamber, is continuous with the endoplasmic reticulum (ER). 11. Surface section of the nuclear pores. Notice
that a central plug is present in each pore (arrow).
Fig. 12. Partly protruded peduncle supported by a large microtubular strand (msp: microtubular strand of the peduncle). The exit
opening is surrounded by a dense collar (arrows), and a dense body is present within the extended peduncle (arrowhead). Numerous
elongated bodies with opaque content (large arrow) are situated close to the msp. The transverse microtubular root extension (TMRE)
close to a collared pit (cp) and the transverse striated collar (TSC) are also visible. Transverse flagellar canal, tfc.
flagellum. It is supported by a microtubular strand and
surrounded by a dense collar where it exits the cell (Figs
12,13,22). Numerous elongated bodies with opaque
contents are located near the microtubular strand. A
dense body was often situated in the basal part of the
protruded peduncle (Fig. 12). The peduncle is similar
to that of G. chlorophorum but peduncles are now being
found in an increasing number of dinoflagellates and
its presence is perhaps more the rule than the excep-
tion in the group, as in thecate species (e.g. Jacobson
1999).
The 3-D-architecture of the flagellar apparatus is
similar to that previously published for G. chlorophorum
(see Hansen & Moestrup 2005, fig. 34).
The two basal bodies insert at an angle of approximately 155° to each other (Figs 15–17). They overlap
slightly, and the transverse basal body (TB) is situated
to the right of the longitudinal basal body (LB)
Figs 13–19. Non-adjacent slightly oblique longitudinal serial sections of the flagellar apparatus. Sectioning is from right to left and the
cell is seen from the outside from the right. Small encircled numbers refer to section number. 13,14. The R3 root and its transverse
microtubular root extension (TMRE). The peduncle is also visible. 15. The transverse basal body (TB). 16,17. The proximal part of
the R4 flagellar root is associated with the TB. The R2 root is close to the longitudinal basal body (LB) and the pusular canal (pu). 18.
The TMRE and associated nuclear fibrous connective (NFC). 19. The R1 root with associated dorsal fiber (df). Notice the long R4 root
with its single microtubule (TSRM). The src2 connective between the R1 and R4 is also visible. The ventral ridge (vr) and associated
microtubular strand (ms) is clearly visible. The longitudinal striated collar (LSC) encircles the flagellar canal.
Ultrastructure and LSU rDNA of Lepidodinium viride
31
32
G. Hansen et al.
Figs 20 and 21. Continuation of
the
series
from
Figures 13–
19. 20. The transverse microtubular root extension (TMRE) has
bended, continuing towards the
nucleus (not visible). Part of the
connective between longitudinal
basal body (LB) and R1, the C2LB/R1,
is also visible. Notice the striation
pattern of the longitudinal striated
collar (LSC). 21. The nuclear
fibrous connective (NFC) with striations.
(Figs 26,27). This is exactly as in G. chlorophorum
(Hansen & Moestrup 2005), but also in a several other
gymnodinioid dinoflagellates, such as G. aureolum and
Gymnodinium acidotum Nygaard (Farmer & Roberts
1990; Hansen 2001). In the type species, G. fuscum,
the TB is situated slightly to the left of the LB (Hansen
et al. 2000).
The flagellar root system consists of a large multimembered microtubular root situated to the left of the
LB, the R1 (previously longitudinal microtubular root)
(Figs 19–21,24–32). It consists of approximately 30
microtubules, but in one cell an extra band of approximately 10 microtubules was observed (Fig. 28). It
might be interpreted as a predivision stage of the R1
root, but the flagellar basal bodies were not duplicated,
which usually occurs prior to the flagellar root formation. Also, the parental R1 root seems to be unaltered during flagellar transformation (Heimann et al.
1995).
A single-membered microtubular root, the R2 (previously SMR, single microtubular root) is associated
with the right proximal side of the LB. It runs in a
Figs 22–27. Non-adjacent transverse serial sections of the flagellar apparatus. Sectioning is from anterior to posterior and the cell is
seen outside from the anterior. The ventral side of the cell is downwards. Small encircled numbers refer to section number. 22. The
peduncle with encircling collar (arrowheads) and elongated bodies (arrows). The R4 root is near the transverse flagellar canal (tfc). 23.
Cross section of the R4 root and its associated microtubule (TSRM). A scale-containing vesicle is associated with the microtubular
root extension (TMRE) (arrowhead). 24. The R1, R3 and src2. 25. The src1 and src2 interconnect the R1 and R4 roots, the former
perhaps also attaching to the transverse basal body (TB) and longitudinal basal body (LB). A microtubular strand (ms) is located to right
(viewers left) of the vr. 26. The starting point of the LB. The bbc2 and bbc3 attach to TB triplet 5 and 6, respectively. 27. The bbc1
attaches to TB triplet 4.
Ultrastructure and LSU rDNA of Lepidodinium viride
33
34
G. Hansen et al.
Figs 28–32. Continuation of the series from Figures 22–27. 28. The transverse microtubular root extension (TMRE) is curved. The
dorsal fiber (df) has a mosaic pattern. Notice also the apparent ‘bifurcation’ of the R1 in this cell (arrows). The bbc 4 links the most
proximal part of transverse basal body (TB) (barely visible) with the longitudinal basal body (LB). 29,30. The striated C2 LB/R1 links the
ventral side of the R1 with one of the LB triplets. Inset: section from a different cell showing the attachment to the LB triplet
(arrowhead). 31. The delicate C1LB/R1 also attaches to one of the LB triplets and the R1 root. 32. The striated nuclear fibrous connective
(NFC) associated with the TMRE. The ventral connective (vc) is attached to the leftmost part of the R1.
posterior direction close to the pusule and more or less
parallel with the R1 (Figs 16,17). A similar root was
observed in G. chlorophorum, but its consistency was
doubted as a result of flagellar root duplication (Hansen
& Moestrup 2005). This is not the case in L. viride, as
the basal bodies had not duplicated. The presence
of an R2 root in gymnodinioids is unusual, as this
root has hitherto been found in certain gonyalacoid
and peridinioid dinoflagellates (Hansen & Moestrup
1998; Calado et al. 1999). However, flagella with two
microtubular roots seem to be the typical situation in
many protist groups and have probably evolved early in
eukaryote evolution (Moestrup 2000). Its phylogenetic
significance might, therefore, not be very important,
Ultrastructure and LSU rDNA of Lepidodinium viride
and the R2 root has probably been lost repeatedly in
the dinoflagellates. It is readily overlooked as it is often
obscured by shrouds of dense material and requires
sections at an appropriate angle to become visible.
A single microtubular root, the R3 (previously transverse microtubular root) is located on the right dorsal
side of the TB (Fig. 24). This root takes an anterior
direction, runs along the transverse flagellar canal
and usually terminates near collared pits. Here the R3
root nucleates numerous microtubules, the TMRE
(Figs 12,13,25). Quite surprisingly, they take a direction back towards the two basal bodies, and at the level
of the LB make a sharp bend to the left to become
closely associated with a fiber, the nuclear fibrous connective (NFC) (Figs 14–21,28–32,36). The TMRE and
NFC continue in a slightly more anterior direction into
the cell, where they finally attach to an extension of the
nucleus (Figs 33–36). It should be noted that the
nuclear extension and the attachment of the TMRE/
NFC were broken in most cells examined, indicating
this arrangement as very susceptible to physical
changes during fixation and/or centrifugation. The NFC
has a striated pattern and is indirectly attached to the
R1 root through dense fibrous material situated at the
dorsal side of the R1 (Figs 21,32,35). This material
consistently shows a peculiar mosaic pattern of dense
and less dense components (Fig. 28). A connection
between the TMRE and the nucleus is very unusual and
has previously only been observed in G. chlorophorum.
The arrangement in this species is very similar to that
observed in L. viride, although there are some notable
exceptions. Thus, the NFC in G. chlorophorum is less
conspicuous and non-striated. Also, the TMRE, rather
than bending to the left as in L. viride, seems to bend
towards the right (Hansen & Moestrup 2005). A nuclear
fibrous connective is a characteristic of the Gymnodinium group, but has also been observed in the heterotrophic species Actiniscus pentasterias (Ehrenberg)
Ehrenberg and Polykrikos kofoidii Chatton (Bradbury
et al. 1983; Hansen 1993), which are related to but
not included in the Gymnodinium sensu stricto group,
in particular because of their peculiar nuclear capsules.
In all examined species the NFC is a dense non-striated
fibrous structure, except in Gymnodinium cryophilum
(Wedemayer, Wilcox et Graham) Gert Hansen et
Moestrup, where the NFC (originally interpretated as a
striated root) also appears to be striated (Wilcox et al.
1982). The presence or absence of striations might,
however, depend on the physiological state of the cell.
For example the striation pattern of the flagellar collars
in Paulsenella sp. disappeared when calcium levels in
the fixative were raised (Schnepf et al. 1985).
The TMRE also seems to play a role in guidance or
transportation of Golgi-derived scale-containing vesicles to the transverse flagellar canal (Figs 23–25). This
seems also to be the case for scale transport in Hetero-
35
capsa rotundata (Lohmann) Gert Hansen (G. Hansen
unpubl. obs., 1988).
A compound root, the R4 (previously TSR + TSRM,
transverse striated root + transverse striated root microtubule) consists of a prominent striated fiber with an
associated single microtubule and is associated with
the left proximal part of the TB (Figs 16–19,23–25).
This root runs parallel to the flagellar canal and continues along the cingulum (Fig. 19). R4 has been found
in practically all dinoflagellates examined, exceptions
being G. fuscum and the zoospores of Noctiluca (Höhfeld & Melkonian 1995; Hansen et al. 2000).
Several fibrous connectives associate with the basal
bodies and the flagellar roots. Thus, two connectives,
the C1(LB/R1) and the C2(LB/R1), attach R1 to adjacent
triplets of the LB (Figs 29–31). The C2(LB/R1) has a
distinct striation pattern and is intimately associated
with the ventral surface of the R1 (Fig. 29, inset).
Except for G. fuscum, these two connectives have been
observed in all gymnodinioid dinoflagellates examined
in detail (i.e. G. aureolum, G. chlorophorum and
G. nolleri) (Ellegaard & Moestrup 1999; Hansen 2001;
Hansen & Moestrup 2005).
A striated ventral connective is associated with the
left-most part of the R1 and is also present in the
aforementioned species, including G. fuscum (Fig. 32).
Four connectives interlink the two basal bodies, the
bbc1, bbc2, bbc3 and bbc4. If the triplets of the TB
(viewed from tip to base) are labeled anticlockwise
starting with the triplet associated with the R3 root,
then the bbc1, bbc2 and bbc3 attach to triplet number
4, 5 and 6, respectively (Figs 26,27). We have not been
able to determine the specific attachment of bbc4
(Fig. 28). Whereas the bbc1 is present in most
dinoflagellates, the presence of four basal body connectors is rather unusual. However, three basal body
connectors were observed in G. chlorophorum and
Prosoaulax lacustris (F. Stein) Calado et Moestrup (Calado et al. 1998 as Amphidinium lacustre F. Stein;
Hansen & Moestrup 2005). A survey of the published
literature has shown that the bbc1 is always attached
to triplet 3, except for P. lacustris where it bifurcates
and attaches to both triplet 3 and 4 (Hansen &
Moestrup 2005). In L. viride, bbc1 appears to be
attached to triplet 4 rather than 3, and the site of
attachment might, therefore, not be as constant as
previously anticipated. The attachment site of bbc2
and bbc3 is, however, similar in L. viride and
G. chlorophorum. The bbc4 was not observed in
G. chlorophorum, but observations of bbc often require
favorable section angles and/or extensive tilting of the
sections.
The R1 and R4 roots are interconnected by two
striated connectives, the src1 and src2 (Figs 19,24,25).
The src1 also seems to be attached to the LB and TB
(Fig. 25). The presence of two src is exactly as in
36
G. Hansen et al.
Figs 33–36. Non-adjacent transverse serial sections of the nuclear connective. Sectioning is from anterior to posterior and the cell is
seen from the outside anterior. Small encircled numbers refer to section number. 33,34. The nuclear extension. 35,36. The transverse
microtubular root extension (TMRE) makes a sharp turn. Notice also the intimate association between the nuclear fibrous connective
(NFC) and the TMRE.
G. chlorophorum and has not been observed in other
Gymnodinium species. In G. chlorophorum the src1
does not appear to attach to the TB but perhaps to the
LB (Hansen & Moestrup 2005; fig. 27).
In G. chlorophorum a small connective also linked
the basal part of the TB with the R1 root, referred to
as C3TB/R1 (Hansen & Moestrup 2005). This connective
was, however, not found in L. viride.
The openings of the flagellar canals are surrounded
by striated collars, the transverse and longitudinal striated collar, respectively (Figs 15,20,21). A ventral
ridge (vr) consisting of alternating dense and less dense
Ultrastructure and LSU rDNA of Lepidodinium viride
layers spans the distance between the exit points of the
two flagella and is associated with the two striated
collars. A microtubular strand is situated on the right
side of the vr (Figs 19,25). A similar arrangement was
found in G. chlorophorum, but is widespread in
dinoflagellates, including gymnodinioid species (Ellegaard & Moestrup 1999; Hansen 2001; Hansen &
Moestrup 2005). Striated collars are, however, missing
in G. fuscum (Hansen et al. 2000).
Molecular phylogeny
Maximum parsimony, maximum likelihood and Bayesian analyses essentially gave the same tree topology,
although the position of G. fuscum relative to other
species varied enough for a multifurcating tree to be
produced with all multiple-search methods. Here we
present the Bayesian analysis (Fig. 37), and unlike parsimony, likelihood or distance bootstraps its tree presents both support values (posterior probability for
clades) and branch lengths. As in previous investigations, the genus Gymnodinium formed a well supported
group, although there is a clear and well-supported
differentiation between a clade containing G.
catenatum and its relatives, and another containing the
rest of the species in this genus. Previously published
phylogenies of gymnodinioid dinoflagellates (Daugbjerg
et al. 2000; de Salas et al. 2003, 2004) show two
additional clades within Gymnodinium, which have
moderate support: one formed by G. aureolum,
G. chlorophorum and G. impudicum, and another
formed by G. fuscum, G. palustre and G. cf. placidum.
Support for this group was low in the analysis presented
here as the relative position of G. fuscum was found to
vary between analyses, and always had low support.
This has translated to the clade containing most species in Gymnodinium sensu Daugbjerg et al. (2000)
becoming collapsed into a polytomy, and accounts for
the missing maximum parsimony and minimum evolution bootstrap support values in Figure 37. However,
the clade formed by G. chlorophorum and L. viride
remains as a group with very high support, and is found
to be closest to G. aureolum and G. impudicum (in that
order) in most analyses performed. G. chlorophorum
from Germany and Tasmania differed in only two bases
of 864, a difference attributable to geographic differentiation or sequencing error. L. viride from South
Africa forms a sister group to G. chlorophorum, and
differed from this species by 3.75% of its partial LSU
sequence. Interestingly, the genetic distance between
L. viride and G. chlorophorum is considerably less than
between many of the Gymnodinium species included in
the analysis.
It seems likely that dinoflagellates with green endosymbionts form a natural group. Uptake of the endosymbiont might have taken place only once, but insight
37
into this event, as well as the phylogenetic affiliation
of the endosymbiont, requires analyses of the symbiont
genes.
Taxonomic considerations
The ultrastructure of L. viride and G. chlorophorum is
more similar than previously anticipated, with the most
pertinent character being the attachment of the TMRE
to the nucleus, but it also has other essentially identical
minor features, such as the various connectives.
The molecular data also demonstrate a close relationship between the two species, even closer than
between many other Gymnodinium species.
Three different taxonomic decisions can be made:
1. Maintain the status quo. This is, in our opinion, not
a satisfactory solution as it hides the close phylogenetic
relationship
between
L. viride
and
G. chlorophorum.
2. Transfer of L. viride to the genus Gymnodinium.
This is also not desirable for the same arguments
as above; in addition, a number of flagellar apparatus details set these two species apart from the
other Gymnodinium species.
3. Transfer G. chlorophorum to the genus Lepidodinium. Although not optimal, this is, in our opinion,
the most satisfactory solution, at least for the time
being. This will require emendation of the genus
Lepidodinium, as G. chlorophorum lacks body
scales. This is an unusual situation, as presence of
scales is normally considered to be an important
generic character.
Because the type species G. fuscum differs considerably from most other Gymnodinium species further
taxonomic changes are anticipated. However, the ultrastructure of many species is insufficiently known and
their phylogenetic interrelationships are not clear.
A close relationship between G. chlorophorum and
L. viride has also been shown using the SSu rRNA gene
(Shalchian-Tabrizi et al. 2006).
Taxonomic appendix
Lepidodinium M. Watanabe, S. Suda, I. Inouye Sawaguchi et Chihara emend. Gert Hansen, Botes et de
Salas
Unarmored dinoflagellates with an NFC and the
TMRE connected to the nucleus. A horseshoe-shaped
apical groove running in a counterclockwise direction
is present. The chloroplast contains chlorophyll a and
chlorophyll b. Body scales present or absent.
Type species: Lepidodinium viride M. M. Watanabe,
S. Suda, I. Inouye Sawaguchi et Chihara
38
G. Hansen et al.
Ultrastructure and LSU rDNA of Lepidodinium viride
39
Fig. 37. Phylogenetic analysis of the genus Gymnodinium including Lepidodinium, using the Bayesian method, with Woloszynskia
pseudopalustris as outgroup. Tree length = 1130, CI = 0.629, RI = 0.691, HI = 0.371. Support values are: Bayesian posterior probability/
PAUP minimum evolution bootstrap/PAUP maximum parsimony bootstrap. Note that although high support is given to the clade formed by
Lepidodinium viride and Lepidodinium chlorophorum, the position of this group within Gymnodinium is unclear and clade posterior
probability values are low.
Other species: Lepidodinium chlorophorum (Elbrächter
et Schnepf) Gert Hansen, Botes et de Salas comb.
nov.
Basionym: Gymnodinium chlorophorum Elbrächter et
Schnepf (1996, p. 382, figs 1–39)
ACKNOWLEDGMENTS
We would like to thank Lisbeth Thrane Haukrogh for
sectioning. Dr B. Price, Mrs M. Waldron and Dr
C. O’Ryan of the University of Cape Town, South
Africa, assisted with laboratory facilities and general
advice on procedures and techniques. We thank Dr
Ø. Moestrup for critical reading and discussions of the
manuscript. This study was financed by the Carlsberg
Foundation (GH) and the Marine and Coastal Management (LB).
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