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European Journal of Phycology
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Mitosis, cytokinesis and multinuclearity in a
Xanthonema (Xanthophyta) isolated from Antarctica
Andrzej Massalski
a
, Igor Kostikov
b
, Maria Olech
c
& Lucien Hoffmann
d
a
Department of Botany, Akademia Swietokrzyska, Biology Institute, Jan Kochanowski
University of Humanities and Science, 15 Swietokrzyska, 25-406 Kielce, Poland
b
Department of Botany, Kyiv Taras Shevchenko National University, 64 Volodymirska
Street, UA-01033 Kyiv, Ukraine
c
Department of Polar Research and Documentation, Institute of Botany, Jagiellonian
University, 27 Kopernika, 31-501 Krakow, Poland; Department of Antarctic Biology, Polish
Academy of Sciences, 10 Ustrzycka, 02-141 Warszawa, Poland
d
Public Research Centre-Gabriel Lippmann, 41 rue du Brill, L-4422 Brill, Grand-Duchy of
Luxembourg
Version of record first published: 08 May 2009.
To cite this article: Andrzej Massalski , Igor Kostikov , Maria Olech & Lucien Hoffmann (2009): Mitosis, cytokinesis and
multinuclearity in a Xanthonema (Xanthophyta) isolated from Antarctica, European Journal of Phycology, 44:2, 263-275
To link to this article: http://dx.doi.org/10.1080/09670260802636274
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Eur. J. Phycol., (2009), 44(2): 263–275
Mitosis, cytokinesis and multinuclearity in a Xanthonema
(Xanthophyta) isolated from Antarctica
ANDRZEJ MASSALSKI1
, IGOR KOSTIKOV2
, MARIA OLECH3
AND LUCIEN HOFFMANN4
1
Department of Botany, Akademia Swietokrzyska, Biology Institute, Jan Kochanowski University of Humanities and Science,
15 Swietokrzyska, 25-406 Kielce, Poland
2
Department of Botany, Kyiv Taras Shevchenko National University, 64 Volodymirska Street, UA-01033 Kyiv, Ukraine
3
Department of Polar Research and Documentation, Institute of Botany, Jagiellonian University, 27 Kopernika, 31-501 Krakow,
Poland; Department of Antarctic Biology, Polish Academy of Sciences, 10 Ustrzycka, 02-141 Warszawa, Poland
4
Public Research Centre-Gabriel Lippmann, 41 rue du Brill, L-4422 Brill, Grand-Duchy of Luxembourg
(Received 12 January 2006; revised 28 February 2008; accepted 3 November 2008)
The ultrastructure of a Xanthonema strain featuring multinucleate cells was investigated by transmission electron
microscopy. An important specific feature of the organisation of the photosynthetic apparatus in this strain is its
association with mitochondrial profiles. The chloroplast girdle is composed of two different U-shaped lamellae, one peripheral
and one subcentral. Multinuclearity is observed as often as the uninucleate state. The transition from the uninucleate to the
multinucleate stage is connected to disturbances in the normal division pattern of the parietal chloroplast-mitochondria
complex during interphase. As a result mitosis is not coordinated with cytokinesis. The return to the uninucleate stage occurs
as a result of asynchronous cytokinesis or by aplanospore formation. Mitosis is of the semi-closed type, as in Tribonema.
Centrioles replicate in early interphase, after the end of karyokinesis and progeny nuclei separate with the aid of CER
invagination. Filament fragmentation takes place between neighbouring cells where two U-shaped segments adjoin, resulting
in fragment ends being rounded rather than ‘zweispitzig’. The taxonomic significance of various ultrastructural features
for the classification of filamentous Xanthophyta is discussed.
Key words: chloroplast, cytokinesis, girdle lamellae, mitochondria, mitosis, multinuclearity, Xanthonema, Xanthophyta
Introduction
Recent investigations (e.g. Bailey & Andersen,
1998; Negrisolo et al., 2004; Zuccarello &
Lokhorst, 2005) have shown that the traditional,
morphologically-based, classification of the
Xanthophyceae, is often incongruent with molecular
data and that a new classification system
is needed. For example, molecular data do not
support the monophyly of the traditional order
Tribonematales, containing Tribonema Derbe` s
et Solier, Xanthonema Silva and Heterococcus
Chodat but ultrastructural investigations have
been important for determining the taxonomic
and phylogenetic relationships in the algae.
Studies in the Xanthophyceae (Hibberd, 1980,
1989) revealed that they are characterized by
chloroplasts with four membranes, photosynthetic
lamellae with three thylakoids, and a layer of
chloroplast ER that is continuous with the outer
membrane of the nuclear envelope. They differ
from other chromophytes by the presence of
a girdle lamella and DNA concentrated in
genophores.
Most ultrastructural investigations of the
Xanthophyceae have dealt with the vegetative cell
(Hibberd & Leedale, 1971), the chromatophore
(Bo¨ ger & Kiermayer, 1974; Hesse, 1980), spermatogenesis
(Ott & Brown, 1978), zoospores
(Massalski & Leedale, 1969; Lokhorst & Star,
2003), and flagellar hair formation (Leedale et al.
1970; Deason, 1971). Mitosis and cytokinesis have,
however, been poorly documented, although the
significance of these features for phylogenetic and
taxonomic understanding has been demonstrated
for other algae, especially chlorophytes. The only
complete ultrastructural studies of the cell cycle
in the Xanthophyceae are of Vaucheria litorea C.
Ag. (Ott & Brown, 1972) and Tribonema regulare
Pascher (Lokhorst & Star, 1988). There are no
published data on mitosis, cytokinesis and cellwall
formation in Xanthonema.
The genus Xanthonema Silva is a filamentous,
unbranched alga lacking obviously H-shaped cell
Correspondence to: Andrzej Massalski. E-mail: amassal@
pu.kielce.pl
ISSN 0967-0262 print/ISSN 1469-4433 online/09/020000263–275 ß 2009 British Phycological Society
DOI: 10.1080/09670260802636274
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wall segments when viewed by light microscopy
(LM; Ettl, 1978). During a preliminary investigation
of a Xanthonema strain isolated from the
Antarctica, we noticed the presence of a relatively
high number of multinucleate cells, despite reports
that Xanthonema is generally considered to be
uninucleate. The objective of this investigation
was to study the ultrastructure of this isolate,
with special emphasis on its mitosis, cytokinesis
and the formation of multinucleate cells. The
results were compared to data on other xanthophycean
genera to evaluate the taxonomic significance
of mitotic and cytokinetic features at genus and
species level.
Materials and methods
Soil samples were collected in 1993 by M. Olech from
the moraines of Ecology Glacier (62
09’S; 58
28’W)
during the XVI Polish Antarctic Expedition to the
Henryk Arctowski Station (King George Island, South
Shetland Islands). Soil samples were first moistened
with distilled water and algal samples were inoculated
onto 1.5% agar solidified with Bristol’s medium
(Bristol, 1920). Cultures were maintained at 20
C
under a 16-h : 8-h light–dark cycle at a light intensity
of 3000 mE.mÀ2
.sÀ1
provided by 40 W cool fluorescent
tubes.
For transmission electron microscopy (TEM), filaments
were removed from the surface of agar cultures
with a spatula and fixed for 1 h at room temperature
with 3% glutaraldehyde in 0.05 M phosphate buffer at
pH 7.2. The filaments were then washed three times for
10 min in phosphate buffer, and fixed for 2 h at room
temperature with 1% osmium tetroxide in 0.05 M phosphate
buffer. After two washes of 10 min in the same
buffer and dehydration in an ethanol series, the cells
were infiltrated overnight with a 1 : 1 mixture of propylene
oxide and Spurr’s medium (Spurr, 1969), and then
embedded in fresh Spurr’s medium. The samples were
polymerized at 70
C for 12 h. Sections were cut with
glass knives on a Reichert–Jung Supernova ultramicrotome
and collected on unsupported 400-mesh copper
grids, which were subsequently double stained for
3 min with 5% uranyl acetate in 50% ethanol, followed
by 1 min with a lead citrate solution (Reynolds, 1963).
Over 200 cells were examined and photographed with
a TESLA BS 500 electron microscope.
Results
Light microscopy observations
In 2-month old agar cultures, filaments are short,
usually 2–4 cells long, and easily disintegrate into
single cells. Filaments are (3.7À)4.2–5.0(À5.3) mm
wide and cylindrical or slightly constricted near the
transverse cell walls. The cell wall is thin, smooth,
and about 0.14–0.25 mm thick.
The terminal cells are rectangular, rounded, lack
the ‘H’ endings but occasionally, display small,
wart-like protrusions at the corners. Two types of
cells are present: (i) short (5.7–9.0 (À11.2) mm long)
cells with 2–4 parietal, plate-like chloroplasts
(Figs 1, 2), and (ii) elongated (10.2–18.1
(À20.0) mm long) cells with 4–8 (À10) chloroplasts,
both parietal and internal (Fig. 3). A variable
number, generally six to 30, of small vacuoles can
be observed near the transverse cell walls or scattered
throughout the cell cytoplasm.
Reproduction is by fragmentation (Fig. 3) or the
formation of akinetes, which are mainly produced
in pairs (Figs 4, 5). Akinete-like cells with thickened
cell walls, which germinate into single nonmotile
cells, were also observed.
Transmission electron microscopy observations
Vegetative cells have a variable number of nuclei,
and can therefore be characterized as mono-, bi- or
multinucleate (with 3–4 nuclei). The mononucleate
cells are always short (cell type ‘a’, Fig. 6), whereas
the multinucleate cells are always long (cell type
‘b’, Fig. 8). The binucleate cells can be either
Figs 1–5. Light microscope drawings of Xanthonema.
Fig. 1. Short filament with uninucleate cells containing
two parietal chloroplasts. Fig. 2. Binucleate cell with four
parietal chloroplasts. Fig. 3. Fragmentation of the filament
with binuclear cells containing parietal and internal chloroplasts.
Fig. 4. Filament with two terminal akinetes. Fig. 5.
Fragmentation of the filament with akinetes. Scale bar:
5 mm.
A. Massalski et al. 264
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short (type ‘a’, Fig. 7) or elongate (type ‘b’, fig. 8).
Bi- and multinucleate cells represent around
20–30% of all cells.
The ultrastructure of mononucleate cells
varies during the cell cycle. In early interphase
(Figs 9, 13), the transverse cell wall is thin and
often wavy. Several small or 3–5 large vacuoles,
occasionally with small aggregates of electron
dense material, were observed near the cell wall.
In young cells, a single nucleus with a central spherical
nucleolus is located in the centre of the cell.
The nucleus has an indentation on one side in
which the Golgi body is situated. A single centriole
is located beside the Golgi body in early interphase.
Some cells with a thin transverse cell wall
have two centrioles, which are parallel to each
other but at different levels in the cell. There are
two parietal plate-like chloroplasts, each of which
has an envelope of four membranes and contains
5–7 lamellae, each of which usually comprises three
thylakoids. A girdle lamella extends under the
plastid envelope. A genophore is present at each
plastid pole. Individual, electron-dense, plastoglobules
are present between the lamellae. The external
chloroplast membrane (CER) of at least one of
the chloroplasts is continuous with the nuclear
membrane. There are two mitochondrial profiles
associated with each chloroplast. These are situated
at the poles of the chloroplasts on the side
facing the centre of the cell. Mitochondria have
tubular cristae.
In middle interphase (Figs 14, 15) all mononucleate
cells have transverse cell walls of equal
thickness. The nucleus is central and the nucleolus
is well visible. Vacuoles accumulate near both
transverse cell walls and some are also located
towards the cell centre. The number of lamellae
in the chloroplast increases to seven to nine. The
mitochondrial profiles elongate, some of them
reaching the connection between the CER and
the nuclear membrane.
In late interphase (Figs 11, 17), the small
vacuoles are irregularly distributed throughout
the internal cell space; the chloroplasts divide
and the cells have four chloroplasts. During
chloroplast division, one mitochondrial profile
overlaps the two progeny chloroplasts, on the
inner side. The division of the mitochondrial
profiles accompanies separation of the progeny
chloroplasts. It is possible that the chloroplast
is involved in the division of the mitochondrial
profiles. The number of chloroplast lamellae
decreases to six to seven. The centrioles move in
opposite directions from a more or less parallel
position towards each side of the Golgi body.
The nucleolus starts to disappear at the end of
late interphase (Fig. 12).
Figs 9–12. Drawings to show uninucleate cells of
Xanthonema during interphase. Fig. 9. Cells in early interphase
with primary transverse cell wall. Fig. 10. Cells in the
middle interphase with almost completed secondary cell
wall. Fig. 11. Late interphase: chloroplast division completed
but mitochondria still dividing; vesicles have
moved. Fig. 12. Cells in late interphase (top) and early
prophase (bottom). Scale bar: 3 mm.
Figs 6–8. Drawings to show different types of cells of
Xanthonema during interphase. Fig. 6. Short uninucleate
cell with two chloroplasts (type a). Fig. 7. Short binucleate
cell with four chloroplasts (type a). Fig. 8. Elongated multinucleate
cell with numerous both parietal and internal
chloroplasts (type b). Scale bar: 2 mm.
Mitosis, cytokinesis and multinuclearity in Xanthonema 265
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Mitosis in mononucleate cells
In prophase, mononucleate cells have four parietal
chloroplasts and eight mitochondrial profiles,
two of which, at the poles of each chloroplast,
face the centre of the cell (Fig. 12). In early prophase
the mitochondrial profiles increase in size,
the parietal vacuoles concentrate in the equatorial
plane of the cell where the future transverse cell
wall will be formed, and the centrioles are positioned
diagonally on opposite sides of the nucleus.
Each centriole is perpendicular to the future mitotic
spindle. At this stage, a Golgi body is positioned
beside each centriole. One of the Golgi
bodies is large, whereas the other is relatively
small. The developmental mechanism of the
second Golgi body remains unknown. A large
perforation, through which the mitotic spindle
microtubules enter the nucleus, appears in the
nuclear membrane near each centriole at each
nuclear pole.
During metaphase (Figs 25, 27) chromosomes
are present on the equatorial plate. The Golgi
bodies are located parallel to the nuclear membrane,
with a centriole at each Golgi pole, at an
angle of about 90
to the cisternae. A bundle of
mitotic spindle microtubules appear in the perforation
zone of the nuclear membrane beside the top
end of the centriole. The spindle microtubules are
intranuclear; mitosis is of the semi-closed type.
Small vesicles can be observed in the space between
the Golgi body and the nuclear membrane. Some
of these appear to originate from the proximal
Golgi cisternae, whereas others originate from the
external nuclear membrane.
Chromosomes diverge towards opposite poles
during anaphase (Figs 28, 29), and a large invagination
appears in the nuclear membrane near its
connection with CER (Fig. 20). In telophase
(Fig. 26), the CER invagination divides the progeny
nuclei and the mitotic spindle disappears.
Vesicles, possibly containing building material for
the cell wall, concentrate between the progeny
nuclei in the plane of the future transverse wall
and cytokinesis begins.
Cytokinesis and the formation of transverse
cell walls
Cytoplasmic division in parent cells takes place as
a result of fusion of lateral vesicles with the plasmalemma
and fusion of central vesicles with each
other. Central vesicles are bigger than lateral ones,
and are characterized by the presence of electron
dense material. During central vesicle fusion their
contents become denser (Fig. 31) and a thin electron
dense plate, the primary transverse cell wall,
appears in the space between future progeny cells
Figs 13–16. Electron micrographs of uninucleate cells of
Xanthonema during interphase. Fig. 13. Cell with two parietal
chloroplasts, four mitochondrial profiles associated
with chloroplasts, and thin transverse cell wall (arrows).
Figs 14, 15. Cell in middle interphase with two chloroplasts
and terminal pyrenoid-like body; mitochondrial profiles
associated with CER, Golgi body adjacent to the nucleus
with well defined nucleolus, thick transverse cell wall.
Fig. 16. Detail of pyrenoid-like body associated with the
chloroplast. Scale bars: 1.5 mm (Figs 13–15); 0.75 mm
(Fig. 16). Abbreviations: Ch: chloroplast; m: mitochondrion;
N: nucleus; Nc: nucleolus; Ps: pyrenoid-like body.
A. Massalski et al. 266
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(Figs 32, 19, 20). Progeny cell protoplasts then
form their plasmalemmae from membranes of
central vesicles and a thin primary transverse cell
wall appears (Figs 33, 21); vesicles originating from
the Golgi body continue to fuse with the plasmalemmae
of new progeny cells, leading to the
appearance of a plate-like structure at the centre
on each side of the primary cell wall (Figs 34, 22,
23). These plate-like structures gradually extend
centrifugally to form the new U-shaped segment
of the progeny cell walls (Figs 35, 36). Each cell
forms one U-shaped segment of secondary cell
wall. The pair of U-shaped segments is joined by
the primary cell wall, thus forming an H-like
structure.
The formation of the secondary cell wall
takes place centrifugally and results in the
elongation of progeny cells. At the same time, the
primary cell wall width decreases, gradually depolymerizing
centripetally (Figs 35, 36, 24). After
depolymerization of the primary cell wall, the
remnants of the primary cell wall hold the progeny
cells together. If the primary cell wall disappears
entirely, the progeny cells separate and fragmentation
of the filaments occurs. In the latter case,
‘H’-shaped cell endings are not formed but there
are small thickenings at the corners of the cells.
Each progeny cell has one nucleus and two
chloroplasts.
Chloroplasts and their movements in the different
phases of the cell cycle
The chloroplast lamella girdle is not closed, but
U-shaped with two arms. One arm is peripheral,
situated just under the chloroplast envelope membrane
(Fig. 37). The second arm is subcentral
situated close to the longitudinal axis of the chloroplast.
The latter is separated from the chloroplast
envelope by the peripheral branch of the second
girdle lamella and also often by one or two free
interior lamellae.
The chloroplast usually possesses five to seven
lamellae in early interphase, but at late interphase,
a constriction appears in the middle of the chloroplast.
The lamellae are fragmented at the constriction,
and progeny genophores may be visible in the
thylakoid-free zones. In early interphase of mononucleate
cells, both arms of the girdle lamellae
usually comprise three thylakoids, whereas in the
curved zone, only one (Fig. 38) or two thylakoids
are present. Free interior lamellae usually contain
three thylakoids, and one of their ends often anastomoses
with the peripheral branch of the girdle
lamella.
In middle interphase, new lamellae can form
through fragmentation, subsequent separation,
growth and reassociation of thylakoids leading to
an increase in the number of lamellae to seven to
nine. During this stage, the curved zones of girdle
lamellae comprise three thylakoids.
Figs 17–20. Electron micrographs of cells from late interphase
to start of primary wall formation. Fig. 17. Cell in
late interphase with four chloroplasts, mitochondrial profiles
divided. Fig. 18. After caryokinesis the different types
of vesicles begin to concentrate in the equatorial plane of
the binucleate cell. Figs 19, 20. Serial sections of the start of
primary cell wall formation. Thin primary cell wall is situated
between the plasmalemmae of daughter cells; vesicles,
some with electron dense contents, are concentrated on
both sides of the primary wall (arrows). Scale bars:
1.5 mm (Figs 17, 18); 0.75 mm (Figs 19, 20). Abbreviations:
Ch: chloroplast; m: mitochondrion; N: nucleus; Nc:
nucleolus.
Mitosis, cytokinesis and multinuclearity in Xanthonema 267
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Figs 21–24. Electron micrographs of cell wall formation.
Fig. 21. Completed primary cell wall spanning the parent
cell walls (arrows) Fig. 22. Beginning of secondary cell
wall formation. Fig. 23. Later stage of secondary cell
wall formation (arrows). Fig. 24. Empty spaces between
the mature cell wall segments of two daughter and one
parent cell appear as the primary cell wall disintegrates
(arrow). Scale bars: 0.75 mm (Figs 21–23); 1.5 mm
(Fig. 24). Abbreviations: C: centriole; Ch: chloroplast; G:
Golgi apparatus; m: mitochondrion; N: nucleus; Nc:
nucleolus.
Figs 25, 26. Drawings of mitosis in Xanthonema. Fig. 25.
Semi-closed mitosis with internal spindle, Golgi body adjacent
to centrioles, chromosomes form metaphase plate.
Fig. 26. Anaphase; invagination of CER begins to separate
daughter nuclei. Scale bar: 0.5 mm
A. Massalski et al. 268
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Figs 27, 28. Electron micrographs of mitosis in
Xanthonema. Fig. 27. Metaphase with chromosomes in
equatorial plane, two Golgi bodies on opposite sides of
nucleus, longitudinally sectioned centriole. Fig. 28. Early
anaphase showing chromosomes being pulled apart by
spindle microtubules (black arrow), intact nuclear envelope
(white arrow) with polar opening (double arrow) near the
oblique, longitudinally sectioned, centriole. Scale bar:
0.75 mm. Abbreviations: C: centriole; Ch: chloroplast;
Chr: chromosome; G: Golgi apparatus; m: mitochondrion.
Figs 29, 30. Electron micrographs of mitosis in
Xanthonema. Fig. 29. Late anaphase. Fig. 30. Telophase,
chromosomes no longer discernable, nucleolus beginning
to appear. Scale bar: 0.75 mm. Abbreviations: Ch: chloroplast;
Chr: chromosome; G: Golgi apparatus; N: nucleus;
Nc: nucleolus.
Mitosis, cytokinesis and multinuclearity in Xanthonema 269
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Formation of multinucleate cells
Abnormal chloroplast or mitochondrial divisions
disturb the translocation of Golgi vesicles in
bi-nucleate cells. As a result, progeny plasmalemmae,
primary transverse cell walls, and U-shaped
segments of the secondary cell wall are not formed.
Thus, karyokinesis is not followed by cytokinesis,
and vegetative cells containing two or more (three
to four) nuclei are formed. We observed several
variants.
Variant A. Mitochondria do not divide after chloroplast
division in late interphase and, as a result,
a mitochondrial profile bridges two progeny chloroplasts
during karyokinesis (Figs 40, 43). When
nuclear division is complete, the undivided mitochondrial
profile is situated on the equatorial plane
of the cell, where the cell wall is usually formed,
thus preventing lateral translocation of vesicles to
the plasmalemma causing cessation of cytokinesis.
An elongated, bi-nucleate vegetative cell with four
parietal chloroplasts is formed. If the nuclei of this
cell undergo new mitoses, in cells with three or four
nuclei and four to seven parietal chloroplasts
result. All chloroplasts are parietal in such cells
Variant B. Chloroplast division is disturbed at the
beginning of late interphase producing multinucleate
cells with internal chloroplasts. One of the
chloroplasts does not divide but continues to
grow in late interphase (Fig. 41), shifting
a progeny chloroplast from a parietal position to
the central area of the cell. When mitosis is finished
the chloroplast moves to the cell centre (we term
such chloroplasts ‘internal’), disturbs the translocation
of vesicles to the equatorial plane of the cell,
and cytokinesis fails (Fig. 44). Further nuclear divisions
lead to the formation of cells with three
(Fig. 46) or four nuclei (Fig. 47).
Figs 37–39. Electron micrographs of chloroplasts in
Xanthonema. Fig. 37. Showing chloroplast-mitochondrion
association and structure of girdle lamella with external and
internal arms and anastamosing lamellae. Fig. 38. Bent
zone of girdle lamella consisting of only one thylakoid
(arrow) at early interphase. Fig. 39. Showing abnormal
internal chloroplast–mitochondrion association, structure
of girdle lamella and presence of plastoglobules. Scale
bars: 0.75 mm (Figs 37, 38); 1.5 mm (Fig. 39). Abbreviations:
Ch: chloroplast; m: mitochondrion; N: nucleus.
Figs 31–36. Schematic drawings of transverse cell wall formation
in Xanthonema. Fig. 31. The vesicles lie in the plane
of the future transverse cell wall; some vesicles contain
granular electron-dense material for primary cell wall formation.
Fig. 32. Medial vesicles fused and primary cell wall
appearing. Fig. 33. Fusion of terminal vesicles with plasmalemma
and separation of descendant cell protoplasts.
Fig. 34. Beginning of formation of U-shaped segment of
secondary cell wall. Figs 35, 36. Growth of U-shaped
segments and disintegration of primary cell wall.
A. Massalski et al. 270
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Variant C. Chloroplasts and mitochondrial
profiles divide abnormally. After chloroplast division
in late interphase or during mitosis, one of
the chloroplasts elongates more rapidly than the
other and, probably as a result of pressure from
the neighbouring chloroplast, begins to bend
towards the cell interior. The outer side of the
chloroplast may touch the mitochondrion of the
neighbouring chloroplast, forming the associated
complex (Figs 39, 42, 45). Normal translocation
of vesicles containing wall-building material
for transverse cell wall formation is prevented by
that part of the chloroplast near the cell centre.
After rearrangement of the mitochondrial profiles,
chloroplast division produces one parietal and one
internal chloroplast. The internal chloroplast does
not interfere with subsequent nuclear divisions,
however, it does interfere with cytokinesis. As
a result, all cells with internal chloroplasts are multinucleate
and contain two, three or four nuclei.
Restitution of the mononucleate stage may
occur as a result of equal or unequal division of
Figs 43–47. Electron micrographs of multinuclear cell formation.
Fig. 43. Undivided mitochondrial profile prevents
normal cytokinesis. Nucleus on the left-hand side
approaching prophase of the next division. Fig. 44.
Progeny chloroplast with two internal mitochondrial profiles
(arrow heads) moving towards the central part of the
cell prevents the next cell division. The nucleus on the right
hand side is starting to divide; two cross-sectioned centrioles
are visible near the interphase nucleus. Fig. 45.
Abnormally elongated chloroplast still undivided and
bent at almost right angles inside the cell; mitochondria
associated with the external side of the bent chloroplast;
centriole and terminal pyrenoid-like body are visible.
Fig. 46. Tri-nucleate cell with two internal chloroplasts;
nucleus on the right hand side appears to be in the early
prophase. Fig. 47. Quadri-nucleate cell with parietal, one
internal and one bent chloroplast. Scale bar: 1.5 mm.
Abbreviations: Ch: chloroplast; N: nucleus; Nc: nucleolus.
Figs 40–42. Variation in multinuclear cell formation.
Fig. 40 After chloroplast division the mitochondrion does
not divide and vesicles cannot move to the plane of the
future transverse cell wall. Fig. 41. One chloroplast does
not divide and the other moves towards the plane of the
future transverse cell wall. Fig. 42. One chloroplast moves
to the centre of the cell and the mitochondrion adjacent
to the neighbouring chloroplast makes contact with
it; two chloroplast-mitochondrial complexes interfere with
normal vesicle migration to the plane of the future transverse
cell wall. Scale bar: 3 mm.
Mitosis, cytokinesis and multinuclearity in Xanthonema 271
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multinucleate cells, or as a result of aplanospore
formation. During division, cytokinesis normally
proceeds only when the vesicles containing building
material for the transverse primary cell wall
are positioned in the transverse cell space, and
when the space is not penetrated by chloroplast
or mitochondrial profiles.
Discussion
Mitosis
Different types of mitosis have been reported for
the Xanthophyta. Ott & Brown (1972) described
closed mitosis with centrioles for V. litorea.
In this species, the spindle was intranuclear and
separated chromosomes were not visible; instead
the chromatin formed a compact chromosome
plate. Centriole replication took place during anaphase
or early telophase, and the telophase nucleus
was dumb-bell shaped. Subsequently, mitosis
and cytokinesis were investigated in T. regulare
(Lokhorst & Star, 1988). This species has semiclosed
mitosis with centrioles, an intranuclear
spindle and lacks the dumb-bell shaped nucleus
in telophase.
In our Xanthonema strain, Vaucheria De
Candolle and Tribonema, an intranuclear spindle
is formed (Ott & Brown, 1972; Lokhorst & Star,
1988) centrioles participate in mitosis, and the
chromosomes form a compact plate during metaphase.
As in Tribonema, the nuclear envelope
forms two large polar perforations and the nucleus
does not acquire a dumb-bell shape in telophase.
Progeny nuclei stay close to each other until the
beginning of cytokinesis. A specific feature of the
Xanthonema from Antarctica is that replication of
centrioles occurs in early interphase, after the end
of karyokinesis. Another peculiarity is the separation
of progeny nuclei with the help of a CER
invagination. The Golgi apparatus is associated
with the polar nuclear perforation and the centrioles.
Association of the Golgi apparatus with
the interphase nucleus has been described and/or
demonstrated in electron micrographs of vegetative
cells and zoospores of unicellular (Andreoli
et al., 1999), filamentous (Falk & Kleinig, 1968;
Massalski & Leedale, 1969; Broady et al., 1997;
Lokhorst, 2003; Lokhorst & Star, 2003) and heterotrichous
(Massalski, 1969; Andersen et al.,
1998) species of Xanthophyta, but was never
found in siphonous Vaucheria.
The similarity of the interphase nucleus of our
Xanthonema strain with nuclei of other non-siphonous
xanthophycean genera suggests that semiclosed
mitosis is most probably more widespread
than closed mitosis in the Xanthophyta.
Multinuclearity
In keys and floras of xanthophycean algae
(Pascher, 1939; Ettl, 1978; Ettl & Ga¨ rtner, 1995),
species of Xanthonema are referred to as uninucleate.
However, Massalski (1969) demonstrated the
presence of vegetative cells with several nuclei in
some Xanthonema strains, but did not analyse
the process of multinucleate cell formation. Our
data show that multinuclearity occurs as often as
the uninucleate state. Multinucleate cells can be
easily recognized even under LM, being characterized
by a higher number of chloroplasts, some of
which are central or sub-central, in addition to
the normal parietal position. The presence of cells
with parietal and internal chloroplasts has been
previously documented for several Xanthonema
species, e.g. X. solidum (Vischer) Silva, X. exile,
X. debile (Vischer) Silva, X. montanum (Vischer)
Silva, X. sessile (Vinatzer) Ettl et Ga¨ rtner
(Pascher, 1932, 1939; Vischer, 1936; Ettl, 1978;
Broady et al., 1997, and others). Thus it seems
that multinuclearity is quite typical for the genus.
The origin of cells with several nuclei is
connected to disturbances in the normal division
pattern of parietal chloroplast-mitochondrion
complexes during interphase. As a result of such
irregularities, mitosis and cytokinesis are no longer
coordinated. It is interesting to note that complexes
with mitochondrial profiles associated with
the internal face of the parietal chloroplast only are
present in all published electron micrographs
of these species (Massalski, 1969; Broady et al.,
1997; Olech et al., 1998; Lokhorst, 2003), but are
absent on micrographs of other filamentous and
coccoid xanthophycean genera. i.e. Tribonema,
Bumilleria Borzi and Bumilleriopsis Printz (Falk
& Kleinig, 1968; Massalski & Leedale, 1969;
Deason, 1971; Hibberd & Leedale, 1971; Bo¨ ger &
Kiermayer, 1974; Lokhorst, 2003). It is known that
multinucleate vegetative cells of Tribonema viride
Pascher can possess up to six nuclei (Falk &
Kleinig, 1968), but the chloroplast-mitochondrion
association has not been documented.
Cytokinesis
It is known that the cell wall in Xanthonema consists
of two U-shaped segments (Massalski, 1969;
Broady et al., 1997; Lokhorst, 2003) and thus
resembles the cell wall of Tribonema (Falk &
Kleinig, 1968; Lokhorst, 2003), despite the absence
of the ‘zweispitzig’ structure at the filament ends.
When a filament of Tribonema ruptures, it breaks
in the middle of a cell, in the zone of overlap of two
cell-wall segments, hence the ‘zweispitzig’ structure
at the filament ends. In contrast, in Xanthonema,
fragmentation occurs between neighbouring
A. Massalski et al. 272
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cells, where two U-shaped segments abut, resulting
in fragmented filaments with rounded ends.
Lokhorst (2003) suggested that the rupture of filaments
without ‘zweispitzig’ structure formation in
Xanthonema (and also in some species of
Tribonema) is caused by the presence of transverse
cell walls of an intercellular lumen, bounded distally
by an outer layer of cell wall. Such lumina are
absent in the majority of Tribonema species, and
because of this, H-shaped ends are formed.
Our data fully support Lokhorst’s model. The
morphology of fragmented filament ends depends
on the behaviour of the primary transverse cell wall
that joins the two U-shaped segments of secondary
wall into H-shaped structures. During secondary
cell-wall formation (which proceeds centrifugally)
in Xanthonema, the primary cell wall disintegrates
centripetally and intercellular lumina appear in
place of the disintegrated primary cell wall
(Lokhorst, 2003). If the primary cell wall completely
or almost completely disintegrates, the
progeny cells separate to give rounded-ended filaments.
If the primary cell wall does not disintegrate,
or only partially disintegrates, the U-shaped segments
of the progeny cells remain joined by the
primary cell wall, and fragmentation occurs in the
middle of the cell, where the U-shaped segments
overlap. In this case the fragmented filament
ends have the ‘zweispitzig’ structure.
It is interesting to note, that during cytokinesis
aplanospore formation of our strain the primary
cell wall is not formed, and aplanospores always
lie singly within sporangia, without filament
formation.
Intercellular lumina are present in published
electron micrographs of other strains of
Xanthonema (Broady et al., 1997, figs 33, 35).
Intercellular plates (primary cell wall) are also present
in micrographs of Tribonema aequale Pascher
(Falk & Kleinig, 1968, fig. 1b), and traces are
visible as electron-dense zones on electromicrographs
of transverse cell walls of T. minus (Klebs)
Hazen, T. regulare, T. affine (Ku¨ tzing) G.S. West,
T. hormidioides (Fischer) Lokhorst (Falk &
Kleinig, 1968, fig. 1a; Lokhorst, 2003, figs
58–60). It is interesting to note that the intercellular
lumina in T. hormidioides (Lokhorst, 2003), in
which filaments can fragment, as in Xanthonema,
are also situated in the zone of primary cell wall
disintegration.
Behaviour of the primary cell wall and of vesicles
with secondary wall building-material may be an
important generic character in the Tribonematales
s.l. In particular, differences between Xanthonema
and Tribonema seem to be related to the behaviour
of the primary cell wall during the cell cycle.
Similarly, these genera differ from Bumilleria in
their secondary cell wall formation. In Tribonema
and Xanthonema, each progeny cell produces only
one U-shaped segment de novo during cytokinesis,
whereas in Bumilleria progeny cells seem to make
both U-shaped segments, and thus the remnants
of the parent cell wall form intercalary H-shaped
structures (Hibberd & Leedale, 1971; Hibberd,
1980).
Organization of the photosynthetic apparatus
Chloroplast-mitochondrion association. An important
feature of the organization of the photosynthetic
apparatus in our strain is its association with
mitochondrial profiles. In early and middle interphase
uninucleate cells, mitochondrial profiles are
adjacent to the internal side of the chloroplast
poles. Prior to mitosis and cytokinesis, the chloroplast
and mitochondrial profiles associated with
the chloroplast divide. To our knowledge the presence
of ‘chloroplast-mitochondrion’ complexes in
Xanthonema has not been described before,
although such complexes can be seen in all
electron micrographs of previously investigated
Xanthonema species (Massalski, 1969; Broady
et al., 1997; Lokhorst, 2003).
Tribonema and Bumilleria present a different
cellular arrangement of chloroplasts and mitochondria.
For example, cells of Bumilleria sicula
Borzı´ have mitochondrial profiles of two types:
(i) peripheral profiles that are situated between
chloroplast and plasmalemma, and (ii) internal
profiles, that, as in Xanthonema, are adjacent to
the internal side of the chloroplast (Massalski,
1969; Massalski & Leedale, 1969). In T. viride
and T. vulgare mitochondrial profiles are numerous
and small. Some of them are situated in free
cytoplasm, whereas others are adjacent to the
nuclear envelope (Falk & Kleinig, 1968;
Massalski, 1969; Massalski & Leedale, 1969).
Mitochondrial profiles are also present in
a Tribonema sp., isolated from Antarctica (unpublished
data). The absence of a chloroplast-mitochondrion
association is also evident in TEM
pictures of T. affine and T. hormidioides
(Lokhorst, 2003).
Girdle lamellae. Like other Xanthonema and
Tribonema species, the chloroplasts of our
Xanthonema strain, possess a girdle lamella.
The presence of a girdle lamella is regarded as
a typical feature of the Xanthophyta (Lefort,
1962; Falk & Kleinig, 1968; Massalski & Leedale,
1969; Lohhorst & Star, 1988; Broady et al., 1997;
Lokhorst, 2003), but details of its structure have
not been discussed.
In our Xanthonema, the girdle is composed of
branches of two different, U-shaped lamellae,
each consisting of one peripheral and one
Mitosis, cytokinesis and multinuclearity in Xanthonema 273
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subcentral arm. The presence and organization of
girdle lamellae may be an important taxonomical
character for filamentous yellow-green algae.
For example, Bumilleria and Bumilleriopsis lack
the girdle lamella (Massalski & Leedale, 1969;
Deason, 1971; Hibberd & Leedale, 1971).
If the girdle lamella is present, its structure can
differ among species with respect to the number of
girdle lamellae per chloroplast, and the mode of
girdle lamella packing. In T. vulgare (Massalski &
Leedale, 1969, figs 7, 13), X. solidum (Lokhorst,
2003, fig. 63), Pseudopleurochloris antarctia
Andreoli et al. (Andreoli et al., 1999, figs 7, 8),
and the closely related genus, Phaeothamnion
Lagerheim (Andersen et al., 1998, figs 8, 9),
a single-girdle lamella is present in the chloroplast.
In T. vulgare, the girdle lamella has a circular
appearance. In X. solidum, the two ends of the
girdle lamella overlap for half the chloroplast
length, resembling the single turn of a helix.
In Ph. confervicola Lagerheim the girdle lamella
forms two irregular helical loops, while in Ps. antarctica,
the girdle lamella forms a regular helix of
several turns.
Tribonema viride has two girdle lamellae per
chloroplast (Falk & Kleinig, 1968, fig. 5), as do
Heterococcus caespitosus Vischer (Massalski, 1969,
fig. 47), Xanthonema sp. strain 395 (Broady et al.,
1997, fig. 35), Xanthonema sp. strain 601 (Broady
et al., 1997, fig. 38) and Xanthonema cf. exile.
In Xanthonema and Heterococcus each girdle
lamella is U-shaped, and consists of a peripheral
arm, situated just under the chloroplast envelope,
and an internal arm, separated from the chloroplast
envelope by the peripheral arm of another girdle
lamella or by the free lamella. In H. caespitosus
and Xanthonema sp. strain 601, free lamellae are
absent between the peripheral and internal arms
of the first and second girdle lamellae, but in our
Xanthonema and Xanthonema sp. strain 395, one or
several free lamellae separate the peripheral and
internal arms of the first and second girdle lamellae.
Tribonema viride has two girdle lamellae, each of
which forms a helix with two loops.
In conclusion, it appears that the presence
or absence of girdle lamellae may be used as
a taxonomic character at genus level in the
Xanthophyta, whereas the number and type of
girdle lamellae (when they are present) may be taxonomically
informative at species level.
Taxonomic conclusions
Published data and our results show that, in addition
to filament end morphology and the presence
of intercalary cell wall segments, the presence of
a girdle lamella, behaviour of the primary transverse
cell wall during cytokinesis and association
of mitochondria with other cell structures are
important diagnostic features at genus level.
Thus, Xanthonema and Tribonema are characterized
by the presence of girdle lamellae, and by the
formation of only one segment per cell of secondary
cell wall during cytokinesis. But Xanthonema
clearly differs from Tribonema by complete distintegration
of the primary transverse cell wall, and
by the presence of a chloroplast–mitochondrion
association. In Tribonema the primary transverse
cell wall does not disintegrate at all, or disintegrates
only slightly, resulting in solid junctions
of U-shaped segments of secondary cell wall of
progeny cells. Furthermore, mitochondria are not
associated with the chloroplast, but sometimes
with the nucleus. Xanthonema, Bumilleria, and
Bumilleriopsis share the chloroplast-mitochondrion
association, but during interphase, mitochondria
of Bumilleria are not only associated with the internal
side, but also with the external side of the
chloroplast, whereas in Xanthonema mitochondria
are associated only with the internal side of the
chloroplast. In contrast to Xanthonema and
Tribonema, Bumilleria and Bumilleriopsis lack the
girdle lamella.
According to Broady et al. (1997), species delimitation
in the genus Xanthonema is problematic.
Based on LM features, our specimen resembles
four species of Xanthonema: X. debile, X. exile,
X. montanum and X. solidum, but differs from all
of them except X. exile by the presence of a slightly
protruding terminal pyrenoid. Our specimen is also
almost identical with Xanthonema sp. strain 395
(Broady et al., 1997), differing only in the absence
of a stigma in the zoospores, in accordance with
Pascher’s (1939) diagnosis of the species.
In conclusion, our investigation demonstrates
that ultrastructural data may significantly improve
the classification system of filamentous yellowgreen
algae at the generic level, and also be relevant
to species-level separations.
Acknowledgements
The authors are grateful to Prof Elliot Shubert,
The Natural History Museum, London, UK, for
critical reading of the manuscript.
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