Journal of Experimental Botany, Vol. 52, Roots Special Issue,
pp. 403±411, March 2001
The peri-cell-cycle in Arabidopsis
Tom Beeckman, Sylvia Burssens and Dirk InzeÂ1
Vakgroep Moleculaire Genetica en Departement Plantengenetica, Vlaams Interuniversitair Instituut voor
Biotechnologie (VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
Received 23 March 2000; Accepted 10 July 2000
Abstract
The root systems of plants proliferate via de novo
formed meristems originating from differentiated
pericycle cells. The identity of putative signals
responsible for triggering some of the pericycle cells
to re-enter the cell cycle remains unknown. Here, the
cell cycle regulation in the pericycle of seedling roots
of Arabidopsis thaliana (L.) Heynh. is studied shortly
after germination using various strategies. Based on
the detailed analysis of the promoter-b-glucuronidase
activity of four key cell cycle regulatory genes, combined with cell length measurements, microdensitometry of DNA content, and experiments with a
cell cycle-blocking agent, a model is proposed for
cell cycle regulation in the pericycle at the onset of
lateral root initiation. The results clearly show that
before the first lateral root is initiated, the pericycle
consists of dissimilar cell files in respect of their cell
division history. Depending on the distance behind
the root tip and on position in relation to the vascular
tissue, particular pericycle cells remain in the G2
phase of the cell cycle and are apparently more
susceptible to lateral root initiation than others.
Key words: Cell cycle, lateral root initiation, pericycle, root
branching.
Introduction
One of the fundamental questions in developmental
biology relates to how cells proliferate and organize
themselves to form discrete organs. Unlike animals, in
which organogenesis occurs primarily in the embryo,
normal growth in plants involves both embryonic and
post-embryonic organogenesis. The primary shoot and
root apical meristems are formed as a part of the
developing embryo and generate the cells that divide to
1
form the shoot and root, respectively. In recent years, the
understanding of the basic molecular mechanisms that
regulate cell division in plants has progressed considerably. In active meristems, cells are driven through the
successive phases of the cell cycle (S, G2, M, and G1) by
the formation and activation of different heterodimeric
serineuthreonine protein kinases (Mironov et al., 1999).
These kinases consist of a catalytic subunit, the cyclindependent kinase (CDK), and an activating subunit,
a cyclin. CDK activity is regulated at various levels, such
as expression, differential subcellular localization, phosphorylation, proteolysis, and interaction with regulatory
proteins. Nevertheless, several questions remain to be
addressed, especially regarding the developmental and
environmental control of cell division. One such question
is how cell division is re-initiated in cells that have left the
cell cycle. To approach this problem at the molecular level,
the choice of an appropriate developmental process is
indispensable. A classic example in plants of such a process
is the initiation of lateral roots. The root system must
proliferate via de novo formed meristems originating from
differentiated pericycle cells. Most studies suggest that
this happens some distance away from the root apical
meristem in the differentiation zone of the root, where
pericycle cells are not actively dividing. Consequently,
lateral root initiation involves re-entry of cells into the
cell cycle. The study of this re-entry process necessitates
a good knowledge of the cell cycle behavior of pericycle
cells, in particular of those that will give rise to new
primordia.
In a previous study, timing and site of the ®rst lateral
root initiation event after germination have been determined using Arabidopsis thaliana plants transformed with
a promoter-b-glucuronidase (GUS) fusion for a mitotic
cyclin (Arath;CycB1;1) (Dhooge et al., 1999). Initiation
of the ®rst lateral root, formed in the acropetal sequence,
was found to occur within the ®rst 48 h after germination
at a relatively constant distance from the root tip.
To whom correspondence should be addressed. Fax : q32 9 264 5349. E-mail: [email protected]
ß Society for Experimental Biology 2001
404
Beeckman et al.
Here, the cell cycle regulation of pericycle cells was
analysed prior to and during the initiation of a new lateral
root primordium. In a ®rst set of experiments, the cell cycle
regulation was de®ned, based on the promoter activity of
four cell cycle genes that are expressed at different intervals of the cell cycle. The promoter-GUS fusions for two
CDKs (Cdc2aAt and Cdc2bAt) and two cyclins (CycB1;1
and CycA2;1) have previously been proven to be elegant
molecular markers for cell cycle studies at the whole
plant level (Hemerly et al., 1993; Ferreira et al., 1994;
de Almeida Engler et al., 1999). The Cdc2aAt gene is
transcribed throughout the cell cycle at a constant level
(Martinez et al., 1992; Hemerly et al., 1993) whereas
Cdc2bAt is preferentially expressed from the S phase to
the G2 (Segers et al., 1996). The CycB1;1 transcript
levels rise during the G2 phase, reaching a maximum at
the G2-to-M transition, whereas the CycA2;1 expression
rises during the S phase, reaches a maximum at the end
of G2, and is down-regulated during the early M phase
(Shaul et al., 1996).
To get a better insight into the cell cycle behaviour of
distinct pericycle cell ®les, the GUS expression data of the
different cell cycle genes were compared with measurements of cell sizes and DNA contents. Furthermore, the
effect on lateral root initiation of an arrest of cell cycle
progression during S phase was studied on hydroxyureacontaining medium. A model for cell cycle regulation in
the pericycle prior to and at the moment of lateral root
initiation is proposed.
Materials and methods
Plant material and growth conditions
Seeds from Arabidopsis thaliana (L.) Heynh. (ecotype C24) and
transgenic lines expressing the bacterial reporter gene GUS
under the control of the Cdc2aAt, Cdc2bAt, Arath;CycA2;1,
and Arath;CycB1;1 promoters were sown on a modi®ed agarsolidi®ed Hoagland medium with 0.3% sucrose (Beemster and
Baskin, 1998). After 48 h of strati®cation at 4 8C, square tissue
agar plates (Greiner Labortechnik, Frickenhausen, Germany)
were placed vertically in a growth chamber under continuous
light (23 8C).
For the hydroxyurea treatments, the same growth medium
was used with addition of 10 or 100 mM hydroxyurea. For all
experiments, except for the hydroxyurea treatments, the
seedlings were harvested 40 h after germination.
Microscopy
Histochemical assays of GUS activity were performed and
stained whole seedlings were analysed as described previously
(Beeckman and Engler, 1994). For detailed anatomical studies,
GUS-stained roots were embedded and sectioned in Technovit
7100 (Heraeus Kulzer, Wehrheim, Germany) (Beeckman
and Viane, 2000). Sections of 5 mm thickness were stained for
20 min in fresh 0.05% ruthenium red (Fluka Chemie, Buchs,
Switzerland), analysed and photographed using an Axioskop
microscope (Carl Zeiss, Jena, Germany).
Cell length measurements
Pericycle cells were measured on longitudinal sections (see
above) using a graphical tablet SummaSketch2 (Summagraphics,
Scottsdale, AZ, USA) and a light microscope M20 (Wild,
Heerbrugg, Switzerland) equipped with camera lucida. Serial
transverse sections of 5 mm thickness (see above) were made
through seedling roots (40 h after germination) from the root tip
up to the base of the hypocotyl. For each pericycle cell ®le,
nuclei were counted, starting from the region where the ®rst two
sieve elements were differentiated. The distance between the
most `proximal' and most `distal' nuclei was determined by
multiplying the number of sections between these two points
by section thickness (5 mm). Mean cell length in each cell ®le
was then calculated by dividing this distance by the number of
nuclei minus 1.
In situ relative DNA content quantification
To quantify relative DNA contents of the discrete pericycle cell
®les, serial transverse sections through roots of 40-h-old
seedlings were made as described above. To increase the
probability to hit complete nuclei in one section, the section
thickness was 6 mm. Sections were ®rst stained for 20 min in
0.05% ruthenium red, followed by a brief rinse in distilled water,
and subsequently stained with 0.1 mg ml±1 49,6-diamidino-2phenylindole (DAPI) (Katsuhara and Kawasaki, 1996). The
prestaining with ruthenium red assured a weak ¯uorescence of
the cell walls, thus allowing a better localization of the discrete
pericycle cells.
After staining, sections were mounted in Vectashield (Vector
Laboratories, Burlingame, UK). The DAPI was visualized using
the ®lter set 02 on an Axioskop ¯uorescence microscope (Carl
Zeiss) and quanti®ed with a microscope photometer (MPM 100;
Zeiss). The measured values were pooled in two groups, namely
from nuclei lying at the phloem poles and at the xylem poles. Per
group the values were ordered in histograms (see Fig. 4) according to their relative position from the root tip. The DAPI ¯uorescence in metaphase cells from the same sections was included
as reference for a 4C value.
Results
Cell cycle gene expression
In a previous study (Dhooge et al., 1999), CycB1;1 was
found to be expressed in the lateral root founder cells at
the advent of the ®rst division. This early GUS expression
pattern allowed the determination of the timing and site
of the ®rst lateral root initiation event after germination.
In brief, the ®rst lateral is initiated in the distal part of the
root approximately 1.40"0.28 mm (SE) from the root
tip, which happens at the earliest 32 h after germination
onwards. In a sample taken 40 h after germination, 29%
of the seedlings had one initiation site, 13% more than
one, and 58% had none. As a result, this stage provided
enough material to study cell cycle regulation in the
pericycle prior to and just at the moment of lateral root
initiation. Therefore, this stage was chosen to analyse the
expression pattern of the four reporter genes, namely
Cdc2a-gus, Cdc2b-gus, CycB1;1-gus, and CycA2;1-gus.
Cell cycle regulation
405
Fig. 1. Promoter activity for the four analysed cell cycle genes at the moment of lateral root initiation in seedlings 40 h after germination. Overview of
whole seedlings (A±D). Arrows indicate the site of lateral root initiation. Detailed view on the site of lateral root initiation using differential
interference contrast microscopy (E±H). Cdc2aAt-gus expression (A, E); Cdc2bAt-gus expression (B, F); CycB1;1-gus expression (C, G); and CycA2;
1-gus expression (D, H). Arrows indicate sites of lateral root initiation. Arrowheads indicate transition zone between root and hypocotyl. FC, founder
cells for lateral root formation in pericycle cells at the xylem poles; RM, apical root meristem; X, xylem strand. Bars ˆ 1 mm (A±D), ˆ 50 mm (E±H).
Cdc2a-gus was strongly expressed in the root tips (all
cell ®les of the meristem) and in the entire vascular
cylinder (pericycle and stelar parenchyma cells) of roots
with (Fig. 1A, E) and without initiation sites (data not
shown). At this stage, Cdc2b-gus was very faintly
expressed in the root tips, in the adventitious root primordia formed at the root±hypocotyl junction, and in
the stomata of the cotyledons (Fig. 1B). It was absent
from pericycle cells that were not experiencing an
initiation event and appeared from the moment lateral
roots were initiated, in the lateral root founder cells
(Fig. 1F).
The CycB1;1-gus expression pattern was comparable
to that of Cdc2b-gus, being present at the root±hypocotyl
junction and the stomata. In the root, it was strongly
expressed in the meristem and absent from the entire
pericycle, where it appeared only when laterals were
initiated (Fig. 1C, G).
The CycA2;1-gus expression pattern seemed more
complicated. It was strongly expressed in the root apical
meristems (Figs 1D; 2E, F) and, in contrast to CycB1;1,
expression continued for a few 100 mm in the central
cylinder above the meristem (Fig. 1D). Serial sectioning
showed the GUS precipitate in this zone was present in
pericycle cells as well as in stelar parenchyma cells
(Fig. 2D). Above this zone, CycA2;1-gus disappeared or
became very faint in the vascular cylinder, including the
pericycle (Fig. 2C). Higher up in the root, irrespective of
the presence of an initiation site, CycA2;1-gus expression
re-appeared in the central cylinder (Fig. 2A). Serial
sections in this zone showed the staining to be localized
in the stelar parenchyma cells, in pericycle cells at the
xylem poles and, interestingly, to be absent from the
other pericyle cells (Fig. 2B). When initiation occurred,
CycA2;1-gus was also strongly expressed during the early
stages (Fig. 1H).
Cell length measurements
The difference in CycA2;1-gus expression pattern between
xylem pericycle cells compared to other pericycle cells and
the peculiar position in lateral root initiation occupied
by these cells, prompted the investigation of possible
406
Beeckman et al.
differences in cell division history between these types of
pericycle cells. Therefore, on longitudinal sections, the
length of pericycle cells in roots of seedlings where no
initiation had taken place was determined (see Materials
and methods).
Cell lengths were divided into three groups depending
on the position of the pericycle cell ®les: at the xylem pole,
at the phloem pole, and in between. Cells at the phloem
poles and the intermediate pericycle cells were similar
in length (77.205"2.356 mm and 74.957"3.837 mm),
whereas the pericycle cells at the xylem poles were shorter
(64.155"4.187 mm) (n ˆ 0±50).
As it is dif®cult to measure enough pericycle cells in
intermediate regions on longitudinal sections, another
Fig. 2. Detailed analysis of CycA2;1-gus expression throughout the root pericycle just prior to lateral root initiation (30 h after germination). (A)
Whole seedling. (B±F) Bright-®eld microscopy of serial sections taken from different zones of a GUS-stained seedling root as shown in (A). Arrows
indicate the positioning of the section along the root. Arrowheads indicate the location of the protoxylem poles. C, cortex; EN, endodermis; EP,
epidermis; P, pericycle. Bars ˆ 1 mm (A) and ˆ 50 mm (B±F).
Cell cycle regulation
method was used to calculate the pericycle cell length
based on the number of nuclei in a series of sections with
known thickness (see Materials and methods). Three
groups of pericycle cell ®les were distinguished depending
on their location with respect to the vascular tissues. The
mean cell length of six roots 40 h after germination is
given in Fig. 3. Again, pericycle cells opposite the xylem
poles were shorter than the other pericycle cells. Mean cell
lengths were also comparable to the values measured on
sections.
DNA measurements
On serial transverse sections from 10 seedling roots at
40 h after germination, the relative DNA contents of the
individual pericycle nuclei at the xylem poles and phloem
poles were determined (see Materials and methods). The
¯uorescence of metaphase cells, in sections of the same
kind through the root apical meristem, were used as
a reference point. Representative histograms are shown
for a root without (Fig. 4A, C) and with lateral initiation
event in the distal region (Fig. 4B, D). In both root types,
G2 values were only found in some cells at the xylem poles
and always in the upper half of the root (Fig. 4A, B). In
all roots analysed, phloem pericycle cells showed predominantly G1 values (Fig. 4C, D). In roots with early
initiation events, groups of G2 cells could clearly be
recognized at the site where the ®rst divisions took place.
407
expression would point out at least the ®rst round of cell
division.
Based upon previous data obtained using the promoter-gus fusion for the CycB1;1 gene, seedlings at 24 h
after germination showed no lateral root initiation in the
pericycle (Dhooge et al., 1999). Therefore, seedlings at this
stage were transferred to 10 and 100 mM hydroxyurea
and stained for CycB1;1-gus expression after 48 h and
72 h of incubation. As control, a part of the seedlings
was transferred to Hoagland medium (see Materials and
methods).
In roots transferred to the control medium, several
stages of lateral root formation could be seen (Fig. 5A).
Hydroxyurea experiments
Hydroxyurea blocks cycling cells during S phase by
inhibiting ribonucleotide reductase (Shaul et al., 1996).
Hydroxyurea was used to elucidate the point in the cell
cycle at which founder cells were arrested. If the founder
cells for lateral root initiation were blocked in a G1 phase
of the cell cycle, incubation of these roots in hydroxyurea
would inhibit the initiation and no CycB1;1-gus expression in the pericycle would be observed. On the other
hand, if the founder cells stayed in the G2 phase before
being triggered to divide, a G1 block would have no effect
on the ®rst round of cell division and CycB1;1-gus
Fig. 3. Length of pericycle cells (in mm) determined with serial sections
from six seedling roots at 40 h after germination as described in
Materials and methods. Pericycle cells opposite xylem (X1, X2), opposite phloem (Ph1, Ph2), and in interjacent regions (interj.). Error
bars ˆ standard error.
Fig. 4. Relative DNA content in pericycle cells 40 h after germination at
the moment (A, C) and just prior to lateral root initiation (B, D).
Histograms show the relative DNA content for each pericycle nucleus of
two representative roots situated at the xylem poles (A, B) and at the
phloem poles (C, D) that was hit during serial sectioning. The ®rst ®ve
values in each histogram depicted in black represent metaphase ®gures
re¯ecting a 4C DNA content charactersitic for the G2 phase of the cell
cycle. Arrows in (A) and (B) indicate individual xylem pericycle cells
with a 4C DNA content. Nuclei of the same kind were not found at
phloem poles.
408
Beeckman et al.
The most developed root primordia reached the point of
emergence from the parent root (Fig. 5B).
In roots that had been incubated on hydroxyurea for
48 h and 72 h, several lateral root initiation sites became
visible because of the CycB1;1-gus expression. Closer
examination of these sites in cleared preparations revealed
that the staining was restricted to only a small group of
founder cells or to their immediate derivatives. In contrast
Fig. 5. Promoter activity of CycB1;1-gus during lateral root formation in the presence of hydroxyurea, a cell cycle inhibitor that blocks the G1-to-S
transition. (A, B) Lateral root primordia in roots of control plants (72 h after germination) that were transferred to the same control medium after 24 h
of germination on control medium. (B) Closer view on an emerging lateral root primordium. (C±E) Initiation sites of lateral root primordia in roots
(72 h after germination) that were transferred to a growth medium containing hydroxyurea after 24 h of germination on control medium. (D, E) Close
view on the GUS-stained sites being composed of pericycle cells at the xylem poles that represent the early stages of lateral root initiation (founder
cells). Arrows in (C) indicate lateral root initiation sites, the arrows with letter D and E show the initiation sites that are depicted at a higher
magni®cation in (D) and (E), respectively. FC, founder cells for lateral root formation lying at the xylem poles in the pericycle; LRP, emerging lateral
root primordium. Bars ˆ 250 mm (A, C) and 50 mm (B, D, E).
Cell cycle regulation
409
to the control plants, no more developed lateral root
primordia were found.
Discussion
The initiation of the ®rst lateral root primordium after
germination was chosen as a model system to study the
cell cycle regulation in the pericycle of Arabidopsis. Forty
hours after germination, the founder cells in the pericycle switch in approximately 30% of the seedlings from
a non-dividing to a dividing state (Dhooge et al., 1999).
In a ®rst set of experiments, the expression patterns of
four cell cycle genes was analysed using the gus reporter
system. Based on these expression patterns combined with
the data from DNA measurements, the model presented
in Fig. 6 is proposed.
In the root tip (Fig. 6, zone A), all four GUS markers
are expressed. Consistently, cell division is active in most
of the cell ®les of the meristem. Although each GUS
marker had its own staining pattern in the root tip, no
differences between the pericycle cells were found.
However, in the mature pericycle, differences in cell
length between pericycle cells at the xylem poles and the
intervening ones were noted. Such differences in length
between mature cells must re¯ect differences in the
number of transverse divisions of the cells from which
they arise in the root tip, because sliding of cells occurs
rarely in root tissues (Webster and MacLeod, 1980;
Casero et al., 1989). Similar differences in pericycle cells
were reported in Allium cepa and Pisum sativum (Lloret
et al., 1989). As in the case of Arabidopsis, pericycle cells
located opposite xylem poles were shorter than cells lying
opposite phloem poles. In both species lateral root
primordia originated opposite xylem poles. In a species
with lateral root initiation occurring at the phloem poles,
shorter pericycle cells were found opposite these poles,
even in regions of the primary root that is located close to
the root tip (Lloret et al., 1989). These observations
indicate that differential cell cycle regulation in the pericycle cell ®les, near or within the root tip, may be crucial
for the patterning of root branching. However, not every
pericycle cell opposite a given protoxylem (or protophloem) pole is involved in lateral root initiation.
Therefore, the observed structural differences are not
suf®cient to understand root branching. Other control
mechanisms on cell cycle regulation must be acting within
these pericycle cell ®les (see also Skene, 2000).
In the provascular cylinder including the pericycle,
GUS staining was observed only for Cdc2aAt and
CycA2;1 in cells above the root meristem (Fig. 6, zone
B), suggesting that these cell ®les leave the cell cycle at a
later time point than the cortical and epidermal cell ®les.
The pericycle cells together with provascular cells move
from G1 to G2 via S phase, as implied by the CycA2;1
Fig. 6. Model for the cell cycle regulation in the pericycle before the
initiation of the ®rst lateral root primordium (40 h after germination).
To describe cell cycle regulation in young seedling roots, four different
zones (A, B, C, D) were distinguished. Zone A coincides with the root
apical meristem where active cell divisons take place in all cell ®les
including the pericycle. Based on CycB1;1-gus expression this zone can
be estimated to cover approximately 0.4 mm of the root tip (without
root cap). In zone B (only 100±200 mm), the outer tissue layers have left
the cell cycle and become differentiated whereas the cells in the central
cyclinder including the pericycle continue to divide. Above this narrow
zone all pericycle cells (and probably other cell types from the central
cyclinder as well) stop dividing and remain in the G1 phase in zone C.
Zone C ends approximately 1 mm above the distal end of the apical
root meristem with the start of zone D where only the cells at the xylem
poles progress via S phase to G2 to become competent to lateral
root initiation.
410
Beeckman et al.
expression. The cortex and epidermis might stop the cell
cycle completely and may enter a G0 phase, or may be
the subject of endoreduplication. Further research is
needed to clarify their cell cycle behaviour in this zone.
The observed cell ®le-dependent exit from the cell cycle
supports earlier views that a root meristem does not have
a sharp distal boundary with the elongation zone, but is
rather composed of proliferation zones of different length
(Barlow, 1984; Casero et al., 1989).
Immediately above this zone (Fig. 6, zone C), CycA2;1
expression diminishes or disappears completely from the
pericycle indicating a progression through the cycle via
mitosis to a G1 phase. This observation is in agreement
with the microdensitometric measurements, which show
predominantly a 2C DNA content in all pericycle cells in
this zone.
Xylem pericycle cells, however, do not leave the cell
cycle completely as is the case for the outer tissue layers.
First, the pericycle shows the presence of Cdc2aAt-gus
expression throughout the whole root, indicating the
maintenance of cell division competence. Secondly,
CycA2;1-gus expression diminishes only in a narrow zone
above the root meristem, but reappears in xylem pericycle
cells in the upper half of the seedling root (Fig. 6, zone
D). Likewise, in sections of Raphanus sativus roots that
were hybridized with a radioactively labelled CycA2;1
mRNA fragment, strong labelling was observed preferentially in pericycle cells at the xylem poles (Burssens
et al., 2000).
Furthermore, two lines of evidence suggest that those
pericycle cells that will give rise to a lateral root remain in
the G2 phase of the cycle. Firstly, when seedling roots, at
a stage before laterals could be initiated, were treated with
hydroxyurea, the very ®rst divisions in the founder cells
could still take place, while further development was
blocked immediately thereafter by the inhibition of the S
phase transition. Secondly, the few 4C values were all
recorded in that part of the root where the initiation of
the ®rst lateral is to be expected.
Although phloem pericycle cells also show Cdc2aAtgus expression throughout the whole root, no CycA2;1
expression could be observed in the distal root part.
Taking into account their 2C DNA content, these cells
most probably remain in the G1 phase of the cell cycle.
These data together with the cell length measurements
allow the conclusion to be drawn that, at least in
Arabidopsis, the pericycle is composed of dissimilar cell
®les. The cells adjacent to the phloem poles reach their
maturation after fewer cell divisions and remain in the G1
phase whereas the xylem pericycle cells undergo more
divisions in the meristem itself, do not remain in the G1
phase, but proceed to the G2 phase in the upper half of
the seedling root where they may receive a signal to divide
and start the initiation of a new primordium. The reason
why xylem pericycle cells seem to be more susceptible to
lateral root initiation in most plants may result from the
fact that these cells have completed DNA synthesis and
remain at the phase that immediately precedes the M
phase. This hypothesis ®ts nicely with the `primed
pericycle model' of Skene (Skene, 2000). In this model,
pericycle cells at the protoxylem poles become primed by
a radial factor. Only a subset of these primed (G2) cells
will be triggered later on by a longitudinally distributed
factor. It has been well documented that auxin has a
promotive effect on lateral root initiation (Blakely et al.,
1982; Boerjan et al., 1995; Celenza et al., 1995; Laskowski
et al., 1995). Therefore, this plant hormone could play a
dominant role in both priming and triggering the
pericycle cells. However, the precise link on the molecular
level between auxin and cell division has still to be
clari®ed. Finally, the presence of both types of pericycle
cells also explains the con¯icting ideas found in literature
in which pericycle cells were ®rst thought to be arrested in
G1 (Corsi and Avanzi, 1970) and later in G2 (Blakely and
Evans, 1979).
Acknowledgements
The authors thank Sandra Dhooge, Ive De Smet, and Roeland
Nieuwborg for help with microdensitometric measurements,
Dr Keith Skene (Department of Biological Sciences, University
of Dundee, UK) for helpful comments, Martine De Cock for
help in preparing the manuscript, and Stijn Debruyne for
®gures.
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