Blackwell Science, LtdOxford, UKBOJBotanical Journal of the Linnean Society0024-4074The Linnean Society of London, 2005? 2005
149?
457464
Original Article
REDUCTION OF CHROMOSOME NUMBER IN
ELEOCHARIS
Botanical Journal of the Linnean Society, 2005, 149, 457–464. With 19 figures
C. R. M. DA SILVA
Et al.
Reduction of chromosome number in Eleocharis
subarticulata (Cyperaceae) by multiple translocations
CARLOS R. M. DA SILVA1, M. SOCORRO GONZÁLEZ-ELIZONDO2
and ANDRÉ L. L. VANZELA1*
1
Laboratório de Biodiversidade e Restauração de Ecossistemas, Departamento de Biologia Geral, CCB,
Universidade Estadual de Londrina, 86051-970, Londrina, PR, Brazil
2
Instituto Politécnico Nacional CIIDIR, Durango, Dgo. 34000, Mexico
Received March 2005; accepted for publication May 2005
Eleocharis subarticulata is recorded as the third species of Cyperaceae with a reduced chromosome number (n = 3),
following reports on Rhynchospora tenuis (n = 2) and Fimbristylis umbellaris (n = 3). For Eleocharis, the numbers
recorded to date vary from 2n = 10 to 2n = c. 196, with x = 5 as the possible basic number. The karyotype of
E. subarticulata was studied using conventional staining (mitosis and meiosis), C-CMA3/DAPI banding, and FISH
with 45S rDNA and telomere probes. The chromosomes showed no primary constrictions, as expected in the holocentric chromosomes of Cyperaceae. The meiotic behaviour was abnormal, with a single multivalent ring of six chromosomes at metaphase I, resulting from multiple translocations. At anaphase I six chromatids migrated to each pole,
evidencing the inverted meiosis, and these groups were also visible at metaphase II. The C-CMA3/DAPI banding
technique showed only four terminal GC-rich blocks. FISH with 45S rDNA probes revealed four terminal signals,
probably associated with GC-rich blocks. The telomeric probe located terminal signals in all the chromosomes,
besides a hybridization site in the middle of the large pair. The occurrence of ectopic telomeric sites has not been
described previously for plants with holokinetic karyotypes and with reduced chromosome numbers. These data
reinforce the hypothesis of the reduction in chromosome number by multiple translocations. © 2005 The Linnean
Society of London, Botanical Journal of the Linnean Society, 2005, 149, 457–464.
ADDITIONAL KEYWORDS: chromosome banding – FISH – holocentric chromosomes – rDNA – telomere.
INTRODUCTION
Eleocharis subarticulata (Nees) Boeckl. is a small species in the Cyperaceae characterized by short, filiform
and opaque culms (5–15 cm), purple sheaths with usually obtuse subinflated apex; spikelets fusiform to lanceolate, achene trigonous to almost planoconvex,
narrowly obovate, shining olivaceous, deeply reticulate, style-base narrow, subulate, grey, a third to a
quarter as long as the achene body, and bristles light
brown, shorter than or exceeding the achene (Svenson,
1929). Svenson (1929) considered the position of
E. subarticulata in the genus Eleocharis to be uncertain. Menapace (1993) included this species in the
series Palustriformes based on the micromorphology of
the achenes. The Palustriformes group is part of the
*Corresponding author. E-mail: [email protected]
subseries Eleocharis, in the subgenus Eleocharis
(Strandhede, 1967; González-Elizondo & Peterson,
1997).
Eleocharis subarticulata differs from other members
of the subseries Eleocharis in several morphological
traits and its taxonomic position in the genus remains
unclear. Some cytogenetic peculiarities confirm the taxonomic uniqueness of this species. Species of Eleocharis
as well as other representatives of the Cyperaceae family possess holocentric chromosomes, post-reductional
meiosis, and lack tetrads (see Faulkner, 1972). The
chromosome numbers previously recorded for Eleocharis ranged from 2n = 10 (Wulff, 1937 and Levitski,
1940, both cited by Strandhede, 1965a, b, c, d, 1966,
1967, 1973; Lewis, Stripling & Ross, 1962) to 2n = c.
196 in E. kuroguwai (Hoshino, 1987). The predominance of numbers that are multiples of n = 5 (2n = 10,
20, 30, 40, 50), suggests that x = 5 is the probable basic
© 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 149, 457–464
457
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C. R. M. DA SILVA ET AL.
number. The karyotype evolution in the family occurs
preferentially by agmatoploidy, symploidy (see Luceño
& Guerra, 1997) and polyploidy (see Vanzela, Luceño &
Guerra, 2000). Other cytogenetic features of Cyperaceae are the occurrence of species with multiple 45S
rDNA sites localized at the terminal positions of the
chromosomes (see Vanzela et al., 1998) and terminal–
interstitial bands revealed by C-Giemsa and C-CMA 3/
DAPI (Vanzela & Guerra, 2000).
Only a few plant species are known to have low
chromosome numbers, but two Cyperaceae are among
them: Rhynchospora tenuis with 2n = 4 (Vanzela,
Guerra & Luceño, 1996) and Fimbristylis umbellaris
with 2n = 6 (Rath & Patnaik, 1981). Other examples of
species with 2n = 4 are Haplopappus gracilis (Jackson, 1973) and Brachycome dichromosomatica (SmithWhite, Carter & Stace, 1970) (both Asteraceae),
Zingeria biebersteiniana and Colpodium versicolor
(Bennett, Leitch & Bennett, 1995) (both Poaceae), and
Ornithogalum tenuifolium (Stedje, 1988) (Hyacinthaceae). Those with 2n = 6 are Brachycome goniocarpa and B. leptocarpa (Smith-White et al., 1970),
Podolepis capillaris (Watanabe et al., 1999), Crepis
capillaris, C. dichotoma, C. fuliginosa, C. polymorpha,
C. reuteriana, C. virens and C. zacintha (Fedorov,
1969), Pterotheca falconeri (Mehra et al., 1965),
Astranthium orthopodum (DeJong, 1965), Hypochoeris
cretensis and H. pinnatifida (Stebbins, Jenkins &
Walters, 1953) (all Asteraceae), Crocus candidus,
C. graveolens, C. olivieri, C. speciosus (Özhatay, 2002)
and several other species of Crocus listed by Fedorov
(1969) (Iridaceae), and Drosera roseana (Kondo, Sheik
& Hoshi, 1994) (Droseraceae).
Eleocharis subarticulata (n = 3) is a new record for
this group of plants with reduced chromosome numbers. It is the third in the family Cyperaceae and has
the lowest chromosome number yet recorded for Eleocharis. Thus, the aims of this study are to describe
the meiotic behaviour and karyotype features of
E. subarticulata, based on conventional staining, chromosome banding and FISH with 45S rDNA and telomeric probes. The organization and the evolution of
this karyotype from the probable basic number x = 5
and the cytotaxonomical position of this species in the
genus Eleocharis will be discussed.
MATERIAL AND METHODS
Samples of Eleocharis subarticulata were collected
from two flooded fields at Ponta Grossa city, Paraná,
southern Brazil, about 50 km apart. Plants were cultivated in pots to produce new roots and anthers.
Vouchers were deposited in the FUEL herbarium. For
the study of somatic chromosomes, roots were pretreated with 2 mM 8-hydroxyquinoline for 24 h and
fixed in absolute ethanol:glacial acetic acid (3 : 1, v : v)
for 12 h and kept at −20 °C until used. Root tips were
digested for 3 h in a mixture of 2% (v/v) cellulase and
20% (v/v) pectinase, further hydrolysed in 1 M HCl at
60 °C for 11 min, dissected in a drop of 45% acetic acid
and squashed. The cover slips were removed after
freezing in liquid nitrogen. The material was stained
with 2% Giemsa. The size of the chromosomes and the
length of the haploid set were measured from ten different metaphases.
For meiotic study, spikelets were dissected and the
anthers fixed in absolute ethanol:glacial acetic acid
(3 : 1, v : v) for 12 h, and kept at −20 °C until used.
Anthers were hydrolysed in 1 M HCl at 60 °C for 5 min
and squashed in a drop of 45% acetic acid. The coverslips were removed after freezing, as described above
and the samples were stained with 2% Giemsa. Slides
were mounted with Entellan.
All photographs were taken on Kodak Imagelink 25
ISO film.
Chromosome banding was performed with root tips
pretreated as described above, but softened in 4% cellulase plus 40% pectinase at 37 °C for 3 h and
squashed in a drop of 45% acetic acid. The coverslips
were removed after freezing and the slides were aged
for three days, incubated in 45% acetic acid, 5% barium hydroxide, and 2× SSC (Schwarzacher, Ambros &
Schweizer, 1980; with modifications). The material
was aged for three more days and sequentially stained
with 0.5 mg/mL CMA3 for 1.5 h, washed in distilled
water and immediately stained with 2 µg/mL DAPI for
30 min. Slides were mounted with a medium composed of glycerol/McIlvaine buffer pH 7.0, 1 : 1, plus
2.5 mM MgCl2. Cells were observed and photographed
on Kodak T-max 100 ISO film.
Fluorescent in situ hybridization (FISH) was performed according to Heslop-Harrison et al. (1991) and
Cuadrado & Jouve (1994), with modifications. Slides
were prepared as described for banding, but without
ageing. The 45S rDNA and telomere probes were
labelled with biotin-14-dATP by nick translation and
utilized for FISH in a mixture of 30 µL composed of
100% formamide (15 µL), 50% polyethylene glycol
(6 µL), 20× SSC (3 µL), 100 ng of Calf thymus DNA
(1 µL), 10% SDS (1 µL), and 100 ng of probe (4 µL).
The material was denatured at 90 °C for 10 min and
hybridization was performed at 37 °C overnight in a
humidified chamber. Post-hybridization washes were
carried out in 2× SSC, 20% formamide in 0.1 × SSC,
0.1× SSC and 4× SSC/0.2% Tween 20, all at 42 °C. The
probes were detected with avidin-FITC conjugate and
post-detection baths were performed in 4× SSC/0.2%
Tween 20 at room temperature. Slides were mounted
in a solution (25 µL) composed of 12.5 µL of antifade
and 12.5 µL of 50% glycerol in McIlvaine buffer,
pH 7.0, with 2.5 mM MgCl2, plus 1 µL of 50 µg/mL propidium iodide.
© 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 149, 457–464
REDUCTION OF CHROMOSOME NUMBER IN ELEOCHARIS
Photographs were taken on Kodak Proimage colour
100 ISO.
RESULTS AND DISCUSSION
CONVENTIONAL
STAINING AND REDUCTION
OF CHROMOSOME NUMBER
Eleocharis subarticulata was found to possess 2n = 6
chromosomes (Fig. 15), comprising a larger (2.65 µm),
an intermediate (2.48 µm) and a smaller chromosome
pair (2.08 µm long), with a size of haploid set of
7.21 µm. Primary constrictions and satellites were not
observed. At mitotic anaphase, the chromatids separated parallel to the equatorial plate, as is expected for
holocentric chromosomes. The occurrence of holocentric chromosomes in Eleocharis was previously demonstrated by Håkansson (1954) in E. palustris after
irradiation of young spikelets, with the observation
that the behaviour of the chromosome fragments during subsequent cellular cycles was normal. Sharma &
Bal (1954), Sanyal & Sharma (1972) and Bir et al.
(1993, cited by Ball, Reznicek & Murray, 2002) found
that several species of Cyperaceae (particularly
Eleocharis) possess chromosomes with localized centromeres; but this seems to be uncommon in the family. Chromosome studies in Eleocharis subseries
Eleocharis have been conducted by Strandhede,
(1965a, b, c, d, 1966, 1967, 1973), who found holocentric chromosomes. The data presented here are also
evidence of the occurrence of holokinetic chromosomes
in Eleocharis.
In the Cyperaceae, karyotype differentiation is often
a manifestation of chromosome rearrangements such
as agmatoploidy and symploidy. These events seem to
be common in Eleocharis, since of the 50 species with
determined chromosome numbers, half possess numbers that deviate from x = 5, which is considered to be
the probable basic number for Eleocharis and some
other Cyperaceae. Several of these species possess
more than one chromosome number, e.g. 2n = 15 and 16
in E. palustris (L.) R & S, 2n = 44, 45 and 46 in
E. ambigens Kuekenthal (Strandhede, 1965a), and
2n = 20 and 21 in E. atropurpurea (Retz.) Presl. (Nijalingappa, 1973) and E. acicularis (L.) R & S (Yano et al.,
2004). Although chromosome fusion and fission are
common events in Eleocharis, as well as in other Cyperaceae (Wahl, 1940; Davies, 1956; Faulkner, 1972;
Luceño & Castroviejo, 1991), other species present
karyotypes that are diploid or polyploid multiples of
x = 5, such as 2n = 10 and 20 in E. geniculata (Nijalingappa, 1973). Agmatoploidy and symploidy have
been associated with the events of chromosome number
reduction in the family, as previously described in
Rhynchospora tenuis, from n = 5 to n = 2 (Vanzela
et al., 1996) and in Fimbristylis umbellaris, from n = 5
to n = 3 (Rath & Patnaik, 1981). Reduction in the chro-
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mosome number has also been reported for other
groups with holocentric chromosomes. Kondo & Segawa (1988) and Kondo et al. (1994) considered that the
chromosome numbers n = 3, 5, 6, 7, 8, 9, 10, 11, 12 and
13 found in Drosera roseana (Droseraceae) are involved
mainly with mechanisms of fission (agmatoploidy) and
fusion (symploidy) of holocentric chromosomes.
The establishment of species whose karyotypes
were modified by loss of DNA segments or diminution
of chromosome numbers by fusions and/or translocations seems not to be just a speciality of plant groups
with holocentric chromosomes. In Crepis (Asteraceae),
a genus with monocentric chromosomes, the most
primitive and least specialized karyotypes with n = 3
occur preferentially in annual plants (see Babcock,
Stebbins & Jenkins, 1942). Another example of chromosome number reductions was reported by Goldblatt
et al. (2002) in the African species of Moraea (Iridaceae). Although this group possesses monocentric
chromosomes, its karyotype evolution is marked by a
series of chromosome numbers (n = 4, 5, 6, 7, 8, 9, 10),
with n = 6 the most frequent. Cytogenetical and
molecular data indicate n = 10 to be the ancestral
chromosome number of the genus, the karyotypes
with reduced numbers being considered to be derived.
In this context, n = 3 in E. subarticulata probably represents a derived condition that has acted as a reproductive barrier. The phenotypic differentiation of the
species does not permit its placement in any of
the subseries of Eleocharis recognized until now. The
micromorphological similarity of the achenes of
E. subarticulata to those of species of the Palustriformes group (Menapace, 1993) represents only a
superficial resemblance. Several characters (e.g. filiform culms, subinflated apex of the sheaths, and
deeply reticulate achenes) separate E. subarticulata
from the members of the Palustriformes (which is now
recognized as a part of the subseries Eleocharis).
The closest approximation would be to recognize
E. subarticulata as a member of the subgenus Eleocharis, and possibly of the section Eleocharis, but both
its morphology and its peculiar karyotype seem to
indicate that this species is not closely related to others in the genus.
MEIOTIC
BEHAVIOUR
The meiotic analysis of 435 cells always showed n = 3,
but there was an unusual meiotic behaviour. The cells
in pachytene exhibited no chromosome ends, or occasionally one (Figs 1, 2), where at least six ends would
be expected, considering that there should have been
three bivalents. In diplotene, the chromosomes
appeared to form a unit, usually contorted into loops.
In cells with chromosomes that were more condensed
it was possible to see united structures of fine fila-
© 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 149, 457–464
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C. R. M. DA SILVA ET AL.
Figures 1–14. Meiosis in Eleocharis subarticulata. Scale bar = 5 µm. Figs 1,2. Early prophases, showing the absence or
rarity of chromosome ends. Figs 3–6. Diplotene stages, showing chromosome associations with loops and absence of visible
chromosome ends. Fig. 7. Polar view of metaphase I, with the six chromosomes organized into a single ring. Fig. 8.
Equatorial view of metaphase I. Fig. 9. Diakinesis. Fig. 10. Anaphase I, with separation of two groups of six sister
chromatids. Fig. 11. Early anaphase II, with two groups of six chromatids each separating into two threes. Fig. 12. Late
anaphase II, with precocious separation of one division into two groups, each with three chromatids (complements
positioned above and to right). The other division (below) is later-separating. Figs 13, 14. Pollen mitosis and lack of tetrad
production. Fig. 13. Three of the four complements produced at meiosis (left) are degenerating and are much smaller than
the fourth complement (right), which is in prophase of pollen mitosis. Fig. 14. Three post-meiotic complements are
degenerating further (left). The remaining complement is in anaphase of pollen mitosis and will eventually produce the
vegetative and generative nuclei.
© 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 149, 457–464
REDUCTION OF CHROMOSOME NUMBER IN ELEOCHARIS
ments (Figs 3–6). At metaphase I, the chromosome
association was more evident as a single multivalent
ring of six. The chromosomes stay associated end-toend, forming a ring on the equatorial plate (Figs 7, 8)
and in a subsequent stage all the chromosomes
appeared to be connected by fine filaments or were
completely disconnected, appearing as six independent structures (Fig. 9). Two groups of six chromatids
were visible at anaphase I and metaphase II, indicating a segregation of sister chromatids and not of
homologous chromosomes (Figs 10, 11). The occurrence of the same chromosome numbers at both
metaphase I and II is in agreement with the proposal
of Wahl (1940) that evidence of post-reductional meiosis is provided by the occurrence of the same number
of structures in the metaphases I and II, which
decreases to half in anaphase II and pollen mitosis.
These data show that E. subarticulata possesses a
typical inverted meiosis. The absence of bivalents
forming a ‘box structure’ is also a criterion indicating
the presence of post-reductional meiosis in holocentric
chromosomes. In the transition from metaphase II to
anaphase II, the initial migration of three of the four
chromatid groups to one of the poles was observed,
leaving one complement isolated (Fig. 12), representing the beginning of the degeneration of three of the
four complements and the failure of tetrad formation.
As expected for the Cyperaceae, E. subarticulata also
showed absence of tetrads, with generation of one pollen grain from each pollen mother cell (Figs 13, 14).
The meiotic behaviour of E. subarticulata causes
doubts about the range of mechanisms involved in
karyotype evolution in the Cyperaceae. Until now,
only agmatoploidy (fission), symploidy (fusion) and
polyploidy have been considered, but this case is very
similar to the formation of multiple translocations
found in Oenothera, involving some or all the chromosomes of the complement (Cleland, 1923). This evidence permits us to propose that chromosome
reduction in these holocentrics is the result of multiple
translocations that involve all the chromosomes of the
complement and form a multivalent ring during meiosis, and not symploidy, because bivalents and
quadrivalents were not observed. According to Luceño
& Guerra (1997), symploidy can be responsible for the
reduction of one or a few chromosomes, which is
represented by the formation of trivalents and/or
quadrivalents. At present, it is not possible to add
more information about the extent of this event in
E. subarticulata as a whole, because the entire geographical range of the species was not sampled.
CHROMOSOME
BANDING AND IN SITU HYBRIDIZATION
The C-CMA3/DAPI banding techniques applied to
E. subarticulata showed only small GC-rich blocks in
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the terminal regions of the intermediate and smallest
chromosome pairs (Fig. 16), very similar to those
found in Rhynchospora tenuis, 2n = 4 (Vanzela &
Guerra, 2000). Information on banding patterns in
holocentrics is scarce in the Cyperaceae. Greilhuber
(1995) reported the occurrence of several GC-rich terminal bands in Carex, probably associated with the
NORs, and Vanzela & Guerra (2000) found heterochromatic blocks varying in composition (CMA 3+/
DAPI0, CMA3+/DAPI– and CMA3–/DAPI+), size and
location in different species of Rhynchospora. The
data obtained here indicate that amplification of
repetitive DNA that appears in some species of
Cyperaceae does not occur in E. subarticulata, being
useful only to differentiate the largest pair, which
does not possess bands.
FISH with 45S rDNA probe revealed terminal signals on the second and third chromosome pairs
(Fig. 17). Vanzela et al. (1998) and Vanzela, Cuadrado & Guerra (2003) also showed karyotypes of
several species of Rhynchospora with multiple and
terminal 45S rDNA sites. These data indicate that
the terminal positioning of 45S rDNA was maintained, in spite of the occurrence of multiple translocations, accompanying the tendency towards the
accumulation of ribosomal genes in terminal positions, observed previously in Cyperaceae by Vanzela
et al. (1998, 2003) and Furuta & Hoshino (1999).
FISH with a telomeric probe localized hybridization
sites in the terminal positions of all the chromosomes, also an additional signal in the middle of the
large pair (Figs 18, 19). This is the first demonstration of ectopic telomeric sites among species
with holocentric chromosomes and reduced number,
and was not observed by Fuchs, Brandes & Schubert (1995) or Vanzela et al. (2003) in Luzula purpurea and Rhynchospora tenuis, respectively.
Interstitial telomeric sites could support the reduction of chromosome number by multiple translocations, but it is important to recall that interstitial
sites appear not only as the result of chromosome
changes, but also due to the random processes of
amplification or slippage replication (Biessmann &
Mason, 1994).
In conclusion, chromosome changes producing a
dramatic reduction in chromosome numbers are not
frequent, as is shown by the low number of plant
species with n = 2 or 3. There are also few examples
where the reduction path has been accompanied by
evolutionary success. Illustrative examples are the
tandem fusion that produced Haplopappus gracilis
with n = 2 from H. ravenii with n = 4 (Ikeda, 1987),
or the series of unequal translocations that resulted
in the emergence of chromosome races with n = 2 in
Ornithogalum tenuifolium (Stedje, 1989). In this
context, it is possible to expect some modification
© 2005 The Linnean Society of London, Botanical Journal of the Linnean Society, 2005, 149, 457–464
462
C. R. M. DA SILVA ET AL.
Figures 15–19. Premetaphase and metaphase of mitosis in Eleocharis subarticulata. Scale bar = 5 µm. Fig. 15. Giemsa
stained premetaphase. Fig. 16. C-CMA3 banding. Note the GC-rich regions dispersed and separated from chromosomes.
Fig. 17. FISH with 45S rDNA probe, showing terminal and dispersed hybridization sites. Note that with dispersion of
terminal 45S rDNA segments, the band can appear distant from the chromosome. Fig. 18. Premetaphase hybridized
with the telomeric probe. Note the interstitial sites and a lack of signal owing to dispersion of 45S rDNA segments (see
also Figs 16, 17). Fig. 19. Metaphase hybridized with the telomeric probe. Note the interstitial chromosome sites in the
first pair.
capable of generating isolation after a dramatic
numerical reduction. In the case of E. subarticulata,
the emergence of the reduced chromosome number
(n = 3) might have caused a reproductive barrier
that provided this species with its singular morphology, which makes it difficult to classify the species.
The results obtained here are new evidence of the
importance of chromosome fusion in karyotype evolution in the Cyperaceae. However, the lack of further chromosome markers does not allow us to
suggest whether other evolutionary forces besides
multiple translocations are responsible for the
reduction, nor which species could be basal to
E. subarticulata. Future studies using genomic in
situ hybridization and chromosome isolation for
painting could aid in this search.
ACKNOWLEDGEMENTS
The authors are grateful to the Brazilian agencies
CAPES and Fundação Araucária/ProPPG-UEL for
financial support
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