Annals of Botany 95: 271–276, 2005

doi:10.1093/aob/mci022, available online at www.aob.oupjournals.org

Variation in Chromosome Numbers, CMA Bands and 45S rDNA Sites in

Species of Selaginella (Pteridophyta)
A D R I A N A B U A R Q U E M A R C O N , I V A C A R N E I R O L E Ã O B A R R O S and M A R C E L O G U E R R A *
Universidade Federal de Pernambuco, Centro de Ciências Biológicas, Departamento de Botânica,
Rua Nelson Chaves s/n, Cidade Universitária, 50.670-420, Recife, Pernambuco, Brasil

Received: 16 January 2004 Returned for revision: 1 May 2004 Accepted: 15 August 2004 Published electronically: 26 November 2004

Background and Aims Selaginella is the largest genus of heterosporous pteridophytes, but karyologically the genus
is known only by the occurrence of a dysploid series of n = 7–12, and a low frequency of polyploids. Aiming to
contribute to a better understanding of the structural chromosomal variability of this genus, different staining
methods were applied in species with different chromosome numbers.

Methods The chromosome complements of seven species of Selaginella were analysed and, in four of them, the
distribution of 45S rDNA sites was determined by fluorescent in situ hybridization. Additionally, CMA/DA/DAPI
and silver nitrate staining were performed to investigate the correlation between the 45S rDNA sites, the hetero-
chromatic bands and the number of active rDNA sites.

Key Results The chromosome numbers observed were 2n = 18, 20 and 24. The species with 2n = 20 exhibited
chromosome complement sizes smaller and less variable than those with 2n = 18. The only species with 2n = 24,
S. convoluta, had relatively large and asymmetrical chromosomes. The interphase nuclei in all species were of the
chromocentric type. CMA/DA/DAPI staining showed only a weak chromosomal differentiation of heterochromatic
bands. In S. willdenowii and S. convoluta eight and six CMA+ bands were observed, respectively, but no DAPI+
bands. The CMA+ bands corresponded in number, size and location to the rDNA sites. In general, the number of
rDNA sites correlated with the maximum number of nucleoli per nucleus. Ten rDNA sites were found in S. plana
(2n = 20), eight in S. willdenowii (2n = 18), six in S. convoluta (2n = 24) and two in S. producta (2n = 20).

Conclusions The remarkable variation in chromosome size and number and rDNA sites shows that dramatic
karyological changes have occurred during the evolution of the genus at the diploid level. These data further suggest
that the two putative basic numbers of the genus, x = 9 and x = 10, may have arisen two or more times
independently. ª 2004 Annals of Botany Company

Key words: Pteridophyta, Selaginella, chromosome number, fluorochrome staining, silver nitrate staining, in situ
hybridization, 45S rDNA.

INTRODUCTION In spite of several cytotaxonomical studies, the karyolo-

gical evolution of this group remains poorly understood.
Selaginella P. Beauv. is a heterosporous genus of the family
Cytological investigations have been based primarily on
Selaginellaceae, comprising approx. 750 species distributed
chromosome counts and, in a few cases, on chromosome
throughout the tropical regions, including 250 species in the
morphology. Meanwhile, in angiosperms and gymnosperms
Americas (Tryon and Tryon, 1982; Kramer and Green,
the karyological studies in the last two decades have turned
1990). They are generally found in humid and shady forests,
to more specific methods such as banding methods and
although they can also be present in dry forests, swamps, on
fluorescent in situ hybridization (FISH) (Greilhuber,
damp rocks along riverbanks or near waterfalls, and even in
1995; Stace and Bailey, 1999). Heterochromatic bands in
cold regions such as the Alps, or in rocky deserts (Tryon and
plants have been analysed mainly by C-banding or by
Tryon, 1982).
base-specific fluorochrome staining (Guerra, 2000).
Polyploidy and hybridization have occurred numerous
Fluorochrome banding has the advantage of being a simpler,
times in the evolution of the pteridophytes, resulting in
more reproducible and less destructive method, as com-
the characteristically high chromosome numbers seen in
pared with C-banding (Guerra, 1993). The fluorochromes
this group (Walker, 1984). Nonetheless, high chromosome
chromomycin A (CMA) and 40 ,6-diamidino-2-phenylindole
numbers are rare among the heterosporous pteridophytes.
(DAPI) exhibit preferential staining for GC and AT-rich
Selaginella, the largest heterosporous genus, has only a few
DNA sequences, respectively, allowing the identification
polyploid species, although a large dysploid variation has
of different types of heterochromatin. Distamycin A
been reported. The chromosome number ranges between
(DA) has also been used to enhance the contrast between
2n = 14 and 2n = 60, and different authors have reported
CMA and DAPI (Schweizer and Ambros, 1994; Marcon
different basic chromosome numbers for the genus: x = 7,
et al., 2003). The 45S rDNA sites are generally positively
8, 9, 10, 11 and 12 (Kuriachan, 1963; Jermy et al., 1967;
stained with CMA and negatively stained with DAPI. In
Takamiya, 1993).
many species, the rDNA sites are the only positively stained
CMA bands (see, for example, Melo and Guerra, 2003). The
* For correspondence. E-mail mguerra@ufpe.br variation in number and position of the rDNA sites has been
Annals of Botany 95/2 ª Annals of Botany Company 2004; all rights reserved
272 Marcon et al. — Cytogenetic Variability in Species of Selaginella
T A B L E 1. Species of Selaginella analysed, with their respective voucher number, provenance, chromosome number and previous
counting

Voucher Chromosome Previous

Species number Provenance number (2n) countings (2n) References

S. muscosa Spring 36588 Bonito 18 – –

S. simplex Baker 36412 Gravatá 18 – –
S. willdenowii 36589 Recife 18 18 Jermy et al. (1967); Fabri (1963);
(Desv. ex Poiret) Baker Kuriachan (1963)
Selaginella sp. – Itamaracá 18 – –
S. producta Baker 36410 Paulista 20 – –
36411 Igarassu 20 – –
36591 Bonito 20 – –
S. plana (Desv. ex Poiret) 36409 Recife 20 20 Jermy et al. (1967); Fabri (1963);
Hieron Kuriachan (1963)
36590 Rio de Janeiro 20 20 Jermy et al. (1967); Fabri (1963);
Kuriachan (1963)
S. convoluta (Arnott) Spring 36408 Petrolina 24 – –

All the samples were collected in the Brazilian state of Pernambuco, except S. plana, which is from the state of Rio de Janeiro.

demonstrated to be an additional important karyotype by 23 h at 10  C. Subsequently, they were fixed in Carnoy

feature to the cytotaxonomic analyses of some angiosperm
genera, such as Clivia (Ran et al., 1999) and Sanguisorba
(Mishima et al., 2002). Silver nitrate staining is another
method that allows the visualization of nucleolus organizer
regions (NORs), which are also related to chromosomal
secondary constrictions and rDNA sites, but after the silver
nitrate staining only those sites activated in an interphase
nucleus are stained in the subsequent prophase or metaphase
(Moscone et al., 1995). In general, the maximum number of
silver-stained sites in prophase or metaphase chromosomes
and the maximum number of nucleoli in interphase nuclei
correspond to the number of 45S rDNA sites (see, for exam-
ple, Moscone et al., 1995). This relationship seems to be
true also for homosporous pteridophytes (Kawakami et al.,
1999; Marcon et al., 2003).
In order to better understand the cytogenetics and evolu-
tion of this genus, the chromosome numbers were analysed
in seven Brazilian species of Selaginella and in four of them
the distribution of 45S rDNA sites was observed. Addition-
ally, the heterochromatic blocks stained with CMA/DA/
DAPI and the number of nucleoli per nucleus were inves-
tigated, as an indication of the number of NORs. The results
are discussed in relation to the chromosomal evolution of
the genus.
MATERIALS AND METHODS
The species investigated, with their respective collection
sites, chromosome numbers and previous chromosome
counts are listed in Table 1. Part of the material collected
was prepared for the herbarium, and the vouchers were
deposited in the UFP herbarium (Federal University of
Pernambuco, Brazil). Another part of the material was
maintained at the experimental garden of the Department
of Botany, at the Federal University of Pernambuco, for
cytogenetic analysis.
Leaf buds were collected and pretreated in 0002 M
8-hydroxyquinoline for 1 h at room temperature, followed
(ethanol : acetic, acid 3 : 1) for 24 h at room temperature, and
stored at 20  C.
For conventional chromosome staining, the fixed leaf
buds were washed twice in distilled water for 5 min, hydro-
lysed in 5 N HCl for 30 min, and then the meristem was
isolated and squashed in 45 % acetic acid (Guerra, 1983).
The preparation was stained with Giemsa 5 % and mounted
with Entellan. Chromosome measurements were performed
on amplified photographs with the aid of a pachymeter.
The triple staining with the fluorochromes CMA, DAPI
and DA was performed according to Schweizer and Ambros
(1994). Leaf buds were washed twice in distilled water for
5 min, digested in 2 % cellulase/20 % pectinase for 2 h at
37  C, and squashed in 45 % acetic acid. After coverslip
removal the slides were aged for 3 d at room temperature,
stained with CMA (05 mg mL1, 1 h), counterstained with
distamycin A (01 mg mL1, 30 min), stained with DAPI
(2 mg mL1, 30 min) and mounted in McIlvaine’s (pH 70)
buffer–glycerol (v/v, 1 : 1) containing 25 mM MgCl2.
CMA/DAPI staining without distamycin A produced a
poorer differentiation.
Leaf buds without pretreatment were fixed directly into
Carnoy and used for silver nitrate staining. Slides were
prepared using the same enzyme treatment employed for
fluorochromes, except that they were not aged before stain-
ing. A small drop of 50 % silver nitrate diluted in formic
acid was placed over the cells, covered with a coverslip and
incubated in a moist chamber at 60  C (Rufas et al., 1987)
for approx. 10 min or until adequate staining was attained.
To locate the 45S rDNA sites, probes SK18S and SK25S
were used, containing 18S and 25S rDNA of Arabidopsis
thaliana (Unfried et al., 1989; Unfried and Gruendler, 1990),
kindly provided by Prof. D. Schweizer of the University
of Vienna, and labelled by nick translation with biotin-
11-dUTP (Sigma) or digoxigenin-11-dUTP (Roche). The
probe was detected with mouse anti-biotin monoclonal
antibody (Dakopatts no. M743) and visualized with rabbit
anti-mouse antibody conjugated to tetramethyl rhodamine
isothiocyanate (TRITC) (Dakopatts no. R270) or detected
 Marcon et al. — Cytogenetic Variability in Species of Selaginella 273

A B C D

E F G H I

J K L

F I G . 1. Chromosome complement (A–G), interphase nuclei (H and I) and number of NORs and nucleoli (J–L) observed in species of Selaginella:
(A) S. willdenowii (2n = 18) with two satellites (arrows); (B) Selaginella sp. (2n = 18) with three satellites (arrows); (C) S. simplex (2n = 18);
(D) S. muscosa (2n = 18); (E) S. plana (2n = 20); (F) S. producta (2n = 20); (G) S. convoluta (2n = 24); (H and I) chromocentric interphase nuclei of
S. plana and S. willdenowii, respectively; (J) prometaphase cell showing NORs (arrows); (K and L) prometaphase cells showing chromosomes associated with
nucleoli (arrows) in S. plana (K) and S. convoluta (L). The bar in A = 5 mm.

with sheep anti-digoxigenin antibody conjugated to
fluorescein isothiocyanate (FITC) (Boehringer Mannheim
no.1207741) and FITC-conjugated rabbit anti-sheep
(Dakopatts F135, DAKO). The technique was based on
Moscone et al. (1996), with some modifications (denaturation
at 80  C and the post-hybridization washes in 01 · SSC at
42  C). The hybridization mixture contained 60 % forma-
mide, 5 % dextran sulfate, 2 · SSC, 001 % salmon sperm
DNA, and probe at a final concentration of 12–30 ng mL1. In
most hybridization experiments, a 5S rDNA probe obtained
from total genomic DNA of Passiflora edulis Sims (Melo and
Guerra, 2003) was added to the mixture but no clear signal was
observed. Slides were stained with DAPI (2 mg mL1) and
mounted in Vectashield (Vector).
The best cells were captured with a CCD Cohu camera on
a Leica DMLB microscope, or photographed using Kodak
Imagelink HQ ASA 25 film for bright field, or Kodak ASA
400 film for fluorescence photography.
RESULTS
The seven species of Selaginella analysed in the present
work showed the following chromosome numbers: 2n = 18
[S. muscosa Spring, S. simplex Baker, S. willdenowii
(Desv. ex Poiret) Baker and Selaginella sp.], 2n = 20
[S. producta Baker and S. plana (Desv. ex Poiret) Hieron.]
and 2n = 24 [(S. convoluta (Arnott) Spring] (Fig. 1A–G).
Among the species with 2n = 18, S. willdenowii and
Selaginella sp. exhibited the largest chromosomes, most
of them meta- to submetacentric. The chromosomes in
S. willdenowii varied between 144 and 248 mm (Fig. 1A),
and showed up to six satellites: two on the short arms and
four on the long arms of submetacentric chromosomes. In
Selaginella sp. the chromosome size varied between 135 and
232 mm and three satellites were observed: two on a meta-
centric pair, and a third on a submetacentric chromosome
(Fig. 1B). On the other hand, S. simplex and S. muscosa had
much smaller chromosomes, varying, respectively, between
085 and 126 mm and 080 and 162 mm (Fig. 1C and D).
The species with 2n = 20 had smaller chromosomes,
ranging from 070 to 104 mm in S. plana, and 078 to
143 mm in S. producta (Fig. 1E and F). The position of
the centromere could not be observed in any of these four
species, mainly due to the small size of their chromosomes.
Satellites were also not clearly identified.
In the only species with 2n = 24, S. convoluta, the
morphology of the chromosomes seemed to vary from
274 Marcon et al. — Cytogenetic Variability in Species of Selaginella
T A B L E 2. Variation in number of chromosomes, rDNA sites and nucleoli per nucleus in four species of Selaginella
Species 2n Number of rDNA sites
S. willdenowii 18 8 1015
S. plana 20 10
S. producta 20 2 308
S. convoluta 24 6 528
metacentric to acrocentric. Chromosome size varied
between 070 and 177 mm and satellites were not observed
(Fig. 1G).
In all species analysed, the interphase nucleus structure
was granulated, with small chromocentres, corresponding to
the chromocentre type, according to the classification
of Tanaka (1971). Some species showed larger and more
sharply outlined chromocentres (Fig. 1H and I).
Staining with silver nitrate revealed that the maximum
number of nucleoli per nucleus varied among species
(Table 2). In S. producta the maximum number of nucleoli
per nucleus was two, whereas in the other three species
investigated, the maximum number ranged between six
and ten, although most nuclei exhibited only three nucleoli.
Selaginella willdenowii exhibited a variation of one to ten
nucleoli per interphase nucleus and up to ten NORs were
observed in prometaphase (Fig. 1J). In S. convoluta and
S. plana, it was not possible to identify NORs, but six to
ten prophase or prometaphase chromosomes associated
with nucleoli were observed (Fig. 1K and L).
A high number of metaphase cells was obtained from
S. willdenowii, S. plana, S. producta and S. convoluta mak-
ing it possible to analyse the chromosomes with fluoro-
chromes and in situ hybridization. Staining with CMA/
DA/DAPI revealed differentiation of bands only in
S. willdenowii and S. convoluta. The chromosomes of the
other species stained deeply and homogeneously with both
CMA and DAPI, even when counterstained with distamy-
cin A. In prometaphase cells of S. willdenowii, up to eight
CMA+/DAPI terminal bands were observed (Fig. 2A and
B). In S. convoluta (2n = 24), six terminal CMA+/DAPI
bands were localized in three chromosome pairs (Fig. 2D
and E). No DAPI+ bands were observed in these two spe-
cies, while the CMA+ bands were often negatively stained
with DAPI.
In situ hybridization of 45S rDNA in chromosomes of
S. willdenowii revealed the presence of terminal sites on the
long arms of two chromosome pairs and on the short arms of
another two chromosome pairs (Fig. 2C). In two populations
of S. plana analysed, five submetacentric pairs were labelled
terminally: three pairs on the long arms and two pairs on the
short arms (Fig. 2H and I). While in one of the populations it
was not possible to observe clearly the smaller site, for this
stains weakly, in the another population analysed the ten
sites of 45S rDNA were clearly labelled (Fig. 2I). In
S. convoluta there were four sites on two apparently acro-
centric pairs and two sites on a submetacentric pair (Fig. 2F).
In S. producta (Fig. 2G and J) only one chromosome pair was
Variation in the number of nucleoli per nucleus
No. of cells analysed Range Most frequent numbers
1–10 3 (29.5 %) and 4 (21.1 %)
800 1–10 2 (27.8 %) and 3 (31.7 %)
1–2 1 (94.2 %)
1–6 2 (28.0 %) and 3 (35.7 %)
labelled, at the terminal position, in the two populations.
These two signals were always remarkably distinct in inter-
phase nuclei (Fig. 2K), whereas up to ten signals were
observed in the interphase nuclei of S. plana (Fig. 2L).
DISCUSSION
The chromosome numbers and the structure of interphase
nuclei reported in the present work are compatible with
the karyological data known for the genus Selaginella
(Kuriachan, 1963; Jermy et al., 1967; Ghatak, 1977;
Takamiya, 1993). Of the seven species examined, only
S. plana and S. willdenowii had been analysed previously.
The chromosome numbers observed for these species
agreed with the previous counts.
With regard to chromosome size and morphology, there
is a strong trend to conserve the karyotype symmetry, with
predominance of metacentric and submetacentric chromo-
somes. The species with 2n = 20 analysed in this sample
were more stable in chromosome size than those with 2n =
18. Takamiya (1993) found similar results among species
with x = 10 and x = 9. A higher number of meta- and
submetacentric chromosomes was observed in the karyo-
type of all species analysed, and appears to be a common
trend in this genus (Takamiya, 1993), and in other hetero-
sporous pteridophytes (Kuriachan, 1979, 1994). On the
other hand, acro- and telocentric chromosomes are more
frequent among homosporous pteridophytes (Kawakami,
1982; Takamiya et al., 1992; Marcon et al., 2003).
In situ hybridization with 45S rDNA revealed the most
prominent structural variation between species. One species
with 2n = 20, S. plana, displayed ten sites of 45S rDNA
whereas another one with the same chromosome number,
S. producta, exhibited only two. On the other hand,
S. willdenowii with 2n = 18, showed more rDNA sites
(eight) than S. convoluta (six) with 2n = 24. In angiosperms
a similar variation has been widely reported. In Passiflora,
for instance, the number of 45S rDNA sites varied from one
to three pairs among diploid species (Melo and Guerra,
2003). In Paeonia (Zhang and Sang, 1999), five diploid
species (2n = 10) exhibited three to ten pairs of rDNA
sites. Variation in the number of rDNA sites among angios-
perm species of the same ploidy level has been attributed to
chromosome rearrangements, transpositional events and
gene silencing (Moscone et al., 1999). Likewise, similar
mechanisms may be acting in Selaginella species.
In S. plana, S. convoluta and S. producta there was
a perfect correlation between the maximum number
 Marcon et al. — Cytogenetic Variability in Species of Selaginella 275

A B C

D E F

G H I

J K L

F I G . 2. Distribution of the CMA/DAPI bands and 45S rDNA sites in species of Selaginella: S. willdenowii (A and B) and S. convoluta (D and E), stained with
CMA (A and D) and DAPI (B and E), respectively. Arrows in A, B, D and E indicate the CMA+/DAPI– bands. 45S rDNA sites (C and F–L) in S. willdenowii
(C), S. convoluta (F), in two populations of S. producta (G and J), and in two populations of S. plana (H and I). Interphase nuclei of S. producta (K), showing the
two sites of 45S rDNA and of S. plana (L), showing up to ten sites. The bar in L = 5 mm.

of nucleoli (10, 6 and 2, respectively) and the number of 45S fusion of nucleoli, as observed in angiosperms (Moscone
rDNA sites. The wide variation in the number of nucleoli et al., 1995).
per nucleus observed in all species examined, with a higher Curiously, S. willdenowii, although having up to
frequency of nuclei with few nucleoli, is probably due to the ten NORs, displayed only eight 45S rDNA sites and six
276 Marcon et al. — Cytogenetic Variability in Species of Selaginella
chromosomes with satellites. This apparent lack of correla-
tion between NORs and rDNA sites may be due to a pair of
rDNA sites very reduced in size, not detected by FISH. The
number of satellites observed, on the other hand, is often
lower than the number of rDNA sites (see, for example,
Guerra et al., 1996), and in some cases they are not observed
at all, as in S. convoluta, S. plana and S. producta.
The CMA/DA/DAPI staining revealed only CMA+/DAPI
bands in S. willdenowii and S. convoluta. The number of
CMA+ bands was positively correlated with the number of
rDNA sites and the maximum number of nucleoli observed
in these species. Similar results were found in the homo-
sporous genus Acrostichum (Marcon et al., 2003), suggest-
ing that these three features are also positively correlated in
pteridophyte chromosomes. No previous report of CMA
bands in pteridophytes was found in the literature.
Jermy et al. (1967) proposed that the primary basic num-
ber of Selaginella would be x = 10, characteristic of species
growing in dense tropical and subtropical forests (see also
Kuriachan, 1963). However, later karyological analyses
(Takamiya, 1993), as well as morphological, anatomical
and paleobotanical data (Mukhopadhyay, 1998) suggest
that the basic number of the genus would be x = 9, gen-
erating successive dysploids with n = 10, 11 and 12 on one
hand and n = 8 and 7 on the other. Takamiya (1993) pro-
posed that dysploidy might have occurred repeatedly in
different evolutionary lines within each subgenus. Indeed,
the remarkable variation in chromosome number and in size
and number of rDNA sites, seems to indicate that structural
variation is rather common in the genus and that its
karyological evolution may be far more complex, with
each basic number arising two or more times.
ACKNOWLEDGEMENTS
The authors are very greatful to Dr Iván Valdespino from
University of Panama, for species identification, and to
Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológico (CNPq) and Fundação de Amparo à Ciência
e Tecnologia do Estado de Pernambuco (FACEPE) for
financial support.
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