Pl. Syst. Evol.

257: 119–127 (2006)

DOI 10.1007/s00606-005-0375-8

Karyotype characterization of five Brazilian species of Echinodorus

(Alismataceae) with chromosomal banding and 45S rDNA FISH
J. Y. Costa1,3, E. R. Forni-Martins1, and A. L. L. Vanzela2
1
Departamento de Bot^anica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP),
Campinas, SP Brazil
2
Departamento de Biologia Geral, Centro de Ci^encias Biológicas, Universidade Estadual de Londrina
(UEL), Londrina, PR Brazil
3
Student from Programa de Pós Graduaç~ao em Biologia Vegetal, UNICAMP, Brazil

Received March 17, 2005; accepted August 21, 2005

Published online: January 17, 2006

Springer-Verlag 2006

Abstract. The karyotypes of five Brazilian species
of Echinodorus; E. bolivianus, E. grandiflorus,
E. longipetalus, E. macrophyllus and E. tenellus
(Alismataceae); were studied using C-banding,
CMA3/DAPI banding and fluorescence in situ
hybridization with a 45S rDNA probe. There were
few differences in the G-C rich regions of the five
species, but marked differences were seen in the
number and position of C-bands, A-T rich regions
and 45S rDNA sites. Overall, these characteristics
were species-specific, with the exception of E. boliv-
ianus and E. tenellus, which were highly similar in
all of the karyotypic characteristics studied.
Key words: Aquatic plants, chromosomal banding,
cytotaxonomy, heterochromatin, heteromorphism,
in situ hybridization.
The family Alismataceae (Judd et al. 1999)
consists of aquatic and semi-aquatic herbs that
grow in muddy or water-logged substrates and
have erect or floating leaves. Members of the
family can be recognized by the presence of
laticifers, basal placentation and fruits that are
mostly achenes (Judd et al. 1999). According
to Cronquist (1988), this family contains 11
genera, with Echinodorus and Sagittaria being
the largest and most representative. Based on a
combined phylogenetic analysis using mainly
the rbcL sequence, Judd et al. (1999) extended
the Alismataceae to include the genera previ-
ously grouped in the family Limnocharitaceae.
The unification of these two families had
already been suggested by Dahlgren and
Clifford (1981) and is supported by a karyo-
type analysis of several species (Forni-Martins
and Calligaris 2002, Costa and Forni-Martins
2003). Currently, the Alismataceae contains 16
genera and about 100 species.
Echinodorus, with 45 species, is still the
most representative of this group and contains
several Neotropical species. Seventeen species
of Echinodorus occur in Brazil (Haynes and
Holm-Nielsen 1994), with six of these in the
State of São Paulo (E. tenellus (Mart.) Buche-
nau, E. grandiflorus (Cham. & Schltdl.) Mic-
heli, E. longipetalus Micheli, E. macrophyllus
(Kunth) Micheli, E. aschersonianus Graebn.
and E. paniculatus Micheli) (M.C.E. do Ama-
ral, pers. comm.). Most of these species had the
chromosome number of 2n=22 and a strong
120 J. Y. Costa et al.: Karyotype characterization of Brazilian Echinodorus species
bimodal karyotype (Costa and Forni-Martins
2003), which had a low symmetry index
because of the predominance of acrocentric
chromosomes. Costa and Forni-Martins (2003)
also found a high karyotypic similarity between
the pairs E. aschersonianus – E. pubescens and
E. grandiflorus – E. macrophyllus.
C-banding and CMA3/DAPI banding data
have been reported only for E. tenellus (Costa
and Forni-Martins 2004), and include two
cytotypes, one diploid (2n=22) and one trip-
loid (2n=33). The diploid cytotype had a
CMA+/DAPI+ region, also banded by C-
banding, on a heteromorphic acrocentric pair
of chromosomes. The triploid cytotype which
had two C/CMA+/DAPI+ bands, indicated
autopolyploid origin for the triploid cytotype.
Heteromorphic C-bands have also been
observed in different species of Alismataceae
(Kenton 1981) and some of the C-bands were
also associated with the satellite (SAT) region
on the short arm of the smallest acrocentric
chromosomes.
Fluorescence in situ hybridization (FISH)
has been successfully used to locate sequences
of nucleic acids in the nucleus and chromo-
somes (Leitch et al. 1994). Sequences of the
rDNA region, especially the 5S and 45S, have
been extensively used to analyze chromosomal
structures and phylogenetic relationships
among species (Tagashira and Kondo 2001,
Vanzela et al. 2002, Fregonezi et al. 2004).
Vanzela et al. (2002) observed that species of
Helianthus (Asteraceae) differed not only in
their level of ploidy, but also in number and
position of the heterochromatic regions, iden-
tified by chromosomal banding, and in the
number and intensity of the 45S rDNA
hybridization signals. The latter finding sug-
gested that rearrangements involving small
heterochromatic and rDNA segments played
a major role in the karyotypic evolution of the
group.
Aquatic plants are widely recognized as a
diverse biological group with many convergent
morphological features and extensive pheno-
typic plasticity (Wooten 1986, Les and Philbrick
1993, Les and Haynes 1995). Furthermore,
asexual reproduction via clonal growth and
a high vagility of vegetative propagules repre-
sent a significant evolutionary catalyst that
allows the perpetuation of hybrid offspring
and anomalous cytotypic variants (Les and
Philbrick 1993). Together, these characters
represent many problems for traditional taxon-
omists, and make it difficult to discern pheno-
typic plasticity in hybrid individuals or
cytotypic variants. Natural hybridization has
been speculated to occur between E. cordifolius
and E. berteroi, but there have been no detailed
studies on interspecific crosses in Echinodorus
(Les and Philbrick 1993). Cytotypic variants
have been described for E. tenellus (Costa
and Forni-Martins 2004). In this work, we
used chromosomal banding (C, CMA3 and
DAPI) and FISH with a 45S rDNA probe to
analyze the karyotypic differences and evolu-
tionary dynamics of five Brazilian species of
Echinodorus.
Material and methods
The five species and populations used in this work
are listed in Table 1. Voucher specimens were
deposited in the Herbarium of the Universidade
Estadual de Campinas (UEC), Brazil. For cytoge-
netic analysis, root tips of the species were collected
in the field and pre-treated with a mixture (2:1, v/v)
of saturated paradichlorobenzene (PDB) solution
and 0.009% cycloheximide for 5 h, at 16–18C.
Roots were then fixed in Carnoy’s solution (etha-
nol:acetic acid, 3:1, v/v) for at least 24 h, and stored
in 70% ethanol at )4C. For banding techniques
and FISH, root tips were digested in an enzymatic
solution containing cellulase (2%), pectinase (20%)
and distilled water. Slides were prepared using a
standard squashing technique.
C-banding and fluorescent CMA3/DAPI band-
ing were done in E. bolivianus, E. grandiflorus, E.
longipetalus and E. macrophyllus. For C-banding,
we used the protocol of Schwarzacher et al. (1980),
and for CMA3/DAPI banding we used that of
Schweizer (1976), the latter preceded by some of the
steps used in C-banding (slides were treated with
45% acetic acid, 5% barium hydroxide and 2xSSC,
J. Y. Costa et al.: Karyotype characterization of Brazilian Echinodorus species 121

Table 1. Karyotype characteristics of Echinodorus species. (-) not analyzed, (*) species maintained in a
greenhouse, (#) see Costa and Forni-Martins (2004). Populations: I) Campinas, SP - S 2254¢20¢¢, W
4703¢39¢¢; II) Casa Branca, SP - 2146¢26¢¢, 4705¢11¢¢; III) Itapetininga, SP - 2335¢30¢¢, 4803¢11¢¢; IV)
Itirapina, SP - 2215¢10¢¢, 4749¢22¢¢; V) Pereira Barreto, SP - 2038¢18¢¢, 5106¢33¢¢; VI) Bonito, MS -
2120¢28¢¢, 5633¢34¢¢
Species Population Voucher 2n Number of bands FISH
C CMA3/DAPI
E. bolivianus (Rusby) VI J.Y.C. 1101 22 1 1 CMA+/DAPI+ –
Holm-Niels.
E. grandiflorus II, IV, V J.Y.C. 1073, 091, 0103 22 2 2 CMA+/DAPI0 2
(Cham. & Schltdl.) Micheli
E. longipetalus Micheli II, IV J.Y.C. 1071, 1072 22 38 4 CMA+/DAPI0 –
14 CMA0/DAPI+
E. macrophyllus I * 22 24 2 CMA+/DAPI0 4
(Kunth) Micheli
12 CMA0/DAPI+
E. tenellus III J.Y.C. 1075 22 1# 1 CMA+/DAPI+ # 3
(Mart.) Buchenau

all at 60C) before staining with the fluorochromes.
Photographs were taken with black and white film
(ISO 25 for C-banding and ISO 100 for CMA3/
DAPI). Color slide film (ISO 400) was also used for
fluorescent banding. We used MicroMeasure 3.3 to
measure chromosomes and identify position of
bands. With aid of previously presented idiograms
(Costa and Forni-Martins 2003) we draw an
idiogram showing the longitudinal characterization
of C/CMA/DAPI bands of E. grandiflorus, E.longi-
petalus and E. macrophyllus. Unfortunately it was
not possible to draw an idiogram for E. bolivianus,
due to poor quality of chromosome spreading.
Fluorescence in situ hybridization was done for
E. grandiflorus, E. macrophyllus and E. tenellus.
The pTa71 probe containing 45S rDNA isolated
from wheat (Gerlach and Bedbrook 1979) was used
for FISH. The probe was labeled with biotin-14-
dATP by nick translation. The slides were incu-
bated in RNase (100 lg/ml), post-fixed in 4% (w/v)
paraformaldehyde, dehydrated in a 70%–100%
graded ethanol series and air-dried. The probe
(20–100 ng) mixture was denatured at 70C for
10 min and immediately chilled on ice. Chromo-
some denaturation/hybridization was done at 90oC
for 10 min, 50C for 10 min and 38C for 5 min
using a thermal cycler (MJ Research), and kept at
37C overnight in a moist chamber. Post-hybrid-
ization washes were done at 80% stringency in
2xSSC, 0.1xSSC/20% formamide, 0.1xSSC, 2xSSC,
and 4xSSC/0.2% Tween 20, at 42C for 5 min
each. The biotin-labeled probe was detected with
an avidin FITC-conjugate, and the slides were
mounted in a solution composed of 50% Antifade
(Vector Labs), 50% glycerol in McIlvaine buffer,
pH 7.0, and 4% propidium iodide (2.5 lg/ml)1).
Photographs were taken with Kodak Proimage ISO
100 color film.
Results
The species studied differed in number, distri-
bution and composition of their heterochro-
matic regions (Table 1). There were also
differences in number of 45S rDNA sites
(Table 1), although in all species these sites
were visualized in the terminal region of the
short arms of acrocentric chromosomes.
The C-banding pattern revealed only one
heterochromatic region in E. bolivianus, and
was located on the short arm of one chromo-
some of a pair of acrocentric chromosomes
(Fig. 2a). A heteromorphic pair of chromo-
somes was also seen in this species. Echinodorus
grandiflorus had two heterochromatic regions
on the short arms of one pair of acrocentric
122 J. Y. Costa et al.: Karyotype characterization of Brazilian Echinodorus species

Fig. 1. Idiograms showing number and position of C/CMA3/DAPI bands in Echinodorus. a) E. grandiflorus, b)
E. longipetalus and c) E. macrophyllus

chromosomes (Figs. 1a, 2b). In E. longipetalus, E. macrophyllus, C-banding revealed 24 het-

38 heterochromatic regions were observed in
the terminal, intercalary and proximal regions.
Both pairs of metacentric chromosomes and
the largest pair of acrocentric chromosomes
had only proximal dots. One pair of acrocentric
chromosomes showed only terminal hetero-
chromatic regions, whereas six pairs of these
chromosomes had intercalary and proximal
C-bands, and one pair had terminal, proximal
and intercalary C-bands (Figs. 1b, 2c). In
erochromatic regions, with 2n=22 chromo-
somes, and a predominance of terminal bands.
Three acrocentric pairs showed intercalary
C-bands. In two of these acrocentric pairs
there was a terminal C-band on the short arms,
while in one of these pairs the satellite region
was also strongly banded. In the metacentric
pair and in six pairs of acrocentric chromo-
somes there were C-bands in the terminal
regions of the long arms (Figs. 1c, 2d).
J. Y. Costa et al.: Karyotype characterization of Brazilian Echinodorus species 123

Fig. 2. C/CMA3/DAPI banding in species of Echinodorus. a, b, c, d) C-banding; e, f, g, h) CMA3 banding; i, j,

k, l) DAPI banding; a, e, i) Echinodorus bolivianus; b, f, j) E. grandiflorus; c, g, k) E. longipetalus; d, h, l) E.
macrophyllus. Bar = 10 lm

The CMA3/DAPI banding in E. bolivianus
revealed only one heteromorphic CMA+/
DAPI+ region, located on the short arms of
one chromosome of an acrocentric pair (Fig. 2e
and 2i), in the same region identified by
C-banding. Only two CMA+/DAPI0 bands
were seen in E. grandiflorus (Figs. 1a, 2f and
2j), on the short arms of an acrocentric pair.
Echinodorus longipetalus had four CMA+/
DAPI0 regions (Figs. 1b, 2g) that were also
located on the short arms of two pairs of
acrocentric chromosomes, and 14 CMA0/
DAPI+ bands (Figs. 1b, 2k) that were always
found in intercalary regions on the long arms of
acrocentric chromosomes; the latter were prob-
ably associated with the intercalary C-bands.
Echinodorus macrophyllus had two CMA+/
DAPI0 regions (Figs. 1c, 2h) located on the
short arms of an acrocentric pair of chromo-
somes, and 12 CMA0/DAPI+ bands (Figs. 1c,
2l), ten of which were located at terminal
positions of the long arms of the metacentric
and acrocentric pairs of chromosomes; the
remaining two bands were at an intercalary
position on the long arms of an acrocentric pair
of chromosomes. The G-C and A-T rich
regions were probably associated with hetero-
chromatic regions.
FISH using the pTa71 probe of 45S rDNA
in E. grandiflorus revealed only two sites
124 J. Y. Costa et al.: Karyotype characterization of Brazilian Echinodorus species
Fig. 3. 45S rDNA in situ hybridization of Echino-
dorus. a) Echinodorus grandiflorus, b) E. macrophyllus,
c) E. tenellus. Bar = 10 lm
hybridized with the probe on the short arms of
one acrocentric pair of chromosomes (Fig. 3a).
In E. macrophyllus, there were four hybridiza-
tion sites for the 45S rDNA probe, all of which
were also located on the short arms of acro-
centric chromosomes (Fig. 3b). Echinodorus
tenellus showed three hybridization sites for
45S rDNA on the short arms of acrocentric
chromosomes, with heteromorphism for one of
these sites (Fig. 3c).
Discussion
There have been few studies of chromosomal
banding in Alismatales, and no investigation
using in situ hybridization. Previous studies
with Hydrocleys nymphoides (Kenton 1981)
and Echinodorus tenellus (Costa and Forni-
Martins 2004) showed that these species had a
reduced number of heterochromatic regions
and an easily observed heteromorphism. As
shown here, some species had a small number
of heterochromatic regions, but this was not a
consistent characteristic of the family. Two
species, Echinodorus longipetalus and E. mac-
rophyllus, had a large number of heterochro-
matic regions. Most of the heterochromatic
terminal bands that were present on the short
arms of acrocentric chromosomes were asso-
ciated with G-C rich regions. However,
E. longipetalus and E. macrophyllus had an
additional terminal heterochromatic region on
the short arm of an acrocentric pair of
chromosomes that was not associated with
G-C rich regions. Guerra (2000) has pointed
out that a combination of C-banding and
fluorescent banding is a reliable method for
identifying different types of heterochromatin,
but that C-bands sometimes react neutrally to
fluorochrome staining. The association of
heterochromatic regions with G-C rich het-
erochromatin, and even with secondary con-
strictions and nucleolus organizer regions
(NORs), has been observed in several plant
genera, including Scilla (Greilhuber et al.
1981), Capsicum (Moscone et al. 1996), Ses-
bania Scop. (Forni-Martins and Guerra 1999),
Clivia (Ran et al. 1999) and Pinus (Jacobs
et al. 2000).
Another pattern observed here was that the
intercalary and terminal heterochromatic
regions present on the long arms of chromo-
somes were usually associated with A-T rich
regions. Only E. macrophyllus showed some
J. Y. Costa et al.: Karyotype characterization of Brazilian Echinodorus species
intercalary and terminal heterochromatic
bands that reacted neutrally during fluorescent
banding. The pattern of heterochromatin asso-
ciated with A-T rich regions suggested that
E. longipetalus and E. macrophyllus could be
closely related, and that chromosomal inver-
sions could have played an important role in
the evolution of these species since proximal
and intercalary C-bands predominated in E.
longipetalus, whereas terminal C-bands pre-
dominated in E. macrophyllus. Furthermore,
these two species were the only ones that had
a large number of C-bands and A-T rich
regions.
As shown here and by Costa and Forni-
Martins (2004), there was a strong similarity
between the banding patterns of E. bolivianus
and E. tenellus. Both E. bolivianus and the
diploid cytotype of E. tenellus had one hetero-
chromatic region associated with a CMA+/
DAPI+ band in the terminal region of only
one pair of heteromorphic chromosomes. The
synonymization of these two species under E.
tenellus was suggested by Jérémie et al. (2001)
based on the examination of several herbarium
specimens and also of the type material during
the description of a new species of Echinodo-
rus. We suggest that these species are at least
closely related since they showed the greatest
similarity in banding patterns among all of the
species studied, and also showed the exclusive
presence of one CMA+/DAPI+ band.
In situ hybridization sites for the 45S
rDNA probe were always observed at terminal
positions on the short arms of acrocentric
chromosomes, and were associated with het-
erochromatic regions in E. grandiflorus and
E. macrophyllus. In E. grandiflorus, the 45S
rDNA sites were also associated with G-C rich
regions. An association between G-C rich
regions and 45S rDNA sites has been observed
in several plant genera, including some Aster-
aceae such as Helianthus (Vanzela et al. 2002),
Crepis, Galinsoga and Chaptalia (Fregonezi
et al. 2004). However, such an association was
not always observed in all of the species
studied here. In E. macrophyllus, two of the
four 45S rDNA sites were not detected by
125
CMA3 banding, even though these sites were
seen by C-banding. In E. tenellus, the in situ
hybridization done here and the banding data
presented by Costa and Forni-Martins (2004)
showed that two of the three 45S rDNA sites
were not detected by either C-banding or
CMA3 banding. The lack of an association
between G-C rich regions and 45S rDNA sites
has also been reported for some of the chro-
mosomal bands in species of Helianthus (Vanz-
ela et al. 2002), although there are usually
more G-C rich regions than 45S rDNA sites.
We suggest that in situ hybridization with the
45S rDNA probe should also be done in E.
bolivianus to allow comparison with the data
obtained for E. tenellus.
Although only a few species were studied
here, the combination of C-banding, fluores-
cent banding and in situ hybridization with the
45S rDNA probe proved to be an efficient
method for detecting differences and similari-
ties among these species. The two species (E.
grandiflorus and E. macrophyllus) that showed
the greatest karyotypic similarity using con-
ventional techniques (Costa and Forni-Mar-
tins 2003) actually had very different banding
and hybridization patterns. In contrast, the
similar banding patterns of E. bolivianus and
E. tenellus suggested a close relationship
between these two species and could represent
further evidence to corroborate the synonimi-
zation suggested by Jérémie et al. (2001). The
application of the techniques used here to a
greater number of Alismataceae species could
be useful in clarifying taxonomical problems in
this group and could help to identify chromo-
somal changes relevant to the evolution of
these species.
The authors thank Maria do Carmo Estanislau
do Amaral and Emerson Ricardo Pansarin for
identifying the species, Iara F. Bressan for helping
with the laboratory techniques and the Laboratório
de Citogenética e Genética Molecular de Plantas
(Universidade Estadual de Londrina), for provid-
ing access to laboratory facilities. J.Y. Costa was
supported by a fellowship from the Fundação de
Amparo à Pesquisa do Estado de São Paulo
(FAPESP, #00/00767–0).
126 J. Y. Costa et al.: Karyotype characterization of Brazilian Echinodorus species
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J. Y. Costa et al.: Karyotype characterization of Brazilian Echinodorus species 127

Addresses of the authors: J.Y. Costa, E.R.
Forni-Martins (e-mail: elianafm@unicamp.br),
Departamento de Botânica, Instituto de Biologia,
Universidade Estadual de Campinas (UNI-
CAMP), CP 6109, CEP 13083-970, Campinas,
SP, Brazil. A. L. L. Vanzela, Departamento de
Biologia Geral, Centro de Ciências Biológicas,
Universidade Estadual de Londrina (UEL), CEP
86051-990, Londrina, PR, Brazil.