CARYOLOGIA
Vol. 61, no. 2: 135-138, 2008
Karyotype similarities among two populations of Geophagus
brasiliensis (Perciformes, Cichlidae) from the Tibagi river
basin/PR/Brazil
Pires Bettin Larissa, Lucia Giuliano-Caetano and Ana Lúcia Dias*
Universidade Estadual de Londrina, Departamento de Biologia Geral, 86051-970, Londrina, Paraná, Brazil.
Abstract — A cytogenetic study of G. brasiliensis was conducted in two populations from the Tibagi river basin/
PR. The diploid number was found to be 48 chromosomes and the fundamental number of 52. An AgNOR was
observed on the short arm of a chromosome pair of the ST-A group, coinciding with the secondary constriction.
This was seen in both populations, where in one of them there was a size heteromorphism of this region. Coincident results were found with chromomycin (CMA3) staining and with fluorescence in situ hybridization using an
18S rDNA probe. Heterochromatin was distributed in the centromeric region in the majority of the chromosomes,
as with the secondary constriction in both populations.
Key words: AgNOR, C Banding, CMA3 staining, FISH, Geophagus.
INTRODUCTION
In the Perciformes, a diploid number (2n) of
48 chromosomes, with a predominance of subteloacrocentric ones, is a highly conservative characteristic in the group. However, the fundamental
number (FN), thus the karyotype formula, shows
a large variation, with the majority of the species
showing a FN equal to or greater than 48 (BRUM
and GALETTI 1997; LOUREIRO 1999; among others).
Cytogenetic studies in cichlids began with Post
in 1965, and they are the most studied in the order
Perciformes. However, among the 1292 species
of cichlids known, only 135 have been analyzed
karyotypically. In the majority of cases, studies
have been limited to the determination of diploid
number (2n), which was found to vary from 38 to
60 chromosomes (FELDBERG et al. 2003).
Among the species of cichlids in the southeast
of Brazil, Geophagus brasiliensis is the most common, known as “acará”, occurring in almost all of
the Brazilian coastal waters. The first cytogenetic
finding for the genus Geophagus was presented by
MICHELE and TAKAHASHI (1977) who described G.
brasiliensis as having a diploid number (2n) of 48
with 3 meta-submetacentric and 45 subtelo-ac-
* Corresponding author: phone: 00 55 (43) 33714417;
fax: 00 55 (43) 33714257; e-mail: [email protected].
rocentric chromosomes, and with a fundamental
number (FN) of 51. Since then, other populations
of G. brasiliensis from different locations have
been analyzed, all showing a diploid number of 48
chromosomes and a fundamental number between
50 and 56, and thereby demonstrating a variation
in karyotypic morphology (FELDBERG et al. 2003).
The aim of the present study was to characterize G. brasiliensis from the Tibagi river basin/PR,
using conventional Giemsa staining and different
chromosome banding techniques.
MATERIALS AND METHODS
Sixteen specimens of Geophagus brasiliensis
(8 males and 8 females) collected in Cambezinho and Três Bocas stream, from the Tibagi river
basin (Paraná state, Brazil) were cytogenetically
studied. Mitotic chromosome preparations were
obtained from kidney using the term curt culture
(FENOCCHIO et al. 1991). Chromosomes classified as metacentric (M) and submetacentric were
considered as biarmed. Subtelocentric (ST) and
acrocentric (A) were considered as uniarmed.
Nucleolar organizer regions (AgNOR) and Cbanding were performed using the methods of
HOWELL and BLACK (1980) and SUMNER (1972),
respectively. Chromomycin A3 (CMA3) staining
followed SCHWEIZER (1976). A rDNA 18S probe
136
pires, giuliano-caetano and dias
(1700 pb), obtained from the nuclear DNA of the
fish Oreochromis niloticus, was used for in situ hybridization, which was carried out as described by
SWARÇA et al. (2001).
RESULTS
All of the individuals of Geophagus brasiliensis, from the Cambezinho and Três Bocas stream
showed a diploid number (2n) of 48 chromosomes
consisting of 2 pairs of submetacentric (SM) and
12 pairs of subtelo-acrocentric (ST-A) chromosomes, and a fundamental number (FN) of 52,
where the secondary constriction was observed in
pair 6 in both populations (Figure 1A). The figure
1B-a and 1B-b show the secondary constriction in
G. brasiliensis from Três Bocas stream, with a size
heteromorphism, and a chromosome pair with
satellites observed in pair 17, respectively.
Through C banding, heterochromatin was visualized in the two populations in the centromeric
region of the majority of the chromosomes, being
usually observed a heterochromatin block in the
secondary constriction region (Figure 2A), where
a size heteromorphism was observed in the population from Três Bocas stream (Figure 2E).
In the two populations examined, an AgNOR
was localized on the short arm of chromosome
pair 6 of the ST-A group, coinciding with the
secondary constriction (Figure 2B), where a size
heteromorphism was seen between the homologous chromosomes only in specimens from Três
Bocas stream (Figure 2E).
Chromomycin A3 treatment showed results
similar to those found with AgNOR staining,
demonstrating fluorescent staining in the secondary constriction region of a ST-A chromosome
(Figure 2C), where a size heteromorphism was also observed only in the population from the Três
Bocas stream (Figure 2E).
In situ hybridization with the 18S rDNA probe
showed the presence of ribosomal cistrons on the
short arm of a ST-A chromosome pair, coincident
with AgNOR and CMA3 staining (Figure 2D),
and also revealing a heteromorphism in the population from the Três Bocas stream (Figure 2E).
DISCUSSION
The species Geophagus brasiliensis studied in
this work showed a diploid number of 48 chromosomes, confirming the high chromosome stability
in the family Cichlidae, since this diploid number
has been found in the majority of species examined (FELDBERG et al. 2003). There were no differences observed with respect to karyotype between the individuals from two locations studied
here, where their karyotype formula consisted of
2 submetacentric and 22 subtelo-acrocentric pairs
of chromosomes, with a fundamental number of
52. These findings differ from the results obtained
by LOUREIRO (1999) for the same species also collected from the Tibagi river basin/PR, although at
different locations, and from the Itajai-Açu river
basin/SC, where the karyotype formula was found
to be 3 submetacentric and 21 subtelo-acrocentric pairs of chromosomes, with a fundamental
number of 54. This small difference can be due
to the condensation of chromosomes resulting
in different measurements. MIZOGUCHI and MARTINS-SANTOS (2000) found the same karyotype as
that observed in the present study in individuals
of G. brasiliensis from the region of the Iguaçu
river/PR.
Fig. 1 — Karyotype pattern of Geophagus brasiliensis from the Cambezinho and Três Bocas stream (A) and on
(B) the chromosome pairs with the secondary constriction (a) and satellites (b) of G. brasiliensis from Três Bocas
stream.
karyotype similarities among populations of geophagus brasiliensis
137
Fig. 2 — Somatic metaphases of Geophagus brasiliensis: (A) C banding; (B) AgNOR; (C) CMA3; (D) FISH, all
belonging to the population from the Cambezinho stream. And the figure (E) corresponds to the chromosome pair
with heteromorphism for the population from the Três Bocas stream.
Other authors found for G. brasiliensis from
different locations the same diploid number of
48 chromosomes, but with different karyotype
formulas, showing a variation in FN of 50 to 56
(FELDBERG et al. 2003). Such findings demonstrate,
as suggested by FELDBERG and BERTOLLO (1985), a
conservative chromosomal evolution in this group
of fish, in relation to diploid number, and with the
occurrence of pericentric inversions that result in
these variations in fundamental number.
In some metaphases of G. brasiliensis, in the
two populations from the Tibagi river basin, it
was possible to detect the secondary constriction
on the short arm of chromosome pair 6 of the STA group, which upon silver nitrate impregnation
was shown to be a nucleolus organizing region
(NOR). MARTINS et al. (1995), BRUM et al. (1998)
and LOUREIRO (1999) studied different populations of G. brasiliensis and also observed that the
secondary constriction was AgNOR positive and
always located on ST-A type chromosomes.
The individuals from the Três Bocas stream
showed variations in the size of AgNORs, showing
a well-evident heteromorphism in the individuals
studied, where in some the NOR was much larger
than in the homologous chromosome. These variations in size of the NOR were also observed by
MARTINS et al. (1995) and BRUM et al. (1998) in G.
brasiliensis for different populations.
Treatment with the fluorochrome chromomycin A3 in G. brasiliensis detected the presence of
fluorescent staining on the short arm of a ST-A
chromosome pair, coincident with NOR staining,
which was also observed in other populations of
G. brasiliensis (LOUREIRO 1999) and other species
of cichlids, such as Crenicichla sp. and C. niederleinii (LOUREIRO et al. 2000), demonstrating that
NOR is rich in GC base pairs. In C. niederleinii
and C. iguassuensis from the Piquiri river and
Iguaçu river, LORSCHEIDER (2004) utilized the
fluorochrome mithramycin to detect regions rich
in GC. The heteromorphism pattern observed for
AgNOR in individuals from the Três Bocas stream
was also observed with CMA3 staining.
In situ hybridization with the 18S rDNA probe
showed evidence of a pair of chromosomes bearing ribosomal cistrons in this species, exhibiting
differences in size of the hybridization signals only
138
pires, giuliano-caetano and dias
in specimens from Três Bocas stream, as observed
for AgNOR and CMA3. Differences in the size of
rDNA sites have been attributed to variation in the
number of rRNA genes per cluster. Mechanisms
such as unequal crossing-over, transpositions or
other rearrangements including deletions and/or
duplications, involve segments of homologous
chromosomes, and are thus frequently attributed
to mechanisms related to structural modifications
of NORs (CASTRO et al. 1998).
C banding analysis showed that heterochromatin in both populations of G. brasiliensis is distributed in the centromeric region of the majority
of the chromosomes. This heterochromatin pattern is the same pattern as that reported by MARTINS et al. (1995), BRUM et al. (1998) and LOUREIRO
(1999), and follows the general pattern of the family Cichlidae. The secondary constriction was C
band positive in the two populations, and in the
specimens from the Três Bocas stream, there was
variation in the size of this heterochromatin region, thus indicating the presence of heterochromatin associated with ribosomal cistrons.
The present study contributes with more cytogenetic information on Geophagus brasiliensis,
confirming its extremely conserved nature, mainly
in relation to diploid number, despite the variations found in karyotype formula resulting in different fundamental numbers. The distribution
pattern of heterochromatin and of NORs also
remains very constant in this species suggesting,
therefore, a conservative karyotype evolution in
this group of cichlids.
Acknowledgments — The authors are grateful to
CNPq/PROSUL and Fundação Araucária for financial
support. We are also thankful to Dr. Albert Leyva for
his help in the preparation of the manuscript.
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Received December 27th 2006; accepted April 25th 2007