Vol. 54, no. 3: 209-215, 2001
CARYOLOGIA
Chromosome R-banding pattern and conservation of a
marker chromosome in four species, genus Astyanax
(Characidae, Tetragonopterinae)
M.F.Z. DANIEL-SILVA and LURDES F. DE ALMEIDA TOLEDO*
Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil
Abstract - The R-banding pattern obtained by 5-Bromodeoxiuridine incorporation in the chromosomes was analyzed in four species from genus Astyanax
(Pisces, Characidae), A. altiparanae, A. fasciatus, A. schubarti and A. scabripinnis
using specimens sampled from populations of the upper Paraná river system,
in Brazil. Chromosome constitution and C-band pattern are also described for
the four species. The occurrence of chromosome homoeologies of several chromosome pairs, including a chromosome marker that is found in almost every
species belonging to family Characidae was observed. These chromosome similarities may indicate a close evolutionary relationship within this group of species.
Key words: Astyanax, 5-BrdU, R-bands.
INTRODUCTION
Genus Astyanax (Tetragonopterinae, Characidae) is composed of a group of Neotropical
freshwater fish species from which about one
hundred species and subspecies inhabit the
Neotropical Region rivers (GARUTTI and BRITSKI
1997). Seven species from this genus, Astyanax
mexicanus, A. altiparanae, A. fasciatus, A. schubarti, A. scabripinnis, A. eigenmanniorum and
A. taeniatus, have already been analyzed cytogenetically (OLIVEIRA et al. 1988) using conventional staining and, in some cases, banding techniques. Populations of Astyanax species sampled
from Brazilian river basins have been thoroughly
studied by many authors, so that cytogenetic data on specimens from 70 different geographic
populations are available (DANIEL-SILVA 1996 for
a review). These data disclosed the occurrence of
both inter- and intraspecific chromosome variability, with diploid numbers ranging from 2n=36
to 2n=50 chromosomes (OLIVEIRA et al. 1988).
Astyanax schubarti presents 2n=36 chromosomes
(MORELLI et al. 1983), A. fasciatus has 2n=46 and
* Corresponding author: fax: ++ 55 11 3818 7553; e-mail:
[email protected]
48 (JIM and TOLEDO 1975; MORELLI et al. 1983;
PAGANELLI 1990), A. scabripinnis has 2n=46, 48
and 50 chromosomes (MORELLI et al. 1983; MOREIRA-FILHO and BERTOLLO 1991; MAISTRO et al.
1992; ROCON-STANGE and ALMEIDA-TOLEDO
1993; SOUZA et al. 1995; MIZOGUCHI et al. 1998;
PORTO-FORESTI et al. 1997); A. altiparanae,
A. eigenmanniorum, and A. taeniatus had a fixed
number of 2n=50 chromosomes in all the populations analyzed (OLIVEIRA et al. 1988). Multiple
nucleolus organizer regions (NORs) and a variable pattern of constitutive heterochromatin
blocks, usually species-characteristic, were found
in these Astyanax species (DANIEL-SILVA 1996).
A striking feature of the chromosome complement of all these species is the presence of a distinct chromosome marker, either metacentric or
submetacentric, usually much larger than the other elements of the karyotype. This chromosome
marker, described by SCHEEL (1973) is present in
almost every Characidae species already analyzed
and may constitute a conserved structure in this
family.
The analysis of chromosome homoeologies
in fish has been hindered by the difficulty of getting good G- or R-bands in fixed chromosomes.
The R-bands induced by “in vivo” incorporation
210
DANIEL-SILVA
of 5-bromodeoxyuridine (5-BrdU) in chromosomes have been proven to be a good alternative
for fish chromosome analysis (ALMEIDA-TOLEDO
et al. 1988). In the present study we analyzed specimens of four species from genus Astyanax, A. altiparanae, A. fasciatus, A. schubarti and A. scabripinnis using C-bands and R-bands induced by an
“in vivo” treatment with 5-Bromodeoxiuridine (5BrdU), in order to compare the banded karyotypes
of these species and contribute to the identification of possible chromosome homoeologies in the
group, with special emphasis on the large biarmed
marker chromosome shared by these species.
and ALMEIDA-TOLEDO
were analyzed: Astyanax altiparanae from Paranapanema river, Salto Grande, SP; A. fasciatus from
Mogi-Guaçu river, Pirassununga, SP; A. schubarti
from Mogi-Guaçu river, Pirassununga, SP; A. scabripinnis, from a tributary of the Paranapanema river,
Botucatu, SP. After chromosome preparation, the
specimens were fixed properly, given identification
numbers and conserved in our laboratory collection.
Specimens of four species from genus Astyanax
sampled from populations of the Paraná river basin
Mitotic chromosome preparations were obtained
according to the following protocol: fish were injected with a 0.02% colchicine solution at a concentration of 0.5ml/100g body weight. After dissection, the
kidney cells were suspended in 0.075M KCl and incubated at 37°C for 30 min. The hypotonized cell
suspension was then centrifuged and the pellet was
resuspended in fixative (methanol:acetic acid 3: 1 solution) and centrifuged twice. The final pellet was
suspended in fresh fixative and dropped onto
warmed slides. The slides were analyzed after Giem-
Fig. 1 – Astyanax altiparanae - a) karyotype after Giemsa
staining; b) C-banding.
Fig. 2 – Astyanax fasciatus - a) karyotype after Giemsa staining; b) C-banding.
MATERIALS AND METHODS
R-BANDING PATTERNS IN ASTYANAX
sa staining and C-banding, obtained by routine cytogenetic techniques. For R-banding, fish were injected, according the protocol of ALMEIDA-TOLEDO
et al. (1988), with a solution of 5-Bromodeoxiuridine
at a final concentration of 25 µg/ml in a proportion
of 1 ml/100g of body weight, 5 to 7 hours before sacrifice. The slides were analyzed after FPG staining
(ISCN 1978).
RESULTS
Chromosomes were classified according to
their centromeric positions and were identified
as metacentrics (M), submetacentrics (SM), subtelocentrics (ST) and acrocentrics (A).
Astyanax altiparanae (Figs. 1a, b) - The
diploid number was 2n=50 chromosomes
(10M;22SM; 6ST;12A). Distinct heterochromatin blocks were detected after C-banding in
the proximal region of the long arm of chromosome pairs 2, 7, 8, 9, 10, 11, 12, 13, 15, 18, 19,
20, 23 and 24.
Fig. 3 – Astyanax schubarti - a) karyotype after Giemsa staining; b) C-banding.
211
Astyanax fasciatus (Figs. 2a, b) - The diploid
number was 2n=46 chromosomes (12M;20SM;
10ST;4A). After C-banding, six heterochromatin
marker blocks were detected in the distal region
of the long arm of chromosomes 8, 10, 11, 18, 19
and 22.
Astyanax schubarti (Figs. 3a, b) - The diploid
number was 2n=36 chromosomes (12M;16SM;
4ST;4A). A few darkly stained marker heterochromatic blocks were observed in the pericentromeric region of chromosomes 2, 3, 8 and
9. All the other chromosomes presented lightly
stained pericentromeric heterochromatin.
Astyanax scabripinnis (Figs. 4a, b) - The
diploid number was 2n=50 chromosomes
(8M;18SM; 10ST;14A). Heterochromatin blocks
Fig. 4 – Astyanax scabripinnis - a) karyotype after Giemsa
staining; b) C-banding.
212
were observed in the distal portion of the long
arms of chromosomes 14, 15, 17, 19, 20, 21, 22,
23, 24 and 25.
5-BrdU treated metaphases were observed in
two patterns: late replication pattern and full banded pattern. The late replication pattern usually
showed mainly regions that were lightly stained
by Giemsa, corresponding to C-positive regions.
Exceptions were the short arms of chromosomes 2 and 14 of A. altiparanae (Fig. 5a), chromosomes 5, 8 and 10 of A. fasciatus (Fig. 5b), chromosomes 5, 11 and 16 of A. schubarti (Fig. 5c) and
chromosomes 2, 4, 13, 16 and 17 of A. scabripinnis
(Fig. 5d) which were entirely late replicating and
C-band negative.
DANIEL-SILVA
and ALMEIDA-TOLEDO
replicating pattern obtained for A. scabripinnis permitted the identification of all the homologues in the
chromosome complement (Figs. 5 a-d and Figs. 6
a-c) and allowed the identification of the Characid
marker chromosome in these species and a comparison of their banding patterns. Fig. 7 shows a
comparison of the banding pattern of these three
species. The marker chromosome of A. scabripinnis
is also shown. The R-band analysis in cells with different levels of BrdU incorporation allowed the establishment of the replication sequence of the marker chromosome bands in A. altiparanae (Fig. 8).
DISCUSSION
Chromosome interspecific variability in Astyanax
The full R-band pattern plus the late replicating
pattern obtained for three of the species, A. altiparanae, A. fasciatus and A. schubarti and the late
The species that form genus Astyanax are not
considered to constitute a natural group from the
Fig. 5 – Karyotypes after incorporation with 5-BrdU; late replicating pattern. a) A. altiparanae; b) A. fasciatus; c) A. schubarti;
d) A. scabripinnis.
R-BANDING PATTERNS IN ASTYANAX
213
Fig. 7 – R-bands of the Characidae marker chromosomes in
A. altiparanae (chromosome 1), A. fasciatus (chromosome 1),
A. schubarti (chromosome 2) and A. scabripinnis (chromosome 1).
Fig. 8 – Replication sequence of chromosome 1 in A. altiparanae.
Fig. 6 – Karyotypes after incorporation with 5-BrdU; full band
pattern. a) A. altiparanae; b) A. fasciatus; c) A. schubarti.
phylogenetic point of view (BRITSKI 1992). Chromosome data, available for six species of this
group, most of them collected in the Paraná, Sào
Francisco and East basins, indicate that this is a
variable group in relation to chromosome number and formula, presenting both inter- and intraspecific chromosome variability. Nevertheless,
species presenting a conserved chromosome
number are also found in this group. This is the
case of A. altiparanae. Samples analyzed from 15
populations of A. altiparanae from three river
basins presented a fixed number of 2n=50 chromosomes (DANIEL-SILVA 1996), even though the
chromosome formula was sometimes quite distinct for different populations of this same nominal species. A. eigenmanniorum and A. taeniatus
also have 2n=50 chromosomes (OLIVEIRA et al.
1988) but only a few samples of these two species
have been analyzed. On the other hand, A. fasciatus and A. scabripinnis presented chromosome
variability among populations, involving both
chromosome number and formula. A. fasciatus
presented 2n=46 in populations from the Paraná
basin, and 2n=48 chromosomes both in populations from the East basins (MORELLI et al. 1983)
and from the São Francisco basin (OLIVEIRA et al.
1988). A. scabripinnis presented two different
diploid numbers, 2n=50 and 2n=48 chromosomes in specimens from the Paraná basin, 2n=46
and 2n=50 chromosomes in populations from
214
São Francisco basin, and 2n=50 chromosomes in
populations from the East basin (DANIEL-SILVA
1996); only one population of A. schubarti was
analyzed from the Paraná basin and presented
2n=36 chromosomes (MORELLI et al. 1983). Similarity in chromosome number may sometimes be
misleading considering Astyanax karyotypes,
since, in many cases, chromosome morphology
varied both inter- and intraspecifically.
The C-bands of A. altiparanae, A. fasciatus,
A. schubarti and A. scabripinnis analyzed in the
present study, show a very variable pattern, since
A. altiparanae presents interstitial blocks,
A. schubarti has a few faint pericentromeric
blocks and both A. fasciatus and A. scabripinnis
have telomeric heterochromatin blocks in the distal region of the long arms of large acrocentric
chromosomes that are not correspondent in the
two species. The C-banding pattern associated
with the characteristics of chromosome number
and formula result in species-specific karyotypes
for these four species.
The high chromosome variability found in
A. scabripinnis has been attributed to the peculiar geographic localization of this species, restricted to the headwaters of small tributaries of
larger rivers. In fact, this profile of high variability of chromosome number and formula is usually found in fish species characterized by small
populations with low capacity of dispersion or
even, as may be the case of Astyanax, medium
sized populations combined with a low capacity
of dispersion.
R-banding pattern
The R-banding patterns obtained for A. altiparanae, A. fasciatus, A. schubarti and A. scabripinnis, showing both late-replicating regions and
entirely banded chromosomes, allowed the pairing of alI the homologues of the chromosome
complement of each species and evidenced chromosome homoeologies among species. In spite of
the intense variability of chromosome number
and morphology, C-banding pattern and NOR
number and location (see DANIEL-SILVA 1996)
that characterizes this group of species, some
chromosome markers detected by R-bands are
very well conserved indicating a close relationship
involving these four species.
DANIEL-SILVA
and ALMEIDA-TOLEDO
The short BrdU treatment that usually reveals
late replicating heterochromatic C-band positive
regions in fish species, revealed in the present fish
species C-band negative regions that were entirely
late replicating. The fact that these regions were
found in morphologically similar chromosomes
in alI the four species analyzed constitutes evidence of a close karyotypic relationship.
This feature was also observed in relation to a
small metacentric that corresponds to chromosome 2 in A. altiparanae, chromosome 5 in A. fasciatus, chromosome 5 in A. schubarti and chromosome 2 in A. scabripinnis. These chromosomes
are comparable in size and morphology and, after the short 5-BrdU treatment, by the late replicating characteristic of their short arm. This latereplicating region is not C-band positive. It is also possible to observe a lightly stained interstitial
band in the long arm of the four corresponding
chromosomes (Figs. 5 a-d), giving further indication of their similarity.
The longer BrdU treatment revealed a high
resolution R-band pattern for A. altiparanae,
A. fasciatus and A. schubarti evidencing a clear
banding pattern for each chromosome, in particular the larger ones, such as the chromosome
marker of family Characidae. The banding pattern of this chromosome marker was also apparent in the late replicating metaphases and this allowed the comparison of its banding pattern for
the four species. The marker chromosome was
found to correspond to chromosome 1 of A. altiparanae, A. fasciatus and A. scabripinnis and to
chromosome 2 of A. schubarti (Fig. 7). Fig. 8
shows the replication pattern of chromosome 1
in A. altiparanae, where it is possible to notice the
early replication character of the main chromosome bands of this marker chromosome. Other
chromosome homoeologies may be found among
these four species and a study involving specifically the chromosome homeologies of all the
chromosome pairs of A. fasciatus and A. schubarti is currently in preparation.
The finding of a conserved pattern in these
marker chromosomes may help in discussions
about the evolutionary relationship among
Characidae species. On the other hand the occurrence of homeologous chromosomes with
characteristic C-negative late replication bands in
these four species points to a close karyotypic re-
R-BANDING PATTERNS IN ASTYANAX
lationship. Based on these characteristics, it is possible to suggest that this group of species deserve
a better analysis in terms of morphological characteristics in order to verify if they could possibly
constitute a separate group in genus Astyanax.
Acknowledgments – This research was supported by CNPq and FAPESP (1999/10441-5). The
authors are grateful to Dr. Ann J. Stocker for the
critical reading of the manuscript.
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Received February 13, 2001; accepted March 12, 2001