Vol. 56, no. 4: 421-425, 2003
CARYOLOGIA
Cytogenetic characterization of Pimelodella aff.
avanhandavae (Siluriformes, Pimelodidae)
from Tibagi River (Paraná State, Brazil)
ANA CLAÚDIA SWARÇA, ANA PAULA VIDOTTO and ANA LÚCIA DIAS*
Universidade Estadual de Londrina, Depto. de Biologia Geral, Londrina, PR, Brazil.
Abstract - Seventeen specimens of Pimelodella aff. avanhandavae from the Tibagi River (Paraná state, Brazil) were analyzed. The diploid number of 52 chromosomes was observed and the karyotype was composed by 30M and 22SM with
fundamental number (FN) of 104. Results of analyses of the nucleolus organizer regions (NORs), obtained by AgNO3 and CMA3, staining and FISH with 18S
rDNA probe showed fluorescence in a terminal position on the short arm of a
pair of subtelocentric chromosomes. The C-banding regions were evidenced
weakly in telomeric and centromeric regions and some chromosomes present conspicuous marks, also staining positively the NORs.
Key words: Pimelodella, C-banding, NOR, CMA3, 18S rDNA.
INTRODUCTION
The Siluriformes show a great diversity, however the total number of the Neotropical species
is an open question, but at least two thousand
species could be identified (LUNDBERG et al.
2000).
This order is composed for more than thirty
families with approximately 412 genera and
2400 species (NELSON 1994). The Pimelodidae
family is an endemic group of the Neotropical
region and presents the greatest number of
species, being Pimelodella one of the more specious genera, with more than 60 species (BURGUESS 1989). In the Tibagi river basin (Paraná
state, Brazil), the Siluriformes, with 22 species
belonging to 7 families, represents around 23%
of the total abundance, and 8,6% correspond to
the fishes of the Pimelodidae family (BENNEMAN et al. 1995). At now in this basin have been
* Corresponding author: e-mail: [email protected]
found two different forms of Pimelodella, P. aff.
avanhandavae and P. aff. meeki.
Due to the difficult to identify correctly the
species of Pimelodella, some taxonomic mistakes could be occurred, attributing cytogenetics characteristics to ambiguous taxonomic entities.
In the Pimelodella genus has been found
three diploid numbers: 2n= 46, 52 and 58, being
the first number found in the greatest number of
species and populations cytogenetically studied,
as in the case of Pimelodella sp. from MogiGuaçu and Pardo Rivers (DIAS and FORESTI
1993; TOLEDO and FERRARI 1976), Pimelodella
avanhandavae from Araquá and Capivara Rivers
(VISSOTTO et al. 1999) and in Pimelodella sp.
from Paraná River (VASCONCELOS and MARTINSSANTOS 2000). The 2n=52 was observed until
this moment in Pimelodella sp. of Paraná River
(VASCONCELOS and MARTINS-SANTOS 2000) and
the 2n=58 in P. kronei and P. transitoria, both of
Iporanga (São Paulo) (ALMEIDA-TOLEDO et al.
1992).
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SWARÇA, VIDOTTO
Karyotypic data on Pimelodidae give some
valuable information but they are scarce to make
generalizations concerning about their phylogenetic and evolutive relationships.
The objective of the present study was to
characterize cytogenetically Pimelodella aff.
avanhandavae from Tibagi River (Paraná state,
Brazil), comparing the results with other available data and also would help to clarify some of
the systematic relationships within the genus.
MATERIALS AND METHODS
Were studied seventeen specimens of Pimelodella
aff. avanhandavae (nine females and eight males) collected from the Tibagi River/PR/Brazil. Mitotic chromosome preparations were obtained according to
and DIAS
BERTOLLO et al. (1978). NOR silver staining was performed using the method of HOWELL and BLACK
(1980). C-banding were obtained using the method
described by S UMNER (1972) and chromomycin
(CMA3) staining as described by (SCHWEIZER 1976 in
VERMA and BABU 1995).
Chromosome morphology was determined on the
basis of arm ratios as proposed by LEVAN et al. (1964).
Metacentric (M), submetacentric (SM) and subtelocentric (ST) chromosomes were considered as
biarmed ones.
The 18S rDNA segment containing 1700pb of the
fish Oreochromis niloticus was used for fluorescence in
situ hybridization (FISH) and labeled with biotin-14dATP by nick translation (Gibco cat Nº 18247-015),
according to the manufacturer’s instructions. The
hybridization technique, post-hybridization washes
and visualization were carried out as reported by
SWARÇA et al. (2001).
Fig. 1 – Giemsa-stained karyotype of Pimelodella aff. avanhandavae. Scale bar 10 µm.
CYTOGENETIC CHARACTERIZATION OF PIMELODELLA AFF. AVANHANDAVAE
RESULTS
The diploid number for the analyzed individuals of Pimelodella aff. avanhandavae was 52
chromosomes with 30 M and 22 SM with a fundamental number (FN) of 104 in both, males
and females. The first metacentric pair of the
complement is the largest one, being around
50% bigger than the second (Fig. 1).
The karyotype of P. aff. avanhandavae was
characterized by one pair of NOR-bearing submetacentric chromosomes, the nucleolar organizers were located at telomeric positions
and showed an evident size heteromorphism
(Fig. 2a), these regions were also CMA3 positive
(Fig. 2b).
423
The FISH technique was employed to localize
18S rDNA in the chromosomes of P. aff. avanhandavae. In all analyzed metaphases the FITC
signals appeared on the short arms of a submetacentric pair (Fig. 2c), which were stained with silver salts and CMA3. FISH and fluorochrome have
not evidenced the size heteromorphism between
the NOR bearing homologues.
The heterochromatin was weakly visualized
with C-banding in telomeric and/or centromeric
regions of few chromosomes and was observed a
metacentric chromosome pair with strong heterochromatic blocks on both telomeres. The
NOR-bearing pair also shows heterochromatic
marks coincident with the nucleolar organizers
(Fig. 2d).
Fig. 2 – (a) AgNOR staining, (b) CMA3 staining, (c) Fluorescence in situ hybridization with biotin-labeled 18S rDNA probe, (d)
C-banded metaphase. The arrows show the nucleolar chromosomic pair and the arrows head shows the markers chromosomes.
424
SWARÇA, VIDOTTO
DISCUSSION
The diploid number 2n=52 observed in P. aff
avanhandavae from the Tibagi River, has been
found in Pimelodella sp. of Paraná River analyzed by VASCONCELOS and MARTINS-SANTOS
(2000). The karyotypes of these two species are
very close, showing a first pair constituted by two
large metacentric chromosomes, uncommon feature in Pimelodidae, leading to suppose that they
could be the same species. However, some differences in the number of biarmed chromosomes
have been observed, Pimelodella. aff. avanhandavae has 30M and 22SM (FN= 104) and
Pimelodella sp. 22M, 22SM, 8ST (FN=104).
Therefore, discordances in the karyotypic formulae could be attributed to differences in chromosome condensation leading to confusion in
classification of chromosome types, specifically
between submetacentric and subtelocentric ones.
In the family Pimelodidae the presence of simple NORs, with only one pair of NOR bearing
chromosomes, is the most frequent situation. In
P. aff avanhandavae, the NOR was observed in
terminal position on the short arm of one pair of
submetacentrics (pair 20), showing a size heteromorphism between homologous chromosomes.
The same situation was observed in Pimelodella
sp. from Paraná River, but according the authors
the NORs are located at the 18th chromosome
pair (VASCONCELOS and MARTINS-SANTOS 2000).
In P. aff. avanhandavae rDNA 18S signals
show correspondence with CMA3 positive marks
and Ag-NOR sites. These results has been
observed in others Pimelodidae species: Pinirampus pirinampu (SWARÇA et al. 1999), Zungaro
zungaro (SWARÇA et al. 2001) and Rhamdia quelen
(FENOCCHIO et al. 2003).
With these techniques, CMA3 and FISH, was
not possible to observe the NOR size heteromorphism evidenced by silver salts. Probably the
heteromorphism evidenced by AgNO3 staining
have a functional origin and is not related to a
greater number of rDNA gene copies in one of
the homologues. This result was also observed by
VIÑAS et al. (1996) in some fish species.
The C-band distribution observed in P. aff.
avanhandavae is a characteristic of other pimelodids. The NORs are heterochromatic, and weak
centromeric and telomeric bands were observed
in several chromosomes. The presence of the
metacentric pair with conspicuous bi-telomeric
C bands (Fig. 2d) seems to be a cytogenetic mark-
and DIAS
er of this specie, being that the same pattern was
observed in Pimelodella sp. of the Paraná River.
This metacentric pair with conspicuous bitelomeric C bands also represent a marker chromosomes sharing by other species of the family
and could be observed as part of the A complement and in others cases as B chromosomes
(HOCHBERG and ERDTMANN 1988; FENOCCHIO
and BERTOLLO 1990; FENOCCHIO et al. 2000;
ABUCARMA and MARTINS-SANTOS 2001).
The comparison of the karyotypes and NOR
position of P. aff. avanhandavae and Pimelodella
sp., suggest that these can be the same species
and clearly indicates the need of a carefully taxonomic identification.
The present paper is the first that report a
complete karyotype description of a Pimelodella
species, including results of treatments with fluorochromes and FISH with rDNA18S. These
results evidence that cytogenetics could help to
clarify the taxonomic relationships.
Acknowledgements – The authors are grateful
to CAPES for their financial support. We are also
thankful to Dr. Oscar A. Shibata for the identification of the specie and Dr. Alberto Sérgio Fenocchio
for his critical reading of this paper.
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Received January 9, 2003; accepted May 19, 2003