INTRODUCTION

The Siluriformes show a great diversity, how-ever the total number of the Neotropical speciesis an open question, but at least two thousandspecies could be identified (LUNDBERG et al.2000).

This order is composed for more than thirtyfamilies with approximately 412 genera and2400 species (NELSON 1994). The Pimelodidaefamily is an endemic group of the Neotropicalregion and presents the greatest number ofspecies, being Pimelodella one of the more spe-cious genera, with more than 60 species (BUR-GUESS 1989). In the Tibagi river basin (Paranástate, Brazil), the Siluriformes, with 22 speciesbelonging to 7 families, represents around 23%of the total abundance, and 8,6% correspond tothe fishes of the Pimelodidae family (BENNE-MAN et al. 1995). At now in this basin have been

found two different forms of Pimelodella, P. aff.avanhandavae and P. aff. meeki.

Due to the difficult to identify correctly thespecies of Pimelodella, some taxonomic mis-takes could be occurred, attributing cytogenet-ics characteristics to ambiguous taxonomic enti-ties.

In the Pimelodella genus has been foundthree diploid numbers: 2n= 46, 52 and 58, beingthe first number found in the greatest number ofspecies and populations cytogenetically studied,as in the case of Pimelodella sp. from Mogi-Guaçu and Pardo Rivers (DIAS and FORESTI

1993; TOLEDO and FERRARI 1976), Pimelodellaavanhandavae from Araquá and Capivara Rivers(VISSOTTO et al. 1999) and in Pimelodella sp.from Paraná River (VASCONCELOS and MARTINS-SANTOS 2000). The 2n=52 was observed untilthis moment in Pimelodella sp. of Paraná River(VASCONCELOS and MARTINS-SANTOS 2000) andthe 2n=58 in P. kronei and P. transitoria, both ofIporanga (São Paulo) (ALMEIDA-TOLEDO et al.1992).

CARYOLOGIA Vol. 56, no. 4: 421-425, 2003

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 Tiba-gi River (Paraná state, Brazil) were analyzed. The diploid number of 52 chro-mosomes was observed and the karyotype was composed by 30M and 22SM withfundamental number (FN) of 104. Results of analyses of the nucleolus organiz-er regions (NORs), obtained by AgNO3 and CMA3, staining and FISH with 18SrDNA probe showed fluorescence in a terminal position on the short arm of apair of subtelocentric chromosomes. The C-banding regions were evidencedweakly in telomeric and centromeric regions and some chromosomes present con-spicuous marks, also staining positively the NORs.

Key words: Pimelodella, C-banding, NOR, CMA3, 18S rDNA.

* Corresponding author: e-mail: anadias@uel.br

Karyotypic data on Pimelodidae give somevaluable information but they are scarce to makegeneralizations concerning about their phylo-genetic and evolutive relationships.

The objective of the present study was tocharacterize cytogenetically Pimelodella aff.avanhandavae from Tibagi River (Paraná state,Brazil), comparing the results with other avail-able data and also would help to clarify some ofthe systematic relationships within the genus.

MATERIALS AND METHODS

Were studied seventeen specimens of Pimelodellaaff. avanhandavae (nine females and eight males) col-lected from the Tibagi River/PR/Brazil. Mitotic chro-mosome preparations were obtained according to

BERTOLLO et al. (1978). NOR silver staining was per-formed using the method of HOWELL and BLACK

(1980). C-banding were obtained using the methoddescribed by SUMNER (1972) and chromomycin(CMA3) staining as described by (SCHWEIZER 1976 inVERMA and BABU 1995).

Chromosome morphology was determined on thebasis of arm ratios as proposed by LEVAN et al. (1964).Metacentric (M), submetacentric (SM) and subtelo-centric (ST) chromosomes were considered asbiarmed ones.

The 18S rDNA segment containing 1700pb of thefish Oreochromis niloticus was used for fluorescence insitu hybridization (FISH) and labeled with biotin-14-dATP by nick translation (Gibco cat Nº 18247-015),according to the manufacturer’s instructions. Thehybridization technique, post-hybridization washesand visualization were carried out as reported bySWARÇA et al. (2001).

422 SWARÇA, VIDOTTO and DIAS

Fig. 1 – Giemsa-stained karyotype of Pimelodella aff. avanhandavae. Scale bar 10 µm.

RESULTS

The diploid number for the analyzed indi-viduals of Pimelodella aff. avanhandavae was 52chromosomes with 30 M and 22 SM with a fun-damental number (FN) of 104 in both, malesand females. The first metacentric pair of thecomplement is the largest one, being around50% bigger than the second (Fig. 1).

The karyotype of P. aff. avanhandavae wascharacterized by one pair of NOR-bearing sub-metacentric chromosomes, the nucleolar or-ganizers were located at telomeric positionsand showed an evident size heteromorphism(Fig. 2a), these regions were also CMA3 positive(Fig. 2b).

The FISH technique was employed to localize18S rDNA in the chromosomes of P. aff. avan-handavae. In all analyzed metaphases the FITCsignals appeared on the short arms of a submeta-centric pair (Fig. 2c), which were stained with sil-ver salts and CMA3. FISH and fluorochrome havenot evidenced the size heteromorphism betweenthe NOR bearing homologues.

The heterochromatin was weakly visualizedwith C-banding in telomeric and/or centromericregions of few chromosomes and was observed ametacentric chromosome pair with strong hete-rochromatic blocks on both telomeres. TheNOR-bearing pair also shows heterochromaticmarks coincident with the nucleolar organizers(Fig. 2d).

CYTOGENETIC CHARACTERIZATION OF PIMELODELLA AFF. AVANHANDAVAE 423

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.

DISCUSSION

The diploid number 2n=52 observed in P. affavanhandavae from the Tibagi River, has beenfound in Pimelodella sp. of Paraná River ana-lyzed by VASCONCELOS and MARTINS-SANTOS

(2000). The karyotypes of these two species arevery close, showing a first pair constituted by twolarge metacentric chromosomes, uncommon fea-ture in Pimelodidae, leading to suppose that theycould be the same species. However, some dif-ferences in the number of biarmed chromosomeshave been observed, Pimelodella. aff. avanhan-davae has 30M and 22SM (FN= 104) andPimelodella sp. 22M, 22SM, 8ST (FN=104).Therefore, discordances in the karyotypic for-mulae could be attributed to differences in chro-mosome condensation leading to confusion inclassification of chromosome types, specificallybetween submetacentric and subtelocentric ones.

In the family Pimelodidae the presence of sim-ple NORs, with only one pair of NOR bearingchromosomes, is the most frequent situation. InP. aff avanhandavae, the NOR was observed interminal position on the short arm of one pair ofsubmetacentrics (pair 20), showing a size hetero-morphism between homologous chromosomes.The same situation was observed in Pimelodellasp. from Paraná River, but according the authorsthe NORs are located at the 18th chromosomepair (VASCONCELOS and MARTINS-SANTOS 2000).

In P. aff. avanhandavae rDNA 18S signalsshow correspondence with CMA3 positive marksand Ag-NOR sites. These results has beenobserved in others Pimelodidae species: Pini-rampus pirinampu (SWARÇA et al. 1999), Zungarozungaro (SWARÇA et al. 2001) and Rhamdia quelen(FENOCCHIO et al. 2003).

With these techniques, CMA3 and FISH, wasnot possible to observe the NOR size hetero-morphism evidenced by silver salts. Probably theheteromorphism evidenced by AgNO3 staininghave a functional origin and is not related to agreater number of rDNA gene copies in one ofthe homologues. This result was also observed byVIÑAS et al. (1996) in some fish species.

The C-band distribution observed in P. aff.avanhandavae is a characteristic of other pimelo-dids. The NORs are heterochromatic, and weakcentromeric and telomeric bands were observedin several chromosomes. The presence of themetacentric pair with conspicuous bi-telomericC bands (Fig. 2d) seems to be a cytogenetic mark-

er of this specie, being that the same pattern wasobserved in Pimelodella sp. of the Paraná River.

This metacentric pair with conspicuous bi-telomeric C bands also represent a marker chro-mosomes sharing by other species of the familyand could be observed as part of the A comple-ment 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 NORposition of P. aff. avanhandavae and Pimelodellasp., suggest that these can be the same speciesand clearly indicates the need of a carefully taxo-nomic identification.

The present paper is the first that report acomplete karyotype description of a Pimelodellaspecies, including results of treatments with flu-orochromes and FISH with rDNA18S. Theseresults evidence that cytogenetics could help toclarify the taxonomic relationships.

Acknowledgements – The authors are gratefulto CAPES for their financial support. We are alsothankful to Dr. Oscar A. Shibata for the identifica-tion of the specie and Dr. Alberto Sérgio Fenocchiofor his critical reading of this paper.

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Received January 9, 2003; accepted May 19, 2003

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