Cytogenetics of Imparfinis schubarti ( Siluriformes : Heptapteridae ) from the Piumhi drainage , a diverted river in Minas Gerais State , Brazil

Specimens of Imparfinis schubarti (Gomes, 1956) collected in the Piumhi river drainage, state of Minas Gerais, Brazil, were studied cytogenetically. The river was diverted from the Rio Grande Basin into the Sao Francisco basin in the early 1960s. All individuals presented 2n = 58 chromosomes, including 18 metacentric, 34 submetacentric and six subtelocentric chromosomes. A secondary constriction was observed in the interstitial region of the long arm of the largest chromosome pair, coinciding with the NOR. A single conspicuous heterochromatic block located in the largest pair of metacentric chromosomes was observed, adjacent to the secondary constriction. A detectable 18S rDNA probe hybridization region occurs in only one chromosome pair and is synthenic with the marking obtained with 5S rDNA probe. These results fit the cytogenetic pattern previously described for the genus Imparfinis Eigenmann & Norris, 1900.

In this paper we describe the karyotype of a population of I. schubarti from the Piumhi River basin.
The River Piumhi, a tributary of the River Rio Grande in state of Minas Gerais, Brazil, was diverted in the 1960s from the Rio Paraná basin into the São Francisco basin. During construction of the Furnas hydroelectric dam in the Rio Grande, a secondary dike was built in the lower course of the River Piumhi in order to raise the water volume of the Furnas Dam and to avoid the flooding of the town of Capitólio. This secondary damming required the deviation of the Piumhi from the Rio Grande drainage into the São Francisco basin (MOREIRA-FILHO & BUCKUP 2005) (Fig. 1). This study is part of a comprehensive investigation on the mixture of Paranean and San Franciscan fish faunas that resulted from the diversion of the River Piumhi.
The mitotic metaphases were obtained according to BERTOLLO et al. (1978) andFORESTI et al. (1993). Chromosome morphology was determined according to the arm size relation proposed by LEVAN et al. (1964). The fundamental number (FN) was established through the sum of the number of chromosome arms, counting two arms for metacentric (m), submetacentric (sm), and subtelocentric (st) chromosomes, and one arm for acrocentric (a) chromosomes. The constitutive heterochromatin was identified using the barium hydroxide method (SUMNER 1972), and the nucleolar organizing regions were detected through silver nitrate staining (HOWELL & BLACK 1980). Each preparation was stained in conventional Giemsa staining, and subsequently submitted to C-banding.
The location of the 18S and 5S rDNA sites in the chromosomes was performed using the Fluorescence in situ Hibridization (FISH) technique (PINKEL et al. 1986), with 77% stringency and probes obtained from Prochilodus argenteus Spix & Agassiz, 1829 (HATANAKA & GALETTI JR 2004) and Leporinus elongatus Valenciennes, 1850(MARTINS & GALETTI JR 1999, respectively. The probes were marked with 14-dATP-biotin by nick translation according to the manufacturer's instructions (Bionick Labelling System -Invitrogen). The chromosomes were counterstained with DAPI (0.2 mg/ml) and analyzed in an Olympus BX50 epifluorescence microscope. The software Image-Pro Plus (Media Cybernetics) was used for image capture.

RESULTS
The samples presented 2n = 58 chromosomes with 18m + 34sm + 6st (Fig. 2) and a fundamental number of 116. No gender-related chromosomal differences were observed. A conspicuous secondary constriction coinciding with the Ag-NOR was observed in the interstitial region of the long arm of the first submetacentric chromosome pair. Differences in the sites obtained through silver nitrate impregnation between the chromosomes of pair 1 were often observed (Fig. 3). The chromosomes have low quantities of constitutive heterochromatin, except for the first pair of submetacentric chromosomes, which has a large heterochromatic block adjacent to the secondary constriction (Fig. 3).
Fluorescence in situ hybridization with 18S rDNA probes produced markings that coincided with the AG-NORs located in the secondary constriction of the first pair of sub metacentric chromosomes. These markings were heteromorphic between homologous chromosomes (Fig. 4). The 5S rDNA FISH markings were synthenic with those for 18S rDNA (Fig. 5).

DISCUSSION
The 2n = 58 karyotype identified in I. schubarti is the most common diploid number among species of Imparfinis (Tab. I), as well as within the entire order Siluriformes (OLIVEIRA et al. 1988). Species of Imparfinis, as most Siluriformes, are also characterized by predominance of metacentric and submetacentric chromosomes and high fundamental number (FN) values. These features correspond to plesiomorphic conditions that are widely distributed throughout the order (OLIVEIRA & GOSZTONYI 2000). The FN = 116, found in I. schubarti, is the most common in the genus, being present also in I. piperatus (VISSOTTO et al. 2001), I. mirini (VISSOTTO et al. 1997) and Imparfinis sp. aff. I. schubarti (STOLF et al. 2004).
Simple nucleolar organizing regions located in the largest chromosome pair coinciding with interstitial secondary constrictions and 2n = 58 are characteristics present in I. schubarti, I. mirini, I. piperatus-cytotypes A and B, and Imparfinis sp. aff. I. schubarti (Tab. I). If 2n = 58 is a plesiomorphic condition as suggested by OLIVEIRA et al. (1988) andFENOCCHIO et al. (2003), a reduction in the number of chromosomes may be a synapomorphy grouping Imparfinis sp. cf. I. piperatus from the Juquiá River (VISSOTTO et al. 2001, FENOCCHIO et al. 2003, I borodini (MARGARIDO & MOREIRA-FILHO 2008) and I. hollandi (VISSOTTO et al. 1999). This hypothesis is corroborated by the repositioning of the nucleolar organizing region in these species from chromosome pair 1 to other pairs in these species. Additionally, the migration of the

5
nucleolar organizing region from an interstitial to a terminal position may represent a synapomorphy further grouping I. hollandi and I. borodini. According to FENOCCHIO et al. (2003), Cetopsorhamdia sp. collected in the River Canta Galo (city of Itirapina, SP, Brazil) has a simple NOR located in the largest pair of metacentric chromosomes and 2n = 58. Cetopsorhamdia iheringi Schubart & Gomes, 1959(VISSOTTO et al. 1999) also has 2n = 58 and simple interstitial Ag-NOR in the largest chromosome pair, although that pair is submetacentric. In this last study, a conspicuous heterochromatic block adjacent to the Ag-NOR was also observed. Within the Heptapteridae, both Imparfinis and Cetopsorhamdia belong to the "Nemuroglanis subclade" (FERRARIS 1988, LUNDBERG et al. 1991a, BOCKMANN 1994. The absence of these characteristics in heptapterids that are not members of the subclade, such as Rhamdia Bleeker, 1858 and Pimelodella Eigenmann & Eigenmann, 1888, suggests that they may be apomorphic characters shared at least by these two genera. The use of the 18S rDNA probe confirmed the data obtained with the silver nitrate staining and revealed heteromorphism in the single NOR-bearing chromosome pair in I. schubarti. STOLF et al. (2004) identified in Imparfinis sp. aff. I. schubarti a pattern identical to the one observed in this study, however with the absence of heteromorphism.
In superior eukaryotes, the 5S and 18S genes are frequently disposed in separate chromosome pairs (LONG & DAVID 1980, LUCCHINI et al.1993, DROUIN & MONIZ DE SÁ 1995. This is the most frequent condition in fishes (MARTINS & GALETTI JR 2001) and represents the ancestral state of the chromosomal organization (MARTÍNEZ et al. 1996). The synthenic location of the 5S and 18S rDNA observed in I. schubarti is therefore an apomorphic condition, and further investigation on the distribution of this trait among species of Imparfinis may provide useful phylogenetic data.
The natural distribution of I. schubarti comprises the upper Paraná River Basin (BOCKMANN & GUAZZELLI 2003, BOCKMANN 2007. However, the transposition of the River Piumhi in the early 1960s introduced several species from the upper Paraná into the São Francisco river basin (Fig. 1) (MOREIRA-FILHO & BUCKUP 2005). The presence of I. schubarti in the region of the transposition channel of the Piumhi River, now belonging to the São Francisco River Basin, emphasizes the importance of cytoge- netic and taxonomical studies involving native species of the São Francisco basin, such as Imparfinis minutes (Lütken, 1874). The present study represents a starting point in the evaluation of possible hybridization of such species with I. schubarti.