Among the anostomid fishes, the genus Leporellus is represented by only three species: L. nattereri, endemic of the Amazon River, L. retropinnis, endemic of the Piracicaba River, and L. vittatus, widely distributed in rivers from Peru, Colombia, Guianas, and different major hydrographic basins of Brazil. A cytogenetic study carried out on specimens of Leporellus vittatus from three major Brazilian hydrographic basins evidenced a karyotype of 54 metacentric and submetacentric chromosomes. C-banding analysis revealed the presence of large pericentromeric heterochromatic segments in all chromosomes and a telomeric block coincident with the NOR sites. Ag, CMA3 or MM staining, and FISH with ribosomal probes located the 45S ribosomal genes on the terminal region of the long arm of the 12th chromosome pair of all populations. Nevertheless, in the specimens from the Paraná and São Francisco Basins the 5S rDNA clusters were interstitially located by FISH on the long arm of the 2nd chromosome pair, while in the specimens from the Tocantins-Araguaia Basin these sites were observed on the long arm of the 9th chromosome pair and on the short arm of the 17th chromosome pair. These data suggest that the species currently named Leporellus vittatus may comprise a complex of cryptic species.
karyotype; C-bands; 45S rDNA; 5S rDNA
Chromosome mapping of 5S rRNA genes differentiates Brazilian populations of Leporellus vittatus (Anostomidae, Characiformes)
Cecilia Teixeira de AguilarI; Pedro Manoel Galetti JuniorII
IDepartamento de Genética, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
IIDepartamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brazil
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Among the anostomid fishes, the genus Leporellus is represented by only three species: L. nattereri, endemic of the Amazon River, L. retropinnis, endemic of the Piracicaba River, and L. vittatus, widely distributed in rivers from Peru, Colombia, Guianas, and different major hydrographic basins of Brazil. A cytogenetic study carried out on specimens of Leporellus vittatus from three major Brazilian hydrographic basins evidenced a karyotype of 54 metacentric and submetacentric chromosomes. Cbanding analysis revealed the presence of large pericentromeric heterochromatic segments in all chromosomes and a telomeric block coincident with the NOR sites. Ag, CMA3 or MM staining, and FISH with ribosomal probes located the 45S ribosomal genes on the terminal region of the long arm of the 12th chromosome pair of all populations. Nevertheless, in the specimens from the Paraná and São Francisco Basins the 5S rDNA clusters were interstitially located by FISH on the long arm of the 2nd chromosome pair, while in the specimens from the TocantinsAraguaia Basin these sites were observed on the long arm of the 9th chromosome pair and on the short arm of the 17th chromosome pair. These data suggest that the species currently named Leporellus vittatus may comprise a complex of cryptic species.
Key words: karyotype, Cbands, 45S rDNA, 5S rDNA.
The Anostomidae family comprises 12 genera of typically Neotropical fishes, occurring from Central America to South America (Garavello and Britski, 2003). According to the current taxonomic classification, based on morphological characters, the genus Leporellus is the smallest one of this family, represented by only three species: L. nattereri, endemic of the Amazon River (Northern Brazil), L. retropinnis, endemic of the Piracicaba River (Southeastern Brazil), and L. vittatus, widely distributed in rivers from Peru, Colombia, Guianas, and three major Brazilian hydrographic basins: Paraná, São Francisco and TocantinsAraguaia (Fowler, 1950; Garavello and Britski, 2003).
Nevertheless, there is little agreement as to the limits of the genus. The wide geographical distribution of L. vittatus has raised questions about the cospecificity of local populations of this species. As the major drainage basins of Brazil began to develop during the Tertiary (Buerlen, 1970) and several teleost fish species of the families Cichlidae, Characidae, and Curimatidae have already been described as endemic of distinct hydrographic basins (Kullander, 1983; Menezes, 1988; Vari, 1988), it is possible that, due to the limited gene flow, some populations have genetically diverged, although the conservative morphology prevents the detection of such differentiation. Therefore, the species known as Leporellus vittatus may be actually representing a complex of species.
Classical population models of chromosome evolution have postulated that small and/or restricted populations may show a higher karyotypic diversity than migratory and/or large populations, which seem to retain more conservative karyotypes, at least at the macrostructure level (Lande, 1979).
Previous cytogenetic studies have determined the diploid number, the constitutive heterochromatin distribution pattern (Cbands), and the location of the nucleolus organizer regions (NORs) by silverstaining on specimens of just one population of L. vittatus from the Paraná Basin (Galetti et al., 1991).
In view of these considerations, the aim of the present study was to investigate the possible geographical variation of Leporellus vittatus through cytogenetic comparisons. Samples collected in three major Brazilian hydrographic basins were cytogenetically analyzed and Giemsa karyotypes, Cbanding patterns, and ribosomal DNA sites (rDNA) studied by silver staining, basespecific fluorochromes (chromomycin A3 or mithramycin, and 4',6diamidino2phenylindole) and fluorescence in situ hybridization were investigated in order to find potential chromosome markers.
Material and Methods
Samples of Leporellus vittatus representing three distinct populations of major Brazilian hydrographic basins were collected. Seventeen specimens from the MogiGuaçu River, Pirassununga, São Paulo State (21° 9' S and 47° 4' W), Paraná Basin, Southeastern Brazil; four from the São Francisco River, Três Marias, Minas Gerais State (18° 2' S and 45° 2' W), São Francisco Basin, Southeastern Brazil; and three from the Araguaia River, Barra do Garças, Mato Grosso State (15° 9' S and 52° 3' W), AraguaiaTocantins Basin, Central Brazil were analyzed (Figure 1).
Chromosome staining techniques
Mitotic chromosomes were obtained from kidney cellular suspensions through the airdrying technique (Bertollo et al., 1978) or, alternatively, by short term solid tissue culture (Fenocchio et al., 1991). Giemsa karyotypes were established for the three populations. The constitutive heterochromatin distribution pattern was investigated by barium hydroxide treatment (Sumner, 1972). The nucleolus organizer regions (AgNORs) were observed by colloidal silverstaining (Howell and Black, 1980). Fluorochrome staining with the GCspecific chromomycin A3 (CMA3) or mithramycin (MM), and the ATspecific 4',6diamidino2phenylindole (DAPI) was carried as described by Schmid (1980) and Schweizer (1978), respectively.
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) was performed basically according to Pinkel et al. (1986), using a cocktail of 18S and 28S cloned fragments of the rDNA of Xenopus laevis (Cortadas and Pavon, 1982), and a 5S rDNA probe of the fish Leporinus elongatus, obtained by PCR (polymerase chain reaction) as described in Martins and Galetti (1999). The probes were labeled with biotin16dUTP by nick translation. The metaphase chromosome slides were incubated with RNase (40 µg/mL) for 1.5 h at 37 °C in a moist chamber. The chromosomal DNA was denatured for 5 min at 70 °C in a solution of 70% formamide in 2 x SSC. After that, 40 µL of hybridization mixture (1 µg of denatured probe, 50% formamide, and 10% dextran sulphate in 2 x SSC) were applied to the slides under a glass coverslip. The hybridization was performed overnight at 37 °C in a moist chamber. The slides were then washed three times at 37 °C, once in a solution of 50% formamide in 2 x SSC, and twice in 2 x SSC, for 15 min each. The probes were detected by avidinFITC conjugate. The signal was enhanced by biotinylated antiavidin and avidinFITC. Afterwards, the chromosomes were counterstained with 70 µL of propidium iodide (100 µg/mL) and the slides were mounted with 25 µL of the antifading Vectashield® Mounting Medium.
A total of 24 specimens were cytogenetically analyzed. Specimens from all populations had a modal diploid number of 54, composed of metacentric and submetacentric chromosomes (FN = 108) (Table 1 and Figure 2a).
The different applied staining techniques produced a pattern common to specimens of both sexes from all the populations. Cbanding analysis revealed the presence of large pericentromeric heterochromatic segments in all chromosomes and a block at the terminal region of a middlesized chromosome pair (Figure 2b).
Silver nitrate and chromomycin A3 or mithramycin staining revealed the presence of two NORbearing chromosomes in this species (Table 2). The NOR sites were located near the telomere of the long arm of the mediumsized chromosome pair 12 and are apparently coincident with the conspicuous secondary constrictions, which appeared positive Cbanded (Figure 3a and 3b). DAPI staining produced negative bands in these regions, while all the remaining chromosomes appear almost uniformly stained (Figure 3c).
The number of NORs and their unique location on chromosome pair 12 were further confirmed by FISH with biotinylated 18S and 28S rDNA (45S rDNA) probes (Figure 3d).
The 5S ribosomal genes were detected by FISH on chromosome pairs distinct from the ones bearing the 45S rDNA clusters. In the specimens from the Paraná and São Francisco Basins the 5S rDNA clusters were interstitially located on the long arm of the 2nd chromosome pair (Figure 4a and b ). The specimens from the TocantinsAraguaia Basin showed two chromosome pairs bearing these sites. In this population the 5S rDNA sites were observed on the long arm of the 9th chromosome pair and on the short arm of the 17th chromosome pair (Figure 4c).
According to Buerlen (1970), the major drainage basins of Brazil began to develop during the Tertiary. Although the precise time of formation of each hydrographic system cannot be determined, several teleost fish species of the families Cichlidae, Characidae, and Curimatidae have been described as endemic of distinct hydrographic basins, reinforcing the supposed vicariant events (Kullander, 1983; Menezes, 1988; Vari, 1988).
The cytogenetic markers studied here included different banding techniques, fluorochrome staining, and in situ hybridization with ribosomal probes, allowing a careful investigation of the constitutive heterochromatin and, particularly, of the ribosomal sites, which are chromosomal regions often described as variable in fishes.
Although the three studied populations of Leporellus vittatus share the same karyotypic structure already described by Galetti et al. (1991) for the population from the Paraná Basin and no differences could be detected in the heterochromatin distribution pattern and in the number and location of the 45S rDNA sites, interpopulation differences were evidenced concerning the number and location of the 5S rDNA clusters.
The association between heterochromatin and NOR sites observed in all the three studied populations seems to be a common feature in fishes and has also been described for many other fish species (Galetti et al., 1991; Rossi et al., 1996; Aguilar and Galetti, 1997; Martins and Galetti, 1997). This NORassociated heterochromatin, positive stained with CMA3, is thus GCrich as originally reported by Amemiya and Gold (1986). DAPI staining, an ATspecific fluorochrome, confirmed the GCrich nature of this heterochromatin, since negative bands coincident to the NOR sites were observed.
The lack of large chromosomal differences among the populations is not surprising since the karyotypic macrostructure of L. vittatus seems to be conserved among the Anostomidae (Galetti et al., 1981; Martins and Galetti, 1997, 1998) and other related Characiformes families, such as Curimatidae (Venere and Galetti, 1989; Feldberg et al., 1992), Parodontidae (Jesus and MoreiraFilho, 2000), and Prochilodontidae (Pauls and Bertollo, 1990), suggesting that the karyotype with 2n = 54, FN = 108, is ancient among the Characiformes.
Nevertheless, subtle changes in the chromosomal microstructure involving distinct rearrangements in the ribosomal regions seem to have occurred during the evolutionary diversification of the Anostomidae and may be strictly related to species differentiation. Intrapopulational chromosomal polymorphisms involving the number of 45S rDNA sites have already been reported in Leporinus friderici and Leporinus trifasciatus (Galetti et al., 1991; Galetti et al., 1995a,b), despite the conserved chromosome structure (2n = 54, FN = 108). In the present study we identified differences in the number and location of the 5S rDNA clusters among distinct populations of Leporellus vittatus from major Brazilian hydrographic basins.
In Neotropical characiform fishes, the 5S rDNA clusters are generally distributed in an interstitial position in two autosomal chromosome pairs and are usually not syntenic to the 45S rDNA sites, suggesting that this could be a common condition for the 5S rRNA gene organization in the genome of these fishes (Martins and Galetti, 1999, 2000, 2001; Born and Bertollo, 2000).
However, variations concerning the number and location of 5S rDNA sites have already been described for many other fish species. Multiple sites have been described for some salmonid species that show up to eight sites located on distinct autosomal chromosome pairs (Fujiwara et al., 1998), and for Astyanax scabripinnis, a characid fish, that presents eight 5S rDNA sites located on four distinct chromosome pairs (Ferro et al., 2000). The 5S rDNA clusters have also been located on the X or Y sex chromosomes of some salmonid species (Moran et al., 1996; Iturra et al., 2001; Stein et al., 2001), and on the Ychromosome of the males of Chionodraco hamatus, an Antarctic fish (Mazzei et al., 2004). Frequently, differences in the number and position of 5S rDNA sites have been reported as good chromosome markers to discriminate closely related fish species, such as in some families of the orders Mugiliformes and Perciformes (Gornung et al., 2001; Molina and Galetti, 2002; Rossi et al., 2005). Accordingly, the interpopulation differences observed in the present study concerning the number and location of the 5S rDNA clusters between Paraná/São Francisco and TocantinsAraguaia populations of L. vittatus suggest that the species currently named Leporellus vittatus may comprise a complex of cryptic species.
The recent discovery that, during the construction of the Furnas hydroelectric power dam in the upper Paraná River Basin in the early 1960s, the Piumhi River drainage outflow was diverted into the headwaters of the São Francisco River Basin has raised questioning about the current São Francisco watershed ichthyofauna structure (MoreiraFilho and Buckup, 2005). As this transposition event allowed the entire fish fauna of the Piumhi River and associated swamps, lakes, and tributaries to intermingle with the fish fauna of the São Francisco Basin, it may have contributed to the chromosomal stability described by many authors for Paraná and São Francisco Neotropical fish populations, as also described in the present study. Therefore, further studies comparing the different populations of L. vittatus and other anostomid species through molecular approaches, such as DNA sequencing of mitochondrial genes, may probably give a more precise answer to the present question Is Leporellus vittatus a complex of cryptic species?
This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Universitária José Bonifácio (FUJB).
Received: August 30, 2006; Accepted:July 10, 2007.
Associate Editor: Lurdes Foresti de AlmeidaToledo
License information: This is an openaccess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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03 June 2008
Date of issue
30 Aug 2006
10 July 2007