Chromosome mapping of 5 S rRNA genes differentiates Brazilian populations of Leporellus vittatus ( Anostomidae , Characiformes )

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 12 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 2 chromosome pair, while in the specimens from the Tocantins-Araguaia Basin these sites were observed on the long arm of the 9 chromosome pair and on the short arm of the 17 chromosome pair. These data suggest that the species currently named Leporellus vittatus may comprise a complex of cryptic species.


Introduction
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 Tocantins-Araguaia (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 (C-bands), and the location of the nucleolus organizer regions (NORs) by silver-staining 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, C-banding patterns, and ribosomal DNA sites (rDNA) studied by silver staining, base-specific fluorochromes (chromomycin A 3 or mithramycin, and 4',6diamidino-2-phenylindole) and fluorescence in situ hybridization were investigated in order to find potential chromosome markers.

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 biotin-16-dUTP 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 avidin-FITC conjugate.The signal was enhanced by biotinylated anti-avidin and avidin-FITC.Afterwards, the chromosomes were counterstained with 70 μL of propidium iodide (100 μg/mL) and the slides were mounted with 25 μL of the anti-fading Vectashield ® Mounting Medium.

Results
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.C-banding analysis revealed the presence of large pericentromeric heterochromatic segments in all chromosomes and a block at the terminal region of a middle-sized chromosome pair (Figure 2b).
Silver nitrate and chromomycin A 3 or mithramycin staining revealed the presence of two NOR-bearing chro- mosomes 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 C-banded (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 190 Chromosomal differences in L. vittatus 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 2 nd chromosome pair (Figure 4a and b).The specimens from the Tocantins-Araguaia Basin showed two chromosome pairs bearing these sites.In this population the 5S rDNA sites were observed on the long arm of the 9 th chromosome pair and on the short arm of the 17 th chromosome pair (Figure 4c).

Discussion
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 NOR-associated heterochromatin, positive stained with CMA 3 , is thus GC-rich as originally reported by Amemiya and Gold (1986).DAPI staining, an ATspecific fluorochrome, confirmed the GC-rich nature of this heterochromatin, since negative bands coincident to the NOR sites were observed.
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 Y-chromosome 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 Tocantins-Araguaia 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 (Moreira-Filho 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?
Figure 2 -Metaphase plate of Leporellus vittatus after Giemsa staining (a) and sequential C-banding (b).Arrows indicate the NOR-bearing chromosomes.