Karyotypic conservatism in five species of Prochilodus (Characiformes, Prochilodontidae) disclosed by cytogenetic markers

The family Prochilodontidae is considered a group with well conserved chromosomes characterized by their number, morphology and banding patterns. Thence, our study aimed at accomplishing a cytogenetic analysis with conventional methods (Giemsa staining, silver staining of the nucleolus organizer regions-AgNOR, and C-banding) and fluorescence in situ hybridization (FISH) with 18S and 5S ribosomal DNA probes in five species of the Prochilodus genus (Prochilodus argenteus, Prochilodus brevis, Prochilodus costatus, Prochilodus lineatus and Prochilodus nigricans) collected from different Brazilian hydrographic basins. The results revealed conservatism in chromosome number, morphology, AgNORs 18S and 5S rDNAs location and constitutive heterochromatin distribution patterns. The minor differences observed in this work, such as an Ag-NOR on a P. argenteus chromosome and a distinct C-banding pattern in P. lineatus, are not sufficient to question the conservatism described for this group. Future work using repetitive DNA sequences as probes for FISH will be interesting to further test the cytogenetic conservatism in Prochilodus.


Introduction
Fishes of the family Prochilodontidae are significant components of the fauna of Neotropical rivers and are considered one of the most important elements of commercial and subsistence freshwater fisheries in South American environments, except in Chile, where they are not found (Lowe-McConnell, 1975;Goulding, 1981;Vari, 1983;Flecker, 1996).
Therefore, the objective of the current study was to conduct a comparative analysis with conventional and molecular cytogenetic markers in five species of the genus Prochilodus collected in different Brazilian hydrographic basins to look for chromosome differences that may have accumulated in these populations over the years allowing their cytogenetic differentiation.

Materials and Methods
We analyzed 20 samples of P. lineatus from the Mogi-Guaçu river, Pirassununga, (São Paulo); 17 individuals of P. nigricans from the Tocantins Araguaia basin (Tocantins); 15 individuals of P. costatus acquired from the Aquicultura Tropical pisciculture, Propiá (Sergipe); six samples of P. argenteus from the São Francisco basin; and five individuals of P. brevis, acquired from the Departamento de Nacional de Obras Contra a Seca (DNOCS) dam, in Natal (Rio Grande do Norte).
Active NORs were identified after silver nitrate staining (Howell and Black, 1980) and the constitutive heterochromatin was detected after C-banding (Sumner, 1972).
The 5S probe was labeled with biotin-dUTP and the 18S probe was labeled with digoxigenin-dUTP (Roche) by PCR, according to the manufacturer's instructions.

Results
All specimens of Prochilodus (P. argenteus, P. brevis, P. costatus, P. lineatus and P. nigricans) collected in the different Brazilian hydrographic basins presented a karyotype with 2n = 54, FN = 108 and metacentric and submetacentric chromosomes (Figure 1a-e). All specimens of P. lineatus had supernumerary chromosomes (Figure 2a), whereas one P. nigricans specimen had a single B chromosome that showed intraindividual variation, with 23 cells out of 30 exhibiting the extra chromosome (Figure 2b).
After Ag-NOR staining, only one homolog of a submetacentric pair presented a NOR in P. brevis, P. costatus, P. lineatus and P. nigricans (Figure 1b-e, highlighted). In P. argenteus, the NOR was observed only on one homologue of the second largest submetacentric pair. This Ag-NORs pattern was found in approximately 40 metaphases of all P. argenteus specimens.
FISH with the 5S and 18S rDNA probes was performed to confirm if there was a karyotypic difference in the 18S gene location in P. argenteus. Synteny between these two genes was observed in all samples tested, including P. argenteus, in which the 18S rDNA labeled both homologues of the second submetacentric pair, which was not observed after Ag-NOR (Figure 3a, b, c, d and e). Furthermore, the location of these genes was identical in all the species, i.e., the 5S gene was located near the terminal region of the long arm of the submetacentric chromosome pair and the 18S gene, in a pericentromeric position, syntenic with the 5S gene. Clusters of these ribosomal sequences were not detected in any of the species studied, nor in the B chromosomes present in the genome of P. lineatus and P. nigricans.
C-banding was performed to identify the distribution of constitutive heterochromatin in all the species. The results allowed us to differentiate among the specimens of P. lineatus and those of the other four Prochilodus species. Conspicuous heterochromatic blocks present only in the centromeric regions of the standard A chromosome set were observed in P. lineatus (Figure 4d). In P. argenteus, P. brevis, P. costatus and P. nigricans, besides the presence of heterochromatic regions in the centromere, a large heterochromatic block on the long arm of a submetacentric chromosome pair was also observed (Fig. 4b-e). The supernumerary chromosomes of P. lineatus and P. nigricans were totally heterochromatic (Figure 4d, e). 348 Voltolin et al.
The presence of supernumerary microchromosomes in some species of Prochilodus enabled us to study aspects concerning their origin, evolution, structure and maintenance. First described in P. lineatus by Pauls and Bertollo (1983), up to two B microchromosomes were also identified in P. brevis (=P. cearensis) by these same authors (Pauls and Bertollo (1990). Venere et al. (1999) described the occurrence of one or two B chromosomes in P. nigricans and Oliveira et al. (2003) identified up to three supernumerary chromosomes in some individuals of P. mariae from the Orinoco river basin in Venezuela.
In P. argenteus, a single AgNOR was observed on the long arm of one homologue of a submetacentric chromosome (inbox in Figure 1a). This result is inconsistent with the literature data for this species, in which the single AgNOR was observed on the second largest submetacentric pair (Hatanaka and Galetti Jr, 2004).
Silver nitrate does not directly bind to rDNA, but to the proteins associated with the nucleolar structure, restricting the identification to the NORs that had been active in the preceding interphase (Miller et al., 1976). This is the most reasonable hypothesis to explain the single AgNOR in P. argenteus.
Neither the 5S nor the 18S ribosomal genes have been found in the B microchromosomes of P. lineatus (Jesus and Moreira-Filho, 2003) and of P. nigricans.
C-banding has proven very useful in cytogenetic studies of fish, permitting the identification of constitutive heterochromatin regions. Differences in the amount or distribution of heterochromatin identified by C-banding are considered important for some fish groups and the distribution of C-bands may characterize genera, species and populations (Montovani et al., 2000).
Several C-banding studies have been performed in Prochilodontidae, especially in Prochilodus lineatus. Maistro et al. (2000) reported centromeric and subtelomeric constitutive heterochromatin in the A complement of curimbatás collected in the Mogi-Guaçu river, Pirassununga, São Paulo. However, telomeric C-bands were absent in the chromosome preparations analyzed herein. but the heterochromatic nature of the supernumerary chromosomes was corroborated (Jesus and Moreira-Filho, 2003;Artoni et al., 2006;Voltolin et al., 2009).
Specimens of Prochilodus are characterized by the presence of conspicuous centromeric heterochromatic blocks in the A complement and in all supernumerary chromosomes (Jesus and Moreira-Filho, 2003;Artoni et al., 2006;Voltolin et al., 2009).
In this study, we observed different patterns of heterochromatin distribution in the genomes of some species of Prochilodus. In P. lineatus, constitutive heterochromatin was observed only in the pericentromeric regions of all chromosomes of the standard (A) complement ( Figure  4d) and all B chromosomes were heterochromatic. In P. argenteus, P. brevis, P. costatus and P. nigricans, in addition to the centromeric region of all A chromosomes, we also observed a large heterochromatic block on a submetacentric chromosome pair (Figure 4a, b, c, e). In addition to the centromeric C-bands in specimens of P. lineatus from the Mogi Guaçu river, Jesus and Moreira-Filho (2003) also evidenced heterochromatic blocks close to the telo- 350 Voltolin et al.  meric region of a submetacentric pair. Despite these small differences in the constitutive heterochromatin distribution in Prochilodus, we can still consider them a cytogenetically conserved group. Oliveira et al. (2003) confirmed the conservative nature of the chromosome number and morphology in Prochilodontidae and reinforced the idea that small structural chromosome rearrangements may be the main cause of karyotypic diversification in this group. In that study, the authors observed that in Prochilodus mariae from the Orinoco river basin, Venezuela, in addition to the occurrence of constitutive heterochromatin in all centromeres of the autosomes, a heterochromatic block was present in a submetacentric pair, as observed herein in P. argenteus, P. brevis, P. costatus and P. nigricans. Oliveira et al. (2003) also identified differing C-banding patterns between Semaprochilodus kneri and Semaprochilodus laticeps. In S. kneri, constitutive heterochromatin was present in the centromeric regions of all A chromosomes and a conspicuous heterochromatic block occurred on the long arm of pair 24. In S. laticeps, heterochromatin was only found in the pericentromeric regions, as we also observed in P. lineatus.
The presence of heterochromatin only in centromeric regions of the standard A chromosome set, as described herein in P. lineatus, differs from the data published by Maistro et al. (2000) and Jesus and Moreira-Filho (2003) for P. lineatus from the Mogi Guaçu River, the same location of our collections. These authors described the presence of heterochromatin in the centromeric region of all autosomes and in the telomeric regions of some pairs of the A complement.
Nevertheless, the four species analyzed (P. argenteus, P. brevis, P. nigricans and P. costatus) exhibited the same heterochromatin distribution pattern, in full agreement with literature data (Pauls and Bertollo, 1993;Venere et al., 1999;Hatanaka and Galetti Jr, 2004). Bertollo (1983, 1990) stated that the family Prochilodontidae, especially the Prochilodus genus, presented a conserved karyotype, resulting from a conservative chromosome evolution. The data obtained herein are in agreement with this proposed conservatism. The small cytogenetic variations found herein, such as the constitutive heterochromatin distribution and the position of the NOR among the studied Prochilodus, are not sufficient to contradict the strong conservatism proposed for the species of this genus by various authors of numerous cytogenetic studies.