Karyotype differentiation and cytotaxonomic considerations in species of Serrasalmidae ( Characiformes ) from the Amazon basin

Six species of Serrasalmidae from the central Amazon, representatives of the genera Serrasalmus (S. elongatus, S. maculatus, S. cf. rhombeus, and S. rhombeus), Pygocentrus (P. nattereri), and Colossoma (C. macropomum), were analyzed regarding the distribution of the Ag-NORs, C-positive heterochromatin and 18S and 5S rRNA genes on the chromosomes. All specimens had 2n = 60 chromosomes, except S. cf. rhombeus, with 2n = 58, and C. macropomum with 2n = 54 chromosomes. The Ag-NORs were multiple and located on the short arms of subtelo-acrocentric chromosomes in all Serrasalmus species and in P. nattereri, but were found on metacentric chromosomes in C. macropomum. The 18S rDNA sites were usually coincident with Ag-NORs, although some species had a higher number and/or a distinct localization of these sites. C-positive heterochromatin was preferentially situated in centromeric regions, remarkably on metacentric pair number 7 in all Serrasalmus species and number 3 in P. nattereri, which beared a conspicuous proximal C-band on the long arms. The 5S rDNA sites were detected in a single chromosomal pair in all species. In Serrasalmus and P. nattereri, this pair was the number 7 and 3, respectively, thereby revealing its co-localization with the conspicuous heterochromatic band. However, in C. macropomum, only one homologue (probably belonging to pair number 12) exhibited 5S rDNA sites on the short arms, close to the centromere. The present data revealed reliable cytotaxonomic markers, enabling the evaluation of karyotype differentiation and interrelationships among Serrasalmidae, as well as the probable occurrence of a species complex in S. rhombeus.


Introduction
Chromosome banding techniques have been useful in evolutionary cytogenetics, as they allow the identification of potential chromosome markers related to karyotype interrelationships.The detection of nucleolar organizer regions (NORs) by silver nitrate staining (Ag-NORs) has been one of the most widely employed procedures in fish, especially from 1980s on, providing important information for cytotaxonomic and evolutionary studies (Galetti Jr. et al., 1984;Gold, 1984; Venere & Galetti Jr., 1989;Feldberg et al., 1992Feldberg et al., , 2003)).This tool is usually poorly informative for groups with single NORs on equivalent chromosomes.Moreover, not all the nucleolar organizer regions are necessarily active in species bearing multiple NOR systems and would therefore remain undetectable by silver nitrate staining (Miller et al., 1976).
Since the 1990s, chromosome studies have improved quickly due to the use of base-specific fluorochromes, such as chromomycin A 3 and mithramycin, and the development of fluorescent in situ hybridization (FISH).These procedures have been successfully applied to NORs and heterochromatin studies in fish metaphase chromosomes, as they are able to detect base richness and both active and inactive nucleolar regions.Fluorescent in situ hybridization (FISH) is the most useful method for mapping NOR sites in a species genome.
The aim of the present study was to determine the location of 5S rDNA and 18S rDNA sites in species of Serrasalmidae from the central Amazon and carry out a comparative analysis with Ag-NORs.These data, along with other peculiar karyotype characteristics, proved to be important tools for understanding the interrelationships within this fish group.

Results
A diploid number of 60 chromosomes was found in all species, except S. cf.rhombeus, which had 2n = 58 chromosomes, and C. macropomum, which had 2n = 54 chromosomes.The fundamental number ranged from 106 to 110 and no sex-related chromosomes were found (Table 1).
All species exhibited multiple Ag-NORs, located in the terminal position on the short arms of acrocentric and subtelocentric chromosomes, with the exception of C. macropomum, in which the Ag-NORs were found on metacentric chromosomes (Fig. 2 a, c, e, g, i, and k).In general, the 18S rDNA sites coincided with Ag-NOR sites.However, in some species, a higher number of 18S rDNA sites were detected (Fig. 2 b, d, f, h, j, and l).
Besides the general location of the C-bands in the centromeric regions (data not shown), a conspicuous heterochromatic block was also evident on the long arms of a metacentric pair close to the centromeres, corresponding to pair number 7 in the species of Serrasalmus and to pair number 3 in P. nattereri (Fig. 3 a, c, e, g, and i).
The 5S rDNA was located interstitially, close to the centromeres on a single chromosome pair.In Serrasalmus and Pygocentrus, this site was co-located with the aforementioned interstitial heterochromatic region on the long arms of pairs 7 and 3, respectively.In C. macropomum, 5S rDNA signals were  found on the short arms of a single chromosome -probably from pair number 12 (Fig. 3 b, d, f, h, j, and l).
The results are summarized in Table 1.Previous data on another three species of Serrasalmus [S.altispinnis, S. gouldingi, and S. serrulatus (Nakayama et al., 2008)] were also included in this table to provide a comparative analysis.

Discussion
Ag-NORs in Serrasalmidae have been analyzed since the 1980s and have invariably been visualized as multiple sites usually located in the terminal region on the short arms of subtelo-acrocentric chromosomes, although some species bear terminal Ag-NORs on the long arms (Galetti Jr. et al., 1985;Almeida-Toledo et al., 1987;Cestari & Galetti Jr. 1992a, b;Martins-Santos et al., 1994;Nakayama et al., 2001Nakayama et al., , 2002;;2008;Centofante et al., 2002a;Nirchio et al., 2003;Gaviria et al., 2005).However, the numerical variation in these regions (in both inter and intra-specific terms) is much more notable than the positional differences, ranging from four sites in C. macropomum to 12 sites in S. rhombeus (Table 1).
This Ag-NOR variability is also supported by the FISH analysis of 18S rDNA sites, indicating that, alongside with differences in diploid number, fundamental number and karyotype formula, Serrasalmidae species have undergone significant modifications in the chromosomal distribution of ribosomal genes during their evolutionary process.However, full correspondence between 18S rDNA and Ag-NOR sites was not constantly observed, since in some species, the number of 18S rDNA sites was greater than the number of Ag-NORs.Moreover, the location of some 18S rDNA sites was not detected by silver nitrate in S. rhombeus and S. serrulatus (Nakayama et al., 2008) (Table 1).Two probable reasons may putatively explain these divergences.Firstly, fluorescent in situ hybridization identifies ribosomal genes regardless of their activity status, whereas silver nitrate impregnation is only able to detect previously active sites (Miller et al., 1976).Therefore, some 18S rDNA sites that were undetectable by silver nitrate may correspond to inactive genomic regions.Secondly, NORs in Serrasalmidae are usually very small, probably due to a small number of gene copies, which does not allow its detection on the chromosomes.On the other hand, the occurrence of NOR transposition between different chromosomes, facilitated by transposable elements and the consequent diversity in their location within the karyotypes, cannot be excluded (Biémont & Vieira, 2006).
In addition to the standard karyotypes with 2n = 60 chromosomes, previous reports in S. rhombeus described a cryptic form (S. cf.rhombeus) bearing 2n = 58 chromosomes (Nakayama et al., 2001), as well as another karyomorph was identified by chromosomal and molecular analyses (Teixeira et al., 2006), suggesting that S. rhombeus is a complex of species.
This hypothesis is strengthened by the present data on 18S rDNA sites, since S. cf.rhombeus and S. rhombeus differed in both number and localization of these sites on the chromosomes (Table 1, Fig. 2 f and h).
In general, the 5S rDNA clusters are interstitially situated on the chromosomes of fish species, usually involving a single pair in the karyotype, which is thought to be a preferential condition for this group (Martins & Wasko, 2004).Similarly, the 5S rDNA in all Serrasalmidae species was interstitially located on the long arms of a metacentric pair, without synteny with NORs.This chromosome corresponds to pair number 7 in Serrasalmus and probably pair number 3 in P. nattereri and pair number 12 in C. macropomum (Fig. 3).
A precise identification of the 5S rDNA-bearing pair in the karyotypes of different species of Serrasalmidae was somewhat difficult to achieve without additional markers, since a great number of chromosomal pairs are morphologically similar.However, based on size, form, 5S rDNA location and conspicuous C-bands, these chromosomes were putatively homeologous between Serrasalmus and Pygocentrus (Fig. 3).Indeed, 5S rDNA-bearing chromosomes in all Serrasalmus species as well as in P. nattereri had a peculiar C-positive band close to the centromere on the long arms, associated with the 5S rDNA genes.In some other Neotropical fish groups, such as Anostomidae, Parodontidae, and Prochilodontidae, 5S rDNA-bearing chromosomes also seem to be conserved (Martins & Galetti Jr., 1999, 2000;Vicente et al., 2001;Centofante et al., 2002b;Jesus & Moreira Filho, 2003;Hatanaka & Galetti Jr., 2004).Meanwhile, the same inference is not valid for Serrasalmidae as a whole, once 5S rDNA sites in C. macropomum were located on clearly different chromosomes when compared to the other studied species.
Based on inner anatomy, miological, and osteological characters, Machado-Allison (1983) subdivided Serrasalmidae into two distinct groups: "pacus" and "piranhas".Among "pacus", Colossoma is thought to represent one of the more derived genera.Among "piranhas", Catoprion is thought to be the most basal group, followed by Pygopristis, Pygocentrus, Pristobryconm, and Serrasalmus, the latter two corresponding to the more derived genera.On the other hand, molecular phylogeny based on mtDNA (Ortí et al., 1996) clustered "pacus" and "piranhas", and placed Colossoma as the basal genus for the family.Within this phylogenetic analysis, Catoprion remains the basal group of piranhas, along with Pristobrycon striolatus, followed by other species of Pristobrycon, Serrasalmus, and Pygocentrus.
All these species have a karyotype composed of m, sm, st, and a chromosomes, thus diverging from C. macropomum, which has 2n =54 m-sm chromosomes.The location of 5S rDNA sites in similar chromosomes also reinforces a closer relationship between Serrasalmus and Pygocentrus as well as the separation of Colossoma.Therefore, a number of karyotype traits corroborate the distance between Colossoma and other species of Serrasalmidae, as proposed by the phylogenetic hypotheses of Machado-Allison (1983) and Ortí et al. (1996).Moreover, the evident co-localization of 5S rDNA and C-bands on probable homeologous chromosomes supports the high proximity between the species of Serrasalmus and P. nattereri, as proposed by Ortí et al. (1996).
Considering C. macropomum as occupying the most basal position within Serrasalmidae, the karyotype differentiation observed in the other species would be derived from chromosome rearrangements, such as centric fissions, Table 1.Karyotype characteristics in the studied species of Serrasalmidae.2n = modal diploid number; m = metacentric, sm = submetacentric, st = subtelocentric, a = acrocentric chromosomes; FN = fundamental number; p = short arm; q = long arm; t = terminal; Ag-NORs = nucleolar organizer regions stained with silver nitrate; 18S rDNA = maximum number of sites detected by FISH; 5S rDNA = number of sites detected by FISH.*S.maculatus = S. spilopleura cytotype A (according to Nakayama et al., 2000;Centofante et al., 2002a).increasing the basal diploid number from 2n = 54 metasubmetacentric chromosomes to 2n = 58 and 60 chromosomes, alongside with the formation of acrocentric chromosomes.
Similarly, the NORs have also undergone numerical and positional modifications during the karyotype evolution of this fish group, changing from a location on m-sm chromosomes, as seen in C. macropomum, to a preferential location on st-a chromosomes, as observed in the other genera.
From this perspective, it is likely that the few m-sm chromosomes bearing 18S rDNA, as detected in S. rhombeus and S. serrulatus, represent an ancestor feature similar to that found in C. macropomum.As for the 5S rDNA clusters, their identical location in all Serrasalmus species and in P. nattereri suggests that, once established from a putative ancestor condition as that found in C. macropomum, their distribution remained conserved.However, while chromosomes bearing 5S rDNA sites are located in pair number 3 in P. nattereri karyotype, they correspond to pair number 7 in Serrasalmus.Therefore, the same location of the 5S rDNA sites on metacentric chromosomes and their co-localization with C-positive heterochromatin per se are not a unique feature of the genus Serrasalmus, since these aspects are also shared by P. nattereri.Instead, the exclusive characteristic of Serrasalmus must refer to the position of the chromosomes bearing such markers in the karyotype (pair number 7).
In conclusion, chromosome characteristics, particularly the analysis of ribosomal genes and C-bands, provided reliable cytotaxonomic markers for evaluating karyotype differentiation and evolutionary relationships among species of Serrasalmidae.