Chromosome mapping of repetitive sequences in four Serrasalmidae species (Characiformes).

The Serrasalmidae family is composed of a number of commercially interesting species, mainly in the Amazon region where most of these fishes occur. In the present study, we investigated the genomic organization of the 18S and 5S rDNA and telomeric sequences in mitotic chromosomes of four species from the basal clade of the Serrasalmidae family: Colossoma macropomum, Mylossoma aureum, M. duriventre, and Piaractus mesopotamicus, in order to understand the chromosomal evolution in the family. All the species studied had diploid numbers 2n = 54 and exclusively biarmed chromosomes, but variations of the karyotypic formulas were observed. C-banding resulted in similar patterns among the analyzed species, with heterochromatic blocks mainly present in centromeric regions. The 18S rDNA mapping of C. macropomum and P. mesopotamicus revealed multiple sites of this gene; 5S rDNA sites were detected in two chromosome pairs in all species, although not all of them were homeologs. Hybridization with a telomeric probe revealed signals in the terminal portions of chromosomes in all the species and an interstitial signal was observed in one pair of C. macropomum.


Introduction
The family Serrasalmidae comprises approximately 80 species distributed among 15 genera, with a number of commercially important species of the genera Colossoma, Mylossoma, and Piaractus, which are valued for fishing or aquaculture in the Amazon region (Araújo-Lima and Goulding, 1998;Oliveira and Araújo-Lima, 1998;Jégu, 2003;Nelson, 2006). The species of this family are popularly known as "pacus", "piranhas" and "tambaqui" (C. macropomum), which have high, laterally compressed bodies with abdominal spines and long dorsal fins. Their distribution is exclusively Neotropical and they inhabit environments such as floodplains, seasonally flooded forests, white-waters, and rivers main channels throughout South America, mainly in the Amazon, Paraguay, and Orinoco river basins (Goulding, 1980;Jégu, 2003).

Material and Methods
Four species belonging to the Serrasalmidae family were analyzed: Colossoma macropomum, Mylossoma aureum, M. duriventre, and Piaractus mesopotamicus. Thirty-eight specimens collected in the central Amazon region or from pisciculture farms (Table 1) were anesthetized with eugenol (a 5 mL stock solution diluted into 12 L of water) and sacrificed to obtain chromosome preparations. Mitotic chromosomes were obtained from kidney cells (Bertollo et al., 1978). Constitutive heterochromatin detection was achieved using the C-banding technique (Sumner, 1972), and the nucleolus organizing regions (NORs) were evidenced by silver nitrate impregnation (Howell and Black, 1980). Total DNA extraction was performed using muscle tissue samples of C. macropomum, M. aureum and M. duriventre following Sambrook et al. (1989), and quantification was performed in agarose gels by comparison with a standard lambda marker. The 18S and 5S rDNA genes were amplified by polymerase chain reaction (PCR), employing the oligonucleotide primers 18Sf (5'-CCG CTT TGG TGA CTC TTG AT-3') and 18Sr (5'-CCG AGGACC TCA CTA AAC CA-3') (Gross et al., 2010), 5Sa (5'-TAC GCC CGA TCT CGT CCG ATC-3'), and 5Sb (5'-CAGGCT GGT ATG GCC GTA AGC-3') (Martins and Galetti Jr., 1999). PCR reactions were performed in a final volume of 25 mL containing genomic DNA (200 ng), 10x buffer with 1.5 mM MgCl 2 , Taq DNA polymerase (5 U/mL), dNTPs (1 mM), the primer pairs (5 mM), and deionized water. Conditions for the 18S rDNA amplification reaction were: 1 min at 95°C, 35 cycles of 1 min at 94°C, 1 min at 56°C, and 90 s at 72°C; with a final extension of 5 min at 72°C. Conditions for the 5S rDNA amplification reaction were: 1 min at 95°C, followed by 30 cycles of 1 min at 94°C, 1 min at 59°C and 90 s at 72°C; with a final extension of 5 min at 72°C. PCR reactions for the telomeric sequences (TTAGGG)n were performed in a final volume of 25 mL containing 10x buffer with 1.5 mM of MgCl 2 , dNTPs (1 mM), 0.2 mL (TTAGGG)5 primer, 0.2 mL (CCCTAA)5 primer, and 2 U of Taq DNA polymerase (Ijdo et al., 1991). The first part of the amplification process was conducted under low stringency (4 min at 94°C; followed by 12 cycles of 1 min at 94°C, 45 s at 52°C and 90 s at 72°C), followed by 35 cycles at high stringency conditions (1 min at 94°C, 90 s at 60°C and 90 s at 72°C).
After FISH, the metaphases were analyzed under an Olympus BX51 epifluorescence microscope. Images were captured using a coupled Olympus DP71 digital camera and the Image-Pro MC 6.3 software. Metaphases were processed with Adobe Photoshop CS3 and the chromosomes were measured with Image J and classified according to the nomenclature proposed by Levan et al. (1964).

Karyotypic descriptions
The four species analyzed had diploid numbers of 2n = 54 chromosomes and fundamental numbers FN = 108 -although their karyotypic formulas differed. A subtelocentric pair was present in M. duriventre. None of the species had heteromorphic sex chromosomes. The karyotypic formulas were: 26m+28sm in Colossoma Ribeiro et al. 47 Table 1 -Data of the Serrasalmidae specimens analyzed.

Mylossoma duriventre
Lago Catalão (confluence of the Negro and Solimões rivers -3º11'59" S and 59º53'59" W) 13 (5 females and 8 males) Nucleolus organizing regions (NOR) were located on the terminal portions of the long arms of metacentric pairs 6 and 12 of C. macropomum (Figure 1a), in terminal regions of chromosome 4 in P. mesopotamicus (Figure1b) and in the short arms of one homologue of pair 12 in species of Mylossoma (Figure 1c, d).

Repetitive sequences mapping
In situ hybridization experiments with 18S sequences as probes were performed numerous times in Mylossoma species but no labeling of ribosomal sites could be detected. This result may reflect methodological problems or may be 48 Repetitive sequences mapping due to the small size of these sites which would prevent their visualization. The 18S rDNA sequences were visualized in the terminal regions of the long arms of metacentric pairs 6, 10 and 12 of C. macropomum (Figure 2a), with pairs 6 and 12 coinciding with the Ag-NOR. In P. mesopotamicus, signals were observed in the terminal regions of the long arms of metacentric pairs 4 and 6 ( Figure  2b). 5S rDNA sequences mapped to euchromatic and heterochromatic regions of two chromosome pairs in each of the four species (Figure 3e, f, g, h). Labeling was observed in an interstitial position of pair 11 and in the terminal region of the short arm of pair 13 in C. macropomum and P. mesopotamicus (Figure 3a, b). In both species, the 5S rDNA sites of pair 11 co-localized with heterochromatic blocks and the 5S rDNA sites of pair 13 were adjacent to heterochromatic regions (Figure 3e, f). The 5S rDNA sites were present in pairs 4 and 12 of Mylossoma aureum (Fig-ure 3c, g) and in pairs 4 and 8 of M. duriventre (Figure 3d). The interstitial signals in the long arms coincided with the heterochromatic blocks in both species. FISH with telomeric probes revealed signals at the terminal portions of both arms of all chromosomes in the four species (Figure 4a, b, c, d). An interstitial telomeric site (ITS) was observed in one metacentric pair of C. macropomum (Figure 4a) and coincided with a positive C-banding region.

Cytogenetic descriptions
According to the phylogeny based on mtDNA data proposed by Ortí et al. (2008), the Serrasalmidae family may be divided into three main clades: "pacus", "Myleus", and "piranhas". All of the species analyzed herein belong to Ribeiro et al. 49 the "pacus" clade, which comprises the Piaractus, Colossoma, and Mylossoma genera, considered basal among Serrasalmidae (Ortí et al., 1996). The evolutionary relationships among Serrasalmidae are not well defined and have been the focus of several studies. Molecular data have been used to supplement morphological studies that confirm the monophyly of this fish group from species to family and/or subfamily levels (Calcagnotto et al., 2005;Freeman et al., 2007).
The karyotypes of Colossoma macropomum and P. mesopotamicus were different from those previously described. The karyotypic formula of our specimens of C. macropomum differed from that described by Nirchio et al. (2003); P. mesopotamicus presented the same karyotypic formula described when this species was still classified as Colossoma mitrei (Almeida-Toledo et al., 1987), but differences were detected in the distribution of heterochromatic blocks, with small quantities of constitutive heterochromatin observed in the present work. These differences may be due to different factors, including environmental influences. The great diversity of habitats may expose organisms to different selection pressures that may activate or inactivate certain genes through epigenetic mechanisms. The appearance of distinct heterochromatic regions in different populations could well reflect adaptive processes that are part of the necessary metabolic fine-tuning for species survival. According to Richards et al. (2010), epigenetic changes induced by hybridization or environmental stress may be passed on for many generations and contribute for the adaptation of these fish to new environments.

Repetitive sequences mapping
Fluorescence in situ hybridization with 18S rDNA sequences showed multiple signals in two species, as previously reported for other Serrasalmidae (Nakayama et al., 2008(Nakayama et al., , 2012. The 18S rDNA distribution in three chromosome pairs of C. macropomum corroborated earlier findings for that same species (Nirchio et al., 2003;Nakayama et al., 2012), in contrast to the Ag-NOR staining that evidenced only two pairs. The pattern observed in P. mesopotamicus (two labeled pairs) differed, however, from that described in other species of the same genera (P. brachypomus) which presented a single 18S rDNA-bearing chromosome pair (Nirchio et al., 2003). The smaller number of NOR evidenced by silver nitrate could be explained by the fact that this agent only interacts with proteins in recently active regions, whereas FISH detects all rDNA sites despite their recent activity.
If the presence of single NOR are considered a plesiomorphic characteristic, as in other fish groups (e.g., Feldberg et al., 2003 -family Cichlidae), a similar situation may well exist in the Serrasalmidae family, with the basal species P. brachypomus (Orti et al., 2008) bearing 18S rDNA sequences in a single chromosome pair (Nirchio et al., 2003), followed by other species of the same genus with two 18S rDNA-bearing pairs and by C. macropomum with these sequences present in three pairs. The "piranhas" clade has five to eight chromosome pairs bearing 18S rDNA sites (Nakayama et al., 2012).
FISH with 5S rDNA sequences resulted in two chromosome pairs labeled in the four species. The pairs bearing the 5S rDNA sites (pairs 11 and 13) in C. macropomum and P. mesopotamicus appeared to be homeologs, as their sizes and morphologies were very similar (Figure 3a, b). Although the Mylossoma species also showed 5S rDNA sites in two chromosome pairs, these did not seem to be intra-or intergeneric homeologs, as the morphology and interstitial labeling location in one of the pairs differed from those observed in the other species (Figure 3c, d). Additionally, the 5S rDNA localization in C. macropomum differed from that previously reported for this same species, in which the single 5S rDNA site coincided with a heterochromatic region (Nakayama et al., 2012). This variation in the number of chromosome pairs with multiple 5S rDNA sites in C. macropomum contradicts Nakayama et al. (2012) observation that the pattern of a single 5S rDNA-bearing site found in Serrasalmus was a characteristic shared by the entire family. Additionally, the synteny of the 5S and 18S rDNA sites previously described in C. macropomum was not observed in the present work, perhaps due to the fact that their chromosomes morphological similarities led to errors in their identification and the5S and 18S rDNA probes were not hybridized simultaneously.
These observations also do not corroborate the hypothesis that the localization of interstitial sites on a single chromosome pair is characteristic of basal species (Martins and Galetti Jr., 1999). In the Serrasalmidae family, for example, the opposite pattern was observed, with the species of the basal genera (Colossoma, Mylossoma, and Piaractus) having two 5S rDNA-bearing chromosome pairs and the most derived species (of the genus Serrasalmus) showing a single chromosome with 5S rDNA sequences (Nakayama et al., 2012). The location of the 5S rDNA sites in heterochromatic regions was the only similarity observed in relation to other serrasalmids.
Telomeric sequences were present in the terminal regions of all the chromosome arms in all the analyzed species, confirming the hypothesis that the observed variations between the karyotype were due to inversions. Interstitial telomeric sequences (ITSs) in one chromosome pair were only observed in C. macropomum and did not seem to result from fusion, as the diploid number was maintained, neither to be due to inversions. These ITS coincided with heterochromatic regions, which could explain their presence. Other repetitive DNA sequences present in constitutive heterochromatin regions have been associated with telomeric sequences . Additionally, ITS observed in centromeric positions (as in C. macropomum) may be related to satellite DNAs transferred from other regions by processes such as unequal crossing-over, transpositions, or duplications in the heterochromatin .
Based on the data presented herein, we conclude that the basal clade of Serrasalmidae, composed of C. macropomum, M. aureum, M. duriventre, and P. mesopotamicus has karyotypic features consistent with the previously published data for these species, including their 2n = 54. This is compatible with the phylogeny of the group. The data presented herein demonstrate the need of additional molecular studies within this clade to aid the identification of the distribution patterns of the repetitive sequences that are effectively determining the evolutionary trend within this group.