B chromosome and NORs polymorphism in Callichthys callichthys (Linnaeus, 1758) (Siluriformes: Callichthyidae) from upper Paraná River, Brazil

B chromosomes are extra chromosomes from the normal chromosomal set, found in different organisms, highlighting their presence on the group of fishes. Callichthys callichthys from the upper Paraná River has a diploid number of 56 chromosomes (26 m-sm + 30 st-a ) for both sexes, with the presence of a sporadically acrocentric B chromosome. Moreover, one individual presented a diploid number of 57 chromosomes, with the presence of a morphologically ill-defined acrocentric B chromosome in all analyzed cells. The physical mapping of 5S and 18S rDNA shows multiple 5S rDNA sites and only one pair of chromosomes with 18S sites in C . callichthys , except for two individuals. These two individuals presented a third chromosome bearing NORs (Ag-staining and 18S rDNA) where 5S and 18S rDNA genes are syntenic, differing only in position. The dispersion of the 18S rDNA genes from the main st-a chromosome pair 25 to one of the chromosomes from the m-sm pair 4 would have originated two variant individuals, one of which with the ill-defined acrocentric B chromosome. Mechanisms to justify the suggested hypothesis about this B chromosome origin are discussed in the present study. um cromossomo B acrocêntrico. Além do mais, um indivíduo apresentou número diploide de 57 cromossomos, com a presença de um cromossomo B acrocêntrico morfologicamente mal definido em todas as células analisadas. O mapeamento físico do DNAr 5S e 18S mostrou múltiplos sítios de DNAr 5S e apenas um par de cromossomos com sítio para o DNAr 18S em C . callichthys , com exceção para dois indivíduos. Estes dois indivíduos apresentaram um terceiro cromossomo portador das RONs (Ag-RONs e 18S rDNA), onde os genes DNAr 5S e 18S são sintênicos, diferindo apenas na posição. A dispersão dos genes DNAr 18S do par de cromossomos principal st - a 25 para um dos cromossomos do par m - sm 4 teria originado dois indivíduos variantes, um dos quais com cromossomo B acrocêntrico mal definido. Mecanismos para justificar a hipótese sugerida sobre a origem deste cromossomo B são discutidos no presente estudo.


Introduction
B chromosomes are extra chromosomes from the normal chromosomal set that follow their own evolutionary pathway . Many studies concerning B chromosomes seek to clarify the molecular organization, ways of transmission Jesus et al., 2003;Jones & Houben, 2003), origin and evolution of these chromosomes Mestriner et al., 2000;Jesus et al., 2003;Poletto et al., 2010); however it is not always possible to precisely determine their origin (Jamilena et al., 1994;Camacho et al., 2000).
In their differentiation process, B chromosomes would develop biologically meaningful but not essential functions (Mião et al., 1991;Plowman & Bougourd, 1994), being considered selfish chromosomes, genomic parasites or accessories (Jones & Houben, 2003;Poletto et al., 2010). Some authors suggest a correlation between their presence and environmental factors (Néo et al., 2000), or the possibility that these chromosomes convert into a reservoir of genetic B chromosome and NORs polymorphism in Callichthys callichthys 604 variability, showing an evolutionary role (Rejón et al., 1987).
Cytogenetic studies in the Callichthyinae subfamily show the presence of B chromosomes only in Callichthys Linnaeus, 1758. These extra chromosomes were observed in different populations of C. callichthys, varying in size, quantity and morphology Shimabukuro-Dias et al., 2005). In the present study, chromosomal structure of one population of C. callichthys was studied focusing the distribution of ribosomal sites and the biology of supernumerary chromosomes in this species, highlighting the occurrence of a morphologically ill-defined B chromosome and a hypothesis about its origin.

Results
The analysis of mitotic cells revealed a diploid number of 56 chromosomes (26 m-sm + 30 st-a) for both sexes, with the sporadic occurrence of an acrocentric B chromosome, which appears in three males e one female individuals (from 2.3% to 7.14% of metaphasic cells frequencies; 100 metaphase plates/ individual analyzed) (Fig. 1a). Also, one male individual had a B chromosome, like a morphologically ill-defined acrocentric one, present in all analyzed metaphases (Fig. 1b).
C-banding revealed centromeric heterochromatin, with pericentromeric and telomeric markings on some chromosomes and coincident markings with the intercalary region of the ribosomal sites in all specimens analyzed. Also, both the B chromosomes were completely heterochromatic (Figs. 2a, b). The intercalary region of the NORs was positive for CMA 3 and negative for DAPI (Figs. 3a, b, c, d); indicating that the intercalary region of the 45S rDNA has a GC rich composition. The ill-defined acrocentric B chromosome was not differentially stained by base-specific fluorochromes (Fig. 3c).
The NORs (Ag-staining and 18S rDNA) were simple, on the short arm of the chromosome pair 25, although two individuals presented an extra marking, located on the short arm of one of  4a); variant 1, 5S rDNA in the terminal position of the short arm, syntenic to interstitial 18S rDNA in only one homologous of the pair (Fig. 4b); variant 2, 5S rDNA in the interstitial position of the short arm syntenic to 18S rDNA in terminal position on only one chromosome, with the occurrence of the ill-defined acrocentric B chromosome (Fig. 4c). chromosome from the m-sm pair 4. This additional marking was interstitial in one individual and terminal on the other (Figs. 1a, b, box and Fig. 4b, c). The individual with the third NOR on terminal position had the ill-defined acrocentric B chromosome.
The physical mapping of ribosomal genes through FISH showed 5S rDNA sites on four m-sm chromosome pairs. However, two individuals showed differences in the number of rDNA sites, one with seven and another with nine chromosomes bearing 5S rDNA. Both individuals also had a cause a fast heterochromatinization of extra elements, constituting the basis for differentiation of B chromosomes that isolates them from the rest of the genome  or just keeps the gene dosage (Amos & Dover, 1981). This segment possibly had its gene organization modified through rearranges like proposed by Jamilena et al. (1994). The presence of this B chromosome in all observed cells suggests that a neo-centromere might have been originated, capable of anchoring proteins from the kinetochore like observed by Depinet et al. (1997), differing from other reports of the occurrence of B chromosomes in some populations of C. callichthys that presented intra-individual variation Sanchez & Fenocchio, 1996), apart from a report of a B chromosome present in all cells of one individual (Shimabukuro-Dias et al., 2005).
On the present study, the presence of intercalary heterochromatin on the NORs might have facilitated the dispersion and origin of the B chromosome. The presence of specific repetitive DNAs in B chromosomes has been described for many fish species (Mestriner et al., 2000;Jesus et al., 2003). However, the composition of the ill-defined acrocentric B chromosome was not similar to its origin (GCrich) (Fig. 3c), suggesting different evolutionary mechanisms for B chromosomes .
A hypothesis was raised about the origin of the third NOR bearing chromosome and the B chromosome. The process would have begun with the amplification of the ribosomal genes on the main acrocentric chromosomes pair (25). It is possible that an event of dispersion to a third chromosome happened, as proposed by Schweizer and Loidl (1987), with the transposition of terminal segments from acrocentric chromosomes to nonhomologous, followed by homogenization. This dispersion carried terminal 5S rDNA to chromosome 4 (m-sm), originating the variant 1, with 5S rDNA in the terminal position of the short arm, syntenic to interstitial 18S rDNA in only one chromosome from the pair (Fig. 4b). Possibly, a paracentric inversion happened afterwards, originating variant 2, with 5S rDNA in the interstitial position of the short arm, syntenic to 18S rDNA in terminal position. In this process, a small segment would have been lost by a fission following the paracentric inversion, corresponding to the additional chromosome (Fig.  4c). Although 18S rDNA sites have already been observed on B chromosomes (Polleto et al., 2010), the B chromosome observed in C. callichthys does not have ribosomal cistrons, which was verified through silver nitrate impregnation and confirmed through 18S rDNA-FISH.
The ill-defined acrocentric B chromosome is completely heterochromatic (Fig. 5). It is possible that the ribosomal cistrons were eliminated, as seen by Jamilena et al. (1994) in Crepis capillaris. This suggests the presence of cell mechanisms which  Terminal NORs were described in many populations of C. callichthys Sanchez & Fenocchio, 1996;Shimabukuro-Dias et al., 2005). On the present study, the analyzed population shows the same pattern, except for two individuals. These individuals showed the presence of a third NOR bearing chromosome, similar to a situation described for one individual by Porto & Feldberg (1993). The presence of an additional chromosome bearing NORs is a recurring condition for C. callichthys. It is possible that these regions have undergone homogenization like the model proposed by Schweizer & Loidl (1987); however, this issue demands further studies. Further studies performing in situ hybridization with probes obtained from the ill-defined acrocentric B chromosome are necessary to confirm the proposed hypothesis for the origin of this chromosome in C. callichthys.