Chromosomal diversity in three species of electric fish (Apteronotidae, Gymnotiformes) from the Amazon Basin

Cytogenetic studies were carried out on samples of Parapteronotus hasemani, Sternarchogiton preto and Sternarchorhamphus muelleri (Apteronotidae, Gymnotiformes) from the Amazon basin. The first two species exhibited both a 2n = 52 karyotype, but differed in their karyotypic formulae, distribution of constitutive heterochromatin, and chromosomal location of the NOR. The third species, Sternarchorhamphus muelleri, was found to have a 2n = 32 karyotype. In all three species the DAPI and chromomycin A3 staining results were consistent with the C-banding results and nucleolar organizer region (NOR) localization. The 18S rDNA probe confirmed that there was only one pair of ribosomal DNA cistron bearers per species. The telomeric probe did not reveal interstitial telomeric sequences (ITS). The karyotypic differences among these species can be used for taxonomic identification. These data will be useful in future studies of these fishes and help understanding the phylogenetic relationships and chromosomal evolution of the Apteronotidae.


Introduction
Apteronotidae is the Gymnotiformes family with the largest number of formally described species: 86 species divided into 15 genera (De Santana, 2007;Vari, 2009, 2010a,b;Albert and Crampton, 2009;De Santana and Crampton, 2010). The species of this family are found in rivers from Panama to northern Argentina, including rivers that flow into the Pacific Ocean (eastern Colombia), the Orinoco, Maracaibo, Magdalena, Guyana shield, the Amazon, and the Paraná-Paraguay and San Francisco basins (Mago-Leccia, 1994). Intra-and interspecific variations in size and shape of the head have been observed, probably related to trophic specialization and/or aggression between males (Cox-Fernandes, 1998;Albert, 2001;Cox-Fernandes et al., 2002).
Although Apteronotidae is the Gymnotiformes family with the largest number of described species, chromosomal information is available only for Apteronotus albifrons, which has 2n = 24 chromosomes (Howell, 1972;Almeida-Toledo et al., 1981;Mendes et al., 2012). Reportedly, Apteronotus albifrons from the Parana River also has B chromosomes, which were found as microchromosomes (Mendes et al., 2012).
Here, we made the cytogenetic characterization of three additional Apteronotidae species, Sternarchorhamphus muelleri, Parapteronotus hasemani and Sternarchogiton preto, in an effort to increase the amount of chromosomal information available for representatives of this family, to allow comparative analyses and provide new insights into the possible mechanisms underlying the diversification of these species.

Results
The species Sternarchorhamphus muelleri was found to have a karyotype of 2n = 32 (28m/sm+4st/a) and a fundamental number (FN) of 60. C-banding showed the presence of constitutive heterochromatin (CH) in the centromeric regions of most chromosomes, except for pairs 7, 11, 12, 13, 14 and 16, where the banding was almost imperceptible (suggesting that there was relatively little CH). Pairs 1, 3 and 10 had pericentromeric heterochromatic blocks, and pair 15 had a heterochromatic block in the proximal region of the long arm. A nucleolar organizer region (NOR) was found in the interstitial region of the long arm of pair 15, and showed heteromorphism between the homologs (Figure 3A, B).
The karyotype of Parapteronotus hasemani was 2n = 52 (36m/sm+16st/a) and FN = 88. CH was observed in the centromeric regions of all chromosomes. Pairs 1, 22 and 23 also had heterochromatic blocks in the proximal regions of their long arms. Pairs 2, 3, 5 and 6 had large heterochromatic blocks that spanned nearly the entire length of their short arms. Pairs 4 and 12 had heterochromatic blocks in their pericentromeric regions. A NOR was found on the distal short arm of pair 3; it coincided with a secondary constriction and showed heteromorphism between the homologs ( Figure 3C, D).
Sternarchogiton preto had a 2n = 52 (38m/sm+14st/a) karyotype and FN = 90. CH was observed in the centromeric region of most chromosomes, except for pairs 5, 9, 12, 16 and 20, where the banding was almost im-Silva et al. 639 perceptible. In addition, pair 1 had a heterochromatic block throughout the long arm, pair 2 had a heterochromatic block in the proximal region of the long arm, and pair 3 had a heterochromatic block on the long arm. Pairs 4, 8, 21, 23 and 24 had heterochromatic blocks on their short arms. A NOR was observed in the distal region of the long arm of pair 3, within the heterochromatic block, and showed heteromorphism between the homologs ( Figure 3E, F). The three studied species showed similar results in the fluorochrome, CMA 3 and DAPI staining, as well as FISH analysis with 18S rDNA and telomere repeat probes. CMA 3 labeled the NORs in all three species, showing that the ribosomal genes were interspersed with GC-rich sequences. DAPI staining revealed that the CH was AT-rich, and FISH with 18S rDNA probes showed that these sites were localized in a single pair per species, coincident with the NOR. Finally, FISH using the telomeric probes did not indicate the presence of any interstitial telomeric sequences ( Figure 4A-L).
Parapteronotus hasemani and Sternarchogiton preto have the same diploid number (2n = 52), but differ in their karyotypic formulae. This difference can be explained by chromosomal inversions, which can change the morphology of chromosomes while maintaining the diploid number (Alves et al., 2003;De Oliveira et al., 2006). Karyotypes of 2n = 52 are also found in other Apteronotidae species (Sternarchorhynchus cramptoni, S. oxyrhynchus, Platyurosternachus macrostomus, Apteronotus bonapartii; unpublished data), while 2n = 50 is seen in two species of genus Adontosternarchus (A. clarkae and A. balaenops; unpublished data). This indicates that a higher diploid number is common in the family. Although they share the same diploid number, P. hasemani and S. preto display considerable differences in their C-banding patterns and the location of their NORs. The latter may be due to translocations of the NOR-bearing chromosomes.
The karyotype of Sternarchorhamphus muelleri (2n = 32) is quite different from those of the other two species in chromosome number and morphology, CH distribution and NOR location. The diploid number reduction appears to have occurred through chromosomal fusion events. Such events are believed to explain the reduction of 2n in genus Ancistrus (Loricariidae) (Alves et al., 2003;De Oliveira et al., 2009), and have been suggested for other groups of fishes (Cipriano et al., 2008;Margarido and Moreira-Filho, 2008). Similarly, fusion events could also explain the diploid number decrease in Apteronotus albifrons. The location of the NORs in this species is quite different from that seen in other species, as it typically appears in the interstitial region of the long arm of an acrocentric chromosome. This variation in the NOR-bearing chromosome may have arisen, in this case, through a pericentric inversion that moved the NOR to the middle of the chromosome arm. Nagamachi et al. (2010) used chromosome painting to show that the degree of chromosomal rearrangement between karyotypes of two cryptic species of Gymnotus cf carapo (2n = 40 and 2n = 42) was much greater than that estimated using classical cytogenetics (Milhomem et al., 2008). Thus, the pericentric inversions and centric fusions that were believed to differentiate the karyotypes of these species may be an underestimate of the actual degree of genomic rearrangement at work. CH was found in the centromeric regions of virtually all chromosomes of the analyzed species. DAPI staining revealed that this CH is AT-rich, which is consistent with reports concerning other Gymnotiformes (Milhomem et al., 2007(Milhomem et al., , 2008(Milhomem et al., , 2012aSilva et al., 2008Silva et al., , 2009Cardoso et al., 2011). The three species analyzed in the present study showed differing CH patterns, with several additional blocks in non-centromeric regions. Processes related to the dynamics of repetitive DNA, such as amplifications and translocations, might have been involved in the develop-640 Cytogenetics of Apteronotidae ment of these blocks. Using microarray analysis, Lippman et al. (2004) showed that in Arabidopsis the heterochromatin is determined by transposable elements and related to tandem repeats. Transposons have been found in the heterochromatin of several fish groups, including Cichla kelberi (Teixeira et al., 2009), Hisonotus leucofrenatus (Ferreira et al., 2011), and Antarctic fishes of the suborder Notothenioidei (Ozouf-Costaz et al., 2004). Furthermore, transposons have been associated with the karyotypic variation observed in Erythrinus erythrinus (Cioffi et al., 2010), and the formation of the Y chromosome in Chionodraco roseofuscus (Capriglione et al., 2000). Souza et al. (2009) analyzed species of genus Peckoltia (Siluriformes: Loricariidae) and proposed possible homeologies among some pairs with similar C-ban-ding patterns, morphologies and NOR localizations. In the karyotypes of P. hasemani, S. muelleri and S. preto, we identified chromosomes (pair 1 in the first two species and pair 2 in the latter) that resembled one another in chromosome morphology, size and HC distribution ( Figure 5).
Heteromorphisms in NOR size (by 2-fold or more) have been described in some Gymnotiformes species, and the NORs of Eigenmannia sp and E. virescens were found to be larger than those of the other examined species. Tandem duplications, unequal crossovers involving repeated regions, and/or accidental duplications might explain these variations .
The NOR regions of the tested species stained positive with CMA 3 , revealing that the ribosomal genes are interspersed with GC-rich sequences (Pendás et al., 1993). Our results resemble those found in other Gymnotiformes and additional fish species (Artoni and Bertollo, 1999;Milhomem et al., 2007Milhomem et al., , 2008Milhomem et al., , 2012aSilva et al., 2008Silva et al., , 2009Cardoso et al., 2011). FISH with telomeric probes failed to show any evidence of interstitial telomeric sequences (ITSs). This may reflect the absence of chromosomal rearrangements involving the telomeres (Silva et al., 2009). Alternatively, telomeric sequences away from the chromosomal ends may have undergone sequence changes that hinder probe hybridization (Albuín et al., 1996).
Accumulating evidence suggests that variability in diploid number and karyotype formula can be explained by chromosome rearrangements, such as fusions (which can decrease the diploid number) and inversions. According to the phylogenies of Alves-Gomes et al. (1995) and Albert (2001), the family Gymnotidae, represented by Gymnotus and Electrophorus, occupies the basal position among the other Gymnotiformes. The species in this family have diploid numbers ranging from 34 to 54, with a higher occurrence of 2n above 50 (for review, see Milhomem et al., 2012a,b). Thus, we believe that the higher diploid numbers (2n = 50 or 52) are basal in Apteronotidae and that the karyotypes of Apteronotus albifrons and Sternarchorhamphus muelleri underwent rearrangements that decreased their 2n values. Their CH patterns are also quite variable, showing additional blocks that are characteristic to each species. The NORs varied in size and location on the chromosome, but all three species had a single NOR.
It is therefore likely that some homeologies exist among these species. Together, the results presented herein add new information that may prove valuable in future studies of this group, facilitating taxonomic identification, and increasing our understanding of the chromosomal evolution and phylogenetic relationships of the Apteronotidae.