Chromosomal analyses in Megalonema platanum (Siluriformes: Pimelodidae), an endangered species from South American rivers

This study presents chromosomal data of Megalonema platanum from rio Tibagi, Paraná, Brazil and from rio Paraná, Argentina. The diploid number was equal 54 with karyotype composition of 24m+16sm+2st+12a in both populations. The AgNOR sites were detected in the terminal position of a submetacentric pair of the two analyzed populations, coinciding with secondary constrictions on the short arm of pair 15. CMA 3 and FISH with 18S rDNA probe displayed fluorescent signals that correspond to the AgNOR sites and secondary constriction. The presence of a small acrocentric supernumerary chromosome can be observed in M . platanum from rio Tibagi, with centromeric heterochromatin. Others heterochromatic blocks were evidenced in the terminal position of some chromosome and one metacentric large chromosome pair, probably the first pair, showed an interstitial heterochromatin. In the population of the rio Paraná were still observed heterochromatic blocks in both ends in some chromosomes. This work brings for the first time cytogenetic date of M . platanum , which is a very rare species in the rio Paraná basin and may be endangered. 15. CMA 3 e FISH com sonda de DNAr 18S exibiram sinais fluorescentes que correspondem aos sítios AgNORs e à constrição secundária. A presença de um pequeno cromossomo supranumerário acrocêntrico foi observado em M . platanum do rio Tibagi, com heterocromatina centromérica. Outros blocos heterocromáticos foram evidenciados na posição terminal de alguns cromossomos e um par cromossômico submetacêntrico grande, provavelmente o primeiro par, mostrou heterocromatina intersticial. Na população do rio Paraná foram observados ainda blocos heterocromáticos em ambas regiões terminais em alguns cromossomos. Este trabalho mostra pela primeira vez dados citogenéticos de M . platanum , que é uma espécie muito rara na bacia do rio Paraná e pode estar ameaçada de extinção.

The genus Megalonema Eigenmann, 1910 is endemic to South America, for which there are few biological data available. Lundberg & Littmann (2003) defined six valid species of Megalonema: M. argentina (MacDonagh, 1938), M. pauciradiatum Eigenmann, 1919 andM. platanum (Günther, 1880) found in the rio Paraná basin; M. platycephalum Eigenmann, 1912 in the basins of the rios Amazonas, Essequibo and Orinoco;M. psammium Schultz, 1944 in the lago Maracaibo basin and M. xanthum Eigenmann, 1912 in the rio Magdalena basin. Recently Lundberg & Dadhul (2008) described more two species: M. amaxanthum n. sp. from the rio Amazonas basin and M. orixanthum n. sp. from the rio Orinoco basin. In M. platanum the body lacks clear and colored spots, with only the back of the head slightly darkened. The fins are clear, with the first ray of the pectoral ossified, and the snout is rounded and the eyes large (Lundberg & Littmann, 2003).
The present study presents the first karyotype description of Megalonem platanum using AgNO R conventional,fluorochrome staining, in additions to C-banding and fluorescence in situ hybridization (FISH) with 18S rDNA probe.
Three individuals of M. platanum from rio Tibagi showed the presence of an additional small acrocentric chromosome (Fig. 1a), indicating the occurrence of a supernumerary chromosomes. This chromosome was found to be present at a high intrapopulation frequency (75%) and a low intraindividual frequency (15.6%), where in a total of 96 metaphases examined in the population, only 15 had this small supernumerary chromosome. The absence of these chromosomes in the samples of lower rio Paraná basin suggested that these populations are isolated which should be confirmed through the analyses of additional specimens.
The C-banding technique allowed the detection in M. platanum of heterochromatic blocks in the terminal position of some chromosome pairs and a pair of large submetacentric chromosomes with interstitial heterochromatin in all populations (Fig. 1b, c). The individuals from rio Paraná also present some chromosomes with bands in both telomeric regions (Fig. 1c). C-banding for most of the family Pimelodidae is similar to that seen in M. platanum with some heterochromatic regions in the terminal position, as for example, in species of the genus Pimelodus (Souza et al., 2004;Garcia & Moreira-Filho, 2005;Treco et al., 2008), Steindachneridion Eigenmann & Eigenmann, 1919(Swarça et al., 2005 and others. In several species and/or populations of the genus Pimelodus, chromosomal marker pairs, with interstitial heterochromatin, could also be seen (Borin & Martins-Santos, 2004;Treco et al., 2008).
Supernumerary chromosomes are generally heterochromatic; however, in this work, the acrocentric B-chromosome of M. platanum showed heterochromatin only in the centromeric region, thus partially heterochromatic (Fig. 1b) The AgNORs sites were detected in the terminal position of a submetacentric pair of the two analyzed populations, coinciding with secondary constrictions on the short arm of pair 15 (Fig. 2a). In some metaphases, only one silver-stained  (Fig. 2b).
In situ hybridization with 18S rDNA probe in M. platanum confirmed the NOR location in the secondary constriction of a pair 15 (Fig. 2c) as also observed in some Pimelodidae species by different authors: Souza et al. (2004), Carvalho & Dias (2007), Swarça et al. (2008), Garcia & Moreira Filho (2008). CMA 3 staining on chromosomes of M. platanum displayed fluorescent signals on the short arm of a pair of sm chromosomes (Fig. 2d), probably coinciding with the AgNOR, showing that this region of secondary constriction is rich in GC base pairs since the fluorochrome CMA 3 preferably binds to chromatin segments rich in these base pairs. In these preparations, it was not possible to observe the supernumerary chromosome. In several species of fish, a positive correlation has been reported between of CMA 3 sites and AgNORs (Garcia & Moreira-Filho, 2005;Treco et al., 2008).
The data show that in terms of diploid number, M. platanum can be regarded as ancestral in relation to other species of Pimelodidae, and suggest that chromosomal rearrangements such as Robertsonian translocation or chromosomal fusion/fission are involved in the evolution of this group of fish. Thus, M. platanum must represent a primitive group within the family, with 2n = 54 that is a feature rare and shared with Pseudopimelodidae that possibly is the sister group of Pimelodidae (Sullivan et al., 2006). Therefore 2n = 56 and 2n = 50, observed in other Pimelodidae species, could be derived karyotype characteristics. As for the microstructure, that is, number and location of the nucleolus organizer regions and the pattern of heterochromatin distribution, M. platanum has a more conservative character, even though individuals from rio Paraná present some peculiarities (i.e., heterochromatin in both telomeric regions).