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Cytogenetic studies in two populations of Astyanax altiparanae (Pisces, Characiformes)

2005; BioMed Central; Volume: 141; Issue: 3 Linguagem: Inglês

10.1111/j.1601-5223.2004.01832.x

1601-5223

Carlos Alexandre Fernandes, Isabel Cristina Martins-Santos,

Fish biology, ecology, and behavior

The genus Astyanax has a wide geographic distribution, comprising more than a hundred species and subspecies in neotropical rivers (Garutti and Britski 1997). The genus also reveals several closely similar forms, forming a highly complex group, from taxonomic point of view. The species identified as Astyanax bimaculatus, of the upper Paraná river, has earlier been described as Astyanax altiparanae by Garutti and Britski (2000). A. bimaculatus and A. altiparanae populations already studied have diploid number 2n=50 chromosomes with differences in their karyotype formulae and in the number of NOR-bearing chromosomes. Multiple NOR systems with telomere markings up to six organizing nucleolus chromosomes have been reported for most A. altiparanae populations (Paganelli 1990; Takahashi 1995; Daniel-Silva 1996). Simple NOR was nonetheless found in the population of A. altiparanae in the stream Sarandi (Pirapó river basin) described by Vale and Martins-Santos (1998). In certain populations, constitutive heterochromatin pattern analysis has marked interstitial blocks in most chromosomes (Paganelli 1990; Daniel-Silva and Almeida-Toledo 2001), whereas others have few heterochromatin blocks with some telomere, interstitial and centromere markings (Takahashi 1995). Two Astyanax altiparanae populations are here compared cytogenetically so that variations may be detected for a better understanding of the "altiparanaecomplex" and of the chromosome rearrangements mechanisms involved during diversification of this group. Eight specimens (4 males and 4 females) of Astyanax altiparanae were collected from Índios river (Ivaí basin), and nine specimens (4 males and 5 females) were colleted from Paraná river, Porto Rico region (Paraná basin). Mitotic chromosomes were obtained from kidney cells following methodology described by Bertollo et al. (1978). C-band technique by Sumner (1972) was employed for the analysis of constitutive heterochromatin and silver nitrate (AgNO3) staining nucleolus organizer regions (NORs) by the method of Howell and Black (1980). Regions rich in GC were detected by fluorochrome chromomycin A3 (CMA3) described by Schmid (1980). Identification of chromosomes was done according to arm ratio criteria (AR), suggested by Levan et al. (1964). The population presented diploid number 2n=50 chromosomes, karyotype comprising 6 M, 30 SM, 4 ST and 10 A, and fundamental number (FN) 90, for both sexes. Silver nitrate analysis showed markings in the terminal regions of the short arm of chromosome pairs 2 (M), 10 (SM), 19 (ST, coinciding with secondary constriction seen in only one homologue), 20 (ST), and in the long arm of chromosome pair 8 (SM). The above presents multiple NOR system totaling 10 Ag-NOR chromosomes (Fig. 1a). Chromomycin A3 analysis showed that most NORs of the population were GC rich (Fig. 2a). Giemsa conventional karyotypes and NOR band of Astyanax altiparanae populations from Índios (a) and Paraná (b) rivers. Somatic metaphases of Astyanax altiparanae populations with Chromomycin A3 treatment in Astyanax altiparanae populations from Índios (a) and Paraná (b) rivers. Arrows indicate fluorescent regions in the chromosomes. The constitutive heterochromatin pattern had weak centromere markings in few chromosomes and evident interstitial markings in a region proximal to the centromere on the long arm of chromosomes 6, 7, 10, 11, 14 and 19. NOR on the short arm of chromosome pair 19 was positive C-band (Fig. 3a). Distribution pattern of constitutive heterochromatin in Astyanax altiparanae populations from Índios (a) and Paraná (b) rivers. Specimens of this population present diploid number 2n=50 chromosomes, karyotype comprising 6 M, 26 SM, 6 ST and 12A, and fundamental number (FN) 88, in both sexes. NOR lies on the short arm of the single acrocentric chromosome pair (pair 20), coinciding with the secondary constriction seen in only one homologue (Fig. 1b). CMA3 analysis shows that other chromosomes have fluorescent bands besides NOR (Fig. 2b). Constitutive heterochromatin has also been detected in the centromere regions in a few chromosomes and in interstitial ones proximal to the centromere on the long arm of chromosomes 6, 7, 9, 10, 11, 18 and 19. NOR on short arm of chromosome pair 20 was positive C-band (Fig. 3b). Although having the same diploid number 2n=50 chromosomes, the two populations under discussion differ in their karyotype formulae and the number of NOR-bearing chromosomes. Whereas the Índios river population had FN=90 and multiple NOR system, with 5 nucleolus organizing pairs, the Paraná river population comprised FN=88 and simple NOR system. Cytogenetic studies in different A. altiparanae populations have shown constant diploid number 2n=50 chromosomes (Table 1). In spite of this trend towards conservatism, differences in karyotype constitution and NOR number has indicated what they constitute a species complex, similar to what has been reported in other species of the same genus. From the cytogenetic point of view, multiple NOR system reported in the Índios river population is the most frequent among A. altiparanae populations (Paganelli 1990; Takahashi 1995; Pacheco et al. 2001; Daniel-Silva and Almeida-Toledo 2001). However, the number and types of chromosomes involved in this region differ in the populations. Simple NOR system has been currently reported in A. bimaculatus populations of the São Francisco, Doce, and Paraguay rivers (Paganelli 1990) and in A. altiparanae of the Paraná river (present paper) and in the Sarandi stream (Vale and Martins-Santos 1998). NOR-size heteromorphism among homologues in populations of the Índios and Paraná rivers, which has been detected respectively in the first subtelocentric and acrocentric chromosome pairs, is a common event in fish. Pacheco (2001) analyzed two Astyanax altiparanae populations from Claro and Tibagi rivers (Limoeiro stretch) and found NOR-size heteromorphism among homologues in the first acrocentric pair. Such difference may be explained by transposition events or unequal crossing-over in this region. The use of chromomycin demonstrated that most Ag-NOR regions are CMA3+ for the population of Índios river and thus rich in GC. Chromomycin has been used for NOR detection in fish independently from its genic activity (Amemiya and Gold 1986; Schmid 1982). In certain cases this fact has shown a CMA3+ number higher than that obtained by silver nitrate, perhaps due to inactivated rDNA by genic regulation processes. In our study, the Índios river population had a higher Ag-NOR+ than CMA3+. Contrastingly, the Paraná river population had a higher CMA3+ number than that of Ag-NOR+. This fact shows that in the case of the former population not all NORs are rich in GC, neither all NORs are active in the latter. Although the number of CMA3+ markings may represent heterochromatic sites, the fluorescent bands fail to coincide since the former are telomeric and the heterochromatic bands are interstitial. CMA3+ data for the Paraná river population suggest the occurrence of more than one pair of NOR-bearing chromosomes. According to Phillips et al. (1989) chromomycin analysis coupled to silver nitrate has been useful in detecting polymorphism of these sites. Galetti Jr. et al. (1995) have also noted variations in data obtained by silver nitrate, CMA3 and in situ hybridization. The same authors attributed this type of polymorphism to genetic regulation or to structural change involving the same chromosome region. In situ hybridization ought to be used so that the true rDNA number in the Paraná river population could be explained satisfactorily. In the case of C-band pattern, both populations had weak centromere markings and several chromosomes with most conspicuous interstitial blocks of constitutive heterochromatin. Secondary constriction in one homologue coincided with NOR and positive C-band in pair 20 for the Paraná river population and in pair 19 in the Índios river one. Paganelli (1990) and Daniel-Silva and Almeida-Toledo (2001) analyzed Astyanax altiparanae populations of the Mogi Guaçu and Paranapanema rivers respectively and observed marked interstitial blocks of constitutive heterochromatin in most chromosomes. This fact shows the group's most frequent pattern. Cytogenetic data above and those in the literature (Table 1) show that the karyotype formulae varies among A. altiparanae populations with FN ranging from 76 to 100, although the group is conservative in its diploid number. These differences show the occurrence of non-robertsonian chromosome rearrangements during the diversification of the "altiparanaecomplex", principally pericentric inversions. However, since diploid number remains constant, B chromosomes are absent and the heterochromatin pattern of the different populations is similar, a higher karyotype stability of this complex is shown when contrasted to the other species of the same genus. This is specially true for A. scabripinnis and A. fasciatus complexes that has presented a variation in diploid number and more pronounced differences in the macro and microstructure of their chromosomes. The authors would like to thank Núcleo de Pesquisa em Limnologia, Ictiologia e Aqüicultura (NUPELIA), Dr. Horácio Ferreira Júlio Jr. and Dayani Bailly for their help in the collection of specimens. Research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).