Cytogenetical analyses in four species of the genus Astyanax (Pisces, Characidae) from Paraíba do Sul River Basin

Transcrição

1 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN: (Print) (Online) Journal homepage: Cytogenetical analyses in four species of the genus Astyanax (Pisces, Characidae) from Paraíba do Sul River Basin Karine Frehner Kavalco & Orlando Moreira-Filho To cite this article: Karine Frehner Kavalco & Orlando Moreira-Filho (2003) Cytogenetical analyses in four species of the genus Astyanax (Pisces, Characidae) from Paraíba do Sul River Basin, Caryologia, 56:4, , DOI: / To link to this article: Published online: 18 Dec Submit your article to this journal Article views: 101 View related articles Citing articles: 2 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 23 May 2017, At: 19:11

2 CARYOLOGIA Vol. 56, no. 4: , 2003 Cytogenetical analyses in four species of the genus Astyanax (Pisces, Characidae) from Paraíba do Sul River Basin KARINE FREHNER KAVALCO and ORLANDO MOREIRA-FILHO* Universidade Federal de São Carlos, Departamento de Genética e Evolução. Rodovia Washington Luís, km 235, C.P. 676, CEP São Carlos, SP, Brazil. Abstract - Cytogenetical data in four species of the genus Asytanax from Paraitinga river, Paraíba do Sul river basin, Brazil, are presented. Astyanax scabripinnis and Astyanax parahybae from other localities were previously cytogenetically described, however the karyotypes of Astyanax intermedius and Astyanax giton are described for the first time. A. scabripinnis, A. intermedius, and A. giton showed 2n=50 chromosomes, which are different from to A. parahybae, that presented 2n=48 chromosomes. The four species revealed multiple rdna sites, as detected by silver nitrate staining and FISH-18S. The present data are discussed based on karyotypes and NOR peculiarities, biological aspects of the analyzed species, and their evolutionary consequences. Key words: Karyotypic diversity; Astyanax fish; Paraíba do Sul river; nucleolar organizer regions (NORs); fluorescent in situ hybridization (FISH). INTRODUCTION The Paraíba do Sul river is situated on the bottom of a tectonic depression formed between the Serra do Mar and Serra da Mantiqueira. This river is originated by the confluence of Paraitinga and Paraibuna rivers, composing an isolated basin that flows towards the Atlantic Ocean on Southeastern Brazil. The headwater region of the Paraitinga river is characterized by mountain formations, with several small tributaries that comprise a rich ichthyofauna, where it is verified the occurrence of populational isolates. Aquatic animals show an increased regional diversity in mountain regions where isolated river systems are present (FUTUYMA 1986). It is probable that, over more than 65 millions of years of Otophysi evolutionary history, a great number of geographical and ecological alterations have occurred in the water systems, providing several * Corresponding author: ; address: omfilho@power.ufscar.br examples of vicariance. These events, associated to allopatric speciation, possibly generated the wider portion of the taxonomical diversity in the South American freshwater ichthyofauna (CAS- TRO 1999). The genus Astyanax, whose representatives are popularly known as characins, are distributed from the South of United States to Northern Argentina (EIGENMANN 1921). This Neotropical genus comprises about 74 species (EIGENMANN 1921), several of them inhabiting headwaters. Yet, this number is not precise, and, most likely, there are many species to be described (MELO 2001). Several Astyanax species, such as A. fasciatus, A. bimaculatus, and A. schubarti, among others, can occupy the main river channels, forming large schools of migratory behavior, moving throughout the extension of the hydrographic basins (GODOY 1975). However, the species complex denominated A. scabripinnis (MOREIRA-FILHO and BERTOLLO 1991) is restricted to headwaters of small streams. Thus, the channels of great rivers act as

3 454 KAVALCO and MOREIRA FILHO biological barriers for populations from distinct tributaries, whether by competition with other characin species or by different predators, restraining the gene flow and reinforcing the allopatric condition among them. Such reproductively isolated populations tend to follow diverse evolutionary pathways, since they are under different selection pressures and, because of their restricted population size, can be affected by genetic drift. The chromosomal variability present within Astyanax species reflects their ecological and genetic characteristics. It comprises variations in the diploid number (2n=36 to 2n=50 chromosomes) and karyotypical macrostructure, occurrence of natural polyploidy, presence of B- chromosomes, polymorphism related to distribution of constitutive heterochromatin, and amount and location of nucleolar organizer regions (NORs) (Table 1). The rdna is organized in two genic families: 45S rdna (coding 18S, 5,8S and 28S rrnas) and 5S rdna. Their location can be determined by in situ hybridization with specific probes. NORs sites can also be detected by silver nitrate staining but, in this case, the transcriptional activity of the 45S rdna during the preceding interphasis is required (HOWELL 1977; HUBBELL 1985), as silver nitrate aggregates to nucleolar proteins and not directly to rdna (MILLER et al. 1976). In fish, the localization of 45S rdna cistrons is used as an important cytogenetical marker, revealing groups that characteristically present single NORs (Curimatidae, Anastomidae, Parodontidae, Prochilodontidae, and Cichlidae), and others that bear multiple NORs (Sternopygidae, Characidae, Lebiasinidae, Loricariidae, Erythrinidae, and Callichthyidae) (ALMEIDA-TOLEDO and FORESTI 1985; GALETTI 1998), sometimes located on sex chromosomes (BERTOLLO and CAVALLARO 1992; BORN and BERTOLLO 2000; ARTONI and BERTOLLO 2002). In the present work, the cytogenetical characterization in four species of the genus Astyanax was performed (A. scabripinnis, A. parahybae, A. intermedius, and A. giton), specially focused on nucleolar organizer regions. MATERIAL AND METHODS Six specimens of A. scabripinnis (6 females) from Macacos river (S 22 o 50,050 / Wo 44 o 50,940 ), 9 specimens of A. parahybae (6 males and 3 females), 28 specimens of A. intermedius (2 males and 26 females) from Paraitinga river (S 22 o 52,225 / Wo 44 o 51,041 ), and 21 specimens of A. giton from two different collection sites, (3 males and 10 females) in the Paraitinga river (S 22 o 52,225 / Wo 44 o 51,041 ) and (3 males, 4 females Fig. 1 Brazil map highlighting Southeastern region and its main rivers. The arrow indicates the Paraitinga river.

4 CYTOGENETICAL ANALYSES IN FOUR SPECIES OF THE GENUS ASTYANAX 455 Table 1 Cytogenetical data in the species Astyanax parahybae and Astyanax scabripinnis. Species Collection Sites Hydrographic Basin 2n Bs NORs Chromosomal Types M SM ST A Reference Astyanax parahybae Paraibuna river (Paraibuna - SP) Eastern Basin 48 3 to Paraibuna river (Paraibuna SP) - Eastern Basin Astyanax scabripinnis Água do Rancho river (Maringá PR) Paraná Basin to a , 5, 6 Ligeiro river (Maringá PR) Paraná Basin to , 6 Yucatan river (Maringá PR) Paraná Basin 50 0 a 1 1 to , 5, 6 Sarandi creek (Maringá PR) Paraná Basin to , 6 Centenário stream (Maringá PR) Paraná Basin to , 8 Marrecas creek (Londrina PR) Paraná Basin to , 8 Pedras creek (Itatinga SP) Paraná Basin to , 10 São Pedro creek (Itatinga SP) Paraná Basin to , 10 Tamanduá creek (Itatinga SP) Paraná Basin to Tamanduá creek (Itatinga SP) Paraná Basin to Grande creek (Pardinho SP) Paraná Basin to , 10 Pardo river (Pardinho SP) Paraná Basin to , 10 Claro river (Botucatu SP) Paraná Basin to , 10 Hortelã stream (Botucatu SP) Paraná Basin to Claro river (Salesópolis SP) Paraná Basin to , 13 Capivara river (Botucatu SP) Paraná Basin to , 14 Cascatinha stream (Botucatu SP) Paraná Basin 50 0 a 1 1 to , 15, 16 Lavapés river (Botucatu SP) Paraná Basin to , 10 Araquá river (Botucatu SP) Paraná Basin 50 0 a 1 1 to , 14, 17 Araquá river (Botucatu SP) Paraná Basin to Bicudos creek (Brotas SP) Paraná Basin Canta Galo stream (Itirapina SP) Paraná Basin to Canta Galo stream (Itirapina SP) Paraná Basin to Monjolinho creek (São Carlos SP) Paraná Basin to , 13 Perdizes creek (Campos do Jordão SP) Paraná Basin 50 0 a Pedras stream (Campos do Jordão SP) Paraná Basin 50 0 a , 20 Pedras stream (Campos do Jordão SP) Paraná Basin to , 21 Casquilho creek (Campos do Jordão SP) Paraná Basin 50 0 a Capivari creek (Campos do Jordão SP) Paraná Basin 50 0 a 1 1 to Fojo creek (Campos do Jordão SP) Paraná Basin 50 0 a 2 1 to Carpas lake (Campos do Jordão SP) Paraná Basin 50 0 a 1 2 to Piracuama river (Tremembé SP) Paraíba do Sul Basin (780m alt.) to Piracuama river (Tremembé SP) Paraíba do Sul Basin (1800m alt.) 50 0 a 1 1 to Ribeirão Grande (Pindamonhangaba SP) Paraíba do Sul Basin (700m alt.) to , 24, 25 Grande creek (Pindamonhangaba SP) Paraíba do Sul Basin (1800m alt.) 50 0 a 2 1 to , 24, 25 Grande creek (Pindamonhangaba SP) Paraíba do Sul Basin (1920m alt.) 50 0 a 2 1 to , 24, 25 Lavrinha Farm stream (Campos do Jordão SP) Paraíba do Sul Basin 50 0 a 1 2 to Viveiro de Mudas stream (Três Marias MG) São Francisco Basin to , 28 Barreiro Grande stream (Três Marias MG) São Francisco Basin to , 13 Curral das Éguas stream (São Gonçalo do Abaeté MG) São Francisco Basin to , 28 Curral das Éguas stream (São Gonçalo do Abaeté - MG) São Francisco Basin Jucu river (Vitor Hugo ES) Jucu river Basin 50 0 a 4 1 to Bs: presence of B-chromosomes; NORs: Nucleolar organizer regions; M: metacentric; SM: submetacentric; ST: subtelocentric; A: acrocentric; 1 MORELLI et al. (1983); 2 JUSTI (1993); 3 HERAS (1998); 4 MIZOGUCHI and MARTINS-SANTOS (1997); 5 - MIZOGUCHI and MARTINS-SANTOS (1998a) 6 - MIZOGUCHI and MARTINS-SAN- TOS (1998b); 7 MOREIRA-FILHO and BERTOLLO (1991a); 8 - MANTOVANI et al. (2000); 9 MAISTRO (1991); 10 - MAISTRO et al. (1998); 11 MAISTRO et al. (2000); 12 DANIEL-SILVA (1996); 13 MOREIRA-FILHO (1989); 14 MAISTRO et al. (1994a); 15 MAISTRO et al. (1994b); 16 - PORTO-FORESTI et al. (1997); 17 MAISTRO et al. (1992); 18 SOUZA et al. (1996); 19 VICENTE et al. (1996); 20 SALVADOR and MOREIRA-FILHO (1992); 21 FAUAZ et al. (1994); 22 - FERRO et al. (2001); 23 SOUZA and MOREIRA-FILHO (1995); 24 NÉO et al. (2000a); 25 - NÉO et al. (2000b); 26 - ABEL (2001); 27 ROCON-STANGE and ALMEIDA-TOLEDO (1993).

5 456 KAVALCO and MOREIRA FILHO and 1 individual of undetermined sex) from Jacuí stream (S 23 o 02,436 / Wo 44 o 56,103 ), all belonging to Paraíba do Sul river basin (Fig. 1), were collected. The specimens were identified and kept in the ichthyological collection of the National Museum of Rio de Janeiro, registered as OMNRJ REG The mitotic chromosomes were obtained according to BERTOLLO et al. (1978). NOR silver nitrate staining (Ag-NORs) followed the procedure described by HOWELL and BLACK (1980). The localization of the ribosomal cistrons was accomplished according to PINKEL et al. (1986), by fluorescent in situ hybridization (FISH), with 18S rdna probe (kindly provided by Dr. Terumi Hanakata and Dr. Pedro M. Galetti Jr.). The classification of chromosomal types followed LEVAN et al. (1964), taking into account the arm ratio. RESULTS Astyanax scabripinnis presented 2n=50 chromosomes, a karyotypic formula composed by 8M+20S+8ST+14A and a fundamental number FN=86 (Fig. 2a). The silver nitrate staining (Fig. 3a) showed multiple NORs, with a modal number of 2 marks (an acrocentric pairs). In situ hybridization with 18S rdna probe (Fig. 4a) revealed up to 7 fluorescent signals, several of them discrete. Astyanax parahybae presented 2n=48 chromosomes, with 8M+18SM+12ST+10A and FN=86 (Fig. 2b). The silver nitrate staining (Fig. 3) evidenced multiple NORs, on metacentric and acrocentric chromosomes, with a modal number of 2 marks on a metacentric pair. Fluorescent in situ hybridization with 18S rdna probe (Fig. 4b) identified up to 8 small positive signals. Astyanax intermedius possess 2n=50 chromosomes and a karyotypic formula composed by 6M+8SM+4ST+32A and FN=68 (Fig. 2c). Multiple silver nitrate marks were also observed (Fig. 3c), comprising up to 6 marks, all on acrocentric chromosomes, at the short or long arms. Fluorescent in situ hybridization with 18S rdna probe (Fig. 4c) revealed up to 12 marks, some dot-like. Astyanax giton presented 2n=50 chromosomes and karyotype composed by 6M+8SM+8ST+28A and FN=72, for populations from both sampled localities (Fig. 2d). The silver nitrate staining (Fig. 3d) showed multiple NORs (up to 6 marks), with a modal value equal to 2 marks (an acrocentric pair). Fluorescent in situ hybridization with 18S rdna probe (Fig. 4d) revealed up to 10 marks, many of them very reduced. DISCUSSION The genus Astyanax have proved to be an interesting group for evolutionary studies because of its increased karyotypical variability. Amongst the four surveyed species, A. scabripinnis, A. giton, and A. intermedius presented the same diploid number, 2n=50 chromosomes, however displaying distinct karyotypes and FN values. On the other hand, A. parahybae presented a karyotype composed by 48 chromosomes. None sex-related heteromorphic chromosome was observed in these species. The chromosomal variability of this genus can be further observed within a same species, such as A. scabripinnis, considered a species complex. According to MOREIRA-FILHO and BERTOLLO (1991), this group seems to have evolved independently in each hydrographic basin, or even within a same hydrographic system, from populational isolates. In agreeing with these data, the karyotype found for this species in the headwaters of Paraitinga river presents remarkable differences to others previously described populations, as karyotipic formula and absence of B chromosomes. Even distinct populations from Paraíba do Sul basin reveal great divergences related to chromosomal macrostructure, although the same diploid number is maintained (2n=50 chromosomes) (SOUZA and MOREIRA-FILHO 1995; NÉO et al. 2000a; NÉO et al. 2000b; FERRO et al. 2001). There is a tendency within A. scabripinnis group for karyotypes with an increased number of submetacentric chromosomes, corroborating the data from the present paper (Table 1). Frequently, B-chromosomes are also observed, and the occurrence of such chromosomal elements in other A. scabripinnis populations from Paraíba do Sul basin have been reported (SOUZA and MOREIRA-FILHO 1995; NÉO et al. 2000a; NÉO et al. 2000b; FERRO et al. 2001), but they were absent in the population herein analyzed. Interestingly, the frequency of B-chromosomes can be related to the altitude where each population is found (NÉO et al. 2000a; NÉO et al. 2000b) and, just as proposed, these accessory chromosomes were not observed in populations situated below 1,000m, since the population presently studied, at Macacos river, is located at 972m of altitude. The diploid number of 50 chromosomes observed in A. scabripinnis, A. intermedius, and

6 CYTOGENETICAL ANALYSES IN FOUR SPECIES OF THE GENUS ASTYANAX 457 A. giton, seems to be a plesiomorphic condition among Characidae, as reported by OLIVEIRA et al. (1988). Therefore, A. parahybae would represent a derived condition by the presence of 48 chromosomes, probably originated by Robertsonian rearrangements. Besides, another general feature observed in the karyotypes of several Characidae, including the four analyzed species, is the presence of a metacentric pair much larger than the other chromosomal pairs (SCHEEL 1973; MORELLI et al. 1983; PORTELA et al. 1988). Cytogenetical studies involving R-bands corroborate the hypothesis that such chromosomal pair is conserved within the family (DANIEL-SILVA 2001), constituting a simplesiomorphic character of Characidae. The karyotypes of A. intermedius and A. giton are being presented for the first time. They show a similar structure and small differences are probably due to non-robertsonian rearrangements, such as pericentric inversions, that modified their karyotypic formulae and fundamental numbers. Morphologically, A. giton is differentiated from A. intermedius by a gradual variation in teeth size, a characteristic not present among other Astyanax species from Paraíba do Sul river basin, with exception of A. hastatus (MELO 2001). These species are collected simpatrically in the Paraitinga river, without any evidence of hybridization, which reinforces their genetic isolation. Based on their high karyotypical similarity, it seems plausible to assume that both species are probably derived from a common ancestor. The evolutionary divergence between A. intermedius and A. giton seems to be restricted to the Paraíba do Sul river basin, as the latter species is endemic to such basin, while the former presents a wider distribution, occurring, also at São Francisco and Paraná river basins (FOWLER 1948). Recent taxonomical review elevates A. parahybae to the species status, apart from the Fig. 2 Giemsa stained karyotypes of a - A. scabripinnis; b - A. parahybae; c - A. intermedius; and d - A. giton.

7 458 KAVALCO and MOREIRA FILHO complex A. fasciatus (MELO 2001). Its diploid number, 2n=48, and the karyotypical organization corroborates previous studies (JUSTI 1993). Therefore, A. parahybae trends to maintain a similar karyotypical structure among distinct populations, contrasting to the accentuated karyotypic diversity of A. scabripinnis. Several multiple rdna sites were observed after silver nitrate staining in the four analyzed species, with occurrence of numerical intraindividual variations. Such variation in the number of Ag-NOR were extensively demonstrated in the genus Astyanax (Table 1). It should be pointed out that A. scabripinnis, A. parahybae, and A. giton present one chromosomal pair that is preferentially stained by silver nitrate, as detected in all metaphases analyzed for each species, i. e., an acrocentric pair in A. scabripinnis and A. giton, and a metacentric pair in A. parahybae, indicating the major NORs sites for this species. The fluorescent in situ hybridization with 18S rdna probe revealed a great intraindividual variation in the four analyzed species, up to 12 signals in A. intermedius, 10 in A. giton, 8 in A. parahybae, and 7 in A. scabripinnis. This variability can be related to the amount of rdna repeats from each site, masking the localization of the entire set of rrna genes in the karyotype. Similar difficulties were reported for other populations of A. scabripinnis, concerned to the small size of ribosomal cistrons as a possible cause of their inter- and intraindividual variation (FERRO et al. 2001). The high number of 18S rdna sites in the analyzed species could be determined by dispersal events, such as translocations and/or transpositions, which seems to occur in sever- Fig. 3 Nucleolar organizer regions after silver nitrate staining. The arrows indicate the NORs sites in: a A. scabripinnis; b A. parahybae; c A. intermedius; and d A. giton.

8 CYTOGENETICAL ANALYSES IN FOUR SPECIES OF THE GENUS ASTYANAX al fish groups (CASTRO et al. 1996; GALETTI JR. et al. 1995; ALMEIDA-TOLEDO et al. 1996). This process would be facilitated by the organization of interphase nuclei (S CHWEIZER and LOIDL 1987) and seems to be common within Astyanax. Indeed, up to 15 silver nitrate marks were observed in A. scabripinnis (table 1), suggesting that this should be the minimum number of ribosomal sites in this population (ROCON-STANGE and ALMEIDA-TOLEDO 1993). The present data strengthen out the accentuated cytogenetical variability of the genus 459 Astyanax, based on the karyotypic macrostructure differences, Ag-NORs and rdna location. Such biodiversity might be maximized by the non migratory behavior of some species, which are able to form isolated populations under specific selection pressure. Acknowledgements The authors thank to Paulo A. Buckup, PhD, for taxonomic identification and Dr. Luiz A. C. Bertollo for his contributions. This work was supported by FAPESP (Proc. 01/ and 01/ ) and CNPq. Fig. 4 Fluorescent in situ hybridization with 18S rdna probe. The arrows indicate the sites in: a A. scabripinnis; b A. parahybae; c A. intermedius; d A. giton.

9 460 KAVALCO and MOREIRA FILHO REFERENCES ABEL, L.D.S., 2001 A variabilidade do complexo de espécies scabripinnis (Pisces, Characidae) como estratégia adaptativa. Estudo da diversidade cariotípica do grupo com ênfase em populações da bacia do rio São Francisco. São Carlos SP: Dissertação de Mestrado, Universidade Federal de São Carlos. ALMEIDA-TOLEDO L.F., BIGONI A.P., BERNARDINO G., FORESTI F. and TOLEDO-FILHO S.A., 1996 Karyotype and NOR conservatism with heterochromatin reorganization in Neotropical Bryconids. Caryologia, 49: ALMEIDA-TOLEDO L.F. and FORESTI F., 1985 As regiões organizadoras do nucléolo em peixes. Ciência e Cultura, 37: ARTONI R.F. and BERTOLLO L.A.C., 2002 Evolutionary aspects of the ZZ/ZW sex chromosome system in the Characidae fish, genus Triportheus. A monophyletic state and NOR location on the W chromosome. Heredity, 89: BERTOLLO L.A.C. and CAVALLARO Z.I., 1992 A highly differentiated ZZ/ZW sex chromosome system in a Characidae fish Triportheus guentheri. Cytogenet. Cell Genet., 60: BERTOLLO L.A.C., TAKAHASHI C.S. and MOREIRA- FILHO O., 1978 Cytotaxonomic considerations on Hoplias lacerdae (Pisces, Erythrinidae). Brazil. J. Genetics, 1, 2: BORN G.G. and BERTOLLO L.A.C., 2000 An XX/XY sex chromosome system in a fish species, Hoplias malabaricus, with a polymorphic NORbearing X chromosome. Chromosome Research, 8: CASTRO R.M.C., 1999 Evolução da ictiofauna de riachos sul-americanos: padrões gerais e possíveis processos causais. In: E.P. Caramashi, R. Mazzoni, and P.R. Peres-Neto, Ecologia de Peixes de Riachos, Série Oecologia Brasiliensis, vol. VI. PPGE-UFRJ, Rio de Janeiro, Brasil. pp CASTRO, J. VIÑAS A. SÁNCHEZ L. and MARTINEZ P., 1996 Characterization of an atypical NOR site polymorphism in Brown trout (Salmo trutta) with Ag- and CMA 3 -staining, and fluorescent in situ hybridization. Cytogenet. Cell Genet., 75: DANIEL-SILVA M.F.Z., 1996 Estudos citogenéticos comparativos em quatro espécies do gênero Astyanax (Pisces, Characidae). São Paulo SP: Dissertação de Mestrado Instituto de Biociências da Universidade de São Paulo., 2001 Análises citogenéticas comparativas em Characidae (Pisces, Characiformes). São Paulo SP: Tese de Doutorado - Instituto de Biociências da Universidade de São Paulo. EIGENMANN C.H., 1921 The American Characidae. Mem. Mus. Comp. Zool., 23: Cambridge. FAUAZ G., VICENTE V.E. and MOREIRA-FILHO O., 1994 Natural triploidy and B chromosomes in the neotropical fish genus Astyanax (Characidae). Brazil. J. Genetics, 17: FERRO D.A.M., NÉO D.M., MOREIRA-FILHO O. and BERTOLLO L.A.C, 2001 Nucleolar organizing regions, 18S and 5S rdna in Astyanax scabripinnis (Pisces, Characidae): populations distribution and functional diversity. Genetica, 110: FOWLER H. W., 1948 Os peixes de água doce do Brasil. Arq. Zool., 6: FUTUYMA D., 1986 Evolutionary Biology. 2nd. edition, Sinauer Associates, Inc. GALETTI JR. P.M., 1998 Chromosome diversity in neotropical fishes: NOR studies. Ital. J. Zool., 65(Suppl.): GALETTI JR. P.M., MESTRINER C.A., MONACO P.J. and RASCH E.M., 1995 Post-zygotic modifications and intra- and interindividual nucleolar organizing region variations in fish: report of a case involving Leporinus friderici. Chromosome Research, 3: GODOY M.P., Peixes do Brasil subordem Characoidei. Vol. I, editora Franciscana, Piracicaba-SP, 216pp. HERAS M.P., 1998 Estudos citogenéticos em Astyanax fasciatus (Pisces, Characidae) de rios do Brasil. São Carlos SP: Dissertação de Mestrado Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos. HOWELL W.M., 1977 Visualization of ribosomalgene activity: silver stin proteins associated with RNA transcribed from oocyte chromosomes. Chromosoma, 62: HOWELL W.M., and BLACK D.A., 1980 Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia, 36: HUBBEL H.R., 1985 Silver staining as an indicator of active ribosomal genes. Stain Technol., 60: JUSTI A.J., 1993 Caracterização cariotípica de populações de Astyanax fasciatus (Cuvier, 1819), Pisces, Characidae, em três bacias hidrográficas. São Carlos SP: Dissertação de Mestrado Universidade Federal de São Carlos. LEVAN A., FREDGA K. and SANDBERG A.A., 1964 Nomenclature for centromeric position on chromosomes. Hereditas, 52: MAISTRO E.L., 1991 Caracterização citogenética e morfológica de populações de Astyanax scabripinnis paranae (Pisces, Characidae) das bacias do

10 CYTOGENETICAL ANALYSES IN FOUR SPECIES OF THE GENUS ASTYANAX 461 Tietê e Paranapanema. Dissertação de Mestrado. Universidade Estadual Paulista, Botucatu SP., 2000 Sympatric occurrence of two cytotypes of Astyanax scabripinnis (Characiformes, Characidae). Genet. and Molec. Biol., 23: MAISTRO E.L., FORESTI F., OLIVEIRA C. and ALMEI- DA-TOLEDO L.F., 1992 Occurrence of macro B chromosomes in Astyanax scabripinnis paranae (Pisces, Characiformes, Characidae). Genetica, 87: MAISTRO E.L., FORESTI F. and OLIVEIRA C., 1994a New occurrence of a macro B-chromosome in Astyanax scabripinnis paranae (Pisces, Characiformes, Characidae). Brazil. J. Genetics, 17: MAISTRO E.L., DIAS A.L., FORESTI F., OLIVEIRA C. and MOREIRA-FILHO O., 1994b Natural triploidy in Astyanax scabripinnis (Pisces, Characidae) and simultaneous occurrence of macro B-chromosomes. Caryologia, 47: MAISTRO E.L., OLIVEIRA C. and FORESTI F., 1998 Comparative cytogenetic and morphological analysis of Astyanax scabripinnis paranae (Pisces, Characidae, Tetragonopterinae). Genet. and Molec. Biol., 21: MANTOVANI M., ABEL L.D.S., MESTRINER C.A. and MOREIRA-FILHO O., 2000 Accentuated polymorphism of heterochromatin and nucleolar organizer regions in Astyanax scabripinnis (Pisces, Characidae): tools for understanding karyotypic evolution. Genetica, 109: MELO F.A.G., 2001 Revisão taxonômica das espécies do gênero Astyanax Barid e Girard, 1854 (Teleostei: Characiformes: Characidae) da região da Serra dos Órgãos. Arq. Mus. Nac., 59: Rio de Janeiro. MILLER D.A., DEV V.G., TANTRAVAHI R. AND MILLER O.J., 1976 Supression of human nucleolus organizer in mouse-human somatic hybrid cells. Exp. Cell Res., 101: MIZOGUCHI S.M.H.N. and MARTINS-SANTOS I.C., 1997 Macro- and microchromosomes B in females of Astyanax scabripinnis (Pisces, Characidae). Hereditas, 127: , 1998a Activation patterns of the nucleolar organizer region in Astyanax scabripinnis populations (Pisces, Characidae). Cytologia, 63: , 1998b Cytogenetic and morphometric differences in populations of Astyanax scabripinnis (Pisces Characidae) from Maringá region, PR, Brazil. Genet. and Molec. Biol., 21: MOREIRA-FILHO O., 1989 Análises cariotípicas e morfológicas sobre a diversidade no complexo Astyanax scabripinnis (Jenyns, 1842) (Pisces, Characidae, Tetragonopterinae). Tese de doutorado. Departamento de Ciências Biológicas - Universidade Federal de São Carlos, São Carlos-SP. MOREIRA-FILHO O. and BERTOLLO L.A.C., Astyanax scabripinnis (Pisces, Characidae): a species complex. Brazil. J. Genetics, 14: MORELLI S., BERTOLLO L.A.C., FORESTI F., MOR- EIRA-FILHO O., ALMEIDA-TOLEDO FILHO S., 1983 Cytogenetic considerations on the genus Astyanax (Pisces, Characidae). I. Karyotypic variability. Caryologia, 36: NÉO D.M., BERTOLLO L.A.C. and MOREIRA-FILHO O., 2000b Morphological differentiation and possible origin of B chromosomes in natural population of Astyanax scabripinnis (Pisces, Characidae). Genetica, 108: NÉO D.M., MOREIRA-FILHO O., and CAMACHO J.P.M., 2000a Altitudinal variation for B chromosome frequency in the characid fish Astyanax scabripinnis. Heredity, 85: OLIVEIRA C., ALMEIDA-TOLEDO L.F., FORESTI F., BRITSKI H.A. and TOLEDO FILHO S.A., 1988 Chromosome formulae of neotropical freshwater fishes. Genet. and Molec. Biol., 11: PINKEL D., STRAUME T. and GRAY J.W., 1986 Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci., 83: PORTELA A.L.B.S., GALETTI JR P.M. and BERTOLLO L.A.C., 1988 Considerations on the chromosome evolution of Tetragonopterinae (Pisces, Characidae). Brazil. J. Genetics, 11: PORTO-FORESTI F., OLIVEIRA C., MAISTRO E.L. and FORESTI F., 1997 Estimated frequency of B chromosomes and population density of Astyanax scabripinnis paranae in a small stream. Brazil. J. Genetics, 20: ROCON-STANGE E.A. and ALMEIDA-TOLEDO L.F., 1993 Supernumerary B chromosomes restricted to males in Astyanax scabripinnis (Pisces, Characidae). Brazil. J. Genetics, 16: SALVADOR L.B. and MOREIRA-FILHO O., 1992 B chromosomes in Astyanax scabripinnis (Pisces, Characidae). Heredity, 69: SCHEEL J.J., 1973 Fish chromosomes and their evolution. Internal Report of Denmarks Akvarium, Charlottenlund, Denmark, 22p. SCHWEIZER D. and LOIDL J., 1987 A model for heterochromatin dispersion and the evolution of C band patterns. Chrom. Today, 9: SOUZA I.L. and MOREIRA-FILHO O., 1995 Cytogenetic diversity in the Astyanax scabripinnis species complex (Pisces, Characidae) I. Allopatric distribution in a small stream. Cytologia, 60: VICENTE V.E., MOREIRA-FILHO O. and CAMACHO J.P.M., 1996 Sex-ratio distortion associated to the presence of a B chromosome in Astyanax scabripinnis (Pisces, Characidae). Cytogenet. Cell Genet., 74: Received February 24, 2003; accepted May 26, 2003