C 1999 The Japan Mendel Society Cytologia 64: 63-67, 1999 Extensive NOR Variability in Fishes of the Genus Bryconamericus (Characidae) A. P. Wasko and P. M. Galetti Jr. Departamento de Genetica e Evolucao, Universidade Federal de SaooCarlos, Rodovia Washington Luiz, Km 235, 13565-905, Sao Carlos, SP, Brazil Accepted December 14, 1998 Summary An extensive NOR variability was described in four Bryconamericus species which are still awaiting more precise taxonomic identification. Nine NOR phenotypes were identified by silver nitrate and mithramycin staining, and cytotaxonomy and phylogenetic relationships were inferred. The wide NOR polymorphism probably related to differences in the number of tandemly repeat ribo- somic cistrons and rDNA transcriptional regulation thus far may characterize a particular situation of higher level of NOR phenotype diversification in Bryconamericus than in other neotropical genus fish. Key words Fish, Characidae, Bryconamericus, NOR. The nucleolar organizer regions have been largely studied in fishes and they can constitute good cytotaxonomic markers for some groups (e.g. Galetti Jr. et al. 1984). Many fish species can show only two homologous NOR-bearing chromosomes in their karyotypes (Gold 1984, Takai and Ojima 1986, Amemyia and Gold 1988, among others), while other ones present multiple NOR- bearing chromosomes (Galetti Jr. et al. 1985, Sola et al. 1990, 1992). Among neotropical fishes, the large Characidae family presents no unique NOR distribution pattern. In some subfamilies, such as Salmininae (Marco 1986) and Bryconinae (Margarido and Galetti Jr. 1996), most species seem to have a single NOR pair. In contrast, multiple NORs have been preferentially observed in Tri- portheinae (Bertollo and Cavallaro 1992), Serrasalminae (Galetti Jr. et al. 1985, Cestari and Galetti Jr. 1992a, b), Cynopotaminae (Falcao and Bertollo 1985) and Tetragonopterinae (Portela et al. 1988, Foresti et al. 1989, Moreira Filho 1989). For the best of our knowledge, only two Bryconamericus species had the NOR sites already identified in their karyotypes (Wasko et al. 1996) and similarly to most tetragonopterins multiple NOR-bearing chromosomes were found. In the present work, an extensive NOR variability was detected in four species of Bryconamer- icus and nine NOR phenotypes might be useful to infer on phylogenetic relationships among them. Material and methods NOR studies were carried out in four species of the genus Bryconamericus, temporarily de- nominated A, B, C and D, as previously suggested in Wasko and Galetti Jr. (1998). All studied specimens were deposited at Museu de Ciencias, Pontificia Universidade Catolica do Rio Grande do Sul (MCP). Bryconamericus sp. A (2 females, MCP 19792) and Bryconamericus sp. B (5 fe- males and 10 males, MCP 19793) were collected in the Piracicaba river (municipality of the same name, Sao Paulo State). Bryconamericus sp. C (3 females and 6 males, MCP 19794) was caught in the Tres Bocas stream, a tributary of the Tibagi river (municipality of Sertanopolis, Parand State), and Bryconamericus sp. D (3 females, MCP 19795) was collected in the Avoadeira stream, a tribu- tary of the Garcas river (municipality of Barra do Garcas, Mato Grosso State). Chromosome preparations were obtained by direct preparations (Bertollo et al. 1978) and by 64 A. P. Wasko and P. M. Galetti Jr. Cytologia 64 short-term solid tissue culture (Fenocchio et al. 1991). The nucleolar organizer regions (NORs) were identified by silver nitrate (Ag-) (Howell and Black 1980) and mithramycin A (MM) staining, a GC-specific fluorochrome countarstained with distamycin A (Schmid 1980). Results All analysed species had their karyotypes previously reported, showing 2n= 52 and FN= 88 to 94 (Wasko and Galetti Jr. 1998). Both silver nitrate and MM staining revealed remarkable intra- and interindividual NOR variations in these fishes. Nine NOR phenotypes were detected and hereafter named by 1 to 6, followed by p (short arm) or q (long arm) regarding to the NOR-bearing chromosome arm. Thus, 1p phenotype=NOR site in the short arm of a medium submetacentric, lq=NOR in the long arm of a medium submetacentric, 2p =NOR in the short arm of a large submetacentric, 2q=NOR in the long arm of a large submeta- centric, 3p =NOR in the short arm of a medium acrocentric, 4p =NOR in the short arm of a medi- um subtelocentric, 4q=NOR in the long arm of a medium subtelocentric, 5p =NOR in the short arm of a medium metacentric, 6p =NOR in the short arm of a small metacentric (Table 1, Fig. 1). Two to three Ag-NOR bearing chromosomes were detected in Bryconamericus sp. A, referring to the 1p and 2p NOR phenotypes. Fluorescent MM± bands were observed coincident with these Ag-NOR sites. In Bryconamericus sp. B, up to 3 Ag-NOR bearing chromosomes were evidenced, representing the 3p and 4p NOR phenotypes. MM analyses were able to detect only the two homologous acro- centric chromosomes related to the 3p phenotype. NOR size differences were also present and, if compared to their size when they were present in all three NOR-bearing chromosomes/cell, NOR sites seemed to be either two or three times larger when detected in only two or one chromosome/cell respectively. Up to 4 Ag-NOR chromosomes/cell were observed in Bryconamericus sp. C. This species showed wider variability, represented by 1p, 1 q, 2p, 2q, 3p, 4q, 5p and 6p NOR phenotypes which were also detected by MM staining. Both Ag- and MM staining also revealed up to 4 NOR sites in Bryconamericus sp. D, corre- sponding to the 3p and 4p phenotypes. Table 1. NOR phenotypes of the analysed species (M =metacentric; SM = submetacentric; ST = subtelocentric; A=acrocentric). 1999 NOR Variability in Bryconamericus 65 Fig. 1. NOR phenotypes of Bryconamericus: a) lq, 5p and 6p phenotypes. b) 2p phenotype . c) 2q phe- notype. d) 3p and 4p phenotypes. e) 1p phenotype. f) 4q phenotype. b-f) partial metaphases. Discussion Despite the relative stability detected in the diploid number of the Bryconamericus species (2n= 52), the karyotype structureand the heterochromatindistribution have been reported diverse and cytotaxonomicallypowerful among these fishes(Wasko and Galetti Jr. 1998).The nucleolaror- ganizerregions were also detectedquite variableand all four species studiedhere showedintra- and interindividualvariations in the numberand locationof the rDNAcistrons , characterizingBrycona- mericusas a group with multipleNORs. ExtensiveNOR polymorphismshave alreadybeen report- ed in other Characidae,such as in Serrasalminae(Galetti Jr. et al. 1985), Triportheinae(Bertollo and Cavallaro1992) and Cynopotaminae(Falcao and Bertollo 1985). Sometetragonopterins, other than Bryconamericus,also showedwide NOR polymorphisms(e.g. Portelaet al. 1988,Foresti et al. 1989),and the NOR characterhas its cytotaxonomicpower diminished for major phylogeneticcon- structions. However, most Bryconamericus species studied here could be characterized by their NOR phe- notypes (Table 1), suggesting that at least within a lower taxon, the NOR character, even if highly polymorph, can be useful for taxonomic and phylogenetic approaches. Bryconamericus sp. C pre- sented larger number of NOR phenotypes. Besides this greater variability, this species was also dis- tinguished by the presence of NORs in the long arm of some chromosomes, while NOR sites were only observed at the end of short arms in the remaining species. It seems that a more general fea- Cytologia 64 66 A. P. Wasko and P. M. Galetti Jr. ture is the NOR located in the short chromosome arms within Bryconamericus. In the other hand, there is no common NOR phenotypes shared by all four studied species. Therefore, the 3p NOR phenotype detected in Bryconamericus sp. B, Bryconamericus sp. C, and Bryconamericus sp. D might be a more ancient NOR feature among these fishes. Additionally, Bryconamericus sp. A and Bryconamericus sp. C, sharing two other phenotypes ( 1p and 2p), ap- pear to be more related to each other than to other Bryconamericus species. Similarly, Bryconamer- icus sp. B and Bryconamericus sp. D, besides the 3p NOR phenotype also share the 4p phenotype and may also represent a more related phylogenetic clade. Structural NOR polymorphisms due to differences in the number of tandemly repeat ribosomic cistrons (Warburton et al. 1976, Foresti et al. 1981) or functional polymorphisms related to rDNA transcriptional regulation (Miller et al. 1976, Galetti Jr. et al. 1985) are no rare events among fishes. In the Bryconamericus genus, the extensive NOR polymorphism is probably related to both struc- tural and functional factors. Futhermore, it may characterize a particular situation of higher level of NOR phenotype diversification occurring in this genus than in other neotropical fish group. Acknowledgements The authors are grateful to CNPq for financial support, to Heraldo A. Britski for identification of the studied species and to Luiz R. Malabarba and Jose E Pezzi for suggestions. References Amemyia, C. T. and Gold, J. R. 1988. Chromosomal NORs as taxonomic and systematic characters in North American cyprinid fishes. Genetica 76: 81-90. Bertollo, L. A. C., Takahashi, C. S. and Moreira Filho, O. 1978. Cytotaxonomic considerations on Hoplias lacerdae (Pisces, Erythrinidae). Brazil. J. Genetics 1(2): 103-120. - and Cavallaro, Z. I. 1992. A highly differentiated ZZ/ZW sex chromosome system in a Characidae fish, Triportheus guenteri. Cytogenet. Cell. Genet. 60: 60-63. Cestari, M. M. and Galetti Jr. P. M. 1992a. Chromosome studies of Serrasalmus spilopleura (Characidae, Serrasalminae) from the Parana-Paraguay rivers: evolutionary and cytotaxonomic considerations. Copeia 1: 108-112. -and - 1992b. Chromosome evolution in the genus Serrasalmus and cytotaxonomic considerations about Serrasalminae (Characidae, Pisces). Brazil. J. Genetics 15(3): 555-567. Falcao, J. N. and Bertollo, L. A. C. 1985. Chromosome characterization in Acestrorhinchinae and Cynopotaminae (Pisces, Characidae). J. Fish Biol. 27: 603-610. Fenocchio, A. S., Venere, P. C., Cesar, A. C. G., Dias, A. L. and Bertollo, L. A. C. 1991. Short term culture from solid tis- sues of fishes. Caryologia 44(2): 161-166. Foresti, F, Almeida-Toledo, L. F. and Toledo, S.