Print version ISSN 1679-6225
Neotrop. ichthyol. vol.7 no.4 Porto Alegre 2009
Cytogenetic analysis of Astyanax laticeps (Cope, 1894) (Ostariophysi: Characidae) from the laguna dos Patos system
Renata da RosaI, II; Marceléia RubertIII; Luiz R. MalabarbaIV; Isabel Cristina Martins-SantosI; Lucia Giuliano-CaetanoII
IDepartamento de Biologia Celular e Genética, Universidade Estadual de Maringá. Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil. firstname.lastname@example.org
IIDepartamento de Biologia Geral, CCB, Universidade Estadual de Londrina, 86051-990 Londrina, PR, Brazil. email@example.com
IIIDepartamento de Genética e Evolução, Universidade Federal de São Carlos, 13565-905 São Carlos, SP, Brazil. firstname.lastname@example.org
IVDepartamento de Zoologia, Universidade Federal do Rio Grande do Sul. Av. Bento Gonçalves, 9500, 91501-970 Porto Alegre, RS, Brazil. email@example.com
The genus Astyanax is prominent among Characiformes, due to the large number of species found and its wide geographic distribution. In this work, Astyanax laticeps specimens from the laguna dos Patos system were cytogenetically analyzed. A diploid number of 2n = 50 chromosomes distributed into 6m+16sm+16st+12a (FN = 88) was found, without differences between males and females. A few small heterochromatin blocks were observed, besides three more conspicuous C-bands, corresponding to NORs, as confirmed by silver nitrate and CMA3 staining, FISH, and DAPI negative staining. These regions were located in a medium-sized subtelocentric and in a large subtelocentric chromosomal pair, probably because of a deletion of this region in one homologous chromosome, or due to a transposition event between them.
Key words: Astyanax scabripinnis species complex, rDNA, Homologous transposition.
O gênero Astyanax é destacado entre os Characiformes, pelo grande número de espécies encontradas e a ampla distribuição geográfica. Neste trabalho, foram analisados citogeneticamente espécimes de Astyanax laticeps do sistema da laguna do Patos. O número diplóide observado foi de 2n = 50 cromossomos distribuídos em 6m+16sm+16st+12a (NF= 88), sem diferenças entre machos e fêmeas. Foram observados poucos blocos de heterocromatina, além de três bandas-C mais conspícuas, correspondentes às NORs, confirmado pela coloração com nitrado de prata,CMA3, FISH, e coloração negativa ao DAPI. Estas regiões foram localizadas em um cromossomo subtelocêntrico de tamanho médio e em um par subtelocêntrico grande, provavelmente devido a deleção desta região em um dos cromossomos homólogos, ou por eventos de transposição entre eles.
Within the family Characidae, the genus Astyanax, with 86 species, is considered the second species-richest genus in this family (Lima et al., 2003). A remarkable karyotypical diversity is observed in the genus Astyanax, involving both chromosomal number and morphology. The diploid number in this genus can range from 2n = 36 in A. schubarti Britski 1964 (Moreira-Filho et al., 2001) to 2n = 50 in A. eigenmanniorum (Cope 1894) (Fauaz et al., 1994), A. scabripinnis (Jenyns 1842) (Mizoguchi & Martins-Santos, 1997; Kavalco et al., 2004; Mantovani et al., 2004), A. altiparanae Garutti & Britski 2000 (Pacheco et al., 2001; Fernandes & Martins-Santos, 2006), among others.
Astyanax scabripinnis, the most cytogenetically studied species, shows a high karyotypical diversity, and is considered a species complex (Moreira-Filho & Bertollo, 1991). In fact, none of the previous studies of the karyotype of Astyanax scabripinnis actually seems to have been developed with this species, but with other populations of morphologically similar species. According to Melo (2001) and Bertaco & Lucena (2006), the occurrence of A. scabripinnis is restricted to Rio de Janeiro State, and the species may be extinct, since no additional specimens have been collected after its description. Indeed, all published information about the karyotype of A. scabripinnis will be referred in our text as to the A. scabripinnis species complex and not to a single species. Bertaco & Lucena (2006) have listed fifteen species that form the A. scabripinnis complex. Astyanax laticeps, formerly regarded as a subspecies of A. scabripinnis, has been recently recognized as a distinct species by Bertaco & Malabarba (2001), based on morphological characters and color patterns.
There are no reports in the literature on the cytogenetic features of the species A. laticeps. In the present work, we report the first cytogenetic analysis in this species, including karyotypical description, heterochromatin distribution pattern, localization of AT- and GC-rich regions, and 45S rDNA sequences.
Material and Methods
Eighteen specimens of A. laticeps (MZUEL 4874, ten females and eight males) from rio Forquetinha (52º19'22.7"W 29º16'48.8"S), a tributary of the rio Taquari, laguna dos Patos system, Forquetinha municipality, Rio Grande do Sul State, Brazil, were cytogenetically analyzed. Metaphases were obtained through the air drying method (Bertollo et al., 1978). The chromosomes were classified according to Levan et al. (1964) into metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a). The heterochromatin distribution pattern was determined using C-banding (Sumner, 1972). Active nucleolar organizer regions (NORs) were detected by silver nitrate staining (Howell & Black, 1980). The AT- and GC-rich blocks were visualized by fluorochrome staining, using 4',6-Diamidino-2-phenylindole (DAPI) and Chromomycin A3 (CMA3) respectively (Schweizer, 1976). Fluorescent in situ hybridization (FISH) was performed according to Heslop-Harrison et al. (1991) and Cuadrado & Jouve (1994), using pTa71 probes carrying the 45S rDNA sequence (Gerlach & Bedbrook, 1979).
The studied samples of A. laticeps (Fig. 1a) showed a diploid number of 2n = 50 chromosomes, distributed into 6m+16sm+16st+12a, with a fundamental number (FN) of 88 (Fig. 1b). No chromosomal differences between males and females were detected. The analysis of the constitutive heterochromatin showed weak marks, located on centromeric and telomeric regions of a few chromosomes. Three chromosomes presented small but more evident terminal heterochromatic blocks, which probably corresponded to NORs (Fig. 2d).
All analyzed specimens presented three chromosomes bearing Ag-NORs at the terminal region, comprising a mediumsized subtelocentric and a single homologous pair of a larger subtelocentric chromosme (Fig. 2a). The interphase nuclei stained with silver nitrate had two nucleoli, usually close to each other (Fig. 2b). FISH with 45S rDNA probes also showed fluorescent signals at the telomeric regions of three chromosomes, equivalent to Ag-NOR location. Similarly, the sequential analysis with CMA3 and DAPI fluorochromes showed positive and negative signals (Figs. 2e and f), respectively, on the three NOR-bearing chromosomes (Fig. 2g).
The precise diagnosis of species in the "A. scabripinnis species complex" is hindered by their high degree of similarity in the body morphology. Moreira-Filho & Bertollo (1991), studying morphometric and cytogenetic features in five populations formerly identified as A. scabripinnis, have reliably discriminated them by both canonical variables analysis and the occurrence of sympatric cytotypes with 48 and 50 chromosomes, without any detectable hybrid forms, thereby suggesting the occurrence of a species complex ("A. scabripinnis complex").
The cytogenetic analysis in A. laticeps from rio Forquetinha showed a diploid number of 2n = 50 chromosomes, distributed into 6m+16sm+16st+12a (FN = 88), demonstrating a similarity in the chromosome number observed for most of the A. scabripinnis species complex populations studied so far (Moreira-filho & Bertollo, 1991; Maistro et al., 1992, 2000; Mantovani et al., 2000; Mizoguchi & Martins-Santos, 1998), as well as in other species of the genus: A. mexicanus (Kirby et al., 1977), Astyanax aff. bimaculatus (Pamponet et al., 2008), A. taeniatus (Paganelli, pers. comm.), A. eigenmanniorum (Fauaz et al., 1994), A. altiparanae (Pacheco et al., 2001), A. intermedius, A. giton (Kavalco & Moreira-Filho, 2003), and A. jacuhiensis (Pacheco, pers. comm.).
Regarding the heterochromatin distribution, A. laticeps presented small blocks at pericentromeric and telomeric regions of few chromosomes, besides three more evident heterochromatic blocks equivalent to NORs. Such reduced amount of heterochromatin is also observed in other species of this genus, such as A. parahybae (Centofante et al., 2003), Astyanax sp. B (Fazoli et al., 2003), A. altiparanae (Fernandes & Martins-Santos, 2004), and A. jacuhiensis (Pacheco, pers. comm.). In the A. scabripinnis species complex, there is a wide variation in the heterochromatin distribution, since populations with large heterochromatic blocks and populations presenting a reduced heterochromatin content have been reported (Moreira-Filho & Bertollo, 2001; Maistro et al., 1994; Mizoguchi & Martins-Santos, 1997).
Analyses of major rRNA genes distribution in Astyanax laticeps were performed by Ag-NOR technique, base-specific fluorochromes and FISH with 45S rDNA probes. Silver nitrate staining showed the presence of three NOR-bearing chromosomes with one medium-sized subtelocentric pair and a single homologous element in a large subtelocentric pair. This finding was supported by FISH with 45S rDNA probes, which may indicate the occurrence of a deletion in this region within one of the large subtelocentric homologous, or a transposition between homologues, once the signal on the single chromosome is considerably conspicuous. A similar hypothesis was previously proposed by Mantovani et al. (2000) for populations of A. scabripinnis species complex from the rios Marrecas and Centenario, based on the chromosomal arrangement in the interphase nucleus corroborated by Swcheizer & Loidl (1987). Considering the observed data, individuals with two chromosomes bearing 45S rDNA genes - (homozygote 45S rDNA), one homologue with rDNA genes (heterozygote), and two homologues without the 45S rDNA genes (homozygote without 45S rDNA) would be expected. However, only heterozygotes were observed. A possible explanation for this fact may be the lethality of the homozygous lethal condition for these chromosomes, as already observed in Oncorhynchus mykiss. In this species, a paracentric inversion involving 18S rDNA bearing chromosomes is observed only in heterozygosis; the homozygous for the inverted chromosomes results in death (Porto-Foresti et al., 2004).
The presence of three 45S rDNA sites is a peculiar feature to A. laticeps. Despite having the same chromosome number, the other species of this genus differ in the distribution pattern of ribosomal cistrons. Differential numbers of NORs have already been seen in A. jacuhiensis with only two marks (Pacheco, pers. comm.), A. altiparanae, with 4 to 7 sites (Fernandes, pers. comm.), A. giton with 10, A. intermedius, with 12 small sites (Kavalco & Moreira-Filho, 2003) and A. scabripinnis species complex, with sites ranging from 4 to 14, the largest variation in number of sites in different populations reported to date (Rocon-Stange & Almeida-Toledo, 1993; Ferro et al., 2001).
The three NOR-bearing chromosomes showed CMA3+ and DAPI- blocks, indicating a GC-rich (AT-poor) composition. Similar results on the chemical composition of NORs have been found in A. scabripinnis species complex (Ferro et al., 2001), A. altiparanae (Fernandes & Martins-Santos, 2004), and A. jacuhiensis (Pacheco, pers. comm.), among others. This region seems to be linked to GC-rich heterochromatin segments. The presence of nucleolar organizer regions rich in GC is a common finding among fish species, and it is postulated that this feature occurs because heterochromatin would be interspersed with NORs in this group (Pendás et al., 1993). The present data, besides reporting the karyotype of this species, corroborates the taxonomic recognition of A. laticeps as a valid species, apart from other karyotyped species from the A. scabripinnis species complex.
The authors are grateful to Ana Paula S. Dufech and UFRGS team in helping field work, to Oscar A. Shibatta in cataloguing specimens at MZUEL, and the Brazilian agencies CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), Fundação Araucária and CAPES for financial support. This research had permission from IBAMA (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis) # 11339, cad. 1947869 for collection and Field work.
Bertaco, V. A. & C. A. S. Lucena. 2006. Two new species of Astyanax (Ostariophysi: Characiformes: Characidae) from eastern Brazil, with a synopsis of the Astyanax scabripinnis species complex. Neotropical Ichthyology, 4(1): 53-60. [ Links ]
Bertaco, V. A. & L. R. Malabarba. 2001. Description of two species of Astyanax (Teleostei: Characidae) from headwater streams of southern Brazil, with comments on the "A. scabripinnis species complex". Ichthyological Exploration of Freshwaters, 12(3): 221-234. [ Links ]
Bertollo, L. A. C., C. S. Takahashi & O. Moreira-Filho. 1978. Cytotaxonomic considerations on Hoplias lacerdae (Pisces, Erythrinidae). Brazilian Journal of Genetics, 1: 103-120. [ Links ]
Centofante, L., L. A. C. Bertollo, A. J. Justi & O. Moreira-Filho. 2003. Correlation of chromosomal and morphologic characters in two Astyanax species (Teleostei, Characidae). Ichthyological Exploration of Freshwaters, 14(4): 361-368. [ Links ]
Cuadrado, A. & N. Jouve. 1994. Mapping and organization of highlyrepeated DNA sequences by means of simultaneous and sequential FISH and C-banding in 6x-Triticale. Chromosome Research, 2: 231-338. [ Links ]
Fauaz, G., V. E. Vicente & O. Moreira-Filho. 1994. Natural triploidy and B chromosomes in the neotropical fish genus Astyanax (Characidae). Revista Brasileira de Genética, 17(2): 157-163. [ Links ]
Fazoli, L. C., V. A. B. Silva, A. L. B. Portela-Castro & H. Júlio Jr. 2003. Chromosome characterization of Astyanax sp. B (Characidae, Tetragonopterinae), and endemic species of the Iguaçu river, Paraná, Brazil. Cytologia, 68: 289-293. [ Links ]
Fernandes, C. A. & I. C. Martins-Santos. 2004. Cytogenetics studies in two populations of Astyanax altiparanae (Pisces, Characiformes). Hereditas, 141: 328-332. [ Links ]
Fernandes, C. A. & I. C. Martins-Santos. 2006. Mapping of the 18S and 5S ribosomal RNA genes in Astyanax altiparanae Garutti & Britski, 2000 (Teleostei, Characidae) from the upper Paraná river Basin, Brazil. Genetics and Molecular Biology, 29(3): 464-468. [ Links ]
Ferro, D. A. M., D. M. Néo, O. Moreira-Filho & L. A. C. Bertollo. 2001. Nucleolar organizing regions, 18S and 5S rDNA in Astyanax scabripinnis (Pisces, Characidae): populations distribution and functional diversity. Genetica, 110: 55-62. [ Links ]
Gerlach, W. L. & J. R. Bedbrook. 1979. Cloning characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Research, 7: 1869-1885. [ Links ]
Heslop-Harrison, J. S., T. Schwarzacher, K. Anamthawat-Jonsson, A. R. Leitch, M. Shi & I. J. Leitch. 1991. In situ hybridization with automated chromosome denaturation. Technique, 3: 106-109. [ Links ]
Howell, W. M. & D. A. Black. 1980. Controlled silver staining of nucleous organizer regions with a protective colloidal developer: a 1-step method. Experientia, 36: 1014-1015. [ Links ]
Kavalco, K. F. & O. Moreira-Filho. 2003. Cytogenetical analyses in four species of the genus Astyanax (Pisces, Characidae) from Paraíba do Sul river basin. Caryologia, 56(4): 453-461. [ Links ]
Kavalco, K. F., R. Pazza, L. A. C. Bertollo & O. Moreira-Filho. 2004. Gene mapping of 5S rDNA sites in eight fish species from the Paraíba do Sul river basin, Brazil. Cytogenetics and Genome Research, 106: 107-110. [ Links ]
Kirby, R. F., K. W. Thompsom & C. L. Hubbs. 1977. Karyotypic similarities between the Mexican and blind tetras. Copeia, 1977(3): 578-580. [ Links ]
Levan, A., K. Fredga & A. A. Sandberg. 1964. Nomenclature for centromeric position on chromosomes. Hereditas, 52: 201-220. [ Links ]
Lima, F. C. T., L. R. Malabarba, P. A. Buckup, J. F. P. Silva, R. P. Vari, A. Harold, R. Benine, O. T. Oyakawa, C. S. Pavanelli, N. A. Menezes, C. A. S. Lucena, M. C. S. L. Malabarba, Z. M. S. Lucena, R. E. Reis, F. Langeani, L. Casatti, V. A. Bertaco, C. Moreira & P. H. F. Lucinda. 2003. Genera Incertae Sedis in Characidae. Pp. 106-113. In: Reis, R. E., S. O. Kullander & C. J. Ferraris Jr. (Eds.). Check List of the Freshwater Fishes of South and Central America. Porto Alegre, Edipucrs, 729p. [ Links ]
Maistro, E. L., F. Foresti, C. Oliveira & L. F. Almeida-Toledo. 1992. Occurrence of macro B chromosomes in Astyanax scabripinnis paranae (Pisces, Characiformes, Characidae). Genetica, 87: 101-106. [ Links ]
Maistro, E. L., F. Foresti & C. Oliveira. 1994. New occurrence of a macro B-chromosome in Astyanax scabripinnis paranae (Pisces, Characiformes, Characidae). Brazilian Journal of Genetics, 17(2): 153-156. [ Links ]
Maistro, E. L., C. Oliverira & F. Foresti. 2000. Sympatric occurrence of two cytotypes of Astyanax scabripinnis (Characiformes, Characidae). Genetics and Molecular Biology, 23(2): 365-369. [ Links ]
Mantovani, M., L. D. S. Abel, C. A. Mestriner & O. Moreira-Filho. 2000. Accentuated polymorphism of heterochromatin and nucleolar organizer regions in Astyanax scabripinnis (Pisces, Characidae): tools for understanding karyotypic evolution. Genetica, 109: 161-168. [ Links ]
Mantovani, M., L. D. S. Abel, C. A. Mestriner & O. Moreira-Filho. 2004. Evidence of the differentiated structural arrangement of constitutive heterochromatin between two populations of Astyanax scabripinnis (Pisces, Characidae). Genetics and Molecular Biology, 27(4): 536-542. [ Links ]
Mizoguchi, S. M. H. N. & I. C. Martins-Santos. 1997. Macro- and microchromosomes B in females of Astyanax scabripinnis (Pisces, Characidae). Hereditas, 127: 249-253. [ Links ]
Mizoguchi, S. M. H. N. & I. C. Martins-Santos. 1998. Cytogenetic and morphometric differences in populations of Astyanax "scabripinnis" (Pisces, Characidae) from Maringá region, PR, Brazil. Genetics and Molecular Biology, 21(1): 55-61. [ Links ]
Moreira-Filho, O. & L. A. C. Bertollo. 1991. Astyanax scabripinnis (Pisces, Characidae): a species complex. Revista Brasileira de Genética, 14(2): 331-357. [ Links ]
Moreira-Filho, O., A. S. Fenocchio, M. C. Pastori & L. A. C. Bertollo. 2001. Occurrence of a metacentric macrochromosome B in different species of the genus Astyanax (Pisces, Characidae, Tetragonopterinae). Cytologia, 66: 59-64. [ Links ]
Pacheco, R. B., L. Giuliano-Caetano & A. L. Dias. 2001. Cytotypes and multiple NORs in an Astyanax altiparanae population (Pisces, Tetragonopterinae). Chromosome Science, 5: 109-114. [ Links ]
Pamponet, V. C. C., P. L. S. Carneiro, P. R. A. M. Affonso, V. S. Miranda, J. C. S. Júnior, C. G. Oliveira & F. A. Gaiotto. 2008. A multi-approach analysis of the genetic diversity in populations of Astyanax aff. bimaculatus Linnaeus, 1758 (Teleostei: Characidae) from Northeastern Brazil. Neotropical Ichthyology, 6(4): 621-630. [ Links ]
Pendás, A. M., P. Morán & E. Garcia-Vázquez. 1993. Ribosomal RNA genes are interspersed throughout heterochromatic chromosome arm in Atlantic salmon. Cytogenetic and Cell Genetics, 63: 128-130. [ Links ]
Rocon-Stange, E. A. & L. F.Almeida-Toledo. 1993. Supernumerary B chromosomes restricted to males in Astyanax scabripinnis (Pisces, Characidae). Revista Brasileira de Genetica, 16(3): 601-615. [ Links ]
Schweizer, D. 1976. Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma, 58: 307-324. [ Links ]
Sumner, A. T. 1972. A simple technique for demonstrating centromeric heterochromatin. Experimental Cell Research, 75: 304-306. [ Links ]
Accepted July 20, 2009
Published December 18, 2009