2005 The Japan Mendel Society Cytologia 70(1): 47 51, 2005 Karyotypic Description of Corumbataia cuestae (Pisces, Loricariidae, Hypoptopomatinae) Fábio Mendes Camilo and Orlando Moreira Filho* Universidade Federal de São Carlos, Departamento de Genética e Evolução, Laboratório de Citogenética Animal, Via Washington Luis, Km 235, Cx. Postal 676, 13565-905 São Carlos, SP, Brasil Received October 25, 2004; accepted December 18, 2004 Summary In the present work, cytogenetic studies were performed in Corumbataia cuestae, a member of the subfamily Hypoptopomatinae (Siluriformes, Loricariidae). The specimens were collected in the Lapa Stream, a tributary of the Passa-Cinco River (Sao Paulo State, Brazil). It presented 2n 54 chromosomes with a karyotype constitution of 14M 10SM 3ST/A. Only 2 nucleolar organizing regions were observed in the terminal region of the short arm of the metacentric pair 2. Both were heteromorphic in size, while sequential analyses of the metaphases in Giemsa/silver and Giemsa/C-banding showed that the secondary constrictions correspond to NORs. The constitutive heterochromatin pattern was clearly observed in the nucleolar chromosome pair with large pericentromeric heterochromatic blocks. In the present study the authors report the hypothesis of conservation in the status of the karyotypic macrostructure of the species as compared to other species studied in the subfamily Hypoptopomatinae. Therefore, this study brings a contribution to base the phylogenetic correlations between the Corumbataia genus and other genera in the group. Key words Corumbataia cuestae, Karyotypic structure. The family Loricariidae groups nearly 600 reported species distributed through 70 genera and 6 subfamilies. This is probably the most taxonomically complex family among the Siluriformes (Isbrücker 1980). The subfamily Hypoptopomatinae is the best-known group among the loricariids and it is represented by nearly 70 fish species and 16 genera largely spread across the freshwater courses of South America (Reis and Schaefer 1992, 1998, Isbrücker 2001). In the last few years, studies concerning taxonomy on the hypoptopomatids have been published, with descriptions of genera and species (Britski 1997, Garavello et al. 1998, Reis and Schaefer 1992, 1998, Schaefer 1996, 1997, Schaefer and Provenzano 1998). Yet recently, 2 species were identified in the genus Corumbataia (Britski 1997); Corumbataia tocantinensis and Corumbataia cuestae. Fish from the subfamily Hypoptopomatinae are of small size (most of them less than 60 mm in length), and usually share an adult physiognomy and peculiar morphology in the pectoral ray skeleton (Schaefer 1991). Also, they choose preferentially lotic waters sites from the streams. This site is usually rich in pebbles and the bottom is filled with sand and forage. Among the fish families Loricariidae species are scarcely reported on the chromosomes. Cytogenetic studies (Andreatta et al. 1994) demonstrate that most of the Hypoptopomatidae present invariable chromosome number in the diploid form (2n 54 chromosomes). Despite this tendency, a diversity concerning chromosome morphology, nucleolar organizing regions (NORs) and constitutive heterochromatin pattern is evident. In the present study the chromosome characteristics of C. cuestae were analyzed, in particular the karyotypic structure, the NOR and the heterochromatin variability. Our results allow us to establish comparisons with the other reports and, therefore, to infer on the karyotypic evolution of this * Corresponding author e-mail: omfilho@power.ufscar.br
48 Fábio M. Camilo and Orlando M. Filho Cytologia 70(1) group. Material and methods Two hundred and thirty-one metaphases of Corumbataia cuestae (17 females and 11 males) from the Lapa Stream, an affluent of the Passa-Cinco River a tributary of the Piracicaba River, all belonging to the Parana River basin, were used. The mitotic chromosomes were obtained according to Bertollo et al. (1978). To detection of the constitutive heterochromatin the protocol developed by Sumner (1972) was followed, and to analysis the nucleolar organizing regions (NORs) the technique of silver staining described by Howell and Black (1980) was used. The chromosome types were classified according to their arm-size ratio, as proposed by Levan et al. (1964). Results Metaphases of 28 specimens of C. cuestae, collected from the Lapa Stream in the municipal district of Itirapina, São Paulo State, Brazil, were analyzed. The diploid number (2n 54) was observed with a karyotype structure of 14 pairs of metacentric chromosomes, 10 pairs of submetacentric chromosomes and 3 pairs of subtelocentric/acrocentric chromosomes (Fig. 1). Two nucleolar organizing regions (NORs) were observed, both located at the terminal region of the short arm in the metacentric pair 2. The markings were heteromorphic in size (Fig. 1, boxes a, b and c). After sequential analyses of the metaphases in Giemsa/silver and Giemsa/C-banding it was observed that the secondary constrictions in the second pair of metacentric chromosomes corresponded to the NORs (Fig. 1, boxes d and e). The constitutive heterochromatin pattern was similar for both sexes. It was clearly observed in the second chromosome pair large heterochromatic blocks close to the centromere. It corresponds to the NOR-bearing chromosome pair. The constitutive heterochromatin region was located adjacent to the secondary constriction region, which appears with negative C-banding (Fig. 1, boxes f and g). No evidence was found in relation to sex or supra-numerary chromosomes. Discussion The shortage of cytogenetic studies in fish, as well as the description of new species, in places of large species variability instigates researchers to develop works on karyotypic characterization and molecular studies. The subfamily Hypoptopomatinae is especially relevant in such concern. Hitherto, the most loricariids, included C. cuestae the object of the present study, was not studied on the cytogenetic point of view. The family Loricariidae presents a large variability in the diploid chromosome number ranging from 2n 36 in Rineloricaria latirostris (Giuliano-Caetano 1998) to 2n 96 in Upsilodus sp. (Kavalco et al. 2003); however, the family Hypoptopomatinae seems to be more conserved since all karyotyped species present 2n 54 chromosomes, except for Otocinclus aff. vestitus (2n 72) and Hisonotus gibbosos (2n 58) (Andreata et al. 2000). The diploid number (2n 54) is also reported in other subfamilies of this group such as Neuplecostominae and Hemipsilichthiinae. C. cuestae presented 2n 54 chromosomes as previously reported by Andreatta et al. (1993, 1994) for other species of the Hypoptopomatini tribe. These results also evidenced the occurrence of simple NORs, which, according to Gold (1984) and Moreira-Filho et al. (1984), have been more frequent among the fish species studied through the present approach. The localization of NORs in a single chromosome pair in this species is a trait also observed in all other species of the subfamily Hypoptopomatinae (Andreatta et al. 1994). The silver nitrate marking located in the interstitial region of a single large metacentric chromosome
2005 Karyotypic Description of Corumbataia cuestae (Pisces, Loricariidae, Hypoptopomatinae). 49 Fig. 1. Karyotypic of Corumbataia cuestae (male and female); in box (a), (b) and (c) chromosomes mitotic by Giemsa; in box (d) and (e) chromosomes by silver nitrate staining (NORs); in box (f) and (g) chromosomes by C-banding. pair coincides with that of other species from different genera of this subfamily, such as Otocinclus affinis, Microlepdogaster leucofrenatus, Microlepdogaster sp. and Pseudotocinclus tietensis (Andreatta et al. 1992). It is assumed that the presence of one pair of NORs or multiple NORs characterize determine fish groups (Almeida-Toledo and Foresti 1985). Another aspect observed was the size of the heteromorphism in the NORs, also reported by Andreatta et al. (1994) in all other species studied in this subfamily. Polymorphic situations that involve size differences of the markings in homologous chromosomes are very frequent among species that present simple NORs (Foresti et al. 1981). According to Markovic et al. (1978), the difference in the NOR sizes between homologous chromosomes is possibly a result a probable tandem duplication of one of the NORs or of uneven cross-overs, possibly being transmitted by inheritance. In this species, the correspondence between NORs and secondary constrictions was evident. This Giemsa staining characteristic is also found in other species of this and other fish groups. Nevertheless, it is important to point out that this kind of relationship, despite very common, is not always present in all fish species (Pa-
50 Fábio M. Camilo and Orlando M. Filho Cytologia 70(1) ganelli 1990). The observable constitutive heterochromatin regions were very tenuous in the centromeric and telomeric regions of some chromosomes, but very clear heterochromatic blocks were restricted to one homologous chromosome pair; this marking corresponding to the NORs. C-banding in karyotypes of other fish species of the subfamily Hypoptopomatinae evidence markings in various chromosomes. In Hypostomus, very evident and pale markings are also frequently found (Artoni and Bertollo 1996). The results from the present study on the cytogenetics of C. cuestae permit to conclude that the karyotypic macrostructure of the subfamily Hypoptopomatinae is very conserved and contributes to establish the phylogenetic bases for the interrelationships between Corumbataia and other genera of this fish group. Acknowledgements Federal University of São Carlos, CNPq and FAPESP supported this work. References Almeida-Toledo, L. F. and Foresti, F. 1985. As regiões organizadoras do nucléolo em peixes. Ciência e Cultura 37: 448 453. Andreatta, A. A., Almeida-Toledo, L. F., Oliveira, C., Toledo-Filho, S. A. 1992. Chromosome studies in Hypoptopomatinae (Pisces, Siluriformes, Loricariinae) I: XX/XY sex chromosome heteromorphism in Pseudotocinclus Tietensis. Cytologia 57: 369 372.,, and 1993. Chromosome studies in Hypoptopomatinae (Pisces, Siluriformes, Loricariinae) II: ZZ/ZW sexchromosome system, B chromosomes, and constitutive heterochromatin differentiation in Microlepdogaster Leucofrenatus. Cytogenet. Cell Genet. 63: 215 220.,, and 1994. Chromosome studies in Hypoptopomatinae (Pisces, Siluriformes, Loricariinae) III: Analysis of seven species. Caryologia 47: 27 37., Alves, A. L., Foresti, F. and Oliveira, C. 2000. Hisonotus gibbosos (Teleostei, Loricariidae) segundo Hypoptopomatineo com número diplóide diferente de 54 cromossomos. Genet. Mol. Biol. 23 (Suppl.): 57. Artoni, R. F. and Bertollo, L. A. C. 1996. Cytogenetic studies on Hypostominae (Pisces, Siluriformes, Loricariidae). Considerations on karyotype evolution in the genus Hypostomus. Caryologia 49: 81 89. Bertollo, L. A. C., Takahashi, C. S. and Moreira-Filho, O. 1978. Cytotaxonomic considerations on Hoplias Lacerdae (Pisces, Erythrinidae). Rev. Bras. Genet. 1: 103 120. Britski, H. A. 1997. Descrição de um novo gênero de Hypoptopomatinae, com duas espécies novas (Siluriformes, Loricariidae). Papéis Avulsos Zool. 40: 231 55. Foresti, F., Almeida-Toledo, L. F. and Toledo-Filho, S. A. 1981. Polymorphic nature of nucleolus organizer regions in fishes. Cytogenet. Cell. Genet. 31: 137 144. Garavello, J. C., Britski, H. A. and Schaefer, S. A. 1998. Systematics of the genus Otothyris Myers 1927, with comments on geographic distribution (Siluriformes: Loricariidae: Hypoptopomatinae). Am. Mus. Novit. 3222: 19. Giuliano-Caetano, L. 1998. Polimorfismo cromossômico Robertsoniano em populações de Rineloricaria Latirostris (Pisces, Loricariinae). Tese de Doutorado. Departamento de Genética e Evolução, Universidade Federal de São Carlos-SP. Gold, J. R. 1984. Silver staining and heteromorphism of chromosomal nucleolus organizer region in North American cyprinid fishes. Copéia 1: 133 139. 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: 1014 1015. Isbrüquer, I. J. H. 1980. Classification and catalogue of the mailed Loricariidae (Pisces, Siluriformes). Verslagen en technische Gegevens 22: 1 181. 2001. Nomemklator der gattungen und arten der harnischwelse, familie Loricariidae Rafinesque, 1815 (Teleostei, Ostariophysi). Datz Sonderheft Hamischwelse 2: 25 32. Kavalco, K. F., Bertollo, L. C. A. and Moreira-Filho, O. 2003. Contribuição citogenética à análise da biodiversidade da ictiofauna das nascentes do Rio Paraitinga. Dissertação de mestrado. Departamento de Genética e Evolução, Universidade Federal de São Carlos-SP. Levan, A., Fredga, K. and Sandberg, A. A. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52: 201 220. Markovic, V. D., Worton, R. G. and Berg, J. M. 1978. Evidence for the inheritance of silver-stained nucleolus organizer re-
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