Cytologia 68(4): 363–368, 2003

Cytogenetic Studies and Correlate Considerations on Rhamdiinae Relationships (Pisces, Siluroidei, Pimelodidae)

Alberto S. Fenocchio1,*, Luiz A. C. Bertollo2, Catarina S. Takahashi3, Ana L. Dias4 and Ana C. Swarça4

1 Universidad Nacional de Misiones, Depto. de Genética, Argentina 2 Universidade Federal de São Carlos, Depto. de Genética e Evolução, Brasil 3 Universidade de São Paulo, Faculdade de Filosoﬁa, Ciências e Letras de Ribeirão Preto, Brasil 4 Universidade Estadual de Londrina, Londrina, PR, Brasil

Received June 11, 2003; accepted September 9, 2003

Summary In Pimelodidae fish the available chromosome numbers range from 2nϭ46 to 2nϭ63, the value 2nϭ56 being the most frequent one. In the present paper 6 species belonging to the Rhamdiinae subfamily were analyzed: Rhamdia hilarii, R. quelen, Cetopsorhamdia sp., C. iheringhi, Imparfinis cf. piperatus and Imparfinis aff. schubarti. Chromosome preparations were obtained by direct and short term culture methods from kidney cells and analysed under standard Giemsa staining, C-banding and NOR silver staining. Rhamdia species present a basic karyotype composed by 58 chromosomes and the NORs are located on the short arm of a subtelocentric chromosome pair. Ce- topsorhamdia sp., C. iheringhi and Imparfinis aff. schubarti. also shows 2nϭ58, while I. cf. piperatus is the only analyzed species presenting 2nϭ56. However, these 4 species show an interstitial NOR location. These chromosomal data agree with the occurrence of subgroups in the Rhamdiinae subfamily, proposed on the basis of their morphological traits. Key words Fish cytogenetics, Cytotaxonomy, Rhamdiinae, Siluriformes.

The Pimelodidae fish family comprises upper 300 neotropical species, showing a wide vari- ability in size and shape. Their systematics and taxonomy need a reanalysis and a better definition in several cases because phylogenetic relationships remains obscure being suggested some internal sub-divisions, i.e. Heptapteridae family, including a number of genera of the classical pimelodids (de Pinna 1998). There are more than fifty Pimelodidae nominal genera and several undescribed species and, at least, 4 major groups can be considered into this family (Lundberg et al. 1988, 1991). Karyotypic data about 40 species, mainly from the Pimelodinae and Sorubiminae subfamilies, are available (Toledo and Ferrari 1976, LeGrande 1981, Hochberg and Erdtmann 1988, Oliveira et al. 1988, Fenocchio and Bertollo 1990, 1992, Dias and Foresti 1993, Martins-Santos et al. 1996, Abucarma and Martins-Santos 2001, Swarça et al. 2000, Vissotto et al. 2001). The diploid number ranges from 2nϭ46 (Pimelodella sp.) to 2nϭ63 (Rhamdia hilarii), the value 2nϭ56 being the most frequent one. About 26 genera can be found in Rhamdiinae, sharing a number of synapomorphies (Lundberg et al. 1991). However, some subgroups can also be considered into this subfamily on the basis of morphological traits (Lundberg et al. 1991). In this study are presented some karyotypic data of 6 Rhamdiinae species and their probable evolutionary relationships in this fish subfamily.

* Corresponding author, e-mail: [email protected], [email protected], Present address: Rua Vila Lobos, 793, Jardim Tucanos, Londrina-PR/Brazil. 364 Alberto S. Fenocchio et al. Cytologia 68(4)

Materials and methods Six species, belonging to three genera of Rhamdiinae (sensu Lundberg et al. 1991), were studied: Rhamdia hilarii (2 females and 2 males from Aguapey river, Corrientes, Argentina), R. quelen (3 females from Paraná river, Posadas, Argentina), Cetopsorhamdia iheringhi (3 unsexed specimens, 2 from São Francisco river, Três Marias, Minas Gerais, and 1 from Rio das Marrecas, Paraná, Brasil), Cetopsorhamdia sp. (2 females and 1 male from Canta Galo streem, Itirapina, São Paulo, Brasil); Imparﬁnis aff. schubarti. (1 female and 4 males from Canta Galo streem, Itirapina, São Paulo, and 2 females and 2 males from Ribeirão Três Bocas, Paraná, Brasil) and I. cf. piperatus (1 female and 2 males from Juquiá river, Carlos Botelho, São Paulo, Brasil). Mitotic preparations were obtained from kidney cells by direct and culture cell methods (Bertollo et al. 1978 and Fenocchio et al. 1991). Nucleolar organizer regions (NORs) were identi- ﬁed by a silver staining method, according to Howell and Black (1980). The chromosomes were grouped into metacentrics (M), submetacentrics (SM), subtelocentrics (ST) and acrocentrics (A) according arm ratios and arranged in decreasing order size in the karyotypes.

Results and discussion The populations of Rhamdia hilarii and R. quelen here analyzed present a basic karyotype composed by 2nϭ58 chromosomes, being 26 M, 16 SM, 8 ST and 8 A (NFϭ108) (Fig. 1A, B). However, some reports show sligth variations around this karyotypic formulae, probably due to dif- ferences in chromosome condensation. On the other hand, both species can present a B chromosome system (Hochberg and Erdtmann 1988, Fenocchio and Bertollo 1990), ranging the diploid number until 2nϭ63 in Rhamdia hilarii (Fenocchio and Bertollo 1990, Fenocchio et al. 2000), but in this case species analyzed have not show these chromosomes. NORs are located on an acrocen-

Fig. 1. Karyotypes and in the boxes nucleolar organizer regions (Ag-NORs) of the Rhamdiinae species studied. A. Rhamdia hilarii; B. Rhamdia quelen. In both karyotypes pair N°27 show satellited NOR bearing chromosomes association. 2003 Correlate Consideration on Rhamdiinae Relationships 365 tric chromosome pair, in a terminal position on the short arm. These regions can show a strong size heteromorphism and frequent chromosomal associations. Cetopsorhamdia iheringhi presents 2nϭ58, with a karyotype composed by 11 M, 8 SM, 5 ST and 5 A chromosome pairs. NORs are located in an interstitial position on the long arm of a ST chromosome (Fig. 2A). Cetopsorhamdia sp. shows 2nϭ58 chromosomes, distributed among 11 M, 8 SM, 5 ST and 5 A pairs. NORs are interstitially located on the long arm of the first M chromosome (Fig. 2B). Imparfinis aff. schubarti. also presents 2nϭ58 and a karyotype composed by 11 M, 9 SM, 5 ST and 4 A chromosome pairs. NORs are located on the long arm of the first M chromosome, on a

Fig. 2. Karyotypes and nucleolar organizer regions (Ag-NORs) of the Rhamdiinae species studied. A) Cetopsorhamdia iheringhi; B) Cetopsorhamdia sp.; C) Imparfinis aff. schubarti.; D. Imparfinis cf. piperatus. In the boxes NOR bearing chromosomes. 366 Alberto S. Fenocchio et al. Cytologia 68(4) secondary constriction that frequently appear extremely decondensed (Fig. 2C). Imparfinis cf. piperatus is the only species showing 2nϭ56 chromosomes, with a karyotype entirely composed by biarmed chromosomes: 12 M, 6 SM and 10 ST pairs. NORs are also located in an interstitial position on the long arm of a ST chromosome (Fig. 2D). In view of the present results and other available data it is evident that Rhamdiinae species share some general karyotypic features, i.e., 2nϭ58 chromosomes, a fundamental number (number of chromosomal arms) beyond to 100, only one pair of NORs (Toledo and Ferrari 1976, LeGrande 1981, Hochberg and Erdtmann 1988, Fenocchio and Bertollo 1990, Abucarma and Martins-Santos 2001, Vissoto et al. 2001). Two exceptions could be seen, the first is represented by Imparfinis cf. piperatus that shows 2nϭ56, as well as the absence of acrocentric chromosomes which occur in the other species, indicating that this karyotype is a derivative one. The second is Heptapterus longi- cauda with only 52 chromosomes and single terminal NORs. Lundberg et al. (1991) suggested that some species could constitute subgroups within the Rhamdiinae subfamily (Group B), on the basis of their synapomorphies, occuping different branch- es in the proposed phylogenetic tree. Rhamdia and some other related genera are seen on the same branch of the proposed cladogram, while Cetopsorhamdia and Imparfinis occupy another one, to- gether with other genera (Fig. 3). The branch including Rhamdia shows other seven nominal genera but is only partly resolved. Likewise, in the other, (Lundberg et al. 1991) included Heptapterus, but

Fig. 3. Correlation between available cytogenetic data (*) and Rhamdiinae subgroups. Modified from Lundberg et al. (1988, 1991) and Fenocchio 1993. 2003 Correlate Consideration on Rhamdiinae Relationships 367 some morphological evidences suggest that this genus represents an independent basal taxon, origi- nating the family Heptapteridae (de Pinna 1998). In accordance, cytogenetic data can also support this proposition. Thus, independently of the diploid numbers, NORs allow to set apart these 2 fish groups. Indeed, all Rhamdia species/populations and related genera studied thus far show a single NOR pair located in a terminal position. In opposition, Cetopsorhamdia and Imparfinis species show these regions in an interstitial position on the chromosomes (Hochberg and Erdtmann 1988, Fenocchio and Bertollo 1990, Fenocchio et al. 2000, Abucarma and Martins-Santos 2001, Vissotto et al. 1999, 2001). It is possible to observe that the species included by Lundberg et al. (1988) on the basis of morphological characters into the Group C had 2nϭ54 (Lophiosilurus, Microglanis) and almost all the cytogenetically studied species of the Group A 56 chromosomes (Swarça et al. 2000). The present study makes clear that cytogenetic data can corroborates with the morphological ones in phylogenetic hypothesis offering additional support. However, although these data represent and interesting approach, specially in a very specious family as Pimelodidae it is necessary to study a larger number of species in each group in order to confirm the observations now reported.

Acknowledgements The authors are grateful to Yoshimi Sato, CODEVASF-CEMIG, for assistance with specimens collection and to Conselho Nacional de Desenvolvimento Cientiﬁco e Tecnologico (CNPq) (Fellow to A.S.F. Ptoc. 305218/02-6) and Secretaría de Políticas Universitarias (SPU) for ﬁnancial support.

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