P.M. Galetti Jr. a, A.C.G. Cesar a & P.C. Venere a a Departamento de Genética, Universidade Federal

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1 This article was downloaded by: [ ] On: 25 March 2014, At: 10:41 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics Publication details, including instructions for authors and subscription information: Heterochromatin and NORs variability in Leporinus fish (Anostomidae, Characiformes) P.M. Galetti Jr. a, A.C.G. Cesar a & P.C. Venere a a Departamento de Genética, Universidade Federal de São Carlos, São Carlos, SP, Brazil. Published online: 31 Jan To cite this article: P.M. Galetti Jr., A.C.G. Cesar & P.C. Venere (1991) Heterochromatin and NORs variability in Leporinus fish (Anostomidae, Characiformes), Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics, 44:3-4, , DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages,

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3 CARYOLOGIA Vol. 44, n. 3-4: , 1991 Heterochromatin and NORs variability in Leporinus fish (Anostomidae, Characiformes) P.M. GALETTI JR., A.C.G. CESAR and P.C. VENERE Departamento de Genetica, Universidade Federal de Sao Carlos, Sao Carlos, SP, Brazil. INTRODUCTION SUMMARY- C-banding studies in four species of Characiformes fish of the genus Leporinus (Anostomidae) identified interspecific differences despite the strict s\milarity in karyotype macrostructure among them. A perceptible reduction in centromeric heterochromatin observed in L. piau seems to be a derived condition. L. amblyrhyncus and L. taeniatus exhibit a centromeric and telomeric heterochromatin pattern which is possibly basic in the group, and L. striatus shows a few exclusive intercalary blocks probably derived from inversions and/or transpositions. The nucleolar organizer regions, which were always found to be associated with heterochromatin segments, appear in different chromosomes or chromosome sites in the species studied, also indicating the occurrence of modifications during karyotype evolution in these fish. Fish of the genus Leporinus (Anostomidae, Characiformes) are typically neotropical and occur from Central America to the southernmost tip of South America. This group presents a marked wealth of shapes, color patterns and body size. Even though it can be recognized subgroups with characteristic color patterns this genus seems to be monophyletic (GARAVELLO 1979). Leporinus shares a very similar karyotype with other genera of the family Anostomidae. All have 2n =54 and an almost identical karyotype structure with meta- and submetacentric chromosomes. Some differentiations, however, occur within the genus. Although most of the species investigated have been found to be homogametic, there are a few that present distinctive sex chromosomes (GALETTI etal. 1981a; GALETTI and FoREST! 1987). Moreover, studies of the location of nucleolar organizer regions have also shown several differences among these fish (GALETTI et al. 1984). In a recent study of C-banding positive Supported by Funda~ao de Amparo a Pesquisa do Estado de Sao Paulo and Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico.

4 288 GALETTI, CESAR and VENERE heterochromatin in the Anostomidae, GALETTI et al. (1991) identified differences conspicuous within Leporinus, Schizodon and Leporellus genera. In some Leporinus species, heterochromatin is practically absent and, when present, does not show bright C bands but, on the contrary, is rather pale. On the other hand, Leporellus shows considerably increased pericentromeric heterochromatin blocks with conspicuous, well-contrasted C-band blocks. In turn, Schizodon shows an intermediate situation, with more heterochromatin (centromeric in particular) than Leporinus but definitely less than Leporellus. These differences are clear evidence of the importance of heterochromatin in the chromosome evolution of these fish. To study heterochromatin and NOR variability in Anostomidae fish chromosomes, four species of the genus Leporinus were now investigated and some considerations about the karyotype evolution and genus diversification were made. MATERIALS AND METHODS The study was conducted on four species of Anostomidae fish (Characiformes) belonging to the genus Leporinus which represent two subgroups (sensu GARAVELLO 1979) of body color patterns: (i) longitudinal bands along the body - L. striatus, 14 specimens (6 males and 8 females) from the Mogi-Guas;u river (SP), Upper Parana basin, L. taeniatus, 8 specimens (3 males and 5 females) and L. amblyrhyncus, 5 specimens (3 males and 2 females), both from the Sao Francisco river, Tres Marias (MG) region, Sao Francisco basin; (ii) three lateral spots over the body - L. piau, 4 specimens (2 males and 2 females) from the Sao Francisco river, Tres Marias (MG) region, Sao Francisco basin. Chromosome preparations were obtained from cell suspensions of the cephalic portion of the kidney according to BERTOLLO et al. (1978). Constitutive heterochromatin was identified with barium hydroxide (SUMNER 1972) and the nucleolar organizer regions were identified by the colloidal silver nitrate technique (HowELL and BLACK 1980). RESULTS Of the species studied here Leporinus striatus already has a defined karyotype described by GALETTI et al. (1981b). Like the remaining species now studied, it shows a diploid number of 54 chromosomes exclusively consisting of biarmed types (Fig. 1). Constitutive heterochromatin shows similar distribution in L. amblyrhyncus and L. taeniatus, which also show preferentially small centromeric and telomeric blocks (Figs. 2 and 3). In L. striatus, in addition to these blocks, a few intercalary C-bands appear, such as those observed in chromosomes 2, 6 and 8

5 HETEROCHROMA'llN AND NORs IN LEPORINUS FISH 289 Fig Leporinus amblyrhyncus (A), L. striatus (B), L. taeniatus (C) and L. piau (D) Giemsa-karyotypes. Fig C-handing (A) and Ag-NOR (B) chromosomes in L. amblyrhyncus. (Fig. 4). In the first, one large heterochromatic block is located on the long arm immediately below the centromere and is crossed by the nucleolar organizer sites identified by silver staining. At times, these sites appear to be duplicated within the heterochromatin. Ag-NOR is terminal on the short arm of a large metacentric chromosome (the 4th in the complement) in L. amblyrhyncus and it is in the terminal portion of a small metacentric (possibly the 23th) in L. taeniatus. Heterochromatin also appears to be associated with these regions in both species. In L. taeniatus, perceptible intraindividual variations are also observed among homologous Ag-NORs and they coincide with variations in

6 290 GALETn, CESAR and VENERE Fig Fig C-banding (A) and Ag-NOR (B) chromosomes in L. taeniatus. C-banding (A) and Ag-NOR (B) chromosomes in L. striatus. Fig C-banding (A) and Ag-NOR (B) chromosomes in L. piau. secondary constrictions visualized by Giemsa staining and by C-banding. In L. piau, heterochromatin appears to be much more reduced (Fig. 5). Positive C bands are practically absent in the centromeres and are quite faint in most of the telomeres, while a few chromosome pairs (2, 17 and 26) present more evident C-hand blocks. Bright C-hand blocks were observed in the terminal portions of both arms of chromosome 2. In the short arm, it is associated with the nucleolar organizer regions. Clear intraindividual variations were observed among the homologous Ag-NOR sites.

7 HETEROCHROMATIN AND NORs IN LEPORINUS FISH 291 DISCUSSION The present results agree with previous data that the Leporinus species share a strictly similar karyotype structure (GALETTI et al. 1981b; GALETTI and FoREST! 1987). However, despite a strong tendency towards the maintenance of a common karyotypic macrostructure, changes in heterochromatin and NORs are observed. Heterochromatin distribution is similar in L. amblyrhyncus and L. taeniatus, with a characteristic occurence in the centromeres and telomeres of the chromosomes. There are indications that this pattern may be basic among Anostomidae (GALETTI et al. 1991). Of the four species studied, Leporinus striatus seems to have more conspicuous heterochromatin segments and is the only species showing intercalary blocks in some chromosomes. These segments may possibly derive from inversions or even transpositions along these chromosomes. In L. piau there is a considerable reduction in the heterochromatin, specially in the centromeric region. This pattern of virtual absence of centromeric heterochromatin has been reported for other species of Leporinus (GALETTI and FoREST! 1987; GALETTI et al. 1991) and seems to represent a derived unit within the genus. It is interesting to point out that the species with reduced heterochromatin have the same body color pattern (three lateral spots) and belong to a common morphological subgroup (GARAVELLO 1979). In contrast, L. amblyrhyncus, L. taeniatus and L. striatus, belong to another subgroup which includes the fish with longitudinal stripes along the body. Thus, it appears that the reduction in heterochromatin in Leporinus with three spots is a mechanism of chromosome evolution that accompanied their morphological differentiation. Even though all species of Leporinus show only two Ag-NORs per cell, as reported for other Anostomids (GALETTI et al. 1984), these sities were present on different chromosomes in each species. Because these chromosome segments are quite susceptible to changes (ScHMID 1980; within others), either chromosome rearrangements or transpositions or both can lead to differences in NOR locations within related species (GoLD 1984; Plm..uPs et al. 1989; VENERE and GALETTI 1989). These mechanisms also may be probable causes of the differences observed among these Leporinus species. The heterochromatin, which appears associated with the NORs, may contribute to NOR dislocations. Homologous Ag-NOR variations are found in the species studied here. These variations are also common in fish chromosomes in general, and either represent structural polymorphism (FoREST! et al. 1981) or are due to genetic regulation of their ribosomal cistrons (GALETTI et al. 1985). The presence of the heterochromatin in these chromosome regions can favour structural changes (MoREIRA Filho et al. 1984) as well as may be an important element in the probable genetic control of these cistrons. Thus, we suggest the occurrence of changes in the chromosome microstructure in Leporinus genus although the karyotype macrostructure remains

8 292 GALETII, CESAR and VENERE unaltered. These changes were shown by heterochromatin and NORs differences among the species. Some of these microstructural changes may have phylogenetic sense and the reduction of the heterochromatin seems to be a chromosome evolutionary path shared by some species in the genus Leporinus. Other chromosome banding methods should provide valuable details. Acknowledgments. - We are grateful to Companhia do Desenvolvimento do Vale do Siio Francisco (Tres Marias, MG) for help in the collection of some species studied, and to Ellen M. RAscH, East Tennessee State University, for valuable comments on the manuscript. REFERENCES BERTOU.O L.A.C., TAKAHAsHI C.S. and MoREIRA Filho 0., Cytotaxonomic considerations Hoplias lacerdae (Pisces, Erythrinidae). Brazil. J. Genet. 1: FoREsn F., ALMEmA ToLEDO L.F. and ToLEDO Filho S.A., Polymorphic nature of nucleolus organizer regions in fishes. Cytogenet. Cell Genet., 31: GALETII JR. P.M. and FoREsn F., Evolution of the ZZ/ZW system in Leporinus (Pisces, Anostomidae). Role of constitutive heterochromatin. Cytogenet. Cell Genet., 43: GALETIIJR. P.M., FoREsn F., BERTOu.o L.A. C. and MoREIRA Filho 0., 1981a. -Heteromorphic sex chromosomes in three species of the genus Leporinus (Pisces, Anostomidae). Cytogenet. Cell Genet., 29: , 1981b. - Karyotypic similarity in three genera (Leporinus, Leporellus and Schizodon) of the family Anostomitlae (Pisces, Teleostei). Brazil. J. Genet. 4: , 1984., - Characterization of eigpt species of Anostomidae. (Cypriniformes) fish on the basis of the nucleolar organizing region. Caryologia, 37: GALE1TI JR. P.M., MESTR.INER C.A., VENERE P.C. and FoREsn F., Heterochromatin and karyotype reorganization in fish of the family Anostomidae (Characiformes). Cytogenet. Cell Genet., 56: GALE1TI JR. P.M., Sn.vA KB. and CERMINARO R.T., A multiple NOR system in the fish Serrasalmus spilopleura (Serrasalminae, Characidae). Brazil. J. Genet., 8: GARAVEll.O J.C., Revistio taxonomica do ghlero Leporinus Spix, 1829 (Ostariophysi, Anostomidae). Unpubl. Doctoral Thesis, Departamento de Zoologia, Institute de Biociencias, Universidade de Siio Paulo. GoLD J.R., Silver staining and heteromorphism of chromosomal nucleolus organizer regions in North American cyprinid fishes. Copeia, 1984, 1: HoWELLW.M. and BLACK D.A., Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia, 36: MoREIRA Filho 0., BERTOu.o L.A.C. and GALE1TI JR. P.M., Structure and variability of nucleolar organizer regions in Parodontidae fish. Can. J. Genet. Cytol., 26: PHiu..n>s R.B., PLEYTE K.A., ERT L.M.V. and HARTLEY S.E., Evolution of nucleolar organizer regions and ribosomal RNA genes in Salvelinus. Physiol. Ecol. Japan, 1: ScHMID M., Chromosome banding in Amphibia. N. Differentiation of GC- and AT-rich chromosome regions in Anura. Chromosoma, 77: SUMNER A.T., A simple technique for demonstrating centromeric heterochromatin. Expl. Cell Res., 74: VENERE P.C. and GALE1TI JR. P.M., Chromosome evolution and phylogenetic relationships of some neotropical Characiformes of the family Curimatidae. Brazil. J. Genet.; 12: Received 20 March 1991; revision accepted 21 July 1991