A Monophyletic ZW Sex Chromosome System in Leporinus (Anostomidae, Characiformes)

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1 _??_ 1995 The Japan Mendel Society Cytologia 60: , 1995 A Monophyletic ZW Sex Chromosome System in Leporinus (Anostomidae, Characiformes) P. M. Galetti Jr1., N. R. W. Lima2 and P. C. Venere3 1 Departmento de Genetica e Evolucao, Universidade Federal de Sao Carlos, Caixa Postal 676, Sao Carlos SP Brazil 2 Setor Eco-Genetica Evolutiva, Laboratorio de Ciencias Ambientais, Universidade Estadual Norte Fluminense, Campos RJ Brazil 3 Departamento de Biologia, ICLMA, Universidade Federal do Mato Grosso, Pontal do Araguaia MT Brazil Accepted September 22, 1995 After the first cases of sex chromosomes were described in fish (Chen and Ebeling 1966, Ebeling and Chen 1970, among others), several studies have demonstrated their occurrence in different fish groups (for a review, see Oliveira et al. 1988, Moreira-Filho et al. 1993). However, contrary to mammals and birds, in which the XY and ZW systems, respectively, appear to have evolved only once, sex chromosomes have arisen several times in fish. It has been described cases ranging from simple XO, XY and ZW systems to multiple sex chromo somes, and with heterogametic males in some cases and heterogametic females in others (Bertollo et al. 1979, 1983, Moreira-Filho et al. 1980, Almeida Toledo et al. 1984, Bertollo and Gavallaro 1992 among others). Leporinus is a fish group rich in species (approximately 70 known thus far) which occurs from Central America to the River Plate basin in South America (Garavello 1979). From a cytologic viewpoint, this group has been characterized by the occurrence of both homogametic and heterogametic species, a fact that makes it highly interesting for the study of sex chromosome evolution. At the time the sex chromosomes of this fish group were first described (Galetti et al. 1981), an XX/XY system was reported for L. lacustris and a ZZ/ZW system for L. obtusidens and L. elongatus (respectively cited as L. silvestrii and L. obtusidens). Recently, the X and Y chromosomes of the L. lacustris were not confirmed by meiotic studies of the synaptonemal complex in combination with more detailed analyses of mitotic chromosomes by different banding methods (Mestriner et al. 1995). However, the W chromosome of L. obtusidens and L. elongatus is a large subtelocentric, the largest in the female complement, and is definitely known to be absent in males. This same chromosome was later detected in L. reinhardti and in Leporinus sp (currently identified as L. macrocephalus) (Galetti and Foresti 1987). Because of the morphological similarity of these chromosomes in these four species, also in terms of distribution of C-positive bands, it has been suggested that the differentiation of these Z and W chromosomes occurred in a probable common ancestor (Galetti and Foresti ). In the present study, we provide further considerations about the origin and distribution of these chromosomes within the genus Leporinus and describe two new occurrences in L. conirostris and L. trifasciatus. Materials and methods Mitotic chromosomes of the following animals were analyzed: L. conirostris, 9 specimens 1 Corresponding author.

2 376 P. M. Galetti Jr., N. R. W. Lima and P. C. Venere Cytologia 60 (6 males and 3 females) from the Paraibuna river, a tributary of the Paraiba do Sul river (East basin) collected in the township of Paraibuna (SP); L. trifasciatus, 6 specimens, 1 male and 2 females from the Madeira river close to the township of Porto Velho (RO), and 3 additional females from the Solimoes river close to the island of Marchantaria, both sites located in the Amazon region. Chromosome preparations were obtained from cell suspensions of the cephalic portion of the kidney by standard methods, basically as described by Bertollo et al. (1978). Constitutive heterochromatin was identified with barium hydroxide (Sumner 1972) and the nucleolar organizer regions were visualized by the silver staining method of Howell and Black (1980). Results L. conirostris and L. trifasciatus have 2n=54 chromosomes in both sexes. The karyotype of these species exclusively consists of meta- and submetacentric chromosomes, except for a large W subtelocentric chromosome present in the female karyotype and absent in males (Fig. 1). A large C band-positive block occupies almost the entire length of this chromosome in both species (Figs. 2a, b, c). The probable Z chromosomes, a single element in the female karyotype and two elements in the male karyotype, have a terminal heterochromatin block occupying approximately 40-60% of the length of the long arm. In L. conirostris a small interstitial heterochromatin block appears on the long arm of the probable Z chromosome proximal to the centromere (Fig. 2d). In both species, two chromosomes/cell bearing Ag-positive sites corresponding to the nucleolar organizer regions were identified in most of the cells analyzed (Figs. 3a, b, c, d, 4a, b). Intraindividual size variations between the homologous NORs might be often observed in L. conirostris (Fig. 3a, b) while in L. trifasciatus some cells presented three chromosomes bearing Ag-NOR sites (Fig. 4c). Fig. 1. a and b refers to the karyotypes of L. trifasciatus and L. conirostris, respectively. Z and W sex chromosomes are shown in the boxes.

3 1995 ZW Sex Chromosomes in Leporinus Fish 377 Fig. 2. Z and W sex chromosomes after C banding. a and b metaphases of L. trifasciatus females. c refers to L. conirostris female and d male individuals. Discussion 1. Origin and evolution of chromosomes Z and W The presence of a large subtelocentric chromosome exclusively detected in the karyotypes of females of both species characterizes new occurrences of the ZZ/ZW system of sex chromosomes in the genus Leporinus. The morphological similarity of chromosomes Z and W between these species and the other ZW Leporinus previously studied (Galetti et al. 1981, Galetti and Foresti 1986, 1987) further supports the hypothesis of a common origin for these chromosomes. Thus, this system of ZW sex chromosomes probably represents a synapomorphy between this group of species within the genus Leporinus. Two major paths have been proposed for the first step of sex chromosomes differentiation in lower vertebrates. First, mechanisms of structural chromosome modifications, as pericentric inversions for certain snakes (Becak and Becak 1969) and translocations for some fishes (Moreira-Filho et al. 1980, Bertollo et al. 1983, Almeida Toledo et al. 1984), may or may not be followed by an ulterior heterochromatinization. In the second, an initial heterochromatin ization due to the incorporation and development of specific satellite DNA sequences (Singh et al. 1976, 1980) may be followed by an accumulation of this segment along the chromosome, as

4 378 P. M. Galetti Jr., N. R. W. Lima and P. C. Venere Cytologia 60 Fig. 3. Arrows showing NOR-bearing chromosomes of L. conirostris submitted to silver nitrate staining. a complete and b, c and d partial metaphases with two NORs/cell. on the W chromosome long arms in Leporinus species (Galetti and Foresti 1986, 1987). The presence of heterochromatin on the long arm of the now existing Z chromosomes strongly suggests that the heterochromatinization has occurred similarly on both original Z chromo somes. After an accumulation of the heterochromatin segment by regional replication or tandem duplication, or both, might develop the present W chromosome (Galetti and Foresti 1986, 1987). This differential heterochromatinization process has been recorded as a decisive factor in the prevention of meiotic exchanges between sex chromosomes (Singh et al. 1980, among others), facilitating the isolation of sexually related DNA segments. 2. Cytotaxonomy and phylogenetic considerations As previously observed in most of the Leporinus species studied (Galetti et al. 1984, Galetti and Foresti 1987, Galetti et al. 1991a), the AgNORs of the two species studied here are located on two chromosomes of the complement, even though the NOR-bearing chromosomes are different between them, i.e., a medium sized metacentric in L. conirostris and a large metacen tric in L. trifasciatus. Interestingly, the NOR-bearing chromosomes are also different among the remaining ZW species described (Galetti et al 1984, Galetti and Foresti 1987), and their identification in the chromosome complement might play an important cytotaxonomic role for these species. It is well known, however, that the method of NOR identification by silver nitrate staining only reveals genetically active sites (Miller et al. 1976, Hsu et al. 1976) and intra- and interindividual variability is a common occurrence, specially related to size differences between homologous NORs (Foresti et al. 1981, Moreira-Filho et al. 1984, Phillips et al. 1989, among

5 1995 ZW Sex Chromosomes in Leporinus Fish 379 Fig. 4. Arrows showing NOR-bearing chromosomes of L. trifasciatus after silver nitrate staining. a complete, and b partial metaphases showing two NORs/cell. c partial metaphase with three NORs/cell. others). About 10-15% of the L. trifasciatus cells analyzed present a third chromosome bearing Ag-NOR sites, suggesting that usually inactivated secondary sites may occur in this species. Similar results have been reported for other Leporinus (Galetti et al. 1991b, Galetti et al. 1995). Thus, when considering the cytotaxonomic aspect of these chromosome regions, it should be accounted their variability. Although they seem to represent a natural group of species, the fish of the genus Leporinus have different body color patterns that may even characterize different subgroups (Garavello 1979). All ZW species detected thus far have the same color pattern (three lateral maculae along the body), although other species having this same pattern have no sex chromosomes (Galetti et al. 1991a, b). If the pattern of three lateral maculae along the body is of a certain phylogenetic significance, the ZW species would be distinct from the remaining ones with the same pattern by the presence of sex chromosomes. The ZW Leporinus species show an interesting distribution (Fig. 5). These system of sex chromosomes is represented in practically all the large hydrographic basins in the Brazilian territory. In the Amazon region it is represented by L. trifasciatus, in the Paraguay river, by L. macrocephalus, in the Upper Parana,

6 380 P. M. Galetti Jr., N. R. W. Lima and P. C. Venere Cytologia 60 Fig. 5. Geographical map indicating the collecting and/or occurrence sites of six ZW Leporinus species. Letters c, e, m, o, r and t meaning L. conirostris, L. elongatus, L. macrocephalus, L. obtusidens, L. reinhardti, and L. trifasciatus, respectively. by L. obtusidens and L. elongatus, in the Sao Francisco river, by L. reinhardti, and in the rivers of the East, by L. conirostris. Although this is an incomplete picture, a trend can be detected. In addition to the color pattern discussed above, these species share a large size and a good swimming ability. If this trend is maintained, most of the Leporinus fish, characteristically small sized, can be excluded and this diagram then becomes close to reality. With the exception of L. elongatus, which has been recorded in the entire Parana-Paraguay system and also in the Sao Francisco river, the remaining ZW species seem to replace each other in the different water basins. Replacement of closely related species has been previously reported in this genus between L. friderici from the Upper Parana and L. piau from the Sao Francisco river, as well as between L. striatus and related species (Garavello 1979). Thus, we believe that it is more probable that this system of ZW sex chromosomes evolved only once in Leporinus and that the ZW ancestor must be quite ancient, preceding the separation of the present species among the large hydrographic systems. Today this cytologic Leporinus unit assembles more related species than the remaining species of the genus, possibly

7 1995 ZW Sex Chromosomes in Leporinus Fish 381 an ecologically equivalent group of species. Summary Leporinus is a fish group rich in species which occurs from Central America to the River Plate basin in South America. From a cytologic viewpoint, this group is characterized by the occurrence of both homogametic and heterogametic species, a fact that renders it highly interesting for the study of sex chromosome evolution. In the present paper we report new occurrences of ZW sex chromosomes in L. trifasciatus from the Amazon region and L. conirostris from the basin of Eastern Brazil. Considerations about the origin and distribution of these sex chromosomes within the genus are also presented. The material was analyzed by standard Giemsa staining, by silver nitrate staining for the localization of nucleolar organizer regions, and by C banding for heterochromatin identification. The two species presented 2n= 54 for both sexes as well as a large subtelocentric chromosome, the largest in the complement, exclusively detected in females, characterizing a W chromosome in both species. The hetero chromatin data obtained permit us to assume that this system involves the same chromosomes not only in the species reported here but also in four others reported in previous studies. Thus the ZW system seems to have arisen only once in Leporinus and currently represent a unit of species more related to one another and cytologically distinct from the remaining species in the genus. Acknowledgements This work was supported by CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) and FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo). References Almeida Toledo, L. F., Foresti, F. and Toledo-Filho, S. A Complex sex chromosome system in Eigenmannia sp (Pisces, Gymnotiformes). Genetica 64: Becak, W. and Berak, M. L Cytotaxonomy and chromosomal evolution in Serpentes. Cytogenetics 8: Bertollo, L. A. C. and Cavallaro, Z. I A highly differentiated ZZ/ZW sex chromosome system in a Characidae fish, Triportheus guentheri. Cytogenet. Cell Genet. 60: , Takahashi, C. S. and Moreira-Filho, O Cytotaxonomy considerations on Hoplias lacerdae (Pisces, Erythrinidae). Rev. Brasil. Genet. I: , - and Karyotypic studies of two allopatric populations of the genus Hoplias (Pisces, Erythrinidae). Rev. Brasil. Genet. II: , - and Multiple sex chromosomes in the genus Hoplias (Pisces, Erythrinidae). Cytologia 48: Chen, T. R. and Ebeling, A. W Probable male heterogamety in the deep-sea fish Bathylagus wesethi (Teleostei: Bathylagidae). Chromosoma 18: Ebeling, A. W. and Chen, T. R Heterogamety in teleostean fishes. Trans. Amer. Fish. Soc. 99: Foresti, F., Almeida Toledo, L. F. and Toledo-Filho, S. A Polymorphic nature of nucleolus organizer regions in fishes. Cytogenet. Cell Genet. 31: Galetti Jr., P. M. and Foresti, F Evolution of the ZZ/ZW system in Leporinus (Pisces, Anostomidae). Role of constitutive heterochromatin. Cytogenet. Cell Genet. 43: and Two cases of ZZ/ZW heterogamety in Leporinus (Anostomidae, Characiformes) and their relationships in the phylogeny of the group. Rev. Brasil. Genet. X: , Foresti, F., Bertollo, L. A. C. and Moreira-Filho, O Heteromorphic sex chromosomes in three species of the genus Leporinus (Pisces, Anostomidae). Cytogenet. Cell Genet. 29: , -, - and Characterization of eight species of Anostomidae (Cypriniformes) fish on the basis of the nucleolus organizer regions. Caryologia 37: , Cesar, A. C. G. and Venere, P. C. 1991a. Heterochromatin and NORs variability in Leporinus fish (Anostomidae, Characiformes). Caryologia 44:

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