A Monophyletic ZW Sex Chromosome System in Leporinus (Anostomidae, Characiformes)
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_??_1995 The Japan Mendel Society Cytologia 60: 375-382 , 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, 13565-905 Sao Carlos SP Brazil 2 Setor Eco-Genetica Evolutiva, Laboratorio de Ciencias Ambientais, Universidade Estadual Norte Fluminense, 28015-620 Campos RJ Brazil 3 Departamento de Biologia , ICLMA, Universidade Federal do Mato Grosso, 78600-000 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 1986. 1987). 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. 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. 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 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 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.