Morphologically Differentiated Sex Chromosomes in Neotropical Freshwater ﬁsh

Morphologically differentiated sex chromosomes in neotropical freshwater ﬁsh

L.F. de Almeida Toledo1 &F.Foresti2 1Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, Caixa Postal 11.461 – 05422- 970, São Paulo, SP, Brasil (E-mail: [email protected]); 2Departamento de Morfologia, Instituto de Biociências, UNESP, Campus de Botucatu, 18618-000, Botucatu, Brasil

Key words: ﬁsh cytogenetics, neotropical ﬁshes, sex-chromosomes, XX-XY, ZZ-ZW

Abstract A general survey of the occurrence of morphologically differentiated sex chromosomes in the neotropical freshwater fishes is presented. The total number of 32 occurrences involving simple XX-XY and ZZ-ZW, and multiple X1X2Y, X Y 1Y2 and ZW1W2 sex chromosome systems is described, with comments on the aspects of sex chromosome evolution in this fish fauna. The occurrence of different sex chromosome systems in related species of the same genus, or in different populations of the same nominal species, involving male and sometimes female heterogamety, and differences in the molecular composition of sex-linked heterochromatin, are considered as indicative of the early stage of sex chromosomes evolution in fish.

Introduction logues, in simple or multiple systems with male or female heterogamety. The neotropical freshwater fish fauna is the most mor- The first evidence of morphologically differenti- phologically diversified among the epicontinental fish ated sex chromosomes in neotropical freshwater fish faunas in the world (Vari & Malabarba, 1998) and was the discovery of clearly visible neo-Y chromo- its number of species may exceed 8,000 (Schaefer, somes in males of two unnamed Mexican fish species 1998). About 10% of these species have been analyzed (Uyeno & Miller, 1971, 1972). In the following years, cytogenetically, that is approximately 900 species, be- with the growing development of cytogenetic tech- longing to 252 genera and 44 families (Oliveira, 2000). niques, new occurrences of differentiated sex chromo- Paralleling their extraordinary number and morpho- somes have been described. Considering our present logical diversity, neotropical freshwater fish species revision, differentiated sex chromosomes may be con- present considerable inter and intraspecific chromo- sidered to occur in a total number of 32 species, that is, some variability (Oliveira et al., 1988) with diploid in about 4.0% of the total number of species already numbers that vary from 2n = 20 found in Pterole- analyzed cytogenetically. A very interesting feature bias longipinnis, a Cyprinodontiformes species (Post, of the sex chromosomes in the neotropical freshwa- 1965), to 2n = 132 in Corydoras aeneus, a Siluri- ter fish is that, in spite of the relatively low number formes species (Scheel, Simonsen & Gyldenholm, of occurrences, a wide range of sex chromosome sys- 1972). tems is present, involving simple XX-XY and ZZ-ZW, Considering the sex chromosomes, fish are char- and multiple X1X2Y, X Y 1Y2 and ZW1W2 systems. acterized by a remarkable variability of sex determin- In most cases, the difference between the sex homo- ation systems. While mammals are characterized by logues is due to heterochromatin addition/deletion to XX-XY and birds by ZZ-ZW sex chromosomes, eight the X, the Y, the Z or the W chromosome. The mor- types of sex chromosome systems have already been phological differentiation of these chromosomes is described in fish (Tave, 1993), involving both mor- very well studied in ZZ-ZW systems in which the W phologically undifferentiated and differentiated homo- chromosome has a heterochromatic region not present 92 in the Z chromosome (Haaf & Schmid, 1984; Galetti Table 1. XX-XY sex chromosomes in neotropical freshwater fish & Foresti, 1986; Feldberg et al., 1987; Bertollo & Species 2n Sex Reference Cavallaro, 1992; Moreira-Filho et al., 1993). Mul- chromosome tiple systems, on the other hand, have originated by system Robertsonian or tandem fusion, sometimes resulting in heterochromatin loss (Almeida-Toledo et al., 2000a) STERNOPYGIDAE or by chromosome fission. Eigenmannia virescens 38 XX-XY 1,5 Differentiated sex chromosomes present a very HYPOPTOPOMATINAE Pseudotocinclus tietensis 54 XX-XY 2 peculiar distribution among fish species. The same POECILIIDAE system may be found in all the species of a group, Poecilia reticulata 46 XX-XY 3 as is the case of the ZZ-ZW system which is found ERYTHRINIDAE in all the species in genus Triportheus (Bertollo & Hoplias malabaricus 42 XX-XY 4 Cavallaro, 1992; Artoni, 1999); or it may be found in part of a larger group, as in six out of more than 20 References: 1. Almeida-Toledo, Foresti and Toledo-Filho, 1988; Andreata et al., 1992; 3. Nanda et al., 1990; 4. Born and Bertollo, cytogenetically analyzed and probably monophyletic, 2000; 5. Almeida-Toledo et al., submitted. ZZ-ZW species from the genus Leporinus (Galetti & Foresti, 1986). Most of the occurrences, however, are sporadic, as in Semaprochilodus, a Characidae genus Other occurrences have been found by meiotic ana- for which sex chromosomes are present only in one lysis, such as in the rainbow trout Oncorhyncus mykiss species of the group, S. taeniurus (Feldberg et al., (Oliveira et al., 1995) and in the tilapia, Oreochromis 1987). The most interesting feature of the sex chro- niloticus, as suggested by Foresti et al. (1993) and mosome distribution in fish groups is that in these confirmed by Carrasco et al. (1999). sporadic occurrences, different systems for example Among the neotropical freshwater species we now male and female heterogamety and simple or mul- list four species for which clearly documented occur- tiple systems, may be found in closely related species rences of XX-XY sex chromosomes were reported of the same genus, or in different populations of (Table 1): Eigenmannia virescens (Almeida-Toledo, the same species. Such is the case in Eigenmannia Foresti & Toledo-Filho, 1988), Poecilia reticulata (L.F. Almeida-Toledo & F. Foresti, submitted) and in (Nanda et al., 1990), Pseudotocinclus tietensis (An- Hoplias (Bertollo et al., 2000) and probably charac- dreata et al., 1992) and Hoplias malabaricus (Born terizes early stages and different strategies of incipient & Bertollo, 2000). The interesting feature in these sex chromosome differentiation. cases is that the differentiation of the homologues was attained either by Y or X heterochromatinization: in both E. virescens and in H. malabaricus the het- XX-XY systems erochromatinization occurred in the X chromosome. For P. reticulata and for P. tietensis, addition of a A reference list of the occurrence of morphologic- heterochromatic block occurred in the Y chromosome. ally differentiated sex chromosomes in gonochoristic In Eigenmannia, an early stage of differentiation teleosteans was provided by Chourrout (1988) and of an XX-XY system with heterochromatin addi- among sixty species reported, 25 had XX-XY sex tion to the X chromosome was detected, involving chromosomes. These first reports include the well- two geographically related populations of Eigenman- documented occurrences of XX-XY in a deep-sea nia virescens (Gymnotiformes, Sternopygidae) from species (Chen & Ebeling, 1966) and in a Salmonidae which the two otherwise identical cytotypes differ by species (Thorgaard, 1977). However, some of the re- the presence or lack of well-differentiated sex chromo- ported occurrences, mainly for neotropical freshwater somes (L.F. Almeida-Toledo et al., submitted). In this species, were put in doubt (Moreira-Filho, Bertollo case, in one of the cytotypes of E. virescens (cytotype & Galetti-Jr, 1993) or discarded (Mestriner, Berto- A), the acrocentric X chromosome presents an addi- llo & Galetti, 1995) by further studies. On the other tional block of heterochromatin in its terminal portion hand, some new occurrences of the XX-XY sex chro- without a corresponding region in the acrocentric Y mosome system have been detected with the use chromosome, while the other cytotype (cytotype B) of banding techniques, as for example in the lake has no differentiated sex homologues (Figure 1). The trout Salvelinus namaycush (Phillips & Ihssen, 1985). additional heterochromatic segments in the sex homo- 93

Figure 1. C-banded metaphases of Eigenmannia virescens. (A) cytotype A female with large heterochromatic blocks in the X-chromosomes (B) cytotype A female with polymorphic heterochromatic blocks in the X-chromosomes; (C) cytotype A male; (D) cytotype B with undifferentiated sex chromosomes. The arrows indicate X-chromosomes (Almeida-Toledo et al., submitted).

logues seem to result from DNA ampliﬁcation. After in all cases males always presented a reduction of BrdU incorporation, it was possible to identify the heterochromatin in relation to females (Figure 1(B)). same banding pattern in the euchromatic region of Hoplias malabaricus, like E. virescens,presentsa X and Y-chromosomes in cytotype A andalsointhe heterochromatic block in the submetacentric X chro- corresponding chromosome pair in cytotype B.There mosome (Born & Bertollo, 2000). In this case also, the is no indication of any alteration in the replication occurrence may be considered to be in an early stage pattern of the euchromatic region of the sex chromo- of diversiﬁcation, since related populations of the somes in the two populations. A polymorphic size same species do not display differentiated sex chro- variation of the heterochromatic segment present in mosomes or present other sex chromosome systems, the sex chromosomes was detected in cytotype A,but as will be discussed later. 94

In Poecilia reticulata (Poeciliidae, Cyprinodon- neotropical region, sex chromosomes were detected tiformes) a heterochromatic region with differential in six of the 20 species already analyzed cytogenetic- accumulation of (GACA)4 repeats was detected in ally. The sex chromosome differentiation in these six specific Y chromosomes of a laboratory strain, in- species is due to the presence of a ZW heteromorphic dicating the occurrence of an XX-XY mechanism of pair in females, in which one small subtelocentric sex determination (Nanda et al., 1990). According chromosome is the Z and one large subtelocentric to the authors, the results obtained for these lineages chromosome with a large C-band positive block that may point to early stages in morphological differen- occupies nearly the entire length of the long arm is the tiation of vertebrate sex chromosomes in general. In W (Galetti & Foresti, 1986, 1987; Galetti, Lima & Pseudotocinclus tietensis (Siluriformes, Loricariidae), Venere, 1995) (Figure 2). The geographic distribution an XY heteromorphic sex chromosome pair was de- of the ZW species of Leporinus including species from tected, and the metacentric Y chromosome presented the upper Paraná river system and the São Francisco a conspicuous heterochromatic region in the short arm river system seems to indicate that this sex chromo- (Andreata et al., 1992). some system evolved only once in this group, from It is important to notice that from the initial de- a very ancient ancestor, and before the separation of scriptions of XX-XY systems, for all the confirmed these two river systems in South America (Galetti, occurrences, the differentiation of the heteromorphic Lima & Venere, 1995). This monophyletic origin is pair was attained by processes of addition/deletion corroborated by the homologies exhibited concerning of heterochromatin. Reported occurrences of differ- relative size, centromere position and amount and dis- entiated XY chromosomes for which the homologue tribution of heterochromatin of the W chromosome in differentiation was attributed to pericentric inversion Leporinus species (Koehler et al., 1997). could not be entirely proved and, in some cases, were In the genus Triportheus (Characiformes, Chara- reconsidered when detailed meiotic chromosome ana- cidae), on the other hand, a ZZ-ZW system is present lysis was performed. This was, for instance, the case in all of the eight species and populations that have of Leporinus lacustris (Mestriner et al., 1995). already been analyzed cytogenetically. A well dif- In E. virescens, the X-linked heterochromatic seg- ferentiated ZW pair is present in this group, the W ment is brightly stained with chromomycin A3 and chromosome being a small almost entirely hetero- mithramycin. It is also late replicating in BrdU treated chromatic metacentric and the Z a large metacentric chromosomes, is digested in situ by the restriction (Bertollo & Cavallaro, 1992; Artoni, 1999). Morpho- enzyme AluI, and resistant to HindIII digestion (L.F. logical differentiation of the W chromosome has also Almeida-Toledo et al., submitted). In Hoplias malab- been observed among populations of one nominal spe- aricus on the other hand, this segment is DAPI positive cies of this genus in samples from different river basins (Born & Bertollo, 2000). (Artoni, 1999), indicating their probable origin from a common ancestor. ZZ-ZW systems Leporinus and Triportheus are the only two genera for which a female sex chromosome heteromorphism The ZZ-ZW simple system with female heterogamety is a fixed characteristic within a group of species or is the most frequent among neotropical freshwater all of the species in a genus. Other ccurrences of fish, with 20 occurrences already reported (Table 2). the ZW system in neotropical freshwater fishes are The ZZ-ZW system differs from the previously dis- sporadic (Table 2). It has been observed in Parodon cussed XX-XY system in being well established in hilarii (Characiformes, Parodontidae) (Moreira-Filho, certain fish genera such as Leporinus and Triportheus. Bertollo & Galetti, 1993), Semaprochilodus tae- This latter genus presents female heterogamety in all niurus (Characiformes, Prochilodontidae) (Feldberg the species already analyzed, a situation that is still et al., 1987), Characidium fasciatum (Characiformes, unique in the neotropical fish fauna, that is, an en- Characidae) (Maistro et al., 1998), Microlepidogaster tire group presenting differentiated sex chromosomes. leucofrenatus (Siluriformes, Loricariidae) (Andreata In these two cases, the presence of such a distinct- et al., 1993), and Poecilia sphenops var. melanistica ive characteristic seems to imply a close phylogenetic (Cyprinodontiformes, Poeciliidae) (Haaf & Schmid, relationship among the species involved. 1984). In all these cases, the W differs from the In the genus Leporinus (Characiformes, Anostom- Z by the presence of conspicuous heterochromatin idae) containing fish with a wide occurrence in the blocks. The only exception to this pattern is found in 95

Table 2. ZZ-ZW sex chromosomes in neotropical freshwater ﬁsh

Species 2n Sex chromosome Reference system

ANOSTOMIDAE Leporinus elongatus 54 ZZ-ZW 1 Leporinus obtusidens 54 ZZ-ZW 1 Leporinus reinhardti 54 ZZ-ZW 2 Leporinus macrocephalus 54 ZZ-ZW 2 Leporinus trifasciatus 54 ZZ-ZW 3 Leporinus conirostris 54 ZZ-ZW 3 Leporinus cf. elongatus 54 ZZ-ZW 4 CHARACIDAE Triportheus albus 52 ZZ-ZW 5 Triportheus signatus 52 ZZ-ZW 5 Triportheus elongatus 52 ZZ-ZW 5 Triportheus cf. elongatus 52 ZZ-ZW 7 Triportheus ﬂavus 52 ZZ-ZW 5 Triportheus guentheri 52 ZZ-ZW 6 Triportheus paranense (MT) 52 ZZ-ZW 7 Triportheus paranense (MS) 52 ZZ-ZW 7 Characidium fasciatum 50 ZZ-ZW 8 PARODONTIDAE Parodon hilarii 54 ZZ-ZW 9 PROCHILODONTIDAE Semaprochilodus taeniurus 54 ZZ-ZW 10 LORICARIIDAE Hypostomus sp. 64 ZZ-ZW 11 Microlepidogaster leucofrenatus 54 ZZ-ZW 12 POECILIIDAE Poecilia sphenops var. melanistica 46 ZZ-ZW 13

References: 1. Galetti et al.,1981; 2. Galetti and Foresti, 1986; 3. Galetti, Lima and Venere, 1995; 4. Molina, Schmid and Galetti, 1998; 5. Falcão, 1988; 6. Bertollo and Cavallaro, 1992; 7. Artoni, 1999; 8. Maistro et al., 1998; 9. Moreira-Filho, Bertollo and Galetti, 1993; 10. Feldberg et al., 1987; 11. Artoni et al., 1998; 12. Andreata et al., 1993; 13. Haaf and Schmid, 1984.

Hypostomus sp. (Siluriformes, Loricariidae) (Artoni and autosomes, either by centric fusion, reciprocal et al., 1998). In this case a ZZ-ZW system is present, translocation between metacentric chromosomes, or but a heterochromatin block occurs in the interstitial centric fission; tandem fusions may also be involved region of the acrocentric Z chromosomes, the W being in this process (White, 1973). a small metacentric with no detectable heterochro- Multiple sex chromosome systems have been de- matin differentiation. tected in seven species of freshwater fish from the The W-linked heterochromatic region is DAPI pos- neotropical region (Table 3), with male X1X1X2X2- itive in Poecilia sphenops var. melanistica (Haaf & X1X2Y and XX-XY1Y2, or female ZZ-ZW1W2 Schmid, 1984), but either G + CrichorA+ Trich heterogamety. In five species, X1X1X2X2-X1X2Y regions have been observed in sex chromosomes of systems were reported: initially, in one Mexican spe- Leporinus (Molina, 1995; Koehler et al., 1997). cies of Cyprinodontidae where the Y chromosome originated through a Robertsonian fusion with some chromatin addition (Uyeno & Miller, 1971) and in one Multiple sex chromosome systems species of Goodeidae in which the Y chromosome also originated by fusion of the sex chromosome and one Multiple sex chromosome systems usually arise as a autosome (Uyeno & Miller, 1972), with no detectable result of rearrangements involving sex chromosomes chromatin addition. Similar occurrences were reported 96

Figure 2. C-banded metaphases of Leporinus macrocephalus. (A) female; (B) male. The arrow indicates the W-chromosome.

Table 3. Multiple systems of sex chromosome differentiation in neotropical freshwater ﬁsh

Species 2n Sex chromosome Reference system

PARODONTIDAE Apareiodon afﬁnis F-55 F-ZW1W2 1,2 M-54 M-ZZ ERYTHRINIDAE Hoplias malabaricus F-40 F-X1X1X2X2 3 M-41 M-X1X2Y Hoplias sp. F-40 F-XX 4 M-41 M-X1X2Y STERNOPYGIDAE Eigenmannia sp.2 F - 32 F - X1X1X2X2 5 M-31 M-X1X2Y HYPOPOMIDAE Brachyhypopomus F-42 F-X1X1X2X2 6 pinnicaudatus M-41 M-X1X2Y CYPRINODONTIDAE unnamed Mexican Sp. F - 48 F - X1X1X2X2 7 M-47 M-X1X2Y GOODEIDAE unnamed Mexican sp F - X1X1X2X2 8 M-X1X2Y F = female; M = male. References: 1. Moreira-Filho, Bertollo and Galetti, 1980; 2. Moreira-Filho, Bertollo and Galetti, 1993; 3. Bertollo, Takahashi and Moreira-Filho, 1983; 4. Dergan and Bertollo, 1990; 5. Almeida-Toledo, Foresti and Toledo-Filho, 1984; 6. Almeida-Toledo et al., 2000b; 7. Uyeno and Miller, 1971; 8. Uyeno and Miller, 1972. 97

Figure 3. Chromosome plates of Eigenmannia sp.2. (A) Giemsa stained metaphase of a female; (B) Giemsa-stained metaphase of a male; (C) Mithramycin-stained male metaphase. The arrows indicate the Y chromosome (Almeida-Toledo et al., 2000a). in Eigenmannia sp.2 (Gymnotiformes, Sternopygidae) sp.2 and the missing region was G + Crich(Fig- (Almeida-Toledo, Foresti & Toledo-Filho, 1984, ure 3) (Almeida-Toledo et al., 2000a). Conversely, Almeida-Toledo et al. 2000a) and in Hoplias sp. in Hoplias malabaricus, no chromosome loss appar- (Erythrinidae, Characiformes) (Bertollo, Takahashi & ently resulted from the tandem translocation involved Moreira, 1983). In Eigenmannia, as been observed in in the neo-Y chromosome formation, as shown by C- the Mexican species, a Robertsonian translocation was banding analysis (Bertollo et al., 1997). The same is the possible origin of the large neo-Y chromosome; in true for Brachyhypopomus pinnicaudatus (Almeida- Hoplias, a complex rearrangement, possibly involving Toledo et al., 2000b). In all these cases, disturbance a pericentric inversion and a chromosome transloca- in the meiotic chromosome pairing process is caused tion seems to have occurred (Bertollo et al. 1997). by the chromosome rearrangement, as seen in H. A second occurrence in Gymnotiformes, and the malabaricus (Bertollo & Mestriner, 1998). fourth in neotropical freshwater fish of X1X1X2X2- X1X2Y sex chromosome systems involving Robertso- nian translocation, was reported more recently in Bra- General comments chyhypopomus pinnicaudatus (Almeida-Toledo et al., 2000b). A general view of the morphologically differentiated Besides these occurrences of X1X2Ysystems,a sex chromosomes in neotropical freshwater fish indic- ZZ-ZW1W2 system and an XX-XY1Y2 system were ates a relatively wide occurrence of the ZZ-ZW system reported. The ZZ-ZW1W2system was detected in as a more constant and fixed presence in two groups of Apareiodon affinis (Characiformes, Parodontidae). In Characiformes, that is, in part of family Anostomidae this species, males have one chromosome less than and in the entire subfamily Triportheinae. Sporadic females, probably as result of a fission process in- occurrences in other fish families of Characiformes, volving the largest chromosome pair, since in a closely (Prochilodontidae and Parodontidae) also occur, in- related species the corresponding pair is homomorphic cluding a unique occurrence in the neotropical fish in both males and females (Moreira-Filho et al., 1980). fauna, of a multiple ZW1W2 system in a Parodontidae The only reported occurrence of an XX-XY1Y2 sys- species. Male heterogamety was not detected in the tem was detected in one cytotype of the Hoplias species already described in the group including famil- malabaricus (Characiformes, Erythrinidae) complex ies Prochilodontidae, Parodontidae and Anostomidae. (Bertollo, Takahashi & Moreira-Filho, 1983; Bertollo This points to a possible tendency for female het- et al., 2000) in which males have one chromosome erogamety that could be a fixed characteristic in this more than females, probably originating through a group. According to Vari (1983), Prochilodontidae fusion/fission process. and Parodontidae are closely related to Anostomidae, Heterochromatin loss in the neo-Y formation, and the presence of the same sex chromosome system probably related to the occurrence of a chromosome in species of this group reinforces their relationship translocation process, was observed in Eigenmannia from the genetic point of view. This tendency is not 98 observed when all the Characiformes species are con- males and females in Eigenmannia sp.2: a CMA3 pos- sidered: in Erythrinidae, a family belonging to this or- itive and G + C rich heterochromatic portion of one of der, only male heterogamety was described (Bertollo the chromosomes involved in the Robertsonian fusion et al., 2000). that gave origin to the bi-armed Y was lost, so that the Most of the reported occurrences of XX-XY sys- female X1 and X2 chromosomes have more G + Crich tems were sporadic, but sometimes multiple sex chro- heterochromatin than the corresponding neo-Y in the mosomes with male heterogamety were detected in male (Almeida-Toledo et al., 2000a). In another Ei- populations or species related to those presenting genmannia species, E. virescens, the heterochromatic XX-XY systems. This was the case of Sternopy- region associated with the X chromosome is G+C rich. gidae (Almeida-Toledo, Foresti & Toledo-Filho, 1984, The detection of sex-chromosomes linked A + Trich 1988) and Erythrinidae (Bertollo et al., 2000). For heterochromatin in the pericentromeric region of the these two fish families, both simple XX-XY and mul- neo-Y chromosome of Eigenmannia sp.2 and B. pin- tiple X1X1X2X-X1X2Y systems were described in nicaudatus (Almeida-Toledo et al., 2000a,b) indicates closely related species. that these regions, probably present in the original In Poeciliidae, a well studied group that presents a translocated acrocentrics, were not lost during the fu- wide variety of sex determination mechanisms which sion process. A + T rich heterochromatin associated span from simple ZZ-ZW or ZZ-ZW systems to with the sex chromosomes has already been reported polyfactorial sex determination (Volff & Schartl, this to occur in Poecilia by Haaf and Schmid (1984) and volume), only early stages of morphologically differ- in the Hoplias malabaricus X-chromosome (Born & entiated sex chromosomes have been detected (Haaf & Bertollo, 2000). Schmid, 1984; Nanda et al., 1990) and both male and The significance of the loss of G + C rich hetero- female heterogamety were found. chromatin in Eigenmannia sp.2 males and the con- Experiments carried out by Nakayama et al. (1994) servation of A + T heterochromatin associated with using sex-specific clones isolated from Leporinus the sex chromosomes in these species remains to be elongatus and hybridized with DNA from 24 adults explained. The finding that, in addition to the oc- of the same species have revealed the presence of currence of different systems of sex chromosomes, one female and two male individuals with opposite differences also occur in the molecular nature of the hybridization, that is, three specimens had atypical heterochromatin associated with the sex-related re- W-chromosomes. The results were probably due to a gions, reinforces the idea that sex chromosomes may possible loss of the sex-related insert used as a probe have evolved independently and in different ways (Nakayama et al., 1994). However these investigators in the various fish lineages, resulting in the rich presented an alternative suggestion that these atyp- variability of sex chromosome systems in the neo- ical Ws could have been the product of a cross-over tropical fish fauna. Transposable elements that are involving the sex-linked region, thereby providing widespread in fish, one of them having been identi- evidence that might explain how male and female het- fied in the sex chromosomes of Xiphophorus (Volff erogamety has appeared several times in different fish et al., 1999) could also be involved in the high de- subtaxa (Nakayama et al., 1994). As further support gree of variability of the sex chromosomes in this of this idea, sex-chromosome crossovers were also re- group. ported in Xiphophorus maculatus in the region of the The occurrence of such variable forms of chro- X and Y chromosomes that encodes several important mosome heteromorphism and of different mechanisms traits, including the determination of sex (Gutbrod & of sex determination in closely related species, most Schartl, 1999). of them at an early stage of evolution, seems to be Fluorochrome analysis of the heterochromatic re- well tolerated in fish, although not the case with gions of sex chromosomes was carried out in some higher vertebrates. An indication of this labiality of species. In the family Anostomidae Z and W chromo- sex determination is found in sex reversal experiments somes of Leporinus obtusidens are characterized by carried out in aquaculture programs (Mair et al., 1991; a high GC content, while AT sequences have accu- Rosenstein & Hulata, 1994 among others). In the mulated in the heterochromatic portions of Z and W neotropical species Leporinus macrocephalus,which chromosomes of L. elongatus (Koehler et al., 1997). presents morphologically well differentiated sex chro- A difference in the amount of heterochromatin associ- mosomes, histologically analyzed male gonads were ated with the sex chromosomes was detected between observed in genetically ZW females that had been 99 submitted to methyltestosterone treatment (E. Tori- Artoni, R.F., P.C. Venere & L.A.C. Bertollo, 1998. A heteromorphic yama & F. Foresti, unpublished). ZZ/ZW sex chromosome system in fish, genus Hypostomus The growing use of a molecular cytogenetic ap- (Loricariidae). Cytologia 63: 421–425. Bertollo, L.A.C., C.S. Takahashi & O. Moreira-Filho, 1983. Mul- proach in the neotropical freshwater fishes, mainly tiple sex chromosomes in the genus Hoplias (Pisces, Erythrin- involving replication banding, in situ hybridization of idae). Cytologia 48: 1–12. selected sequences, and other high resolution cyto- Bertollo, L.A.C., G.G. Born, J.A. Dergam, A.S. Fenocchio & O. Moreira-Filho, 2000. A biodiversity approach in the neotrop- genetic techniques, may help provide a better under- ical Erythrinidae fish, Hoplias malabaricus. Karyotypic survey, standing of the chromosome evolutionary trends in geographic distribution of cytotypes and cytotaxonomic consid- fishes of the neotropical region, and may also allow erations. Chrom. Res. 8: 603–613. a closer view of the molecular organization of fish sex Bertollo, L.A.C. & Z.I. Cavallaro, 1992. A highly differentiated ZZ/ZW sex chromosome system in a Characid fish Triportheus chromosomes. guentheri. Cytogenet. Cell Genet. 60: 60–63. Bertollo, L.A.C., M.S. Fontes, A.S. Fenocchio & J. Cano, 1997. The X1X1Y sex chromosome system in the fish Hoplias malab- Acknowledgements aricus. I. G-, C- and chromosome replication banding. Chrom. Res. 5: 493–499. Bertollo, L.A.C. & C.A. Mestriner, 1998. The X1X2Ysexchro- The authors are grateful to Maria de Fátima Z. Daniel- mosome system in the fish Hoplias malabaricus: II. meiotic Silva for her helpful assistance and to Dr Ann Stocker analysis. Chromosome Res. 6: 141–147. for the critical reading of the manuscript. Funds sup- Born, G.G. & L.A.C. Bertollo, 2000. An XX/XY sex chromosome system in a fish species, Hoplias malabaricus with porting this study were provided by FAPESP and a polymorphic NOR-bearing X-chromosome. Chrom. Res. 8: CNPq. Copyright permission was obtained for public- 111–118. ation of Figure 3(C). Carrasco, L.A.P., D.J. Penman & N. Bromage, 1999. Evidence for the presence of sex chromosomes in the Nile tilapia (Oreo- chromis niloticus) from synaptonemal complex analysis of XX, XY and YY genoptypes. Aquaculture 173: 207–218. Chen, T.R. & A.W. Ebeling, 1966. Probable male heterogamety in References the deep-sea fish Bathylagus wesethi (Teleostei, Bathylagidae). Chromosoma 18: 88–96. Almeida-Toledo, L.F., F. Foresti, E.V. Péguignot & M.F.Z. Daniel- Chourrout, D., 1988. Genetic sex determination in teleostean fish – Silva, XX-XY sex chromosome system with X heterochromatin- a review. B. Soc. Zool. Fr. 113: 123–144. ization: an early stage of sex chromosome differentiation in the Dergam, J.A. & L.A.C. Bertollo, 1990. Karyotypic diversification neotropic electric cel Eigenmannia virescens (submitted). in Hoplias malabaricus (Osteichthyes, Erythrinidae) of the São Almeida-Toledo, L.F., F. Foresti & S.A. Toledo-Filho, 1984. Francisco and Alto Paraná basins, Brazil. Braz. Journ. Genet. 13: Complex sex chromosome system in Eigenmannia sp (Pisces, 755–766. Gymnotiformes). Genetica 64: 165–169. Falcão, J.N., 1988. Caracterização Cariotípica em Peixes do Gênero Almeida-Toledo, L.F., F. Foresti & S.A. Toledo-Filho, 1988. Triportheus (Teleostei, Characiformes, Characidae). PhD Thesis. An early stage of sex chromosome differentiation in the fish Universidade Federal de São Carlos, SP. Eigenmannia virescens (Sternopygidae). Genome 30 (suppl1): Feldberg, E., L.A.C. Bertollo, L.F. Almeida-Toledo, F. Foresti, 132. O. Moreira-Filho & A.F. Santos, 1987. Biological aspects of Almeida-Toledo, L.F., F. Foresti, M.F.Z. Daniel-Silva & S.A. Amazonian fishes. IX Cytogenetic studies in two species of Toledo-Filho, 2000a. Sex chromosome evolution in fish: the the genus Semaprochilodus (Pisces, Characidae). Genome 29: formation of the neo-Y chromosome in Eigenmannia (Gymno- 1–4. tiformes). Chromosoma 109: 19–200. Foresti, F., C. Oliveira, P.M. Galetti-Jr & L.F. Almeida-Toledo, Almeida-Toledo, L.F., M.F.Z. Daniel-Silva, C.E. Lopes & S.A. 1993. Synaptonemal complex analysis in spermatocytes of Toledo-Filho, 2000b. Sex chromosome evolution in fish: II. tilapia, Oreochromis niloticus (Pisces, Cichlidae). Genome 36: 1124–1128. second occurrence of an X1X2Y sex chromosome system in Gymnotiformes. Chrom. Res. 8: 335–340. Galetti-Jr, P.M. & F. Foresti, 1986. Evolution of the ZZ/ZW sys- Andreata, A.A., L.F. Almeida-Toledo, C. Oliveira & S.A. Toledo- tem in Leporinus (Pisces, Anostomidae). Role of constitutive Filho, 1992. Chromosome studies in Hypoptopomatinae (Pisces, heterochromatin. Cytogenet. Cell Genet. 43: 43–46. Siluriformes, Loricariidae): I. sex chromosome heteromorphism Galetti-Jr, P.M. & F. Foresti, 1987. Two new cases of ZZ/ZW het- in Pseudotocinclus tietensis. Cytologia 57: 369–372. erogamety in Leporinus (Anostomidae, Characiformes) and their Andreata, A.A., L.F. Almeida-Toledo, C. Oliveira & S.A. Toledo- relationships in the phylogeny of the group. Rev. Brasil. Genet. Filho, 1993. Chromosome studies in Hypoptopomatinae (Pisces, X: 135–140. Siluriformes, Loricariidae): II. ZZ/ZW sex chromosome system, Galetti-Jr. P.M., F. Foresti, L.A.C. Bertollo & O. Moreira-Filho, B-chromosomes, and constitutive heterochromatin differenti- 1981. Heteromorphic sex chromosomes in three species of the ation in Microlepidogaster leucofrenatus. Cytogenet. Cell Genet. genus Leporinus (Pisces, Anostomidae). Cytogenet. Cell Genet. 63: 215–220. 29: 138–142. Artoni, R.F., 1999. Citogenética do sistema de cromossomos sexuais Galetti-Jr, P.M., N.R.W. Lima & P.C. Venere, 1995. A monophyletic ZZ/ZW no gênero Triportheus (Pisces, Characidae). PhD Thesis. ZW sex chromosome system in Leporinus (Anostomidae, Chara- Universidade Federal de São Carlos, SP. ciformes). Cytologia 60: 375–382. 100

Gutbrod, H. & M. Schartl, 1999. Intragenic sex-chromosomal cros- Oliveira, C., L.F. Almeida-Toledo, F. Foresti., H.A. Britski & sovers of Xmrk oncogene alleles affect pigment pattern forma- S.A. Toledo-Filho, 1988. Chromosome formulae of neotropical tion and the severity of melanoma in Xiphophorus. Genetics 151: freshwater fishes. Rev. Brasil. Genet. 11: 577–624. 773–783. Oliveira, C., F. Foresti, M.G. Rigolino & Y.A. Tabata, 1995. Syn- Haaf, T. & M. Schmid, 1984. An early stage of ZW/ZZ sex chro- aptonemal complex analysis in spermatocytes and oocytes of mosome differentiation in Poecilia sphenops var. melanistica rainbow trout Oncorhynchus mykiss (Pisces, Salmonidae): the (Poeciliidae, Cyprinodontiformes). Chromosoma 89: 37–41. process of autosome and sex chromosome synapsis. Chrom. Res. Koehler, M.R., D. Dehm, M. Guttenbach, I. Nanda, T. Haaf, W.F. 3: 182–190. Molina, P.M. Galetti-Jr & M. Schmid, 1997. Cytogenetics of the Phillips, R.B. & P.E. Ihssen, 1985. Identification of sex chromo- genus Leporinus (Pisces, Anostomidae): 1. karyotype analysis, somes in the lake trout (Salvelinus namaycush). Cytogenet. Cell heterochromatin distribution and sex chromosomes. Chrom. Res. Genet. 39: 14–18. 5: 12–22. Post, A., 1965. Vergleichende Untersuchungen Chromosomenzah- Mair, G.C., A.G. Scott, D.J. Penman, J.A. Beardmore & D.O.F. len bei Süsswasser Teleosten. Z. Zool. Syst. Evol. Forsch. 3: Skibinski, 1991. Sex determination in the genus Oreochromis: 47–93. I. sex reversal, gynogenesis and triploidy in O. niloticus (L.). Rosenstein, S. & G. Hulata, 1994. Sex reversal in the genus Oreo- Theor. Appl. Genet. 82: 144–152. chromis: optimization of feminization protocol. Aquacult. Fish. Maistro, E.L., E. Prieto-Mata, F. Foresti & C. Oliveira, 1998. Manage. 25: 329–339. Unusual occurrence of a ZZ/ZW sex-chromosome system and Schaefer, S., 1998. Conflict and resolution: Impact of the new supernumerary chromosomes in Characidium cf. fasciatum taxa on phylogenetic studies of the neotropical cascudinhos (Pisces, Characiformes, Characidiinae). Genetica 87: 101–106. (Siluroidei: Loricariidae), pp. 375–400 in Phylogeny and Clas- Mestriner, C.A., L.A.C. Bertollo & P.M. Galetti-Jr, 1995. Chromo- sification of Neotropical Fishes, edited by L.R. Malabarba, R.E. some banding and synaptonemal complexes in Leporinus lacus- Reis, R.P. Vari, Z.M.S. Lucena & C.A.F. Lucena. Edipucrs, Porto tris (Pisces, Anostomidae): analysis of a sex system. Chrom. Res. Alegre, RS. 3: 440–443. Scheel, J.J., V. Simonsen & A.O. Gyldenholm, 1972. The karyo- Molina, W.F., 1995. Cromossomos sexuais e polimorfismo cro- types and some electrophoretic patterns of fourteen species of mossômico no gênero Leporinus (Pisces, Anostomidae). PhD genus Corydoras. Z. Zool. Syst. Evol. Forsch. 10: 144–152. Thesis. Universidade Federal de São Carlos. Tave, D., 1993. Genetics for Fish Hatchery Managers. Van Nostrand Molina, W.F., M. Schmid & P.M. Galetti-Jr., 1998. Heterochromatin Reinhold, New York, 2nd edn. and sex chromosomes in the neotropical fish genus Leporinus Thorgaard, G.H., 1977. Heteromorphic sex chromosomes in male (Characiformes, Anostomidae). Cytobios 94: 141–149. rainbow trout. Science 196: 900–902. Moreira-Filho, O., L.A.C. Bertollo & P.M. Galetti-Jr, 1980. Evid- Uyeno, T. & R.R. Miller, 1971. Multiple sex chromosomes in a ences for multiple sex chromosome system with female hetero- Mexican Cyprinodontid Fish. Nature 231: 452–453. gamety in Appareiodon affinis (Pisces, Parodontidae). Caryolo- Uyeno, T. & R.R. Miller, 1972. Second discovery of multiple sex gia 33: 83–91. chromosomes among fishes. Experientia 28: 223–225. Moreira-Filho, O., L.A.C. Bertollo & P.M. Galetti-Jr, 1993. Dis- Vari, R.P., 1983. Phylogenetic relationships of the families tribution of sex chromosomes mechanisms in neotropical fish Curimatidae, Prochilodontidae, Anostomidae and Chilodon- and description of a ZZ/ZW system in Parodon hilarii (Parodon- tidae (Pisces, Characiformes). Smithsonian Contrib. Zool. 378: tidae). Caryologia 46: 115–125. 1–60. Nakayama, I., F. Foresti, R. Tewari, M. Scharti & D. Chourrout, Vari, R.P. & R.L. Malabarba, 1998. neotropical Ichthyology: an 1994. Sex chromosome polymorphism and heterogametic males overview. pp, 1–10 in Phylogeny and Classification of Neotrop- revealed by two molecular probes in the ZZ/ZW teleost fish ical Fishes, edited by L.R. Malabarba, R.E. Reis, R.P. Vari, Leporinus elongatus. Chromosoma 103(1): 31–39. Z.M.S. Lucena & C.A.F. Lucena. Edipucrs, Porto Alegre, RS. Nanda, I., W. Feichtinger, M. Schmid, J.H. Schröder, H. Zischler Volff J.N., C. Körting, K. Sweeney & M. Schartl, 1999. The & J.T. Epplen, 1990. Simple repetitive sequences are associated non-LTR retrotransposon Rex3 from the fish Xiphophorus is with differentiation of the sex chromosomes in the guppy fish. J. widespread among teleosts. Mol. Biol. Evol. 16: 1427–1438. Mol. Evol. 30(5): 456–462. Volff, J.N. & M. Schartl, 2001. Variability of genetic sex determin- Oliveira, C., 2000. Síntese dos estudos citogenéticos em peixes neo- ation in poeciliid fishes. Genetica 111: 101–110. tropicais. Resumos do VIII Simpósio de Citogenética e Genética White, M.J.D., 1973. Animal Cytology and Evolution. Cambridge de Peixes Neotropicais, p. 108. University Press, Cambridge, 3rd edn.