Introduction

The majority of Neotropical fish species that have been studied cytogenetically do not have differentiated sex chromosomes (Moreira-Filho et al. 1993; Centofante et al. 2002). However, a variety of differentiated sex chromosome systems have been described for this ichthyofauna (e.g. Galetti et al. 1981; Bertollo et al. 1997; Bertollo and Mestriner 1998; Almeida-Toledo et al. 2001; Centofante et al. 2002; Venere et al. 2004; Alves et al. 2006; de Oliveira et al. 2006). These systems may involve only one pair of originally homologous chromosomes (XX/XY, XX/XO and ZZ/ZW), referred to as simple systems, or several of the chromosomes of the complement (X1X1X2X2/X1X2Y, XX/XY1Y2 and ZZ/ZW1W2), called multiple systems.

XX/XY systems have been documented in species of Gymnotiformes, Eigenmannia virescens (Almeida-Toledo et al. 2001), Cyprinodontiformes, Poecilia reticulata (Nanda et al. 1990), and Characiformes, Hoplias malabaricus (Born and Bertollo 2000). ZZ/ZW systems are more frequent and occur in a diversity of families, such as Anostomidae (Galetti et al. 1981; Venere et al. 2004), Prochilodontidae (Feldberg et al. 1987), Characidae (Bertollo and Cavallaro 1992; Maistro et al. 1998) and Parodontidae (Moreira-Filho et al. 1993; Centofante et al. 2002) in Characiformes, and Poeciliidae in Cyprinodontiformes (Haaf and Schmid 1984). Multiple systems have been documented in Characiformes of the families Parodontidae (Moreira-Filho et al. 1980) and Erythrinidae (Bertollo et al. 1983; Dergam and Bertollo 1990; Bertollo et al. 1997; Bertollo and Mestriner 1998) and in Gymnotiformes of the families Sternopygidae (Almeida-Toledo et al. 1984) and Hypopomidae (Almeida-Toledo et al. 2000).

Although cytogenetic studies of the family Loricariidae have revealed the predominance of homomorphic sex chromosomes (Artoni and Bertollo 2001; Alves et al. 2005; Kavalco et al. 2005), cases of simple and multiple systems of sex chromosomes have been described for this group. In the subfamily Hypostominae, XX/XY (Michelle et al. 1977; Mariotto and Miyazawa 2006), XX/XO (Alves et al. 2006), and ZZ/ZW (Artoni et al. 1998; Mariotto et al. 2004; de Oliveira et al. 2006) sex chromosome systems have been found. XX/XY (Andreata et al. 1992) and ZZ/ZW (Andreata et al. 1993; 1994) systems have been described for species of Hypoptopomatinae, and ZZ/ZW (Scavone and Júlio Jr. 1995) and XX/XY1Y2 (Centofante et al. 2006) systems have been described for Loricariinae.

Here we describe two cases of multiple sex chromosome systems found in two species of loricariids of the genus Ancistrus from the Brazilian Amazonia.

Material and methods

All specimens were collected from small streams in the state of Amazonas, Brazil. Since the species studied here are most likely new to science (S. Fish-Muller, pers. comm.), we present a short description of color pattern and include images of a preserved specimen for each species. Voucher specimens were deposited in the fish collection at the National Institute for Amazonian Research (INPA).

Ancistrus sp.1 “Balbina” (INPA 25633): 12 specimens (six males, six females) collected in the Barretinho Stream, an affluent of the Uatumã River in the municipality of Presidente Figueiredo, approximately 180 km north of Manaus (S 01°58′20′′, W 59°29′48′′). Specimens have a brown dorsal region with olive spots a little larger than the pupilla; ventral region dark with large, well-defined light olive spots; dorsal fin with diffuse, alternating black and white stripes (Fig. 1a).

Fig. 1
figure 1

Lateral, dorsal and ventral views of Ancistrus sp.1 “Balbina” (INPA 25633), 83 mm SL (a), and Ancistrus sp.2 “Barcelos” (INPA 25627), 92 mm SL (b)

Full size image

Ancistrus sp.2 “Barcelos” (INPA 25627): 11 specimens (seven males, four females) collected from the Demeni River, middle portion of Negro River basin, in the municipality of Barcelos (S 00°25′19′′, W 062°54′42′′). This species has a black dorsal region finely stippled with tiny white spots; ventral region dark with well-defined pale spots, larger than those on dorsal region; dorsal fin black with white stipples and a white border (Fig. 1b).

Chromosomal preparations were obtained from kidney cells, following the “air-drying” technique of Bertollo et al. (1978). The constitutive heterochromatin was identified according to Sumner (1972) and the nucleolar organizing regions (NORs) were detected using the technique described by Howell and Black (1980). Chromosomes were classified based on arm ratios as metacentrics (m), submetacentrics (sm), subtelocentrics (st), and acrocentrics (a) as proposed by Levan et al. (1964). The fundamental number (FN) or arm number was determined by considering meta-, submeta- and subtelocentric chromosomes with two arms and acrocentrics with only one.

Results

Males of Ancistrus sp.1 “Balbina” have a modal number of 2n = 39 chromosomes, fundamental number FN = 78, and karyotypic formula of 27 m + 10 sm + 2 st; females have a modal number of 2n = 38 chromosomes, FN = 76, and karyotypic formula of 26 m + 10 sm + 2 st (Fig. 2a and b). NORs are located in the terminal region of the long arms of pair 12 (Fig. 2c).

Fig. 2
figure 2

Karyotypes of male (a) and female (b) Ancistrus sp.1 “Balbina” after conventional Giemsa-staining, evidencing the sex chromosomes (box) and the NOR-bearing chromosomes after silver-staining (c). m = metacentric; sm = submetacentric; st = subtelocentric

Full size image

Ancistrus sp.2 “Barcelos” has a modal number of 2n = 52 chromosomes in both sexes and FN = 80 for males and FN = 79 for females. The karyotypic formula is 12 m + 12 sm + 4 st + 24a for males, and 11 m + 12 sm + 4 st + 25a for females (Fig. 3a and b). Heteromorphic NORs are found in the short arms of pair 23 (Fig. 3c).

Fig. 3
figure 3

Karyotypes of male (a) and female (b) Ancistrus sp.2 “Barcelos” after conventional Giemsa-staining, evidencing the sex chromosomes (box) and the NOR-bearing chromosomes after silver-staining (c). m = metacentric; sm = submetacentric; st = subtelocentric; a = acrocentric

Full size image

Ancistrus sp.1 “Balbina” has interstitial blocks of constitutive heterochromatin in the short arms of pairs 3, 4, and 14, and in the long arms of pair 6; pericentromeric blocks in pairs 8 and 9; distal blocks in the long arms of pair 12; and proximal blocks in long arms of pair 19 (Fig. 4a). Ancistrus sp.2 “Barcelos” has small pericentromeric blocks in pairs 15, 16, and 17; conspicuous blocks in the short arms of pairs 14 and 23, in an interstitial position in the long arms of pairs 1 and 11, and in the short arms of pair 2 (Fig. 4b). In both species, NORs are coincident with heterocromatin blocks, which are absent in sex chromosomes (Fig. 4a and b).

Fig. 4
figure 4

Metaphase and Idiogram showing the distribution of constitutive heterochromatin after C-banding technique in Ancistrus sp.1 “Balbina” (a) and in Ancistrus sp.2 “Barcelos” (b). Arrows indicate the NOR-bearing chromosomes with positive C-banding. m = metacentric; sm = submetacentric; st = subtelocentric; a = acrocentric

Full size image

Discussion

Moreira-Filho et al. (1993) summarized five sex chromosome systems that have been described for Neotropical freshwater fishes: ZZ/ZW, XX/XY, X1X1X2X2/X1X2Y, XX/XY1Y2, and ZZ/ZW1W2. Most recently, Alves et al. (2006) described a sixth system, XX/XO. Thus far, 63 occurrences of heteromorphic sex chromosomes have been described for this group of fish. Sixty-three and a half percent of these represent cases in which females are heterogametic, 36.5% in which males are heterogametic, and 19% are cases of multiple systems (Centofante et al. 2002; Mariotto et al. 2004; Venere et al. 2004; Alves et al. 2006; Centofante et al. 2006; de Oliveira et al. 2006; Mariotto and Miyazawa 2006).

In the genus Ancistrus, as in Loricariids in general, few cases of heteromorphic sex chromosomes have been described, all of them representing simple sex determination systems. ZZ/ZW and XX/XY systems were found in distinct populations of Ancistrus cf. dubius of the Brazilian wetlands of the Pantanal in Mato Grosso State (Mariotto et al. 2004; Mariotto and Miyazawa 2006). The only XX/XO system ever documented in a Neotropical fish was found in Ancistrus n. sp.1 of the Vermelho River in Goiás State, Brazil (Alves et al. 2006).

We found two types of multiple sex chromosome mechanisms in Ancistrus species from the central Amazonia. We suggest a XX/XY1Y2 mechanism for Ancistrus sp.1 “Balbina”, based on the presence of an additional chromosome in the male karyotype. In this species, the X chromosomes are represented by large metacentric chromosomes (pair 2 of the complement) and Y chromosomes by small metacentrics (pair 14 of the male complement). We propose a Z1Z1Z2Z2/Z1Z2W1W2 mechanism for Ancistrus sp.2 “Barcelos”, based on heteromorphism in the female karyotype: chromosome Z1 is a small metacentric (pair 5 of the complement), chromosome Z2 is the largest submetacentric of the complement (pair 7), W1 is a small acrocentric, and W2 is a medium-sized submetacentric.

The multiple mechanism of the XX/XY1Y2 type found in Ancistrus sp.1 “Balbina” represents the second documented occurrence of this type of multiple sex chromosomes for Loricariidae, and the third for Neotropical fishes overall. This type of system has been described for the erythrinid Hoplias malabaricus (Bertollo et al. 1983) and for the loricariid Harttia carvalhoi (Centofante et al. 2006). The ZZ/ZW1W2 multiple mechanism, in which the female is the heterogametic sex, has previously been described only in the parodontid Apareiodon affinis (Moreira-Filho et al. 1980). The occurrence of centric fissions seems to be the most plausible explanation for the origin of these sexual systems (Moreira-Filho et al. 1980; Bertollo et al. 1983). However, Centofante et al. (2006) believe that, in Harttia carvalhoi, sex chromosomes have been originated by centric fusions or translocations, since the other species in the genus show higher diploid numbers. Furthermore, karyotypes of these species show that all chromosome pairs are relatively the same size, whereas the first pair of chromosomes in H. carvalhoi is much larger than the other pairs in the complement.

Alves et al. (2003) suggested that centric fusion predominate among the rearrangements that occurred in the evolution of karyotypes of Ancistrus species. Therefore it is likely that centric fusions, followed by pericentric inversions in the chromosomes Y1Y2, are responsible for the differentiation of sex chromosomes in Ancistrus sp.1 “Balbina”. This species has a lower diploid number compared to the supposedly basal condition among the Ancistrini (namely, 2n = 52 chromosomes, the majority meta/submetacentric; Artoni and Bertollo 2001; Alves et al. 2003). We can hypothesize two pathways for the origin of the karyotype observed in males of Ancistrus sp.1 “Balbina”: (1) fusion of a set of acrocentric chromosomes, followed by centric fission of one of the homologues of pair 2, generating two small acrocentrics; subsequent pericentric inversions in these chromosomes would have generated the Y1Y2 small metacentrics; or (2) the initial chromosome fusion event would have involved just one of the homologues of pair 2; then translocations in the two remaining acrocentrics would have generated the Y1Y2 small metacentrics. In females, the complete fusion of the corresponding set of acrocentrics would have generated the X chromosomes and the karyotype of 2n = 38.

The multiple sex chromosome mechanism Z1Z1Z2Z2/Z1Z2W1W2 we propose for Ancistrus sp.2 “Barcelos” is the first observation of this type of system in fish. A similar mechanism was found in a species of flea (Nosopsyllus fasciatus; Aphaniptera), but where the male is the heterogametic sex (Bayreuther 1969). Since the karyotype of Ancistrus sp.2 “Barcelos” is made up of 2n = 52 chromosomes and about half its complement consists of acrocentric chromosomes, we suggest that pericentromeric inversions and translocations have been involved in both the karyotypic evolution as well as the differentiation of sex chromosomes in this species.

The most common mechanism involved in the differentiation of simple sex chromosome systems is the accumulation of heterochromatin. However, structural events are also important and are often associated with sequences of repetitive DNA, as proposed by Artoni et al. (2001) and Artoni and Bertollo (2002) for differentiation of the W chromosome in some Triportheus species (Characiformes: Characidae). Multiple sex chromosome systems are believed to originate from pre-existing simple systems, arising mainly through rearrangements involving sex and autosomic chromosomes (Guerra 1988). The rearrangements most commonly proposed are: (1) translocations, such as those found in populations of Hoplias malabaricus (Bertollo et al. 1983; Bertollo et al. 1997); (2) centric fusion or Robertsonian translocation, as in Eigenmannia sp. 2 (Almeida-Toledo et al. 1984) and Brachyhypopomus pinnicaudatus (Almeida-Toledo et al. 2000); and (3) centric fissions, like those observed in Apareiodon affinis (Moreira-Filho et al. 1980). The absence of a heterochromatin accumulation in the differentiation of multiple chromosome systems in Ancistrus supports our hypothesis of an origin through a simple system.

Congeneric species and even different populations of the same species can exhibit different sex chromosome systems and vary in which sex is heterogametic, which point out to different stages of sex chromosome differentiation (Almeida-Toledo and Foresti 2001; Mariotto et al. 2004; Mariotto and Miyazawa 2006). Therefore, the presence of different sex chromosome systems in species of Ancistrus indicates a probable independent origin of these systems, and suggests that the differentiation of sex chromosomes is evolutionarily recent among species in this genus.