Karyotype characterization and ZZ / ZW sex chromosome heteromorphism in two species of the catfish genus Ancistrus Kner , 1854 ( Siluriformes : Loricariidae ) from the Amazon basin

We present karyotypic characteristics and report on the occurrence of ZZ/ZW sex chromosomes in Ancistrus ranunculus (rio Xingu) and Ancistrus sp. “Piagaçu” (rio Purus), of the Brazilian Amazon. Ancistrus ranunculus has a modal number of 2n=48 chromosomes, a fundamental number (FN) of 82 for both sexes, and the karyotypic formula was 20m+8sm+6st+14a for males and 19m+9sm+6st+14a for females. Ancistrus sp. “Piagaçu” presented 2n=52 chromosomes, FN= 78 for males and FN= 79 for females. The karyotypic formula was 16m+8sm+2st+26a for males and 16m+9sm+2st+25a for females. The high number of acrocentric chromosomes in karyotype of Ancistrus sp. “Piagaçu” differs from the majority of Ancistrini genera studied so far, and may have resulted from pericentric inversions and translocations. The lower number of chromosomes in A. ranunculus indicates that centric fusions also occurred in the evolution of Ancistrus karyotypes. We conclude that karyotypic characteristics and the presence of sex chromosomes can constitute important cytotaxonomic markers to identify cryptic species of Ancistrus. However, sex chromosomes apparently arose independently within the genus and thus do not constitute a reliable character to analyze phylogenetic relations among Ancistrus species.

Here we present the karyotypic structure and record the occurrence of ZZ/ZW sex chromosomes of two species of the genus Ancistrus and discuss possible mechanisms involved in the differentiation of this sex chromosomes.Muller et al., 1994: five specimens (three males and two females) acquired from ornamental fish exporters (Turkys Aquarium, Manaus), from the rio Xingu, Altamira, Pará (03º15'21"S, 52º12'45"W) (Fig. 1a).

Ancistrus ranunculus
Ancistrus sp."Piagaçu": seven specimens (three males and four females) collected in lago Aiapuá, on the left bank of the rio Purus, in the Piagaçu-Purus Sustainable Development Reserve (04º27'26"S, 62º11'56"W).This apparently undescribed species (S.Fish-Muller, pers.comm.)presents the following color pattern: dorsal region dark grey with small white spots; ventral region yellowish grey with well-defined stipples, that in smaller specimens resemble ocelli; dorsal fin dark grey with pale stipples (Fig. 1b).
Chromosomal preparations were obtained from kidney cells, following the "air-drying" technique of Bertollo et al. (1978).Approximately 30 metaphasic plates were analysed for each individual.The constitutive heterochromatin was identified according to Sumner (1972) and the nucleolar organizing regions (NORs) were detected using the technique described by Howell & Black (1980).Chromosome morphology was based on arm ratios (long arm length divided by short arm length), as proposed by Levan et al. (1964).Chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st), or acrocentric (a).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
Ancistrus ranunculus has a modal number of 2n=48 chromosomes and a fundamental number of 82 for both sexes; however, the karyotypic formula was different for males (20m+8sm+6st+14a) and females (19m+9sm+6st+14a).Sex chromosomes of the type ZZ/ZW were found in this species, with the Z being a small metacentric, and the W a medium sized submetacentric.The NORs are located in the region proximal to the long arms of pair 16.We also observed a size heteromorphism of the NORs between the homologues (Fig. 2).Ancistrus sp."Piagaçu" has a modal number of 2n=52 chromosomes and a fundamental number of 78 for males and 79 for females.The karyotypic formula is 16m+8sm+2st+26a for males and 16m+9sm+2st+25a for females.ZZ/ZW chromosomes were also found in this species, the Z chromosome being a large acrocentric, and the W a small submetacentric.The NORs are found on the short arms of pair 26 (Fig. 3).
Ancistrus ranunculus has conspicuous blocks of heterochromatin in the distal region of pairs 18, 20, 21, and 24; smaller blocks in the proximal region of pair 16; and a small pericentromeric block on pair 9.One of the chromosomes of pair 10 has, along with the small pericentromeric blocks observed in its homologue, a considerable portion of heterochromatin on the long arm in the female karyotype (Fig. 4a).In Ancistrus sp."Piagaçu" we found small pericentromeric blocks on pairs 1, 9, 14, 15, 17, 22, 23, 24, and 25; interstitial blocks on the long arms of pairs 2 and 4 and on the short arm of pair 2; small distal blocks on the short arms of pair 1; and conspicuous distal blocks on the short arms of pair 26 (Fig. 4b).In this species, sex chromosomes did not exhibit heterochromatin blocks.In both species, the NORs were coincident with blocks of heterochromatin (Fig. 4).

Discussion
The Ancistrini has the supposedly ancestral diploid number of 2n=52 chromosomes, the majority of which are meta-and submetacentric (Artoni & Bertollo, 2001;Alves et al., 2003).We found Ancistrus sp."Piagaçu" to have the supposedly ancestral diploid number, yet its karyotype is composed of a high number of acrocentric chromosomes, a characteristic different from the majority of the Ancistrini species that have been studied so far.Pericentric inversions and translocations may be the principal rearrangements responsible for the differentiation of the karyotype in this species.The presence of 2n=48 chromosomes in A. ranunculus corroborates the hypothesis of Alves et al. (2003), who suggest that centric fusions, like chromosome rearrangement, predominate in the evolution of Ancistrus karyotypes.
In a comparative karyotypic analysis of males and females, we found morphologically differentiated sex chromosomes of the type ZZ/ZW in both studied species.In A. ranunculus the Z chromosome is represented by a small metacentric (corresponding to one of the homologues of pair 10 of the complement), and the W chromosome by a medium sized submetacentric.In Ancistrus sp."Piagaçu" the Z chromosome is a large acrocentric (pair 16) and the W is a small submetacentric.
The initial heterochromatization seems to be the principal factor in the differentiation of the majority of ZZ/ZW systems in fish, generally resulting in an increase in the size of the W chromosomes.Examples of this can be found in some species in the anostomid genus Leporinus (Galetti et al., 1981;Galetti & Foresti, 1986;Galetti et al., 1995;Venere et al., 2004); in the prochilodontid Semaprochilodus taeniurus (Feldberg et al.,Fig. 2. Karyotypes of male (a) and female (b) Ancistrus ranunculus 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.1987) and in the Hypoptopomatinae loricariid Microlepidogaster leucofrenatus (Andreata et al., 1993); and in two species of the parodontid genus Parodon (Moreira-Filho et al., 1993;Centofante et al., 2002).However, past structural rearrangements seem to have occurred and many times are associated with repetitive sequences of DNA, as suggested for the differentiation of the W chromosome in species of the genus Triportheus (Artoni et al., 2001;Artoni & Bertollo, 2002).
In the genus Ancistrus, the heterochromatization process is involved in the differentiation of the sex chromosome system of Ancistrus cf.dubius (Mariotto et al., 2004) and A. ranunculus (present study), however, the suggested pathway for these two differentiations seems to be distinct.In Ancistrus cf.dubius, there was an accumulation of heterochromatin over chromosome Z, followed by the loss of this heterochromatin segment in W, in such a way that it appears much smaller and only possesses homology with the euchromatic segment of the Z chromosome.In A. ranunculus, the W chromosome is almost completely heterochromatic, with a considerable portion of heterochromatin on the long arm, while the Z chromosome is much smaller and has only a small block of heterochromatin in the centromeric position.
The differentiation of sex chromosomes may also be linked to structural rearrangements, resulting in the prevention of meiotic recombination (Beçak & Beçak, 1969;Almeida-Toledo et al., 2000).Nevertheless, these events are more related to the emergence of multiple sex chromosome systems, as seen in the parodontid Apareiodon affinis (Moreira-Filho et al., 1980), in the sternopygid Eigenmania sp.(Almeida-Toledo et al., 1984), and in some populations of the erythrinid Hoplias malabaricus (Bertollo et al., 1997).While uncommon among species that have the simple ZZ/ZW mechanism, structural rearrangements seem to play an important role in the differentiation of sex chromosomes in the loricariids Loricariichthys platymetopon (Scavone & Júlio Jr., 1995) and Hypostomus sp.(Artoni et al., 1998).For these species, it has been suggested that a pericentric inversion in an acrocentric chromosome similar to the Z chromosome, followed by a loss of chromosomic material, could have originated the W chromosome.We believe that a similar process has occurred in Ancistrus sp."Piagaçu" since we did not find any heterochromatin blocks on the Z (large acrocentric) or W (small submetacentric) sex chromosomes.
The differentiation of sex chromosomes in Neotropical freshwater fish seems to have resulted from independent events, and its presence does not relate to species' phylogenetic relationships (Almeida-Toledo & Foresti, 2001).Nevertheless, in Triportheus (Characidae) all species analyzed share a ZZ/ZW mechanism that may have originated in a common ancestor for this genus (Artoni et al., 2001;Artoni & Bertollo, 2002).The occurrence of ZZ/ZW sex chromosomes in Ancistrus species probably do not represent a synapomorphy for the group, since they involve different chromosome pairs that vary in both size and morphology and probably resulted from distinct differentiation processes.Only A. ranunculus shows the most common mechanism observed among fish with ZZ/ZW sex chromosomes, characterized by an increase in the size of chromosome W through the accumulation of heterochromatin.
Ancistrus is a species-rich loricariid genus that seems to contain a large number of morphologically similar, undescribed species (S.Fish-Muller, pers.comm.).The results presented herein indicate that chromosome macrostructure, and especially the presence of sex chromosomes, represent important cytotaxonomic markers, which can help in the identification of cryptic species of that genus.However, sex chromosomes apparently arose independently within the genus and thus do not constitute a reliable character to analyze phylogenetic relations among Ancistrus species.