B-chromosomes in two Brazilian populations of Dendropsophus nanus ( Anura , Hylidae )

We report on the presence of B-chromosomes in two populations of Dendropsophus nanus (= Hyla nana Boulenger, 1889) from São Paulo State, Brazil. Such chromosomes were observed in 4 out of 43 specimens (9.3%) and in 9 out of 15 specimens (60%) from the municipalities of Nova Aliança and Botucatu, respectively. The karyotype 2n = 30 + 1B found in D. nanus was similar to that of other species with 2n = 30 chromosomes, except for the presence of an additional small telocentric chromosome. In one specimen from Botucatu, cells with one to three extra chromosomes were observed. These B-chromosomes appeared as univalent in meiosis I and did not bear a nucleolar organizer region or exhibit constitutive heterochromatin.


Introduction
B-Chromosomes are extra chromosomes that occur in animals and plants and are generally considered dispensable for normal development, since they have no apparent function (Jones and Rees, 1982). B-chromosomes have been found in approximately 15% of living species (Beukeboom, 1994), and described in 26 species of salamanders and frogs (Green, 2004). As a rule, carriers of these chromosomes are phenotypically indistinguishable from those individuals without them (Clark and Wall, 1996). B-chromosomes bear no similarity to the autosomes, are inherited according to a non-Mendelian pattern, and occur as univalents in meiosis (Jones and Rees, 1982;Green, 1991Green, , 2004. The number of B-chromosomes can vary among populations of the same species, among individuals in a population and among cells in an individual. In the latter case, this variation results from anaphase lag, with subsequent elimination of B-chromosomes from some cells or tissues, or, alternatively, it is caused by mitotic non-disjunction, with sister chromatids migrating to the same pole (Clark and Wall, 1996).
Among vertebrates, one of the largest variations in the number of B-chromosomes has been described in Leiopelma hochstetteri, a frog endemic to New Zealand, in which individuals with up to 16 of these chromosomes have been observed (Green, 1988). This variation has been attributed to mitotic non-disjunction resulting from instability during cell division.
The specimens were identified according to Medeiros et al. (2003), and were deposited in the "Prof. Adão José Cardoso" Museum of Natural History (ZUEC), of Universidade Estadual de Campinas, SP, Brazil, and in the Zoology Department ( Chromosome preparations were obtained from intestinal and testicular cell suspensions, as described by Schmid (1978) and Schmid et al. (1979) and analyzed after routine staining with a 10% Giemsa solution, C-banding (King, 1980), Ag-NOR staining (Howell and Black, 1980) and fluorescence in situ hybridization (FISH) (Viegas-Péquignot, 1992) with an rDNA probe. The FISH probe consisted of a recombinant HM123 plasmid containing a fragment of Xenopus laevis rDNA (Meunier-Rotival et al., 1979), which was biotin-labelled by nick translation reaction using a BioNick TM Labeling System (Invitrogen). The probe was detected using goat anti-biotin and fluorescein anti-goat IgG (Vector Laboratories). Analysis was done with an Olympus BX60 photomicroscope. The chromosomal nomenclature regarding the position of centromere followed the classification proposed by Green and Sessions (1991).

Results
The karyotype of 39 specimens of D. nanus from Nova Aliança and of five specimens from Botucatu showed 2n = 30 chromosomes ( Figure 1A), as previously described for a population from Nova Aliança (Medeiros et al., 2003). However, in four males from Nova Aliança (ZUEC 11673, 11651, 11652 and DZSJRP 1111) and nine males from Botucatu (ZUEC 12242, 12245, 12246, 12247, 12249, 12251, 12254, 12256 and 12261), the karyotype was 2n = 31 chromosomes (Table 1). This karyotype differed 258 Medeiros et al. from that with 2n = 30 by the presence of an extra small telocentric chromosome ( Figure 1B). In one specimen from Botucatu, we observed cells with 2n = 30 + 1B, 30 + 2B and 30 + 3B chromosomes (Figure 1B-D; Table 1). These additional chromosomes were all of the same size and morphology. The small extra chromosome was observed in meiosis I metaphases that had 15 bivalents and one, two or three small univalents (Figure 2A-C). Hence, the B-chromosomes did not pair with the A-chromosomes. Individuals with 2n = 30 chromosomes had 15 bivalents, and no univalents were observed.
The two populations of D. nanus shared a common C-banding pattern characterized by small amounts of heterochromatin in the pericentromeric region of all chromosomes ( Figure 1E), except for the extra chromosomes ( Figure 1F-H), in which no C-bands were observed. These chromosomes did not bear a NOR, as demonstrated by Ag-NOR staining and/or FISH in four specimens with 2n = 30 + 1B chromosomes from Nova Aliança and in all specimens from Botucatu with 2n = 30 + 1B and 2n = 30 + 1 -3B. Chromosome pair 13 was identified as the NORbearing chromosome in all specimens analyzed ( Figures 1I  and 3A, B).

Discussion
The extra chromosomes found in specimens of D. nanus with 2n = 31 and in the specimen from Botucatu that had cells with 2n = 31 to 2n = 33 chromosomes can be considered as B-chromosomes, since they showed some of the characteristics usually attributed to these chromosomes. This small extra element, which differs morphologically from autosomes, always occurs as a univalent in cells in metaphase I (reviewed in Jones and Rees, 1982).
Previous descriptions of the karyotype of D. nanus from other populations (Rabello, 1970;Bogart, 1973;Skuk and Langone, 1992) did not mention the presence of B-chromosomes.
Since individuals carrying B-chromosomes are generally indistinguishable from those without these chromosomes, the conclusion is that B-chromosomes must not carry genes with important phenotypic effects. However, the maintenance of B-chromosomes in certain populations has been taken as indicative of their role in conferring some advantage to transcriptional activity or to the genetic variability of the species, as pointed out by Belcheva and Sofianidou (1990), when describing B-chromosomes in Rana temporaria. Indeed, several studies have shown that some B-chromosome genes are expressed (Green, 1990;Jones, 1995;Covert, 1998;Camacho, 2000;Green, 2004), and their phenotypic influence may be dependent on the number of these chromosomes present in the cell, although this has not yet been documented for amphibians (Green, 1991). The deleterious effect can be a decrease in fertility (Hewitt et al., 1987) or an abnormal meiosis (Parker et al., 1981). An interesting case of B-chromosomes in amphibians involves Leiopelma hochstetteri (Anura), which may have up to 16 extra chromosomes. In this species, the B-chromosomes in the lampbrush state have small lateral loops indicating transcriptional activity, although lower than that of autosomes (Green et al., 1987;Green, 1988 The B-chromosomes of D. nanus did not bear a NOR, as demonstrated by silver staining and FISH). Ribosomal genes are rarely present on B-chromosomes, and in Anura they have been detected only in Scaphiopus hammondi (Green, 1988) and Gastrotheca espeletia (Schmid et al., 2002). In the former species, the NOR contributed to increase the number of nucleoli in the cell, indicating that B-chromosomes can carry functional genes of great importance (Green, 1991).
Various mechanisms have been proposed to explain the origin and maintenance of B-chromosomes. Traditionally, these chromosomes are believed to be derived from autosomes (Jones and Rees, 1982). Since pairing or chiasmata between B-chromosomes and A-chromosomes are not observed in meiosis, it is probable that the original homology between them was rapidly lost. Modifications in the structure and in the pairing behavior during meiosis would prevent association with the ancestral A-chromosomes (Camacho et al., 2000). Interestingly, in the anuran Leiopelma hochstetteri, the B-chromosomes are most probably derived from the sex chromosome (Green, 2004), a conclusion supported by the sequence homology between the B-chromosomes and the univalent W chromosome (Sharbel et al., 1998;Green, 2004).
Heterochromatization is a common process in the differentiation of B-chromosomes, since many are completely heterochromatic in numerous species (Venere et al., 1999). Although the heterochromatic condition of B-chromosomes is not a general pattern, these chromosomes may originate from centromeric fragments, but this hypothesis has received little support (Green et al., 1987;Camacho et al., 2000). This process of B-chromosome differentiation is not applicable to D. nanus, since in this species the B-chromosomes showed no heterochromatic band, and hence their derivation must have involved different mechanisms.
Another possibility to explain the origin of B-chromosomes is that they derive from A-chromosomes of closely related species through interspecific hybridization, as suggested for hybrid species of the fish Poecilia formosa (Schartl et al., 1995) and of the wasp genus Nasonia (McAllister and Werren, 1997). Dendropsophus sanborni Schmidt, 1944, is morphologically (Cei, 1980;Medeiros et al., 2003) and ecologically (Rossa-Ferres and Jim, 2001) very similar to D. nanus, and both belong to the same intrageneric group (Frost, 2004). Considering their great similarity and syntopic occurrence in several localities 260 Medeiros et al.  ( Langone and Basso, 1987), we cannot rule out the hypothesis of interspecific hybridization in the origin of D. nanus karyotypes with B-chromosomes.