The karyotype of Adenomera diptyx (Boettger 1885) (Anura, Leptodactylidae) from northeastern Argentina

In this work we analyzed the karyotype of five populations of Adenomera diptyx from Argentina after conventional staining, Ag-NOR and C-banding. All specimens presented 2n = 26 and FN = 34. The karyotype was formed by three submetacentric, one metacentric and nine telocentric pairs. Silver staining revealed that the NOR was located on a secondary constriction in pair 7. C- banding evidenced constitutive heterochromatin at the pericentromeric region of all chromosomes. The karyotype of A. diptyx was similar to that of A. hylaedactyla (2n = 26, FN = 34) and different from that of A. andreae (2n = 26, FN = 40) in the fundamental number and secondary constriction position. It also differed from the karyotypes of A. marmorata (2n = 24, FN = 34 and 36) and of A. aff. bokermanni (2n = 23, FN = 34) in diploid number. Until a comprehensive cytogenetic analysis of all the species of the genus is performed, their chromosome evolution will remain poorly understood.

The genus Adenomera Fitzinger in Steindachner, 1867 was revalidated by Heyer (1974) to include the species of Leptodactylus belonging to the marmoratus group (Heyer, 1969). At present, the relationships between Adenomera and Leptodactylus are subject to debate and several authors advanced different taxonomic proposals. Frost et al. (2006) considered Adenomera as a synonym of Leptodactylus, suggesting the creation of the subgenus Leptodactylus (Lithodytes), which would include the species of the former genera Lithodytes and Adenomera. Some authors refer to the species of Adenomera as the Leptodactylus marmoratus species group sensu Heyer (1973) (Almeida and Angulo, 2006;Angulo and Reichle, 2008;Campos et al., 2009), whereas others recognize Adenomera as a valid genus (Kwet, 2007;Kwet et al., 2009;Ponssa and Heyer, 2007). In this paper, we adopted the latter systematic approach because there is morphological, ethological, and bioacoustic evidence suggesting that all species of Adenomera may form a natural group with a single ancestral lineage (Kwet et al., 2009).
The karyotypes of A. aff. bokermanni, A. hylaedactyla and of taxa belonging to the A. marmorata species-complex were recently analyzed after silver staining of the NORs, C-banding, fluorochrome staining and FISH (Campos et al., 2009). The telocentric pairs 6, 7 and 11 were reported as NOR-bearing chromosomes and all chromosomes presented pericentromeric C-bands (Campos et al., 2009).
The species identity in the genus Adenomera is hard to resolve because of the intra-and inter-populational morphological variation and due to the existence of cryptic species (Heyer, 1984;de la Riva, 1996;Angulo et al., 2003). To clarify the systematics of the genus, it is necessary to use non-morphological characters, such as advertisement calls, cytogenetic and molecular data (Heyer, 1984).
In this work we describe the karyotype, Ag-NOR location and C-banding patterns of five Adenomera populations from northeastern Argentina currently recognized as A. diptyx. This taxon was revalidated by de la Riva (1996), including the populations of Adenomera from the oriental region of Paraguay, southeastern Bolivia, Mato Grosso (Brazil), and northern Argentina, but the identity of these populations remains poorly investigated (Lavilla and Cei, 2001).
Chromosome spreads were obtained from intestinal epithelium and testes following Schmid (1978). Conventional staining was performed with Giemsa diluted in phosphate-buffered saline solution, pH 6.8. Silver staining of the NORs (Ag-NOR) and C-banding were obtained following Howell and Black (1980) and Sumner (1972), respectively.
After silver staining, theAg-NORs were located in the proximal region of both homologues of telocentric pair 7, at the same site of the secondary constriction, in seven specimens from Corrientes (Figure 1a). Three specimens (UNNEC 8294,8354,9704) showed a stronger silver impregnation on both homologues due to tandem duplications ( Figure 1a). C-banding revealed the presence of constitutive heterochromatin at the pericentromeric region of all chromosomes (Figure 1b).
Current cytogenetic data, available for less than 50% of the recognized species of Adenomera, revealed five different karyotypes ( Table 2), suggesting that Adenomera   (Bogart, 1974;Silva et al., 2000;Amaro-Ghilardi et al., 2006;Lourenço et al., 2007). The specimens of A. diptyx from five localities of northeastern Argentina presented the same karyotype previously reported for Adenomera with 2n = 26 and FN = 34. The similarities in 2n, FN and NOR distribution between A. diptyx and A. hylaedactyla are shown in Table 2, as well as the differences in relation to the other species.
The presence of a single Ag-NOR-bearing chromosome pair, as observed in A. diptyx, and of tandem duplication involving the ribosomal DNA in one homologue are common in anurans (Schmid et al., 1990). Nevertheless, some of our specimens of A. diptyx exhibited duplications in both homologues.
The C-banding pattern observed in A. diptyx, with heterochromatin at the pericentromeric regions of all chromosomes, is similar to that of other species of Adenomera already analyzed and is the most common pattern found among anurans (Campos et al., 2009).
Two alternative hypotheses have been proposed to explain the chromosome evolution of Adenomera. According to Bogart (1974), Heyer and Diment (1974) and Campos et al. (2009), the karyotype evolution in Adenomera could have involved centric fusions and pericentric inversions of uni-armed chromosomes from an ancestral karyotype with 2n = 26 and a large number of telocentrics. Under this hypothesis, the primitive condition would be represented by A. hylaedactyla and A. diptyx and the karyotypes of A. andreae, of the A. marmorata species-complex and of A. aff. bokermanni would be derived. The typical large metacentric pair 1 and the lower diploid numbers of the A. marmorata species-complex (2n = 24) and of A. aff. bokermanni (2n = 23) may be a result of centric fusions (Bogart, 1974;Campos et al., 2009). Pericentric inversions involving one or three pairs of telocentric chromosomes could explain the two different karyotypes reported for the A. marmorata species-complex (2n = 24, FN = 34 and 2n = 24, FN = 36) and the distinctive fundamental number of A. andreae (2n = 26, NF = 40), respectively (Bogart, 1974, Campos et al., 2009).
An alternative hypothesis assumes that a diploid number of 2n = 24, also found in Leptodactylus silvanimbus and in the Leptodactylus sister-clade formed by Paratelmatobius-Scythrophrys, would be the ancestral condition. In this case, the karyotypes of most Leptodactylus (2n = 22), Lithodytes (2n = 18) and Adenomera species (2n = 26) would represent derived conditions (Amaro-Ghilardi et al., 2006). 86 Karyotype of Adenomera diptyx Adenomera seems to be a monophyletic clade (Heyer, 1974;de Sa et al., 2005) without karyotypic uniformity, therefore representing an interesting group for the study of chromosome evolution. Until a comprehensive cytogenetic survey of all the species of the genus is performed, any hypothesis of chromosome evolution will remain poorly supported.

Acknowledgments
We are grateful to EO Lavilla and WR Heyer for their valuable suggestions and comments. Dirección de Fauna, Parques y Ecología, Dirección de Fauna y Parques, and Dirección de Recursos Naturales from the Argentinean provinces of Chaco, Formosa and Corrientes, respectively, provided the permits for collecting the specimens analyzed in this paper. This work was supported by Secretaría General de Ciencia y Técnica (Universidad Nacional del Nordeste) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.