Cytogenetic characterization and B chromosome diversity in direct-developing frogs of the genus Oreobates (Brachycephaloidea, Craugastoridae)

Abstract

Oreobates Jiménez de la Espada, 1872 is a large group of South American frogs with terrestrial reproduction and direct development, located in the superfamily Brachycephaloidea. About 260 brachycephaloidean species have been cytogenetically studied so far, at least with standard techniques. However, this information represents fewer than 17% species of the family Craugastoridae Hedges, Duellman & Heinicke, 2008, where the genus Oreobates is included. In the present work, using a diversity of standard and molecular techniques, we describe the karyotype of Oreobates barituensis Vaira & Ferrari, 2008, Oreobates berdemenos Pereyra, Cardozo, Baldo & Baldo, 2014 and Oreobates discoidalis (Peracca, 1895), from northwestern Argentina. The three species analyzed showed a diploid karyotype with 2n = 22 biarmed chromosomes, fundamental number (FN) = 44, nucleolus organizer regions (NORs) located pericentromerically on pair 7, and a centromeric and pericentromeric C-banding pattern. We observed variations in the chromosome number in Oreobates barituensis due the presence of two morphs of B chromosomes, one medium-sized telocentric (BT) and another subtelocentric and smaller (Bst). Both B chromosomes are mitotically stable and were recorded in all somatic and germinal cells analyzed. The BT chromosome occurred at a maximum of one per individual (2n = 22+BT), and the other one was observed single (2n = 22 + Bst) or as a pair in two doses (2n = 22 + 2BT). We additionally observed other supernumerary chromosomes in the three species analyzed, all of them euchromatic, small, dot-shaped and with instability during mitoses, showing a frequency of occurrence below 50% in studied specimens. The occurrence of polymorphic and spontaneous chromosomal rearrangements and supernumerary chromosomes is a recurrent feature reported in frogs with terrestrial habits (Brachycephaloidea and Hemiphractidae Peters, 1862), which suggests that Brachycephaloidea may be a promising group for studying the origin and maintenance of B chromosomes in anurans.

Keywords: Anura; Cytogenetics; accessory elements; ribosomal DNA.

Figure 1.

Karyotypes of Oreobates barituensis (A, D), Oreobates berdemenos (B, E), Oreobates discoidalis (C, F), the large telocentric B_T and the small subtelocentric B_st supernumerary chromosomes in Oreobates barituensis (Boxes G and H). Giemsa stained (A–C, G), C- banding (D–F, H). The insets (*) shows interstitial C-bands. Arrowheads point to C positive bands. Bar = 10 µm.

Figure 2.

Mitotic metaphases of Oreobates barituensis (A, B), Oreobates berdemenos (C, D) and Oreobates discoidalis (E, F) stained with DAPI (A, C, E) and CMA₃ (B, D, F), arrowheads point pairs 7. Bar = 10 µm.

Figure 3.

NORs bearing chromosome pairs (A–C) and rDNA (D–F), in Oreobates barituensis (A, D), Oreobates berdemenos (B, E) and Oreobates discoidalis (C, F). B_T chromosome in Oreobates barituensis showing positive NORs (G), active and inactive rDNA (H), CMA₃ (I) and DAPI (J). Two interphase cells with three active NORs after silver staining (K). Black arrows indicate Ag-NORs, white arrows and arrowheads shows positive and negative fluorescent marks, respectively. Bars = 10 µm.

Figure 4.

The small B_st chromosome (arrowhead) in Oreobates barituensis with conventional staining (A), DAPI (B) and CMA₃ (C). B_d chromosome (arrow) in Oreobates berdemenos evidenced after Giemsa (D) and C-banding (E). Bar = 10 µm.

Figure 5.

Meiosis I in specimens of Oreobates barituensis. Metaphase I with conventional staining showing Bs as univalent: one B_T (A) and two B_st with negative pyknosis (B). Diakinesis after C- banding with a bivalent B_st (C). Arrow and arrowheads indicate the B_T and B_st respectively.