Research Article |
Corresponding author: Juan Martín Ferro ( ferrojm@gmail.com ) Academic editor: Nikolay Tzankov
© 2016 Juan Martín Ferro, Alberto Taffarel, Darío Cardozo, Jimena Grosso, María Pía Puig, Pablo Suárez, Mauricio Sebastián Akmentins, Diego Baldo.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Ferro JM, Taffarel A, Cardozo D, Grosso J, Puig MP, Suárez P, Akmentins MS, Baldo D (2016) Cytogenetic characterization and B chromosome diversity in direct-developing frogs of the genus Oreobates (Brachycephaloidea, Craugastoridae). Comparative Cytogenetics 10(1): 141-156. https://doi.org/10.3897/CompCytogen.v10i1.5718
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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 O. barituensis Vaira & Ferrari, 2008, O. berdemenos Pereyra, Cardozo, Baldo & Baldo, 2014 and O. 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 O. 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.
Cytogenetics, accessory elements, ribosomal DNA, Anura
Superfamily Brachycephaloidea includes a large group of frogs with terrestrial reproduction and direct development, with more than a thousand species assigned to three families: Brachycephalidae Günther, 1858, Craugastoridae, and Eleutherodactylidae Lutz, 1954 (
Within Craugastoridae (subfamily Holoadeninae Hedges, Duellman & Heinicke, 2008), Oreobates Jiménez de la Espada, 1872 is a South American genus with 23 species that occurs on the lower slopes of the Andes from the upper Amazon basin in southern Colombia to northern Argentina, reaching eastwards some areas in western Brazil (
B chromosomes (Bs) are dispensable extra chromosomes in the standard karyotype (As) present in many taxa, and usually lack phenotypic effects on their hosts. Their prevalence in animal populations is highly variable, being one of the main causes of chromosomal polymorphism in eukaryotes (
In order to complement the karyotypic information available for Oreobates, in the present work we studied three species (O. barituensis, O. berdemenos, and O. discoidalis), from several localities in northwestern Argentina. Chromosome morphology, heterochromatin distribution and composition, and location of nucleolar organizer regions are described. We discuss and evaluate the apparent chromosomal homogeneity observed for the genus Oreobates, in contrast to the variability reported by the presence of supernumerary chromosomes.
We studied 64 specimens of both sexes of O. barituensis (N = 40), O. berdemenos (N = 14), and O. discoidalis (N = 10). Chromosome preparations were obtained from bone marrow, gut epithelium and testes in males (
The advertisement call of O. discoidalis remains unknown, and those described by
Oreobates barituensis, O. berdemenos, and O. discoidalis shared diploid karyotypes with 22 bi-armed chromosomes (2n = 22; FN = 44). Pairs 1, 2, 5, 6, 8–11 were metacentric, while 3, 4 and 7 submetacentric (Fig.
Karyotypes of Oreobates barituensis (A, D), O. berdemenos (B, E), O. discoidalis (C, F), the large telocentric BT and the small subtelocentric Bst supernumerary chromosomes in O. 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.
Mitotic metaphases of O. barituensis (A, B), O. berdemenos (C, D) and O. discoidalis (E, F) stained with DAPI (A, C, E) and CMA3 (B, D, F), arrowheads point pairs 7. Bar = 10 µm.
NORs bearing chromosome pairs (A–C) and rDNA (D–F), in Oreobates barituensis (A, D), O. berdemenos (B, E) and O. discoidalis (C, F). BT chromosome in O. 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.
Chromosome morphology in the three species of Oreobates. Chromosome types according to
Chromosome number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
---|---|---|---|---|---|---|---|---|---|---|---|
Oreobates barituensis | |||||||||||
%Set | 15% | 12% | 11% | 11% | 10% | 9% | 8% | 7% | 6% | 6% | 6% |
CI ± SD | 0.43 ± 0.02 | 0.38 ± 0.03 | 0.37 ± 0.05 | 0.28 ± 0.06 | 0.45 ± 0.02 | 0.43 ± 0.03 | 0.34 ± 0.04 | 0.46 ± 0.02 | 0.46 ± 0.02 | 0.45 ± 0.03 | 0.47 ± 0.03 |
Type | m | m | sm | sm | m | m | sm | m | m | m | m |
Oreobates berdemenos | |||||||||||
%Set | 15% | 12% | 11% | 11% | 10% | 9% | 8% | 7% | 6% | 6% | 6% |
CI ± SD | 0.42 ± 0.02 | 0.38 ± 0.02 | 0.35 ± 0.05 | 0.25 ± 0.04 | 0.44 ± 0.02 | 0.45 ± 0.02 | 0.33 ± 0.04 | 0.45 ± 0.05 | 0.47 ± 0.02 | 0.45 ± 0.03 | 0.46 ± 0.03 |
Type | m | m | sm | sm | m | m | sm | m | m | m | m |
Oreobates discoidalis | |||||||||||
%Set | 15% | 12% | 11% | 10% | 10% | 9% | 8% | 7% | 6% | 6% | 6% |
CI ± SD | 0.44 ± 0.02 | 0.39 ± 0.02 | 0.37 ± 0.04 | 0.29 ± 0.04 | 0.45 ± 0.02 | 0.44 ± 0.03 | 0.27 ± 0.05 | 0.46 ± 0.02 | 0.46 ± 0.03 | 0.43 ± 0.05 | 0.48 ± 0.01 |
Type | m | m | sm | sm | m | m | sm | m | m | m | m |
We found variations in the chromosome number in eight specimens of Oreobates barituensis as a consequence of two different B chromosomes (Fig.
The small Bst chromosome (arrowhead) in O. barituensis with conventional staining (A), DAPI (B) and CMA3 (C). Bd chromosome (arrow) in O. berdemenos evidenced after Giemsa (D) and C-banding (E). Bar = 10 µm.
In addition, eight specimens of O. barituensis (LGE 4785; 6202; MSA 127–8, 161, 164, 174, 177), four O. berdemenos (FML 24626, MSA 138, 142–3), and one O. discoidalis (FML 24513), showed variations in the chromosome number attributable to dot-shaped and mitotically unstable supernumerary chromosomes, named here Bd. These elements share a similar shape with no evident primary constrictions, and no more than one per metaphase was observed (i.e. 2n = 22; 22 + Bd), with an occurrence below 50% per individual. C-banding and DAPI staining showed mostly a euchromatic nature of these elements (Fig.
The Meiosis I analyses on males of O. barituensis with supernumerary chromosomes BT and Bst evidenced the presence of 11 bivalents, corresponding to the A standard complement in addition to Bs. The BT chromosome occurred as a single univalent with not differentiable pyknosis from other chromosomes (Fig.
The genus Oreobates is composed of 23 species (
Within the subfamily Holoadeninae, the chromosomes of only 8 of 119 recognized species were studied (
B chromosomes are widespread among eukaryotes (
NOR-bearing B chromosomes were reported in 27 species of plants and 25 of animals [three of them anurans: Eleutherodactylus gundlachi, Gastrotheca espeletia Duellman & Hillis, 1987, and Spea hammondii (Baird, 1859)], showed to be supernumerary chromosomes carrying rDNA detected by silver staining and/or FISH with a rDNA probe (
Two other types of supernumerary chromosomes observed in O. barituensis were a small subtelocentric and heterochromatic Bst that occurs in a high prevalence in the locality of Tiraxi, and the euchromatic dot-like Bd, which is mitotically unstable. Interestingly, specimens of the two other species of Oreobates analyzed herein also showed supernumeraries Bd of similar size, and smaller than the smallest pair of the A complement. B chromosomes, which lack a functional centromere would be lost by drift (
Finally, the available cytogenetic data points to the Brachycephaloidea as an extremely diverse group, with 2n ranging from 16 to 38, and FN = 26–52 (
The authors thank C. Martín, C. Guerra, and F. Burgos, for providing some specimens used in this work and C. Borteiro, F. Vera Candioti, and M. Pereyra for valuable corrections and comments on the manuscript. We also give thanks to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). JMF, AT, JG, and MSA are grateful to Carrera del Doctorado en Ciencias Biológicas de la FCEFyN de la UNC. JMF, AT and DB are grateful to Comité Ejecutivo de Desarrollo e Innovación Tecnológica (CEDIT) of Misiones Province. PICTs 1524/2011, 1895/2011, 2687/2012, 0404/2013, and PIP 1112008010 2422 Secter-UNJu D-084, and PICTO-UNJu (grant number 153) provided financial support. Specimen collection permits were issued by Administracion de Parques Nacionales (DISPOSICIÓN N°: 20/2013), Secretaría de Gestión Ambiental, Dirección Provincial de Biodiversidad, Gobierno de la Provincia de Jujuy (RESOLUCIÓN N° 111/2012 DPB), and by Ministerio de Ambiente y Desarrollo Sustentable, Gobierno de la Provincia de Salta (RESOLUCIÓN N° 000480).
Specimens of Oreobates examined
Data type: specimens data