Revisiting the polyploidy in the genus Odontophrynus (Anura: Odontophrynidae)

. The genus Odontophrynus , composed of ten species, is found in practically the entire 15 south of South America. Odontophrynus americanus was the first vertebrate registered to 16 present natural polyploidy, considering that most individuals have 2N = 4x = 44 chromosomes, 17 although having 2N = 2x = 22 chromosomes is considered the ancestral condition for all genera 18 of the family Odontophrynidae. The present study aimed to analyze the karyotype of O. 19 americanus , providing a detailed and comparative description of conventional chromosomal 20 markers, with focus on a possible diploidization process operating in this polyploid genome. 21 The individuals were collected in a fragment of Atlantic Forest in the south-central region of 22 Paraná State, Brazil. The analyzed individuals presented the tetraploid pattern, with biarmed 23 chromosomes. The C-band showed heterochromatic regions restricted to centromeres and 24 telomeres. Among homologous chromosomes of the same quartet, small differences were

preparations were performed directly from bone marrow, according to Baldissera et al. (1993). ml/g body weight) 1% per 6 h, and then subjected to deep sedation euthanasia by dermal 78 absorption of Lidocaine 5% pomade, following the recommendations of the Ethical Committee 79 in Animal Use from Universidade Estadual do Paraná. 80 Conventional staining was performed using 5% Giemsa in sodium-phosphate buffer (pH 81 7.0, for 10 min). Detection of the constitutive heterochromatin was accomplished according to 82 Sumner (1972). Silver staining technique (Ag-NOR detection) was carried out according to 83 Howell and Black (1980). The mitotic metaphases were analyzed under a Carl Zeiss Axiolab 84 A1 microscope equipped with the software Zen Lite and a Zeiss AxioCam ICc1 camera with a 85 resolution of 1.4 megapixels (Carl Zeiss, Oberkochen, Germany). Chromosomes were 86 classified based on the centromeric index according to Green and Sessions (1991) and were 87 arranged in decreasing size of karyotypes.

88
The specimens of O. americanus showed a karyotype of 2N = 4x = 44 chromosomes, 89 distributed in eight metacentric quartets (1, 5-11) and three submetacentric quartets (2-4), thus 90 presenting a fundamental number (FN) = 88 (Fig. 1). There was no variation among the 91 specimens karyotyped. Exclusively between homologous chromosomes of quartets 2, 3, and 4, 92 small differences were observed in terms of chromosomal morphology, which often made it 93 difficult to organize these quartets. The centromeric indexes were established confirming the 94 morphology discrepancies between homologs of the same quartet ( Fig. 1). According to the 95 relative size of the chromosomes, the species has a karyotype with four different sizes of 96 chromosomes: one large quartet (1), three medium quartets (2-4), four small (5-8), and three 97 very small (9-11).
Such regions are coincident with secondary constrictions (Fig. 1). A NOR size heteromorphism 100 between homologous chromosomes of the quartet was frequently observed.

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The C-banding showed the presence of constitutive heterochromatin in the centromeric 102 and telomeric regions of almost all quartets (absence of centromeric bands in quartets 8 and 9), 103 and coincident with Ag-NOR staining (quartet 11) (Fig. 2).

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The family Odontophrynidae was first established as a tribe within the (then) huge 105 family Leptodactylidae (Lynch, 1971). The karyotype with 2N = 2x = 22 chromosomes is 106 considered the ancestral condition, given its high frequency in all three genera. This  Table   120 1). In this sense, several studies have indicated that it could consist of a complex of species

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The presence of constitutive heterochromatin on centromeric and telomeric regions is 157 an expected pattern in Odontophrynus. The eventual variation involves the additional presence 158 of interstitial bands that characterize some species/populations (see Table 1). The variation in

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In conclusion, the intra-and interpopulation chromosomal variability in Odontophrynus 165 is a consequence of its wide geographic distribution throughout South America. Regarding 166 polyploidy within the group, its origin via autopolyploidization seems to be the most accepted, 167 mainly due to the presence of multivalents at meiosis (Beçak et al., 1966;Schmid et al., 1985;168 Lanzone et al., 2008). However, multivalent formation can also be observed in some We would like to express our thanks to two anonymous reviewers for helpful comments that