Acta Herpetologica 18(1): 61-67, 2023 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.36253/a_h-12539 Revisiting the polyploidy in the genus Odontophrynus (Anura: Odontophrynidae) André Luis de Souza, Mayara Aparecida das Neves Micalichen, Roger Alves da Rocha, Rafael Bueno Noleto* Departamento de Ciências Biológicas, Universidade Estadual do Paraná, 84600-185, União da Vitória, Paraná, Brazil *Corresponding author. E-mail: rafael.noleto@unespar.edu.br Submitted on: 2022, 4th January; Revised on: 2022, 6th August; Accepted on: 2022, 23rd October Editor: Marcello Mezzasalma Abstract. The genus Odontophrynus, composed of ten species, is found in practically the entire south of South Amer- ica. Odontophrynus americanus was the first vertebrate registered to present natural polyploidy, considering that most individuals have 2N = 4x = 44 chromosomes, although having 2N = 2x = 22 chromosomes is considered the ancestral condition for all genera of the family Odontophrynidae. The present study aimed to analyze the karyotype of O. amer- icanus, providing a detailed and comparative description of conventional chromosomal markers, with focus on a pos- sible diploidization process operating in this polyploid genome. The individuals were collected in a fragment of Atlan- tic Forest in the south-central region of Paraná State, Brazil. The analyzed individuals presented the tetraploid pattern, with biarmed chromosomes. The C-banding showed heterochromatic regions restricted to centromeres and telom- eres. Among homologous chromosomes of the same quartet, small differences were observed in morphology, possibly the result of differentiation after the polyploidization event. Finally, the 45S rDNA (Nucleolar Organizer Regions) was mapped in the short arm of quartet 11, showing the nucleolus organizing regions active in the four homologous chro- mosomes. This genome, although structurally polyploid, may be undergoing a process of diploidization, by becoming functionally equivalent to a diploid genome, via chromosomal rearrangements, epigenetic mechanisms, and/or repeti- tive DNA dynamics. Keywords. Amphibian, diploidization, heterochromatin, rDNA. According to Frost (2023), the family Odontophry- nidae currently contains 55 species distributed in three genera Macrogenioglottus Carvalho, 1946, Odontophrynus Reinhardt and Lütken, 1862, and Proceratophrys Miran- da-Ribeiro, 1920. Earlier phylogenies validate the mono- phyly of the family, as well as that Macrogenioglottus and Odontophrynus are sister taxa (Pyron and Wiens, 2011; Feng et al., 2017). The genus Odontophrynus is composed of eleven species widely distributed in southern and east- ern South America. Odontophrynus americanus (Dumé- ril and Bibron, 1841), a small fossorial anuran with no apparent sexual dimorphism (Quiroga et al., 2015), has the greatest distribution, its range extends to central and southern Argentina, southern Paraguay, southern Brazil, and Uruguay (Frost, 2023). Odontophrynus americanus was the first case of natu- ral polyploidy found in vertebrates (Beçak et al., 1966). The Odontophrynus americanus species group is a com- plex of morphologically indistinguishable diploid and tetraploid species. It includes currently four diploid spe- cies: O. cordobae Martino and Sinsch, 2002, O. juquinha Rocha, Sena, Pezzuti, Leite, Svartman, Rosset, Baldo, and Garcia, 2017, O. lavillai Cei, 1985 and O. maisuma Rosset, 2008 with 2N = 2x = 22 chromosomes, and one widely distributed tetraploid species (O. americanus) with 2N = 4x = 44 chromosomes (Beçak et al., 1966; Ruiz et 62 André Luis de Souza et alii al., 1981; Martino and Sinsch, 2002; Rosset et al., 2006; Rosset, 2008). Martino et al. (2019) established the exist- ence of cryptic diversity and overestimation of species richness by combining molecular, morphological, and bioacoustic data. Populations known as O. americanus comprise at least three species. Polyploidy plays an important role in speciation and evolution in anurans, with about 50 polyploid species described in several families (Bogart, 1980; Mable et al., 2011; Evans et al., 2012; Schmid et al., 2015). Polyploids originate by autopolyploidization (intraspecies whole- genome duplication) or allopolyploidization (associ- ated with interspecific hybridization). Thus, individuals with an autotetraploid genome can originate by fusion of unreduced (i.e., diploids) gametes, or by suppression of the first mitotic division in fertilized eggs (Schmid et al., 2015). In recently evolved autopolyploids, the homolo- gous chromosomes of a quartet are expected to exhibit identical chromosome banding patterns in somatic meta- phases, leading to the multivalent formation during the first meiotic division. On the other hand, in an allopoly- ploid genome, if there are differences among the karyo- types of the parental species, the banding techniques or the genomic in situ hybridization (GISH) allow chromo- somes from parental species to be distinguished (Schwar- zacher et al., 1989), which will form bivalent configura- tions in meiosis (Schmid et al., 2015). In this study, the structure of polyploid karyotype O. americanus from a southern Brazilian population is described and subjected to comparative analysis in order to add new data regarding the speculated species complex. Additionally, the data are placed in an evolutionary context, thus contributing to a better understanding of the evolu- tionary scenario concerning ploidy levels in this group. Cytogenetic analyses were carried out on six juveniles of O. americanus collected in União da Vitória, Paraná State, Brazil (26º13’48”S and 51º05’09”W). Chromosome preparations were performed directly from bone marrow, according to Baldissera et al. (1993). Briefly, the animals received intraperitoneal injection of aqueous solution of colchicine (0.01 ml/g body weight) 1% per 6 h, and then subjected to deep sedation euthanasia by dermal absorp- tion of Lidocaine 5% pomade, following the recommen- dations of the Ethical Committee in Animal Use from Universidade Estadual do Paraná. Conventional staining was performed using 5% Giemsa in sodium-phosphate buffer (pH 7.0, for 10 min). Detection of the constitutive heterochromatin was accomplished according to Sumner (1972). Silver staining technique (Ag-NOR detection) was carried out accord- ing to Howell and Black (1980). The mitotic metaphases were analyzed under a Carl Zeiss Axiolab A1 microscope equipped with the software Zen Lite and a Zeiss Axi- oCam ICc1 camera with a resolution of 1.4 megapixels (Carl Zeiss, Oberkochen, Germany). Chromosomes were classified based on the centromeric index according to Green and Sessions (1991) and were arranged in decreas- ing size. The specimens of O. americanus showed a karyo- type of 2N = 4x = 44 chromosomes, distributed in eight metacentric quartets (1, 5–11) and three submetacentric quartets (2–4), thus presenting a fundamental number (FN) = 88 (Fig. 1). There was no variation among the specimens karyotyped. Exclusively between homologous chromosomes of quartets 2, 3, and 4, small differences were observed in terms of chromosomal morphology, which often made it difficult to organize these quartets. The centromeric indexes were established confirming the morphology discrepancies between homologs of the same quartet (Fig. 1). According to the relative size of the chromosomes, the species has a karyotype with four different sizes of chromosomes: one large quartet (1), Fig 1. Giemsa-stained karyotype of O. americanus. Highlighted the Ag-NORs site localized on the quartet 11. CI: centromeric index; CT: chromosome type; m: metacentric; sm: submetacentric; st: subtelocentric. Bar = 10 µm. 63Incipient diploidization process in Odontophrynus three medium quartets (2–4), four small (5–8), and three very small (9–11). Nucleolus Organizer Regions (NORs) were observed on the short arm of quartet 11. Such regions are coinci- dent with secondary constrictions (Fig. 1). A NOR size heteromorphism between homologous chromosomes of the quartet was frequently observed. The C-banding showed the presence of constitu- tive heterochromatin in the centromeric and telomeric regions of almost all quartets (absence of centromeric bands in quartets 8 and 9), and coincident with Ag-NOR staining (quartet 11) (Fig. 2). The family Odontophrynidae was first established as a tribe within the (then) huge family Leptodactylidae (Lynch, 1971). The karyotype with 2N = 2x = 22 chro- mosomes is considered the ancestral condition, given its high frequency in all three genera. This characteristic karyotype is believed to have arisen from the differentia- tion of the primitive chromosome number of 2N = 26 chromosomes present in the family Leptodactylidae, fol- lowed by centric fusions (Bogart, 1973). Karyotype descriptions of the genus Odontophry- nus reveal so far a very similar and conserved karyotype, which is composed exclusively of biarmed chromosomes, reflecting in fundamental numbers always twice the 2N, with some constant pairs in morphology between the species (Table 1). These small variations are a conse- quence of chromosomal rearrangements that only modify the chromosome morphology, such as pericentric inver- sions, although the centromere repositioning, which alters the chromosome morphology without any accom- panying chromosomal rearrangements (Rocchi et al., 2012), could be an alternative pathway leading to chro- mosomal remodeling. A special interest has been devoted to the study of the occurrence of diploid (2N = 2x = 22) and tetraploid (2N = 4x = 44) constitutions in the O. americanus spe- cies group (see Table 1). In this sense, several studies have indicated that it could consist of a complex of spe- cies (Rosset et al., 2006; Lanzone et al., 2008; Cianciarullo et al., 2019; Martino et al., 2019) and thus, the O. ameri- canus listed with 22 chromosomes are expected to prob- ably be other distinct species. The difficulty in organizing some quartets (i.e., 2–4) in conventional staining, due to small differences in the position of centromeres, may represent a prognosis for an incipient process of diploidization, as observed in other populations (Ruiz et al., 1981; Schmid et al., 1985). A structural heterogeneity must be created between homol- ogous of quartets in the polyploid karyotype, which can originate even from small rearrangements such as peri- centric inversions (Ohno, 1970; Ohno, 1974). Therefore, the differences within the quartets in question can be interpreted as post-polyploid events, indicating a dip- loidization process operating in this polyploid genome (Ohno, 1970; Schmid et al., 1985; Beçak, 2014). The variation of NORs location in species of Odon- tophrynus is the result of translocations (Beçak and Beçak, 1974) and/or transposable elements-mediated transposi- tions events (Gray, 2000; Mandrioli, 2000), which switched these ribosomal genes to other pairs promoting karyotype diversification. The karyotype with NORs on pair 11 is considered as the plesiomorphic condition, found in dip- loid species from three species groups of Odontophrynus, as well as in most individuals studied from tetraploid pop- ulations of O. americanus (see Table 1). A size heteromorphism between homologous was frequently observed. The presence of NOR-associated heterochromatin demonstrated that this heteromorphism between homologous of quartet 11 comprises both func- tional and structural aspects. This condition may have facilitated breaks and transpositions of rRNA genes to other sites in different species and populations of Odon- tophrynus (Wiley et al. 1989; Carvalho et al. 2014). Fig 2. C-banding karyotype of O. americanus. Bar = 10 µm. 64 André Luis de Souza et alii Heteromorphic NORs could also be related to differ- ences in genetic activity (Amaro-Ghilardi et al., 2008). In fact, in polyploids, while the number of 45S rDNA citrons is proportional to the degree of ploidy, gene expression may be equivalent to a diploid genome (Schmidtke and Engel, 1976). Epigenetic mechanisms are responsible for modulat- ing gene expression through chemical modifications of his- tones, via methylation, acetylation, and/or phosphorylation (Furey and Sethupathy, 2013). Equalization of gene activity between 2x and 4x species could be at the transcriptional level, probably by rDNA methylation (Hashimshony et al., 2003). Indeed, Ruiz and Brison (1989) found high levels of methylation of ribosomal genes in tetraploid genomes of O. americanus. It has been validated by Cianciarullo et al. (2000), who found only 25–30% more ribosomes in O. americanus tetraploid than do 2N cells. Therefore, polyploid genomes may become functionally diploid throughout evo- lution (Schmid et al., 2015). The presence of constitutive heterochromatin on cen- tromeric and telomeric regions is an expected pattern in Odontophrynus. The eventual variation involves the additional presence of interstitial bands that characterize some species/populations (see Table 1). The variation in the distribution pattern of constitutive heterochromatin is generally associated with the dynamics of different classes of repetitive DNA. Heterochromatin is normally rich in repetitive sequences, which can have important functions in speciation and/or adaptation, as they are less subject to selective pressures, favoring the accumulation of dif- ferences throughout the evolutionary process (Martins, 2007; Böhne et al., 2008). In conclusion, the intra- and interpopulation chro- mosomal variability in Odontophrynus is a consequence of its wide geographic distribution throughout South America. Regarding polyploidy within the group, its origin via autopolyploidization seems to be the most accepted, mainly due to the presence of multivalents at meiosis (Beçak et al., 1966; Schmid et al., 1985; Lanzone et al., 2008). However, multivalent formation can also be observed in some allopolyploids, because the structure of chromosomes from different species (i.e., homeolo- gous) can be sufficiently conserved to permit multivalent associations. Autopolyploids, on the other hand, might also have mechanisms that prevent multivalent con- Table 1. Summary of the chromosome findings of the species of Odontophrynus: diploid number (2N), centromeric heterochromatin (Ⓒ), telomeric heterochromatin (Ⓣ), interstitial heterochromatin ⓘ, Nucleolus Organizer Region (NOR), short arm (p), long arm (q), funda- mental number (FN), *Artificial hybrid. Species group Species Locality 2N Ploidy level C-banding NOR FN Reference O. americanus O. americanus Brazil 22 2x - 4p 44 Ruiz et al. (1981) Argentina 22 2x ⒸⓉⓘ 4p 44 Ruiz et al. (1981) Brazil 22 2x ⒸⓉⓘ 4p, 11p 44 Ruiz et al. (1981) Argentina 33 3x - - 66 Grenat et al. (2018) Brazil 44 4x - 11p 88 Beçak et al. (1966) Argentina 44 4x - 11q 88 Bogart (1967) Uruguay 44 4x - 4p 88 Ruiz et al. (1981) Argentina 44 4x - - 88 Grenat et al. (2018) Brazil 44 4x ⒸⓉⓘ 11p 88 Ruiz et al. (1981) Uruguay 44 4x ⒸⓉⓘ 4p, 11p 88 Ruiz et al. (1981) Argentina 44 4x ⒸⓉⓘ 11p 88 Schmid et al. (1985) Brazil 44 4x ⒸⓉ 11p 88 Present study Uruguay 66* 6x - 11p 132 Ruiz et al. (1981) O. cordobae Argentina 22 2x - 11- 44 Martino and Sinsch (2002) Argentina 22 2x - 4p 44 Salas and Martino (2007) O. juquinha Brazil 22 2x - 4p 44 Rocha et al. (2017) O. lavillai Argentina 22 2x - 4p 44 Rosset et al. (2006) O. maisuma Uruguay 22 2x - 4p 44 Rosset (2008) Uruguay 22 2x Ⓒⓘ 4p 44 Borteiro et al. (2010) O. reigi Argentina, Brazil, Paraguay 22 2x - 4p 44 Rosset et al. (2021) O. cultripes O. cultripes Brazil 22 2x ⒸⓉⓘ 11p 44 Ruiz and Beçak (1976) O. carvalhoi Brazil 22 2x ⒸⓉⓘ 8p 44 Ruiz et al. (1981) O. occidentalis O. occidentalis Argentina 22 2x ⒸⓉⓘ 9q, 11p 44 Ruiz et al. (1981) 65Incipient diploidization process in Odontophrynus figuration and thus form bivalents (Gregory and Mable, 2005). Therefore, distinguishing between auto- and allopolyploidization is difficult, since the scenario pos- sibly involves a combination of both mechanisms. The disjunct tetraploid populations are closely associated with several diploid species, which suggests that polyploidy has multiple origins, with putative older lineages accu- mulating more chromosomal changes within the homolo- gous quartets. The evidence suggests that the benefits of polyploidization are stabilized by epigenetic mechanisms, small structural rearrangements, and repetitive DNA dynamics, which lead the tetraploid genomes to become functionally diploid (diploidization). 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