ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah Acta Herpetologica 9(1): 109-113, 2014 DOI: 10.13128/Acta_Herpetol-12658 Meiotic behavior of two polyploid species of genus Pleurodema (Anura: Leiuperidae) from central Argentina Nancy E. Salas1, Julián A. Valetti2, Pablo R. Grenat1,3,*, Manuel A. Otero1, Adolfo L. Martino1 1Ecología-Educación Ambiental, Departamento de Ciencias Naturales, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Uni- versidad Nacional de Río Cuarto, Ruta Nacional N° 8 - km 601, (X5804BYA) Río Cuarto, Argentina. *Corresponding author. Email: pgrenat@exa.unrc.edu.ar 2Genética de Poblaciones, Departamento de Ciencias Naturales, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional N° 8 - km 601, (X5804BYA) Río Cuarto, Argentina 3CONICET Fellowships Submitted on: 2013, 22nd February; revised on: 2013, 19th December; accepted on: 2014, 11th January Abstract. Polyploidy is an important evolutionary force but rare in vertebrates. However, in anurans, the genus Pleu- rodema has polyploid species, two of them tetraploid and one octoploid. The manner in which the chromosomes join in diakinesis can vary among species and, crucially, if they differ in their ploidy levels. In this work, we describe the meiotic configurations in two cryptic species from central Argentina, with different ploidy levels, Pleurodema kriegi (tetraploid) and P. cordobae (octoploid). A total of 306 diakineses from 19 individuals were analyzed. In meiosis, P. kriegi form 22 bivalents, whereas P. cordobae exhibits variation in meiotic figures. We discuss the possible allo- and autopolyploid origin of these species, and we consider that the autopolyploid origin of P. cordobae from P. kriegi might be the most feasible. Keywords. Pleurodema cordobae, polyploidy, cytogenetics, diakinesis. Polyploidy is an important evolutionary force in some plant groups, (White, 1973; Lacadena, 1996; Otto and Whitton, 2000), but this mechanism is rarer in verte- brates (Orr, 1990; Holloway et al., 2006). However, poly- ploidy in anuran amphibians has been documented in numerous families (Barrio and Rinaldi de Chieri, 1970; Bogart, 1980; Kawamura, 1984; Kuramoto, 1990; Beçak and Beçak, 1998; Otto and Whitton, 2000; Martino and Sinsch, 2002; Stöck et al., 2002; Rosset et al., 2006; Mable et al., 2011; Bogart and Bi, 2013). The frog genus Pleurodema Tschudi, 1838 is distrib- uted from Panama throughout South America to south- ern Chile and Argentina and currently is represented by 15 species, of which 10 have been recorded in Argen- tina (Valetti et al., 2009; Kolenc et al., 2009; Maciel and Nunes, 2010; Faivovich et al., 2012). This genus is excep- tional in including species with three levels of ploidy. According to cytogenetic studies conducted so far, Pleu- rodema includes three polyploid species, P. kriegi (Mül- ler 1926) and P. bibroni Tschudi 1838 which are 2N = 4X = 44, and P. cordobae Valetti, Salas and Martino 2009 which is 2N = 8X = 88, while the remaining species are diploid (2N = 2X = 22) (Brum-Zorrilla and Sáez, 1968; Barrio and Rinaldi de Chieri, 1970; Veloso et al., 1972; Duellman and Veloso, 1977; Schmid et al., 1993; Louren- ço et al., 2006; Valetti et al., 2009). During meiosis, chromosome pairing and cross- ing over of homologous chromosomes occurs in pro- phase I, and is in diakinesis as can be seen by the more condensed chromosomes and the formation of chiasmata (Macgregor, 1993). The way in which chromosomes are joined forming chiasmata may vary between species, par- ticularly if they are of different ploidy levels, resulting in different meiotic configurations (Salas and Martino, 2009). 110 N.E. Salas et alii In this paper, we analyze the meiotic figures of Pleu- rodema kriegi (tetraploid) and Pleurodema cordobae (octoploid). Pleurodema kriegi and P. cordobae are two polyploid cryptic species endemic to the Sierra Grande of Cordo- ba, Argentina. Despite having different ploidy levels, they maintain a high degree of morphological and acoustical similarity (Valetti et al., 2009). The size of adult individu- als in both species varies between 28 and 41 mm, P. cor- dobae is slightly larger than P. kriegi and, in both cases, females are larger than males (Valetti, 2012). These spe- cies breed from November to March (austral spring-sum- mer) and are crepuscular and nocturnal (Valetti et al., 2009). Samples of P. kriegi from two localities of Sierra de Achala (31º36’46’’ S, 64º52’29’’ W and 31º36’23’’ S, 64º52’6’’ W) and P. cordobae from two sites of Sierra de Comechingones (32º23’58’’ S, 64º55’35’’ W and 32°22’13” S, 64°55’55” W) were collected during spring and sum- mer months between 2006 and 2010. The Sierra de Córdoba constitutes the eastern group of the Sierras Pampeanas system embracing the Sierra Grande, Sierra Norte and Sierra Chica. Sierra Grande includes the Sierra de Achala to the north and the Sier- ra de Comechingones to the south (Miró, 1999; Ramos, 1999). This region is characterized by a humid temperate climate with snow in winter. The annual rainfall is 800– 900 mm, distributed mainly in the spring-summer period (Capitanelli, 1979). The environment both in the Pampa de Achala and the Sierra de Comechingones is very simi- lar. The water bodies in which the species were sampled were semi-permanent ponds in highland pastures. After treatment in vivo with 0.3% colchicine, individ- uals were anesthetized and sacrificed by immersion in a 1% solution of MS-222 (Pukhta and Blazhek, 2004). Mei- otic chromosomes, prepared from testis, were studied in 64 spermatocytes in diakinesis of four P. kriegi individuals and 242 spermatocytes in diakinesis from 15 P. cordobae individuals. All techniques, hypotonic treatment, fixation of cells and staining were performed according to Schmid, (1978a, b); Schmid et al., (1979) and Schmid et al., (1990). Chromosomal preparations, when dried and stained, were examined using a Zeiss Axiophot D-7082 fluorescence microscope with a Zeiss Axiocam HRC digital camera. Images were captured using the software AxioVision ver- sion 4.8. The diakineses were examined using the image analysis program Adobe ® Photoshop ™ CS2. The analysis of spermatocytes in diakinesis revealed that all individuals of the tetraploid species P. kriegi formed 22 bivalents rings (Fig. 1A, 2A). In contrast, the analysis of diakinesis in individuals of P. cordobae revealed different meiotic configurations (Table 1). The most common configuration (68%) comprised an octava- lent (VIII), 15 tetravalents (IV) and 10 bivalents (II) (Fig. 1B, 2b). However, other figures were observed. In 23% of the cases two types of configurations were observed: 1) an octavalent, 14 tetravalent and 12 bivalents; and 2) an octavalent, 16 tetravalents and eight bivalents. A small number (9%) of cells analyzed correspond to other types of configurations (Table 1). Fig. 2. Karyotype of the diakinesis of (A) Pleurodema kriegi, La Posta, Pampa de Achala, ((2N = 4x = 44) and (B) Pleurodema cor- dobae, Los Tabaquillos, Sierra de Comechingones, (2N = 8x = 88). Fig. 1. Spermatocytes in diakinesis. (A) Pleurodema kriegi, La Posta, Pampa de Achala, (2N = 4x = 44). (B) Pleurodema cordobae, Los Tabaquillos, Sierra de Comechingones, (2N = 8x = 88). Table 1. Patterns of meiotic figures in Pleurodema cordobae. Meiotic configurations Total observed diakineses 1 VIII 15 IV 10 II 165 1 VIII 14 IV 12 II 30 1 VIII 16 IV 8 II 25 15 IV 14 II 6 13 IV 18 II 6 1 VIII 13 IV 14 II 5 1 VIII 14 IV 12 II 5 111Meiotic behavior of two polyploid Pleurodema Diakinetic analysis demonstrated that the chromo- somes of tetraploid P. kriegi join forming bivalent instead of tetravalent figures, leading to 22 rings in all cases ana- lyzed. Barrio and Rinaldi de Chieri (1970) interpreted these same results as reflecting an allopolyploid origin. Barrio (1977) suggested that the ancestral species of P. kriegi and P. bibroni (both tetraploid) would have origi- nated by allopolyploidy from P. thaul and related spe- cies (possibly extinct) in semiarid western Argentina, and subsequently spread eastward from the plains to the Atlantic coast. Finally, in the Pleistocene, the ancestral species would have undergone cladogenesis, giving rise to the extant species P. kriegi in the central area of Argen- tina and P. bibroni in Uruguay. In the analysis of P. cordobae diakinesis, chromosomes showed no consistent pattern. In contrast, we observed dif- ferent numbers of rings in their cells, including multivalent meiotic configurations. Multivalent configurations have been described in several polyploid anuran species (Beçak et al., 1966, 1967, 1970; Schmid et al., 1985; Salas and Mar- tino, 2009). Although multivalent formations can occur in an allopolyploid if the hybridizing species are closely related (Chenuil et al., 1999), the multivalent formations observed in P. cordobae could be evidence of autopoly- ploid origin. Moreover, recently Faivovich et al., (2012) performed a phylogenetic analysis of the genus Pleurodema based on sequences of nuclear and mitochondrial genes. These authors obtained a clade comprising the three poly- ploid species, where P. cordobae is the sister of P. kriegi and these two species together form the sister taxon of P. bibro- ni. As the authors demonstrate, these results also would be consistent with an autopolyploid origin of P. cordobae from P. kriegi, although they make clear that cytogenetic studies and a population-level approach will be required to confirm this. Therefore, under the hypothesis of a recent autopolyploid origin of P. cordobae from P. kriegi, both geographically close, it could be expected that the chro- mosomes are paired forming tetrads, in diakinesis. This is because in the potential ancestral species the chromosomes are paired forming dyads. However, as noted above, the behavior of the chromosomes was not as expected. Boga- rt and Bi (2013) conclude that polyploid species generally have higher levels of genetic diversity than related diploid species and that these polyploids accumulate genes from such diploid associations by having additional linkage groups. Therefore, meiotic aberrations are necessary for the initial evolution of polyploids and may persist to main- tain the genomic integrity (Bogart and Bi, 2013; Stenberg and Saura, 2013). Consequently, the origin of P cordobae by polyploidy could be relatively recent and the meiotic behavior to be responding to the same fact. Although in P. cordobae we did not observe a consistent pattern, the pres- ence of an octavalent ring in all diakinesis tested is nota- ble. However, in accordance with expected pattern and the studies realized to date, we cannot interpret such meiotic behavior. Cytogenetic studies by GISH technique in cells of P. cordobae hybridized with probe of total genomic DNA of P kriegi, would determine the number of chromosome complements shared by the two species. This method would enable evaluate the hypothesis of an autopolyploid origin of P. cordobae from P. kriegi. The polyploid individuals produce viable gametes if these are balanced in chromosome number; otherwise the gametes would be infertile. Macgregor (1993) stated that this problem could be solved by mechanisms to prevent unequal multivalent formation, or by strengthening the effective meiosis diploidization yielding only bivalents. The results obtained from our analysis of meiotic con- figurations of P. cordobae may indicate that the gametes formed in this species have low viability. However, popu- lations of P. cordobae are quite numerous and biological data on the species indicate that these populations are reproducing without major problems (Valetti et al., 2011). 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