ARTÍCULOS ORIGINALES

C-Banding patterns and meiotic behavior in Hypsiboas pulchellus and H. Cordobae (anura, hylidae)

 

Baraquet M.^1,2, Salas N.E.¹, Martino A.L.¹

¹ Ecología, Departamento de Ciencias Naturales, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto. Ruta Nacional N° 36 - km 601, (X5804BYA) Río Cuarto, Argentina.
² CONICET
mbaraquet@exa.unrc.edu.ar

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ABSTRACT

In this work, we observed and evaluated C-banding patterns and meiotic behaviour of Hypsiboas pulchellus and H. cordobae. Twenty metaphasic cells per individual were analyzed, which were obtained from intestinal and testis cells using conventional and C-banding techniques. The two species presented 2n= 24 chromosomes. Pairs 1 and 8 to 12 were metacentric whereas pairs 2 to 7 were submetacentric. The centromeric region of all chromosomes was positively C-banded but the C-banding pattern varied between species. At meiosis, cells with 12 bivalents were observed. Bivalents usually presented two terminal chiasmata, although the largest pair presented only one terminal chiasma. The cytogenetic data reconfirms the diploid somatic chromosome number of H. pulchellus and H. cordobae, species that exhibited identical chromosome morphology. The remarkable similarities between the two species are an indication of the close relationship among members of the H. pulchellus group; however C-banding patterns of Hypsiboas species are distinct and may be also referred to as species-specific. Meiotic behavior is quite similar to the behaviour of most frogs, which possess only one or two -usually terminal- chiasmata, resulting in the typical linear or ring appearance of bivalents.

Key words: Karyotypes; C-banding; Meiosis; Hypsiboas pulchellus; Hypsiboas cordobae.

RESUMEN

Este trabajo describe el patrón de Bandas-C y comportamiento meiótico de Hypsiboas pulchellus e H. cordobae. Se analizaron 20 figuras metafásicas por individuo, obtenidos de intestinos y testículos utilizando técnicas convencionales y de bandeo-C. Para ambas especies se determinó un número cromosómico de 2n= 24. Los pares 1 y 8 a 12 mostraron morfología metacéntrica, y los pares 2 a 7 mostraron morfología submetacéntrica. Para las dos especies se encontró el mismo patrón de heterocromatina constitutiva en la región centromérica de todos los pares, pero el patrón de bandeo varió entre ellas. En meiosis se observaron células con 12 bivalentes. Los bivalentes mostraron normalmente dos quiasmas, pero en el bivalente mayor también se observó un único quiasma terminal. Los datos citogenéticos reconfirman el número diploide de H. pulchellus e H. cordobae, y muestran una morfología de los cromosomas idéntica para ambas especies, siendo esta similitud un indicio de la estrecha relación entre las especies del grupo H. pulchellus; sin embargo los patrones de Bandas-C de las dos especies de Hypsiboas son distintos, pudiendo ser esto reflejo de un carácter especie-específico. El comportamiento meiótico es similar al encontrado en otras especies de anuros por diversos autores.

Palabras clave: Cariotipos; Bandas-C; Meiosis; Hypsiboas pulchellus; Hypsiboas cordobae.

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INTRODUCTION

The genus Hypsiboas Wagler (1830), which belongs to the Hylidae family, Hylinae subfamily and Cophomantini tribe, contains 84 species, most of them included in seven species groups. One of these is the Hypsiboas pulchellus group, which currently contains 36 species (Faivovich et al., 2004; Faivovich et al., 2005; Frost, 2012; Köhler et al., 2010; Lehr et al., 2010; Lehr et al., 2011). This group includes Hypsiboas pulchellus (Duméril and Bibron, 1841) and Hypsiboas cordobae (Barrio, 1965), which are the object of the present study.
H. pulchellus is a widely distributed amphibian, occurring from Santa Catalina to Rio Grande do Sul in Brazil and in Uruguay, and also in the Argentinian provinces of Misiones, Corrientes, Entre Ríos, Santa Fe, La Pampa, Chaco, Córdoba, Buenos Aires and northern Río Negro. Its sister species, H. cordobae, is also found in Argentina, in the hills of Córdoba and San Luis provinces (Barrio, 1962; 1965; Cei, 1980; Gallardo, 1974; 1987; Basso and Basso, 1987; Basso, 1990; Martori and Ávila, 1992; Bridarolli and di Tada, 1994; di Tada et al., 1996; Ávila et al., 1999; di Tada, 1999; Langone and Lavilla, 2002; Faivovich et al., 2004; Kwet et al., 2004), but the exact limits of its range remain unknown, especially in the contact area with H. pulchellus (Barrio, 1965; Cei, 1980; Gallardo, 1987; Bridarolli and di Tada, 1994; Stuart, 2006).
These two species are similar in morphology and ecological features, and have been cytogenetically analyzed by various authors (Morescalchi, 1973; King, 1990; Barale et al., 1991). Although the number of cytogenetic studies seems reasonable, the majority of them are reports of chromosome numbers and morphological phenotypes. Because chromosome studies may be of help to clarify phylogenetic and taxonomic relationships (Reyes Valdéz et al., 2000), in the present study we redescribe the karyotypes H. pulchellus and H. cordobae with the aim of providing new data for the analysis of meiotic behavior and C-banding patterns.

MATERIALS AND METHODS

Our sample of H. pulchellus comprises 18 males specimens collected from different populations in Córdoba province: Río Cuarto (33º 06' 40.78" S, 64º 18' 16.88" W), Las Acequias (33º 15' 26.16" S, 63º 55' 15.10" W), Alejandro Roca (33º 21' 06" S, 63º 42 ‘10" W). Thirty-two males specimens of H. cordobae were collected from different populations in Córdoba and San Luis provinces: Achiras (33º 09' 28.64" S, 64º 58' 55.13" W), Las Guindas (32º 35' 35.22" S, 64º 42' 38.92" W), Pampa de Achala (31º 49' 41.8" S, 64º 51' 44.9" W), Los Linderos (32º 00' 54.05" S, 64º 56' 42.97" W), Los Tabaquillos (32º 23' 59.75" S, 64º 55' 33.69" W), La Carolina (32º 48' 43.94" S, 66º 05' 48.15" W).
Chromosome preparations were obtained as described by Baraquet et al. (2011), and by Schmid (1978), Schmid et al. (1979), Salas (2006) and Salas and Martino (2007). C-banding was done according to Sumner (1972).
Chromosomes were visualized using a Zeiss Axiophot-Axiolab and photographed using Axiocam HRc Zeiss. We analyzed 20 metaphasic cells per individual. On the metaphases the total length of each chromosome and the length of the four arms were measured using image analysis by Adobe® Photoshop® 9.0.
Data were processed using Microsoft Excel® 2000. Length of p and q arms, centromeric index, arm ratio, relative chromosome length and relative arm length were calculated using the following formulas:
Average length of arms p y q: q = (q¹ + q²)/2
p = (p¹ + p²)/2
Centromeric index (i): i = (length of the short arm of a chromosome (p) / total length of the chromosome (p + q) ) × 100
Arm ratio (r): r = q/p
Relative chromosome length (rl): rl = (total length of chromosome / Σ of length of chromosomes of the haploid set) × 100
Relative arm length (rlq o rlp): rlq or rlp = (length of the arms (q o p) / Σ of length of chromosome of the haploid set) × 100
The data were processed to get the average for each species. The ideograms were carried out with these data, which was necessary because in the karyotypes the differences between chromosomes are not observable to the naked eye.
The chromosomes were classified according to Aiassa et al. (2001).

RESULTS

All populations of H. cordobae and H. pulchellus had 2n= 24 chromosomes with a fundamental number NF= 48 (Figure 1). The twelve chromosome pairs can be classified into three groups. The chromosomes of pair 1 are large with a relative chromosome length of 16.20 % for H. cordobae and 12.26 % for H. pulchellus. Pairs 2 to 6 comprise a group of medium chromosomes with a relative chromosome length between 12.45 % and 7.58 % for H. cordobae and 10.63 % and 8.79 % for H. pulchellus. The remaining six chromosome pairs comprise a group of small chromosomes with a relative chromosome length between 6.57 % and 4.04 % for H. cordobae and 8.03 % and 3.90 % for H. pulchellus (karyotype formula 1: 5: 6) (Table 1, Figures 1, 2).

Figure 1. Karyotypes of H. pulchellus (A) and H. cordobae (B). (Bar=10 μm).

Figure 2. Karyotypes of H. pulchellus (A) and H. cordobae (B). (Bar=10 μm).
Ideograms based on the parameters presented in Table 1: H. pulchellus (A) and H. cordobae (B). Black areas indicate C-bands.

Table 1. Karyotype parameters for constructing H. pulchellus and H. Cordobae ideograms

P: number of chromosome pairs; rl: chromosome relative length; r: arm ratio; i: centromeric index; T: Type, M: metacentric, SM: submetacentric.

The chromosome morphology was always metacentric or submetacentric in the two species studied. Pairs 1 and 8-12 were metacentrics whereas the pairs 2-7 were submetacentrics.
C-banding showed the same pattern of constitutive heterochromatin at the centromeric regions of all pairs in the H. pulchellus and H. cordobae.
In H. pulchellus pericentromeric heterochromatin in all pairs, except pair 8, was observed. C-banding in the interstitial regions of the long arms of the pair 2 was observed. C-banded regions also appeared on the telomeric regions of the short arms of the pairs 1, 3, 5, 7 and 8, and in the long arms of the pairs 1-10 (Figure 3A y Table 2).

Figure 3. C-banded karyotypes of H. pulchellus (A) and H. cordobae (B). (Bar=10 μm)

Table 2. Distribution of heterochromatin in H. pulchellus and H. cordobae.

P: number of chromosome pairs; C: bands in centromeric regions; PC: bands in pericentromeric regions; BIp: bands in interstitial regions of short arms; BIq: bands in interstitial regions of long arms, Tp: bands in telomeric regions of short arms; Tq: bands in telomeric regions of long arms.

In H. cordobae revealed positively stained pericentromeric heterochromatin in the chromosomes of pairs 4, 5 and 8-12. C-banding in the interstitial regions of the short arms of the pair 1 was observed (Figure 3B y Table 2).
The meiotic analysis of males showed that the two species studied had 12 bivalents at diakinesis and 12 chromosomes at metaphase II cells (Figure 4A-D). The bivalents generally had two terminal chiasmata (ring-shaped bivalents).

Figure 4. Meiotic cells of Hypsiboas pulchellus (A, B) and H. cordobae (B, C). (A) 12 bivalents with two terminal chiasmata. (B) Metaphase II with n= 12. (C) 11 ring-shaped bivalents and one bivalent with a terminal chiasma (arrow). (D) Metaphase II with n= 12 (D). (Bar=10 μm).

In H. cordobae in most of the cells analyzed (78.50 %) the larger bivalent had one terminal chiasma (Figure 4C). This meiotic configuration was observed in the six populations studied; however, in the population of Pampa de Achala many of the cells exhibit the twelve bivalents ring-shaped (two terminal chiasmata). However, in H. pulchellus, most of the cells analyzed (69.23 %) exhibit all ring-shaped bivalents (Figure 4A) and only in Río Cuarto population the larger bivalent had one terminal chiasma in half of the cells (Table 3).

DISCUSSION

It is well known that most of Hylinae subfamily species have diploid chromosome numbers between 22 and 34 but most of the species within the genus Hypsiboas have a diploid complement 2n= 24 (Morescalchi, 1973; King, 1990).
Despite the differences in morphology and advertisement call between H. pulchellus and H. cordobae (Barrio, 1962; 1965; Cei, 1980; Basso and Basso, 1987; Gallardo, 1987; Faivovich et al., 2004; Baraquet et al., 2007), these species show the same diploid number (2n= 24) and a conserved chromosomal morphology.
Moreover, a comparison of known karyotypes of species of three of the five genus included in Cophomantini, Aplastodiscus, Bokermannohyla, and Hypsiboas corroborates the remarkable similarity among them (Catroli et al., 2011).
The diploid number of H. pulchellus and H. cordobae is largely in accordance with those previously described in the literature for these species (Bogart, 1973; Morescalchi, 1973; Barale et al., 1991; Ananias, 1996) and was identical to other species of Hypsiboas pulchellus group (H. caingua, H. prasinus, H. joaquini, H. semiguttatus, H. marginatus, H. guentheri, H. bischoffi ) (Baldissera et al., 1993; Ananias et al., 2004; Raber et al., 2004), and Hypsiboas faber group (H. albomarginatus and H. faber) (Carvalho et al., 2009). In Hypsiboas the chromosome number and the morphology of the pairs is similar among species, with few or almost no differences among most of them. The only known species with a different chromosome number is H. albopunctatus with 2n= 22 (Gruber et al., 2007; Catroli et al., 2011).
About the chromosome morphology, metacentric or submetacentric for both species studied, our results are in agreement with King (1990). This author reported that Hypsiboas species with diploid complement 2n= 24 have metacentric or submetacentric karyotypes.
These results obtained here show that the two species have common features, however small differences in the chromosome size were observed.
The chromosome morphology of both species was similar to those reported by Barale et al. (1991) for H. cordobae. However, the metacentric morphology of pair 2 and subterminal morphology of pairs 4 and 6 reported by those authors is not observed for us in H. cordobae. For H. pulchellus, Ananias (1996) indicated submetacentric morphology in the pairs 9 and 10, and acrocentric morphology of the pair 6. However, our results did not show this structure.
According to Schmid et al. (1990), in the chromosomes of the Anura the centromeric/pericentromeric regions and telomeres are the preferential locations of heterochromatin. H. cordobae and H. pulchellus show the anuran pattern of constitutive heterochromatin: at centromeric regions in all chromosome pairs and at pericentromeric regions in some of them. We observed that the most important distinction between H. pulchellus and H. cordobae karyotypes was in the C-banding pattern.
Also, the comparison of the C-banding patterns of the two species studied with those previously described for the Hypsiboas pulchellus group and related species reveal that there are some common C-bands in most of them. H. prasinus (Baldissera, et al. 1993; Ananias, 1996), H. joaquini (Ananias, 1996), H. guentheri and H. bischoffi (Raber et al., 2004), H. marginatus and H. semiguttatus (Ananias et al., 2004) have a telomeric band in the long arm of chromosome 1 as in H. pulchellus. The C-banded chromosomes of H. pulchellus revealed heterochromatin at the pericentromeric regions of all pairs (except the pair 8) as in H. guentheri and H. bischoffi (Raber et al., 2004). However, the telomeric heterochromatic band on the long arm of pair 10 observed in H. pulchellus, H. guentheri and H. bischoffi (Raber et al., 2004), H. marginata and H. semiguttata (Ananias et al., 2004), is not observed in H. cordobae.
Chromosome banding is a very important tool in comparative cytogenetics (Baldissera et al., 1993). Interspecific comparisons of C-band patterns are a basic importance for cytotaxonomic studies (Schmid, 1978; Schmid et al., 1990; Baldissera et al., 1993). It has been shown that although many species of several amphibian genuses have a shared similarity in chromosome number and morphology, as we observed among the studied species, could be extensive differences in the position and amount of heterocromatin (Schmid, 1978; Schmid, et al., 1990).
Regarding to meiosis, at diakinesis in both species 12 ring-shaped bivalents are observed, but frequently the larger bivalent present only one terminal chiasmata. These meiotic configurations have also been described by Baldissera et al. (1993) for four Brazilian Hyla species; by Martirosyan and Stepanyan (2007) for Hyla savignyi; and in diplotene cells of Hypsiboas albopunctatus (Gruber et al., 2007). Also, Lourenço et al. (2003) observed in Paratelmatobius cardosoi (Leptodactylidae) that a high percentage of diakinesis showed the larger bivalent with only one terminal chiasmata and open configuration, as observed in this study. These authors explain that this open configuration probably is the result of a terminal association of the long arms of pair 1. The two species studied are quite similar from most frogs, which possess only one or two, usually terminal, chiasmata, resulting in the typical linear or ring appearance of bivalents (Morescalchi, 1973).
The cytogenetic data obtained in this paper reconfirms the diploid number of H. pulchellus and H. cordobae and show identical chromosome morphology; however we find that these species differ slightly in the heterochromatin pattern.
It has been noticed that most members of Cophomantini, and specifically members of the H. pulchellus group (Ananias et al., 2004; Raber et al., 2004), share a similar chromosome morphology (Gruber et al., 2007; Catroli et al., 2011), and therefore, the remarkable similarities are no more than an indication of near relationship between H. cordobae and H. pulchellus. Even these similarities could be indication of a relationship between any member of the H. pulchellus group or with almost any other species of Hypsiboas, with the exception of some members of the H. albopunctatus group (Bogart, 1973; Gruber et al., 2007).
This work represents a significant contribution to redescription of the karyotype of H. pulchellus and H. cordobae, and a contribution with new data of meiotic behavior and C-banding patterns, not made so far.

ACKNOWLEDGEMENTS

The first two authors thank the National Scientific and Technical Research Council (CONICET) for support. The Secretary of Science and Technology of National University of Río Cuarto (SECyT-UNRC) provided funds by Grant PPI 18C/225. We thank P. Grenat and J. Valetti for their help in the field and sample. Our study was authorized by Córdoba Environmental Agency (A.C.A.S.E.).

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