Cytogenetics and sperm ultrastructure of Atelopus spumarius (Anura, Bufonidae) from the Brazilian Amazon

The current taxonomy of most Atelopus species is based on morphological and color data only. Recent studies suggest that A. spumarius may represent a species complex assigned under the same name. Karyotypic data and description of sperm ultrastructure for 13 specimens of A. spumarius are presented here for the first time. A chromosomal analysis revealed 2n = 22 chromosomes, with centromeric heterochromatin in all pairs and a nucleolar organizer region (NOR) on the telomere of pair 7. The sperm was of the bufonoid type, presenting a filiform nucleus covered by an acrosomal complex and a mitochondrial collar in the neck region. The tail was composed of an axoneme, an undulating membrane and an axial rod. A karyotype analysis of A. spumarius showed the same chromosome number and similar chromosomal morphology as described for congeneric species, with slight differences probably resulting from pericentric inversions. The NOR location (on pair 7) was the same as that observed for species belonging to the genus Rhinella. The spermatological findings indicate a close relationship between Atelopus and the bufonoid lineage. The present data are useful for reference in future studies to determine whether more than one species are assigned to A. spumarius.

The harlequin frog, Atelopus spumarius Cope, 1871, is found in the Amazon region throughout Ecuador, Peru, the Guianas and Brazil (Amazonas, Para and Amapá states) (Frost, 2013). Cocroft et al. (1990) and Lötters et al. (2002) suggested that this name may refer to a species complex, with the true A. spumarius being found only in the upper Amazon Basin (Peru, Colombia and Brazil). This suggestion implies that the populations in southern Peru, Ecuador and central Brazil may represent undescribed taxa. Furthermore, Lötters et al. (2002) suggested that the populations from Guiana and the eastern Amazon may belong to yet another species complex: Atelopus hoogmoedi.
The current taxonomy of this genus is solely based on morphological and color data (Ramos et al., 2002), with the presence of toxins also being considered a taxonomic tool (Kim et al., 1975). Other potentially useful criteria, such as osteology, larval morphology, molecular data, cytogenetics and sperm ultrastructure, have not been applied to this genus. In anurans in general, cytogenetics has been used increasingly as a tool to investigate chromosomal evolution and to make taxonomic inferences, indicating the existence of new species and differentiating between cryptic ones Aguiar-Jr 1998, Medeiros et al. 2003;Lourenço et al., 2006;Siqueira et al., 2008), and data from sperm ultrastructure have been used as additional characters in systematic studies (see Jamieson and Leung, 1991;Garda et al., 2002, Aguiar-Jr et al., 2006Veiga-Menoncello et al. 2006. The only cytogenetic studies in Atelopus done so far are those on A. varius (Duellman, 1967;Schmid, 1980), A. ignescens and A. guanujo (Barrera et al. 1984), and A. zeteki (Ramos et al., 2002), which described 2n = 22 chromosomes with highly similar morphology. No differential staining (C-banding or Ag-NOR) studies have been done in this genus. Sperm ultrastructure data are available for other genera in the family Bufonidae, but not for Atelopus. Species within the genera Ansonia, Bufo, Nimbaphrynoides and Melanophriniscus have been studied using sperm ultra-structure data (Jamieson, 2003) and show a morphological pattern that characterizes the superfamily Bufonoidea.
We present unique chromosomal data, including banding patterns, for A. spumarius and the first ultrastructural sperm characteristics for the genus Atelopus. Our aim was to expand the number of characteristics available for making taxonomic inferences in future studies.
Nine males and four females of A. spumarius from Reserva Florestal Adolpho Ducke, Manaus, Amazonas, were analyzed. The frogs were collected with the authorization of the Instituto Brasileiro do Meio Ambiente e Recursos Naturais Renováveis (IBAMA -Proc. 02010.000025/2005-51). Voucher specimens were deposited in the Museu de Zoologia "Prof. Adão José Cardoso" of Instituto de Biologia at Universidade Estadual de Campinas, Brazil, under the following accession numbers: 13274-13298.
Mitotic metaphases were obtained from testicular and intestinal epithelial cell suspensions, as described by King and Rofe (1976) and Schmid (1978). The chromosomes were stained with 10% Giemsa solution and subjected to C-banding (Sumner, 1972), with slight modifications as suggested by Siqueira et al. (2008), and to Ag-NOR (Howell and Black, 1980) techniques. All slides were analyzed with an Olympus BX60 microscope, and images were processed using Image Pro-Plus 5.1 software. The chromosomes of at least five metaphases were measured and classified as described by Green and Sessions (1991).
Testes were removed, cut into small pieces and fixed overnight at 4°C in 0.1 M sodium cacodylate buffer, pH 7.2, containing 2% paraformaldehyde, 2% glutaraldehyde, 3% sucrose and 5 mM CaCl 2 . Postfixation was performed for 1 h in the same buffer containing 1% osmium tetroxide, 0.8% potassium ferricyanide and 5 mM CaCl 2 . The tissues were subsequently rinsed in sodium cacodylate buffer and incubated en bloc with 0.5% uranyl acetate. After rinsing in buffer, the tissue fragments were dehydrated in an increasing ethanol series and embedded in Epon 812 resin. Ultrathin sections were stained with uranyl acetate and lead citrate (Watson, 1958;Venable and Coggeshall, 1965) and examined with a LEO 906 transmission electron microscope. Scanning electron microscopy was performed following the protocol of Veiga-Menoncello et al. (2007).
Atelopus spumarius (Figure 1) presented a diploid number of 22 chromosomes; pairs 1, 2, 5-7 and 9-11 were metacentrics, and pairs 3, 4 and 8 were submetacentrics (Figure 2A-C). This species has the same chromosome number and chromosomal morphology similar to A. varius, A. zeteki, A. ignescens and A. guanujo (Duellman, 1967;Schmid, 1980;Barrera et al. 1984;Ramos et al., 2002). Pairs 8 and 9 have a very similar size, accounting for their inverted position when different karyotypes are compared. Such inverted position and some variation in chromosomal morphology between the karyotyped species may be due to pericentric inversions, a mechanism suggested for chromosomal evolution in Atelopus (Ramos et al., 2002).
According to Kuramoto (1990), the majority of Bufonidae species have 2n = 22 chromosomes, suggesting that chromosome number is highly conserved in this family. This assumption was corroborated by Bush et al. (1977), who postulate that the bufonids have a slow rate of Siqueira et al. 529  chromosomal evolution in comparison with other vertebrate groups. This hypothesis is supported by karyotype analyses of other bufonid taxa, including Rhinella crucifer, R. icterica and R. schneideri (Kasahara et al., 1996), Melanophryniscus species (King, 1990;Kuramoto, 1990), and the species of the genus Bufo studied by Baldissera et al. (1999), whose karyotypes are very similar to each other and to those of Atelopus.
All the karyotypes described so far in this genus had only been analyzed by conventional Giemsa staining. The C-banding technique here employed detected heterochromatin only in the centromeres of all pairs ( Figure 2B). The lack of C-banding patterns for other Atelopus species precludes intrageneric comparisons. Nonetheless, the patterns described here are very similar to those found for Chaunus species by Amaro-Ghilardi et al. (2008). The shared centromeric-pericentromeric banding pattern in these species reinforces the idea of a conserved karyotypic nature of this pattern, but the significance of such similarity cannot be assessed until the evolutionary state of such character (heterochromatin distribution) is defined.
NOR regions were detected on the telomeres of the long arm of pair 7 ( Figure 2C). In A. zeteki (Ramos et al., 2002), the pericentromeric constriction observed in pair 7 is probably the NOR site, which coincides with the location found in A. spumarius.
On the basis of studies of NOR location, Baldissera et al. (1999) found three groups within six Bufo species analyzed. The first two groups were composed of species with Ag-NOR labeling on pair 5 and/or 10 (B. arenarium, B. rufus, B. ictericus and B. paracnemis). The third group included B. marinus and B. crucifer, where NORs were located on pair 7, which is characteristic of the crucifer and marinus groups. The last two species (B. ictericus and B. paracnemis) were later separated from the other Bufo species and included in the new genus Rhinella (Chaparro et al., 2007;Frost, 2013) The shared presence of a NOR in pair 7 could indicate a degree of proximity between these taxa and the Atelopus species.
Under scanning electron microscopy, the spermatozoon of Atelopus spumarius presented a 19 mm long filiform head and a 40 mm long tail. The tail had a very large undulating membrane ( Figure 3A). In longitudinal sections the nucleus appeared cylindrical, with the anterior portion covered by the acrosomal complex ( Figure 3B). The acrosome consists of a conical electron-dense vesicle covering 6 mm of the anterior portion of the nucleus.
A subacrosomal cone filled with a diffuse, thick material is observed under the acrosome ( Figure 3B). In transverse sections the acrosomal complex appeared circular and became progressively narrower posteriorly, together with the subacrosomal cone ( Figure 3D-H). Also in these sections the nucleus appeared circular with a 0.9 mm width at its base (Fig. 3I). The chromatin was highly electron dense and had a fibrillar-like structure, with differentially condensed areas ( Figure 3H-J).
The features of the acrosomal complex here observed (i.e., the nucleus covered by an acrosomal vesicle above the subacrosomal cone) are characteristic of the neobatrachian bufonoid lineage and have been described for most species of the previous genus Bufo (Jamieson, 2003). 530 Karyotype and sperm of A. spumarius In the midpiece, a nuclear fossa contained the proximal and distal centrioles, which lie at an angle of 90 o to each other ( Figure 3C). The axoneme originates from the distal centriole. A cytoplasmic expansion containing mitochondria extended from the nuclear base to the initial portion of the flagellum, forming a collar-like structure also observed in transverse sections ( Figure 3C and K).
In transverse sections, the flagellum consisted of an axoneme, an undulating membrane and a highly electrondense axial fiber with a width similar to that of the axoneme ( Figure 3K and L). The axoneme and axial fiber merge in the distal portion of the tail ( Figure 3M). The fiber appeared as a protein bridge that narrows at the final portion of the tail ( Figure 3N). In the distal portion of the flagellum, the axial fiber became closer to the axoneme and then disappeared together with the undulating membrane (Figure 3N). At the end of the flagellum, only the axoneme was observed ( Figure 3O).
The spermatological patterns of the flagellum (i.e., composed of an axoneme and axial rod, presence of a mitochondrial collar and undulating membrane) are also characteristic of the neobatrachian bufonoid lineage, as found by Jamieson (2003) in most species of the previous genus Bufo.
Using molecular data, Grant et al. (2006) presented a cladogram indicating as the Atelopus sister groups the genus Melanoprhyniscus and the three genera now included in the family Hylodidae (Hylodes, Crossodactylus and Megaelosia). These proposed relationships are supported by the present data, as Melanophryniscus cambaraensis (Báo et al., 2001) and the hylodids Hylodes phyllodes, Crossodactylus sp. and Megaelosia massarti display the same sperm ultrastructural pattern observed in A. spumarius.
Taken together, the sperm ultrastructural characteristics of A. spumarius and the karyotype data support its close proximity to the bufonoid lineage.
The present findings better characterized the species Atelopus spumarius and allow future comparative studies to determine whether more than one species is assigned under this name. The unique C-banding, Ag-NOR and ultrastructural data of A. spumarius corroborate its position within the family Bufonidae. In addition, the numerically and morphologically conserved karyotypes within Atelopus species studied so far attest to the well-documented monophyly postulated by Lötters et al. (2010).