Zoologischer Anzeiger 250 (2011) 205–214

Comparative cytogenetics of eight species of (Anura, ) Rafael Bueno Noletoa,∗, Renata Cecília Amarob,c, Vanessa Kruth Verdaded, João Reinaldo Cruz Campose, Luiz Fernando Kraft Gallegof, André Magnani Xavier de Limag, Marta Margarete Cestarif, Sanae Kasaharae, Yatiyo Yonenaga-Yassudac, Miguel Trefaut Rodriguesb, Luís Felipe Toledoh aDepartamento de Biologia, Universidade Estadual do Paraná, Campus de União da Vitória, Pra¸ca Cel. Amazonas, Caixa Postal 291, CEP: 84600-000, União da Vitória, Paraná, Brazil bDepartamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Travessa 14, 101, CEP: 05508-900, São Paulo, São Paulo, Brazil cDepartamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 277, CEP: 05508-900, São Paulo, São Paulo, Brazil dCentro de Ciências Naturais e Humanas, Universidade Federal do ABC, Rua Santa Adélia, 166, CEP: 09210-170, Santo André, São Paulo, Brazil eDepartamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista, Av 24A, 1515, CEP: 13506-900, Rio Claro, São Paulo, Brazil f Departamento de Genética, Universidade Federal do Paraná, Centro Politécnico, Caixa Postal 19071, CEP: 81531-980, Curitiba, Paraná, Brazil gPós-gradua¸cão em Ecologia e Conserva¸cão, Universidade Federal do Paraná, Centro Politécnico, Caixa Postal 19031, CEP: 81531-980, Curitiba, Paraná, Brazil hMuseu de Zoologia “Prof. Adão José Cardoso”, Instituto de Biologia, Universidade Estadual de Campinas, Caixa Postal 6109, CEP: 13083-970, Campinas, São Paulo, Brazil

Received 30 October 2010; received in revised form 14 March 2011; accepted 5 April 2011 Corresponding Editor: C. Lueter.

Abstract

Several aspects of the biology of Cycloramphus species of the Atlantic Forest are still poorly known, which makes it difficult to understand their historical relationships. Therefore, we were stimulated to promote a comparative cytogenetic analysis of several species of the genus Cycloramphus. The study of Cycloramphus acangatan, C. boraceiensis, C. brasiliensis, C. carvalhoi, C. eleutherodactylus, C. fuliginosus, C. lutzorum, and C. rhyakonastes, revealed that these eight species share a diploid number 2n = 26. Cycloramphus fuliginosus presented the most distinct karyotype, due to the presence of subtelocentric chromosomes in pairs 1 and 4. The main diagnostic feature observed in the other species was the presence of one pair of telocentric chromosomes in C. boraceiensis, C. carvalhoi, and C. eleutherodactylus, while the remaining species presented karyotypes composed exclusively of biarmed chromosomes. Constitutive heterochromatin was predominantly located in pericentromeric regions in all species,

∗Corresponding author. E-mail addresses: [email protected] (R.B. Noleto), [email protected] (R.C. Amaro), [email protected] (V.K. Verdade), [email protected] (J.R.C. Campos), [email protected] (L.F. Toledo).

0044-5231/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.jcz.2011.04.001 206 R.B. Noleto et al. / Zoologischer Anzeiger 250 (2011) 205–214 although additional C-bands detected on telomeric and/or interstitial regions were partially species-specific. Silver staining revealed Ag-NORs located on the pair 6 in six species, whereas C. acangatan presented it on pair 1 and a multiple pattern was observed in C. fuliginosus with three Ag-NOR bearing chromosomes. Fluorescent in situ hybridization using rDNA probe was performed in specimens of C. eleutherodactylus from Paraná, C. lutzorum, and C. rhyakonastes, which did not reveal inactive NOR. Despite the apparent highly conserved diploid number, data on the karyotype microstructure characterize the cytogenetic profile of the genus and may contribute to clarify the phylogenetic relationships among Cycloramphus, the Cycloramphinae, or even the family Cycloramphidae. © 2011 Elsevier GmbH. All rights reserved.

Keywords: cytogenetics; Cycloramphus; Karyotypes; Ag-NORs; C-banding; FISH

1. Introduction Saez, 1968; Bogart, 1970; Bec¸ak et al., 1970; Silva et al., 2001; Lima et al., 2010). The data for most reveal karyotypes The genus Cycloramphus (Tschudi, 1838) comprises 27 with 2n = 26 chromosomes and a fundamental number (FN) species of , all restricted to the Atlantic forest domain equal to 50 due to the presence of one pair of telocentric (Ab’Sáber, 1970), occurring from the southern portion of the chromosomes, or FN = 52, with all biarmed chromosomes. State of Bahia down to the State of Santa Catarina, reaching In order to contribute to the general knowledge on and their highest diversity in the mountain chains in southeastern evolution of the genus we present a cytogenetic analysis and southern Brazil (Heyer, 1983, 1988; Haddad and Sazima, and comparison of eight species: Cycloramphus acangatan, 1989; Verdade and Rodrigues, 2003, 2008; Brasileiro et al., Cycloramphus boraceiensis (Heyer, 1983), Cycloramphus 2007). brasiliensis (Steindachner, 1864), Cycloramphus carvalhoi, Despite the lack of available data for the most Brazilian Cycloramphus eleutherodactylus, Cycloramphus fuliginosus cycloramphids, natural history coupled with morphol- (Tschudi, 1838), Cycloramphus lutzorum (Heyer, 1983), ogy can be used to classify Cycloramphus species into and Cycloramphus rhyakonastes (Heyer, 1983) based on two ecomorphological groups, which so far have not conventional staining, Ag-NOR detection, C-banding, and been tested by an explicit phylogeny. The forest litter fluorescent in situ hybridization using rDNA probes. dwellers comprising Cycloramphus acangatan (Verdade and Rodrigues, 2003), Cycloramphus bolitoglossus (Werner, 1897), Cycloramphus carvalhoi (Heyer, 1983), Cyclo- 2. Materials and methods ramphus diringshofeni (Bokermann, 1957), Cycloramphus eleutherodactylus (Miranda-Ribeiro, 1920), Cycloramphus 2.1. Specimens faustoi (Haddad Sawaya and Sazima, 2007), (Heyer, 1988), and Cycloramphus stejnegeri (Noble, Cytogenetic analyses were carried out on 23 specimens 1924), lay their eggs in the moist forest floor, and present of eight species of Cycloramphus collected in the states of terrestrial endotrophic tadpoles (Heyer and Crombie, 1979; Bahia, Paraná, Rio de Janeiro, and São Paulo (Table 1). Verdade, 2005; Brasileiro et al., 2007). The stream dweller Voucher specimens are deposited in the Célio F.B. Haddad group includes the remaining 18 species, which breed in Amphibian Collection, Universidade Estadual Paulista, in streams in forested areas, lay their eggs on rocks in the splash Rio Claro, São Paulo (CFBH); Capão da Imbuia National zone, (except for Cycloramphus bandeirensis (Heyer, 1983) History Museum, in Curitiba, Paraná (MHNCI); and Museum that live in high open areas and lay their eggs under rocks), of Zoology, Universidade São Paulo, São Paulo (MZUSP), and present semiterrestrial exotrophic tadpoles (Heyer, 1983; all in Brazil. Haddad and Sazima, 1989; Giaretta and Cardoso, 1995; Giaretta and Facure, 2003; Lima et al., 2010; Verdade et al., 2.2. Chromosome preparation and techniques unpublished data). Their distribution, usually associated with areas of sharp The procedures used in the current study were in accor- relief, and the stealthy habits of both leaf litter and stream dance with the Experimentation Ethics Committee dwellers, make it difficult to find and collect them. This, recommendations (UFPR 01/03BL) and the current Brazilian in turn, might explain the scarce information on vocaliza- legislation (CONCEA 1153/95). Chromosome preparations tion, tadpoles, and distribution (see Heyer and Maxon, 1983; were made directly from bone marrow and liver according Verdade,2005; Lingnau et al., 2008), so most of these species to Baldissera et al. (1993), or from intestinal epithelium as are placed into the data deficient category of IUCN (IUCN, described by Schmid (1978). Chromosomes were classified 2008). Such scarcity of data is also reflected in chromoso- by visual inspections according to the nomenclature proposed mal information, since only four species of Cycloramphus by Green and Sessions (1991). had their karyotypes described and most of them were lim- Conventional staining was performed using 5.0% Giemsa ited to the description of diploid number (Brum-Zorrilla and diluted in sodium-phosphate buffer (pH 7.0, for 10 min). R.B. Noleto et al. / Zoologischer Anzeiger 250 (2011) 205–214 207

Table 1. Cycloramphus species, number of individuals, sex, and sampled localities. F: female; M: male; SP: São Paulo state; RJ: Rio de Janeiro state; PR: Paraná state; BA: Bahia state.

Species Sample Locality

Cycloramphus acangatan 2M, 1F Piedade, SP (23◦42S, 47◦25W) 1F Paranapiacaba, SP (23◦46S, 46◦18W) 1F Ribeirão Grande, SP (24◦05S, 48◦21W) Cycloramphus boraceiensis 2M, 1F São Sebastião, SP (23◦45S, 45◦24W) 1F Ubatuba, SP (23◦22S, 44◦50W) Cycloramphus brasiliensis 2M, 1F Guapimirim, RJ (22◦42S, 42◦58W) Cycloramphus carvalhoi 1M Campos do Jordão, SP (22◦42S, 45◦28W) Cycloramphus eleutherodactylus 1M Ribeirão Grande, SP (24◦05S, 48◦21W) 1M Iporanga, SP (24◦35S, 48◦35W) 1M Salesópolis, SP (23◦32S, 45◦51W) 1M Sengés, PR (24◦05S, 49◦29W) Cycloramphus fuliginosus 1M São José da Vitória, BA (15◦09S, 39◦18W) Cycloramphus lutzorum 1M Morretes, PR (25◦35S, 48◦48W) Cycloramphus rhyakonastes 2M, 2F Morretes, PR (25◦23S, 48◦51W)

The Ag-NOR and C-banding techniques were carried out 6(Fig. 1b). Cycloramphus brasiliensis and C. rhyakonastes according to Howell and Black (1980) and Sumner (1972), showed similar karyotypes with metacentric pairs 1, 5 to 13, respectively. Fluorescent in situ hybridization (FISH) was and submetacentric pairs 2, 3 and 4 (Fig. 1c and i, respec- performed using 18S rDNA probe from the fish Prochilo- tively). Cycloramphus carvalhoi and C. eleutherodactylus dus argenteus (Spix and Agassiz, 1829) (Hatanaka and have a karyotype similar to those observed in C. brasilien- Galetti, 2004). The probes were labeled with biotin 14-dATP sis and C. rhyakonastes, except for the submetacentric pair by nick translation, following the manufacturer’s instruc- 5inC. eleutherodactylus and the telocentric pair 13 in both tions (BionickTM DNA Labeling System – Invitrogen). The species (Fig. 1d–f, respectively). The karyotype of C. lutzo- detection and amplification of hybridization signals were rum is also similar to C. brasiliensis and C. rhyakonastes, carried out using conjugated streptavidin-FITC (Molecular except the pair 11 which is submetacentric (Fig. 1h). C. ProbesTM – Invitrogen). The metaphases were examined on fuliginosus presented the most distinctive karyotype within a Zeiss Axiophot epifluorescence microscope and the chro- the genus, with subtelocentric pairs 1 and 4, submetacentric mosome images were captured using the software Case Data pairs 2, 3, 5, 8 and 12 and metacentric pairs 6, 7, 9 to 11 Manager Expo 4.0 (Applied Spectral Imaging), or viewed and 13 (Fig. 1g). Heteromorphic sex chromosomes were not under an Olympus BX51 microscope with a digital camera identified in the present samples including males and females. Olympus D71, and the images captured using the software Secondary constrictions were observed at proximal regions DPController. of the long arm of telocentric pair 6 in C. boraceiensis,inan interstitial region in the long arm of the pair 6 in C. brasilien- sis, and in the interstitial regions of short arms of pair 6 in 3. Results C. eleutherodactylus and C. lutzorum (Fig. 1b, c, e, f, and h, respectively). 3.1. Karyotype

All species analyzed showed karyotypes composed of 26 3.2. Nucleolar organizer region (NOR) chromosomes (Fig. 1), the first six pairs being medium and large-sized chromosome pairs, and the remaining seven pairs Except for C. fuliginosus, which showed Ag-NOR in the of small sizes. Cycloramphus acangatan, C. brasiliensis, interstitial region of long arms of pairs 1 and 4, the other C. fuliginosus, C. lutzorum, and C. rhyakonastes presented species presented only one Ag-NOR bearing chromosome karyotypes that consist exclusively of biarmed chromosomes pair (Fig. 2). Cycloramphus acangatan showed Ag-NOR in with FN = 52, while C. boraceiensis, C. carvalhoi, and C. the interstitial region of the short arm of pair 1 and the remain- eleutherodactylus have one pair of telocentric chromosomes ing species presented Ag-NOR in pair 6 in the interstitial showing FN = 50 (Fig. 1, Table 2). region of the short arm in C. eleutherodactylus, C. lutzorum, The karyotype of Cycloramphus acangatan presented and C. rhyakonastes, and interstitial and proximal region metacentric pairs 1, 5, 7 to 10, submetacentric pairs 2, 3, 4, of the long arm in C. boraceiensis, C. brasiliensis, and C. 6 and 12 and subtelocentric pairs 11 and 13 (Fig. 1a). Cyclo- carvalhoi, respectively (Fig. 2 and Table 2). ramphus boraceiensis has metacentric pairs 1, 5, 7 to 11 and One specimen of C. eleutherodactylus from Estac¸ão 13, submetacentric pairs 2, 3, 4 and 12, and telocentric pair Biológica de Boracéia (Salesópolis, São Paulo) presented 208 R.B. Noleto et al. / Zoologischer Anzeiger 250 (2011) 205–214

Fig. 1. Giemsa-stained karyotypes of (a) Cycloramphus acangatan, (b) C. boraceiensis, (c) C. brasiliensis, (d) C. carvalhoi, (e) C. eleuthero- dactylus from São Paulo, (f) C. eleutherodactylus from Paraná, (g) C. fuliginosus, (h) C. lutzorum, and (i) C. rhyakonastes.Bar=10␮m.

Table 2. Karyotypic features in Cycloramphus species (m: metacentric; sm: submetacentric; st: subtelocentric; t: telocentric; p: short arm; q: long arm).

Species 1 2345678910111213NORs FN

C. acangatan msmsmsmmsmmmmm stsmst1p52 C. aspera msmsmsmmmmmmmmmm– 52 C. boraceiensis msmsmsmmt mm mm m smm 6q50 C. brasiliensis msmsmsmmmmmmmmmm6q52 C. carvalhoi msmsmsmmmmmmmmmt 6p52 C. dubiusa msmsmsmmt mm mm smmsm– 50 C. eleutherodactylus msmsmsmsmmmm mm m m t 6p50 C. fuliginosusb msmsmsmmm mm mm m m sm–52 C. fuliginosus st sm sm st sm m m sm m m m sm m 1q 4q 52 C. lutzorum msmsmsmmm mm mm smmm 6p52 C. rhyakonastes msmsmsmmmmmmmmmm6p52

a Bec¸ak et al. (1970). b Bogart (1970). only one active Ag-NOR in one homologue of pair 6. Het- 3.3. Constitutive heterochromatin eromorphism in size of Ag-NOR was frequently observed in C. boraceiensis, C. brasiliensis, C. eleutherodactylus, and C. The C-banding in C. acangatan and C. lutzorum had lutzorum and the secondary constrictions detected in these an exclusively centromeric and pericentromeric heterochro- species corresponded to the Ag-NOR sites. matin pattern (Fig. 4a and g, respectively); in C. boraceiensis FISH using 18S rDNA probes was carried out in C. the constitutive heterochromatin is distributed in pericen- eleutherodactylus from Morretes, Paraná, C. lutzorum and tromeric region on all chromosomes and additional C-bands C. rhyakonastes, confirming the previous results detected by were detected in the telomeric regions of pairs 3 and 4, in the silver staining and not revealing the presence of inactive NOR short arms of pair 13 and in the same region of secondary (Fig. 3). constriction of the pair 6 (Fig. 4b). Besides the pericen- R.B. Noleto et al. / Zoologischer Anzeiger 250 (2011) 205–214 209

three locations in the state of São Paulo showed the peri- centromeric pattern and positive C-bands in the short arms of pair 6, that correspond to the secondary constriction (Fig. 4e), while a specimen from Sengés, in the state of Paraná, showed Fig. 2. Ag-NOR bearing chromosomes of (a) Cycloramphus acan- conspicuous telomeric bands in the short arm of pair 2 gatan, (b) C. boraceiensis, (c) C. brasiliensis, (d) C. carvalhoi, (e) C. (Fig. 4f). The C-banded idiograms, including Ag-NOR bear- eleutherodactylus from Paraná, (f) C. lutzorum, (g) C. rhyakonastes, ing chromosomes, of all species are shown in Fig. 5. and (h) C. fuliginosus.

4. Discussion tromeric pattern, C. brasiliensis showed an additional C-band in the long arms in pair 3 (Fig. 4c). Cycloramphus carvalhoi Considering the data available for Cycloramphus species, presented a similar pattern of C. brasiliensis and interstitial all presented 26 chromosomes with two distinct fundamen- C-bands in the long arm of pairs 4 and 6 (Fig. 4d). In C. tal numbers. C. acangatan, Cycloramphus asper (Werner, rhyakonastes faint interstitial C-bands were also detected in 1899), C. brasiliensis, C. fuliginosus, C. lutzorum, and the proximal region of long arm of pairs 1, 2 and 3, as well C. rhyakonastes have a FN = 52 composed exclusively of as telomeric faint bands on both arms of pair 1 (Fig. 4h). biarmed chromosomes, whereas a FN = 50 is characteris- In C. eleutherodactylus intraspecific variation of hete- tic of C. boraceiensis, C. carvalhoi, rochromatin distribution was observed. Individuals from (Miranda-Ribeiro, 1920), and C. eleutherodactylus, due to

Fig. 3. Metaphase chromosome spreads of after FISH with an 18S probe: (a) Cycloramphus eleutherodactylus from Paraná, (b) C. lutzorum and (c) C. rhyakonastes. The arrows indicate in the hybridization signals in the short arm of pair 6. Bar = 10 ␮m.

Fig. 4. C-banded karyotypes of: (a) Cycloramphus acangatan, (b) C. boraceiensis, (c) C. brasiliensis, (d) C. carvalhoi, (e) C. eleutherodactylus from São Paulo, (f) C. eleutherodactylus from Paraná, (g) C. lutzorum and (h) C. rhyakonastes. Bar=10␮m. 210 R.B. Noleto et al. / Zoologischer Anzeiger 250 (2011) 205–214

Fig. 5. Idiograms of the karyotypes of: (a) Cycloramphus acangatan, (b) C. boraceiensis, (c) C. brasiliensis, (d) C. carvalhoi, (e) C. eleutherodactylus from PR, (f) C. lutzorum and (g) C. rhyakonastes. Solid blocks: dark C-bands; Gray blocks: faint C-bands; Circles: Ag-NORs. the occurrence of one telocentric pair (Brum-Zorrilla and in the present study, and this latter species is morpholog- Saez, 1968; Bec¸ak et al., 1970; Bogart, 1970; Silva et al., ically similar to C. fuliginosus occurring also in Rio de 2001; Lima et al., 2010; present study). Most karyological Janeiro. Another hypothesis is that these differences might studies on the genus have described the chromosomal num- be indicative of two distinct closely related taxa presently ber and morphology based only on conventional staining. The identified under the name C. fuliginosus, or either that kary- only exception is the study carried out by Silva et al. (2001), otypic differentiation occurs among different populations of where chromosomal banding patterns in C. boraceiensis were this species. The external and internal morphology of speci- described. mens belonging to these populations were studied by Heyer The individuals of C. boraceiensis studied here showed a (1983) and Verdade (2005), who did not find significant karyotype similar to those described by Silva et al. (2001). differences. Additional studies are needed to address these However, our specimen of C. fuliginosus was obtained at issues. São José da Vitória, Bahia, in the furthest northern part Bogart (1973) and Heyer and Diment (1974) suggested that of the species distribution area. It has a karyotype distinct a2n = 26 was the primitive chromosome number for the fam- from that described by Bogart (1970) for individuals from ily “” (sensu Lynch, 1971), which included Tijuca forest, Rio de Janeiro. The main differences between the current family Cycloramphidae. However, amphibian these specimens are due to the presence of two pairs of sub- systematics has recently undergone major changes and the telocentric chromosomes (pairs 1 and 4) in the individual former Leptodactylidae are now spread into many dif- from Bahia, while in all specimens from Rio de Janeiro ferent families (Frost et al., 2006; Grant et al., 2006; these pairs are submetacentric. It is presently admitted that Heinicke et al., 2007; Hedges et al., 2008). The lack C. fuliginosus, a stream-adapted species has a disjunct dis- of a complete phylogeny for Cycloramphidae limits our tribution throughout its area of occurrence, confined to the interpretation on the origin of 2n = 26. Cycloramphus is cur- states of Bahia, Espírito Santo, and Rio de Janeiro (Heyer rently included in the subfamily Cycloramphinae, which and Maxon, 1983). Based on the above considerations, there also includes Crossodactylodes (Cochran, 1938) (2n = 26), are two possibilities to explain the karyological differences Rhinoderma (Duméril and Bibron, 1841) (2n = 26), and among these specimens. The first one is that the specimen Zachaenus (Cope, 1866) (2n = 26) (Bec¸ak et al., 1970; karyotyped by Bogart (1970) may have been misidenti- Bogart, 1970, 1973; Formas, 1976; Campos, 2010). Cyclo- fied because the karyotype described by Bogart (1970) is ramphinae is the sister-group to subfamily Alsodinae, very similar to the karyotype described for C. brasiliensis including Alsodes (Bell, 1843) (2n = 22, 26, 30, 34), Eup- R.B. Noleto et al. / Zoologischer Anzeiger 250 (2011) 205–214 211 sophus (Fitzinger, 1843) (2n = 28, 30), Hylorina (Bell, Cycloramphus boraceiensis presented differences in the 1843) (2n = 26), Insuetophrynus (Barrio, 1970) (2n = 26), pattern of distribution of constitutive heterochromatin. Limnomedusa (Fitzinger, 1843) (2n = 26), Macrogenioglot- Besides pericentromeric C-bands, the assessed individuals tus (Carvalho, 1946) (2n = 22), Odontophrynus (Reinhardt had additional bands in the telomeric regions of pairs 3 and and Lütken, 1862) (2n = 22, 44), Proceratophrys (Miranda- 4, in the short arm of pair 13 and in the region of secondary Ribeiro, 1920) (2n = 22), and Thoropa (Cope, 1865) (2n = 26) constriction of pair 6 (Figs. 4b and 5b). Specimens described (King, 1990; Formas, 1991; Formas et al., 2002; Cuevas and by Silva et al. (2001) showed additional interstitial bands in Formas, 1996, 2001, 2003; Silva et al., 2003; Cuevas, 2008; pairs 2 and 5 and a much more conspicuous telomeric band in Campos, 2010). This intrafamily variation in diploid number the short arm of pair 4. Interpopulation differences of distri- precludes characterizing an ancestral karyotype hypothesis bution of constitutive heterochromatin have been described for this group. for some anurans, e.g. in Bufo japonicus (Temminck and Although the diploid number is conserved in the genus Schlegel, 1838), Haddadus binotatus (Spix, 1824), Eupem- Cycloramphus, a difference in chromosome formulae is phix nattereri (Steindachner, 1863), latrans observed among the karyotypes. Cycloramphus acangatan, (Steffen, 1815) and Leptodactylus fuscus (Schneider, 1799) C. asper, C. brasiliensis, C. lutzorum, and C. rhyakonastes (Miura, 1995; Silva et al., 2000; Amaro-Ghilardi et al., 2004; (FN = 52) present karyotypes composed only of biarmed Ananias et al., 2007; Campos et al., 2008) and were related to chromosomes. In contrast, karyotypes with one telocentric processes of population differentiation, or even an indicative chromosome pair are found in C. boraceiensis, C. carvalhoi, of a species complex. C. dubius, and C. eleutherodactylus. One possible explana- Interpopulation polymorphism of C-bands was also tion for these differences would be the occurrence of gross observed in C. eleutherodactylus (Fig. 4e and f): the indi- chromosomal rearrangements mainly pericentric inversions, vidual from Sengés, Paraná exhibited a conspicuous C-band although other rearrangements, like deletions, transpositions, in the telomeric region of the short arms of pair 2, absent in amplifications of satellite DNAs, among others, must also populations of this species from São Paulo. Cycloramphus have occurred leading to differentiation of the chromosome eleutherodactylus is the species with the widest distribution morphology (Fig. 1, Table 2). in the genus (Heyer, 1983). The differences observed in C- The karyological differences detected in Cycloramphus are band pattern must be added to the morphological variation difficult to interpret considering the phylogenetic information considered so far to be a cline (Heyer, 1983; Verdade, 2005; available (Heyer, 1983; Heyer and Maxon, 1983; Verdade, Matos, unpublished data). 2005). As for the current phylogenies, there is low taxon and The C-banding pattern of pair 3 also deserves to be character sampling (Heyer, 1983; Heyer and Maxon, 1983) highlighted. Cycloramphus brasiliensis and C. rhyakonastes and species groups are poorly supported, except for the rela- showed a proximal C-band in the long arm, while C. bora- tionships between the forest litter dwellers (Verdade, 2005). ceiensis showed C-bands in the short arm of this pair, The species of the genus Cycloramphus are divided into five indicating the occurrence of pericentric inversion events. A groups according to morphological similarities (sensu Heyer, similar pattern was observed in the genus Alsodes (Cuevas, 1983). Wehave studied the species from three of these groups: 2008), reinforcing the importance of the role of peri- C. acangatan and C. carvalhoi which are allocated in the centric inversions in the differentiation of karyotypes of C. bolitoglossus group, C. eleutherodactylus allocated in the cycloramphids, either followed or not by heterochromatin C. eleutherodactylus group, and the remaining species that amplification. are allocated in the C. fuliginosus group. Neither the species According to the results using FISH and chromosome of the C. bolitoglossus group, nor those of the C. eleuthero- banding, the NORs were found to be adjacent or embed- dactylus group, which are forest litter dwellers, or the species ded in C-banded heterochromatin (Figs. 2–5). In six out of the C. fuliginosus group that are stream dwellers have of eight Cycloramphus species analyzed here, chromo- shown chromosomal differences correlated with these habits. some 6 was identified as the Ag-NOR bearing, but the The differences observed in chromosome morphology, in position of these Ag-NORs varied: interstitial portion of the pattern of localization of Ag-NORs, and distribution of the short arm of C. eleutherodactylus, C. lutzorum, and constitutive heterochromatin are not consistent with any of C. rhyakonastes; interstitial region of the long arm of C. these groups, morphologically or eco-morphologically deter- brasiliensis and C. carvalhoi and in the proximal region mined. Thus, this framework does not allow us to draw of the long arm of C. boraceiensis. These changes in the definite conclusions. localization of Ag-NORs could be explained by intrachro- Different staining techniques indicated the occurrence of mosomal rearrangements, like pericentric and paracentric species-specific markers in Cycloramphus (Figs. 2, 4 and 5). inversions. Except for C. acangatan and C. lutzorum, which showed A different situation was observed in C. acangatam, only a pericentromeric pattern of distribution of constitu- which showed Ag-NOR in the short arm of pair 1, while tive heterochromatin, the other species exhibited interstitial C. fuliginosus showed multiple Ag-NOR in pairs 1 and 4 and telomeric C-bands that characterize each species (Fig. 2a and h, respectively). This condition could be due (Figs. 4 and 5). to interchromosomal rearrangements, e.g. transposition or 212 R.B. Noleto et al. / Zoologischer Anzeiger 250 (2011) 205–214 translocation, which may lead to the ribosomal gene loci to Amaro-Ghilardi, R.C., Silva, M.J.J., Rodrigues, M.T., Yonenaga- other chromosomes, mainly when there is constitutive hete- Yassuda, Y., 2008. Chromosomal studies in four species of genus rochromatin associated with NOR. This situation has already Chaunus (Bufonidae, Anura): localization of telomeric and ribo- been reported for the majority of cases of NOR variability in somal sequences after fluorescence in situ hybridization (FISH). the genome of several vertebrates groups (Ruiz et al., 1981; Genetica 134, 159–168. Reed and Phillips, 1995; Woznicki et al., 2000; Datson and Amemiya, C.T., Gold, J.R., 1988. Chromosomal NORs as system- atic characters in North cyprinid fishes. Genetica 76, 81–90. Murray, 2006). Ananias, F., Bombeiro, A.L., Silva, K.D.B., Silva, A.P.Z., Haddad, A single pair of Ag-NOR in the genome represents the C.F.B., Kasahara, S., 2007. Cytogenetics of Eupemphix nattereri primitive condition for most vertebrate species (Hsu et al., (Anura, Leiuperidae) and karyotypic similarity with species of 1975; Schmid, 1978; Amemiya and Gold, 1988). Therefore, related genera: taxonomic implications. Acta Zoologica Sinica the occurrence of more than one Ag-NOR-bearing chromo- 53, 285–293. some pair, as observed in C. fuliginosus can be considered Baldissera Jr., F.A., Oliveira, P.S.L., Kasahara, S., 1993. Cyto- an apomorphy (Hsu et al., 1975). Additional analysis by genetics of four Brazilian Hyla species (Amphibia–Anura) FISH will be useful to confirm the number and location of and description of a case with a supernumerary chromo- the true NOR in the species. Besides, size heteromorphism some. Rev. Brasil. Genet. (Brazilian Journal of Genetics) 16, between homologous is frequent in some species. Rearrange- 335–345. ments, such as spontaneous deletions, duplications or unequal Bec¸ak, M.L., Denaro, L., Bec¸ak, W., 1970. Polyploidy and mech- anisms of karyotypic diversification in Amphibia. Cytogenetics recombination are important evolutionary processes that act 9, 225–238. over repetitive sequences in tandem (Dover, 1986), which Bogart, J.P., 1970. Systematic problems in the amphibian family may originate heteromorphisms. Heteromorphic NORs could Leptodactylidae (Anura) as indicated by karyotypic analysis. also be related to differences in genetic activity (Schmid, Cytogenetics 9, 369–383. 1978; Amaro-Ghilardi et al., 2008). Bogart, J.P., 1973. Evolution of anuran karyotypes. In: Vial, The apparently highly conserved diploid number within J.L. (Ed.), Evolutionary Biology of Anurans. Missouri Press, Cycloramphus species would fit as an outcome of kary- Columbia, pp. 337–349. otypic orthoselection evolution, characterized by pericentric Brasileiro, C.A., Haddad, C.F.B., Sawaya, R.J., Sazima, I., 2007. inversions, which has conserved the basic karyotype in the A new and threatened island dwelling species of Cycloramphus species. Our comparative study, including various Cycloram- (Anura: Cycloramphidae) from southeastern Brazil. Herpetolog- phus species, revealed species-specific karyotypes both in ica 63, 501–510. Brum-Zorrilla, N., Saez, F.A., 1968. Chromosomes of Leptodactyl- conventional and differential staining, which improves our idae (Amphibia, Anura). Experientia 24, 969. understanding of the karyotype evolution within this genus Campos, J.R.C., 2010. Constituic¸ão cariotípica em leptodactilídeos and may contribute to future phylogenetic studies. do gênero Leptodactylus e em espécies de famílias relacionadas à Leptodactylidae (Amphibia: Anura). PhD Thesis, Universidade Estadual Paulista. Acknowledgments Campos, J.R.C., Ananias, F., Haddad, C.F.B., Kasahara, S., 2008. Karyotypic similarity among Barycholos ternetzi and five The authors thank Dr. Roberto F. Artoni for helpful species of the genus Eleutherodactylus from southeastern Brazil comments on the manuscript. This work was supported (Anura, Brachycephalidae). Micron 39, 151–159. by Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Cuevas, C.C., Formas, J.R., 1996. Heteromorphic sex chromosomes Superior (CAPES), Conselho Nacional de Desenvolvimento in insularis (Amphibia, Anura, Leptodactylidae). Científico e Tecnológico (CNPq) (grant No. 151591/2009-1), Chromosome Research 4, 467–470. Fundac¸ão Araucária de Apoio ao Desenvolvimento Científico Cuevas, C.C., Formas, J.R., 2001. A new species of Alsodes (Anura: e Tecnológico do Estado do Paraná and Fundac¸ão de Amparo Leptodactylidae) from central Chile. Amphibia–Reptilia 22, 187–198. à Pesquisa do Estado de São Paulo (FAPESP) (grant Nos. Cuevas, C.C., Formas, J.R., 2003. Cytogenetic analysis of four 2001/05470-8, 2006/01266-0, 2006/56193-8, 2008/50325-5, species of the genus Alsodes (Anura, Leptodactylidae) with com- 2008/52847-9). We thank also Dante Pavan, Jose Cassimiro, ments about the karyological evolution of the genus. Hereditas and Felipe F. Curcio for help during field work. 138, 138–146. Cuevas, C.C., 2008. A new species of the genus Alsodes (Anura: Neobatrachia) from the Nothofagus forest, coastal range, south- References ern Chile, identified by its karyotype. Zootaxa 1771, 43–53. Datson, P.M., Murray, B.G., 2006. Ribosomal DNA locus evolution Ab’Sáber, A.N., 1970. Províncias geológicas e domínios morfo- in Nemesia: transposition rather than structural rearrangement climáticos no Brasil. Geomorfologia 20, 1–25. as the key mechanism? Chromosome Research 14, 845–857. Amaro-Ghilardi, R.C., Rodrigues, M.T., Yonenaga-Yassuda, Y., Dover, G.A., 1986. Molecular drive in multigene families: how bio- 2004. Chromosomal studies after differential staining and flu- logical novelties arise, spread and are assimilated. Trends in orescence in situ hybridization using telomeric probe in three Genetics 2, 159–165. Leptodactylus species (Leptodactylidae, Anura). Caryologia 57, Formas, J.R., 1976. New karyological data of Rhinoderma: the chro- 53–65. mosomes of Rhinoderma rufum. Experientia 32, 1000–1003. R.B. Noleto et al. / Zoologischer Anzeiger 250 (2011) 205–214 213

Formas, J.R., 1991. The karyotypes of the Chilean frogs Eupsophus Howell, W.M., Black, D.A., 1980. Controlled silver-staining of emiliopugini and E.vertebralis (Amphibia: Anura: Leptodactyl- nucleolus organizer regions with a protective colloidal devel- idae). Proceedings of the Biological Society of Washington 104, oper: a 1-step method. Experientia 36 (8), 1014–1015. 7–11. Hsu, T.C., Pathak, S., Chen, T.R., 1975. The possibility of latent Formas, J.R., Cuevas, C.C., Brieva, L., 2002. A new species centromeres and a proposed nomenclature system for total chro- of Alsodes (Anura: Leptodactylidae) from Cerro Mirador, mosome and whole arm translocations. Cytogenetics and Cell Cordillera Pelada, southern Chile. Proceedings of the Biological Genetics 15, 41–49. Society of Washington 115, 708–719. IUCN, 2008. The IUCN Red List for threatened Species. Amphib- Frost, D.R., Grant, T., Faivovich, J., et al., 2006. The amphibian tree ians. International Union for Conservation of Nature and Natural of life. Bulletin of the American Museum of Natural History 297, Resources, Cambridge, Electronic database available from: 1–370. http://www.iucnredlist.org/amphibians (accessed 22.07.10). Giaretta, A.A., Cardoso, A., 1995. Reproductive behavior King, M., 1990. Amphibia. In: John, B., Gwent, C. (Eds.), Animal of Cycloramphus dubius Miranda–Ribeiro (Amphibia, Cytogenetics. Amphibia 4 Chordata 2. Gebruder Borntraeger, Anura, Leptodactylidae). Revista Brasileira de Zoologia 12, Berlin, pp. 1–241. 229–232. Lima, A.M.X., Garey, M.V.,Noleto, R.B., Verdade,V.K.,2010. Nat- Giaretta, A.A., Facure, K.G., 2003. Clutch attendance: Cycloram- ural history of the Lutz’s Cycloramphus lutzorum Heyer, phus boraceiensis. Herpetological Review 34, 50. 1983 (Anura: Cycloramphidae) in the Brazilian Atlantic Forest. Grant, T., Frost, D.R., Caldwell, J.P., et al., 2006. Phylogenetic Description of the Advertisement Call, Tadpole, and Karyotype systematic of dartpoison frogs and their relatives (Amphibia: Journal of Herpetology 44 (3), 360–371. Athesphatanura: Dendrobatidae). Bulletin of the American Lingnau, R., Solé, M., Dallacorte, F., Kwet, A., 2008. Descrip- Museum of Natural History 269, 1–262. tion of the advertisement call of Cycloramphus bolitoglossus Green, D.M., Sessions, S.K., 1991. Nomenclature for chromo- (Werner 1897), with comments on other species in the genus somes. In: Green, D.M., Sessions, S.K. (Eds.), Amphibian from Santa Catarina, south Brazil (Amphibia, Cycloramphidae). Cytogenetics and Evolution. Academic Press, San Diego, North-Western Journal of Zoology 4, 224–235. pp. 431–432. Lynch, J.D., 1971. Evolutionary relationship, osteology and zoo- Haddad, C.F.B., Sazima, I., 1989. A new species of Cycloramphus geography of leptodactylid frogs. Miscellaneous Publication from southeastern Brazil (Amphibia: Leptodactylidae). Herpeto- – University of Kansas, Museum of Natural History 53, logica 45, 425–429. 1–238. Hatanaka, T., Galetti, P.M., 2004. Mapping of the 18S and 5S Miura, I., 1995. The late replication banding patterns of chromo- ribosomal RNA genes in the fish Prochilodus argenteus Agas- somes are highly conserved in the genera Rana. Hyla and Bufo siz, 1829 (Characiformes, Prochilodontidae). Genetica 122 (3), (Amphibia–Anura). Chromosoma 103, 567–574. 239–244. Reed, K.M., Phillips, R.B., 1995. Molecular cytogenetic analysis of Hedges, S.B., Duellman, W.E., Heinicke, M.P., 2008. New World the double-CMA3 chromosome of lake trout. Salvelinus namay- direct-developing frogs (Anura: Terrarana): molecular phy- cush. Cytogenetics and Cell Genetics 70, 104–107. logeny, classification, biogeography, and conservation. Zootaxa Ruiz, R.G., Soma, M., Bec¸ak, W., 1981. Nucleolar organizer regions 1737, 1–182. and constitutive heterochromatin in polyploidy species of the Heinicke, M.P., Duellman, W.E., Hedges, S.B., 2007. Major genus Odontophrynus (Amphibia, Anura). Cytogenetics and Caribbean and Central American Frog Faunas originated by Cell Genetics 29, 84–98. ancient oceanic dispersal. Proceedings of the National Academy Schmid, M., 1978. Chromosome banding in Amphibia I. Consti- of Sciences of the United States of America 104 (24), tutive heterochromatin and nucleolus organizer regions in Bufo 10092–10097. and Hyla. Chromosoma 66, 361–388. Heyer, W.R., 1983. Variation and systematics of frogs of the Silva, A.P.Z., Haddad, C.F.B., Kasahara, S., 2000. Chromosomal genus Cycloramphus (Amphibia, Leptodactylidae). Arquivos de studies on five species of the genus Leptodactylus Fitzinger, Zoologia 30, 235–339. 1826 (Amphibia–Anura), using differential staining. Cytobios Heyer, W.R., 1988. A notable collection of Cycloramphus 103, 25–38. (Amphibia, Leptodactylidae) from Bahia, Brazil, with a descrip- Silva, A.P.Z., Haddad, C.F.B., Kasahara, S., 2001. Cytogenetic tion of a new species (Cycloramphus migueli). Proceedings of analysis of Cycloramphus boraceiensis Heyer (Anura, Lepto- the Biological Society of Washington 10, 151–154. dactylidae). Revista Brasileira de Zoologia 18 (1), 111–115. Heyer, W.R., Crombie, R.I., 1979. Natural history notes on Craspe- Silva, A.P.Z., Haddad, C.F.B., Kasahara, S., 2003. Chromo- doglossa stejnegeri and Thoropa petropolitana (Amphibia: some banding in Macrogenioglottus alipioi Carvalho, 1946 Salientia, Leptodactylidae). Journal of the Washington Academy (Amphibia, Anura, Leptodactylidae), with comments on its tax- of Science 69, 17–20. onomic position. Boletim do Museu Nacional do Rio de Janeiro Heyer, W.R., Diment, M.J., 1974. The karyotype of Vanzolin- 499, 1–9. ius discodactylus and comments on usefulness of karyotypes Sumner, A.T., 1972. A simple technique for demonstrating cen- in determining relationships in the Leptodactylus-complex tromeric heterochromatin. Experimental Cell Research 75, (Amphibia, Leptodactylidae). Proceedings of the Bio Society 304–306. of Washington 87, 327–336. Verdade, V.K., 2005. Relac¸ões filogenéticas entre as espécies dos Heyer, W.R., Maxon, L.R., 1983. Relationships, Zoogeography gêneros Cycloramphus e Zachaenus (Anura, Leptodactylidae). and Speciation mechanisms of frogs of the genus Cyclo- PhD Thesis, Universidade de São Paulo. ramphus (Anura, Leptodactilidae). Arquivos de Zoologia 30, Verdade, V.K., Rodrigues, M.T., 2003. Description of a 341–373. new species of Cycloramphus (Anura: Leptodactyli- 214 R.B. Noleto et al. / Zoologischer Anzeiger 250 (2011) 205–214

dae) from Atlantic Forest, Brazil. Herpetologica 59, Woznicki, P., Sanchez, L., Martinez, P., Pardo, B.G., Jankun, M., 513–518. 2000. A population analysis of the structure and variability of Verdade, V.K., Rodrigues, M.T., 2008. On the identity of Cyclo- NOR in Salmo Trutta by Ag, CMA3 and ISH. Genetica 108 (2), ramphus jordanensis Heyer, 1983 (Anura: Cycloramphidae). 113–118. Herpetologica 64, 452–457.