Zoologischer Anzeiger 250 (2011) 205–214 Comparative cytogenetics of eight species of Cycloramphus (Anura, Cycloramphidae) 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: Amphibian 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 frogs, 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), Cycloramphus migueli (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 Animal 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)