Comparative cytogenetics of eight species of Cycloramphus (Anura, Cycloramphidae

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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, Praca Cel. Amazonas, Caixa Postal 291, CEP:84600-000, União da Vitória, Paraná, BrazilbDepartamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Travessa 14, 101, CEP: 05508-900, SãoPaulo, São Paulo, BrazilcDepartamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 277, CEP: 05508-900,São Paulo, São Paulo, BrazildCentro de Ciências Naturais e Humanas, Universidade Federal do ABC, Rua Santa Adélia, 166, CEP: 09210-170, Santo André, São Paulo,BrazileDepartamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista, Av 24A, 1515, CEP: 13506-900, Rio Claro, São Paulo,Brazilf Departamento de Genética, Universidade Federal do Paraná, Centro Politécnico, Caixa Postal 19071, CEP: 81531-980, Curitiba, Paraná,BrazilgPós-graduacão em Ecologia e Conservacão, Universidade Federal do Paraná, Centro Politécnico, Caixa Postal 19031, CEP: 81531-980,Curitiba, Paraná, BrazilhMuseu 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 2011Corresponding Editor: C. Lueter.

Abstract

Several aspects of the biology of Cycloramphus species of the Atlantic Forest are still poorly known, which makes it difficult tounderstand their historical relationships. Therefore, we were stimulated to promote a comparative cytogenetic analysis of severalspecies 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 number2n = 26. Cycloramphus fuliginosus presented the most distinct karyotype, due to the presence of subtelocentric chromosomes inpairs 1 and 4. The main diagnostic feature observed in the other species was the presence of one pair of telocentric chromosomes inC. boraceiensis, C. carvalhoi, and C. eleutherodactylus, while the remaining species presented karyotypes composed exclusivelyof 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 stainingrevealed Ag-NORs located on the pair 6 in six species, whereas C. acangatan presented it on pair 1 and a multiple pattern wasobserved in C. fuliginosus with three Ag-NOR bearing chromosomes. Fluorescent in situ hybridization using rDNA probe wasperformed in specimens of C. eleutherodactylus from Paraná, C. lutzorum, and C. rhyakonastes, which did not reveal inactiveNOR. Despite the apparent highly conserved diploid number, data on the karyotype microstructure characterize the cytogeneticprofile of the genus and may contribute to clarify the phylogenetic relationships among Cycloramphus, the Cycloramphinae, oreven the family Cycloramphidae.© 2011 Elsevier GmbH. All rights reserved.

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

1. Introduction

The genus Cycloramphus (Tschudi, 1838) comprises 27species of frogs, all restricted to the Atlantic forest domain(Ab’Sáber, 1970), occurring from the southern portion of theState of Bahia down to the State of Santa Catarina, reachingtheir highest diversity in the mountain chains in southeasternand southern Brazil (Heyer, 1983, 1988; Haddad and Sazima,1989; Verdade and Rodrigues, 2003, 2008; Brasileiro et al.,2007).

Despite the lack of available data for the most Braziliancycloramphids, natural history coupled with morphol-ogy can be used to classify Cycloramphus species intotwo ecomorphological groups, which so far have notbeen tested by an explicit phylogeny. The forest litterdwellers comprising Cycloramphus acangatan (Verdadeand Rodrigues, 2003), Cycloramphus bolitoglossus (Werner,1897), Cycloramphus carvalhoi (Heyer, 1983), Cyclo-ramphus diringshofeni (Bokermann, 1957), Cycloramphuseleutherodactylus (Miranda-Ribeiro, 1920), Cycloramphusfaustoi (Haddad Sawaya and Sazima, 2007), Cycloramphusmigueli (Heyer, 1988), and Cycloramphus stejnegeri (Noble,1924), lay their eggs in the moist forest floor, and presentterrestrial endotrophic tadpoles (Heyer and Crombie, 1979;Verdade, 2005; Brasileiro et al., 2007). The stream dwellergroup includes the remaining 18 species, which breed instreams in forested areas, lay their eggs on rocks in the splashzone, (except for Cycloramphus bandeirensis (Heyer, 1983)that live in high open areas and lay their eggs under rocks),and present semiterrestrial exotrophic tadpoles (Heyer, 1983;Haddad and Sazima, 1989; Giaretta and Cardoso, 1995;Giaretta and Facure, 2003; Lima et al., 2010; Verdade et al.,unpublished data).

Their distribution, usually associated with areas of sharprelief, and the stealthy habits of both leaf litter and streamdwellers, make it difficult to find and collect them. This,in turn, might explain the scarce information on vocaliza-tion, tadpoles, and distribution (see Heyer and Maxon, 1983;Verdade, 2005; Lingnau et al., 2008), so most of these speciesare placed into the data deficient category of IUCN (IUCN,2008). Such scarcity of data is also reflected in chromoso-mal information, since only four species of Cycloramphushad their karyotypes described and most of them were lim-ited to the description of diploid number (Brum-Zorrilla and

Saez, 1968; Bogart, 1970; Becak et al., 1970; Silva et al.,2001; Lima et al., 2010). The data for most reveal karyotypeswith 2n = 26 chromosomes and a fundamental number (FN)equal to 50 due to the presence of one pair of telocentricchromosomes, or FN = 52, with all biarmed chromosomes.

In order to contribute to the general knowledge on andevolution of the genus we present a cytogenetic analysisand comparison of eight species: Cycloramphus acangatan,Cycloramphus boraceiensis (Heyer, 1983), Cycloramphusbrasiliensis (Steindachner, 1864), Cycloramphus carvalhoi,Cycloramphus eleutherodactylus, Cycloramphus fuliginosus(Tschudi, 1838), Cycloramphus lutzorum (Heyer, 1983),and Cycloramphus rhyakonastes (Heyer, 1983) based onconventional staining, Ag-NOR detection, C-banding, andfluorescent in situ hybridization using rDNA probes.

2. Materials and methods

2.1. Specimens

Cytogenetic analyses were carried out on 23 specimensof eight species of Cycloramphus collected in the states ofBahia, Paraná, Rio de Janeiro, and São Paulo (Table 1).Voucher specimens are deposited in the Célio F.B. HaddadAmphibian Collection, Universidade Estadual Paulista, inRio Claro, São Paulo (CFBH); Capão da Imbuia NationalHistory Museum, in Curitiba, Paraná (MHNCI); and Museumof Zoology, Universidade São Paulo, São Paulo (MZUSP),all in Brazil.

2.2. Chromosome preparation and techniques

The procedures used in the current study were in accor-dance with the Animal Experimentation Ethics Committeerecommendations (UFPR 01/03BL) and the current Brazilianlegislation (CONCEA 1153/95). Chromosome preparationswere made directly from bone marrow and liver accordingto Baldissera et al. (1993), or from intestinal epithelium asdescribed by Schmid (1978). Chromosomes were classifiedby visual inspections according to the nomenclature proposedby Green and Sessions (1991).

Conventional staining was performed using 5.0% Giemsadiluted 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 deJaneiro state; PR: Paraná state; BA: Bahia state.

Species Sample Locality

Cycloramphus acangatan 2M, 1F Piedade, SP (23◦42′S, 47◦25′W)1F Paranapiacaba, SP (23◦46′S, 46◦18′W)1F Ribeirão Grande, SP (24◦05′S, 48◦21′W)

Cycloramphus boraceiensis 2M, 1F São Sebastião, SP (23◦45′S, 45◦24′W)1F Ubatuba, SP (23◦22′S, 44◦50′W)

Cycloramphus brasiliensis 2M, 1F Guapimirim, RJ (22◦42′S, 42◦58′W)Cycloramphus carvalhoi 1M Campos do Jordão, SP (22◦42′S, 45◦28′W)Cycloramphus eleutherodactylus 1M Ribeirão Grande, SP (24◦05′S, 48◦21′W)

1M Iporanga, SP (24◦35′S, 48◦35′W)1M Salesópolis, SP (23◦32′S, 45◦51′W)1M Sengés, PR (24◦05′S, 49◦29′W)

Cycloramphus fuliginosus 1M São José da Vitória, BA (15◦09′S, 39◦18′W)Cycloramphus lutzorum 1M Morretes, PR (25◦35′S, 48◦48′W)Cycloramphus rhyakonastes 2M, 2F Morretes, PR (25◦23′S, 48◦51′W)

The Ag-NOR and C-banding techniques were carried outaccording to Howell and Black (1980) and Sumner (1972),respectively. Fluorescent in situ hybridization (FISH) wasperformed using 18S rDNA probe from the fish Prochilo-dus argenteus (Spix and Agassiz, 1829) (Hatanaka andGaletti, 2004). The probes were labeled with biotin 14-dATPby nick translation, following the manufacturer’s instruc-tions (BionickTM DNA Labeling System – Invitrogen). Thedetection and amplification of hybridization signals werecarried out using conjugated streptavidin-FITC (MolecularProbesTM – Invitrogen). The metaphases were examined ona Zeiss Axiophot epifluorescence microscope and the chro-mosome images were captured using the software Case DataManager Expo 4.0 (Applied Spectral Imaging), or viewedunder an Olympus BX51 microscope with a digital cameraOlympus D71, and the images captured using the softwareDPController.

3. Results

3.1. Karyotype

All species analyzed showed karyotypes composed of 26chromosomes (Fig. 1), the first six pairs being medium andlarge-sized chromosome pairs, and the remaining seven pairsof small sizes. Cycloramphus acangatan, C. brasiliensis,C. fuliginosus, C. lutzorum, and C. rhyakonastes presentedkaryotypes that consist exclusively of biarmed chromosomeswith FN = 52, while C. boraceiensis, C. carvalhoi, and C.eleutherodactylus have one pair of telocentric chromosomesshowing FN = 50 (Fig. 1, Table 2).

The karyotype of Cycloramphus acangatan presentedmetacentric pairs 1, 5, 7 to 10, submetacentric pairs 2, 3, 4,6 and 12 and subtelocentric pairs 11 and 13 (Fig. 1a). Cyclo-ramphus boraceiensis has metacentric pairs 1, 5, 7 to 11 and13, submetacentric pairs 2, 3, 4 and 12, and telocentric pair

6 (Fig. 1b). Cycloramphus brasiliensis and C. rhyakonastesshowed similar karyotypes with metacentric pairs 1, 5 to 13,and submetacentric pairs 2, 3 and 4 (Fig. 1c and i, respec-tively). Cycloramphus carvalhoi and C. eleutherodactylushave a karyotype similar to those observed in C. brasilien-sis and C. rhyakonastes, except for the submetacentric pair5 in C. eleutherodactylus and the telocentric pair 13 in bothspecies (Fig. 1d–f, respectively). The karyotype of C. lutzo-rum is also similar to C. brasiliensis and C. rhyakonastes,except the pair 11 which is submetacentric (Fig. 1h). C.fuliginosus presented the most distinctive karyotype withinthe genus, with subtelocentric pairs 1 and 4, submetacentricpairs 2, 3, 5, 8 and 12 and metacentric pairs 6, 7, 9 to 11and 13 (Fig. 1g). Heteromorphic sex chromosomes were notidentified in the present samples including males and females.

Secondary constrictions were observed at proximal regionsof the long arm of telocentric pair 6 in C. boraceiensis, in aninterstitial region in the long arm of the pair 6 in C. brasilien-sis, and in the interstitial regions of short arms of pair 6 inC. eleutherodactylus and C. lutzorum (Fig. 1b, c, e, f, and h,respectively).

3.2. Nucleolar organizer region (NOR)

Except for C. fuliginosus, which showed Ag-NOR in theinterstitial region of long arms of pairs 1 and 4, the otherspecies presented only one Ag-NOR bearing chromosomepair (Fig. 2). Cycloramphus acangatan showed Ag-NOR inthe interstitial region of the short arm of pair 1 and the remain-ing species presented Ag-NOR in pair 6 in the interstitialregion of the short arm in C. eleutherodactylus, C. lutzorum,and C. rhyakonastes, and interstitial and proximal regionof the long arm in C. boraceiensis, C. brasiliensis, and C.carvalhoi, respectively (Fig. 2 and Table 2).

One specimen of C. eleutherodactylus from EstacãoBiológica de Boracéia (Salesópolis, São Paulo) presented



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 2 3 4 5 6 7 8 9 10 11 12 13 NORs FN

C. acangatan m sm sm sm m sm m m m m st sm st 1p 52C. aspera m sm sm sm m m m m m m m m m – 52C. boraceiensis m sm sm sm m t m m m m m sm m 6q 50C. brasiliensis m sm sm sm m m m m m m m m m 6q 52C. carvalhoi m sm sm sm m m m m m m m m t 6p 52C. dubiusa m sm sm sm m t m m m m sm m sm – 50C. eleutherodactylus m sm sm sm sm m m m m m m m t 6p 50C. fuliginosusb m sm sm sm m m m m m m m m sm – 52C. fuliginosus st sm sm st sm m m sm m m m sm m 1q 4q 52C. lutzorum m sm sm sm m m m m m m sm m m 6p 52C. rhyakonastes m sm sm sm m m m m m m m m m 6p 52

aBecak et al. (1970).bBogart (1970).

only one active Ag-NOR in one homologue of pair 6. Het-eromorphism in size of Ag-NOR was frequently observed inC. boraceiensis, C. brasiliensis, C. eleutherodactylus, and C.lutzorum and the secondary constrictions detected in thesespecies corresponded to the Ag-NOR sites.

FISH using 18S rDNA probes was carried out in C.eleutherodactylus from Morretes, Paraná, C. lutzorum andC. rhyakonastes, confirming the previous results detected bysilver staining and not revealing the presence of inactive NOR(Fig. 3).

3.3. Constitutive heterochromatin

The C-banding in C. acangatan and C. lutzorum hadan exclusively centromeric and pericentromeric heterochro-matin pattern (Fig. 4a and g, respectively); in C. boraceiensisthe constitutive heterochromatin is distributed in pericen-tromeric region on all chromosomes and additional C-bandswere detected in the telomeric regions of pairs 3 and 4, in theshort arms of pair 13 and in the same region of secondaryconstriction of the pair 6 (Fig. 4b). Besides the pericen-



Fig. 2. Ag-NOR bearing chromosomes of (a) Cycloramphus acan-gatan, (b) C. boraceiensis, (c) C. brasiliensis, (d) C. carvalhoi, (e) C.eleutherodactylus from Paraná, (f) C. lutzorum, (g) C. rhyakonastes,and (h) C. fuliginosus.

tromeric pattern, C. brasiliensis showed an additional C-bandin the long arms in pair 3 (Fig. 4c). Cycloramphus carvalhoipresented a similar pattern of C. brasiliensis and interstitialC-bands in the long arm of pairs 4 and 6 (Fig. 4d). In C.rhyakonastes faint interstitial C-bands were also detected inthe proximal region of long arm of pairs 1, 2 and 3, as wellas telomeric faint bands on both arms of pair 1 (Fig. 4h).

In C. eleutherodactylus intraspecific variation of hete-rochromatin distribution was observed. Individuals from

three locations in the state of São Paulo showed the peri-centromeric pattern and positive C-bands in the short arms ofpair 6, that correspond to the secondary constriction (Fig. 4e),while a specimen from Sengés, in the state of Paraná, showedconspicuous telomeric bands in the short arm of pair 2(Fig. 4f). The C-banded idiograms, including Ag-NOR bear-ing chromosomes, of all species are shown in Fig. 5.

4. Discussion

Considering the data available for Cycloramphus species,all presented 26 chromosomes with two distinct fundamen-tal numbers. C. acangatan, Cycloramphus asper (Werner,1899), C. brasiliensis, C. fuliginosus, C. lutzorum, andC. rhyakonastes have a FN = 52 composed exclusively ofbiarmed chromosomes, whereas a FN = 50 is characteris-tic of C. boraceiensis, C. carvalhoi, Cycloramphus dubius(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. lutzorumand (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. eleutherodactylusfrom São Paulo, (f) C. eleutherodactylus from Paraná, (g) C. lutzorum and (h) C. rhyakonastes. Bar = 10 �m.



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 andSaez, 1968; Becak et al., 1970; Bogart, 1970; Silva et al.,2001; Lima et al., 2010; present study). Most karyologicalstudies on the genus have described the chromosomal num-ber and morphology based only on conventional staining. Theonly exception is the study carried out by Silva et al. (2001),where chromosomal banding patterns in C. boraceiensis weredescribed.

The individuals of C. boraceiensis studied here showed akaryotype similar to those described by Silva et al. (2001).However, our specimen of C. fuliginosus was obtained atSão José da Vitória, Bahia, in the furthest northern partof the species distribution area. It has a karyotype distinctfrom that described by Bogart (1970) for individuals fromTijuca forest, Rio de Janeiro. The main differences betweenthese specimens are due to the presence of two pairs of sub-telocentric chromosomes (pairs 1 and 4) in the individualfrom Bahia, while in all specimens from Rio de Janeirothese pairs are submetacentric. It is presently admitted thatC. fuliginosus, a stream-adapted species has a disjunct dis-tribution throughout its area of occurrence, confined to thestates of Bahia, Espírito Santo, and Rio de Janeiro (Heyerand Maxon, 1983). Based on the above considerations, thereare two possibilities to explain the karyological differencesamong these specimens. The first one is that the specimenkaryotyped by Bogart (1970) may have been misidenti-fied because the karyotype described by Bogart (1970) isvery similar to the karyotype described for C. brasiliensis

in the present study, and this latter species is morpholog-ically similar to C. fuliginosus occurring also in Rio deJaneiro. Another hypothesis is that these differences mightbe indicative of two distinct closely related taxa presentlyidentified under the name C. fuliginosus, or either that kary-otypic differentiation occurs among different populations ofthis species. The external and internal morphology of speci-mens belonging to these populations were studied by Heyer(1983) and Verdade (2005), who did not find significantdifferences. Additional studies are needed to address theseissues.

Bogart (1973) and Heyer and Diment (1974) suggested thata 2n = 26 was the primitive chromosome number for the fam-ily “Leptodactylidae” (sensu Lynch, 1971), which includedthe current family Cycloramphidae. However, amphibiansystematics has recently undergone major changes and theformer Leptodactylidae are now spread into many dif-ferent families (Frost et al., 2006; Grant et al., 2006;Heinicke et al., 2007; Hedges et al., 2008). The lackof a complete phylogeny for Cycloramphidae limits ourinterpretation on the origin of 2n = 26. Cycloramphus is cur-rently included in the subfamily Cycloramphinae, whichalso includes Crossodactylodes (Cochran, 1938) (2n = 26),Rhinoderma (Duméril and Bibron, 1841) (2n = 26), andZachaenus (Cope, 1866) (2n = 26) (Becak et al., 1970;Bogart, 1970, 1973; Formas, 1976; Campos, 2010). Cyclo-ramphinae is the sister-group to subfamily Alsodinae,including Alsodes (Bell, 1843) (2n = 22, 26, 30, 34), Eup-



sophus (Fitzinger, 1843) (2n = 28, 30), Hylorina (Bell,1843) (2n = 26), Insuetophrynus (Barrio, 1970) (2n = 26),Limnomedusa (Fitzinger, 1843) (2n = 26), Macrogenioglot-tus (Carvalho, 1946) (2n = 22), Odontophrynus (Reinhardtand Lütken, 1862) (2n = 22, 44), Proceratophrys (Miranda-Ribeiro, 1920) (2n = 22), and Thoropa (Cope, 1865) (2n = 26)(King, 1990; Formas, 1991; Formas et al., 2002; Cuevas andFormas, 1996, 2001, 2003; Silva et al., 2003; Cuevas, 2008;Campos, 2010). This intrafamily variation in diploid numberprecludes characterizing an ancestral karyotype hypothesisfor this group.

Although the diploid number is conserved in the genusCycloramphus, a difference in chromosome formulae isobserved among the karyotypes. Cycloramphus acangatan,C. asper, C. brasiliensis, C. lutzorum, and C. rhyakonastes(FN = 52) present karyotypes composed only of biarmedchromosomes. In contrast, karyotypes with one telocentricchromosome pair are found in C. boraceiensis, C. carvalhoi,C. dubius, and C. eleutherodactylus. One possible explana-tion for these differences would be the occurrence of grosschromosomal rearrangements mainly pericentric inversions,although other rearrangements, like deletions, transpositions,amplifications of satellite DNAs, among others, must alsohave occurred leading to differentiation of the chromosomemorphology (Fig. 1, Table 2).

The karyological differences detected in Cycloramphus aredifficult to interpret considering the phylogenetic informationavailable (Heyer, 1983; Heyer and Maxon, 1983; Verdade,2005). As for the current phylogenies, there is low taxon andcharacter sampling (Heyer, 1983; Heyer and Maxon, 1983)and species groups are poorly supported, except for the rela-tionships between the forest litter dwellers (Verdade, 2005).The species of the genus Cycloramphus are divided into fivegroups according to morphological similarities (sensu Heyer,1983). We have studied the species from three of these groups:C. acangatan and C. carvalhoi which are allocated in theC. bolitoglossus group, C. eleutherodactylus allocated in theC. eleutherodactylus group, and the remaining species thatare allocated in the C. fuliginosus group. Neither the speciesof the C. bolitoglossus group, nor those of the C. eleuthero-dactylus group, which are forest litter dwellers, or the speciesof the C. fuliginosus group that are stream dwellers haveshown chromosomal differences correlated with these habits.The differences observed in chromosome morphology, inthe pattern of localization of Ag-NORs, and distribution ofconstitutive heterochromatin are not consistent with any ofthese groups, morphologically or eco-morphologically deter-mined. Thus, this framework does not allow us to drawdefinite conclusions.

Different staining techniques indicated the occurrence ofspecies-specific markers in Cycloramphus (Figs. 2, 4 and 5).Except for C. acangatan and C. lutzorum, which showedonly a pericentromeric pattern of distribution of constitu-tive heterochromatin, the other species exhibited interstitialand telomeric C-bands that characterize each species(Figs. 4 and 5).

Cycloramphus boraceiensis presented differences in thepattern of distribution of constitutive heterochromatin.Besides pericentromeric C-bands, the assessed individualshad additional bands in the telomeric regions of pairs 3 and4, in the short arm of pair 13 and in the region of secondaryconstriction of pair 6 (Figs. 4b and 5b). Specimens describedby Silva et al. (2001) showed additional interstitial bands inpairs 2 and 5 and a much more conspicuous telomeric band inthe short arm of pair 4. Interpopulation differences of distri-bution of constitutive heterochromatin have been describedfor some anurans, e.g. in Bufo japonicus (Temminck andSchlegel, 1838), Haddadus binotatus (Spix, 1824), Eupem-phix nattereri (Steindachner, 1863), Leptodactylus latrans(Steffen, 1815) and Leptodactylus fuscus (Schneider, 1799)(Miura, 1995; Silva et al., 2000; Amaro-Ghilardi et al., 2004;Ananias et al., 2007; Campos et al., 2008) and were related toprocesses of population differentiation, or even an indicativeof a species complex.

Interpopulation polymorphism of C-bands was alsoobserved in C. eleutherodactylus (Fig. 4e and f): the indi-vidual from Sengés, Paraná exhibited a conspicuous C-bandin the telomeric region of the short arms of pair 2, absent inpopulations of this species from São Paulo. Cycloramphuseleutherodactylus is the species with the widest distributionin the genus (Heyer, 1983). The differences observed in C-band pattern must be added to the morphological variationconsidered so far to be a cline (Heyer, 1983; Verdade, 2005;Matos, unpublished data).

The C-banding pattern of pair 3 also deserves to behighlighted. Cycloramphus brasiliensis and C. rhyakonastesshowed a proximal C-band in the long arm, while C. bora-ceiensis showed C-bands in the short arm of this pair,indicating the occurrence of pericentric inversion events. Asimilar pattern was observed in the genus Alsodes (Cuevas,2008), reinforcing the importance of the role of peri-centric inversions in the differentiation of karyotypes ofcycloramphids, either followed or not by heterochromatinamplification.

According to the results using FISH and chromosomebanding, the NORs were found to be adjacent or embed-ded in C-banded heterochromatin (Figs. 2–5). In six outof eight Cycloramphus species analyzed here, chromo-some 6 was identified as the Ag-NOR bearing, but theposition of these Ag-NORs varied: interstitial portion ofthe short arm of C. eleutherodactylus, C. lutzorum, andC. rhyakonastes; interstitial region of the long arm of C.brasiliensis and C. carvalhoi and in the proximal regionof the long arm of C. boraceiensis. These changes in thelocalization of Ag-NORs could be explained by intrachro-mosomal rearrangements, like pericentric and paracentricinversions.

A different situation was observed in C. acangatam,which showed Ag-NOR in the short arm of pair 1, whileC. fuliginosus showed multiple Ag-NOR in pairs 1 and 4(Fig. 2a and h, respectively). This condition could be dueto interchromosomal rearrangements, e.g. transposition or



translocation, which may lead to the ribosomal gene loci toother chromosomes, mainly when there is constitutive hete-rochromatin associated with NOR. This situation has alreadybeen reported for the majority of cases of NOR variability inthe genome of several vertebrates groups (Ruiz et al., 1981;Reed and Phillips, 1995; Woznicki et al., 2000; Datson andMurray, 2006).

A single pair of Ag-NOR in the genome represents theprimitive condition for most vertebrate species (Hsu et al.,1975; Schmid, 1978; Amemiya and Gold, 1988). Therefore,the occurrence of more than one Ag-NOR-bearing chromo-some pair, as observed in C. fuliginosus can be consideredan apomorphy (Hsu et al., 1975). Additional analysis byFISH will be useful to confirm the number and location ofthe true NOR in the species. Besides, size heteromorphismbetween homologous is frequent in some species. Rearrange-ments, such as spontaneous deletions, duplications or unequalrecombination are important evolutionary processes that actover repetitive sequences in tandem (Dover, 1986), whichmay originate heteromorphisms. Heteromorphic NORs couldalso be related to differences in genetic activity (Schmid,1978; Amaro-Ghilardi et al., 2008).

The apparently highly conserved diploid number withinCycloramphus species would fit as an outcome of kary-otypic orthoselection evolution, characterized by pericentricinversions, which has conserved the basic karyotype in thespecies. Our comparative study, including various Cycloram-phus species, revealed species-specific karyotypes both inconventional and differential staining, which improves ourunderstanding of the karyotype evolution within this genusand may contribute to future phylogenetic studies.

Acknowledgments

The authors thank Dr. Roberto F. Artoni for helpfulcomments on the manuscript. This work was supportedby Coordenacão de Aperfeicoamento de Pessoal de NívelSuperior (CAPES), Conselho Nacional de DesenvolvimentoCientífico e Tecnológico (CNPq) (grant No. 151591/2009-1),Fundacão Araucária de Apoio ao Desenvolvimento Científicoe Tecnológico do Estado do Paraná and Fundacão de Amparoà Pesquisa do Estado de São Paulo (FAPESP) (grant Nos.2001/05470-8, 2006/01266-0, 2006/56193-8, 2008/50325-5,2008/52847-9). We thank also Dante Pavan, Jose Cassimiro,and Felipe F. Curcio for help during field work.

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Comparative cytogenetics of eight species of Cycloramphus (Anura, Cycloramphidae

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