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Author's personal copy
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
a
Departamento de Biologia, Universidade Estadual do Paraná, Campus de União da Vitória, Praça Cel. Amazonas, Caixa Postal 291, CEP:
84600-000, União da Vitória, Paraná, Brazil
b
Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Travessa 14, 101, CEP: 05508-900, São
Paulo, São Paulo, Brazil
c
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 277, CEP: 05508-900,
São Paulo, São Paulo, Brazil
d
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Rua Santa Adélia, 166, CEP: 09210-170, Santo André, São Paulo,
Brazil
e
Departamento 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
g
Pós-graduação em Ecologia e Conservação, Universidade Federal do Paraná, Centro Politécnico, Caixa Postal 19031, CEP: 81531-980,
Curitiba, Paraná, Brazil
h
Museu 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
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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
The genus Cycloramphus (Tschudi, 1838) comprises 27
species of frogs, all restricted to the Atlantic forest domain
(Ab’Sáber, 1970), occurring from the southern portion of the
State of Bahia down to the State of Santa Catarina, reaching
their highest diversity in the mountain chains in southeastern
and 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 Brazilian
cycloramphids, natural history coupled with morphology can be used to classify Cycloramphus species into
two ecomorphological groups, which so far have not
been tested by an explicit phylogeny. The forest litter
dwellers comprising Cycloramphus acangatan (Verdade
and Rodrigues, 2003), Cycloramphus bolitoglossus (Werner,
1897), Cycloramphus carvalhoi (Heyer, 1983), Cycloramphus diringshofeni (Bokermann, 1957), Cycloramphus
eleutherodactylus (Miranda-Ribeiro, 1920), Cycloramphus
faustoi (Haddad Sawaya and Sazima, 2007), Cycloramphus
migueli (Heyer, 1988), and Cycloramphus stejnegeri (Noble,
1924), lay their eggs in the moist forest floor, and present
terrestrial endotrophic tadpoles (Heyer and Crombie, 1979;
Verdade, 2005; Brasileiro et al., 2007). The stream dweller
group includes the remaining 18 species, which breed in
streams in forested areas, lay their eggs on rocks in the splash
zone, (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 sharp
relief, and the stealthy habits of both leaf litter and stream
dwellers, make it difficult to find and collect them. This,
in turn, might explain the scarce information on vocalization, tadpoles, and distribution (see Heyer and Maxon, 1983;
Verdade, 2005; Lingnau et al., 2008), so most of these species
are placed into the data deficient category of IUCN (IUCN,
2008). Such scarcity of data is also reflected in chromosomal information, since only four species of Cycloramphus
had their karyotypes described and most of them were limited to the description of diploid number (Brum-Zorrilla and
Saez, 1968; Bogart, 1970; Beçak et al., 1970; Silva et al.,
2001; Lima et al., 2010). The data for most reveal karyotypes
with 2n = 26 chromosomes and a fundamental number (FN)
equal to 50 due to the presence of one pair of telocentric
chromosomes, or FN = 52, with all biarmed chromosomes.
In order to contribute to the general knowledge on and
evolution of the genus we present a cytogenetic analysis
and comparison of eight species: Cycloramphus acangatan,
Cycloramphus boraceiensis (Heyer, 1983), Cycloramphus
brasiliensis (Steindachner, 1864), Cycloramphus carvalhoi,
Cycloramphus eleutherodactylus, Cycloramphus fuliginosus
(Tschudi, 1838), Cycloramphus lutzorum (Heyer, 1983),
and Cycloramphus rhyakonastes (Heyer, 1983) based on
conventional staining, Ag-NOR detection, C-banding, and
fluorescent in situ hybridization using rDNA probes.
2. Materials and methods
2.1. Specimens
Cytogenetic analyses were carried out on 23 specimens
of eight species of Cycloramphus collected in the states of
Bahia, Paraná, Rio de Janeiro, and São Paulo (Table 1).
Voucher specimens are deposited in the Célio F.B. Haddad
Amphibian Collection, Universidade Estadual Paulista, in
Rio Claro, São Paulo (CFBH); Capão da Imbuia National
History Museum, in Curitiba, Paraná (MHNCI); and Museum
of 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 accordance with the Animal Experimentation Ethics Committee
recommendations (UFPR 01/03BL) and the current Brazilian
legislation (CONCEA 1153/95). Chromosome preparations
were made directly from bone marrow and liver according
to Baldissera et al. (1993), or from intestinal epithelium as
described by Schmid (1978). Chromosomes were classified
by visual inspections according to the nomenclature proposed
by Green and Sessions (1991).
Conventional staining was performed using 5.0% Giemsa
diluted in sodium-phosphate buffer (pH 7.0, for 10 min).
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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
1F
1F
2M, 1F
1F
2M, 1F
1M
1M
1M
1M
1M
1M
1M
2M, 2F
Piedade, SP (23◦ 42# S, 47◦ 25# W)
Paranapiacaba, SP (23◦ 46# S, 46◦ 18# W)
Ribeirão Grande, SP (24◦ 05# S, 48◦ 21# W)
São Sebastião, SP (23◦ 45# S, 45◦ 24# W)
Ubatuba, SP (23◦ 22# S, 44◦ 50# W)
Guapimirim, RJ (22◦ 42# S, 42◦ 58# W)
Campos do Jordão, SP (22◦ 42# S, 45◦ 28# W)
Ribeirão Grande, SP (24◦ 05# S, 48◦ 21# W)
Iporanga, SP (24◦ 35# S, 48◦ 35# W)
Salesópolis, SP (23◦ 32# S, 45◦ 51# W)
Sengés, PR (24◦ 05# S, 49◦ 29# W)
São José da Vitória, BA (15◦ 09# S, 39◦ 18# W)
Morretes, PR (25◦ 35# S, 48◦ 48# W)
Morretes, PR (25◦ 23# S, 48◦ 51# W)
Cycloramphus boraceiensis
Cycloramphus brasiliensis
Cycloramphus carvalhoi
Cycloramphus eleutherodactylus
Cycloramphus fuliginosus
Cycloramphus lutzorum
Cycloramphus rhyakonastes
The Ag-NOR and C-banding techniques were carried out
according to Howell and Black (1980) and Sumner (1972),
respectively. Fluorescent in situ hybridization (FISH) was
performed using 18S rDNA probe from the fish Prochilodus argenteus (Spix and Agassiz, 1829) (Hatanaka and
Galetti, 2004). The probes were labeled with biotin 14-dATP
by nick translation, following the manufacturer’s instructions (BionickTM DNA Labeling System – Invitrogen). The
detection and amplification of hybridization signals were
carried out using conjugated streptavidin-FITC (Molecular
ProbesTM – Invitrogen). The metaphases were examined on
a Zeiss Axiophot epifluorescence microscope and the chromosome images were captured using the software Case Data
Manager Expo 4.0 (Applied Spectral Imaging), or viewed
under an Olympus BX51 microscope with a digital camera
Olympus D71, and the images captured using the software
DPController.
3. Results
6 (Fig. 1b). Cycloramphus brasiliensis and C. rhyakonastes
showed similar karyotypes with metacentric pairs 1, 5 to 13,
and submetacentric pairs 2, 3 and 4 (Fig. 1c and i, respectively). Cycloramphus carvalhoi and C. eleutherodactylus
have a karyotype similar to those observed in C. brasiliensis and C. rhyakonastes, except for the submetacentric pair
5 in C. eleutherodactylus and the telocentric pair 13 in both
species (Fig. 1d–f, respectively). The karyotype of C. lutzorum 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 within
the genus, with subtelocentric pairs 1 and 4, submetacentric
pairs 2, 3, 5, 8 and 12 and metacentric pairs 6, 7, 9 to 11
and 13 (Fig. 1g). Heteromorphic sex chromosomes were not
identified in the present samples including males and females.
Secondary constrictions were observed at proximal regions
of the long arm of telocentric pair 6 in C. boraceiensis, in an
interstitial region in the long arm of the pair 6 in C. brasiliensis, and in the interstitial regions of short arms of pair 6 in
C. eleutherodactylus and C. lutzorum (Fig. 1b, c, e, f, and h,
respectively).
3.1. Karyotype
All species analyzed showed karyotypes composed of 26
chromosomes (Fig. 1), the first six pairs being medium and
large-sized chromosome pairs, and the remaining seven pairs
of small sizes. Cycloramphus acangatan, C. brasiliensis,
C. fuliginosus, C. lutzorum, and C. rhyakonastes presented
karyotypes that consist exclusively of biarmed chromosomes
with FN = 52, while C. boraceiensis, C. carvalhoi, and C.
eleutherodactylus have one pair of telocentric chromosomes
showing FN = 50 (Fig. 1, Table 2).
The karyotype of Cycloramphus acangatan presented
metacentric pairs 1, 5, 7 to 10, submetacentric pairs 2, 3, 4,
6 and 12 and subtelocentric pairs 11 and 13 (Fig. 1a). Cycloramphus boraceiensis has metacentric pairs 1, 5, 7 to 11 and
13, submetacentric pairs 2, 3, 4 and 12, and telocentric pair
3.2. Nucleolar organizer region (NOR)
Except for C. fuliginosus, which showed Ag-NOR in the
interstitial region of long arms of pairs 1 and 4, the other
species presented only one Ag-NOR bearing chromosome
pair (Fig. 2). Cycloramphus acangatan showed Ag-NOR in
the interstitial region of the short arm of pair 1 and the remaining species presented Ag-NOR in pair 6 in the interstitial
region of the short arm in C. eleutherodactylus, C. lutzorum,
and C. rhyakonastes, and interstitial and proximal region
of the long arm in C. boraceiensis, C. brasiliensis, and C.
carvalhoi, respectively (Fig. 2 and Table 2).
One specimen of C. eleutherodactylus from Estação
Biológica de Boracéia (Salesópolis, São Paulo) presented
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Fig. 1. Giemsa-stained 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. 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
C. aspera
C. boraceiensis
C. brasiliensis
C. carvalhoi
C. dubiusa
C. eleutherodactylus
C. fuliginosusb
C. fuliginosus
C. lutzorum
C. rhyakonastes
m
m
m
m
m
m
m
m
st
m
m
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
sm
st
sm
sm
m
m
m
m
m
m
sm
m
sm
m
m
sm
m
t
m
m
t
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
sm
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
st
m
m
m
m
sm
m
m
m
sm
m
sm
m
sm
m
m
m
m
m
sm
m
m
st
m
m
m
t
sm
t
sm
m
m
m
1p
–
6q
6q
6p
–
6p
–
1q 4q
6p
6p
52
52
50
52
52
50
50
52
52
52
52
a Beçak
b Bogart
et al. (1970).
(1970).
only one active Ag-NOR in one homologue of pair 6. Heteromorphism in size of Ag-NOR was frequently observed in
C. boraceiensis, C. brasiliensis, C. eleutherodactylus, and C.
lutzorum and the secondary constrictions detected in these
species corresponded to the Ag-NOR sites.
FISH using 18S rDNA probes was carried out in C.
eleutherodactylus from Morretes, Paraná, C. lutzorum and
C. rhyakonastes, confirming the previous results detected by
silver staining and not revealing the presence of inactive NOR
(Fig. 3).
3.3. Constitutive heterochromatin
The C-banding in C. acangatan and C. lutzorum had
an exclusively centromeric and pericentromeric heterochromatin pattern (Fig. 4a and g, respectively); in C. boraceiensis
the constitutive heterochromatin is distributed in pericentromeric region on all chromosomes and additional C-bands
were detected in the telomeric regions of pairs 3 and 4, in the
short arms of pair 13 and in the same region of secondary
constriction of the pair 6 (Fig. 4b). Besides the pericen-
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Fig. 2. Ag-NOR bearing chromosomes of (a) Cycloramphus acangatan, (b) C. boraceiensis, (c) C. brasiliensis, (d) C. carvalhoi, (e) C.
eleutherodactylus from Paraná, (f) C. lutzorum, (g) C. rhyakonastes,
and (h) C. fuliginosus.
209
three locations in the state of São Paulo showed the pericentromeric 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
conspicuous telomeric bands in the short arm of pair 2
(Fig. 4f). The C-banded idiograms, including Ag-NOR bearing chromosomes, of all species are shown in Fig. 5.
4. Discussion
tromeric pattern, C. brasiliensis showed an additional C-band
in the long arms in pair 3 (Fig. 4c). Cycloramphus carvalhoi
presented a similar pattern of C. brasiliensis and interstitial
C-bands in the long arm of pairs 4 and 6 (Fig. 4d). In C.
rhyakonastes faint interstitial C-bands were also detected in
the proximal region of long arm of pairs 1, 2 and 3, as well
as telomeric faint bands on both arms of pair 1 (Fig. 4h).
In C. eleutherodactylus intraspecific variation of heterochromatin distribution was observed. Individuals from
Considering the data available for Cycloramphus species,
all presented 26 chromosomes with two distinct fundamental numbers. C. acangatan, Cycloramphus asper (Werner,
1899), C. brasiliensis, C. fuliginosus, C. lutzorum, and
C. rhyakonastes have a FN = 52 composed exclusively of
biarmed chromosomes, whereas a FN = 50 is characteristic 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. 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.
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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
Saez, 1968; Beçak et al., 1970; Bogart, 1970; Silva et al.,
2001; Lima et al., 2010; present study). Most karyological
studies on the genus have described the chromosomal number and morphology based only on conventional staining. The
only exception is the study carried out by Silva et al. (2001),
where chromosomal banding patterns in C. boraceiensis were
described.
The individuals of C. boraceiensis studied here showed a
karyotype similar to those described by Silva et al. (2001).
However, our specimen of C. fuliginosus was obtained at
São José da Vitória, Bahia, in the furthest northern part
of the species distribution area. It has a karyotype distinct
from that described by Bogart (1970) for individuals from
Tijuca forest, Rio de Janeiro. The main differences between
these specimens are due to the presence of two pairs of subtelocentric chromosomes (pairs 1 and 4) in the individual
from Bahia, while in all specimens from Rio de Janeiro
these pairs are submetacentric. It is presently admitted that
C. fuliginosus, a stream-adapted species has a disjunct distribution throughout its area of occurrence, confined to the
states of Bahia, Espírito Santo, and Rio de Janeiro (Heyer
and Maxon, 1983). Based on the above considerations, there
are two possibilities to explain the karyological differences
among these specimens. The first one is that the specimen
karyotyped by Bogart (1970) may have been misidentified because the karyotype described by Bogart (1970) is
very similar to the karyotype described for C. brasiliensis
in the present study, and this latter species is morphologically similar to C. fuliginosus occurring also in Rio de
Janeiro. Another hypothesis is that these differences might
be indicative of two distinct closely related taxa presently
identified under the name C. fuliginosus, or either that karyotypic differentiation occurs among different populations of
this species. The external and internal morphology of specimens belonging to these populations were studied by Heyer
(1983) and Verdade (2005), who did not find significant
differences. Additional studies are needed to address these
issues.
Bogart (1973) and Heyer and Diment (1974) suggested that
a 2n = 26 was the primitive chromosome number for the family “Leptodactylidae” (sensu Lynch, 1971), which included
the current family Cycloramphidae. However, amphibian
systematics has recently undergone major changes and the
former Leptodactylidae are now spread into many different families (Frost et al., 2006; Grant et al., 2006;
Heinicke et al., 2007; Hedges et al., 2008). The lack
of a complete phylogeny for Cycloramphidae limits our
interpretation on the origin of 2n = 26. Cycloramphus is currently included in the subfamily Cycloramphinae, which
also includes Crossodactylodes (Cochran, 1938) (2n = 26),
Rhinoderma (Duméril and Bibron, 1841) (2n = 26), and
Zachaenus (Cope, 1866) (2n = 26) (Beçak et al., 1970;
Bogart, 1970, 1973; Formas, 1976; Campos, 2010). Cycloramphinae is the sister-group to subfamily Alsodinae,
including Alsodes (Bell, 1843) (2n = 22, 26, 30, 34), Eup-
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sophus (Fitzinger, 1843) (2n = 28, 30), Hylorina (Bell,
1843) (2n = 26), Insuetophrynus (Barrio, 1970) (2n = 26),
Limnomedusa (Fitzinger, 1843) (2n = 26), Macrogenioglottus (Carvalho, 1946) (2n = 22), Odontophrynus (Reinhardt
and Lütken, 1862) (2n = 22, 44), Proceratophrys (MirandaRibeiro, 1920) (2n = 22), and Thoropa (Cope, 1865) (2n = 26)
(King, 1990; Formas, 1991; Formas et al., 2002; Cuevas and
Formas, 1996, 2001, 2003; Silva et al., 2003; Cuevas, 2008;
Campos, 2010). This intrafamily variation in diploid number
precludes characterizing an ancestral karyotype hypothesis
for this group.
Although the diploid number is conserved in the genus
Cycloramphus, a difference in chromosome formulae is
observed among the karyotypes. Cycloramphus acangatan,
C. asper, C. brasiliensis, C. lutzorum, and C. rhyakonastes
(FN = 52) present karyotypes composed only of biarmed
chromosomes. In contrast, karyotypes with one telocentric
chromosome pair are found in C. boraceiensis, C. carvalhoi,
C. dubius, and C. eleutherodactylus. One possible explanation for these differences would be the occurrence of gross
chromosomal rearrangements mainly pericentric inversions,
although other rearrangements, like deletions, transpositions,
amplifications of satellite DNAs, among others, must also
have occurred leading to differentiation of the chromosome
morphology (Fig. 1, Table 2).
The karyological differences detected in Cycloramphus are
difficult to interpret considering the phylogenetic information
available (Heyer, 1983; Heyer and Maxon, 1983; Verdade,
2005). As for the current phylogenies, there is low taxon and
character sampling (Heyer, 1983; Heyer and Maxon, 1983)
and species groups are poorly supported, except for the relationships between the forest litter dwellers (Verdade, 2005).
The species of the genus Cycloramphus are divided into five
groups 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 the
C. bolitoglossus group, C. eleutherodactylus allocated in the
C. eleutherodactylus group, and the remaining species that
are allocated in the C. fuliginosus group. Neither the species
of the C. bolitoglossus group, nor those of the C. eleutherodactylus group, which are forest litter dwellers, or the species
of the C. fuliginosus group that are stream dwellers have
shown chromosomal differences correlated with these habits.
The differences observed in chromosome morphology, in
the pattern of localization of Ag-NORs, and distribution of
constitutive heterochromatin are not consistent with any of
these groups, morphologically or eco-morphologically determined. Thus, this framework does not allow us to draw
definite conclusions.
Different staining techniques indicated the occurrence of
species-specific markers in Cycloramphus (Figs. 2, 4 and 5).
Except for C. acangatan and C. lutzorum, which showed
only a pericentromeric pattern of distribution of constitutive heterochromatin, the other species exhibited interstitial
and telomeric C-bands that characterize each species
(Figs. 4 and 5).
211
Cycloramphus boraceiensis presented differences in the
pattern of distribution of constitutive heterochromatin.
Besides pericentromeric C-bands, the assessed individuals
had additional bands in the telomeric regions of pairs 3 and
4, in the short arm of pair 13 and in the region of secondary
constriction of pair 6 (Figs. 4b and 5b). Specimens described
by Silva et al. (2001) showed additional interstitial bands in
pairs 2 and 5 and a much more conspicuous telomeric band in
the short arm of pair 4. Interpopulation differences of distribution of constitutive heterochromatin have been described
for some anurans, e.g. in Bufo japonicus (Temminck and
Schlegel, 1838), Haddadus binotatus (Spix, 1824), Eupemphix 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 to
processes of population differentiation, or even an indicative
of a species complex.
Interpopulation polymorphism of C-bands was also
observed in C. eleutherodactylus (Fig. 4e and f): the individual from Sengés, Paraná exhibited a conspicuous C-band
in the telomeric region of the short arms of pair 2, absent in
populations of this species from São Paulo. Cycloramphus
eleutherodactylus is the species with the widest distribution
in the genus (Heyer, 1983). The differences observed in Cband pattern must be added to the morphological variation
considered so far to be a cline (Heyer, 1983; Verdade, 2005;
Matos, unpublished data).
The C-banding pattern of pair 3 also deserves to be
highlighted. Cycloramphus brasiliensis and C. rhyakonastes
showed a proximal C-band in the long arm, while C. boraceiensis showed C-bands in the short arm of this pair,
indicating the occurrence of pericentric inversion events. A
similar pattern was observed in the genus Alsodes (Cuevas,
2008), reinforcing the importance of the role of pericentric inversions in the differentiation of karyotypes of
cycloramphids, either followed or not by heterochromatin
amplification.
According to the results using FISH and chromosome
banding, the NORs were found to be adjacent or embedded in C-banded heterochromatin (Figs. 2–5). In six out
of eight Cycloramphus species analyzed here, chromosome 6 was identified as the Ag-NOR bearing, but the
position of these Ag-NORs varied: interstitial portion of
the short arm of C. eleutherodactylus, C. lutzorum, and
C. rhyakonastes; interstitial region of the long arm of C.
brasiliensis and C. carvalhoi and in the proximal region
of the long arm of C. boraceiensis. These changes in the
localization of Ag-NORs could be explained by intrachromosomal rearrangements, like pericentric and paracentric
inversions.
A different situation was observed in C. acangatam,
which showed Ag-NOR in the short arm of pair 1, while
C. fuliginosus showed multiple Ag-NOR in pairs 1 and 4
(Fig. 2a and h, respectively). This condition could be due
to interchromosomal rearrangements, e.g. transposition or
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R.B. Noleto et al. / Zoologischer Anzeiger 250 (2011) 205–214
translocation, which may lead to the ribosomal gene loci to
other chromosomes, mainly when there is constitutive heterochromatin associated with NOR. This situation has already
been reported for the majority of cases of NOR variability in
the genome of several vertebrates groups (Ruiz et al., 1981;
Reed and Phillips, 1995; Woznicki et al., 2000; Datson and
Murray, 2006).
A single pair of Ag-NOR in the genome represents the
primitive 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 chromosome pair, as observed in C. fuliginosus can be considered
an apomorphy (Hsu et al., 1975). Additional analysis by
FISH will be useful to confirm the number and location of
the true NOR in the species. Besides, size heteromorphism
between homologous is frequent in some species. Rearrangements, such as spontaneous deletions, duplications or unequal
recombination are important evolutionary processes that act
over repetitive sequences in tandem (Dover, 1986), which
may originate heteromorphisms. Heteromorphic NORs could
also be related to differences in genetic activity (Schmid,
1978; Amaro-Ghilardi et al., 2008).
The apparently highly conserved diploid number within
Cycloramphus species would fit as an outcome of karyotypic orthoselection evolution, characterized by pericentric
inversions, which has conserved the basic karyotype in the
species. Our comparative study, including various Cycloramphus species, revealed species-specific karyotypes both in
conventional and differential staining, which improves our
understanding of the karyotype evolution within this genus
and may contribute to future phylogenetic studies.
Acknowledgments
The authors thank Dr. Roberto F. Artoni for helpful
comments on the manuscript. This work was supported
by Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) (grant No. 151591/2009-1),
Fundação Araucária de Apoio ao Desenvolvimento Científico
e Tecnológico do Estado do Paraná and Fundação de Amparo
à 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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