CARYOLOGIA Vol. 56, no. 3

Vol. 56, no. 3: 253-260, 2003
CARYOLOGIA
Cytogenetics of two central Amazonian species
of Colostethus (Anura, Dendrobatidae)
with nidicolous tadpoles
ANA CRISTINA P. VEIGA-MENONCELLO1, ALBERTINA P. LIMA2 and SHIRLEI M. RECCO-PIMENTEL1, *
1
Departamento de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6109,
13084-971, Campinas, SP, Brasil
2 Coordenação de Pesquisas em Ecologia, Instituto Nacional de Pesquisas da Amazônia (INPA), CP 478, 69011-970, Manaus,
AM, Brasil
Abstract - A cytogenetic study of two central Amazonian species of Colostethus
with nidicolous tadpoles indicated that Colostethus stepheni had 2n=24 chromosomes, whereas Colostethus nidicola had 2n=22 chromosomes. These species
also differed in their NOR localization and C-banding pattern. These results suggest that karyotypic variation with a probable reduction in chromosome number
and the presence of nidicolous tadpoles are independent events that may have
occurred more than once during dendrobatid evolution.
Key words: Anura; Dendrobatidae; Colostethus; nidicolous tadpoles; cytogenetic.
INTRODUCTION
Compared to dendrobatids of the genera Allobates, Cryptophyllobates, Dendrobates, Epipedobates, and Phyllobates all of which have toxic skin
secretions and usually have bright coloration
(FROST 2002), frogs of the genus Colostethus are
rather drab in appearance and usually do not
have potent skin toxins (DUELLMAN and SIMMONS 1988). Many species are similar in morphology and therefore difficult to distinguish
from one another.
According to MYERS et al. (1991), current
members of the genus Colostethus are basal to the
lineage leading to the toxic dendrobatids.
Colostethus is extremely speciose, with 113 recognized species (F ROST 2002) distributed
throughout neotropical forests from Costa Rica
and the Antilles to Bolivia and Brazil (COLOMA
1995). One of us (APL) discovered four unde* Corresponding author: fax +55 19 3788 6111; e-mail:
[email protected]
scribed species of Colostethus in central Amazon, including one of the species studied here,
which was recently named as C. nidicola (CALDWELL and LIMA 2003). Another of these four
species has been described by LIMA and CALDWELL (2001) as C. caeruleodactylus.
Social behavior and parental care apparently
occur in all dendrobatids and are considered
synapomorphies of the family (W EYGOLDT
1986). Usually, the males call from close to
potential nest sites to attract females. Amplexus
is cephalic or absent, and the eggs are laid on
leaf-litter on the forest floor. One of parents
guards the eggs until they hatch, after which the
tadpoles climb onto the parent’s back and are
carried to a nearby pool or bromeliad where they
develop (ZUG 1993). Only four Colostethus larvae are known to be endotrophic; C. degranvillei
(LESCURE 1975), C. chalcopis (KAISER and ALTIG
1994), C. stepheni (JUNCÁ 1996) and C. nidicola.
Colostethus stepheni has tadpoles with a terrestrial development, and does not require an
aquatic habitat for reproduction, although the
254
VEIGA-MENONCELLO, LIMA
and RECCO-PIMENTEL
Table 1 – Morphometric analysis of the chromosomes of two Colostethus species.
Colostethus stepheni
CH
1
RL%
17.9
CI
0.437
CC
M
2
13.8
0.396
M
3
12.6
0.238
ST
4
12.0
0.333
SM
5
10.7
0.367
SM
6
9.9
0.369
SM
7
5.3
0.464
M
8
4.6
0.458
M
9
4.1
0.474
M
10
3.7
0.467
M
11
3.4
0.455
M
Colostethus nidicola
CH
1
RL%
16.5
2
13.6
3
11.8
4
12.0
5
10.6
6
9.8
8
5.4
9
5.0
10
4.3
11
3.8
CI
0.443
0.422
0.354
0.286
0.383
0.405
0.488
0.466
0.452
0.452
CC
M
M
SM
SM
M
M
7
7.8
8.4
0.354
0.440*
SM
M*
M
M
M
M
12
2.1
T
CH: chromosome, CI: centromeric index, RL: relative length (%), CC: centromeric classification, m: metacentric, sm: submetacentric,
st: subtelocentric. (*): values obtained for one of the homologs of the respective pairs that showed heteromorphism in the C-banding and NOR size.
leaf litter must remain moist throughout incubation since the tadpoles are not carried to water
to complete their development (JUNCÁ et al.
1994; JUNCÁ 1998). Tadpoles of C. nidicola develop in leaf-litter nests similar to those of C.
stepheni (CALDWELL and LIMA 2003), but this
species does not appear to be closely related to
C. stepheni. According to JUNCÁ (1998), terrestrial development is a derived character in dendrobatid frogs.
The phylogenetic relationships of many genera within the family Dendrobatidae are problematic (see M YERS et al. 1991; F ORD 1993;
CLOUGH and SUMMERS 2000). In Colostethus,
most species groups are defined by combinations
of character states variously present in other
groups, rather than by unambiguous synapomorphies (GRANT et al. 1997). Despite the great
number of Colostethus species, there is little information on their cytogenetics which could contribute to our understanding of the relationships
between or within of dendrobatid genera.
In this paper, we described the chromosome
number, NOR localization and C-banding pattern of the two central Amazonian species of
Colostethus with nidicolous tadpoles.
MATERIAL AND METHODS
Specimens
Fourteen specimens of Colostethus stepheni (five
males and nine females) from the Reserva Florestal
Adolfo Ducke (RFAD), located 25 km from Manaus,
Amazonas, (03º,08’ S, 60º,04’ W) and six specimens of
C. nidicola from the municipality of Careiro, at km 12
on the road to Autazes, state of Amazonas, Brazil
(03º,37’,10.4” S, 59º,86’,78.4” W) were studied. All
specimens were collected by A. P. Lima under a permit
issued by the Instituto Brasileiro de Meio Ambiente e
Recursos Naturais Renováveis (IBAMA) (Proc. no.
02005.001367/99-58-AM). The animals were deposited
in the Museu de Historia Natural “Professor Adão José
Cardoso” (ZUEC) at the Universidade Estadual de
Campinas or in the herpetological collections of the
Instituto Nacional de Pesquisas da Amazonia (INPA),
Manaus, under the following accession numbers: ZUEC
11450, 11609, 11612-14, 11616, 11619, 11621 and
INPA 7200-04, 7207 (C. stepheni) and ZUEC 11694,
11699-702 and 11704 (C. nidicola).
Chromosome preparations and techniques
Chromosome preparations were obtained from a
suspension of intestinal epithelial and testicular cells
from animals pretreated with 2% colchicine for at
least 4 h, as described by KING and ROFE (1976) and
SCHMID (1978a).
The techniques used were conventional Giemsa
staining, Ag-NOR staining as described by HOWELL
and BLACK (1980), and C-banding following the techniques of SUMNER (1972), with slight modifications
in the duration of treatment with 0.2 N hydrochloric
acid. The chromosomes were classified according to
GREEN and SESSIONS (1991).
RESULTS
All specimens of C. stepheni had 2n=24 chromosomes, whereas the specimens of C. nidicola
sp. had 2n=22 chromosomes (Table 1, Figs. 1
and 4). These number were confirmed by meiotic chromosome analysis, where 12 and 11 bivalents respectively were seen (Fig. 2).
255
CYTOGENETICS OF COLOSTETHUS
The karyotype of C. stepheni consisted of
seven pairs of metacentric chromosomes (1, 2, 7,
8, 9, 10 and 11), three submetacentrics (4, 5 and
6), one subtelocentric (3) and one telocentric
pair (12), whereas the karyotype of C. nidicola
consisted of eight pairs of metacentric chromosomes (1, 2, 5, 6, 8, 9, 10 and 11) and three pairs
of submetacentrics (3, 4 and 7) (Table 1 and
Fig. 4).
A bimodal structure of the karyotype was
observed in both species, although the reduction
in chromosome size in C. nidicola was less evident
than in C. stepheni.
Silver nitrate staining revealed only one
NOR-bearing pair in each species. In C.
stepheni, the NOR site was detected in the pericentromeric region on the long arms of pair 1,
coincident with the secondary constriction,
which was sometimes seen in Giemsa-stained
metaphases (Fig. 3A). In C. nidicola, the NOR
site was detected on the short arm of pair 7, but
not coincident with the secondary constriction.
The C-banding revealed interspecific variation
in the location of constitutive heterochromatin. In
C. stepheni, large amounts of constitutive heterochromatin were located in the centromeric
regions of all chromosomes (Fig. 3D). A C-block
occurred coincident with the NOR (Fig. 3A) in
the pericentromeric region on the long arm of
pair 1. Faint C-bands occurred on the long arms
of pairs 3 and 5 (Figs. 3D and 4A). In C. nidicola,
the centromeric region of all chromosomes also
contained C-banded heterochromatin. Telomeric
and interstitial bands were also observed in this
karyotype. A darkly stained heterochromatic
block occurred on the short arm of pair 7, within
the secondary constriction closely adjacent to
NOR. In the same chromosome pair, faintly
stained heterochromatin occurred in the interstitial region on the long arm (Fig. 3D and 4B).
Fig. 1 – Karyotypes after conventional Giemsa staining. Colostethus stepheni (A) and
Colostethus nidicola (B). Bar 10 µm.
256
VEIGA-MENONCELLO, LIMA
and RECCO-PIMENTEL
Fig. 2 – Giemsa-stained meiotic metaphase I. Colostethus stepheni showing 12 bivalents (A) and Colostethus nidicola showing 11 bivalents (B). Bar 10 µm.
Fig. 3 – Silver-stained NOR-bearing chromosome pairs and C-banded karyotypes. Colostethus stepheni (A), the arrow indicates the NOR and the arrowhead indicates the same pair 1 showing the C-band coincident with the NOR. Colostethus nidicola (B), the arrows indicate the homomorphic and heteromorphic NOR sites adjacent to a secondary constriction which is
not stained (arrowhead). Colostethus stepheni (C), the arrows indicate a faint band on the long arm of pairs 3 and 5.
Colostethus nidicola (D). The inset, shows pair 7 with heteromorphic C-blocks. The arrow indicates a faint band on the long
arm of pair 7. Bar 10 µm.
CYTOGENETICS OF COLOSTETHUS
In one specimen of C. nidicola, the C-bands and
the NOR located adjacent to the C-block on the
short arm of pair 7 were heteromorphic in size
(Fig. 3B and D).
DISCUSSION
The terrestrial nonpoisonous dendrobatid
frogs of the genus Colostethus have a wide distribution in the neotropics and 113 species are currently recognized (FROST 2002). However, chromosomal data are available only for species that
have tadpoles which complete their development
in water. One species analysed by R ADA DE
MARTÍNEZ (1976) and eight species analysed by
BOGART (1991) had 2n=24 chromosomes, whereas three species from central Amazonia analyzed
by VEIGA-MENONCELLO (2000) had 2n=22 chromosomes. The karyotype of C. stepheni differs
from the other 24-chromosome species analysed
by R ADA DE M ARTÍNEZ (1976) and B OGART
(1991) in the morphology of some chromosome
pairs and the location of the secondary constriction. However, the Colostethus species studied
by BOGART (1991) were examined using only conventional staining methods. This makes it difficult
to relate the NOR location in C. stepheni to the
secondary constrictions detected in the karyotypes of the species studied by BOGART (1991)
since, according to SCHMID (1978a) and KING
(1980), not all secondary constrictions are nucleolus organizers.
The usual structure of the karyotypes of dendrobatids analysed by LEÓN (1970), RADA DE
MARTÍNEZ (1976), BOGART (1991), RASOTTO et
al. (1987), V EIGA -M ENONCELLO (2000) and
AGUIAR Jr. et al. (2002) included six large chromosome pairs followed by a variable number of
small pairs: six or five in species of Colostethus,
six pairs in species of Epipedobates and three or
four pairs in species of Dendrobates.
The karyotype of C. nidicola sp. was similar to
other 22-chromosome species of Colostethus,
with the only marked difference being in the size
and morphology of pair 7, which altered the common structure of the karyotype since this pair
was larger than in other species of Colostethus.
Our results suggest that chromosomal rearrangements and heterochromatin and NOR-related
events may be involved in this alteration of karyotype structure. In addition, in other species
related to C. marchesianus analyzed by VEIGA-
257
MENONCELLO (2000), the NOR sites were not
located on the same chromosome pair. The presence of an additional NOR site in one of these
species (C. caeruleodactylus) reinforces the suggestion that chromosomal rearrangements
occurred during the differentiation of these
species.
In addition to C. nidicola, one species of
Minyobates (recently synonymized with Dendrobates, FROST 2002) analyzed by BOGART (1991)
also had seven large and four small chromosome
pairs. A cytogenetic study of a larger number of
species of this genus and the application of different banding techniques may be helpful in
understanding the unusual karyotype structure
of M. opisthomelas.
Colostethus nidicola are easily distinguished
from C. stepheni and all 24-chromosome species
already karyotyped by their diploid number of 22
chromosomes, and by the difference in the morphology of pair 7. In addition, C. nidicola can be
Fig. 4 – Representative ideograms of the chromosome numbers, NOR locations and C-banding patterns of Colostethus
stepheni (A) and Colostethus nidicola (B). Solid blocks: dark
C-bands. Gray blocks: faint C-bands. Open regions: secondary constrictions. Gray circles: NORs. The parentheses
indicate the NOR coincident with heterochromatin (A) and
heteromorphic C-band and heteromorphic NOR site (B).
258
distinguished from other species of Colostethus
with 2n=22 described from the Brazilian Amazon
by the NOR localization and C-banding pattern.
The presence of 22 chromosomes in another
species of Colostethus as shown here reinforces
the hypothesis of VEIGA-MENONCELLO (2000)
that this chromosome number could be characteristic of a species complex within the genus
Colostethus.
Constitutive heterochromatin associated with
the nucleolus organizer region (NOR), as
observed in C. stepheni, has also been reported in
other anuran species (S CHMID 1978b, 1982;
KASAHARA et al. 1996; SILVA et al. 1999), although
in these cases the NOR site is located in segments
adjacent to the constitutive heterochromatin
(SCHMID 1978a, 1982). In C. nidicola, the NOR
site was not coincident with the secondary constriction, which also showed darkly stained constitutive heterochromatin in metaphases submitted to C-banding. According to S CHMID
(1978a,b), a nucleolar constriction is a secondary
constriction that reacts specifically with silver
(Ag). Although chromosomal regions in which
constitutive heterochromatin accumulates occasionally also have the appearance of secondary
constrictions, they never exhibit the black Agblocks characteristic of NORs.
K ING (1980) distinguished five structural
classes of secondary constrictions in species of
Litoria. According to this classification, the secondary constriction seen in C. nidicola would be
considered as type 3 since it exhibited a dark Cband. However, this constriction lacks a silver
staining reaction and is not as a nucleolar organizer region. According to KING (1990), the type
3 secondary constriction varies in expression
between homologues and among cells and individuals. This author further suggested that type 3
constrictions appeared to be artifacts induced by
certain characteristics of chromosome structure,
such as a close proximity to C-bands, and may
also be induced by variations in the methods of
sample preparation. Although SCHMID (1978a)
found some constrictions that were not NOR,
and which KING (1980) suggested to be type 3,
no mention was made as to whether the constriction was visible after a silver staining reaction,
as in C. nidicola.
Although most anurans have a single NOR
site, there is considerable heteromorphism in its
size. The small heteromorphism in NOR size
observed in C. nidicola apparently involved an
VEIGA-MENONCELLO, LIMA
and RECCO-PIMENTEL
amplification of rDNA sequences, since our
results did not indicate complete amplification of
the NOR. According to SCHMID (1982) and KING
et al. (1990), the probable mechanisms involved
are unequal crossing over and sister chromatid
exchange.
As mentioned above, the two species studied here were also distinguishable from each other by their C-banding pattern. Despite the different chromosome numbers, no unambiguous
homeology was observed among the chromosomes. That could be indicative of a close relationship among these species. However, the faint
C-band located on the long arm of pair 7 in C.
nidicola was also found in other 22-chromosome
species of Colostethus (V EIGA -M ENONCELLO
2000), and suggests a close relationship among
the species. Considering the difference in the
morphology of pair 7 in C. nidicola relative to
other 22-chromosome species, the presence of
the faint C-band on the long arm of pair 7 corroborates the hypothesis that chromosomal
rearrangements involving constitutive heterochromatin and the NOR occurred in this
species. Although some species of Colostethus
in the Brazilian Amazon share the same chromosome number, they differ in their distribution of heterochromatin. According to KING
(1991), it is very uncommon to find two related
species which are alike in their heterochromatin
distribution. However, in some species of Bufo,
the C-banding pattern does not differ, indicating
that the karyotypes of these species are indistinguishable in their heterochromaitn distribution
(KASAHARA et al. 1996).
B OGART (1991) analysed eight species of
Colostethus and concluded that the variation
observed in these species did not involve changes
in chromosome number or severe karyotypic
restructuring such as seen in species of Dendrobates. The results of the present study indicate
karyotypic restructuring in the small chromosome pairs of C. nidicola They also indicate that
although Colostethus is considered a less specialized genus, the chromosome number is not conserved. In addition, karyotypic variability, with a
probable reduction in chromosome number, and
the presence of nidicolous tadpoles, are independent events that may have occurred more
than once in dendrobatid evolution, specially
considering that there are 24- and 22-chromosome species that do not show this mode of
reproduction.
CYTOGENETICS OF COLOSTETHUS
Acknowledgments – The authors thank W. E.
Magnusson and O. Aguiar-Jr for reading drafts of
the manuscript and providing valuable comments.
This research was supported by Conselho Nacional
de Desenvolvimento Científico e Tecnológico
(CNPq, grant 460233/00-9 to A. P. Lima), Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP, grant 97/12675-8 to S. M. ReccoPimentel), and Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES, fellowship to
A. C. P. Veiga-Menoncello). The authors also thank
the Instituto Brasileiro de Meio Ambiente e Recursos Naturais Renováveis (IBAMA) for issuing the
collection permit (Proc. no. 02005.001367/99-58AM).
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Received October 17, 2002; accepted January 15, 2003