
Conserved karyotypes in the Hyla pulchella species group (Anura,
Hylidae)

FERNANDO ANANIAS1, PAULO C. A. GARCIA2 and SHIRLEI M. RECCO-PIMENTEL3

1Programa de Pós-Graduação em Biologia Celular e Estrutural, Instituto de Biologia, Universidade Estadual de

Campinas (UNICAMP), Campinas, SP, Brasil and Universidade São Francisco, Bragança Paulista, SP, Brasil
2Programa de Pós-Graduação em Zoologia,Universidade Estadual Paulista (UNESP), Rio Claro, SP, Brasil
3Departameno de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP),

Campinas, SP, Brasil

Ananias, F., Garcia, P. C. A. and Recco-Pimentel, S. 2004. Conserved karyotypes in the Hyla pulchella species group (Anura,

Hylidae). */ Hereditas 140: 42�/48. Lund, Sweden. ISSN 0018-0661. Received August 19, 2003. Accepted

November 18, 2003

Cytogenetic analyses were done on specimens of Hyla marginata and on three populations of H. semiguttata differing in

morphology and in the physical parameters of their advertisement call, as well as in individuals of Hyla sp. (aff. semiguttata ).

All specimens had 2n�/24 chromosomes with a morphology very similar to that of other 24-chromosome Hyla species. Hyla

semiguttata and H. marginata showed the same C-banding pattern but were distinguished by the location of the NOR on

pair 1 in H. semiguttata (in the three populations) and Hyla sp. (aff. semiguttata ), and on pair 10 in H. marginata. The H.

semiguttata populations did not differ cytogenetically, despite variations in their morphology and advertisement calls.

Similarly, H. semiguttata and H. p. joaquini studied previously had identical C-banding patterns and NOR locations,

suggesting that they are very closely related.

Shirlei M. Recco-Pimentel, Departameno de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas

(UNICAMP), 13084-971 Campinas, SP, Brasil. E-mail: shirlei@unicamp.br

According to LUTZ (1973), the Hyla pulchella group,

previously known as Hyla raddiana , occurs in Brazil,

Uruguay and Argentina. The group was defined by

DUELLMAN et al. (1997) as consisting of the following

species, excluding the related species of the Hyla

circumdata group: Hyla pulchella pulchella Duméril

and Bibron, 1841; H. pulchella joaquini Lutz, 1968; H.

pulchella cordobae Barrio, 1965; H. pulchella riojana

Koslowski, 1895; H. andina Müller, 1924; H. semi-

guttata Lutz, 1925; H. marginata Boulenger, 1887; H.

prasina Burmeister, 1856; H. cymbalum Bokermann,

1963; H. albonigra Nieden, 1923; H. balzani Boulen-

ger, 1898; H. marianitae Carrizo, 1992; H. melano-

pleura Boulenger, 1912 and H. palaestes Duellman,

De La Riva and Wild, 1997. FAIVOVICH (1996) and

CARAMASCHI and CRUZ (2000) included H. caingua

Carrizo, 1990 and H. ericae Caramaschi and Cruz,

2000 in the pulchella group. In addition, GARCIA et al.

(unpubl.) raised the subspecies H. p. joaquini to the

full species category because of its larger size, robust

arms and distinct acoustic parameters compared to

Hyla p. pulchella .

The characterization of the pulchella group is

difficult. The species considered to be part of this

group have the following characters: (1) a moderately

robust body and a proportionally long, wide head, (2)

flanks and inner thigh areas with a pale coloration and

black bars or reticulations, or dark thighs and flanks

with pale spots, (3) males with hypertrophied forearms

but a well developed prepolex terminating in a spine,

(4) an advertising call consisting of a series of ‘‘bell

type’’ notes, (5) reproduction in flowing water, and

(6) a brown, green or gray dorsal color, generally

with dark spots, reticulations or transversal bars

(DUELLMAN et al. 1997).

In an attempt to clarify the relationships within the

large pulchella group, GARCIA et al. (2001) suggested

that the species H. marginata , H. semiguttata , H. p.

joaquini and H. ericae form a subgroup within the

Hyla pulchella group based on certain common

characteristics, including the absence of stains or

dark bars on the flanks and on the inner surface of

the thighs, long, multi-pulsed acoustic notes, and

reproduction in creeks.

Hyla marginata is found in the southern Brazilian

states of Rio Grande do Sul and Santa Catarina

(GARCIA et al. 2001) and H. semiguttata Lutz in the

states of Rio Grande do Sul, Santa Catarina and

Paraná in Brazil, as well as in northwestern Argentina

(LUTZ 1925; CEI and ROIG 1961; LUTZ 1973; BRAUN

and BRAUN 1980). The relationship between H.

marginata Boulenger and H. semiguttata Lutz within

the pulchella group is unclear. LUTZ (1973) suggested

that H. marginata was similar to H. p. joaquini in some

characters. LANGONE (1993) considered H. semigut-

tata and H. p. joaquini synonymous with H. margin-

Hereditas 140: 42�/48 (2004)


ata . Morphological differences have been observed in

specimens of H. semiguttata from southern of Brazil

and northwestern Argentina (P. C. A. Garcia, pers.

obs.).
GARCIA and HADDAD (1999) reported the existence

of different populations which they referred to as

belonging to the marginata/semiguttata complex.

Analysis of the advertising calls of populations of H.

semiguttata and H. marginata showed that H. margin-

ata had call parameters that differed from those of H.

semiguttata. All populations of H. semiguttata studied

so far (Misiones, Argentina; Cambará do Sul and São
Francisco de Paula, RS, and Piraquara, PR, Brazil)

show significant differences in their call patterns,

which suggests the existence of more than one species

under the same name.

Considering the difficulty in defining H. marginata

and H. semiguttata , as well as the uncertain relation-

ships among species of the pulchella group and

between this and other groups (polytaenia and cir-

cumdata ), the aim of this study was to compare

cytogenetically three populations of H. semiguttata

and one population of H. marginata in order to clarify

some of these issues.

MATERIAL AND METHODS

Specimens of H. marginata , H. semiguttata (popula-

tions of Cambará do Sul, São Francisco de Paula and

Piraquara, Brazil) and Hyla sp. (aff. semiguttata ) from
Argentina were collected and deposited in the collec-

tion of the Dept of Zoology of the State University of

São Paulo (UNESP), Rio Claro, SP, Brazil (Table 1).

Chromosomal preparations were obtained after

intraperitonial injection of aqueous solution of 2%

colchicine (0.02 ml g�1 of body weight). After at least

4 h the intestines and testes were removed to prepare

the cell suspensions (SCHMID 1978; SCHMID et al.
1979). Metaphase preparations stained with 10%

Giemsa solution were photographed with an Olympus

BX60 microscope. The chromosomes were classified

according to their centromere position based on the

nomenclature and centromeric index proposed by

GREEN and SESSION (1991).

The constitutive heterochromatin pattern and NOR

localization were assessed using the C-banding
(SUMNER 1972) and Ag-NOR (HOWELL and BLACK

1980) techniques, respectively.

RESULTS

Karyotypes

All populations of H. marginata , H. semiguttata , and

Hyla sp (aff. semiguttata ) had 2n�/24 chromosomes.

Pairs 1, 2, 8, 11 and 12 were metacentric, pairs 3, 5, 7,

9 and 10 were sub-metacentric, and pairs 4 and 6 were

sub-telocentric (Fig. 1 and 4; Table 2). H. marginata

had secondary constrictions in the centromeric region

of the long arms of pair 10. In H. semiguttata (three
populations) and Hyla sp. (aff. semiguttata ), such

constrictions occurred in the telomeric region of the

short arm of pair 1 in some metaphases (Fig. 1 and 4).

C-banding pattern

The same heterochromatin pattern was observed in H.

marginata , H. semiguttata and Hyla sp. (aff. semi-

guttata ) (Fig. 2 and 4) using the C-banding method.

The centromeric regions of all chromosomes were

labeled. A strong heterochromatic band was observed
on the long arm of pair 10, as well as in the telomeric

region of the long arm in pair 1.

Nucleolar oganizing region �/ (NOR)

The NOR in H. marginata was located on the long

arm of pair 10, coincident with the secondary

constriction and the heterochromatin block. In H.

semiguttata and Hyla sp. (aff. semiguttata ), the NOR

occurred in the telomeric region on the short arm in
pair 1 (Fig. 3 and 4).

DISCUSSION

The diploid chromosome number of 2n�/24 is com-

mon in the order Anura and occurs in species of

Table 1. Number of specimens, collection site in Brazil and Argentina and Museum catalogue number of the

examined specimens. RS�/Rio Grande do Sul State; SC�/Santa Catarina State; PR�/Paraná State; BR�/Brazil.

Species Number of specimens Collection site Museum acession numbers

H. semiguttata 12 males Cambará do Sul, SC; BR 3114�/3122 and 3126�/3128
7 males São Francisco de Paula, RS, BR 3139�/3145
4 males Piraquara, PR; BR 3704�/3707

Hyla sp. (aff. semiguttata ) 4 males Misiones, Argentina 4908, 3446, 4909 and 4910

H. marginata 8 males São Francisco de Paula, RS, BR 3090�/3094 and 3819�/3821

Hereditas 140 (2004) Conserved karyotypes in the Hyla 43


several families (KURAMOTO 1990). Most species

belonging to the genus Hyla show 2n�/24 or

2n�/30 chromosomes (BEÇAK 1968; RABELLO 1970;

BOGART 1973; KURAMOTO 1990; ANDERSON 1991;

SKUK and LANGONE 1992; BALDISSERA et al. 1993;

KAISER et al. 1996), which suggests a dichotomy

within the genus Hyla, despite the fact that there are

also other diploid numbers such as 2n�/18, 20, 26, 30,

32 and 34 (KURAMOTO 1990; BALDISSERA et al.

1993). According to MIURA (1995), the appearance

of Hyla species with 2n�/24 chromosomes can be

related to a common ancestor with 2n�/26, and

BOGART (1973) suggested that species with morpho-

logically similar karyotypes can be considered to share

a common ancestor. One of the possible mechanism to

explain the change from 2n�/26 to 2n�/24 chromo-

somes may be related to centric fusion (MORESCALCHI

1990). The Hyla species with 2n�/18, 20 and 22

chromosomes probably had their origin in the kar-

yotype with 2n�/24 chromosomes (BOGART 1973).

For the 30-chromosomes Hyla , centric dissociation

probable is responsible for the increase in number and

pericentric inversion have shifted the position of the

centromeres in many cases (BOGART 1973; KING

1990). As stated by BOGART (1973) the 30-chromo-

somes Hyla and the 24-chromosome Hyla were

probably independently derived from a 26-chromo-

some ancestor.

Despite the differences in external morphology

among H. semiguttata populations, H. marginata

and Hyla sp. (aff. semiguttata ), these species show a

very conserved chromosomal morphology. The kar-

yotypes of H. marginata , H. semiguttata and Hyla sp.

(aff. semiguttata ) were very similar to other species of

the pulchella group (H. p. pulchella, H. caingua, H.

prasina, H. p. joaquini ) (BALDISSERA et al. 1993;

ANANIAS 1996) and to H. guentheri and H. bischoffi

(RABER 2000), as well as to some neotropical and

holoartic Hyla species (BOGART 1973; ANDERSON

1991).

Comparison of the constitutive heterochromatin

pattern of the three species studied with those

previously described for the pulchella group and

related species revealed that there were some common

C-bands in most of them. H. prasina (BALDISSERA et

al. 1993; ANANIAS 1996), H. p. joaquini (ANANIAS

1996), H. guentheri and H. bischoffi (RABER 2000)

had the same telomeric band in pair 1 also found in

Fig. 1a�/e. Karyotypes of H. marginata (a), H. semiguttata from São Francisco de Paula, RS. (b), Cambará do Sul, RS.
(c) and Piracuara, PR. (d) and Hyla sp. (aff. semiguttata ) (e) after Giemsa staining. Bar�/5 mm.

44 F. Ananias et al. Hereditas 140 (2004)


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Hereditas 140 (2004) Conserved karyotypes in the Hyla 45


H. marginata , H. semiguttata and Hyla sp. (aff.

semiguttata ). However, this band is absent in H.

caingua and H. p. pulchella (ANANIAS 1996).

Although H. marginata , H. semiguttata and Hyla

sp. (aff. semiguttata ) are morphologically different,

their constitutive heterochromatin patterns were the

same, confirming that they are very closely related. In

addition, H. p. joaquini (São Joaquim �/ type location)

(ANANIAS 1996) had the same heterochromatin pat-

tern observed in H. marginata , H. semiguttata and

Hyla sp. (aff. semiguttata ), suggesting that they are

more closely related to each other than to other

species of the group. KASAHARA et al. (1996) reported

similar results in three species of Bufo which had

striking morphological differences but indistinguish-

able C-band pattern, typical of species of the marinus

group, but different from species in other groups

(SCHMID 1978, 1980, 1982; MATSUI et al. 1985;

SCHMID and ALMEIDA 1988; SCHMID and

GUTTENBACH 1988; HERRERO et al. 1993). H. margin-

ata differed from H. semiguttata and Hyla sp. (aff.

semiguttata ) in the localization of the NOR. The

Fig. 2a�/e. C-banded karyotypes of H. marginata (a), H. semiguttata from São Francisco de Paula, RS. (b), Cambará do
Sul, RS. (c) and Piracuara, PR. (d) and Hyla sp. (aff. semiguttata ) (e). Bar�/5 mm.

Fig. 3a�/e. Silver-stained NOR of H. marginata (a), H. semiguttata from São Francisco de Paula, RS. (b), Cambará do
Sul, RS. (c) and Piracuara, PR. (d) and Hyla sp. (aff. semiguttata ) (e).

46 F. Ananias et al. Hereditas 140 (2004)


Ag-NOR staining in the telomere of pair 1 in H.

semiguttata and Hyla sp. (aff. semiguttata ) and in the

near of pericentromeric area of pair 10 in H. margin-

ata may have arisen through translocations. H. p.

joaquini also had an NOR in the telomeric region of

pair 1, suggesting a similarity to H. semiguttata and

Hyla sp. (aff. semiguttata ).

According to GARCIA and HADDAD (1999), there

are differences in the advertisement call among the

three populations of H. semiguttata. However, cyto-

genetic analysis of these populations provides no

evidence to support the hypothesis of their belonging

to different taxa . On the other hand, the synonymiza-

tion of H. semiguttata and H. p. joaquini to H.

marginata , as suggested by LANGONE (1997), was

not confirmed since only H. semiguttata and H. p.

joaquini can be mistaken cytogenetically.

In conclusion, the biological differences within the

Hyla species and populations of H. semiguttata

studied here were not reflected in the chromosomal

structure of these species, even though GOLDMAN and

BARTON (1992) suggested that genetic changes should

be seen in the populational structure and should

influence speciation and diversification. However,
this lack of chromosomal variation does not mean

that there is currently no speciation in progress. Our

results also show that the relationship among H.

marginata , H. p. joaquini and H. semiguttata and

their populations may be better understood through

molecular DNA analysis.

Acknowledgements �/ The authors thank Dr. Célio F. B.
Haddad for his help in collecting some specimens and Klélia
Aparecida de Carvalho for her technical expertise. P.C.A.G.
was supported by a scholarship from FAPESP (process No.
98/06087-9).

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