C 2000 The Japan Mendel Society Cytologia 65: 359-363, 2000 Cytogenetics of Edalorhina perezi (Anura, Leptodactylidae) L. B. Lourengol, A. J. Cardoso" and S. M. Recco-Pimentel1 1 Departamento de Biologia Celular and 2 Departamento de Zoologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP) 13083-970, Campinas, SP, Brasil Accepted June 25, 2000 Summary Specimens of Edalorhina perezi Jimenez de la Espada from the Brazilian Amazon were studied cytogenetically using Giemsa staining, silver staining and C-banding. The diploid comple- ment of 2n=22 agreed with the chromosome number previously described for Edalorhina sp. Mor- phologically, the E. perezi karyotype resembled those of Physalaemus and Pleurodema species, but NOR localization and C-banding were unable to detect accurate homologies between E. perezi kary- otype and those of the Physalaemus and Pleurodema species studied to date. Key words Edalorhina, Leptodactylidae, cytogenetics. Currently, the genus Edalorhina (Leptodactylidae, Leptodactylinae) encompasses 2 species, E. perezi Jimenez de la Espada and E. nasuta Boulenger, as originally proposed by Dunn (1949). E. perezi is widespread in lowland tropical rainforest of the Amazon Basin in Ecuador, Peru, Colom- bia and Brazil, whereas E. nasuta is known only from Peru (Duellman and Morales 1990). Based on morphological and ecological characteristics, Duellman and Morales (1990) described geo- graphic variation in E. perezi and suggested the need for more studies on this species. The phylogenetic relationships of Edalorhina are still not clear. Dunn (1949) considered this genus closely related to Pleurodema, while Lynch (1971) placed Edalorhina between Lithodytes and Physalaemus. The proximity between Edalorhina and Pseudopaludicola was suggested by Heyer (1975), and Bogart (1976) proposed the inclusion of Edalorhina in the genus Physalaemus. In Heyer's study the chromosome number of Edalorhina sp. (Bogart 1973) was considered to- gether with ecological and morphological characters. Bogart (1976) also considered karyological information in his analysis, although no description of the Edalorhina karyotype, other than its chromosome number, has been reported. In this report we describe a more complete cytogenetic analysis of E. perezi in order to contribute for the assembly of chromosomal data for the study of the subfamily Leptodactylinae. Materials and methods Eight specimens (2 females, 5 males and 1 tadpole) of E. perezi from the Alto Jurua Reserve, in Acre State, Brazil, collected in February 1994 and January 1996 were examined. All adult ani- mals were deposited in the "Ada) Jose Cardoso" Zoology Museum at the State University of Camp- inas (ZUEC). The chromosomal preparations were obtained from suspensions of intestinal and testis cells from animals treated with colchicine for at least 4 h, as described by Schmid (1978) and Schmid et al. (1979). Conventional staining with Giemsa solution, silver staining (Howell and Black 1980) and C-banding (King 1980) were used to study the chromosomes, which were classified according to Green and Sessions (1991). * Dr . Adao Jose Cardoso died before this work was finished. 360 L. B. Lourenco, A. J. Cardoso and S. M. Recco-Pimentel Cytologia 65 Results All specimens had a full diploid chromosome complement of 2n=22 consisting of 6 pairs of metacentric chromosomes (pairs 1, 5, 6, 9-11), 4 pairs of submetacentric chromosomes and 1 pair of subacrocentric chromosomes (pair 3) (Fig. 1, Table 1). No heteromorphic sex chromosomes were detected. The nucleolus organizer region (NOR) was located on the long arm of chromosome 8 (Fig. 1C,D), in a region that appeared as a secondary constriction in Giemsa stained metaphases A B C D E F Fig. 1. Karyotype of Edalorhina perezi after Giemsa staining (A, B), silver staining (C, D) and C- banding (E, F). The arrows in A-D indicate the NOR. In F, the small arrows indicate faint C-bands and the large arrow points a conspicuous band of chromosome pair 8. Bar= 5 ƒÊm. 2000 Cytogenetics of Edalorhina perezi 361 Table 1. Morphometric analysis of the Edalorhina perezi karyotype. The values are the meant S.D. of measurements for 29 metaphases from 8 specimens M = metacentric, SM = submetacentric, SA= subacrocentric. (Fig. 1A, B). Neither intra- nor interindividual variability was observed in the occurrence of NOR based on silver staining. The C-banding pattern of the E. perezi karyo- type was determined in 2 specimens. The cen- tromeric region of all the chromosomes and an interstitial segment adjacent to the NOR stained strongly by the C-banding technique. Two other regions, one located interstitially on the short arm of chromosome 1 and the other in a pericen- tromeric segment of the long arm of chromo- some 2, stained faintly in C-banded metaphases Fig. 2. Idiogram of the Edalorhina perezi karyotype based on the morphometric data of Table 1. Solid blocks: (Fig. 1E, F). dark C-bands. Gray blocks: faint C-bands. Open area: sec- ondary constriction. Circles: NOR. Discussion The chromosome number of 2n=22 observed in this population of E. perezi from the Brazilian Amazon is the same as that described by Bogart (1973) for Edalorhina sp. Comparing Edalorhina with the genera considered to be closely related, we can observe that only Lithodytes lineatus (2n=18) (Bogart 1970), the unique species of this genus to be studied karyologically, Pseudopalu- dicola ameghini (2n=20) (Becak 1968), and some cytotypes of Pseudopaludicola falcipes (2n=16, 18, 20) (Batistic et al. 1969) have a diploid chromosome number different from 22. The karyotypes of all Physalaemus and Pleurodema species described to date (Kuramoto 1990, Lourenco et al. 1999) and one cytotype of Pseudopaludicola falcipes (Saez and Brum 1960) have diploid comple- ments of 22 chromosomes, as observed for E. perezi. In addition to their chromosome number, the chromosomal morphology of the karyotypes of E. perezi, Pleurodema species (Barrio and Rinaldi de Chieri 1970, Duellman and Veloso 1977, Schmid et al. 1993) and most Physalaemus species (Becak et al. 1970, De Lucca et al. 1974, Denaro 1972) also share similarities. Karyotype I of Physalaemus petersi (Lourenco et al. 1999) re- sembles that of E. perezi, although the former has heteromorphic sex chromosomes which are ab- sent in E. perezi. In contrast, karyotype II of P petersi (Lourenco et al. 1999) differs considerably from that of E. perezi. 362 L. B. Lourenco, A. J. Cardoso and S. M. Recco-Pimentel Cytologia 65 The apparent divergence in the morphology of some chromosomes in the E. perezi karyotype and those of Pleurodema species (Barrio and Rinaldi de Chieri 1970, Duellman and Veloso 1977, Schmid et al. 1993) may reflect the different classification of these chromosome pairs based on their length. Thus, homologous chromosomes are probably identified by different numbers in differ- ent karyotypes. Alternatively, small changes in chromosome length may have resulted in some karyotypic divergence. On the other hand, the morphology of pair 8 differs greatly between E. perezi and P brachyops (Schmid et al. 1993). A comparative analysis of E. perezi and Physalaemus karyotypes, except for karyotype II of P petersi, revealed smaller divergences in chromosome ordering by length than those observed be- tween E. perezi and Pleurodema karyotypes. However, there was again a clear difference in the morphology of pair 8 between the E. perezi karyotype and P petersi karyotype I (Lourenco et al. 1999). Silver staining showed that pair 8 was the only NOR-bearing pair in E. perezi. In the kary- otypes of Pleurodema species, except P brachyops, pair 8 also bears an NOR constriction (Barrio and Rinaldi de Chieri 1970, Duellman and Veloso 1977, Schmid et al. 1993). Otherwise, the intra- chromosomal localization of these sites apparently differs between E. perezi and those Pleurodema species karyotypes. The P petersi karyotype I studied by silver staining has one of its multiple NOR sites located on pair 8 (Lourenco et al. 1998). However, the very distinct morphology between this pair and pair 8 of E. perezi precludes the recognition of any homology between these chromosomes. C-band analyses of E. perezi could not reveal any chromosomal homologies between this kary- otype and those of Pleurodema and Physalaernus species already studied by this technique (Schmid et al. 1993, Lourenco et al. 1999). Probably more useful information can arise after the cytogenetic study of a greater number of species of these genera. Acknowledgements This work was supported by the Brazilian agency CNPq (Conselho Nacional de Desenvolvi- mento Cientifico e Tecnologico). References Barrio, A. and Rinaldi de Chieri, P. 1970. Estudios citogeneticos sobre el genero Pleurodema y sus consecencias evolutivas (Amphibia, Anura, Leptodactylidae). Physis 30: 309-319. Batistic, R. F., Becak, M. L. and Vizotto, L. D. 1969. Variacao cromosomica no género Pseudopaludicola (Anura). Ciéncia e Cultura 21: 260. Becak, M. L. 1968. Chromosomal analysis of eighteen species of Anura. Caryologia 21: 191-208. ― , Denaro, L. and Becak, W. 1970. Polyploidy and mechanisms of karyotypic deversification in Amphibia. Cytogenetics 9: 225-238. Bogart, J. P. 1970. Systematics problems in the amphibian family Leptodactylidae (Anura) as indicated by karyotypic analy- sis. Cytogenetics 9: 369-383. •\ 1973. Evolution of Amuran Karyotypes. In: Vial J. L. (ed.). Evolutionary Biology of the Anurans: Contemporary Re- search on Major Problems. Univ. Missouri Press, Columbia. pp. 337-349. 1976. Ethology, karyology and taxonomic position of the South American•\ leptodactylid frog genus Edalorhina. Her- petol. Rev. 7: 75. De Lucca, E., Jim, J. and Foresti, F 1974. Chromosomal studies in twelve species of Leptodactylidae and one Brachy- cephalidae. Caryologia 27: 183-192. Denaro, L. 1972. Karyotypes of Leptodactylidae anurans. J. Herpetol. 6: 71-74. Duellman, W E. and Morales, V R. 1990. Variation, distribution, and life history of Edalorhina perezi (Amphibia, Anura, Leptodactylidae). Stud. Neotrop. Fauna Environ. 25: 19-30. and Veloso, M. A. 1977. Phylogeny of Pleurodema (Anura: •\ Leptodactylidae): a biogeographic model. Occas. Papers Mus. Nat. Hist. Univ. Kansas 64: 1-46.