Short Notes Cytogenetics of Brachycephalus Ephippium (Anura, Brachycephalidae) with Comments on Its Relationship to the Bufonida

Short Notes

Cytogenetics of Brachycephalus ephippium (Anura, Brachycephalidae) with comments on its relationship to the Bufonidae

Fernando Ananias1, Ariovaldo A. Giaretta2 and Shirlei M. Recco-Pimentel3

The family Brachycephalidae, which is en- an indented maxilla and a reduced the number demic to the eastern Brazilian rainforest and of digits (Duellman and Trueb, 1994). These includes leaf-litter frogs (Izecksohn, 1988; Gi- frogs also have a dorsal bony plate, fused to the aretta and Sawaya, 1998; Giaretta et al., 1999), vertebral column, that is a characteristic of some contains only two genera, Brachycephalus hylids, leptodactylids and pelobatids (Ruibal Fitzinger and Psyllophryne Izecksohn (Frost, and Shoemaker, 1984; Zatz et al., 1996). Adults 2002). Sminthillus brasiliensis Parker, 1926 specimens of B. ephippium have a yellow to was originally considered a member of the orange color and present a toxic tetrodotoxin Brachycephalidae Family (Parker, 1926; Lutz, (Sebben et al., 1986), a sodium channel blocker. 1954; Cochran, 1955), but was later trans- Although the genus Brachycephalus had been ferred to genus Euparkerella of the Leptodactyl- formerly included in the family Atelopodi- idae (Izecksohn, 1988). Four species are cur- dae, which is now part of the Bufonidae, rently recognized in Brachycephalus: B. ephip- the lack of the Bidder’s organ exclude it pium, B. nodoterga, B. pernix and B. vertebralis from this family (McDiarmid, 1971). Brachy- (Frost, 2002). Brachycephalus ephippium has a cephalus has been considered to be more snout vent length of 13.2-17.9 mm and occurs closely related to the genus Atelopus (Bu- at 750-1.200 m above sea level (Sebben et al., fonidae) based on similarities in the pectoral 1986; Pombal et al., 1994; Giaretta et al., 1999). girdle (Griffths, 1959; Lynch, 1973). Accord- Some features that are considered synapo- ing to Giaretta and Sawaya (1998), the presence morphies of the family Brachycephalidae in- of the ﬁfth digit in Psyllophryne hermogenesie clude the absence of a sternum and pre-maxilla, (Brachycephalidae) decreases the differences between the Brachycephalidae and the genus

1 - Curso de Ciências Biológicas, Universidade São Fran- Euparkerella, currently included in the sub- cisco (USF), 12916-900 Bragança Paulista, SP, Brasil family Eleutherodactylinae (Leptodactylidae). Corresponding author’s e-mail: This conclusion reinforces the hypothesis of [email protected] a close phylogenetic relationship between the 2 - Instituto de Biologia, Universidade Federal de Uberlân- dia (UFU), 38400-902 Uberlândia, MG, Brasil Brachycephalidae and Leptodactylidae (Izeck- e-mail: [email protected] sohn, 1988). Based on the DNA sequences of 3 - Departamento de Biologia Celular, Instituto de Biolo- mitochondrial 12S and 16S, Darst and Canatella gia, Universidade Estadual de Campinas (UNICAMP), CP 6109, 13084-971 Campinas, SP, Brasil (2004) recently inferred that Brachycephalus is e-mail: [email protected] closely related to leptodactylids of the subfam-

Figure 1. Giemsa-stained (A), NOR-bearing chromosome pair in the inset (B) and C-banded (C) of Brachycephalus ephippium karyotype. Bar = 4 µm. Short Notes 123

Table 1. Morphometric data of mitotic chromosomes of B. ephippium. Classiﬁcation by Green and Session (1991).

Chromosome A.R. R.L. C.I. Type number

01 1,47 18,85 0,404 M 02 1,53 13,16 0,400 M 03 1,87 12,45 0,349 SM 04 4,83 10,71 0,175 ST 05 1,20 10,20 0,455 M 06 1,11 8,50 0,472 M Figure 2. Idiogram of the karyotype of Brachycephalus 07 4,29 7,02 0,190 ST ephippium. Solid blocks: dark C-bands. Open region: sec- 08 1,40 6,28 0,416 M ondary constriction. Gray blocks: NORs. 09 1,91 5,09 0,344 SM 10 1,21 4,32 0,457 M 11 1,17 3,33 0,463 M man and Trueb (1994) for B. ephippium.The karyotype of B. ephippium consisted of seven A.R. = arm ration, R.L. = relative length, C.I. = centromeric index, m = metacentric, sm = submetacentric, st pairs of metacentric chromosomes (1, 2, 5, 6, = subtelocentric. 8, 10 and 11), two submetacentrics (3 and 9) and two subtelocentrics (4 and 7) (figs 1A ily Eleutherodactylinae from Central America and 2). Although 22 chromosomes is the and Mexico but not to the Bufonidae. Together, most common number among species of Bufo these studies indicate that the phylogenetic re- (Ullerich, 1966; Beçak, 1968; Bogart, 1972; lationships of the Brachycephalidae with other Formas, 1978; Schmid, 1978, 1980; Birstein groups are still controversial. and Mazin, 1982; Kuramoto, 1990; Foote et al., The lack of cytogenetic data for the Brachy- 1991; Miura, 1995; Kasahara et al., 1996; Bald- cephalidae precludes a detailed comparison issera et al., 1999), and in Melanophryniscus with the Bufonidae, of which several species moreirae (De Lucca et al., 1974) and Atelo- have been analyzed cytogenetically. In this re- pus zeketi (Ramos et al., 2002), they differed port, we describe the karyotype of B. ephippium from B. ephippium in the morphology of some and compare it with those available for species chromosomes. Pairs 4 and 7 are subtelocentric of Bufonidae. in B. ephippium, but submetacentric in Bufo and Atelopus species. In addition, B. ephippium Ten males of B. ephippium were collected in the munic- ipality of Atibaia, São Paulo State, Brazil (ca. 23◦S, with showed a secondary constriction on the larger a precipitation of 1,400-1,700 mm). The specimens were arm of pair 8 (fig. 2), while in Bufo species, deposited in the Museu de História Natural “Prof. Adão the position varied considerably, occurring in José Cardoso” (ZUEC), at the State University of Camp- inas (UNICAMP), under the accession numbers 11476, pairs 1, 5, 6, 10 or 11 (Beçak, 1968; Bog- 11477 and 11479 to 11486. All of the frogs received an in- art, 1972; Formas, 1978; Schmid, 1978, 1980; traperitoneal injection (0,01 ml/g of body weight) of a 1% Miura, 1995; Kasahara et al., 1996; Baldissera colchicine solution 4-6 h before they were sacrificed. Mi- totic chromosomes were obtained from intestinal and testic- et al., 1999). ular cell suspensions, as described by Schmid (1982). Con- The NOR in B. ephippium was located on ventional staining with a 10% Giemsa solution was used the long arm of pair 8, coincident with the sec- to analyze the chromosomal morphology, and silver nitrate ondary constriction (figs 1B and 2). In Bufo labeling of the nucleolar organizer regions (Ag-NOR) was performed according to Howell and Black (1980). The C- species, the NOR is located on other chromo- banding pattern were obtained by the technique of King and somes, such as pair 1 in B. terrestris (Foote John (1980). The chromosome classification followed that et al., 1991) and B. americanus (Schmid, 1980), of Green and Sessions (1991). pair 6 in B. garmani (Schmid, 1980) and The diploid number of 22 chromosomes B. bufo (Birstein and Mazin, 1982), and pair 7 agreed with that previously reported by Duell- in B. marinus and B. arenarum (Schmid, 124 Short Notes

1980), B. crucifer, B. ictericus and B. paracne- Leptodactylidae, including some members of mis (Kasahara et al., 1996). In closely related the subfamily Eleutherodactylinae. Thus, based species, the dispersion of the NOR throughout on the cytogenetic data of B. ephippium and in the genome indicates that chromosomal re- on the data for Bufo, Melanophryniscus and arrangements involving the segment that car- Atelopus, it was not possible to establish a close ries the NOR occur frequently across an evo- relationship between the Brachycephalidae and lutionary time-span. The C-banding technique Bufonidae in this study. The cytogenetic analy- showed the association of heterochromatin with sis of other species of Brachycephalus, Psyl- the NOR (ﬁgs 1C and 2). All of the chromo- lophryne (Brachycephalidae) and Euparkerella somes had a large amount of centromeric het- (Leptodactylidae: Eleutherodactylinae) should erochromatin that, in almost all chromosomes, allow a more conclusive comparative analy- was detected as an interstitial band adjacent to sis of the relationship among these fami- the cetromere. A small band of euchromatin lies. was present between the centromeric and interstitial heterochromatin (ﬁgs 1C and 2). The amount of heterochromatin in the B. ephip- References pium genome was clearly greater than in some Baldissera Jr., F.A., Batistic, R.F., Haddad, C.F.B. (1999): species of Bufo (Schmid, 1978, 1980; Kasahara Cytotaxonomic considerations with the description of et al., 1996; Baldissera et al., 1999). However, two new NOR locations for South American toads, in B. japonicus (Miura, 1995) there are also genus Bufo (Anura: Bufonidae). Amphibia-Reptilia 20: 413-420. interstitial bands, including a marked quantity Beçak, M.L. (1968): Chromosomal analysis of eighteen of juxta-centromeric heterochromatin. Accord- species of Anura. Caryologia 21: 191-208. ing to King (1991), an increase in the hete- Birstein, V.J., Mazin, A.L. (1982): Chromosomal polymorphism of Bufo bufo: karyotype and C-banding pattern of rochromatin of the genome probably represents B. b. verrucosissima. Genetica 59: 93-98. a derived characteristic in amphibians. How- Bogart, J.P. (1972): Karyotypes. In: Evolution in the Genus ever, this characteristic is not suitable for as- Bufo, pp. 171-195. Blair, W.F., Ed., Austin Texas, Uni- sessing the proximity between Brachycephalus versity of Texas Press. Cochran, D.M. (1955): Frogs of southeastern Brazil. Bull. and Bufo since species closely related to each U.S. Nat. Mus. 206, XVI + 423 pp. other from the same genus and, with similar Darst, C.R., Cannatella, D.C. (2004): Novel relationships karyotypes may show a marked difference in among hyloid frogs inferred from 12S and 16S mitochondrial DNA sequences. Mol. Phylogen. Evol. 31: the amount of heterochromatin. This has al- 462-475. ready been shown for Pleurodema, in which the De Lucca, E.J., Jim, J., Foresti, F. (1974): Chromosomal amount of heterochromatin in P. brachyops is studies in twelve species of Leptodactylidae and one almost six-fold greater than in P. thaul (Schmid Brachycephalidae. Caryologia 27: 183-192. Duellman, W.E., Trueb, L. (1994): Biology of Amphibians. et al., 1993) and for Mixophyes fasciolatus and McGraw-Hill Book Company, New York. M. schevilli (Schmid et al., 2002). Foote, D.L., Wiley, J.E., Little, M.L., Meyne, J. (1991): Chromosomal rearrangements and an in- Ribosomal RNA genes site polymorphism in Bufo terrestris. Cytogenet. Cell Genet. 57: 196-199. crease in the amount of heterochromatin may Formas, J.R. (1978): The chromosomes of Bufo rubropunc- have contributed to the differentiation of tatus and Bufo chilensis (Anura, Bufonidae) and other B. ephippium. With the exception of the chro- species of the spinulosus group. Experientia 34: 452- 454. mosome number (2n = 22) and the morpho- Frost, D.R. (2002): Amphibian species of the world: an on- logical similarity of some chromosomes, we line reference. V2.21 (15 July 2002). Electronic data- have found no irrefutable markers to indi- base available at http://research.amnh.org/herpetology/ cate a close relationship between B. ephippium amphibia/index.html. Giaretta, A.A., Sawaya, R.J. (1998): Second species of and the Bufonidae. The chromosomal number Psyllophryne (Anura: Brachycephalidae). Copeia 1998: of 2n = 22 also occurs in several species of 985-987. Short Notes 125

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