Animal Cytogenetics and Comparative Mapping Cytogenet Genome Res 103:128–134 (2003) DOI: 10.1159/000076300 Chromosomal studies on sphaerodactyl lizards of genera Gonatodes and Coleodactylus (Squamata, Gekkonidae) using differential staining and fragile sites analyses R.M.L. dos Santos,a C.E.V. Bertolotto,a,b K.C.M. Pellegrino,c M.T. Rodriguesd and Y. Yonenaga-Yassudaa a Departamento de Biologia, Universidade de Sa˜o Paulo, Sa˜o Paulo; b Faculdade de Medicina Veterina´ria, Universidade Santo Amaro, Sa˜o Paulo; c Departamento de Biomedicina, Universidade Cato´ lica de Goia´s, Goiânia; d Departamento de Zoologia, Universidade de Sa˜o Paulo, Sa˜o Paulo (Brazil) Abstract. The karyotypes of three species of sphaerodactyl NORs. Fragile sites were detected on two medium-sized chro- gekkonid lizards are described after conventional and differ- mosome pairs in the karyotype of G. humeralis, most of them ential staining. Karyotypes of Gonatodes humeralis and G. obtained in BrdU-treated culture preparations. These sites may hasemani are formed by a gradual series of 32 acrocentric chro- represent a putative fission/fusion spot involved in the differ- mosomes, similar to those already published for other species entiation of G. humeralis-like 2n = 32 and C. amazonicus-like of the genus. G. humeralis shows multiple Ag-NORs with 2n = 36 karyotypes. Our results, especially on the location of intra-individual variability, and positive C-bands located at Ag-NORs and the description of fragile sites, are relevant in centromeric and telomeric regions of several chromosome improving our knowledge about the events of chromosome pairs. Coleodactylus amazonicus, the first non-Gonatodes evolution in this extremely variable and poorly known group of sphaerodactyl studied so far karyologically, exhibits 36 acro- lizards. centric/subtelocentric chromosomes and a single pair of Ag- Copyright © 2003 S. Karger AG, Basel The geckos comprise about 1,000 species of lizards distrib- konidae: the widespread speciose and nocturnal gekkonins, and uted in approximately 110 genera. The systematics of Gekkoni- the sphaerodactyls. Differently to the heterogeneous gekkonins, dae is in a permanent state of change and we follow the recent sphaerodactyl geckos are diurnal and restricted to Central and classification of Zug et al. (2001), that recognized four subfam- South America and some Caribbean islands. This monophylet- ilies: Diplodactylinae, Eublepharinae, Gekkoninae and Pygo- ic assemblage of about 150 recognized species is distributed podinae. Apart from the other subfamilies, which are relatively into five genera (Coleodactylus, Gonatodes, Lepidoblepharis, homogeneous, there are two distinctive assemblages in Gek- Pseudogonatodes and Sphaerodactylus) with unresolved phylo- koninae, sometimes recognized as different subfamilies of Gek- genetic relationships (Vanzolini, 1968; Huey and Dixon, 1970; Hoogmoed, 1985; Kluge, 1995). Even though these studies dif- fer considerably in the placement of some taxa, they do agree that Gonatodes and Coleodactylus are basal and terminal groups, respectively. Supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´ gico (CNPq) and Fundaça˜o de Amparo à Pesquisa do Estado de Sa˜o Paulo (FA- In general, the geckos are chromosomally poorly character- PESP). ized and less than 5% of the extant forms were studied (Olmo, Received 12 May 2003; revision accepted 29 July 2003. 1986). Up to now, only five species of sphaerodactyl geckos, all Request reprints from Msc Rodrigo Marques Lima dos Santos from the genus Gonatodes, were karyotyped: G. taniae, 2n = 16 Departamento de Biologia, Universidade de Sa˜o Paulo (Schmid et al., 1994), G. ceciliae, 2n = 22 and 2n = 26 (Mc Bee Sa˜o Paulo, SP, C.P. 11.461, CEP 05422-970 (Brazil) telephone: +55 11 3091 7574; fax: +55 11 3091 7553 et al., 1987), G. humeralis, 2n = 32 (Mc Bee et al., 1984), G. e-mail: [email protected] vittatus, 2n = 32 (Rada De Martı´nez, 1980; Mc Bee et al., 1987; Fax + 41 61 306 12 34 © 2003 S. Karger AG, Basel Accessible online at: ABC E-mail [email protected] 0301–0171/03/1032–0128$19.50/0 www.karger.com/cgr www.karger.com Schmid et al., 1994) and a still undescribed species referred to Here we describe for the first time the Ag-NORs, C- and as G. sp n., 2n = 32 and 40 (Mc Bee et al., 1984). In these stud- RBG-banding patterns for Coleodactylus amazonicus and Go- ies, differential staining (C-banding, fluorescent staining and natodes humeralis, and the conventional karyotype of G. hase- FISH) is only presented for G. taniae. This species has an mani. We emphasize that this is the first study involving karyo- exceptionally low diploid number (2n = 16), the lowest known type description of a non-Gonatodes species, Ag-NOR location for reptiles, which is suggested to be due to a series of centric in sphaerodactyl geckos and the report of fragile sites on liz- fusions from an acrocentric 2n = 32 ancestral karyotype, simi- ards. lar to that found in other species of Gonatodes (Schmid et al., 1994). The variation in diploid number found in the limited num- Materials and methods ber of species that have been studied so far, suggests that the Specimens of Coleodactylus amazonicus, Gonatodes hasemani and G. genus Gonatodes represents a suitable group to investigate cur- humeralis were cytogenetically studied (Table 1) and deposited at the Museu rent models of chromosome evolution. In this scenario, the de Zoologia of the Universidade de Sa˜o Paulo (MZUSP), State of Sa˜o Paulo, analysis of additional sphaerodactyl lizards, using banding Brazil. techniques, would allow identification of rearrangements that Mitotic metaphases were obtained from bone marrow, spleen, intestine took place during the chromosomal evolution within the genus, and liver after in vivo treatment of animals with colchicine according to rou- tine techniques (Kasahara et al., 1987), or from fibroblast cultures of tail and should provide some relevant phylogenetic information muscle (Yonenaga-Yassuda et al., 1988). Meiotic analyses were also per- about this poorly studied group. formed on male specimens. The diploid chromosome number and morphology were established after conventional staining. Mitotic chromosomes were analyzed after CBG and Ag-NOR staining following routine protocols. RBG-banding was obtained after in vitro treatment of cells with 25 Ìg/ml 5-bromodeoxyuridine (5- Table 1. Summary of the information on sampling of sphaerodactyl liz- BrdU) for 16 h, followed by FPG staining (Dutrillaux and Couturier, 1981). ards a b Species Specimen number Sex Locality Results C. amazonicus LG 742 F Aripuanã (MT) (10º10'S, 59º27'W) Coleodactylus amazonicus (2n = 36) G. hasemani LG 1489 M Aripuanã (MT) The karyotype consists of at least two subtelocentric chro- LG 1490 F (10º10'S, 59º27'W) mosome pairs (pairs 1 and 2) and 16 acrocentric pairs, in a total G. humeralis LG 400 M Tucuruí (PA) of 80 metaphases analyzed (Table 2). No heteromorphic chro- (03º45'S, 49º40'W) mosomes were detected (Fig. 1a). The Ag-NORs were located LG 754 E Vai Quem Quer (PA) at the distal region of the long arm of a medium-sized chromo- (01º30'S, 55º50'W) some pair (Fig. 2a). RBG-banding pattern was obtained from LG 822 F São João da Baliza (RR) LG 824 M (00º57'S, 59º54'W) cultured cells, and allowed us to identify precisely pairs 1 to 7 LG 828 M and some small-sized pairs (Fig. 3a). LG 1488 F Aripuanã (MT) (10º10'S, 59º27'W) Gonatodes hasemani (2n = 32) This species presents a karyotype with a gradual series of 32 Total 9 4F, 4M, 1E 4 acrocentric chromosomes (Fig. 1b), without heteromorphic a M = male; F = female; E = embryo. chromosomes, in at least 20 metaphases analyzed per speci- b MT = state of Mato Grosso; PA = state of Pará; RR = state of Roraima. men. Table 2. Number of chromosomes bearing fragile sites and Ag-NORs in different chromosome preparations of Coleodactylus amazonicus and Gonatodes humeralis Species Specimen Chromosome Number of chromosomes with Number of Ag-NORs preparation fragile sites 0 1–2 3 4 Total No. 1 2 3 4 5 6 7 8 Total No. of cells of cells Coleodactylus amazonicus LG 742 Standard Culture 50 0 0 0 50 6 40 0 0 0 0 0 0 46 Culture + BrdU 30 0 0 0 30 Gonatodes humeralis LG 754 In vivo 41 0 0 0 41 Standard Culture 21 4 0 0 25 6 7 5 7 4 5 2 0 36 Culture + BrdU 13 9 4 4 30 LG 822 In vivo 40 2 0 0 42 Standard Culture 14 1 3 2 20 2 3 5 9 6 7 4 4 40 Culture + BrdU 9 4 6 11 30 Cytogenet Genome Res 103:128–134 (2003) 129 Fig. 1. Conventionally stained karyotypes of (a) Coleodactylus amazonicus, female 2n = 36, (b) Gonatodes hasemani, female 2n = 32 and (c) G. humeralis, male 2n = 32. Bar = 10 Ìm. 130 Cytogenet Genome Res 103:128–134 (2003) Fig. 2. Ag-NORs in Coleodactylus and Gonatodes. (a) Metaphase of C. amazonicus with Ag-NOR in a medium-sized chromo- some pair (arrows). (b) Intra-individual variation in number of Ag-NORs (8 to 6) in three different metaphases of G. humeralis. Fig. 3. R-banding patterns. (a) C. amazonicus, 2n = 36. (b) G. humeralis, 2n = 32. (c) Fragile sites in the pairs 4 and 5 from another BrdU culture of G. humeralis. Cytogenet Genome Res 103:128–134 (2003) 131 Fig. 4. C-banding pattern of G. humeralis. Note the centromeric and telomeric C-bands in some chromosome pairs. Gonatodes humeralis (2n = 32) (1977) suggested it as ancestral for all subfamilies of Gekkoni- The G. humeralis karyotype is very similar to that of G. dae, and considered centric fusions as the major mechanism of hasemani, with also 32 acrocentric chromosomes (Fig. 1c), karyotypic evolution in lizards.