Of Genera Gonatodes and Coleodactylus (Squamata, Gekkonidae) Using Differential Staining and Fragile Sites Analyses

Home , Coleodactylus, Diplodactylus vittatus, Gonatodes, Phyllodactylus ventralis

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 Saõ Paulo, Saõ Paulo; b Faculdade de Medicina Veterina´ria, Universidade Santo Amaro, Saõ Paulo; c Departamento de Biomedicina, Universidade Cato´ lica de Goia´s, Goiânia; d Departamento de Zoologia, Universidade de Saõ Paulo, Saõ 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 differ 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 Fundaçaõ de Amparo à Pesquisa do Estado de Saõ 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 Saõ Paulo (Schmid et al., 1994), G. ceciliae, 2n = 22 and 2n = 26 (Mc Bee Saõ 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 Martıńez, 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 routine 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 chromosome 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. counted in at least 20 metaphases analyzed per specimen. In However, the putative ancestral 2n = 38 karyotype has not some metaphases from in vivo preparations and fibroblast cul- been described for sphaerodactyls. Based on the presence of a tures of two specimens (LG 754 and LG 822) one to four 2n = 32 karyotype in G. vittatus, G. humeralis and G. sp.n., and medium-sized chromosomes probably from pairs 4 and 5 pre- in the outgroups Phyllodactylus ventralis and P. marmoratus sented a proximal secondary constriction (Fig. 3c). The R × C (Gekkoninae), and Coleonyx variegatus (Eublepharinae), Mc test of independence using G-test indicated that frequencies of Bee et al. (1987) implied this karyotype as ancestral for sphae- these constrictions increase significantly (df = 6, G = 26.9601, rodactyls. Moreover, the authors conclude that sphaerodactyl P ! 0.001 for LG 754 and df = 6, G = 32.4491, P ! 0.001 for LG geckos must be considered as a distinct family within the infra- 822) depending on growth conditions of the cells (in vivo, stan- order Gekkota. dard fibroblast culture, and fibroblast culture with 5-BrdU; The 2n = 36 karyotype described for Coleodactylus amazo- Table 2), and might represent fragile sites. nicus in the present paper indicates that considerations on the An intra-individual variation in number of Ag-NORs (1–8; ancestral karyotype for Sphaerodactylinae are quite specula- Table 2) located at the telomeric region of the long arms was tive, until we have a better karyological coverage and an explic- detected (Fig. 2b). All chromosomes also presented faintly it phylogenetic framework for this lizard assemblage as well as stained heterochromatic blocks predominantly at the centro- for their close relatives. meric regions; some showed also faint telomeric C-bands The secondary constrictions at the proximal region of chro- (Fig. 4). The RBG-banding patterns allowed us to characterize mosome pairs 4 and 5, observed in metaphases from in vivo all pairs of G. humeralis (Fig. 3b). and in vitro (fibroblast cultures) preparations of G. humeralis, might represent fragile sites. As defined by Sutherland (1979), fragile sites are heritable loci, expressed as gaps or breaks when Discussion cells are exposed to specific culture conditions or certain chem- ical agents, as bromodeoxyuridine, reported as fragile sites Gonatodes humeralis and G. hasemani share a common inducer in humans (Sutherland et al., 1985), cattle (Di Berardi- karyotype with a gradual series of 32 acrocentric chromosomes, no et al., 1983), canine (Stone and Stephens, 1993), buffalo also observed in some other species of Gonatodes. Heteromor- (Mahrous and Ahmed, 2000) and bat (Faria and Morielle-Ver- phic sex chromosomes were not detected. Among geckos there sute, 2002). Common fragile sites, which are probably homozy- are few examples of sexual determination mechanisms with gous in all individuals, are characteristic of the normal struc- heteromorphic sex chromosomes, including female (ZZ:ZW) ture of the chromosome (Sutherland and Richards, 1999) but and male (XX:XY) heterogamety (Moritz, 1990). In sphaero- unfamiliar in respect to the molecular structure and the func- dactyl geckos, an XX:XY sex determination mechanism was tional significance (Ruiz-Herrera et al., 2002). only detected in G. ceciliae, which consists on a large biarmed In a recent study, Ruiz-Herrera et al. (2002) showed that the X chromosome and a small acrocentric Y (Mc Bee et al., breakpoints found at the chromosomes of the primate Macaca 1987). fascicularis, which are implicated in the evolutionary rearran- Based on the widespread occurrence of a 2n = 38 karyotype gements that would have led to human chromosomes, are pref- in geckos with exclusively acrocentric chromosomes, King erentially located at fragile sites. Consequently, the authors

132 Cytogenet Genome Res 103:128–134 (2003) consider the fragile sites as targets for evolutionary rearrange- nyx variegatus (Porter et al., 1994), Hemidactylus mabouia ments because of their special tendency to break and reorgan- (Pellegrino et al., 1995) and Heteronotia binoei (Hillis et al., ize, mostly during intrachromosomal rearrangements, such as 1991). Therefore, the differential distribution of Ag-NORs inversions and centromeric shifts. These results agree with pre- between G. humeralis (multiple Ag-NORs) and C. amazonicus vious studies that show conservation of fragile sites in chromo- (single Ag-NOR) could represent an additional phylogenetic somes of related species of rodents (Djalali et al., 1987) and character within this group. Localization of NORs by fluores- primates (Yunis and Soreng, 1984; Schmid et al., 1985; Smeets cence in situ hybridization (FISH) with rDNA probes in other and Klundert, 1990); some of them were indeed related to rear- Gekkonidae and G. humeralis is necessary to corroborate these rangements that took place during chromosome evolution assumptions. (Miro´ et al., 1987; Fundia et al., 2000). Localization of Ag-NORs and description of fragile sites The occurrence of fragile sites at two chromosome pairs of could improve our knowledge about the events of chromosome G. humeralis (2n = 32) cannot be discarded as related to the evolution in this extremely variable and poorly known group of process of karyotypic differentiation from a C. amazonicus-like lizards. Moreover, the RBG-banding description could reveal 2n = 36 karyotype, where these sites might represent possible which chromosome pairs would be involved in the complex fission/fusion spots involving the disappearance or de novo for- process of 16 centric fusions which likely occurred in the evolu- mation of centromeres. Unfortunately, the lack of additional tion of the karyotype of G. taniae. It is important to point out cytogenetic data for other sphaerodactyls precludes us from that the homologies established between G. taniae (2n = 16) suggesting any robust hypothesis. The RBG-pattern presented and G. vittatus (2n = 32) by Schmid et al. (1994) were only here, although permitting precise identification of most pairs, based on chromosomal measurements after conventional stain- does not allow further comparison of the karyotypes of G. ing and FISH of telomere probes. These techniques are insuffi- humeralis and C. amazonicus. Nevertheless, the occurrence of cient for the definition of karyotypic differentiation in Gona- fragile sites at the chromosomes of G. humeralis reinforces the todes. The occurrence of interstitial telomeric sequences (ITS) idea that sphaerodactyl lizards are a suitable group with excel- has been subject of many recent studies in a variety of verte- lent possibilities to study current models of chromosome evolu- brate species, including lizards (Pellegrino et al., 1999; Berto- tion as proposed by many authors. lotto et al., 2001) and the meaning of this atypical location of Data on NOR locations in lizards are rare, although they telomere sequences remains unclear. provide systematic characters relevant to elucidate the phylogenetic relationships in a range of reptiles (Porter et al., 1991, 1994). In Gekkonidae, multiple Ag-NORs have been described Acknowledgements in Phyllopezus (Pellegrino et al., 1997) and Gehyra (Moritz, The authors are grateful to Vinı´cius Xavier da Silva, Gabriel Skuk, Dante 1986) and are presented here for G. humeralis. Otherwise, Pavan, José Manuel Martins, Celso Morato de Carvalho and Maria J. J. Silva NORs are located at the telomeric region of a single medium- for their help in field, Fabiana E. Casarin for statistical support, and to Dr. sized pair as here described for C. amazonicus and for Coleo- Tien Hsi Chu and Mrs. Miriam Romeo for technical assistance.

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