Journal of Herpetology, Vol. 37, No. 1, pp. 161±168, 2003 Copyright 2003 Society for the Study of Amphibians and Reptiles Mitochondrial Restriction-Site Characterization of a Brazilian Group of Eyelid-Less Gymnophthalmid Lizards M. L. BENOZZATI1,2 AND M. T. RODRIGUES3 1Departamento de Biologia, Instituto de BiocieÃncias, Universidade de SaÄo Paulo, SaÄo Paulo, Brasil 3Departamento de Zoologia, Instituto de BiocieÃncias, and Museu de Zoologia, Universidade de SaÄo Paulo, SaÄo Paulo, Brasil ABSTRACT.ÐPhylogenetic relationships among eight Brazilian genera of the lizard family Gymnophthal- midae were analyzed using mitochondrial DNA restriction-site data. The intergeneric relationships inferred from a cladistic analysis of 32 restriction sites were widely congruent with a recent phylogenetic analysis based on morphology. Assuming a molecular clock of 2% MY, these gymnophthalmid lizards may have diverged from a common ancestor some 7±8 MYA. The monophyly of the ®ve eyelid-less microteiid genera from the Rio SaÄo Francisco Quaternary sand dunes was also supported by mitochondrial data, and the time estimated for this radiation was about 2±3 MYA. The mtDNA data also suggest that the unisexual population of Gymnophthalmus underwoodi from Roraima is not originated by hybridization between its two closely related sympatric bisexual species Gymnophthalmus leucomystax and Gymnopohthalmus vanzoi, in accor- dance with previous biochemical and karyotypic data. The exclusively Neotropical Teiioidea com- monophyletic based on their marked tendencies prises two families: the Teiidae and the Gym- for forelimb reduction and lack of eyelids. Tre- nophthalmidae (Estes et al., 1988). It includes 36 tioscincus is the only genus of the group having genera and about 170 species of small lizards eyelids, and Micrablepharus the oldest most di- that live in leaf litter or ground vegetation of vergent taxon, diagnosed by the absence of eye- forests and open areas, in rocky habitats, or are lids and a rudimentary, or absence of the ®rst semiaquatic. Although phylogenetic relation- ®nger. Gymnophthalmus, including the unisexual ships within Gymnophthalmidae are still poor- Gymnophthalmus underwoodi, was considered by ly known, limb reduction seems to have origi- Rodrigues (1991c) a more derived genus, fol- nated several times independently in the family lowed by the other ®ve genera found on SaÄo (Presch, 1980; Hoyos, 1998). One of the limb- Francisco dunes. A more extensive analysis con- reduced gymnophthalmid groups shows a ten- sidering 71 osteological and morphological dency toward a pronounced reduction of fore- characters of these lizards (Rodrigues, 1995) limbs in relation to hind limbs, and eyelid loss, corroborated its monophyly and showed Procel- among other presumable fossorial adaptations losaurinus and Vanzosaura as sister groups. The (Rodrigues, 1991a). Five new genera from the topology obtained from those data was (Tretios- continental dunes on both banks of the middle cincus (Micrablepharus (Gymnophthalmus ((Procel- Rio SaÄo Francisco, in the morphoclimatic do- losaurinus, Vanzosaura)(Psilophthalmus (Nothoba- main of the semiarid Brazilian Caatinga, have chia, Calyptommatus)))))). It was proposed that been recently described by Rodrigues: Nothoba- the radiation of the group on the continental chia (1984), Calyptommatus (1991a), Psilophthal- dunes derived from a Procellosaurinus-Vanzosau- mus (1991b), Procellosaurinus and Vanzosaura ra Amazonian ancestor. In a period previous to (1991c). Among these new genera, Vanzosaura the origin of psammophily and fossorial adap- was the only one described to reallocate the for- tations, the fauna of the middle Rio SaÄo Fran- mer Gymnophthalmus rubricauda and its synonym cisco lived in typical caatinga depressions with Gymnophthalmus multiscutatus, whereas the other inselbergs and isolated hills (Rodrigues, 1993). four genera were described for new species. As proposed by Ab'Saber (1969) and Tricart Gymnophthalmus, Micrablepharus,andTretioscin- (1974), the river drained into a continental lake cus were previously known genera related to until the end of the last glacial period, when it this radiation. Rodrigues (1991c) proposed that connected to the sea. In humid periods during these eight genera of microteiid lizards are the phase of interior drainage, sand accumulat- ed in lacustrine depressions, isolating elements 2 Corresponding Author. Present address: Departa- of the fauna in disjunct hills. In the onset of a mento de Biologia, Instituto de BiocieÃncias, Universi- semiarid climate, those hills were vanishing fau- dade de SaÄo Paulo, Rua do MataÄo, 277, Cidade Univer- nal refuges in a sandy desert, and species with sitaÂria, SaÄ o Paulo, SaÄo Paulo, Brasil, CEP: 05508-090; preadaptations for life on sand would have ex- E-mail: [email protected] panded on the recent dune ®elds. Present 162 M. L. BENOZZATI AND M. T. RODRIGUES TABLE 1. Sample size and location of the analyzed gymnophthalmid lizard populations and the outgroup Colobosaura modesta. Species Location Sample size Calyptommatus leiolepis Queimadas (Bahia) 6 Gymnophthalmus leucomystax Fazenda Salvamento (Roraima) 2 Gymnophthalmus vanzoi Fazenda Salvamento (Roraima) 2 Gymnophthalmus underwoodi Fazenda Salvamento (Roraima) 2 Ilha de Maraca (Roraima) 2 Microblepharus maximiliani Sta. Rita do Araguaia (GoiaÂs) 3 Nothobachia ablephara Alagoado (Bahia) 5 Procellosaurinus erythrocercus Queimadas (Bahia) 3 Psilophthalmus paeminosus Sto. InaÂcio (Bahia) 5 Tretioscincus agilis PocËaÄo (ParaÂ) 2 Vanzosaura rubricauda Vacaria (Bahia) 5 Colobosaura modesta Alto Araguaia (Mato Grosso) 1 Barra do GarcËas (Mato Grosso) 1 knowledge of microteiid phylogeny (Rodrigues, men of Iphisa elegans from Reserva FaunõÂstica 1995) suggests that Procellosaurinus, Vanzosaura, Cuyabeno, Sucumbios Province, Ecuador Psilophthalmus,andNothobachia were already (AMNH-LJV5189) was also initially included as present in those sand dunes at the time the an- outgroup, but it was discontinued because sin- cestors of the most derived genus of this radi- gle-site pro®les were detected for most of the ation, Calyptommatus, were isolated by the river, endonucleases assayed, and trials to precisely separating populations in opposite margins of map these sites failed with the available DNA the Rio SaÄo Francisco. sample. In this paper, mitochondrial DNA restriction- DNA was extracted from ®eld-collected spec- site pro®les were used to construct a molecular imens stored at 21968C, following Hillis and phylogeny for these gymnophthalmids and to Davis (1986) and Dowling et al. (1990), modi®ed estimate divergences among different genera. by Passoni et al. (2000). The enzymes used in We evaluate the congruence of these molecular the analyses were BamH I, EcoR I, Hind III, Pvu data with the phylogenies based on morpholog- II and Xba I. Additional 6bp (Ava I, BstE II, Dra ical and allozymic analyses. The unisexual G. I, EcoR V, Kpn I, Pst I, and Sal I) and 4bp-rec- underwoodi from the Amazonian state of Rorai- ognizing endonucleases (Hinf I, HinP1 I and ma and its sympatric bisexual species Gymno- Rsa I) were used in initial screening but were phthalmus leucomystax and Gymnophthalmus van- discontinued because they failed to cut the DNA zoi were also analyzed, and the origin of the samples consistently or produced restriction- parthenogenetic lineage is discussed. site pro®les of ambiguous interpretations. Hind MATERIALS AND METHODS III lDNA digests and pBR 328 digested with BamH I, Bgl I and Hinf I were used as molec- Intrageneric relationships were not consid- ular size standards, with fragment sizes, in base ered in the present mtDNA study, as they were not considered in the previous morphological pairs, of 23,130; 9,416; 6,557; 4,907; 4,361; 2,322; analyses (Rodrigues, 1995). Only one species of 2,176; 2,023; 1,766; 1,230 and 1,033. Restriction each eyelid-less gymnophthalmid genus was as- fragments were transferred from 0.8% agarose sayed for mtDNA phylogenetic purposes: No- gels to nylon membranes (``Nylon Membrane thobachia, Psilophthalmus,andVanzosaura are Positive Charged,'' Boehringer Mannheim) and monospeci®c genera, Micrablepharus and Procel- probed with heterologous mtDNA obtained losaurinus are composed of two species, and Ca- from human placenta (Hauswirth et al., 1987) lyptommatus and Gymnophthalmus each has more and labeled with digoxigenin (``DIG DNA la- than three species. Three species of Gymno- beling and detection kit,'' Boehringer Mann- phthalmus collected in Roraima were sampled, heim). the unisexual species G. underwoodi and its sym- Each taxon was assigned a composite mtDNA patric bisexual species G. leucomystax and G. van- genotype, compiled from the restriction site zoi, to investigate a putative maternal ancestor pro®les. Site data were assembled in a pres- of the parthenogenetic lineage. Two specimens ence/absence matrix, from which sequence di- of Colobosaura modesta were assayed as an out- vergence indices (p) were estimated by the group. Sample sizes and locations of the ana- ``site'' approach of Nei and Li (1979). Taxa were lyzed species are indicated in Table 1, and their clustered by neighbor-joining (Saitou and Nei, geographic distribution in Figure 1. One speci- 1987) and by parsimony using the computer MOLECULAR CHARACTERIZATION OF GYMNOPHTHALMID LIZARDS 163 FIG. 1. Map of the localities sampled: 1, Ilha de Maraca (Roraima); 2, Fazenda Salvamento (Roraima); 3, PocËaÄo (ParaÂ); 4, Barra do GarcËas (Mato Grosso); 5, Alto Araguaia (Mato Grosso); 6, Santa