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 Biocieˆncias, Universidade de Saõ Paulo, Saõ Paulo, Brasil 3Departamento de Zoologia, Instituto de Biocieˆncias, and Museu de Zoologia, Universidade de Saõ Paulo, Saõ Paulo, Brasil

ABSTRACT.—Phylogenetic 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 ﬁve eyelid-less microteiid genera from the Rio Sa˜o 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 accordance 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 first semiaquatic. Although phylogenetic relation- finger. 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 five genera found on Saõ (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 Saõ 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 Saõ 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 Biocieˆncias, Universi- semiarid climate, those hills were vanishing fau- dade de Saõ Paulo, Rua do Mataõ, 277, Cidade Univer- nal refuges in a sandy desert, and species with sita´ria, Sa˜ o Paulo, Saõ Paulo, Brasil, CEP: 05508-090; preadaptations for life on sand would have ex- E-mail: [email protected] panded on the recent dune fields. 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 (Goia´s) 3 Nothobachia ablephara Alagoado (Bahia) 5 Procellosaurinus erythrocercus Queimadas (Bahia) 3 Psilophthalmus paeminosus Sto. Inaćio (Bahia) 5 Tretioscincus agilis Poçaõ (Para´) 2 Vanzosaura rubricauda Vacaria (Bahia) 5 Colobosaura modesta Alto Araguaia (Mato Grosso) 1 Barra do Garças (Mato Grosso) 1 knowledge of microteiid phylogeny (Rodrigues, men of Iphisa elegans from Reserva Faunı´stica 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 profiles 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 Saõ Francisco. sample. In this paper, mitochondrial DNA restriction- DNA was extracted from field-collected spec- site profiles were used to construct a molecular imens stored at Ϫ196ЊC, following Hillis and phylogeny for these gymnophthalmids and to Davis (1986) and Dowling et al. (1990), modified 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 profiles of ambiguous interpretations. Hind MATERIALS AND METHODS III ␭DNA 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 monospecific 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 profiles. 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, Poçaõ (Para´); 4, Barra do Garças (Mato Grosso); 5, Alto Araguaia (Mato Grosso); 6, Santa Rita do Araguaia (Goia´s); 7, Alagoado (Bahia); 8, Queimadas (Bahia); 9, Vacaria (Bahia); 10, Santo Inaćio (Bahia). In detail, the paleoquaternary dune fields on the middle Rio Saõ Francisco (Bahia)—the stippled area corresponds to the river flood plain. 164 M. L. BENOZZATI AND M. T. RODRIGUES

striction profiles could be accounted for by changes involving single site losses or gains. Re- striction sites of difficult identification were mapped by analysis of double digests from pairs of the employed enzymes. Origins of the Unisexual Gymnophthalmus underwoodi.—In Gymnophthalmus, an identical site profile was detected between the two specimens of the unisexual species G. underwoodi from Ilha do Maraca´ and the two specimens from Fazenda Salvamento (Roraima). This site profile was compared to those of the bisexual congeners G. leucomystax and G. vanzoi from Fazenda Salva- mento (Roraima). Mitochondrial DNA infor- mation about a possible hybrid origin of the unisexual species was obtained from 23 restriction sites that were unequivocally characterized in those three species of Gymnophthalmus. Remark- able differences were found among them, as FIG. 2. Mitochondrial DNA profiles of Xba I (A) shown in Figure 2 for Pvu II and Xba I restric- and Pvu II (B) endonucleases from eyelid-less gym- tion-site profiles. Nucleotide divergences were nophthalmid lizards: Micrablepharus maximiliani (1); estimated between G. underwoodi and G. leuco- the unisexual species Gymnophthalmus underwoodi (2); mystax (P ϭ 0.0677), G. underwoodi and G. vanzoi the bisexual species Gymnophthalmus leucomystax (3); the bisexual Gymnophthalmus vanzoi (4); and Procello- (P ϭ 0.1012), and between G. leucomystax and G. saurinus erythrocercus (5). [S-molecular weight stan- vanzoi (P ϭ 0.1223). These divergence values are dard]. similar to some estimated for pairs of microteiid genera (see Table 3). Phylogeny.—A presence/absence matrix with program PAUP (vers. 4.0; D. L. Swofford, Sin- the haplotypes of the eight microteiid genera auer Associates, Sunderland, MA, 1999). Boot- was constructed (Table 2), and nucleotide diver- strapping (Felsenstein, 1985) was applied to es- gences were calculated (Table 3). From Gymno- timate confidence in monophyletic groups. phthalmus, the same bisexual species considered MacClade software (vers. 3.03, W. P. Maddison in the previous morphological study (Ro- and D. R. Maddison, Sinauer Associates, Sun- drigues, 1995), G. leucomystax, was included in derland, MA, 1992) was used in the analysis of the analyses. In a phylogenetic assay using the character state changes. A sequence divergence Exhaustive Search option of PAUP, a single most of 2% per million years was considered in a pre- parsimonious tree was obtained, with 54 steps liminary estimation of divergence times be- and a consistency index of 0.56 (Fig. 3A). The tween haplotypes (Brown et al., 1979). five most derived genera according to Ro- drigues (1995) were grouped in this cladogram RESULTS by one unambiguous restriction-site gain and Enzyme assays of DNA samples from the one loss. Procellosaurinus and Vanzosaura were eight genera of the studied gymnophthalmids grouped as sister taxa by four site gains, which revealed a total of 32 sites with the endonucle- increases the confidence of the phylogenies ases BamH I, EcoR I, Hind III, Pvu II and Xba (Templeton, 1983, 1987). Gymnophthalmus was I. No within-species variation was detected in the most divergent genus, with six autapomor- the analyzed samples, and most of different re- phies, four of which were site gains. In the con-

TABLE 2. Presence/absence matrix of restriction sites of BamHI,EcoRI,Hind III, Pvu II, and Xba I endonucleases. Haplotypes are designated by genus names (1—presence; 0—absence).

Colobosaura 10101100001001110100101000001010 Tretioscincus 10000100100001110100000000001000 Micrablepharus 10110100010101110100110010000000 Gymnophthalmus 10111101000000000000100101010110 Procellosaurinus 11101111000110110111110000000001 Vanzosaura 11110100100010010111111000011001 Psilophthalmus 11011100000100110001110100011000 Nothobachia 11011110000110010000110000011000 Calyptommatus 00001110000110111000110000010100 MOLECULAR CHARACTERIZATION OF GYMNOPHTHALMID LIZARDS 165

TABLE 3. Estimates of nucleotide sequence divergence (p) for the eight Gymnophthalmidae genera and the outgroup Colobosaura from mitochondrial DNA restriction-site haplotypes.

Colob Tretio Micra Gymno Procel Vanzo Psilo Notho Calyp Colob — Tret io 0.0676 — Micra 0.0809 0.0933 — Gymno 0.1155 0.2597 0.1459 — Procel 0.1048 0.1527 0.0851 0.1412 — Vanzo 0.1048 0.1223 0.1048 0.1412 0.0580 — Psilo 0.1095 0.1314 0.0872 0.0966 0.0730 0.0730 — Notho 0.1289 0.1609 0.1032 0.1155 0.0676 0.0676 0.0341 — Calyp 0.1527 0.2007 0.1223 0.1388 0.0795 0.1471 0.0809 0.0547 —

sensus cladogram of 200 bootstrap replicates, an attempt to get a better resolution for the sand using the Branch-and-Bound option (Fig. 3B), dune radiation, the Gymnophthalmus haplotype Micrablepharus and Gymnophthalmus formed a was excluded from some of the analyses, and polytomy with the other five genera, and Pro- only Colobosaura, Tretioscincus,andMicrablepha- cellosaurinus and Vanzosaura were grouped as rus were taken as outgroups. Using the Exhaus- sister taxa. In a neighbor-joining phenogram, tive Search option of PAUP, only one most par- these two genera were also grouped, and No- simonious tree was obtained, with 44 steps and thobachia and Calyptommatus were shown as sis- a consistency index of 0.64 (Fig. 5), in which ter taxa (Fig. 4). Higher genetic distances were Procellosaurinus and Vanzosaura were grouped as found for Gymnophthalmus in those analyses. In sister taxa by two site gains, and Nothobachia and Calyptommatus by one unambiguous site gain; the five most derived genera were grouped by one unambiguous site gain and one loss. The same phylogeny was obtained in a consensus cladogram of 200 bootstrap replicates (Branch-and-Bound option), in which sand dune genera formed a monophyletic group with a bootstrap value of 70% approximately (see Fig. 5). The neighbor-joining phenogram had the same topology (not shown). Assuming a mtDNA evolutionary rate of 2% MY (Brown et al., 1979), the divergence time for the radiation of this microteiid group has been preliminary estimated as 7–8 MY, and the differentiation of genera endemic of the Rio Saõ Francisco dunes as 2–4 MY. Divergence times of twice this magnitude would be obtained if the average rate of 1% MY calibrated for reptile mtDNA (Zamudio

FIG. 3. (A) Single most parsimonious tree for the eight studied gymnophthalmid genera (PAUP, ‘‘Ex- haustive’’ search), with 54 steps and a Consistency In- dex of 0.56; the number of changes is indicated on each branch. (B) The strict consensus cladogram of 200 FIG. 4. Neighbor-joining phenogram for the stud- bootstrap replicates in a ‘‘Branch-and-Bound’’ search ied gymnophthalmid genera. Divergence indices were (PAUP vers. 4.0.) with the bootstrap values over 50%. estimated by the ‘‘site’’ approach of Nei and Li (1979). 166 M. L. BENOZZATI AND M. T. RODRIGUES

siderably higher than those reported for par- thenogens and representatives of their respec- tive maternal bisexual ancestor species in lizards (Vyas et al., 1990; Moritz et al., 1992; Ki- zirian and Cole, 1999). Nevertheless, a hybrid origin of the unisexual of Roraima should not be excluded, since a strong correlation between parthenogenesis and hybridity has been estab- lished in vertebrate unisexual species (Moritz et al., 1989). The apparent homozygous genetic na- ture could be the result of a hybridization involving two genetically similar bisexual ances- tors, of which the maternal one was not includ- FIG. 5. Single most parsimonious tree for the eye- ed among the samples analyzed. This interpre- lid-less group of gymnophthalmids, excluding the tation is a plausible one since Gymnophthalmus Amazonian genus Gymnophthalmus (44 steps; C.I.ϭ probably includes still undescribed cryptic spe- 0.64). The number of changes is indicated on each cies. branch and bootstrap values from 200 replicates Phylogeny of the Eyelid-Less Gymnophthalmid (Branch-and-Bound search, PAUP vers. 4.0.) are shown Lizards.—The phylogenetic analysis of mito- below. chondrial DNA restriction-site data supported the deep basal positions of Tretioscincus and Mi- and Greene, 1997) had been considered. There- crablepharus in the group and recovered Vanzo- fore, dates presented here must be considered saura as the sister taxon to Procellosaurinus,as as approximate estimates only. previously evidenced by the morphological analyses (Rodrigues, 1995). The Amazonian ge- DISCUSSION nus Gymnophthalmus was the most divergent, as Origins of the Unisexual Gymnophthalmus un- it was in the allozymic analyses (Martins, 1997). derwoodi.—Great differences in mtDNA site In the single most parsimonious tree (Fig. 3A), profiles of the three species of Gymnophthalmus this genus was basal to a clade composed of the were found, and nucleotide divergences of ap- five most derived genera of the radiation, al- proximately 7% and 10% were estimated be- though in bootstrap analyses (Fig. 3B) Gymno- tween G. underwoodi/G. leucomystax and G. un- phthalmus has been grouped with those genera derwoodi/G. vanzoi, respectively, comparable to in a polytomy. Bootstrap values were quite low some microteiid intergeneric distances (see Ta- (Ͻ 50%) for the terminal clade branches, prob- ble 3). The G. underwoodi population from Ro- ably because of few informative sites, and so the raima was reported by Vanzolini and Carvalho relationships among the most derived genera (1991) as a lineage different from that of Trini- are weakly supported. In neighbor-joining anal- dad and Suriname studied by Cole et al. (1990). ysis the topology matched exactly that of the From karyotypic and protein electrophoretic morphological parsimony tree (Rodrigues, data (Cole et al., 1990, 1993), and mitochondrial 1995), confirming the basal positions of Tretios- DNA sequences (Kizirian and Cole, 1999), this cincus and Micrablepharus, and grouping Procel- Trinidad and Suriname lineage was considered losaurinus and Vanzosaura as the sister taxa to a a unique clone, with a hybrid origin. The Bra- clade comprising the three most derived genera. zilian G. underwoodi was demonstrated to be In this last clade, Nothobachia and Calyptommatus karyotypicaly very different from the hybrid-or- were also grouped as sister taxa. In mitochon- igin lineage studied by Cole et al. (1989, 1990), drial DNA phylogenies from which the most ge- as well as from its sympatric species G. leuco- netically differentiated genus Gymnophthalmus mystax and G. vanzoi, and no chromosomal het- was excluded, the topology was also coincident eromorphisms were detected in the unisexual with that of morphological data, in both parsi- population (Yonenaga-Yassuda et al., 1995). Low mony and neighbor-joining analyses, although levels of heterozygosity were also reported for it was weakly supported by bootstrap analyses. this population in allozymic analyses (Martins, All mitochondrial DNA topologies grouped 1991). In those biochemical and chromosomal the Rio Saõ Francisco sand dunes genera, Pro- reports a spontaneous, nonhybrid origin of par- cellosaurinus, Vanzosaura, Psilophthalmus, Notho- thenogenesis has been considered for G. under- bachia,andCalyptommatus, and showed them as woodi from Roraima. The mtDNA data present- a sister clade of the more ancestral microteiid ed here also suggest that this unisexual lineage genera Tretioscincus, Micrablepharus, and, in most is not a hybrid between its two closely related analyses, Gymnophthalmus, in accordance with sympatric bisexual species G. leucomystax and G. the previous morphological and osteological hy- vanzoi, since nucleotide divergences were con- pothesis (Rodrigues, 1995). Rodrigues proposed MOLECULAR CHARACTERIZATION OF GYMNOPHTHALMID LIZARDS 167 that strict psamophily could have evolved in the chondrial probe. This work was supported by ancestor of the five most derived genera, assum- FAPESP (Fundaçaõ de Amparo a` Pesquisa do ing the occurrence of a secondary ecological re- Estado de Saõ Paulo). version in Vanzosaura, which expanded in a range throughout the open areas in South LITERATURE CITED America. An alternative hypothesis was also AB’SABER, A. N. 1969. Participaçaõ das superfıćies considered in which strict psamophily would be aplainadas nas paisagens do nordeste brasileiro. synapomorphic only for Psilophthalmus, Notho- Geomorfologia (Inst. Geogra´fico, Univ. Sa˜ o Paulo) bachia,andCalyptommatus, and, in this case, the 19:1–39. restriction to sandy habitats would have evolved BARRETO, A. M. F. 1996. Interpretaçaõ paleoambiental do sistema de dunas fixadas do me´dio Rio Saõ convergently in Procellosaurinus. Francisco, Bahia. Unpubl. thesis, Univ. of Sa˜ o Pau- The restriction sites we analyzed are probably lo, Saõ Paulo, Brazil. revealing variations in conserved regions of the BROWN, W. M., M. GEORGE JR., AND A. C. WILSON. mtDNA genome, since intraspecific site poly- 1979. Rapid evolution of animal mitochondrial morphisms were not detected in the assayed DNA. Proceedings of the National Academy of Sci- samples. This kind of data would strengthen the ences USA 76:1967–1971. probability of coalescence of molecular and COLE,C.J,C.R.TOWNSEND,H.C.DESSAUER, AND L. morphological trees and support the inference M. HARDY. 1989. A lizard foretold. Natural His- that these phylogenies may be recovering the tory 5:12–17. COLE, C. J., H. C. DESSAUER,C.R.TOWNSEND, AND M. true phylogenetic relationships. G. ARNOLD. 1990. Unisexual lizards of the genus To the extent that a general evolutionary rate Gymnophthalmus (Reptilia: Teiidae) in the neotrop- of 2% for vertebrate mtDNA (Brown et al., 1979; ics: genetics, origin, and systematics. American Moritz et al., 1987) may be considered, the di- Museum Novitates 2994. vergence of these gymnophthalmids from a COLE, C. J., H. C. DESSAUER, AND A. L. MARKEZICK. common ancestor could be preliminarily esti- 1993. Missing link found: the second ancestor of mated as 7–8 MYA, and the subsequent radia- Gymnophthalmus underwoodi (Squamata: Teiidae), a tion of genera endemic of the continental dunes South American unisexual lizard of hybrid origin. of Rio Saõ Francisco as 2–3 MYA. This would American Museum Novitates 3055. DOWLING,T.E.,C.MORITZ, AND J. D. PALMER. 1990. reinforce the previous suggestion that the recent Nucleic acids II: restriction site analyses. In D. M. history of the Brazilian eyelid-less gymno- Hillis and C. Moritz (eds.). Molecular Systematics, phthlmid lizards has been restricted to those pp. 250–317. Sinauer Associares, Sunderland, MA. sands (Rodrigues, 1995). Divergence times of ESTES, R., K. DE QUEIROZ, AND J. GAUTHIER. 1988. twice this magnitude are estimated if we con- Phylogenetic relationships within Squamata. In R. sider the mean ‘‘reptile mtDNA rate’’ of 1% MY Estes and G. Pregill (eds.). Phylogenetic Relation- calibrated for medium-sized ectotherms, with ships of Lizard Families, pp. 119–281. Stanford adults weighting 3–5 kg (Zamudio and Greene, Univ. Press, Stanford, CA. 1997). Considering that absolute rate-heteroge- FELSENSTEIN, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution neity is associated with body size and metabolic 39:783–792. rate, the rate of mtDNA evolution being nega- HAUSWIRTH, W. W., L. O. LIM,B.DUJON, AND G. TURN- tively correlated to body size (Martin and Pal- ER. 1987. Animal and plant mitochodrial DNA. In umbi, 1993; Rand, 1994), the commonly used V. M. Darley-Usmar, D. Rickwood, and M. T. Wil- vertebrate rate of 2% MY may be more appro- son (eds.). Mitochondria—A Practical Approach, priate for the 1–2 g microteiids. pp. 171–224. IRL Press, Oxford. Mitochondrial DNA divergence rates present- HILLIS,D.M.,AND S. K. DAVIS. 1986. Evolution of ed here, despite being approximate estimates, ribosomal DNA: fifty million years of recorded history in the frog genus Rana. Evolution 40:1275– suggest that the vicariant events involved in 1288. gymnophthalmid differentiation on middle Rio HOYOS, J. M. 1998. A reappraisal of the phylogeny of Saõ Francisco sand dunes predate the last gla- lizards of the family Gymnophthalmidae (Sauria, cial period by some millions of years, contrary Scincomorpha). Revista Espanõla de Herpetologia to what was previously proposed (Rodrigues, (1998) 12:27–43. 1993, 1995). The geological and biological his- KIZIRIAN,D.A.,AND C. J. COLE. 1999. Origin of the tories of those dunes are poorly understood unisexual lizard Gymnophthalmus underwoodi (Barreto, 1996; Rodrigues, 1996), and these pre- (Gymnophthalmidae) inferred from mitochondrial DNA nucleotide sequences. Molecular Phylogenet- liminary date estimates may be useful in for- ics and Evolution 11:394–400. mulating biogeograhic hypotheses for the evo- MARTIN,A.P.,AND S. R. PALUMBI. 1993. Body size, lution of these lizards, and the identification of metabolic rate, generation time, and the molecular important features of that region’s history. clock. Proceedings of the National Academy of Sci- ences USA 90:4087–4091. Acknowledgments.—We are grateful to F. G. MARTINS, J. M. 1991. An eletrophoretic study of two No´brega for assistance in obtaining the mito- sibling species of the genus Gymnophthalmus and 168 M. L. BENOZZATI AND M. T. RODRIGUES

its bearing on the origin of the parthenogenetic G. Rio Saõ Francisco: Bahia: Brasil. III. Procellosauri- underwoodi (Sauria: Teiidae). Revista Brasileira de nus: um novo geˆnero de microteiideo sem pa´lpe- Gene´tica 14:691–703. bra, com a redefiniçaõ do geˆnero Gymnophthalmus . 1997. Estudo alozı´mico de um grupo de la- (Sauria, Teiidae). Papeís Avulsos de Zoologia, Saõ gartos gimnoftalmı´deos (Squamata: Gymno- Paulo 37:329–342. phthalmidae). Unpubl. thesis, Univ. of Sa˜ o Paulo, . 1993. Herpetofauna of the palaeoquaternary Saõ Paulo, Brazil. sand dunes of the middle Saõ Francisco river: Ba- MORITZ, C., T. E. DOWLING, AND W. M. BROWN. 1987. hia: Brazil. VI. Two new species of Phimophis (Ser- Evolution of animal mitochondrial DNA: relevance pentes, Colubridae) with notes on the origin of for population biology and systematics. Annual psammophilic adaptations. Papeís Avulsos de Revue of Ecology and Systematics 18:269–292. Zoologia, Saõ Paulo 38:187–198. MORITZ, C., W. M. BROWN, L.D. DENSMORE,J.W. . 1995. Filogenia e histo´ria geogra´fica de uma WRIGHT,D.VYAS,S.DONNELLAN,M.ADAMS, AND radiaçaõ de lagartos microteı´deos (Sauria, Teiioi- P. B AVERSTOCK. 1989. Genetic diversity and the dea, Gymnophthalmidae). Unpubl. Ph.D. Diss., dynamics of hybrid parthenogenesis in Cnemido- Univ.ofSa˜ o Paulo, Saõ Paulo, Brazil. phorus (Teiidae) and Heteronotia (Gekkonidae). In R. . 1996. Lizards, snakes and amphisbaenians M. Dawley and J. P. Bogart (eds.), Evolution and from the Quaternary sand dunes of the middle Rio Ecology of Unisexual Vertebrates, pp. 87–112. Bul- Saõ Francisco: Bahia: Brazil. Journal of Herpetolo- letin 466, New York State Museum, Albany. gy 30:513–523. MORITZ,C.,J.W.WRIGHT,V.SINGH, AND W. M . SAITOU,N.,AND M. NEI. 1987. The neighbor-joining BROWN. 1992. Mitochondrial DNA analyses and method: a new method for reconstructing phylo- the origin and relative age of parthenogenetic genetic trees. Molecular Biology and Evolution 4: Cnemidophorus.V.Thecozumela species group. Her- 406–425. petologica 48:417–424. TEMPLETON, A. R. 1983. Phylogenetic inference from NEI,M.,AND W. H. LI. 1979. Mathematical model for restriction endonuclease cleavage site maps with studying genetic variation in terms of restriction particular reference to the humans and apes. Evo- endonucleases. Proceedings of the National Acad- lution 37:221–244. emy of Sciences USA 76:5269–5273. . 1987. Nonparametric inference from restric- PASSONI, J. C., M. L. BENOZZATI, AND M. T. RODRIGUES. tion cleavage sites. Molecular Biology and Evolu- 2000. Mitochondrial DNA polymorphism and het- tion 4:315–319. eroplasmy in populations of the three species of TRICART, J. 1974. Existence de pe´riodes se`ches au Tropidurus of the nanuzae group (Squamata, Tro- Quaternaire en Amazonie et dans les re´gions vois- piduridae). Genetics and Molecular Biology 23: ines. Revue de Geomorphologie Dynamique 4: 351–356. 148–158. PRESCH, W. 1980. Evolutionary history of the South VANZOLINI,P.E.,AND C. M. CARVALHO. 1991. Two American microteiid lizards (Teiidae: Gymno- sibling and sympatric species of Gymnophthalmus phthalmidae). Copeia 1980:36–56. in Roraima, Brazil (Sauria, Teiidae). Papeís Avul- RAND, D. M. 1994. Thermal habit, metabolic rate and sos de Zoologia, Saõ Paulo 37:173–226. the evolution of mitochondrial DNA. Trends in VYAS, D., C. MORITZ,D.PECCININI-SEALE,J.W. Ecology and Evolution 9:125–131. WRIGHT, AND W. M. BROWN. 1990. The evolution- RODRIGUES, M. T. 1984. Nothobachia ablephara:um ary history of parthenogenetic Cnemidophorus lem- novo geˆnero e espećie do nordeste do Brasil (Sau- niscatus (Sauria: Teiidae). II. Maternal origin and ria, Teiidae). Papeís Avulsos de Zoologia, Sa˜ o Pau- age inferred from mitochondrial DNA analysis. lo 35:361–366. Evolution 44:922–932. . 1991a. Herpetofauna das dunas interiores do YONENAGA-YASSUDA, Y., P. E. VANZOLINI,M.T.RO- Rio Saõ Francisco: Bahia: Brasil. I. Introduçaõ a` DRIGUES, AND C. M. CARVALHO. 1995. Chromo- a´rea e descriçaõ de um novo geˆnero de microtei- some banding patterns in the unisexual microteiid ideos (Calyptommatus) com notas sobre sua ecolo- Gymnophthalmus underwoodi and two related sibling species (Gymnophthalmidae, Sauria). Cyto- gia, distribuiçaõ e especiaçaõ. Papeís Avulsos de genetics and Cell Genetics 70:29–34. Zoologia, Saõ Paulo 37:285–320. ZAMUDIO,K.R.,AND H. W. GREENE. 1997. Phylo- . 1991b. Herpetofauna das dunas interiores do geography of the bushmaster (Lachesis muta:Vi- Rio Saõ Francisco: Bahia: Brasil. II. Psilophthalmus: peridae): implications for neotropical biogeogra- um novo geˆnero de microteiideos sem pa´lpebra phy, systematics, and conservation. Biological Jour- (Sauria, Teiidae). Papeís Avulsos de Zoologia, Saõ nal of Linnean Society 62:421–442. Paulo 37:321–327. . 1991c. Herpetofauna das dunas interiores do Accepted: 17 July 2002.