Mitochondrial Restriction-Site Characterization of a Brazilian Group of Eyelid-Less Gymnophthalmid Lizards

Journal of Herpetology, Vol. 37, No. 1, pp. 161 168, 003 Copyright 003 Society for the Study of Amphibians and Reptiles Mitochondrial Restriction-Site Characterization of a Brazilian Group of Eyelid-Less Gymnophthalmid Lizards M. L. BENOZZATI 1, AND M. T. RODRIGUES 3 1 Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brasil 3 Departamento de Zoologia, Instituto de Biociências, and Museu de Zoologia, Universidade de São Paulo, São Paulo, Brasil ABSTRACT.Phylogenetic relationships among eight Brazilian genera of the lizard family Gymnophthalmidae were analyzed using mitochondrial DNA restriction-site data. The intergeneric relationships inferred from a cladistic analysis of 3 restriction sites were widely congruent with a recent phylogenetic analysis based on morphology. Assuming a molecular clock of % 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 São Francisco Quaternary sand dunes was also supported by mitochondrial data, and the time estimated for this radiation was about 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 comprises two families: the Teiidae and the Gymnophthalmidae (Estes et al., 1988). It includes 36 genera and about 170 species of small lizards that live in leaf litter or ground vegetation of forests and open areas, in rocky habitats, or are semiaquatic. Although phylogenetic relationships within Gymnophthalmidae are still poorly known, limb reduction seems to have originated several times independently in the family (Presch, 1980; Hoyos, 1998). One of the limbreduced gymnophthalmid groups shows a tendency toward a pronounced reduction of forelimbs in relation to hind limbs, and eyelid loss, among other presumable fossorial adaptations (Rodrigues, 1991a). Five new genera from the continental dunes on both banks of the middle Rio São Francisco, in the morphoclimatic domain of the semiarid Brazilian Caatinga, have been recently described by Rodrigues: Nothobachia (1984), Calyptommatus (1991a), Psilophthalmus (1991b), Procellosaurinus and Vanzosaura (1991c). Among these new genera, Vanzosaura was the only one described to reallocate the former Gymnophthalmus rubricauda and its synonym Gymnophthalmus multiscutatus, whereas the other four genera were described for new species. Gymnophthalmus, Micrablepharus, andtretioscincus were previously known genera related to this radiation. Rodrigues (1991c) proposed that these eight genera of microteiid lizards are Corresponding Author. Present address: Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 77, Cidade Universitária, São Paulo, São Paulo, Brasil, CEP: 05508-090; E-mail: benozzat@usp.br monophyletic based on their marked tendencies for forelimb reduction and lack of eyelids. Tretioscincus is the only genus of the group having eyelids, and Micrablepharus the oldest most divergent taxon, diagnosed by the absence of eyelids and a rudimentary, or absence of the first finger. Gymnophthalmus, including the unisexual Gymnophthalmus underwoodi, was considered by Rodrigues (1991c) a more derived genus, followed by the other five genera found on São Francisco dunes. A more extensive analysis considering 71 osteological and morphological characters of these lizards (Rodrigues, 1995) corroborated its monophyly and showed Procellosaurinus and Vanzosaura as sister groups. The topology obtained from those data was (Tretioscincus (Micrablepharus (Gymnophthalmus ((Procellosaurinus, Vanzosaura) (Psilophthalmus (Nothobachia, Calyptommatus)))))). It was proposed that the radiation of the group on the continental dunes derived from a Procellosaurinus-Vanzosaura Amazonian ancestor. In a period previous to the origin of psammophily and fossorial adaptations, the fauna of the middle Rio São Francisco lived in typical caatinga depressions with inselbergs and isolated hills (Rodrigues, 1993). As proposed by Ab Saber (1969) and Tricart (1974), the river drained into a continental lake until the end of the last glacial period, when it connected to the sea. In humid periods during the phase of interior drainage, sand accumulated in lacustrine depressions, isolating elements of the fauna in disjunct hills. In the onset of a semiarid climate, those hills were vanishing faunal refuges in a sandy desert, and species with preadaptations for life on sand would have expanded on the recent dune fields. Present

16 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 Gymnophthalmus leucomystax Gymnophthalmus vanzoi Gymnophthalmus underwoodi Microblepharus maximiliani Nothobachia ablephara Procellosaurinus erythrocercus Psilophthalmus paeminosus Tretioscincus agilis Vanzosaura rubricauda Colobosaura modesta Queimadas (Bahia) Fazenda Salvamento (Roraima) Fazenda Salvamento (Roraima) Fazenda Salvamento (Roraima) Ilha de Maracá (Roraima) Sta. Rita do Araguaia (Goiás) Alagoado (Bahia) Queimadas (Bahia) Sto. Inácio (Bahia) Poção (Pará) Vacaria (Bahia) Alto Araguaia (Mato Grosso) Barra do Garças (Mato Grosso) 6 3 5 3 5 5 1 1 knowledge of microteiid phylogeny (Rodrigues, 1995) suggests that Procellosaurinus, Vanzosaura, Psilophthalmus, and Nothobachia were already present in those sand dunes at the time the ancestors of the most derived genus of this radiation, Calyptommatus, were isolated by the river, separating populations in opposite margins of the Rio São Francisco. In this paper, mitochondrial DNA restrictionsite profiles were used to construct a molecular phylogeny for these gymnophthalmids and to estimate divergences among different genera. We evaluate the congruence of these molecular data with the phylogenies based on morphological and allozymic analyses. The unisexual G. underwoodi from the Amazonian state of Roraima and its sympatric bisexual species Gymnophthalmus leucomystax and Gymnophthalmus vanzoi were also analyzed, and the origin of the parthenogenetic lineage is discussed. MATERIALS AND METHODS Intrageneric relationships were not considered in the present mtdna study, as they were not considered in the previous morphological analyses (Rodrigues, 1995). Only one species of each eyelid-less gymnophthalmid genus was assayed for mtdna phylogenetic purposes: Nothobachia, Psilophthalmus, and Vanzosaura are monospecific genera, Micrablepharus and Procellosaurinus are composed of two species, and Calyptommatus and Gymnophthalmus each has more than three species. Three species of Gymnophthalmus collected in Roraima were sampled, the unisexual species G. underwoodi and its sympatric bisexual species G. leucomystax and G. vanzoi, to investigate a putative maternal ancestor of the parthenogenetic lineage. Two specimens of Colobosaura modesta were assayed as an outgroup. Sample sizes and locations of the analyzed species are indicated in Table 1, and their geographic distribution in Figure 1. One specimen of Iphisa elegans from Reserva Faunística Cuyabeno, Sucumbios Province, Ecuador (AMNH-LJV5189) was also initially included as outgroup, but it was discontinued because single-site profiles were detected for most of the endonucleases assayed, and trials to precisely map these sites failed with the available DNA sample. DNA was extracted from field-collected specimens stored at 196 C, following Hillis and Davis (1986) and Dowling et al. (1990), modified by Passoni et al. (000). The enzymes used in the analyses were BamH I, EcoR I, Hind III, Pvu II and Xba I. Additional 6bp (Ava I, BstE II, Dra I, EcoR V, Kpn I, Pst I, and Sal I) and 4bp-recognizing endonucleases (Hinf I, HinP1 I and Rsa I) were used in initial screening but were discontinued because they failed to cut the DNA samples consistently or produced restrictionsite profiles of ambiguous interpretations. Hind III DNA digests and pbr 38 digested with BamH I, Bgl I and Hinf I were used as molecular size standards, with fragment sizes, in base pairs, of 3,130; 9,416; 6,557; 4,907; 4,361;,3;,176;,03; 1,766; 1,30 and 1,033. Restriction fragments were transferred from 0.8% agarose gels to nylon membranes ( Nylon Membrane Positive Charged, Boehringer Mannheim) and probed with heterologous mtdna obtained from human placenta (Hauswirth et al., 1987) and labeled with digoxigenin ( DIG DNA labeling and detection kit, Boehringer Mannheim). Each taxon was assigned a composite mtdna genotype, compiled from the restriction site profiles. Site data were assembled in a presence/absence matrix, from which sequence divergence indices (p) were estimated by the site approach of Nei and Li (1979). Taxa were clustered by neighbor-joining (Saitou and Nei, 1987) and by parsimony using the computer

MOLECULAR CHARACTERIZATION OF GYMNOPHTHALMID LIZARDS 163 FIG. 1. Map of the localities sampled: 1, Ilha de Maracá (Roraima);, Fazenda Salvamento (Roraima); 3, Poção (Pará); 4, Barra do Garças (Mato Grosso); 5, Alto Araguaia (Mato Grosso); 6, Santa Rita do Araguaia (Goiás); 7, Alagoado (Bahia); 8, Queimadas (Bahia); 9, Vacaria (Bahia); 10, Santo Inácio (Bahia). In detail, the paleoquaternary dune fields on the middle Rio São Francisco (Bahia)the stippled area corresponds to the river flood plain.

164 M. L. BENOZZATI AND M. T. RODRIGUES FIG.. Mitochondrial DNA profiles of Xba I (A) and Pvu II (B) endonucleases from eyelid-less gymnophthalmid lizards: Micrablepharus maximiliani (1); the unisexual species Gymnophthalmus underwoodi (); the bisexual species Gymnophthalmus leucomystax (3); the bisexual Gymnophthalmus vanzoi (4); and Procellosaurinus erythrocercus (5). [S-molecular weight standard]. program PAUP (vers. 4.0; D. L. Swofford, Sinauer Associates, Sunderland, MA, 1999). Bootstrapping (Felsenstein, 1985) was applied to estimate confidence in monophyletic groups. MacClade software (vers. 3.03, W. P. Maddison and D. R. Maddison, Sinauer Associates, Sunderland, MA, 199) was used in the analysis of character state changes. A sequence divergence of % per million years was considered in a preliminary estimation of divergence times between haplotypes (Brown et al., 1979). RESULTS Enzyme assays of DNA samples from the eight genera of the studied gymnophthalmids revealed a total of 3 sites with the endonucleases BamH I, EcoR I, Hind III, Pvu II and Xba I. No within-species variation was detected in the analyzed samples, and most of different restriction profiles could be accounted for by changes involving single site losses or gains. Restriction 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 Maracá 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 Salvamento (Roraima). Mitochondrial DNA information about a possible hybrid origin of the unisexual species was obtained from 3 restriction sites that were unequivocally characterized in those three species of Gymnophthalmus. Remarkable differences were found among them, as shown in Figure for Pvu II and Xba I restriction-site profiles. Nucleotide divergences were estimated between G. underwoodi and G. leucomystax (P 0.0677), G. underwoodi and G. vanzoi (P 0.101), and between G. leucomystax and G. vanzoi (P 0.13). These divergence values are similar to some estimated for pairs of microteiid genera (see Table 3). Phylogeny.A presence/absence matrix with the haplotypes of the eight microteiid genera was constructed (Table ), and nucleotide divergences were calculated (Table 3). From Gymnophthalmus, the same bisexual species considered in the previous morphological study (Rodrigues, 1995), G. leucomystax, was included in the analyses. In a phylogenetic assay using the Exhaustive Search option of PAUP, a single most parsimonious tree was obtained, with 54 steps and a consistency index of 0.56 (Fig. 3A). The five most derived genera according to Rodrigues (1995) were grouped in this cladogram by one unambiguous restriction-site gain and one loss. Procellosaurinus and Vanzosaura were grouped as sister taxa by four site gains, which increases the confidence of the phylogenies (Templeton, 1983, 1987). Gymnophthalmus was the most divergent genus, with six autapomorphies, four of which were site gains. In the con- TABLE. Presence/absence matrix of restriction sites of BamH I, EcoR I, Hind III, Pvu II, and Xba I endonucleases. Haplotypes are designated by genus names (1presence; 0absence). Colobosaura Tretioscincus Micrablepharus Gymnophthalmus Procellosaurinus Vanzosaura Psilophthalmus Nothobachia Calyptommatus 10101100001001110100101000001010 10000100100001110100000000001000 10110100010101110100110010000000 10111101000000000000100101010110 11101111000110110111110000000001 11110100100010010111111000011001 11011100000100110001110100011000 11011110000110010000110000011000 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 Tretio Micra Gymno Procel Vanzo Psilo Notho Calyp 0.0676 0.0809 0.1155 0.1048 0.1048 0.1095 0.189 0.157 0.0933 0.597 0.157 0.13 0.1314 0.1609 0.007 0.1459 0.0851 0.1048 0.087 0.103 0.13 0.141 0.141 0.0966 0.1155 0.1388 0.0580 0.0730 0.0676 0.0795 0.0730 0.0676 0.1471 0.0341 0.0809 0.0547 sensus cladogram of 00 bootstrap replicates, using the Branch-and-Bound option (Fig. 3B), Micrablepharus and Gymnophthalmus formed a polytomy with the other five genera, and Procellosaurinus and Vanzosaura were grouped as sister taxa. In a neighbor-joining phenogram, these two genera were also grouped, and Nothobachia and Calyptommatus were shown as sister taxa (Fig. 4). Higher genetic distances were found for Gymnophthalmus in those analyses. In an attempt to get a better resolution for the sand dune radiation, the Gymnophthalmus haplotype was excluded from some of the analyses, and only Colobosaura, Tretioscincus, and Micrablepharus were taken as outgroups. Using the Exhaustive Search option of PAUP, only one most parsimonious tree was obtained, with 44 steps and a consistency index of 0.64 (Fig. 5), in which Procellosaurinus and Vanzosaura were grouped as 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 00 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 % 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 São Francisco dunes as 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, Exhaustive search), with 54 steps and a Consistency Index of 0.56; the number of changes is indicated on each branch. (B) The strict consensus cladogram of 00 bootstrap replicates in a Branch-and-Bound search (PAUP vers. 4.0.) with the bootstrap values over 50%. FIG. 4. Neighbor-joining phenogram for the studied gymnophthalmid genera. Divergence indices were estimated by the site approach of Nei and Li (1979).

166 M. L. BENOZZATI AND M. T. RODRIGUES FIG. 5. Single most parsimonious tree for the eyelid-less group of gymnophthalmids, excluding the Amazonian genus Gymnophthalmus (44 steps; C.I. 0.64). The number of changes is indicated on each branch and bootstrap values from 00 replicates (Branch-and-Bound search, PAUP vers. 4.0.) are shown below. and Greene, 1997) had been considered. Therefore, dates presented here must be considered as approximate estimates only. DISCUSSION Origins of the Unisexual Gymnophthalmus underwoodi.great differences in mtdna site profiles of the three species of Gymnophthalmus were found, and nucleotide divergences of approximately 7% and 10% were estimated between G. underwoodi/g. leucomystax and G. underwoodi/g. vanzoi, respectively, comparable to some microteiid intergeneric distances (see Table 3). The G. underwoodi population from Roraima was reported by Vanzolini and Carvalho (1991) as a lineage different from that of Trinidad and Suriname studied by Cole et al. (1990). From karyotypic and protein electrophoretic data (Cole et al., 1990, 1993), and mitochondrial DNA sequences (Kizirian and Cole, 1999), this Trinidad and Suriname lineage was considered a unique clone, with a hybrid origin. The Brazilian G. underwoodi was demonstrated to be karyotypicaly very different from the hybrid-origin lineage studied by Cole et al. (1989, 1990), as well as from its sympatric species G. leucomystax and G. vanzoi, and no chromosomal heteromorphisms were detected in the unisexual population (Yonenaga-Yassuda et al., 1995). Low levels of heterozygosity were also reported for this population in allozymic analyses (Martins, 1991). In those biochemical and chromosomal reports a spontaneous, nonhybrid origin of parthenogenesis has been considered for G. underwoodi from Roraima. The mtdna data presented here also suggest that this unisexual lineage is not a hybrid between its two closely related sympatric bisexual species G. leucomystax and G. vanzoi, since nucleotide divergences were considerably higher than those reported for parthenogens and representatives of their respective maternal bisexual ancestor species in lizards (Vyas et al., 1990; Moritz et al., 199; Kizirian 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 established in vertebrate unisexual species (Moritz et al., 1989). The apparent homozygous genetic nature could be the result of a hybridization involving two genetically similar bisexual ancestors, of which the maternal one was not included among the samples analyzed. This interpretation is a plausible one since Gymnophthalmus probably includes still undescribed cryptic species. Phylogeny of the Eyelid-Less Gymnophthalmid Lizards.The phylogenetic analysis of mitochondrial DNA restriction-site data supported the deep basal positions of Tretioscincus and Micrablepharus in the group and recovered Vanzosaura as the sister taxon to Procellosaurinus, as previously evidenced by the morphological analyses (Rodrigues, 1995). The Amazonian genus Gymnophthalmus was the most divergent, as it was in the allozymic analyses (Martins, 1997). In the single most parsimonious tree (Fig. 3A), this genus was basal to a clade composed of the five most derived genera of the radiation, although in bootstrap analyses (Fig. 3B) Gymnophthalmus has been grouped with those genera in a polytomy. Bootstrap values were quite low ( 50%) for the terminal clade branches, probably because of few informative sites, and so the relationships among the most derived genera are weakly supported. In neighbor-joining analysis the topology matched exactly that of the morphological parsimony tree (Rodrigues, 1995), confirming the basal positions of Tretioscincus and Micrablepharus, and grouping Procellosaurinus and Vanzosaura as the sister taxa to a clade comprising the three most derived genera. In this last clade, Nothobachia and Calyptommatus were also grouped as sister taxa. In mitochondrial DNA phylogenies from which the most genetically differentiated genus Gymnophthalmus was excluded, the topology was also coincident with that of morphological data, in both parsimony and neighbor-joining analyses, although it was weakly supported by bootstrap analyses. All mitochondrial DNA topologies grouped the Rio São Francisco sand dunes genera, Procellosaurinus, Vanzosaura, Psilophthalmus, Nothobachia, and Calyptommatus, and showed them as a sister clade of the more ancestral microteiid genera Tretioscincus, Micrablepharus, and, in most analyses, Gymnophthalmus, in accordance with the previous morphological and osteological hypothesis (Rodrigues, 1995). Rodrigues proposed

MOLECULAR CHARACTERIZATION OF GYMNOPHTHALMID LIZARDS 167 that strict psamophily could have evolved in the ancestor of the five most derived genera, assuming the occurrence of a secondary ecological reversion in Vanzosaura, which expanded in a range throughout the open areas in South America. An alternative hypothesis was also considered in which strict psamophily would be synapomorphic only for Psilophthalmus, Nothobachia, and Calyptommatus, and, in this case, the restriction to sandy habitats would have evolved convergently in Procellosaurinus. The restriction sites we analyzed are probably revealing variations in conserved regions of the mtdna genome, since intraspecific site polymorphisms were not detected in the assayed samples. This kind of data would strengthen the probability of coalescence of molecular and morphological trees and support the inference that these phylogenies may be recovering the true phylogenetic relationships. To the extent that a general evolutionary rate of % for vertebrate mtdna (Brown et al., 1979; Moritz et al., 1987) may be considered, the divergence of these gymnophthalmids from a common ancestor could be preliminarily estimated as 7 8 MYA, and the subsequent radiation of genera endemic of the continental dunes of Rio São Francisco as 3 MYA. This would reinforce the previous suggestion that the recent history of the Brazilian eyelid-less gymnophthlmid lizards has been restricted to those sands (Rodrigues, 1995). Divergence times of twice this magnitude are estimated if we consider the mean reptile mtdna rate of 1% MY calibrated for medium-sized ectotherms, with adults weighting 3 5 kg (Zamudio and Greene, 1997). Considering that absolute rate-heterogeneity is associated with body size and metabolic rate, the rate of mtdna evolution being negatively correlated to body size (Martin and Palumbi, 1993; Rand, 1994), the commonly used vertebrate rate of % MY may be more appropriate for the 1 g microteiids. Mitochondrial DNA divergence rates presented here, despite being approximate estimates, suggest that the vicariant events involved in gymnophthalmid differentiation on middle Rio São Francisco sand dunes predate the last glacial period by some millions of years, contrary to what was previously proposed (Rodrigues, 1993, 1995). The geological and biological histories of those dunes are poorly understood (Barreto, 1996; Rodrigues, 1996), and these preliminary date estimates may be useful in formulating biogeograhic hypotheses for the evolution of these lizards, and the identification of important features of that region s history. Acknowledgments.We are grateful to F. G. 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