Received 16 November 1998; received in revised form and accepted for publication by M. Schmid 8 March 1999

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1 Chromosome Research 7: 247±254, # 1999 Kluwer Academic Publishers. Printed in the Netherlands 247 Chromosomal polymorphisms due to supernumerary chromosomes and pericentric inversions in the eyelidless microteiid lizard Nothobachia ablephara (Squamata, Gymnophthalmidae) Katia Cristina Machado Pellegrino 1, Miguel Trefaut Rodrigues 2 & Yatiyo Yonenaga-Yassuda 1 1 Departamento de Biologia, Instituto de BiocieÃncias, Universidade de SaÄo Paulo, C.P CEP ; SaÄo Paulo, SP, Brazil; Tel: (55) (11) ; Fax: (55) (11) ; kpelleg@usp.br; 2 Departamento de Zoologia, Instituto de BiocieÃncias and Museu de Zoologia, Universidade de SaÄo Paulo, SaÄo Paulo, SP, Brazil Received 16 November 1998; received in revised form and accepted for publication by M. Schmid 8 March 1999 Key words: chromosomal polymorphisms, microteiid lizard, Nothobachia, pericentric inversion, supernumerary Abstract Cytogenetic studies were performed on eight specimens of the monotypic microteiid lizard Nothobachia ablephara, endemic of the sand dunes of the middle SaÄo Francisco river, in the semiarid caatinga, State of Bahia, Brazil. Chromosomes from broblast cultures were analysed after conventional, Ag-NOR staining, C-, and replication R- banding. A basic karyotype of 2n ˆ 62, consisting mostly of subtelocentric and acrocentric chromosomes of decreasing size, was found in ve specimens. Diploid number variation (2n ˆ 63 and 2n ˆ 64) occurred in two specimens due to the presence of one and two medium-sized subtelocentric supernumerary chromosomes (Bs). The Bs were not clearly distinguishable from the autosomes in Giemsa-stained metaphases and C-banding, but showed late replication after R-banding. Polymorphisms of pairs 1 and 5, observed in three different combinations, including acrocentrics, subtelocentrics, submetacentrics and metacentrics, were interpreted as the result of small pericentric inversions. Variation in the number of Ag-NORs was also reported. A chromosomal mechanism of sex determination of the XX:XY type is present in this species. Our data add more evidence to con rm the remarkable chromosomal variability that has been found in Gymnophthalmidae. Introduction The Gymnophthalmidae are an assemblage of small to medium-sized lizards, informally referred to as microteiids, which occur in South and Central America. Currently, the family encompasses 35 genera, 27 exclusive to South America, with about 95 species. Although there is no appropriate scheme of the phylogenetic relationships for the whole family, the monophyletism of a group including eight genera was recently admitted (Rodrigues 1996). The relationships for this monophyletic radiation are: (Tretioscincus (Micrablepharus (Gymnophthalmus (Procellosaurinus, Vanzosaura) (Psilophthalmus (Calyptommatus and Nothobachia))))). Tretioscincus and Gymnophthalmus occur in the Amazon; Micrablepharus and Vanzosaura inhabit open areas of Brazilian cerrados and caatingas; and all the other genera are endemic to a small area of Quaternary sand dunes of the middle SaÄo Francisco river, in the semiarid caatinga of the State of Bahia, Brazil. An interesting feature of this group is the progressive limb reduction and body elongation, associated with psamophily and fossoriality.

2 248 K. C. M. Pellegrino et al. The genus Nothobachia is monotypic, and the type species N. ablephara has an extremely elongated body with reduced limbs and the absence of eyelids and external ear openings. The latter two characteristics are thought to be derived conditions among microteiids (Rodrigues 1984). Little karyotypical information has been obtained from cultured broblasts for gymnophthalmids due to the rarity of specimens, their extremely small body size and dif culties in culturing their cells. Until now, chromosomal data were restricted to: three species of Gymnophthalmus presenting three different karyotypes with 2n ˆ 44, including 20 macrochromosomes (M) and 24 microchromosomes (m) (Yonenaga-Yassuda et al. 1995), two species of Procellosaurinus and one of Vanzosaura (monotypic) with 2n ˆ 40 (16M 24m), but showing distinct karyotypes (Yonenaga-Yassuda et al. 1996a), two species of Micrablepharus with diploid numbers varying from 2n ˆ 50 to 53 without a clear distinction between macrochromosomes and microchromosomes (Yonenaga-Yassuda & Rodrigues 1999), and three Leposoma species from the tropical rain forest displaying distinct karyotypes with 2n ˆ 44 (20M 24m) and 2n ˆ 52 with chromosomes of decreasing size (Pellegrino et al. submitted). Herein, we report the karyotypical data of Nothobachia ablephara after conventional staining and banding techniques. This paper follows three articles about the monophyletic group of microteiid lizards, that, in association with other ongoing studies, add signi cant evidence to con rm the remarkable chromosomal variability that has been described for Gymnophthalmidae. Material and methods Four males and four females of Nothobachia ablephara from Alagoado (098299S, W), State of Bahia, Brazil, were cytogenetically analyzed. Voucher specimens were deposited in the herpetological collection of the Museu de Zoologia, Universidade de SaÄo Paulo (MZUSP), State of SaÄo Paulo, Brazil. Metaphase chromosomes were prepared from - broblast cultures obtained from muscle biopsies and cultured at 298C in Dulbecco's modi ed Eagle's medium containing 20% fetal calf serum, according to Yonenaga-Yassuda et al. (1988). For replication R- banding, the cells were treated with 5-BrdU ( nal concentration 25 ìg=ml) for 8±9 h before harvesting, followed by FPG staining (Dutrillaux & Couturier 1981). C-banding and Ag-NOR staining were based on routine protocols. Meiotic spreads from three male specimens were also performed. Results The seven specimens of Nothobachia ablephara presented diploid numbers varying from 2n ˆ 62 to 64, due to the presence of extra medium-sized subtelocentric chromosomes considered as supernumeraries (Bs), and polmorphisms in morphology of pairs 1 and 5 (Table 1). Homologues of pair 1 were observed as acrocentrics (a), subtelocentrics (st) or submetacentrics (sm) and those of pair 5 as subtelocentrics, submetacentrics and metacentrics (m). Pairs 2 to 4, 6 to 26 and 28 to 30 are subtelocentrics and acro- Table 1. Karyotype variability found in conventionally stained metaphases Nothobachia ablephara from Alagoado (BA) based on Specimen Sex 2n B Pair 1 Pair 5 Sex chromosomes No. of metaphases LG 468 F 62 0 st=a sm=st m=m 17 LG 469 F 62 0 st=a st=st m=m 42 LG 470 M 63 1 st=sm sm=m st=m 36 LG 446 M 64 2 st=a sm=st st=m 50 LG 457 M 62 0 st=sm sm=st st=m 34 LG 448 F 62 0 st=sm sm=st m=m 28 LG 461 F 62 0 a=a st=st m=m 34 Total 241 2n ˆ diploid number; F ˆ female; M ˆ male; B ˆ supernumerary chromosome; a ˆ acrocentric; st ˆ subtelocentric; sm ˆ submetacentric; m ˆ metacentric. Specimen LG 447 not included; only meiotic spreads were available.

3 Polymorphisms of supernumerary chromosomes and pericentric inversions in Nothobachia 249 centrics of decreasing size. The small pair 27 is a clearly recognizable metacentric. A chromosomal mechanism of sex determination of the XX:XY type was also detected. The X is a medium-sized metacentric and the Y a medium-sized subtelocentric (Figure 1a). Five out of the seven specimens presented a 2n ˆ 62 karyotype with three different combinations of the chromosomes that constitute pair 1, in homomorphic and heteromorphic combinations (Table 1). The polymorphism of pair 1 was interpreted, after R- banding analysis, as the result of small pericentric inversions (Figure 2a,b). C-banding analysis did not reveal evidence supporting addition or deletion of constitutive heterochromatin (Figure 2a,b). The distal end of the long arm of pair 1 was always strongly stained after C-banding and late-replicating, in all its morphologies (Figure 2a,b,c). Besides, two combinations of pair 5 (one homomorphic and one heteromorphic) were detected in these specimens (Table 1, Figure 2d,e). One specimen exhibited a 2n ˆ 63 karyotype due to the presence of a medium-sized subtelocentric B which was not clearly distinguishable from the autosomes after Giemsa staining (Figure 1b) or C-banding (Figure 3a). However, the late-replicating nature of the B was revealed after R-banding (Figures 1b and 3b). This specimen presented heteromorphic pairs 1 (st=sm) and 5 (sm=m). The remaining specimen displayed a 2n ˆ 64 karyotype characterized by two medium-sized subtelocentric B chromosomes of different sizes, not easily distinguished from the autosomes (Figure 1c), undetected after C-banding, but always late-replicating Figure 1. (a) Karyotype of Nothobachia ablephara, female, 2n ˆ 62, with a subtelocentric/acrocentric pair 1, a subtelocentric pair 5, and the XY male pair (inset). (b±c) Subtelocentric B chromosomes after Giemsa-staining and RBG-banding. (b) 1B from the specimen with 2n ˆ 63. (c) 2Bs from the specimen with 2n ˆ 64. Bar ˆ 10 ìm.

4 250 K. C. M. Pellegrino et al. Figure 2. Polymorphisms of autosome pairs of Nothobachia ablephara, after conventional staining, RBG- and CBG-banding. (a±c) Different combinations of pair 1. (a) Subtelocentric/acrocentric (st=a). (b) Subtelocentric/submetacentric (st=sm). (c) Acrocentric (a=a). (d± e) Different combinations of pair 5. (d) Subtelocentric (st/st). (e) Submetacentric/subtelocentric (sm=st). (f) Submetacentric/metacentric (sm=m). ˆ centromere position. after R-banding (Figures 1c and 3c). The larger B is very similar to that found in the specimen with 2n ˆ 63, probably representing the same chromosome. Heteromorphism of pair 1 (st=a) and pair 5 (sm=st) was noted. A successful analysis of testicular chromosome spreads, performed in the specimen LG 447, revealed 31 bivalents in diplotene cells and 31 chromosomes in metaphase II (not shown). The analysis of meiotic phases in specimens bearing B chromosomes was inconclusive. A total of 106 metaphases analysed from the seven specimens of N. ablephara revealed variability in the number of presumptive Ag-NORs. Positive AgNo 3 signs, weakly stained, varied from 2 to 7, and were located at the telomere regions of different-sized chromosomes (Figure 4a±e). The presence of positive signs at one or both telomeres of the homologs of the small metacentric pair 27 was the most frequent pattern (Figure 4d,e). Discussion A considerable amount of chromosome variability involving the presence of Bs and polymorphisms of autosome pairs was detected in the seven specimens of N. ablephara from Alagoado, State of Bahia, Brazil, con rming our previous ndings of remarkable karyotype variation in microteiid lizards. The fact that the extra chromosomes found in the two specimens of N. ablephara were late-replicating reinforces our suggestion of their presumptive nature of supernumerary (B). However, the Bs exhibit neither a C-banding pattern nor an intermediate staining among the strongest and the weakest C-positive blocks of the autosomes. The function and composition of B chromosomes is still a controversial question. One widespread heterochromatin feature is its late-replication, and, based on this assumption, the Bs present in the 2n ˆ 63 and 2n ˆ 64 karyotypes could be considered Figure 3. CBG- and RBG-banding in Nothobachia ablephara. (a) CBG-banded karyotype of a 2n ˆ 63 male specimen with one B chromosome, pair 1 st/sm and pair 5 sm/m. (b) RBG-banded karyotype of the 2n ˆ 63 male specimen, showing one late-replicating B chromosome. (c) Partial RBG-banded metaphase from the male with 2n ˆ 64, displaying two late-replicating B chromosomes (arrows). Bar ˆ 10 ìm.

5 Polymorphisms of supernumerary chromosomes and pericentric inversions in Nothobachia 251

6 252 K. C. M. Pellegrino et al. Figure 4. Ag-NOR staining in Nothobachia ablephara. (a) 2 Ag-NORs at one of the telomers of metacentric pair 27. (b) 4 Ag-NORs at both telomeres of pair 27. (c) 4 Ag-NORs at the telomeric region of the long arm of one large subtelocentric and at one and both telomeres of pair 27, respectively. (d) 6 Ag-NORs at telomere regions of a medium-sized acrocentric pair and at both arms of pair 27. (e) 7 Ag-NORs at the telomeric regions of the long arm of three large subtelocentrics, of a medium-sized acrocentric pair and at both arms of one homologue of pair 27. as composed by a speci c class of heterochromatin undetected by routine C-banding procedures. Recently, Silva & Yonenaga-Yassuda (1998) reported a conspicuous heterogeneity of size, morphology, constitutive heterochromatin patterns and localization of telomeric sequences of B chromosomes for the rodent Nectomys, which allowed them to suggest differences in the composition of these chromosomes. Reports on the occurrence of B chromosomes in lizard are still scarce, although they have been found in many organisms. The B chromosomes were recently described in the microteiid Micrablepharus atticolus and M. maximiliani (Yonenaga-Yassuda & Rodrigues 1999). The diploid number variation in M. atticollus (2n ˆ 50 to 53) was attributed to a supernumerary system including one to three Bs of different morphologies and sizes, with heterogeneity in their constitutive heterochromatin patterns but always late-replicating. The high chromosomal variability detected in pair 1ofN. ablephara seemed to be the result of small pericentric inversions. The same kind of rearrangement might be involved in the origin of the polymorphism of pair 5. It is interesting that, in a sample of seven specimens, only one exhibited homomorphic pairs 1 and 5, and, considering the different combinations of these autosome pairs, our sample characterizes ve distinct cytotypes within Nothobachia, although about 36 different combinations of pairs 1 and 5 could occur. We found a considerable variation in the number of positive signs after AgNO 3 treatment in the genus Notobachia, with very weakly stained regions in some chromosomes. The small pair 27 was entirely stained in some metaphases and completely heterochromatic after C-banding. At the distal regions of the larger autosomes long arm, the weakly AgNO 3 signs coincided with the faint C-bands. Thus, we suspect that some of the positive Ag-staining in Nothobachia is representing C-band regions rather than NORs. Further data from a larger sample is still necessary for complete understanding of this Agstaining pattern. Single and multiple NOR-bearing chromosome pairs have already been reported for species of Micrablepharus, presenting a conspicuous variability in their number and location, and for Gymnophthalmus, Procellosaurinus and Vanzosaura species, all of them belonging to the same monophyletic radiation of microteiids (Yonenaga-Yassuda et al. 1995, 1996a, Yonenaga-Yassuda & Rodrigues 1999). Although NOR localization has proved to be an important marker for lizards, we agree with Yonenaga-Yassuda & Rodrigues (1999) that more data on NORs varia-

7 Polymorphisms of supernumerary chromosomes and pericentric inversions in Nothobachia 253 bility of Gymnophthalmidae are still necessary to understand their role in the chromosomal evolution of the family. The striking correlation between the presence of a subtelocentric/metacentric heteromorphic pair and the male sex phenotype, even though only seven specimens had been analyzed, led us to suggest that this pair is related to an XX:XY system of sex determination in N. ablephara. Our suggestion was based on the analysis of Giemsa-stained metaphases rather than on banded karyotypes. In all male standard-stained metaphases, an odd medium-sized metacentric, clearly distinguishable from all the other autosomes, was found along with an unpaired medium-sized subtelocentric. On the other hand, female metaphases always presented two of the mediumsized biarmed chromosomes and none of the subtelocentric seemed to be unpaired. The X and Y chromosomes of Nothobachia differ slightly in their centromere position, due probably to a small pericentric inversion. Their almost identical size could be re ecting the early stage of differentiation of an XY condition. It has been pointed out that, in lineages of lizards, sex chromosomes have arisen recently and independently (Bickham 1984). An XX:XY system among gymnophthalmids was also reported for M. atticolus, M. maximiliani and Gymnophthalmus pleei. In Micrablepharus, the male sexual pair is represented by an acrocentric/subtelocentric heteromorphic pair, and the heterochromatic and the late-replicating Y is the smallest chromosome of the complement (Yonenaga-Yassuda & Rodrigues 1999). In Gymnophthalmus pleei (2n ˆ 34, 12M 22m), a subtelocentric/telocentric heteromorphic pair was considered to be involved in sex determination, based on non-differentially stained metaphases (Cole et al. 1990). The basic diploid number of 2n ˆ 62 and its variants of 2n ˆ 63 and 64 found in N. ablephara are the highest diploid numbers so far reported for microteiid lizards, while the lowest one (2n ˆ 32, 18M 14m) was found in Bachia dorbignyi (Pellegrino 1998). According to the phylogenetic scheme suggested by Rodrigues (1996), Nothobachia is closely related to Calyptommatus, which presents 2n ˆ 57 in males and 2n ˆ 58 in females (Yonenaga- Yassuda et al. 1996b), and both are considered the most derived genera of the microteiid eyelidless radiation. The two genera are also characterized by showing the most striking adaptations to fossoriality, especially observed in limb reduction. Calyptommatus lacks external vestiges of forelimbs; in Nothobachia, forelimbs are still present but reduced to a styliform appendage. Nothobachia has only two toes in the hind limb which is styliform in Calyptommatus. It is noteworthy that the highest chromosome numbers in this radiation are shared by Nothobachia and Calyptommatus, while all other genera are characterized by lower diploid numbers. Our current knowledge on cytogenetics of microteiid species reveals two distinct types of chromosome complements: those with a clear distinction between macrochromosomes and, quite often, 24 microchromosomes, and those exhibiting chromosomes of decreasing size. As observed in Nothobachia (present data), Calyptommatus (Yonenaga- Yassuda et al. 1996b), Micrablepharus (Yonenaga- Yassuda & Rodrigues 1999) and Leposoma (Pellegrino et al. submitted), the highest diploid numbers are not associated with the presence of macro- and microchromosomes. The occurrence of karyotypes showing sharp differences between macro- and microchromosomes and those with decreasing size chromosomes in the same monophyletic radiation, questions the evolutionary importance of these two kinds of chromosomal complements in these gymnophthalmids. Our cytogenetic ndings in N. ablephara clearly indicate the need to gather further chromosomal data from other microteiid genera. The high level of chromosomal variation and polymorphisms in the family strongly contrasts with the conservative karyotypes of other lizards, suggesting that gymnophthalmids may be under an intense process of karyotypical differentiation. Acknowledgments The authors are grateful to Dr. Tien Hsi Chu and Mrs. MõÂriam Romeo for technical assistance, and to Gabriel Skuk, Jose Manoel Martins, Rosana Moraes and Pedro Bernardo da Rocha for collecting specimens. We are indebted to Dr. Marta Svatman, Dr. Daniela Calcagnotto and Dr. ValeÂria Fagundes for critical review of the manuscript. Grants to support this study were provided by Conselho Nacional de Desenvolvimento Cientõ co e TecnoloÂgico (CNPq), FundacËaÄo de Amparo aá Pesquisa do Estado de SaÄo Paulo (FA- PESP), Coordenadoia de AperfeicËoamento de Pessoal

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