Cytogenet Cell Genet 74:203-210 (1996) Cytogenetics and CellGenetics

Chromosomal banding patterns in the eyelid-less microteiid radiation: Procellosaurinus and Vanzosaura (, )

Y. Yonenaqa-Yassuda.' L. Mori.' T.H. ChU,l and M.T. Rodriques?

J Departamento de Biologia and 2 Departamento deZoologia, Institut o de Biocienci as, Universidade de Sao Paulo, Sao Paulo (Brazil)

Abstract. Cytogenetic studies were performed on three spe­ staining. Despite similarities in chromosome number and mor­ ciesofeyelid-less microteiid s, Procellosaurinus erythrocerus, P. phology, each can be differentiated by the position and tetradactylus. and (Squamata, Gym­ amount ofC-heterochromatin. Our cytogenetic and DNA con­ nophthalmidae), all with a diploid number of 2n = 40. The tent data indicate that there are more similarities between the specimens were collected in the palaeoquartenary dune fields of two species of Procellosaurinus than exist between either spe­ the middle Rio Sao Francisco in the State of Bahia, Brazil. cies and V rubricauda, reinforcing the importance of banding Chromosomes from fibroblast cultures were studied after rou­ techniques for the characterization of reptilian species. tine Giemsa staining, CBG- and RBG-banding, and Ag-NOR

The fam ily Gymnophthalmidae currently comprises 35 mi­ nians and 25 species of snakes, totalling 20 new endemic rep­ croteiid genera dwelling in Central and South America . Al­ tiles (Rodrigues, 1991a-d, 1993, 1996; Vanzolini, 1991). The though a satisfactory phylogenetic scheme is still not available new genera of eyelid-less microteiid described in this region are for the whole family (Harris , 1985; Rodrigues, 1991a), a small Calyptom matus (three new species), Nothobachia (monotypic), group, characterized by the presence of scincoid scales and the Psilophthalmus (monotypic), and Procellosaurinus (two new absence of eyelids, has been admitted as monophyletic since species) (Rodrigues, 1991a-c). Another important change was the past century (Boulenger, 1885; Presch, 1980; Rodrigues, the redefinition of the Gymnophthalmus and the inclu­ 1991a-c) . sion of the former Gymnophthalmus rubricauda and its junior The systematics of this eyelid-less microteiid rad iation have synonym G. multiscutatus in the genus Vanzosaura (Rodrigues, undergone major changes in the last years. Until recently, only 1991c). With the exception of Vanzosaura (which, although twogenera, Gymnophthalmus and Micrablepharus, were recog­ sympatric with the other genera, is widespread in open areas), nized (Presch, 1983). Since then, several new genera were all the other genera are restricted to this sandy dune region. described as inhab iting the sand dunes of the middle Rio Sao As this seven genera radiation of eyelid-less gymnophthal­ Francisco in the State of Bahia, Brazil. The area, characterized mid is monophyletic (Rodrigues, 1991c), we and some by an open xeromorphic vegetation, harbors an extraordinary colleagues started to gather morphological , karyotypic , alozym­ fauna, comprising 36 species of lizards and amphisbae- ie, and mitochondrial DNA data to obtain a better understand­ ing of their evolution. The present paper describes the karyo­ typic patterns oftwo of these genera and follows a previous one dealing with the karyotypes of Gymnophthalmus, the first study on microtei id karyotypes to include banding patterns and DNA Receivcd 13 October 1995; revision accepted 28 May 1996. replication bands (Yonenaga-Yassuda et al., 1995). Supported by FAPESP, CNPq, and FIN EP. The majority of cytogenetic studi es on Squamata have been Requcst reprints from Dr. Yatiyo Yonenaga-Yassuda, Depart amen to de Biologia. 1nstituto dc Biociencias, Uni vcrsidad e de Sao Paulo, Sao Paulo, c.P. 11461, performed using con ventionall y (i.e., Giemsa) stained karyo­ CEP 05422-970 (Brazil); teleph on e: 0 11-818-7574; fax: 055-011-818-7553. types; some of these studies have also included the results of

E-mail kargcr@kargcLch © \ 996 S. Karger AG, Basel KARGER Fax +4 \ 6 \ 306 \2 34 030 1-0\ 71/96/0743- 0203S10.00/0 htt p.r/ www. ka rgcr.ch Table I Species, sex, local ity. and speci men num bers of the mi croteiids equidis ta nt from the borders of each ch rom atid was measured. Mcasurs, studie d men ts between th e two chromatids of eac h chro moso me were averaged in determi ni ng the length of each arm. The relat ive length of each chromoso me Species Sex' Local ity" Specimen number expressed as a percentage of the length of the total haploid set, was deter: mined in 10 co nventionally sta ined metaphases fro m P.erythrocercugand V Procellosaurinus 3F Ibiraba LG349,LG356, LG362 rubricauda. T he size of the chromoso mes, includ ing the lengths of constinj, Ibiraba LG279, LG35 7, LG363 erythrocercus 3 M tivc heteroch rom atin blocks. was measured on C-banded karyotypes of 10 P. tetradactylus 3M Alagoado LG444, LG466, LG486 and 8 metaphase cell preparations from P. erythrocercus and V. l"unZOSuura IF LG438 respecti vely. T he lengths of all C-bands were then expressed as a p erc cnt ag~ Vanzosaura 4M Ibiraba LG352, LG366, LG374 , LG376 of the length of the total haploid set for both species. Th e same preparations rubricauda 6M Vacaria LG257, LG264 , LG354, LG360. were used . after C-ha nd ing treatm ent. to qu antify the relative amount of het­ LG 370, LG38 2 erochrom atic DNA in the interphase nucleu s. 1M Santo Inacio LG353 1M Capim Verde LG284 Nuclear DNA call lent 4F Ibiraba LG351. LG364, LG375, LG377 Nuclear D NA content measurements were performed on conventional 3 F Vacaria LG371, LG372. LG37 3 cyto logical preparat ion s. Slides were fixed for 3- 4 min in 3 part s etha nol, 1 part acet ic ac id, a nd I part chloroform and then sta ined by the Feulgen reac­ F = female; M = male. tion (hydrolysis in HCI for 12 min at 60 ° C followed by Schiff's reagent for 60 All localitiesare in the State of Bahia, Brazil. min at roo m temperature). Cyto pho to metric mea surements of stained cells were carried out with a Zeiss microspectrophotometer equ ipped with a 0.5-1101 step scanning stage (Zeiss), which was interfaced to a mic rocomputer. The measurem ents were made at 570 urn , using a 100 x 1.30 N.A. oil-immersion objective. At least 30 C-banding and/or Ag-NOR stairung. Only a few reports of nuelei fro m eac h P. erythrocercus and V rubricauda specimen were quant i­ fied. Human cultu red fibroblast cells were prepared at the same lime and replication banding patterns in reptilian karyot ypes have been used as a standa rd to determ ine the relat ive D NA conte nt of the microteiid published (Yonenaga-Yassuda et aI., 1988, 1995; Volobuev and nuclei. Pasteur, 1988; Volobuev et aI., 1993). Cytogeneti c studies based on banding techniques and DNA analysis using restric­ tion enzymes have been performed on lacertid lizards (Capri­ Results glione et aI., 1989; Olmo et aI., 1990). Here we report the results ofa variety of cytogenetic studies Procellosaurinus erythrocercus (2n =40) utilizing conventional and banding techniques-G-banding, The karyotype comprises 8 pairs ofmeta- and submctaccnt­ C-banding, Ag-NOR staining, and R-banding followi ng incor­ ric macrochromosomes and 12 pairs of acroce ntric, subtelo­ poration of 5-bromodeoxyuridine (BrdU)-on three species of centric, and submetacentric microchromosomes (Fig. I a). eyelid-less microteiids: Proce/losaurinus erythrocercus, P. te­ RBG-banding revealed distinct macrochromosomal band tradactylus, and Vanzosaura rubricauda. Use of these banding patterns. All of the macrochromosomes could be paired , and techniques helped establish species-specific karyotypes and each homologous pair was unequivocally identified by the made it possible to compare them in order to evaluate the chro­ appearance of its replication banding pattern. Positi ve CBG­ mosomal and systematic relationships among these species. We bands showed late-replicating DNA (Fig. Ib). also present measurements ofthe length and nucl ear DNA con­ C-banding disclosed conspicuous blocks of heterochroma­ tent ofthe chromosomes. tin . Prominent telomeric C-bands were present in many chro­ mosomes. In pairs I, 4, 6, 7, and 8, C-bands were detected in the two telomeric regions, as well as in the centromeric regions. Materials and methods In pairs 3 and 5, C-bands were present in the telomeric region ofthe short arms and in the centromeric regions. Pair 2 present­ Specimens ed only a single block of heterochromatin in the telomeric Cytogenetic anal yses were performed on 6 specimens ofP. erythrocercus (3 males and 3 fema les), 4 speci mens of P. tetradactylus (3 males and I region of the long arm. Among the microchromosomes, con­ fem ale), and 19 specimens of V. rubricauda (12 males and 7 fema les). Vou ch­ spicuous C-bands were detected in the distal regions of the long er specimens were deposited in the collection of the Mu seu de Zoo logia da arms ofpairs 9 through 19and also in the short arms of pairs 9, Unive rsida de de Sao Paulo (MZUSP). Tab le I shows the localities and 10, 11 , 14, and 15. The smallest microchromosome pair (pair mu seum catalog numbers ofthe specimens that were studied . All of the chromosome dat a were obtained from fibro blast cultures. Th e 20) showed a slight C-band in the centromeric region. Practical­ cell lines were grown at 29 °C in Du lbecco's modifi ed Eagle's medium sup­ ly all of the chromosomal pairs could be identified by their C­ plem ented with 20 % fetal calfserum. For R-banding, BrdU (final con centra­ banding patterns (Fig. 3a). tion, 25 11g1ml) was added to the cell cult ures for 7-12 h prior to harvesting. Silver staining of 31 metaphases from three specimens C-bands and Ag-NORs were obtained using rout ine meth od s. In some males, revealed Ag-NORs in the short arms ofone pair of microchro­ testicular mat erial was analyzed to chara cterize meiot ic phases. Cultured cells, frozen in liquid nit rogen , a re stored in the Cell Collection of the Dep ar­ mosomes. In well-defined conventionally (Giemsa) stained me­ tamento de Biologia, IBUSP, Sao Paulo, Brazil. The difficulty in establishing taphases, these NORs app eared as secondary constrictions pr imary microteiid cultures ca n be appreciated by the great number ofbio p­ (Fig. 4) . sies needed to obtain one good culture (about 20 specimens). Anal ysis of the testicul ar material showed bivalents of eight Length measurements macrochromosomes and 12 microchromosomes in diplotene Chromosome contours wer e dr awn from projected photograp hic nega­ cells (Fig . 5a) and 20 chromosomes at metaphase II (Fig. 5b). tive images. Then, using a spur-wheel line measurer, the length of a line

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Fig. 1. (a, b) Procellosaurinus erythrocercus male; (e, d) P. tetradactylus female. (a, c) Con ventional Gicmsa-staining: (b, d) RBG-banding.

Procellosaurinus tetradactylus (2n =40) C-banding showed constitutive heterochromatic regions All the specimens of this species also exhibited 40 chromo­ mainly in the telomeric regions of the chromosomes. Promi­ somes, which could be arranged into 8 pairs ofmetacentric and nent telomeric C-bands were detected in the short and long submetacentric macrochromosomes and 12 pairs of acrocent­ arms ofpairs 4, 7, and 8; in the short arms ofpairs 3 and 5; and ric, subtelocentric, and submetacentric microchromosomes in in the long arms of pair 6. Pair 3 presented a weak C-band in decreasing size (Fig . Ie). the distal region ofthe long arm. In pair I, a weak C-band was Following BrdU incorporation, each chromosomal pair had observed in the telomeric region ofboth the short and long arm. a characteristic replication banding pattern, permitting un­ Pair 2 showed weakly stained interstitial and telomeric C­ equivocal identification (Fig. Id). bands in the long arm . The distal regions ofthe long arms ofthe

Cytogenet Cell Genet 74:203-210 (1996) 205 Ag-NORs occurred in the secondary constriction ofan acro­ centric pair of microchromosomes, based on observations of 33 metaphases from three (Fig. 4).

Vanzosaura rubricauda (2n = 40) Thi s karyotype exhibited 8 pairs of biarmed (metacentric and submetacentric) macrochrornosomes and 12 pairs of acro­ centric, subtelocentric, and submetacentric microchromo­ somes (Fig. 2). Twenty bivalents (including eight macroch--; mosomes and 12 microchromosomes) were found in diplotene .:,.... 1~ " .-,' ~ ~ ' .. " cells. 6 7 8 9 10 The RBG-banding patterns allowed precise identification of the majority of the chromosomes. Some very-late-replicating "A' ( ~. ~: {i'i bands were detected in the regions of the few C-positive bands , ~; ' " '-.. '- - 11 12 14 15 observed (Fig. 2b). C-banding patterns appeared in a few chromosomes. In the . ~ -. .'~-~ .. _ :j_ 'I I .\ ~ ' macrochromosomes, a distinctive pattern occurred only in the ~ - - _ . ', -- \~c~' <~- ~ , 16 17 19 " 20 distal region of the long arm ofpair 3. The telomeric regions of a both arms of pairs 1,2, and 4 showed very weakly stained C­ bands. These weak bands were also detected in the tclomeric region ofthe long arm of pair 5. Only three pairs of microchro­ mosomes presented a C-positive band: the interstitial region of the long arm of pair 9, almost the whole long arm of pair 13, and the short arm of pair 16 (Fig. 3c). Ag-NORs were observed in the telomeric region of the long arm ofa submetacentric macrochromosome (pair 6) and in the short arm of an acrocentric microchromosome pair. In an anal­ ysis of nine specimens, four Ag-NORs were found among 152 1 2 3 4 5 metaphases (Fig. 4). Some high-resolution banding patterns were obtained after l- BrdU treatment in prometaphase chromosomes of V rubricau­ da. A much larger number ofbands could be seen in metaphase /,", ( . chromosomes. In many cases, however, these prometaphases ·'_ _"--r. were less informative because the main bands were often indis­ 6 7 8 10 tinct and because the chromosomes frequently overlapped. Dif­ ficulties also occurred in the analysis of extremely long BrdU­ i -C' banded chromosomes. The primary constrictions in the cen­ I ' I tromeric regions were not evident in some cases, and some dif­ 12 13 14 15 ferences between homologs were also observed in their replica­ ! ,j'~ ' tion banding patterns.

17 18 20 b '.'. Comparative REG-banding patterns Chromosome homoeologies were established among all the Fig. 2. Vanzosaura rubricauda male. (a) Conventional Giemsa-staining; macrochromosomes of P. erythrocercus, P. tetradactylus, and (b) RBG-banding. V rubricauda, showing the same replication banding patterns despite differences in their C-banding patterns (see, e.g., pairs 3 and 7). A composite RBG partial karyotype of the three species is presented in Fig. 6. microchromosomes (pairs 9 to 19) displayed especially large quantities of heterochromatin. In addition, C-bands were evi­ Length measurements and analysis ofDNA content dent in the short arms ofmicrochromosome pairs 9, 10, II , and Chromosomal measurements in P. erythrocercus and V rub­ 13. An interstitial band was apparent in pair 14. The smallest ricauda were performed on conventional and C-banded karyo­ pair (pair 20) did not show constitutive heterochromatin types in metaphase cells with good chromosome definition due (Fig.3b). Careful comparisons of the C-banded homologs to the differential quantity of constitutive heterochromatin. A showed minor variations in the size of some homologous het­ total of 38 karyotypes (both standard and C-banded) ofthe two erochromatic regions, but all chromosomal pairs could still be species was measured. Chromosomal arm lengths were ex­ identified by this kind ofbanding. pressed as percentages of the length of the total haploid chro-

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: ~:,; -,. · ~': tlli'; , i ~~../ 11 12 13 14 15 , I ~ : . -- - ' ;i\l ...... - 16 17 18 19 20 C Fig. 3. CBG-banding patterns. (a) Procellosaurinus erythrocercus female; (b) P. tetradactylus fema le; (c) Vanzosaura rubricauda female.

Fig. 4. NOR-bearing chro mosome s. (a) Asso­ ciation ofa conventionally sta ined rnicrochromo­ some pair in Procellosaurinus erythrocercus. (b, c) r i Ag-NOR-positive chromosome pairs in P. tetra­ 1 i dactylus showing association of the NOR-b earing chromoso mes. (d) Th e NOR-bear ing chromo­ somes in Van zosaura rubricauda include one rna­ a b c d crochromosome pair and one microchromosome pair (Ag-NOR staining).

Cytogenet Cell Genet 74:203-21 0 (1996) 207 a b

Fig. 5. Meioti c cells in Procellosaurinus erythroccrcus male specimens. (a) Dipl otene with 20 bival­ ents; (b) metaphase II with 20 chromosomes.

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Fig. 6. RBG-banded macrochromosomes sho wing replication band homoeologie s among the three species: Procellosaurinus erythrocercus(left), P. tetradactylus(center), and Vanzosaurarubricauda (right).

mosome set. In C-banded karyotypes the sizes of positive C­ times that observed in the second species. These data indicate bands were also determined. that the higher DNA content in P. erythrocercus results from an The mean relative length of conventionally stained chromo­ excess of heterochromatin, which, in most cases, is related to a somes of P. erythrocercus varied from 11.39 to 1.72, while those greater number of C-bands in the microchromosomes. The rel­ of V rubricaudavaried from 12.39 to 1.43 (Table II). ative lengths of the chromosomes showed similar results, the P. Analysis of DNA content showed that P..erythrocercus has erythrocercus microchromosomes being longer than those of V. about 1.95 times more DNA than Vanzosaura. The amount of rubricauda (Table II). constitutive heterochromatin in the former species is 1.72

208 Cytogenet Cell Genet 74:203-210 (1996) Table II. Measurements of relative length as a percentage of the haploid saurinus. Certainly, the small amount of constitutive hetero­ set (in a total of 20 metaphases), percent constitutive heterochromatin, and chromatin in both the macrochromosomes and microchromo­ DNA content somes of V rubricauda, as compared to the distinct large het­ Procellosaurinus erythrocercus Vanzosaura rubricauda erochromatic blocks in Procellosaurinus chromosomes, is a dis­ tinctive pattern that can be evidenced even by simple C-band­ zc DNA(pg) 5.60 ± 1.36' 2.87 ± 0.73 % Heterochromatin 39.0 22.7 ing. None of the macrochromosomes of V. rubricauda showed Chromosome pericentromeric C-bands , and only pair 3 showed a clear telo­ 1 11.39 ± 0.48' 10.39 ± 0.78 meric C-band in the long arm. Furthermore, the conspicuous 2 10.62 ± 0.62 10.92 ± 0.79 3 9.04 ± 0.50 10.20 ± 0.67 C-bands of the microchromosomes of Procellosaurinus do not 4 8.70 ± 0.47 9.40 ± 0.50 exist in the majority of the microchromosomes of Vanzosaura 5 8.30 ± 0.52 8.99 ± 0.63 (except in pairs 9, 13, and 16), which were smaller due to the 6 7.60 ± 0.42 8.31 ± 0.57 7 7.60 ± 0.44 7.54 ± 0.55 absence ofheterochromatin. 8 6.58 ± 0.54 6.65 ±0.48 Each ofthe three microteiid species examined in this report 9 3.37 ± 0.39 2.91 ± 0.49 has a specific karyotype, based on its C-banding pattern, which 10 3.15 ± 0.50 2.37 ± 0.32 11 3.01 ± 0.50 2.21 ± 0.40 differs in both the localization and quantity of constitutive 12 2.62 ± 0.34 2.08 ± 0.21 heterochromatin. The additional blocks of C-heterochromatin 13 2.46 ± 0.49 2.06 ± 0.26 in Procellosaurinus correspond to an increase in nuclear DNA 14 2.36 ± 0.23 2.30 ± 0.48 15 2.50 ± 0.43 2.37 ± 0.41 content, when compared with that of Vanzosaura. 16 2.57 ± 0.39 2.14 ± 0.37 Interspecific comparisons ofC-banding patterns are ofbasic 17 2.29 ± 0.29 1.89 ± 0.28 importance for cytotaxonomic studies. It has been shown that 18 2.09 ±0.23 1.90 ± 0.34 19 2.09 ±0.18 1.70 ± 0.26 the three species of Gymnophthalmidae have a striking similar­ 20 1.72 ± 0.30 1.43 ± 0.30 ity in chromosome number and morphology, but can be differ­ entiated by the position and amount of heterochromatin in , Mean ± standard deviation. probably homoeologous chromosomes. Differences between species in the quantity and position of heterochromatic blocks based on C-banding patterns are found Discussion in all groups of organisms. Closely related species may differ not only in the amount of heterochromatin but also in their All three species of microteiids exhibit the same diploid staining properties and DNA composition (Sumner, 1990). number of 2n =40, with a clear-cut distinction between 8 pairs Among lacertids, Olmo et al. (1990) showed that constitutive of macrochromosomes and 12 pairs of microchromosomes. heterochromatin ranges from a minimum of only 4% of the Superficial examination of conventionally stained metaphase total genome to a maximum ofmore than 20%. A detailed anal­ preparations suggests that there are no important karyotypic ysis of the genome of Podarcis sicula revealed the presence of differences among these species. But each of them presents a two highly repetitive satellite DNAs, evidenced by restriction unique, species-specific karyotype when subjected to routine sites localized at the level of the centromeric C-bands (Capri­ chromosomal banding techniques. glione et aI., 1989) and interspersed on the chromosomes (Car­ An exceptional C-banding pattern was found in both species done et al., 1990). The use of C-band variations in cytotaxon­ of Procellosaurinus, which displayed a great amount of consti­ omic studies could be debated once there are plenty ofcases of tutive heterochromatin. Comparative chromosomal analyses intraspecific variation, showing up mainly as changes in the showed that all eight macrochromosome pairs present a dis­ amount of heterochromatin between homologs within a given tinct pattern. Pairs I to 8 of P. erythrocercus have pericentro­ individual, between individuals of the same population, and meric C-bands that are absent in the same eight chromosome between different populations of the same species. The differ­ pairs of P. tetradactylus. Pair 2 of both species also presents a ent C-band patterns in the three microteiid species described differential C-banding pattern: the telomeric block in the long here, however, represent a discontinuous variation in hetero­ arm of pair 2 is more prominent in P. erythrocercus, and this chromatin distribution patterns, resulting in characteristic spe­ pair in P. tetradactylusshows an interstitial band in its long arm cies-specific C-bands. that is absent from P. erythrocercus. The C-bands in pair I ofP. C-band studies in conservative karyotypes with apparently erythrocercus are also more conspicuous than those in the same similar chromosomal morphologies also showed a remarkable pair of P. tetradactylus. The C-banding patterns of the micro­ interspecific variability among four lacertid species (Odierna et chromosomes of both species are very similar. Both species al., 1985) and among six tropidurid lizards, when associated probably share the same NOR-bearing microchromosome with detailed characterization of the macrochromosomes and pair. microchromosomes and identification of Ag-NOR locations The karyotype of V rubricauda can be readily differentiated (Pellegrino et aI., 1994). from those of the two species of Procellosaurinus due to the Ag-NOR staining is an essential part of the characterization morphology of the microchromosomes, even in conventionally of a species karyotype. Using this method, researchers have stained preparations, and due to the presence offour Ag-NORs now localized NORs in a large number of species. It was consid­ (in macrochromosome pair 6 and in a microchromosome pair) ered in some cases that the species that belong to the same in place of the two Ag-NORs found in both species of Procello- defined taxonomic unit usually have an NOR on the same pair

Cytogcnet Cell Genet 74:203-210 (1996) 209 of chromosomes, but in other cases the similarity in the loca­ cation banding patterns. All macrochromosomes of the three tion of the NOR could be a result of convergent evolution species arc homoeologous along their ent ire length, based On (Amemiya and Gold, 1988). their RBG-banding patterns (Fig. 6), despit e the rcmarkable The localization of NORs may be of some diagnostic rele­ differences in their C-banding patterns. vance in the family Gymnophthalmidae. No two species in this Our cytogenetic and cytophotometric DNA measurements famil y that have been stud ied by silver staining have the same indicate that there are more similarities between the two spe­ type of NOR-bearing chromosomes: in the unisexual species cies of Procellosaurinus than either species shares with Vanzo­ Gym nophthalmus underwoodi (2n = 44), two Ag-NORs are saura. Thi s corroborates the idea of two separated genera based located in the telomeric region ofan acrocentric macrochromo­ on morphological characterist ics. Th e use of bandin g tech­ some pair; in G. leucomystax (2n = 44) and Gymnophthalmus niques clearly allows these species to be characterized cytogene­ sp. n. (2n = 44), multiple Ag-NORs are located on microchro­ tically and lays the groundwork for evalu ation of the mecha­ mosome pairs (Yonenaga-Yassuda et aI., 1995); in P. erythro­ nisms involved in the process of their chromosomal evolution. cercus and P. tetradactylus, a single pair of NOR-bearing Morphological, ecological, and biochemical features associated microchromosomes have been observed (this stud y); and in V with our cytogenetic dat a will undoubtedly contribute to the rubricauda. Ag-NORs are present on one pair of microchro­ establishm ent of ph ylogenetic relationships in these eyelid-less mosomes and one pair of macrochromosomes (this study). microteiid species. Heteromorphic sex chromosomes were not found in any of the three species of microteiids in either conventionally stained karyotypes, C-banded or R-banded metaphases, or meiotic Acknowledgements cells. Th e a utho rs a re gra te ful to D r. Regina C. Mingroni Nc tto and Marta The use of BrdU in cultures allowed us to unequivo­ Svartman , M.S., for criticall y reviewing th e m anusc ript , to Mr s. Miriam cally characterize each chromosome pair of the three species Romeo for technical assistance, and to Dr. P aulo A. Otto and Alexandre and to perform some comparative analyses on the basis ofrepli- Uarth Chr isto ff, M .S., for sta tistical ass ista nce.

References

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