Cytogenet Cell Genet 74:203-210 (1996) Cytogenetics and CellGenetics Chromosomal banding patterns in the eyelid-less microteiid radiation: Procellosaurinus and Vanzosaura (Squamata, Gymnophthalmidae) 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 species can be differentiated by the position and tetradactylus. and Vanzosaura rubricauda (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 genus 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 lizards is monophyletic (Rodrigues, 1991c), we and some by an open xeromorphic vegetation, harbors an extraordinary colleagues started to gather morphological , karyotypic , alozym­ reptile 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.