Hereditas 125: 257-264 (1996) Comparative cytogenetic analysis with differential staining in three species of Liulaemlcs (Squamata, Tropiduridae) CAROLINA ELENA VIfiA BERTOLOTTO’ , MIGUEL TREFAUT RODRIGUES’, GABRIEL SKUK~,AND YATIYO YONENAGA-YASSUDA’ ‘ Departamento de Biologia, Instituto de Biocigncias, Universidade de SLio Paulo, S6o Paulo, SP, Brad ’Departamento de Zoologia, Instituto de Biocigncias, Universidade de SLio Paulo, SLio Paulo, SP, Brasil BERTOLOITO,C.E.V., RODRIGUES, M T . SKUK,G. and YONENAGA-YASSUDA,Y. 1996. Comparative cytoge- netic analysis with differential staining in three species of Liolaemus (Squamata, Tropiduridae). ~ Hereditas 125 257-264. Lund, Sweden. ISSN 0018-0661. Received July 31, 1996. Accepted November 24, 1996 Comparative cytogenetic studies based on conventional staining, CBG-banding, RBG-banding, and Ag- NOR staining, were performed on three species of Liolaemus: L. occipitalis, L. [utzue and L. wiegmannii. The three species have 2n=34, with closely similar karyotypes and an XX:XY mechanism of sex determination. The cytogenetic analysis allowed the detection of differences among the macrochromosomes of the three species and some other features characteristic of each species. The karyotype of L. occipitalis and the banding patterns of L. lufzae and L. wiegmannii have not been reported previously. Yariyo Yonenaga-Yassuda, Departamento de Bwlogia, IS-USP, Sao Paula, Brazil, C. P. 11461, CEP 05422-970. E-mail: yyassuda @ spider.usp.br The Liolaeminae is a large and diverse L. occipitalis had differentiated from a common monophyletic group of tropidurid lizards endemic ancestor because of isolation of coastal popula- to South America. The approximately 135 species tions after the last glacial period. of Liolaeminae have traditionally been classified The former “iguanids” are nowadays grouped into several different genera, but currently only into the families Tropiduridae, Polychrotidae, three genera are recognized: Ctenoblepharis, Phy - Phrynosomatidae, Opluridae, Iguanidae, Hoplo- maturus, and Liolaemus (FROSTand ETHERIDGE cercidae, Crotaphytidae, and Corytophanidae 1989; ETHERIDGE1995). Liolaemus is, by far, the (FROSTand ETHERIDGE1989). Within this group- most diverse genus, including 124 species ranging ing, the karyotype with 2n = 36 chromosomes (12 from Tierra del Fuego, the southernmost part of metacentric or submetacentric macrochromosomes South America, throughout Argentina, Chile, Bo- plus 24 microchromosomes) has been considered livia, Peru, and coastal regions of Uruguay and primitive (GORMAN1973; PAULLet al. 1976). southern of Brazil. Along this enormous area, the Karyologically, the genus Liolaemus is more di- genus occurs in very different habitats, from the verse than other tropidurid genera. Among the highest Andean mountains to sand dunes at sea former “iguanids”, chromosomal variation in Lio - level. Of the three species described, Liolaemus laemus involves diploid numbers ranging from wiegmannii is the most widespread, occurring 2n = 30 to 2n = 44 (NAVARROet al. 1981; LAM- in regions of sandy soils from northwestern BOROT and ALVAREZ-SARRET1989). This varia- Argentina to coastal regions in the province of tion is only comparable to that of the North and Buenos Aires (CEI 1993). This species also occurs Central American phrynosomatids of the genus on coastal sand dunes of Uruguay. The other two Sceloporus. species are restricted to sand dunes in the Brazilian The Liolaemus genus encompasses species like L. states of Rio Grande do Sul and Santa Catarina alticolor (2n = 30, 12M + 18m), L. capillitas (2n = (Liolaemus occipitalis) and Rio de Janeiro (Liolae- 32, 12M + 20m), L. nitidus (2n = 32, 12M + 20m), mus Zutzae) (Fig. 1). These three species together and L. pictus (2n = 34, 12M + 22m). These species with L. cranwelli, L. multimaculatus, L. rabinoi, L. exhibit a relatively conserved karyotype, with six riojanus, L. salinicola, and L. scapularis were in- pairs of macrochromosomes which are similar in cluded in the monophyletic wiegmannii group by size and shape to those found in the primitive ETHERIDGE(1995). VANZOLINIand AB’SABER karyotype (2n = 36, 12M + 24m). The karyotypes (1968) also suggested that the species L. lutzae and of these species differ from the ancestral form in 258 c. E. v. BERTOLOTTO ET AL. Hereditat I25 (1996) N t Liolaemus occipitalis Liolaemus wiegmannii Fig. 1. Approximate distribution of the species studied of Liolaemus from southern South America and collecting localities: 1. Cab0 Frio, RJ (Brazil); 2. Laguna, SC (Brazil); 3. Playa Pascual (Uruguay), and 4. Villa Gesell (Argentina). that they have a reduced number of microchromo- metacentric or submetacentric macrochromosomes somes, probably due to centric fusions (ESPINOZA and a second group characterized by a larger num- and FORMAS1976; LAMBOROTet al. 1979; LAM- ber of macrochromosomes with extensivc kary- BOROT and ALVAREZ-SARRET1989; NAVARRO otypical variation (LAMBOROTet al. 1981). 1992). On the other hand, karyotypes with a large GORMANet al. (1967) used conventional stain- number of macrochromosomes were observed in L. ing to describe a 2n = 34 karyotype for L. lutzae. nigromaculatus (2n = 40, 16M + 24m), L. platei NAVARRO( 1992) reported, without photographic platei (2n = 42, 16M + 26m), L. sp. (2n = 44, with- documentation, the chromosome number 2n = 32 out a clear distinction between macro- and mi- in one specimen of L. wiegmannii from northwest- crochromosomes), and a northern chromosomal ern Argentina. In a table in the same paper, how- polymorphic race of Liolaemus monticola monticola ever, the species was reported as having 2n = 34. from central Chile, which presents three variants: In the present paper we describe and compare the 2n = 38 (14M + 24m), 2n = 39 (15M + 24m) and karyotypes of L. occipitalis, L. lutzae, and L. wieg- 2n = 40 ( 16M + 24m) ( LAMBOROTand ALVAREZ- mannii. The analysis is based on conventionally SARRET1989; LAMBOROT1991). In all these cases, stained and banded karyotypes plus meiotic data. the increase in the number of macrochromosomes could be explained by centric fission events. The presence of telocentric and acrocentric macrochro- Materials and methods mosomes supports this hypothesis. In this way, it is possible to divide the genus Four specimens of Liolaemus occipitalis, five speci- Liolaemus into two groups: one containing species mens of L. lutzae, and four specimens of L. wieg- that retain a relatively primitive karyotype with 12 mannii were karyotypically studied (Table 1). The Heredifas 125 (1996) CHROMOSOME BANDING IN THREE TROPIDURID LIZARDS 259 Table 1. Species, specimen number, sex, locality, 2n, and total of metaphases studied with conventional and differential staining for the three species of Liolaemus Species Specimen Sex Locality 2N Total of number metaphases Lwlaemus occipitalis LG3 F Laguna, Santa Catarina, Brazil LG4 M 34 56 LG5 M (28"28'S, 48"46W) LG7 F Liolaemus lutzae LG667 M Cab0 Fno, Rio de Janeiro, Brazil LG668 F LG673 M (22"52'S, 42"OI'W) 34 24 LG67Y F LG680 M Liolaemur wiegmunnii LG75Y M Playa Pascual, Uruguay (34"45'S, 56"35'W) 34 38 LG760 M Villa Gesell, Argentina LG762 F (37"15'S, 56W'W) LG764 M M = male; F = female specimens were identified and deposited at the wiegmannii and submetacentric in L. occipitalis Museu de Zoologia da Universidade de Sgo Paulo (Fig. 2). Pair 5 is metacentric in L. lutzae and (MZUSP). submetacentric in L. occipitalis and L. wiegmannii Chromosomal spreads were obtained from bone (Fig. 2). It was difficult to establish the exact marrow, intestine, liver, and spleen preparations morphology of the microchromosomes. They are after in vivo treatment with a solution of Fleisch- usually acrocentric, but in some metaphases of L. mann yeast (COLEand LEAVENS 1971) and col- occipitalis some pairs are metacentrics. chicine, according to the methodology described in In L. occipitalis, a proximal constriction was KASAHARAet al. (1987). Mitotic preparations of observed in the short arm of pair 1 (Fig. 3). A distal culturea fibroblasts from muscle biopsies were ob- constriction in the long arm of pair 2 was detected tained for one specimen of L. lutzae following the in the three species, but in L. wiegmannii it is more procedure described by YONENAGA-YASSUDAet al. distally located (Fig. 4). One specimen of L. wieg- (1988). mannii exhibits this constriction in only one homo- Diploid numbers and chromosome morphologies logue of pair 2 (Fig. 2c). were determined after Giemsa staining. The chro- An XX:XY chromosome mechanism of sex deter- mosomes were also analyzed after C-banding (SUM- mination occurs in the three species. The Y is a NER 1972), Ag-NOR staining (HOWELLand dot-like microchromosome, present only in male BLACK 1980), and replication R-banding after in cells (Fig. 2 and 3) and the X is probably a large vitro incorporation of 5-BrdU followed by FPG microchromosome morphologically indistinguish- staining (DUTRILLAUXand COUTURIER1981). able from some of the other microchromosomes. Meiotic analyses were carried out on males of L. Diplotene cells of L. occipitalis and L. wiegmannii occipitalis and L. wiegmannii. show six macrobivalents, one bivalent correspond- ing to pair 7, and ten microbivalents (Fig. 5a, c, d). A heteromorphic microbivalent was detected in Results some meiotic cells, representing the sexual pair XY (Fig. 5a, d). Metaphases I1 with 17 chromosomes Conventional staining were observed in L. occipitalis (Fig. 5f). The karyotypes of Liolaemus occipitalis, L. lutzae, and L. wiegmannii have 2n =