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 = 34 (12M 22m). Pairs + Nucleolus organizer regions (NORs) 1, 3, and 6 are metacentrics, pair 2 is submetacen- tric, and pair 7 is acrocentric with an intermediate The three species present Ag-NORs in the distal size between the macro and microchromosomes secondary constriction in the long arm of pair 2 (Fig. 2). Pair 4 is metacentric in L. lutzae and L. (Fig. 4). In L. wiegmannii they are more distally 260 c. E. v. BERTOLOTTO ET AL. Hereditar 125 (1996)

Fig. 2s-c. Karyotypes after conventional staining. a Male Liolaems occipifalis. b Female L. htzae. c Male L. wiegmannii. All with 2n = 34 (12M + 22m). Insets: sex chromosomes of the opposite sex. Bar = IOpm. located, corresponding to the terminal secondary bands at the pericentromeric regions of all constriction observed in routine Giemsa staining. macrochromosomes (Fig. 6). These regions in L. occipitalis are larger than those of L. lutzae (Fig. 6) C-banding Some microchromosomes present slight C-bands in L. occipitalis and L. lutzae show conspicuous C- the same regions. The proximal constriction of pair Hereditas 125 (1996) CHROMOSOME BANDING IN THREE TROPIDURID LIZARDS 261

1 in L. occipitalis is C-band negative (Fig. 6a, b). included in the first group proposed by LAMBOROT Meiotic cells from L. occipitalis and L. wiegmannii et al. (1981), which encompasses species with show evident heterochromatic blocks in the macro- a relatively primitive karyotype, retaining six bivalents (Fig. 5b, e). In L. occipitalis these blocks metacentric or submetacentric macrochromosome are larger than those of L. wiegmannii (Fig. Sb, e). pairs. L. lemniscatus, L. platei curicense, L. pictus, and R-banding L. cyanogaster also exhibit 2n = 34 (12M + 22m) (ESPINOZAand FORMAS1976; LAMBOROTand Replication R-banding after 5-BrdU in vitro incor- ALVAREZ-SARRET1989), with six macrochromo- poration was obtained in L. lutzae and allowed the some pairs which are morphologically similar to exact identification of the first seven chromosome those of the karyotypes considered to be primitive pairs (Fig. 7). (2n = 36, 12M + 24m). The 2n = 34 karyotypes studied by us differ from the 2n = 36 form by having only 22 mi- Discussion crochromosomes and by the presence of an inter- mediate acrocentric pair. This pair could have Cytogenetic investigations on lizard chromosomes resulted from centric fusion or from tandem fusion using differential staining, are scarce in the litera- between microchromosomes, leading to lower ture. For instance, some of them have been per- diploid number. It is probable that the reduction in formed on tropidurids (KASAHARAet al. 1987; the number of microchromosomes is ancestral for YONENAGA-YASSUDAet al. 1988; PELLEGRINOet the whole genus, because until now no species is al. 1994). Previous reports on Liolaemus include known with 2n = 36 (12M + 24m). The condition only conventional staining. of 2n = 34 (12M + 22m) and 2n = 32 (12M + 20m) The karyotypes of L. occipitalis, L. lutzae, and is also found within many of the groups of Liolae- L. wiegmannii are very similar. All specimens mus considered by ETHERIDGE(1995). present 2n = 34 (12 metacentric or submetacentric Pair 7 of Liolaemus Iutzae was described as a macrochromosomes and 22 microchromosomes). metacentric by GORMANet al. (1967). In our According to these data, the three species could be analysis it is clearly acrocentric. 262 c. E. v. BERTOLOTTO ET AL Hereditas 125 (1996)

Fig. 5a-f. Meiotic cells from Liolaemus occipitalis (a, b, c, and f) and L. wiegmannii (d and e). a and d Diplotene cells with 17 bivalents, including the XY sex bivalent (arrows). b and e C-banding patterns in bivalents. c Diplotent bivalents in decreasing sizes. f Metaphase I1 with 17 chromosomes.

Among the other species of the wiegmannii the figure and text) with contradictory data for this group, there are only chromosomal data for latter species (NAVARRO1992). L. saIinicoIa (12M + 20m) and L. scapdaris Although the karyotypes of L. occipitalis, L. (12M + 20m in the table and 12M + 22m in both Iutzae, and L wiegmannii seem to be conserved, a Hereditas 125 (1996) CHROMOSOME BANDING IN THREE TROPIDURID LIZARDS 263

Fig. 6a-c. C-banded chromosomes. a Karyotype of a male Liolaemus occipitalis. b Pair 1 of an other metaphase of L. occipitalis with the proximal secondary constriction at the short arm. c Partial karyotype of a female L. lutzae. careful analysis of chromosome morphologies and wiegmannii; (b) pair 5 is metacentric in L. lutzae banding patterns allowed us to establish species- and submetacentric in L. occipitalis and L. wieg- specific characteristics: (a) pair 4 is submetacentric mannii; (c) a proximal constriction at the short in L. occipitalis and metacentric in L. lutzae and L. arm of pair 1 is only present in L. occipitalis; (d)

Fig. 7. R-banding pattern after BrdU incorporation in the macrochromosomes and in the intermediate pair 7 of Liolaemus lutzue female. 264 c. E. v. BERTOLOTTO ET AL. Herediras 125 (1996)

Ag-NORs occur at the distal region of the long COLE,C. J. and LEAVENS,C. R. 1971. Chromosome preparations arm of pair 2 in the three species, but in L. of amphibians and reptiles: improved technique. - Herpefol. Rev. 3: 102 wiegmannii they are more distally located; (e) DUTRILLAUX,B. and COUTURIER,J. 1981. La pratique de

the pericentromeric C-bands of the macro- I’analyse chromosomique. ~ Musson, Paris chromosomes of L. lutzae were smaller than those ESPINOZA,N. D. and FORMAS,J. R. 1976. Karyological pattern of two Chilean lizards species of the genus Lioluemus (Sauna; of L. occipitalis; (0 the heterochromatic blocks in Iguanidae). - Experientia 32: 299-301 the pericentromeric regions of the macrobivalents ETHERIDGE,R. 1995. Redescription of Ctenoblephuris udspersu of L. occipitalis were larger than those of L. wieg- Tschudi, 1845, and the taxonomy of Liolaeminae (Reptilia: Squamata: Tropiduridae). ~ Am. Mus. Nouitutes 3142: 34 pp mannii. FROST,D. and ETHERIDGE, R. 1989. A phylogenetic analysis and A dot-like microchromosome, present only in taxonomy of iguanians lizards (Reptilia: Squamata). -- Misc. Publ. Univ. Kansas. Mus. Nut. Hist. 81: 65 pp male metaphases, and a heteromorphic microbiva- GORMAN,G. C. 1973. The chromosomes of the Reptilia, a

lent (sexual pair XU) in diplotene cells, suggest an cytotaxonomic interpretation. ~ Cyroruxonomy and Vertebrute Euolution (eds A. B. CHIARELLI and E. CAPPANA),Acad. Press, XX:XY mechanism of sex determination in the New York, p. 349-424 three species. The X is probably a large microchro- GORMAN,G. C., ATKINS,L. and HOLZINCER,T. 1967. New mosome not morphologically recognizable. Other karyotypic data on 15 genera of lizards in the family Iguanidae, with a discussion of taxonomic and cytological implications. - tropidurids such as Tropidurus hispidus ( KASA- Cytogenetics 6 286-299 HARA et a]. 1987) and Uranoscodon superciliosus HOWELL,W. M. and BLACK,D. A. 1980. Controlled silver-stain- (PELLEGRINOet al. 1994) exhibit an XX:XY ing of nucleolus organizer regions with a protective colloidal developer: a I-step method. - Experientiu 36 1014-1015 mechanism, with a dot-like Y microchromosome ITURRA,P., VELOSO,A,, ESPEJO, P. and NAVARRO,J. 1994. and a heteromorphic microbivalent. In the genus Karyotypic and meiotic evidence for a robertsonian chromo- Liolaemus, a mechanism of sex determination was some polymorphism in the lizard Lioluemus fuscus (Tropiduri- dae, Sauria). - Rev. Brusil. Genet. 17: 171-174 only described in L. fuscus (ITURRAet al. 1994), KASAHARA,S., YONENAGA-YASSUDA,Y. and RODRIGUES,M. which presents a heteromorphic pair 8 in males, T. 1987. Geographical karyotypic variations and chromosome banding patterns in Tropidurus hispidus (Sauria, Iguanidae) with a metacentric X and a telocentric Y. from Brazil. - Curyologiu 40 43-57 Although the species studied in this paper show LAMBOROT,M. 1991. Karyotypic variation among populations of a similar karyotype in conventional staining, the Lioluemus monticola (Tropiduridae) separated by riverille barri- ers in the Andean range. - Copeiu 4: 1044- 1059 application of banding techniques and the good LAMBOROT,M. and ALVAREZ-SARRET,E. 1989. Karyotypic definition of the chromosome morphologies al- characterization of some Lioluemus lizards in Chile to (Iguanidae). - Genome 32: 393-403 lowed us detect differences between them and to LAMBOROT,M., ESPINOZA,A. and ALVAREZ,E. 1979. Kary- characterize each species. otypic variation in Chilean lizards of the genus Lsoluemus These results reinforce the importance of using (Iguanidae). - Experientiu 35 593-595 LAMBOROT, M., ALVAREZ,E., CAMPOS,I and ESPINOZA,A. differential staining to characterize species kary- 1981. Karyotypic characterization of three Chilean subspecies otypes in order to contribute to the understanding of Lioluemur monticolu. -- J. Hered. 72: 328-334 of the relationship among the lizard species. NAVARRO,J. 1992. Cariotipos de trece especies de lagattijas del noroeste argentino de 10s grupos Lioluemus, Euluemus y Ortho-

laemus. ~ Actu. Zool. Lillouna. 41: 225-230 NAVARRO,J., SALLABERRY,M., VELOSO,A. and VALEUCIA,J. Acknowledgements. ~ We are grateful to Katia Cristina Machado 1981. Diversidad cromosomica en lagartos (Squamata-Sauria). Pellegrino and Marta Svartman for their comments and valuable 1. Avances citotaxonomicos. Perspectiva de estudios evolutivos suggestions on the manuscript, to Alessandra F. Bizerra, Paulo E. en Iguanidae. - Medio Ambiente 5: 28-38 Vanzolini, Francisca C. Val, and Willian R. Heyer for collecting PAULL,D., WILLIAMS, E. E. and HALL,W. P. 1976 Lizard the animals, to Dr. Tien Hsi Chu for the fibroblast cultures, and karyotypes from the Galapagos Islands: chromosomes in pby- to Miriam Romeo for technical assistance. logeny and evolution. -Brevioru 441: 1-31 This work was supported by Conselho Nacional de Desenvolvi- PELLEGRINO,K. C. M., YONENACA-YASSUDA,Y. and Ro- mento Cientifico e Tecnolbgico (CNPq) and FundasHo de Am- DRIGUES, M. T. 1994. Cytogenetic studies in six species of par0 a Pesquisa do Estado de SHo Paulo (FAPESP) and Tropiduridde (Sauria). - Rev. Brasil. Genet. 17: 401-408 Financiadora de Estudos e Projetos (FINEP). SUMNER,A. T. 1972. A simple technique for demonstrating centromeric heterochromatin. - Exp. Cell. Res. 75: 304-306 VANZOLINI,P. E. and AB’SABER,A. N. 1968. Divergence rate in References South American lizards of the genus Liolaemus ( Sauria, Iguanidae). - Papiis Avulsos Zool. 21: 205-208 CEI, J. M. 1993. Reptiles del noroeste, nordeste y este de la YONENAGA-YASSUDA,Y., KASAHARA,S., CHU, T. H. and Ro- Argentina. Herpetofauna de las selvas subtropicales, Puna y DRIGIJEs, M. T. 1988. High-resolution RBG-banding pattern in Pampas. - Mus. Regionale di Scienze Nururufi, Torino, the genus Tropidurus (Sauria, Iguanidae). - Cytogenet. Cell Monogr. XIV: 949 pp Genet. 48: 68-71