Karyotype, constitutive heterochromatin and nucleolus organizer regions in two species of Liolaemus (Squamata, Tropiduridae)

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1 CARYOLOGIA Vol. 56, no. 3: , 2003 Karyotype, constitutive heterochromatin and nucleolus organizer regions in two species of Liolaemus (Squamata, Tropiduridae) ALEJANDRA HERNANDO Departamento de Biología, Fac. Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste, Avenida Libertad 5700 C.P Corrientes, Argentina. fax ; ahernando@infovia.com.ar. Abstract - A comparative cytogenetic analysis, based on chromosome morphology, Ag- NOR staining and CBG- banding was undertaken on two species of Liolaemus: L. chacoensis and Liolaemus azarai, a new undescribed species. The intestines, bone marrow and testes were sampled. Both species showed a karyotype with 12 biarmed macrochromosomes and 22 microchromomes. No sexual chromosomal heteromorphism was found. The patterns of C and NOR bands were similar. Constitutive heterochromatin was located in the pericentromeric regions of macrochromosomes and nucleolar organizer regions were detected on the long arm of the pair 2. Key words: Liolaemus; karyotypes; CBG banding; Ag-NOR. INTRODUCTION Liolaemus is a monophyletic lizard genus distributed only in austral South America from Peru to Tierra del Fuego. This genus lives in a variety of habitat from a sea level to over 5000 meters (CEI 1993; ETHERIDGE 2000). About 160 species of Liolaemus are recognized and less than 50% from this genus have already been studied cytogenetically after Giemsa standard staining. Liolaemus species exhibit extensive interespecific chromosome diversity with a diploid number ranging from 2n = 30 to 2n= 44 (LAM- BOROT and ALVAREZ-SARRET 1989). The centric fissions events are considered to be the most widespread mechanism for chromosomes change in Liolaemus (NAVARRO et al. 1988; LAMBOROT 1991; ITURRA et al. 1994). LAMBOROT and ALVAREZ-SARRET (1989) divided the Liolaemus species from west of the Andes into two groups: species with conservative karyotypes that have three different diploid numbers, 2n = 30 ( ), 2n = 32 ( ) or 2n = 34 ( ). The karyotype with six pairs of metacentric macrochromosomes is proposed to be primitive within Chilean Liolaemus. The microchromosomes number variations could be explain by centric fussions events from the primitive iguanid karyotype 2n= 36 ( ) (GORMAN et al. 1967; ESPINOSA and FOR- MAS 1976; LAMBOROT et al. 1979; NAVARRO et al. 1981); species with fissioned karyotypes and a increased diploid number, 2n = 38 ( ), 2n = 40 ( , ), 2n = 42 ( ), or 2n = 44 ( ). These karyotypes do not show great differences between macro- and micro-chromosomes. The largest telocentric chromosomes are assumed as result of centric fissions from biarm macrochromosomes. (NAVARRO et al. 1981; ITURRA et al. 1994; LAMBOROT and ALVAREZ-SARRET 1989). In Liolaemus species from west of the Andes chromosome polymorphism involving fissions, pericentric inversions and heterochromatin additions were described (LAMBOROT et al. 1979; LAMBOROT 1991; ITURRA et al. 1994). Triploid individual have been also reported (LAMBOROT and ALVAREZ-SARRET 1989; LAMBOROT and VÁSQUEZ 1998).

2 270 HERNANDO Karyological investigations on Liolaemus from east of the Andes are scarce. Species analysed using conventional staining show less chromosome diversity than Chilean species and have conservative karyotypes (2n = 30, 32 or 34) (GOR- MAN et al. 1967; NAVARRO 1992; AIASSA et al. 1998, 1999). Cytogenetic data based on Ag-NOR and C-banding were presented for only three Liolaemus species (VIÑA BERTOLOTTO et al. 1996). In the present paper, I describe the karyotypes of L. chacoensis and Liolaemus sp. n. using conventional staining and banding techniques. The former specie is a typical inhabitant of Chacoan region. Liolaemus azarai is a sand lizard confined to sandy areas of aeolean origin in northeast of Argentine and bordering areas of Paraguay (AVI- LA 2003) MATERIAL AND METHODS Material - Liolaemus chacoensis: UNNEC ( ),01984 ( ), ( ) Ingeniero Juárez Formosa; UNNEC ( ) El Rosillo; UNNEC ( ) Paraje San Lorenzo; UNNEC ( ), ( ) Paraje Santa Cruz (Argentina). Liolaemus azarai: UNNEC ( ); ( ); ( ); ( ); ( ) Curuzú Laurel; UNNEC-150 ( ); 151 Fig. 1 Collecting localities: 1 Ingeniero Juárez, 2 El Rosillo, 3 Paraje San Lorenzo, 4 Paraje Santa Cruz, 5 Isla Yacyretá, 6 Curuzú Laurel, 7 Estancia San Antonio, 8 Estancia Rodeo Porá. ( ) Estancia San Antonio; UNNEC -158 ( ) Estancia Rodeo Porá (Argentina); UNNEC ( ); ( ); ( ) Isla Yacyretá (Paraguay). Voucher specimens were deposited in the collection of the Facultad de Ciencias Exactas y Naturales, Universidad Nacional del Nordeste (UNNEC). Fig. 2 Standard karyotypes (a) L. chacoensis (2n = 34) and (b) Liolaemus azarai (2n = 34). Bar 10 µm.

3 COMPARATIVE CYTOGENETIC ANALYSIS ON TWO SPECIES OF LIOLAEMUS 271 Metaphase chromosomes were obtained by squash technique of intestinal epithelium, following incubation of small fragments of intestine in distilled water and fixation on 50% acetic acids. Routine air-dried preparation from cell suspensions of bone marrow and testes were prepared after hypotonic treatment with M KCl and fixation in methanol-acetic acid (3: 1). Slides were stained with Giemsa ph 8 to determine the diploid numbers and chromosomes morphologies. The C- banding was performed according to Sumner (1972). The NORs were detected by the Ag-NOR staining (HOWELL and BLACK 1980). RESULTS Conventional staining The diploid number of Liolaemus chacoensis and Liolaemus azarai was 2n = 34. The karyotypes consisted of 12 macrochromosomes and 22 microchromosomes. Pairs 1, 4 and 6 were metacentric, and pair 2, 3 and 5 were submetacentric. At the microchromosome domain, pair 7 was acrocentric with an intermediate size between macro and microchromosomes. The remaining microchromosomes had a dot shape and were morphologically not distinguishable. In metaphases of Liolaemus azarai it was possible to observe a distal secondary constriction in the long arm of pair 2 (Fig. 2). Diplotene cells from males showed 17 bivalents in diplotene. Metaphases II with 6 macrochromosomes and 11 microchromosomes were observed (Fig. 3). Karyotypic differences between males and females were not detected. Fig. 3 Meiotic cells after Giemsa staining. Metaphase II and diplotene bivalent from Liolaemus azarai (a and c) and L. chacoensis (b and d). Nucleolus organizer regions (NORs) The two species showed positive silver staining in the distal region in the long arm of pair 2. In Liolaemus azarai NORs occupied the position of the secondary constriction (Fig. 4). CBG- banding L. chacoensis and Liolaemus azarai had conspicuous pericentromeric regions of constitutive heterochromatin. C- bands were located in all macrochromosomes. The pair 7 showed less evident heterochromatic blocks. C- bands obtained Fig. 4 Pair 2 after Ag-NOR staining.(a) L. chacoensis y (b) Liolaemus azarai.

4 272 HERNANDO in meiotic cells of males of both species were available (Fig. 5). DISCUSSION Few species of Liolaemus from east of Andes have been studied until now. Based on the present cytogenetic analysis, the karyotypes of L. chacoensis and Liolaemus azarai were the most common observed in Liolaemus species from east of Andes (GORMAN et al. 1967; NAVARRO 1992; VIÑA BERTOLOTTO et al. 1996; A IASSA 1998, 1999). The features of karyotype considered to be conservative tend to be maintained: 6 metacentric or submetacentric macrochromosomes pairs, 22 microchromosomes and the pair 7 with intermediate size between macro and microchromosomes (LAMBOROT et al. 1981). The lowest diploid number from the primitive iguanid karyotype: 2n = 36 (12 M + 24 m) is due by a pair less of microchromosomes. The available data suggest the pair 2 seems to be a chromosome shared by the Liolae- mus species with conservative karyotype. A secondary constriction was described frequently in the long arm of this submetacentric pair after conventional Giemsa staining (NAVARRO et al. 1981; NÚÑEZ and FOX 1989; NAVARRO 1992; VIÑA BERTOLOTTO et al. 1996; AIASSA et al. 1998, 1999). In the present study a satellite was evident in Liolaemus azarai but not in L. chacoensis. However, the silver staining showed active nucleolus organizers occur at the distal region of the long arm of pair 2 in both species. At this region Ag- NORs are located in L. occipitalis, L. lutzae and L. wiegmannii (VIÑA BERTOLOTTO et al. 1996). Liolaemus azarai and L. chacoensis could not be identified on the ground of chromosome heterochromatin distribution. The C- bands pattern in the pericentromeric regions of all macrochromosomes showed no differences between the karyotypes studied here and the heterochromatin distribution previously reported in three Liolaemus species from east of the Andes (V IÑA BERTOLOTTO et al. 1996). Fig. 5 C-banding patterns in spermatogonial metaphase (a) and diplotene cells of males (c) of L. chacoensis, intestinal cells (b) and bivalents (d) in Liolaemus azarai.

5 COMPARATIVE CYTOGENETIC ANALYSIS ON TWO SPECIES OF LIOLAEMUS 273 In the genus Liolaemus an XX:XY mechanism of sex determination was described. L. fuscus have a heteromorphic pair 8 (ITURRA et al. 1994) while males of L. occipitalis, L. lutzae and L. wiegmannii exhibit a heteromorphic microbivalente in diplotene cells (VIÑA BERTOLOTO et al. 1996). In the present study, no heteromorphic was found in the male karyotype. REFERENCES AIASSA D., GORLA N., AVILA L. and MARTORI R., 1998 Cariotipo de Liolaemus darwinii (Bell), Squamata: Tropiduridae. Facena, 15: AIASSA D., GORLA N., AVILA L. and MARTORI R., 1999 Cariotipo de Liolaemus saxatilis (Squamata: Tropiduridae): definición y comparación con otros taxones del grupo chiliensis. Rev. Esp. Herp., 12: AVILA L., 2003 A new species of Liolaemus (Squamata: Liolaemidae) from northeastern Argentina and southern Paraguay. Herpetologica, 59: CEI J.M., 1993 Reptiles del noroeste, nordeste y este de la Argentina. Herpetofauna de las selvas subtropicales, Puna y Pampas. Mus. Regionale di Scienze Naturali Torino, Monogr., 14. ETHERIDGE R., 2000 A review of lizards of the Liolaemus wiegmannii group (Squamata, Iguania, Tropiduridae), and a history of morphological change in the sand- dwelling species. Herpetological Monographs, 14: ESPINOZA N.D. and FORMAS J.R., 1976 Karyological pattern of two Chilean lizards species of the genus Liolaemus (Sauria; Iguanidae). Experientia, 32: GORMAN G.C., ATKINS L. and HOLZINGER T., 1967 New karyotypic data on 15 genera of lizards in the family Iguanidae, with a discussion of taxonomic and cytological implications. Cytogenetics, 6: HOWELL W.M. and BLACK D.A., 1980 Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1- step method. Experientia, 36: ITURRA P., VELOSO A., ESPEJO P. and NAVARRO J., 1994 Karyotypic and meiotic evidence for a Robertsonian chromosome polymorphism in the lizard Liolaemus fuscus (Tropiduridae, Sauria). Rev. Brasil. Genet., 17: LAMBOROT M., 1991 Karyotypic variation among populations of Liolaemus monticola (Tropiduridae) separated by riverine barriers in the Andean range. Copeia, 4: LAMBOROT M. and ALVAREZ-SARRET E., 1989 Karyotypic characterization of some Liolaemus lizards in Chile (Iguanidae). Genoma, 32: LAMBOROT M., ALVAREZ E., CAMPOS I. and ESPINOZA A., Karyotypic characterization of three Chilean subspecies of Liolaemus monticola. J. Hered., 72: LAMBOROT M., ESPINOSA A. and ALVAREZ E., 1979 Karyotypic variation in Chilean lizards of the genus Liolaemus (Iguanidae). Experientia, 35: LAMBOROT M. and VÁSQUEZ M., 1998 A triploid lizard (Liolaemus gravenhorsti) from Chile. Journal of Herpetology 33: NAVARRO J., 1992 Cariotipos de trece especies de lagartijas del noroeste argentino de los grupos Liolaemus, Eulaemus y Ortholaemus. Acta zool. Lilloana, 41: NAVARRO J., ESPEJO P., ITURRA P. and VELOSO A., 1988 Un caso extremo de variación cariotípica en Liolaemus (Squamata- Iguanidae). Arch. Biol. Med. Exp., 21: 524. NAVARRO J., SALLABERRY M., VELOSO A. and VALENCIA J., 1981 Diversidad cromosómica en lagartos (Squamata- Iguanidae). I. Avances citotaxonómicos. Perspectivas de estudios evolutivos en Iguanidae (Squamata- Sauria). Medio Ambiente, 5: NUÑEZ H. and FOX S.F., 1989 Liolaemus puritamensis, a new species of iguanid lizard presiously confused with Liolaemus multiformis (Squamata: Iguanidae). Copeia, 2: SUMNER A.T., 1972 A simple technique for demonstrating centromeric heterochromatin. Exp. Cell. Res., 75: VIÑA BETOLOTTO C.E., RODRIGUES M.T., SKUK G. and YONENAGA-YASSUDA Y., 1996 Comparative cytogenetic analysis with differential staining in three species of Liolaemus (Squamata, Tropiduridae). Hereditas, 125: Received October 31, 2002; accepted January 29, 2002