Comparative Cytogenetics and Supernumerary Chromosomes In

Hereditas 136: 51–57 (2002)

Comparative cytogenetics and supernumerary chromosomes in the Brazilian lizard genus Enyalius (Squamata, Polychrotidae) CAROLINA ELENA VIN0A BERTOLOTTO1,3, KATIA CRISTINA MACHADO PELLEGRINO1, MIGUEL TREFAUT RODRIGUES2 and YATIYO YONENAGA-YASSUDA1 1 Departamento de Biologia, Instituto de Biocieˆncias, Uni6ersidade de Saõ Paulo, Saõ Paulo, SP, Brasil 2 Departamento de Zoologia, Instituto de Biocieˆncias and Museu de Zoologia, Uni6ersidade de Saõ Paulo, Saõ Paulo, SP, Brasil 3 Uni6ersidade Santo Amaro, Faculdade de Medicina Veterina´ria, Saõ Paulo, SP, Brasil

Bertolotto, C. E. V., Pellegrino, K. C. M., Rodrigues, M. T. and Yonenaga-Yassuda, Y. 2002. Comparative cytogenetics and supernumerary chromosomes in the Brazilian lizard genus Enyalius (Squamata, Polychrotidae).—Hereditas 136: 51–57. Lund, Sweden. ISSN 0018-0661. Received August 13, 2001. Accepted January 24, 2002

Cytogenetical analyses based on conventional and differential staining were performed for the first time on five species of the Brazilian lizard genus Enyalius: E. bibronii, E. bilineatus, E. iheringii, E. leechii,andE. perditus. The species share a similar 2n=36 (12M+24m) karyotype, comprised of 12 metacentric or submetacentric macrochromosomes, except for an acrocentric pair 6 that characterizes E. bibronii. The 24 microchromosomes were acrocentrics, but in E. perditus two meta/submetacentric microchromosome pairs were unambiguously identified. Karyotypes with 2n=37 and 2n=37/38 chromosomes were also observed in some specimens of E. bilineatus as a result of the presence of supernumerary chromosomes (Bs). Ag-NORs were always located at the distal region of the long arm of the submetacentric pair 2. The constitutive heterochromatin was mostly restricted to the pericentromeric regions of some macrochromosomes and microchromosomes. A XX:XY mechanism of sex determination with a dot-like Y microchromosome occurs in E. bilineatus, E. leechii,andE. perditus.

Carolina E. V. Bertolotto, Departamento de Biologia, Instituto de Biocieˆncias, Uni6ersidade de Sa˜o Paulo, Sa˜o Paulo, SP, C.P. 11.461, CEP 05422-970, Brasil. E-mail: [email protected]

Lizards of the genus Enyalius are predominantly in- the other extreme, there are species from several habitants of eastern and central forested habitats of different families that share the same 2n=36 Brazil. JACKSON (1978) recognized a total of six (12M+24m) karyotype with very similar macrochro- species, two of them polytypic: E. bilineatus, E. mosome complements, which has been considered as brasiliensis (2 ssp.), E. catenatus (3 ssp.), E. iheringii, ancestral for pleurodont Iguania, which includes the E. leechii, and E. perditus. A more recent inspection family Polychrotidae (OLMO 1986). However, the use of additional specimens by one of us (MTR) suggests of differential staining reveal that these convention- that a new revision for the genus is needed, and ally-stained conservative karyotypes are distinct with although we here adopt Jackson’s scheme, we ignore respect to C-banding patterns, Ag-NORs localiza- subspecies and consider tentatively E. bibronii (a sub- tion, morphology of microchromosomes, among species of E. catenatus) as a good species. Except for other cytogenetic aspects (BERTOLOTTO et al. 1996; E. leechii, which occurs in the Amazon basin, all the KASAHARA et al. 1996). Here, we describe the kary- others species are distributed throughout a wide otypes of five species of the Brazilian lizard Enyalius range of the Brazilian Atlantic Forest, extending based on banding patterns, and compare them with from state of Rio Grande do Norte (northeast) to those reported for other pleurodont Iguania taxa. Rio Grande do Sul (southern) (JACKSON 1978; ETHE- RIDGE and DE QUEIROZ 1988). Occasionally, some MATERIAL AND METHODS populations occur in gallery forests in the Cerrados of Central Brazil (E. bilineatus) or in isolated patches A total of 20 individuals from five species of Enyalius of forest in the Caatingas (E. bibronii ). collected from different Brazilian localities were cyto- Cytogenetic studies of pleurodont Iguania (former genetically studied (Table 1), and deposited at the Iguanidae, ESTES et al. 1988; FROST and ETHERIDGE Museu de Zoologia of the Universidade de Saõ Paulo 1989) suggest that there are two distinct trends in (MZUSP), Brazil. Chromosome spreads were ob- chromosome evolution in these lizards. Considerable tained from bone marrow, liver, spleen, testes accord- karyotype variability is found in species of the highly ing to routine techniques, or from fibroblast cultures diverse genus Anolis (2n=25 to 2n=48), and among (YONENAGA-YASSUDA et al. 1988). Mitotic and mei- those of the genus Liolaemus (2n=30 to 2n=44). At otic chromosomes were studied after standard 52 C. E. V. Bertolotto et al. Hereditas 136 (2002)

Table 1. Species, specimen number, sex, locality, diploid number and number of metaphases analysed for ﬁ6e species of Enyalius in this study. The regions of Brazil are: BA=Bahia, SP=Sa˜o Paulo, MG=Minas Gerais, DF=Distrito Federal; MT=Mato Grosso. M=male and F=female;2n=diploid number

Species Specimen Sex Locality 2n Number of number metaphases

E. bibronii LG 1377 F Serra da Jiboía (BA) (12°56%S, 39°31%W) 36 28 E. bilineatus LG 427 FSaõ Jose´ do Rio Preto (SP) (20°49%S, 49°22%W) 36 21 LG 814 F3769 LG 815 M Nova Ponte (MG) (19°08%S, 47°40%W) 36 34 LG 816 F3652 LG 919 M37/38 59/7 LG 1467 F Bras´ılia (DF) (15°46%S, 47°55%W) 36 26 E. iheringii LG 421 F Santana de Parna´ıba (SP) (23°26%S, 46°55%W) 36 12 LG 622 F Picinguaba (SP) (23°22%S, 44°50%W) 36 39 LG 625 F 35 LG 929 F Jureía, Peru´ıbe (SP) (24°19%S, 46°59%W) 36 20 LG 1383 F Pilar do Sul (SP) (23°48%S, 47°42%W3615 LG 1222 M Apiaca´s (MT) (09°32%S, 57°26%W) 4 LG 1223 M365 E. leechii LG 1249 F14 E. perditus LG 1135 F Piraju (SP) (23°11%S, 49°23%W) 36 33 LG 1500 F 10 LG 1501 F Serra da Cantareira (SP) (23°22%S, 46°36%W) 24 LG 1502 F367 LG 1505 M25 Total 5M;15F 539

Giemsa and Ag-NOR staining, and C-banding. 2n=38 (two Bs) lineages, the latter representing Replication R-banding (RBG), after in vitro treat- about 10 % of its cells (Table 1). ment with 5-bromodeoxyuridine (BrdU) and 5- A mechanism of sex determination of XX:XY type fluorodeoxyuridine (FudR) followed by FPG staining was found in E. bilineatus, E. leechii and E. perditus. (DUTRILLAUX and COUTURIER 1981) was carried The Y is a dot-like microchromosome and the X is a out on metaphases of E. bilineatus medium-sized microchromosome which is indistin- guishable from the other chromosomes of the same RESULTS size (Fig. 1b, d, e and Fig. 2a). An evident heteromorphic microbivalent corresponding to the pairing of Comparati6e analyses after con6entional staining the X and Y was observed in some diakinesis cells A similar karyotype of 2n=36 (12M+24m) com- from males of E. bilineatus and E. perditus (Fig. 2b). prised of 12 macrochromosomes and 24 microchro- At metaphase II cells, 6 macrochromosomes and 12 mosomes characterized the five species of Enyalius microchromosomes were visualized. In the aberrant (Fig. 1a–e, Table 1). The macrochromosome pairs 1, specimen of E. leechii (LG 1223), metaphase II cells 3, 4, and 5 are metacentrics, and the pairs 2 and 6 are contained either the submetacentric or the acrocentric submetacentrics in all species, except for E. bibronii macrochromosome 6. Analysis of seven diakinesis which has an acrocentric pair 6 (Fig. 1a). An aber- cells from the mosaic specimen LG 919 showed the rant pair 6 formed by a submetacentric and an acro- presence of 6 macrobivalents, 12 microbivalents (one centric was detected in one specimen (LG 1223) of E. of them representing the pairing XY), and a probable leechii (data not shown). Pair 2 exhibits a typical univalent (Fig. 2b). secondary constriction at the distal end of the long Analyses after differential staining arm. Some microchromosomes seem to be acrocentrics, and there are indubitably two distinct metacen- The Ag-NORs were detected at the distal end of the tric or submetacentric pairs in E. perditus (Fig. 1e). long arm of pair 2, corresponding to the conspicuous Supernumerary chromosomes (Bs) were observed secondary constriction in all five species of Enyalius in two specimens of E. bilineatus (Fig. 2a). The (Fig. 3b–e). Out of 45 Ag-stained metaphases of one specimen LG 814 had a karyotype of 2n=37 chro- specimen of E. bilineatus (LG 815), 37 were hetero- mosomes, while the specimen LG 919 was a mosaic morphic with respect to the size of the NOR (Fig. composed of a major 2n=37 (one B) and a minor 3b). Hereditas 136 (2002) Comparati6e cytogenetics in Enyalius 53

Fig. 1a–e. Karyotypes of species of Enyalius after conventional staining (2n=36, 12M+24m). a E. bibronii female; b E. bilineatus male; c E. iheringii female; d E. leechii female; e E. perditus female. For the sex chromosomes of the opposite sex, see the inset in b, d and e.Bar=10 mm. 54 C. E. V. Bertolotto et al. Hereditas 136 (2002)

Fig. 2a and b. Cells of the mosaic specimen of Enyalius bilineatus, male, after conventional staining. a Mitotic metaphase with 2n=37 (12M+24m+1B). Note the supernumerary chromosome (small arrow) and the dot-like Y microchromosome (large arrow); b Diakinesis cell presenting 6 macrobivalents, 12 microbivalents (one of them heteromorphic: small arrow), and one univalent (B chromosome: large arrow).

Fig. 3a–e. Chromosomes of species of Enyalius after differential staining. a C-banded metaphase of E. bilineatus. The labeled arrows indicate: (a) interstitial constitutive heterochromatin on pairs 1 and 2; (b) constitutive heterochromatin at the distal end of the long arm of pair 2. The supernumerary chromosome is indicated by a B; b Ag-NORs in E. bilineatus. Note the heteromorphism of size (arrows); c–e Pairs 2 of E. perditus, E. iheringii and E. leechii after Ag-staining, respectively.

All ﬁve species of Enyalius were characterized by a ously paired (Fig. 4a). A late replicating region at the small amount of constitutive heterochromatin after distal end of the long arm of pair 2 was also ob- C-banding, which was mostly restricted to the peri- served. The B chromosome is late-replicating (Fig. centromeric region of some chromosomes (Fig. 3a). 4a, b), and, interestingly, in two metaphases of the The region of the secondary constriction of pair 2 mosaic specimen LG 919 (2n=37/38; Table 1) bear- was also positively stained in some metaphases of E. ing two Bs, only one of them was late replicating bibronii, E. bilineatus, and E. perditus. A proximal (Fig. 4c). C-band equidistantly located on pair 1 and 2 was detected in the karyotypes of E. bilineatus and E. DISCUSSION bibronii (Fig. 3a). The B chromosome detected in two specimens of E. bilineatus exhibits an intermediate The ﬁve species of Enyalius share a common 2n=36 staining between the darkest and the lightest C-posi- (12M+24m) karyotype which has been described tive blocks of the autosomes (Fig. 3a). among several taxa within pleurodont Iguania (BICK- RBG pattern was obtained for E. bilineatus, and HAM 1984; KASAHARA et al. 1996; PELLEGRINO et al. allowed all its macrochromosomes to be unambigu- 1999). There is similarity among the macrochromo- Hereditas 136 (2002) Comparati6e cytogenetics in Enyalius 55

Fig. 4a–c. RBG pattern from the mosaic specimen of E. bilineatus. a Partial karyotype showing the six pairs of macrochromosomes. A late-replicating B chromosome from a different metaphase is shown (inset); b Metaphase with one B chromosome (arrow); c Metaphase with two Bs (arrows), with only one of them late-replicating (large arrow). somes of the Enyalius species, with the exception of al. 1999), and Phrynosomatidae (Sceloporus,PORTER E. bibronii that present an acrocentric pair 6, making et al. 1994), indicating that it is a conservative region it distinct from its congeners, all other Polychrotidae, shared by these related families. and related families within pleurodont Iguania that Studies involving comparative analyses of mi- share the conservative karyotype usually with meta- crochromosomes are usually limited because their centric or submetacentric pair 6. We also detected the reduced size prevents precise deﬁnition of their mor- presence of a heteromorphic pair 6 in both somatic phology in most cases, with few exceptions (PINNA- and meiotic cells of one specimen of E. leechii. This SENN et al. 1987; PELLEGRINO et al. 1994; chromosome aberration could be explained by a dele- BERTOLOTTO et al. 1996; KASAHARA et al. 1996). In tion affecting the short arm of one homologue of this all species of Enyalius, the 24 microchromosomes pair. It seems that the macrochromosome pair 6 seem to be acrocentrics, but E. perditus has at least might be a ‘‘hot spot’’ for karyotypic evolution in the two biarmed pairs. Increase in quality of the chromo- genus Enyalius. A parallel example was reported in some preparations should allow these chromosomes the genus Tropidurus (Tropiduridae) with three to be used more often in comparative analyses, and closely related species: T. nanuzae and T. amathites indeed facilitate the detection of mechanisms of sex having a secondary constriction and NORs in the determination that involve these microchromosomes. long arm of pair 6, while in T. di6aricatus these A mechanism of the XX:XY type was detected in regions are located in the short arm of the same pair due to the occurrence of a pericentric inversion E. bilineatus, E. leechii,andE. perditus. A heteromorphic microbivalent, representing the pairing of X and (KASAHARA et al. 1987). All ﬁve species of Enyalius were very similar with Y, was observed in meiotic cells of E. bilineatus and respect to location of the NORs and the amount and E. perditus, and allowed us to evaluate the size of the distribution of constitutive heterochromatin. The dis- X chromosome. In pleurodont Iguania, both simple tal end of the long arm of pair 2 bearing the NORs (XX:XY) and multiple (X1X1X2X2:X1X2Y) mecha- and a positive C-band has been described across nisms of sex determination involving microchromo- several species of pleurodont Iguania families: somes occur, including the polychrotids Pristidactylys Tropiduridae (Tropidurus,KASAHARA et al. 1987, (PELLEGRINO 1993), Anolis (GORMAN and ATKINS 1996; Strobilurus,RODRIGUES et al. 1989; Liolaemus, 1968; GORMAN and STAMM 1975; our unpublished BERTOLOTTO et al. 1996), Polychrotidae (Pristidacty- data), Polychrus (BERTOLOTTO et al. 2001) and lus,PELLEGRINO 1993; Urostrophus,PELLEGRINO et Urostrophus (PELLEGRINO et al. 1999). 56 C. E. V. Bertolotto et al. Hereditas 136 (2002)

Supernumerary chromosomes of intermediate size Bickham JW, (1984). Patterns and modes of chromosomal between the microchromosomes and macrochromo- evolution in reptiles. In: Chromosomes in evolution of somes are present in two specimens of E. bilineatus. eukaryotic groups (eds A Sharma and A Sharma). CRC Press, Florida, p. 13–40. They do not exhibit a heterochromatic pattern, but Brinkman JN, Sessions SK, Houben A and Green DM, they are almost entirely late-replicating after R-band- (2000). Structure and evolution of supernumerary chro- ing, which is typical for the heterochromatin of the mosomes in the Pacific giant salamander, Dicamptodon Bs. Few reports of occurrence of supernumerary tenebrosus. Chromosome Res. 8: 477–485. Dutrillaux B and Couturier J, (1981). La pratique de chromosomes have been made among lizards, com- l’analyse chromosomique. Masson, Paris. pared to those from plants and other animals. Besides Estes R, de Queiroz K and Gauthier J, (1988). Phylogenetic the present study, these chromosomes have been relationships within Squamata. In: Phylogenetic rela- found in ten other genera, which represent eight tionships of the lizard families (eds E Estes and G different families including the polychrotid Anolis Pregill). Stanford Univ., California, p. 119–281. Etheridge R and de Queiroz K, (1988). A phylogeny of (GORMAN et al. 1968). Iguanidae. In: Phylogenetic relationships of the lizard The function, composition, and origin the supernu- families (eds R Estes and G Pregill). Stanford Univ., merary chromosomes are not completely known, with California, p. 283–367. the most intriguing questions regarding their origin. Frost D and Etheridge R, (1989). A phylogenetic analysis and taxonomy of iguanian lizards (Reptilia: Squamata). Several molecular cytogenetic studies utilizing restric- Misc. Publ. Univ. Kansas. Mus. Nat. Hist. 81: 65. tion endonucleases, fluorochromes, in situ hybridiza- Gorman GC and Atkins L, (1968). Confirmation of an X-Y tion, and chromosome microdissection techniques sex determining mechanism in lizards (Anolis). Copeia 1: have been used to elucidate the composition, struc- 159–160. ture, origin and evolution of the supernumerary chro- Gorman GC and Stamm B, (1975). The Anolis lizards of Mona, Redonda, and La Blanquilla: chromosomes, rela- mosomes (LOPEZ-LEON et al. 1994; BRINKMAN et al. tionships, and natural history notes. J. Herpet. 9: 197– 2000; MAISTRO et al. 2000). 205. The present study extends the knowledge on kary- Gorman GC, Thomas R and Atkins L, (1968). Intra- and otypes of the yet poorly known lizard family Poly- interspecific chromosome variation in the lizard Anolis cristatellus and its closest relatives. Breviora 293: 1–13. chrotidae. Considering that the relationships within Jackson JF, (1978). Differentiation in the genera Enyalius this family are still unresolved, studies employing and Strobilurus (Iguanidae): implications for Pleistocene banding techniques on the remained taxa should climatic changes in eastern Brazil. Arq. Zool. S. Paulo allow the identification of synapomorphies that can 30: 1–79. be useful in a phylogenetic context. Kasahara S, Yonenaga-Yassuda Y and Rodrigues MT, (1987). Geographical karyotypic variations and chromosome banding patterns in Tropidurus hispidus (Sauria, Iguanidae) from Brazil. Caryologia 40: 43–57. ACKNOWLEDGEMENTS Kasahara S, Pellegrino KCM, Rodrigues MT and Yone- naga-Yassuda Y, (1996). Comparative cytogenetic stud- The authors are grateful to Maria Jose´ de Jesus Silva, ies of eleven species of the Tropidurus torquatus group Vin´ıcius Xavier da Silva, Gabriel Skuk, Dante Pavan, (Sauria, Tropiduridae), with banding patterns. Hereditas Patr´ıcia Narvaes, Jose´ Manoel Martins, Claudia Silva, 125: 37–46. Cristiano Nogueira, Paula Valdujo and Sandra Favorito Lopez-Leon MD, Neves N, Schwarzacher T, Heslop-Har- for collecting specimens, and to Dr. Tien Hsi Chu and Mrs. rison TS, Hewitt GM and Camacho JPM, (1994). Possi- Miriam Romeo for technical assistance. We thank Dr. ble origin ofaBchromosomededuced from its DNA Elizabeth Sinclair for helpful comments in the early ver- composition using double FISH technique. Chromo- sions of the manuscript. Grants for this study were pro- some Res. 2: 87–92. vided by Conselho Nacional de Desenvolvimento Cient´fiı co Maistro EL, Oliveira C and Foresti F, (2000). Cytogenetic e Tecnolo´gico (CNPq), Fundaçaõ de Amparo a` Pesquisa analysis of A- and B-chromosomes of Prochilodus linea- do Estado de Saõ Paulo (FAPESP) and Conso´rcio Na- tus (Teleostei, Prochilodontidae) using different restric- cional de Engenheiros Consultores (CNEC). tion enzyme banding and staining methods. Genetica 108: 119–125. Olmo E, (1986). Reptilia. In: Animal Cytogenetics (ed. B REFERENCES John). Gebruder Borntraeger, Berlin, Vol. 4/3A, p. 1– 100. Bertolotto CEV, Rodrigues MT, Skuk G and Yonenaga- Pellegrino KCM, (1993). Caracterizaçaõ cromossoˆmica em Yassuda Y, (1996). Comparative cytogenetic analysis 16 espećies das fam´ılias Tropiduridae, Polychrotidae e with differential staining in three species of Lio- Gekkonidae (Sauria) pela aplicaçaõdetećnicas de col- laemus (Squamata, Tropiduridae). Hereditas 125: 257– oraçaõ diferencial. Rev. Bras. Genet. 16: 1140– 264. 1141(Thesis abstract). Bertolotto CEV, Rodrigues MT and Yonenaga-Yassuda Y, Pellegrino KCM, Yonenaga-Yassuda Y and Rodrigues (2001). Banding patterns, multiple sex chromosomes sys- MT, (1994). Cytogenetic studies in six species of tem and localization of telomeric (TTAGGG)n se- Tropiduridae (Sauria). Brazil. J. Genetics 17: 401–408. quences by FISH on two species of Polychrus Pellegrino KCM, Bertolotto CEV, Rodrigues MT and (Squamata, Polychrotidae). Caryologia 54: 217–226. Yonenaga-Yassuda Y, (1999). Banding patterns, hetero- Hereditas 136 (2002) Comparati6e cytogenetics in Enyalius 57

morphic sex chromosomes and Ag-stained NORs after in chromosomes of Squamata reptiles. Copeia 2: 302– pachytene stage in the meiosis of the Brazilian lizard 313. Urostrophus vautieri (Squamata, Polychrotidae). Cary- Rodrigues MT, Yonenaga-Yassuda Y and Kasahara S, ologia 52: 21–26. (1989). Notes on the ecology and karyotypic description Pinna-Senn E, Tada IE and Lisanti JA, (1987). Polymor- of Strobilurus torquatus (Sauria, Iguanidae). Rev. Bras. phism of the microchromosomes and the nucleolar orga- Genet. 12: 747–759. nizer region in Pristidactylus achalensis (Sauria: Yonenaga-Yassuda Y, Kasahara S, Chu TH and Rodrigues Iguanidae). Herpetological 43: 120–127. MT, (1988). High-resolution RBG-banding pattern in Porter CA, Haiduk MW and Queiroz K, (1994). Evolution the genus Tropidurus (Sauria, Iguanidae). Cytogenet. and phylogenetic signiﬁcance of ribosomal gene location Cell Genet. 48: 68–71.