KARYOTYPE AND EVOLUTION OF THE Tropidurus nanuzae SPECIES GROUP (SAURIA, IGUANIDAE)

Rev. Brasil. Genet. X, 2,185-197 (1987) (Brasil. J. Genetics) KARYOTYPE AND EVOLUTION OF THE Tropidurus nanuzae SPECIES GROUP (SAURIA, IGUANIDAE) Sanae Kasahara!, Yatiyo Yonenaga-Yassuda? and Miguel Trefaut Rodrigues" ABSTRACT The karyotypes of Tropidurus nanuzae, T. amathites and T. divaricatus, whichform the nanuzae group, are presented. Data were obtained by Giemsa and NOR staining, C-banding and R-banding after in vivo treatment with 5-bromodeoxyuridine (BrdU). All females have 2n =36 with 12 metacentric or submetacentric macrochromosomes and 24 microchromosomes, while males have 2n =35. The mechanism of sex determination is of the XXAA :XAY/ A type. The only chromosome difference among these species is related to the position of a secondary constriction and to the NOR localization on macrochromosome pair 6, as the result of a pericentric inversion. These data support the idea ofmonophyly in the species of the nanuzae group. Morphological and ecological evidence indicates that allopatric speciation is the most likely explanation for the differentiation of T. amathites and T. divaricatus, and the cytogenetic data support this mode of speciation. INTRODUCTION The genus Tropidurus comprises heliophilous lizards distributed throughout open areas in South America and the Galapagos Islands. Although several problems related to the systematics of this genus, and of the continental forms in particular, 1 Departamento de Biologia, Instituto de Biocicncias, UNESP, Caixa Postal 178, 13500 Rio Claro, SP, Brasil. Send correspondence to S.K. 2 Departamento de Biologia, lnstituto de Biociencias, USP, Caixa Postal 11461,05499 Sfio Paulo, SP, Brasil. 3 Departamento de Zoologia, Instituto de Biocicncias, USP, Caixa Post,.;'l120520, 01498 Sao Paulo, SP Br:J,il

186 Kasahara etal. still exist some taxonomic revisions have been made over the last ten years (Dixon and Wright, 1975; Ortiz, 1980; Cei, 1982; Gudynas and Skuk, 1983; Rodrigues, 1984a, 1987). At present, five relatively homogeneous species groups are accepted to belong to the genus Tropidurus: occipitalis, melanopleurus, peruvianus, torquatus and nanuzae (Rodrigues, 1986). The nanuzae group, recently proposed by Rodrigues (1986) based on scalation, sternal morphology and geographical distribution, comprises three species: Tropidurus nanuzae, T. amathites and T. divaricatus. This group is exclusively cisandean, occurring near or along the Serra do Espinhaco, a quartzitic mountain range of Eastern Brazil extending from the States of Minas Gerais to Bahia. T. nanuzae is predominantly saxicolous and occurs in several localities in the Southernmost and middle portions of the Espinhaco range at altitudes near or above 900 m. T. amathites and T. divaricatus are psammophilous species known only to occur at their type localities at lower elevations, in the Northern extreme of the Espinhaco range and in the surrounding areas, respectively. More detailed discussions about their distribution, ecology, relationships and mode of origin have been presented elsewhere (Rodrigues, 1981, 1984b, 1986).. In this paper we report cytogenetic data on the three species in the nanuzae group, obtained by standard staining, C-banding and silver staining of nucleolus organizer regions (NORs) as well as R-banding after in vivo treatment with 5-bromodeoxyuridine (BrdU). These findings support the hypothesis of a monophyletic origin of the species or the nanuzae group and are suggestive that chromosome variation may have contributed to some extent to the morphological differentiation or speciation of this group. MATERIAL AND METHODS A sample of 6 specimens of T. nanuzae, 6 specimens of T. amathites and 8 specimens of T. divaricatus was studied cytogenetically. The sites at which the lizards were collected and the sex of the specimens are listed in Table I. The map in Figure 1 shows the collecting sites in Brazil. All the voucher specimens are deposited in the Museu de Zoologia, Universidade de Sao Paulo, Brazil. The methods and techniques for obtaining the mitotic and meiotic chromosomes and the chromosome bandings have been described elsewhere (Kasahara et al., 1983; Kasahara et al., in press). Some specimens were injected intraperitoneally with a solution of 5-bromodeoxyuridine (BrdU) and 5-fluordeoxyuridine (FudR). 18 to 20 h before they were killed. R-banding patterns were obtained following the technique of Dutrillaux and Couturier (1981), with the chromosomes being differen tially stained with 33258 Hoechst-Giemsa (FPG staining).

Karyotype and Evolution of Lizard Species Table I -Collection site in Brazil and sex of the specimens of Tropidurus nanuzae, T. amathites and T. divaricatus studied. Site Males Females T. nanuzae Pedra Menina State of Minas Gerais 18 008'S, 43006'W Diamantina State of Minas Gerais 18 015'S,43036'W 3 1 1 T. amathites Santo Inacio State of Bahia 11 006'S,42044'W 4 2 T. divaricatus Arraial do Paulista (Xique-Xique) State of Bahia 10 046'S,42046W 5 3 RESULTS All the females in our sample of T.' nanuzae, T. amathites and T. divaricatus showed 2n = 36 while the males had 2n = 35 (Figure 2A, B and C).;The odd diploid number in male specimens is probably due,to a centric fusion between two microchromosomes. The small metacerttric ot~,submetacentric element resulting from this rearrangement (Figure 2B and C) is rib! always clear iii the mitotic metaphases. However, the presence of a trivalent in < the 'lfieiotic cells (Figure 6A, B and C) confirms unequivocally the occurrence of such rearrangement in males. Possibly the Y chromosome is involved in the centric fusion so that a mechanism of the XXAA:XAY/A type is responsible for sex determination'in the three species. Although the same basic karyotype with 12 metacentric or submetacentric macrochrornosomes and 24 microchrornosomes was found in ' all specimens, some

188 Kasahara etal. \ - -, I ~ \ ~ \. ~--. I :'~: - \",\ )...,.,./~"i ~\. ~... -, I,) :. 1/ Y //r-, /'-,, C 7 -,_... ---; I ' I, I. \ \, '.: I \ \.;-. '-,-.> " \ \... L,," (' - \ \ I ( I I -":<, v-, (.......; \,,r- / \,... 1. DIAMANTINA 2.PEDRA MENINA 3.SANTO INACIO 4. ARRAIAL DO PAULISTA Figure 1 Collecting sites of specimens of T. nanuzae, T. amathites and T. divaricatus in the States of Minas Gerais and Bahia. differences occur. T. nanuzae and T. amathites have allprominent secondary constriction near the centromere on the tong arm of macrochromosome pair 6 (Figures 2A and B). In T. divaricatus the secondary constriction is displaced to the short arm of the same chromosome (Figure 2C) rendering it more metacentric than in the two other species (Figure 3A, C and E). In the three species, the rnacrochromosome pair 6 presents NOR located just at the secondary constriction (Figure 3B, D and F). In some individuals, heteromorphisms of both secondary constriction and NOR sizes were observed (Figure 3A and B).

Karyotype and Evolution of Lizard Species 189 A \I'~ 1\! I \ ' ' f' \ _, t ' i, I, I J t J! J.~ ~ 1 ;,,,.\ 41) 7, a j a'j 2',4) ~) 8 ~} ~~ ) -,1\ ", 3,, "J:'~\' 9. " 1'''; ' I' I ~" ', i f 1 4 ". 5 6 '. ; - )... A, 10 11 12.' 16 17 18 13 - ' 14 15 ' 1,,'. ',.~.,:' -.)'.,.' r- ',. >;., / ',. '.;" ',,, 7 ',",,., "8': 14 ).. 3 4 A ",. 9 10.;.-'.. 15 16 a ' a.... 11 12 17 1 18... n, -,,'J' ',' "\"8 1 I", i'. -. \!.. \ ;, t,' I ', j 1- l..'.! ~ ',.! s 1_ f ".'... ~., "'-.......,.,. 3 4 5 6 -A 9 10 11 12.....-.'. a, " : 15, 16 17' '/18 Figure 2 - Karyotypes after Giemsa staining. A, female of T. nanuzae with 2n = 36; B, male of T. amathites with 2n =35 ; C, male of T. divaricatus with 2n =35. Macrochromosome pair 6 presents a secondary constriction in the long arm (A and B) or in the short arm (C). In B and C the arrow indicates an odd submetacentric microchromosome. Bar =10 Jim,

190 Kasahara et al. B Figure 3 - Macrochrornosorne pair 6 after Giern sa (upper row) and NOR sta ining (lower row). A and B, T. nanuzae; C and D, T. amathites; E and F, T. divaricatus. Heterornorphisrns of both secondary constriction and NOR size are seen in A and B, respectively. Bar =10 J-Lrn. The C-banding patterns are similar in T. nanuzae, T. amathites and T. divaricatus, the microchromosomes showing a large amount of heterochromatin in the pericentromeric region (Figure 4). The macrochromosomes have less evident C-bands and the 6th pair presented in some metaphases a characteristic pattern with C-bands stained on both sides of the secondary constriction (Figure 4). The spermatogonial metaphases of one specimen of T. divaricatus showed well stained C-bands also in the macrochromosomes. In almost all C-banded specimens of the three species a characteristic microchromosome without C-bands or with a less stained heterochromatic region was noticed (Figure 4). C-banding in meiotic cells was obtained from male specimen s of T. nanuzae and T. divaricatus (Figure SA and B). Meiotic analysis of males of the three species showed 16 bivalents plus one small trivalent at diplotene and metaphase I cells (Figures SA, B, 6A, B and C). The trivalent corresponds to the pairing of the rearranged chromosome with two other microchromosomes. Metaphase II cells showed either 6 macrochromosomes and 12 rnicrochromosomes or 6 macro chromosomes and 11 rnicrochromosomes (Figure 6D). R-banding was obtained by BrdU incorporation in T. amathites and T. divaricatus. A precise identification of the macrochromosomes was possible, each pair having the same pattern in both species, with the exception of the 6th (Figure 7). A pericentric inversion may account for the different morphology of this chromosome in the two species.

Karyotype and Evolution of Lizard Species 191 1 2 3 4 5 6 7 13 8 14 9 '." 15 16 11 12, " 17 18 a 6 Figure 4 - C-banding patterns in a T. amathites male with 2n =35. One of the microchromosornes has no C-bands (arrow). Inset: macrochromosome pair 6 from T. nanuzae with characteristic C-bands. Bar =10 J.l.m. The same karyotype was found for the specimens of T nanuzae collected at two different sites in the State ofminas Gerais. DISCUSSION In spite of the difference in diploid number among males and females of T nanuzae, T amathites and T divaricatus, the same basic karyotype occurs in all of

192 Kasahara et al:, '., ", \,", " Figure 5 - C-banding patterns in diplotene cells of males with 2n =35. A, T. nanuzae; B, T. divaricatus. The arrow indicates the trivalent. Bar =10 Mm, these species. The presence of 12 metacentric or submetacentric macrochromosomes and 24 microchromosomes has been described as the typical iguanid karyotype and, in fact, it occurs in several species of the Family Iguanidae (Gorman et al., 1967; Paull et al., 1976). The karyotypic difference found in males and females is due to a chromosomal rearrangement and may be related to a mechanism of sex chromosome determination of the XXAA :XAY/A type. This mechanism is not so rare in lizards and even in the genus Tropidurus we have already found a species of the torquatus group with similar sex chromosomes (Kasahara et al., 1983). A remarkable chromosome difference found in the species of the nanuzae group is in the morphology of the macrochromosome pair 6, which has a secondary constriction in the long arm in T. nanuzae and T. amathites and in the short arm in T. divaricatus. This is probably due to a pericentric inversion and one of the break points may have occurred in the short arm close to the centromere and the other at the distal portion of the secondary constriction in the long arm of chromosome 6 of T. amathites, thus originating the corresponding chromosome of T. divaricatus. The banding patterns in the distal region of the long arms are similar for the chromosomes 6 of both species. As a consequence of this chromosome rearrangement the NOR localization is also distinct in the species of the nanuzae group. Other chromosomal characteristics, such as the C-banding patterns, are quite similar although in one

Karyotype and Evolution of Lizard Species 193 I,,. ",.... t '"., \ ~ -.d.. B c Figure 6 - Meiotic cells of males with 2n =35 after Giemsa staining. A, B and C, diplotene with 16 bivalents and one trivalent (arrow) in T. nanu zae, T. amathites and T. divaricatus, respectively; D, metaphase II with 6 macrochromosomes and 11 micro chromosomes in T. divaricatus. Bar = 10 ui«.

194 Kasahara et ai. 1 2.1 '.''.;'\; '" 1'..". ' r....1 ~..,., :., ~, 3. 1.\, /. ' '\ 4 6 Figure 7 - R-banding patterns after in vivo BrdU incorporation and FPG staining in the macrochromosomes of T. amathites (right) and T. divaricatus (left). Inset: macrochromosomes 6 of T. amathites (right) and T. divaricatus (left) differing by a pericentric inversion. Bar =10 Jim. specimen of T. divaricatus conspicuous C-bands were noticed in the macrochromosome pairs. No well stained C-bands were found either in another individual of this species or in the representatives of T. nanuzae and T. amathites. The R-banding patterns are the same for T. amathites and T. divaricatus. Identical patterns were also observed in the macrochromosomes of T. hispidus, a species of the torquatus group (Kasahara et al., in press) as well as in the species of other tropidurine genera (Kasahara et al, unpublished data). The presence of NORs in macrochromosome pair 6 seems to be characteristic of the three species of the nanuzae group. None of the nine species of Tropidurus belonging to the torquatus group studied cytogenetically thus far, nor any of the five species belonging to related genera such as Plica, Tapinurus and Strobilurus, have NORs located in the corresponding macrochromosome pair 6 (Kasahara et al., 1983; Kasahara et al., 1985, 1986a,b; Kasahara et al., in press; Kasahara et al., unpublished data). This fact strongly supports the previous statement of Rodrigues (1986) that T. nanuzae, T. amathites and T. divaricatus are a monophyletic group of species. In

Karyotype and Evolution of Lizard Species 195 spite of a marked morphological and ecological differentiation, these species belong to the same radiation and the two closest relatives are thought to be T. amathites and T. divaricatus. T. divaricatus is a very atypical species having some characteristics (i.e. absence of neck and antehumeral mite pockets and presence of strongly keeled ventral scales) not found in the species belonging to Tropidurus and to the other tropidurine genera, which are characterized by an enlarged occipital scale. Regarding other traits, T. divaricatus is close toz. amathites: there are no differences in scale counts and both species are very similar in body size and color pattern, sharing a typical pair ofbrown spots-in the supraocular area not present in T. nanuzae. On the other hand, T. nanuzae and T. amathites have antehumeral and neck folds as well as smooth ventral scales, which are primitive characteristics for the genus, but these species differ strongly in color pattern, scalation and in ecology. Although the localities where T. amathites and T. divaricatus occur are near, i.e, less than 20 km apart, their habitats are physiognomically distinct and geographically isolated. T. amathites is found on the right side ofthe Sao Francisco river, in patches of white sands in an area dominated by the "campos rupestres", a quite characteristic open plant formation. T. divaricatus occurs in a white-sand dune habitat on the left side of the Sao Francisco river. The morphological and ecological evidence supports a close relationship between T. amathites and T. divaricatus and Rodrigues (1986) advanced the hypothesis of an allopatric mode of differentiation to explain their origin. This hypothesis is based on paleoclimatic data (Ab'Saber, 1969; Tricart, 1974) which indicate that a continuity of sand habitats existed in the past 12000 years and was interrupted when the Sao Francisco river found its way out to the sea. Since then the ancestral populations ofpsammophilous lizards have differentiated into two new species. The presence of the same pattern of secondary constriction and NOR localization on the long arm of macrochromosome pair 6 in T. nanuzae and T. amathites is independent supporting evidence for the primitiveness of these two species, primarily inferred on morphological grounds. The presence of the secondary constriction and NOR in the short arm of macrochromosome pair 6 in T. divaricatus is certainly a more advanced condition. The occurrence of a pericentric inversion in T. divaricatus leads us to consider the role of this chromosomal rearrangement in the differentiation of T. amathites and T. divaricatus. The karyotypic data are in accordance to the proposed allopatric mode of speciation and in this case, it is probable that chromosome differentiation was established after geographic isolation, accelerating (or not) the process of speciation. Another possibility exists whereby the chromosome differentiation itself can explain the speciation, but unfortunately we have no means to date historically the appearance of this chromosome pattern. Although the allopatric mode of speciation seems to be more likely at present, the

196 Kasahara et al importance of the chromosomal rearrangement in the process cannot be ruled out. We cannot, for example, eliminate the possibility of a relationship of this chromosomal rearrangement with the high level of morphological differentiation of T. divaricatus. ACKNOWLEDGMENTS We are indebted to Antonio Pascon, Cristina Maria de Lourdes Barnabe, Oriel S. Farias and Rosany Carvalho do Prado for technical assistance. We are also grateful to Dr. Nanuza L. Menezes for assistance in the field trips. This research was supported by Fundacao de Amparo as Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPql PIG). RESUMO Sao apresentados os dados cromossomicos de Tropidurus nanuzae, T. amathites e T. divaricatus, que formam 0 grupo nanuzae, com coloracao pelo Giemsa e pelo nitrato de prata (RON), banda C e banda R apos tratamento in vivo com 5-bromodeoximidina (BrdD). Todas as femeas apresentarn 2n =36 com 12 macrocromossomos metacentricos ou subrnetacentricos e 24 microcromossomos enquanto que os machos apresentam 2n = 35. 0 mecanismo de determinacao sexual e do tipo XXAA:XAY/A. A iinica diferenca cromossomica entre essas especies esta relacionada a posicao de uma constricao secundaria e alocalizacao das RONs no par 6 de macrocromossomos, resultante de uma inversao pericentrica, Tais dados apoiam a ideia de monofilia das especies do grupo nanuzae. Evidencias morfologicas e ecologicas indicam que uma especiacao alopatrica e mais provavel para explicar a diferenciacao de T. amathites e T. divaricatus e os dados citogeneticos apoiam este modelo de especiacao. REFERENCES Ab'Saber, A.N. (1969). Participacao das superficies aplainadas nas paisagens do nordeste brasileiro. Universidade de Sao Paulo, Instituto de Geografia. Geomorfologia 19: 1-38. Cei, J.M. (1982). A new species of Tropidurus (Sauria, Iguanidae) from the arid chacoan and Western regions of Argentina. Occas. Papers Mus. Nat. Hist. Univ. Kansas 97: 1-10. Dixon, J.R. and Wright, J.W. (1975). A review of the lizards of the iguanid genus Tropidurus in Peru. Nat. Hist. Mus. Los Angeles Contrib. Sci 271: 1-39. Dutrillaux, B. and Couturier, J. (1981). La Pratique de l'analyse Chromosomique. Masson, Paris. 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: 286-299.

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