c- and Rbandirig patterns and nucleolus organizer regions in the e of Hemidactylus mabouia (Sauria, Gekkonidae)

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1 ruad Jonlal of a.llellcl Z1 531 (1995) ltnlota anallelra de CeD6tc:al c- and Rbandirig patterns and nucleolus organizer regions in the karyo e of Hemidactylus mabouia (Sauria Gekkonidae) Katia Cristina Machado Pellegrino' Maria Cecilia Araripe Sucupira 2 Sanae Kasahara 2 Miguel Trefaut Rodrigues 3 and Yatiyo Yonenaga-Yassuda' ABSTRACf ClfOmosomes 01 HemidaCl}llus mabouia (2n-42) from the states of Paraiba and Slo Paulo Brazil Wefe studied by ronvcntional and dilfefenlial staining (C end R banding and Ag NORs). Moat of tho chrornoeomes am subtelooontrica or aaooenutcs with gradation in size. Banding patterns and Ag NORs location n H. mllbouja are described here (or tho first time. NTRODUCTON The Gekkonidae is the most diverse and ancient group of reptiles and has a worldwide distlibution (King 1990). t constitutes the largest family of lizards comprising 1()() genera and 943 species (Kluge 1991). n spite of the great number of species n the family only a few karyotypes have been reported. except for the Australian species that are fairly well studied and exhibit a high level of chromosomal variation. The diploid number among gekkonid lizards ranges from 2n"16 to 2n=46 (Gorman. 1973: Schmid et a. 1994). The typical karyotype consists of a gradual series of acrocentric chromosomes and there is no distinction between macro and microchromosomes. When biarmed chromosomes are present the centromere is often subterminal (Gorman 1973). 1 Departamemo de Biologla nsliluto de Biociencias USP. Celleo Postal sao Paulo. SP Brasil. send correspondence 10 y.y.y 2 Departamento de BJo!ogla. lnsututo de B~ncias UNESP. Cebca Postal RiO Claro SP BrBBU. 3 Depalamento de Zoologla. nstiluto de Biod1!nciaS. USP sao Paulo. SP. llas11 According to the most recent checklist the genus Hemidaetylus includes 74 species and several subspecies (Kluge 1991). Taxonomically it is the most diverse group of gekkonidlizards (Darevsky eta. 1984). Karyological analyses of the genus have been performed on some diploid and triploid species. Among the diploid species studied are ncluded: H. fenams H. broocii and H. tlaviridis (all with 2n=40) H. turcicus (2n=44) and H. bowligii With 2n=46 (Singh et a. 1970; Gorman 1973; King 1978; De Smet 1981; Kupriyanova and Darevsky 1989). The triploid species which are all parthenogenetic. include: H. vieulamensis (2n=3x-60) H. gamolii (2n=3x=70) and H. stejnegeri with 2n-3x-56 (Kluge and Eckardt. 1969; Darevsky et a. 1984; Ota and Hikida 1989ab; Ota eta. 1989). nall these papers only conventionally stained karyotypes were described t has been postulated that most of the relatively few endemic New World gekkonine genera evolved from Old World ancestors. According to Kluge (1969) Hemidaetylus mabouia is derived from west African coast stock that reached the New World by natural trans-atlantic dispersal. This hypothesis was criticized by Vanzolini (1978) who argued that the

2 528 PeUegnno 61 ai species probably arrived in the New World on several different occasions both by natural and human means oftransportanon.rn the eastern coast of SouthAmerica H. mabouia is known to occur rom Uruguay to Guyana; along most ot the length 01 the Amazon river n BraZil; from its headwaters in Ecuador and Peru; and from the Antilles islands (Kluge 1969). t was also reported that the species was not restricted to a narrow margin along the eastern coast. being found far inland in the upper Amazon the upper Madeira and the edges of the Pantanal in the State of Mato Grosso (Vanzolini 1978). indicating that this expansion has a clear association with human settlements. Until now the data on the karyotype 01 H. mabouia are restricted to Giemsa stained preparations from populations of Sao Paulo Brazil and Tlinidad (Becak et al: 1972: MacBee et al. 1987). MATERlALAND METHODS Cytogenetic studies were carried out on ten specimens of H. mabouia (five males and five females) collected in: Fazenda Bravo Cabaceias State of Paralba ('f'2s's 'W); anct tatiba (23 0C)()'S 4sOs1'W) Rio Claro (22~4'S 4~30'W) and Sao Paulo (23~'S 46~l'W) an in the State of Siio Paulo. All tbe specimens were deposited in the Museum of Zoology University of Siio Paulo (MZUSP). Chromosomal preparations were obtained uem bone marrew liver and fibroblast cultures (according to Yonenaga-Yassuda at a. 1988). Meiotic studies were performed on male specimens following tbe technique described by Eicher (1966). Chromosomes were analyzed after conventional and differential staining which included C'banding (Sumner 1972) and R-banding after in vitro incorporation of 5-bromodooxyuridine (Dutrillaux and Couturier 1981) and Ag-NORs (Howell and Black. 1980) RESULTS HemidactyJus mabouia presented a diploid number of 2n=42 and a karyotype consisting of: eight submetacentric chromosomes (pairs 5 7 and 15) 28 Bubtelocentricsor acrocentrics (pairs ' ) and six small metacentrics (pairs 19-21). Macro and microchromosomes were indistinguishable and no sex chromosome heteromorphism could be observed (Figure la). The silver stained metaphases showed that the Ag-NORs occur in the short arms of one medium sized subtelocentric or acrocentric pair which is not morphologically distinctive. The Ag-NOR-bearing pair was frequently observed inassociation (Figure 1b) The C-bancling pattern revealed that only lightly stained heterochromatic blocks are present in the telomeric region of most chromosomes. Some smaller chromosomes were almost devoid of positive C-bands (Figure 2a). The R-banding pattern allowed the identification of most chromosomal pairs (Figure 2b). Meiotic studies revealed 21 bivalents in diplotene cells (Figure 3a) and 21 chromosomes in metaphase (Figure 3b). No heteromorphic bivalent was detected. DSCUSSON The diploid number of found in H. mabouia is situated within tbe range of the family. <atyorypes with no distlnctlon betweenmacro and microchromosomes as the one we found in H. mabouia are also typical of the Gekkonidae. i ' t~ tl.t i a ~ t T t t t..1 " a e t la A A a e ~ < '....' '.' ""'" 'C.' ~ \. ~"."4'- 6 _.. ~ Jl'.' '. too '. ;;'. Figure 1. (a) KaYOlype of HemidJcl)'Jus lllllbouia female (2n-42).[ter conventionel staining. (b) Metaphose with the Ag-NOlls stained in D mediwn size subteloccnuiczacrocenmc pair (anows). nset: the All-NOR-bearing pair in associlllion (orrow). Bar - 10 'm.

3 ". i j 1' e b L.. _ Figwe3. Meiotic relb of f~us mllbouil rnakl (Zo='ll. (3) Diplotene with twenly'olo bivolents. (b) Mcllp!lase with twenly-one c!homrsllmes lilt a 10 Jm ~ J: ~ J j ti : L A. : - '0 " U ~ - S : ' < t f u tf ;i ; t i t. Ji.. i t "1 t i An essentially identical karyotype was previously reported by~ er al. (1972) and MacBee «al. (1987) fill the same species. 111er8 are some slight differences between the kayotype described by the!lst authors and the one presented here: thepairs 4 and 5 6 and 7 in our study correspond to pails and 6. respectively. in their kayotypes and pair 15 differs n morphology. We consider the latter pair a submetaeentncwhileitwasdesaibedasa metaeentnc by them. Tlese discrepancies ae probably due to "! -- Figure 2. Oandlno Pllt1.emS n /f~fllldoclylu.~ mabouia [emilie Zn-42). (0) C'bonding. (b)ll bonding nncr 5 BrdU ncorporation in wllo. 8a m. - : differences in the quality of the cytological preparations and to differential condsnsauon of the chromosomes. Heteromorphic sex chromosomes were not observed in H. mabouia. This was corroborated by the absen<:e ofa distinctive heteromorphic bivalent n male cells This result is in agreement with that previously found by Beqak 9t a1. (1972) The presence of heteromorphic sex chromosomes in Gekkonidae s known only from 10 species belonging to five genera (Ota et al. 1992) and as far as we know th«. is no reference to sexual chromosomes n Hemid8C1Ylus. Most gekkonlds have a chromosomal mecbanism ofsex determination of the ZZ:ZW type. in which the heterogametic sex s the female such as inharsronotia binoei (Moritz 1984a; 1990) Gehyra ausflalis (King. 1963). Gehyra purpuacens (Moritz. 1984b) and Cyrtodactylus pubisujcus (Ota at sj. 1992). However n a few species the mechanism is of the XX:XY type. such as in Gekko gekko (Solleder and Schmid 1984) Gekko japonicus (Yoshida and Masahiro 1974 apud Moritz. 1990) and Gonatodes cecl1iae (MacBee et a ). cmomoscmel studies basqd on differential staining techniques were mainly performed in Australian genera such as Oehyra (King 1984; Moritz 19Mb; 1986) PbyHodacty1us (King and Rofe 1976) lind Hewroootia (Moritz. 1984a; 1990). n these cases. banding techniques helped to clarify aspects 0( the chromosomal variability of these lizards. C- and R-banding patterns. as well as Ag MaRs location are presented here for the first time for the genus Hemidaetylus 'There ae only a few reports of R-banding patterns of gekkonid lizards. For instance n LepidodaetY!uslugubris (Volobouev and Pasteur 1988; Volobouev at a. 1993) and Ailuronyx seychellensis (Volobouev and netch 1994) a-banding patterns were also estabushed with prsparatlons obtained from fibroblast cultures. Although most kayotoa1cal studies on Jjzards have been perfonned with conventionally stained chromosomes the application of banding patterns represents an essential tool to precisely characterize the complements. as well as to elucidate the evolutionay kayotypic nends of the group. ACKNOWLEDGMENTS 'Clio auuuls wish 10 thank 10 Dr. Tien Hsi Chu lor lhe brobaal wkwes llld 10 Mirialll Rcmeo lor leehntcel l&itonce. We am deeply grolefullo Mllllll Svlrlmlln lor e aitic8lr9lldjng 01 lhe 1ll8lJ8C!pl

4 Pellegrino el a. Ths work was supported by Conselho Nacional de DoscnvolYlmento CicnUftco 0 Tocno6g1co (CNPq) and Furxa\;ao de Ampuo 6 Pel;quso do Estodo de S'lo Paulo (FAPESP). PublicoUon Slppllfled by FAPESP. RESUMO Hemidadylus mabouij (2n-42) provenlonlo doo eslados da Paralba 0 S'lo Paulo Brosi o! estudado citogenelicomenlo atravtls de oolora\;lkl convoncional e diferenclal (bandas C- R- e Ag RONa). A malaria doe aomossomoe 6 do sublolooontricoa au aa0c6ntricoa com varla\;lkl grbdativa do tomsnho. Os padriies de bandamento e a 1ocollzaCiio lias Ag.RONa (om urn par lrbtoloo6ntrico au aaoc6ntrico de tomanho roodio) sao descailcs aqu polo primeira vez. REFERENCES ~ J.L.~ W. and DeDaro L. (1972). Chromosomo polymorphism. goographicol varlauon and karyotypos n. Souria. Quyologia 25: DarevUy LS~ Xuprlyuova LA and Rc.hc:hn V.V. (1984). A new all-femalo triploid species 01 gekko and karyological dalo on the bisexual Homk1BClylus fretl8lus from Vietnam. J. HetperoL 18: zn 284. De Smet W.H.O. (1981). DescripUon of the orcein stalnod karyotypes of V lizards species (Laoartllla Reptilia) belonging to the famllies guanidao Agamidao Chernaleontidao and Ookkonldao. Act Zooogica el Plllhologica AntvetpJensJa 76: Dubillauz B. and Couturl_ J. (1981). La Pratique del' Anayse CllrOf!ltl'lOOlique. M8lJiKi"4 Paris. pp. 88. Ecber E. (1966). An mproved air-drying tocllnique for recovery of all S18ges of melosts n thememmallan testis. MammaL Chrcm News}. 2fJ: rmaD G.C. (1973). Tha chromosomes of the Reptilia a cytotaxonomlc nterpretation. n: CylO8Xonomy snd Venebmre Evo/uliaJ (Chiarelli. A.B. and Cappall8. E. eels.). Academic Press New York P Hown W... and Blcr D.A. (1900). Controlled si!vef-s18lning of nucleolus organizer regions with a protective colloidal dovoloper: a t-step method. ExperJen1J8 36: XDg. (1978). A now caromoeome lorm 01 HomJdaClylus!enalUs (Durner1and Bilxon). HetpetologiCll 34: KDg. (1983). Koryolyplc ewjution n Oohyra (Gekkonidae: Reptilia). ll. Tho Oohyra australis compox. Ausl J. Zoo/. 31: XDg M. (1984). Karyotypic evolution in Ooh)'Ts (Gekkonidao: Reptilia). V. Chromosome change and speciation. Qenetic 64: KDg. (1990). Chromosomal and immunogenetic data: a new perspective on tho origin of Australla's reptiles. n: Cytogenetics of Amphibians and Repl/Jes (Olmo E. ed.). BlrkhiilJBll( Verlag Switzodand P KDg. androle R. (1976). Karyotypic variation in the Australian gekko PhyllodaClYlus U81lDOlatus (Gray) (Gekkonldao: Reptilia). Chromosoma 54: 75-f!l. X1ulJ8 A.G. (1969). Tho evolution and geographical origin 01 tho New World HemJdacrylus mabouja-brookh complex (Ookkonldae Sourla). Mise. Publ. Mus. ZooJ. UnJv. MJchiQan 138: Kluge A.G. (1991). Checklist 01 gekkonotd lizards. SmJrb90nJan Heq:>e.%gical1nformaliaJ SetviaJ 85: Klug.. A.G. and Edwdt MoJ. (1969). Homidac!yus gamolij Dumeril and Bibron a triploid au lemale species of liekkonid lizard. CopeJa 1969: Xupriyanova L.A. and Dare".ky S. (t989). Karyotypic unilorrniry n oost Asan populations of Hemidaclylus!0Tl1Us (Sawia: Gekkonidae). J. Herpetd. 23: Jlacbee t. Blc:dwD J.W. and Dxoa Jr. (1987). Malo heterogamoty and chromosomal variation n caribbeangeckos. J. Hetpeto!. 21: Mortz C. (19840). The ortgtn and evolution of parthenogenesis n HeteronorJa bjnoej (Ookkonidae).. Chromosome banding studies. Chromosoma 89: Moritz C. (1984b). The evolution of a highly variable sex chromosome n Geh)'Ta purpurascons (Gekkonldao). Chromosoma 90: lll-ll9. Mortz C. (1986). The population biology of Oohyra (Ookkonldas): chromosome change and speciotlon. Sysl ZooJ. 35: Moritz C. (1990). Patterns and processes of sex chrolllljllollll evolution n gekkonid lizards (Saurla: Reptilia). n: CylogenerJcs 01 Amphibians and Ropliles (Olmo. E.. od.). BirkMusor VOag Switzerland. pp fJ. Ota H. and Hdda TA (19890). A record ol a triploid gecko HomJdaetylus slbjnegeri. from the northern Phlllppines. Jpn. J. Herpstol. 13: Ota H. and Hdda TA (1989b). A now triploid HomJdaety/us (Souria: Gekkonldae) from Taiwan. with comments on morphological and karyologlcal variation n the H. (}/llolij-violnamonsb complox (Saurla: Gekkonldae). J. HetpOto. 23: Ota H. Hlldda T. ond Lue J. Y. (1989). Poyc!ony in a triploid gecko HemJdaety/us slbjnegeri from Taiwan. with notes on lis bearing on the chromosomel divlllslty of the H. gamoajvielnamensis complex (Sourla: oekkonidao). Genetlal 79: Ota H. Hldda T. Matsui. and Mart A. (1992). Karyotypos 01 two species 01 the genus Cyrtodaclylus (Squamata: Ookkonidael from Sarawok Malayslo. Calyologia 45: c:hmd M~ r.k:htdger W~ HUlda. SChalowU R. Garda R.V~ Puppo J.M. add 8dUo #0:1. (1994). An extraordinary low diploid chromosolllll number n the repue GaJalodes llnlae (Squamata Gekkonldae). J. Heredity 85: Singh L. Sharma T. and Ray Cbaudhurl S.P. (1970). Chromoeomo numbers and sex cnrcmoeomee in a few ndian species of emphibla and reptiles. Mammal. Chrom. NowsJ. :

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