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TheAuk 114(3):516-520, 1997

Sex Identification of South American (, Aves) Using the Human Minisatellite Probe 33.15

CRISTINAY. MIYAKI,• J. MAURICIOB. DUARTE,2 RENATOCAPARROZ, •'2 ADAUTO L. V. NUNES,3 AND ANITA WAJNTALTM •Departamentode Biologia, Universidade de Sao Paulo, C.P. 11,461, CEP 05422-970,Sao Paulo, SP, ; 2DepartamentodeMelhoramento Gen•tico , FCAVJ, UNESP, RodoviaCarlos Tonanni Km5--Jaboticabal, CEP 014870-000,St•o Paulo, SP, Brazil; and 3ParqueZooldgico de Sorocaba, Sorocaba, SP, Brazil

Many speciesof South Americanparrots are en- appliedin a varietyof wild ,including birds dangered,and captive breeding has becomea stan- (Burke and Bruford 1987, Wetton et al. 1987). It was dard procedure for speciesconservation. Because usedto monitorgenetic variability in captivePuerto most South American parrots are not sexually di- Rican Parrots (Amazona vittata; Brock and White morphic, an efficientmeans of determining the sex 1992)and to establishpaternity in endangeredspe- of individuals is an important tool in establishing cies(Math6 et al. 1993). Recently,we used DNA fin- and maintaining a viable breeding population in gerprintingto identifythe sexof Peach-fronted(Ar- captivity. atingaaurea) and Golden (Guaruba[] guar- DNA fingerprinting(Jeffreys et al. 1985)has been ouba)parakeets (Miyaki et al. 1992)and suggested that fingerprintingalso couldbe used to determine sex in other psittacines(Miyaki et al. 1993, 1995). Addresscorrespondence to this author.E-mail: Here, we presentresults on sex determinationusing [email protected] the human minisatelliteprobe 33.15 (Jeffreyset al. July1997] ShortCommunications andCommentaries 517

TABLE1. Patternof intensebands in previouslysexed and unsexed parrots. F andM arenumber of females and males;N and Nb are number of individuals and number with intensebands, respectively.

Sexed Unsexed Species F M Methoda Bandsb N Nb Total Amazona aestivaaestiva 5 8 k - 10 0 23 Amazonaaestiva xanthopteryx 1 3 k - 0 0 4 Amazona amazonica 2 2 k - 2 0 6 Amazona autumnalis diadema 1 0 k - 1 0 2 Amazona brasiliensis 2 2 k, 1 - 7 0 11 Amazonadufresniana rhodocorytha 0 0 4 0 4 Amazonafarinosa 0 2 k - 3 0 5 Amazonafestiva 0 2 k - 4 0 6 Amazonaochrocephala xantholaema 1 1 b - 5 0 7 Amazonapretrei 3 3 d - 4 0 10 Amazona vinacea 2 4 k, b - 6 0 12 Amazonaxanthops 1 0 k - 4 0 5 Anodorhynchushyacinthinus 3 5 k + 55 32 63 Anodorhynchusleari 1 1 k, 1 + 1 1 3 auricollis 2 1 k + 1 1 4 Ara ararauna 3 2 k, b + 12 8 17 Arachloroptera 2 5 k, b + 15 4 22 Ara macao 2 5 k + 9 4 16 Ara manilata 2 1 k + 7 4 10 Ara maracana 4 1 k + 2 0 7 Ara nobilis 2 4 k + 11 3 17 Aratingaacuticaudatta 1 1 k + 0 - 2 Aratingaaurea 2 2 k + 32 14 36 Aratingaleucophthalmus 1 1 b + 10 5 12 Aratingamitrata 0 0 2 1 2 Aratingasolstitialis auricapilla 0 0 8 2 8 Aratingasolstitialis jandaya 0 0 4 4 4 Cyanopsittaspixii 3 4 k + 0 - 7 Deroptyusaccipitrinus 1 1 b - 0 - 2 Guarubaguarouba 3 3 k, b + 19 9 25 Nandayusnenday 2 0 b + 21 10 23 Pionitesleucogaster 1 1 k - 4 0 6 Pionopsittapileata 1 1 d - 6 0 8 menstruus 2 2 b - 7 0 Pyrrhuraegregia 1 0 b - 1 0 2 Pyrrhurafrontalis 1 1 b - 2 0 4 Pyrrhurapicta 1 1 b - 3 0 5 Triclaria malachitacea 1 1 d - 0 - 2 k, karyotypeanalysis; 1, laparoscopy; b, breeding behavior; d, sexualdimorphism. Female-specificbands: -, absent;+, present.

1985)in 36 speciesbelonging to 13 generaof South tion enzymeMboI (for Amazona)or HaelII (for the American parrots. othergenera). Fragments were separated by electro- Methods.--Bloodsamples were collected from phoresisthrough a 30-cmlong 1% horizontalaga- birds belongingto aviculturistsand officialestab- rose gel. Electrophoresiswas stoppedwhen the lishments in Brazil. For some individuals, sex was 2-kilobase(kb) markerband had migratedto thebot- determinedby karyotyping,laparoscopy, or breed- tom of thegel. The fractionated DNA fragmentswere ing behavior.Whenever possible, growing transferredonto a nylonmembrane by standardcap- (ca.25 daysold) werecollected from birdswhose sex illary Southernblotting (Sambrook et al. 1989). wasunknown and processedfor karyotypeanalysis. The membranewas hybridizedwith minisatellite Chromosomepreparation and analysisfollowed 33.15probe (Jeffreys et al. 1985),which was labeled Duarte and Caparroz(1995). The speciesand num- with [ct-32P]dCTP or [ct-32P]dATP. Pre-hybridization ber of individuals studied are shown in Table 1. in 0.263M Na2 HPO4, 1 mM EDTA, 7% SDS,and 1% The protocolsused to obtain multilocusfinger- BSA at 65øClasted for 4 h, and the probewas added printsfollowed Bruford et al. (1992).For each , 5 and left overnight at 65øC. The membrane was •g of genomicDNA were digestedwith the restric- washed in 0.25 M Na2HPO4,1% SDS, 2XSSC, 0.1% 518 ShortCommunications and Commentaries [Auk, Vol. 114

A B C D E F G H I

kbFFMMMFFFFMMFFMFFMFFMMFFMM kb FM F F MMM 23-

4.4-

FIG. 1. ParrotDNA hybridizedwith human minisatelliteprobe 33.15. Female-specific bands are shown with arrows. (A) Ara auricollLs,(B) Am chloroptera,(C) Ara macao,(D) Ara manilata,(E) Am maracana,(F) Ara nobills,(G) Anodorhynchushyacinthinus, (H) Anodorhynchusleari, (I) Cyanopsittaspixii. F = female,M = male.

TABLE 2. Molecular size of the sex-linked bands in SDS and in IxSSC, 0.1% SDS at 65øC. The filter was SouthAmerican parrots. then autoradiographedat -70øC using Kodak RX film with one or two intensifyingscreens. Number Molecular size Results.--Apattern of two to four intensesex-spe- Species of bands (kb) cific bands was detectedwith the 33.15 probe in all femalesstudied in the generaAra and Aratingaand hyacinthinus 3 3.1; 5.1; 5.2 alsoin femalesof Anodorhynchushyacinthinus, A. leari, Anodorhynchusleari 2 3.1; 18.5 CyanopsittaspixiL Guarubaguarouba, and Nandayus Ara ararauna 2 1.9; 2.5 nenday(see Fig. 1). These intense bands also were Ara auricollis 4 2.7; 3.7; 3.9; 4.8 Ara chloroptera 4 2.7; 3.7; 4.0; 4.1 presentin somepreviously unsexed individuals of Ara macao 4 2.5; 4.0; 4.1; 4.3 Aratingasolstitialis auricapilla, A. s.jandaya, and A. mi- Ara manilata 3 2.7; 3.5; 4.0 trata(Table 1). Eachspecies showed unique patterns Ara maracana 3 2.7; 3.9; 4.6 of female-linkedbands according to their molecular Ara nobilLs 3 2.5; 4.7; 11.5 size(Table 2); thesespecies-specific profiles are a po- Amtingaaurea 2 3.7; 7.6 tential tool for speciesidentification. Female-linked Aratingaacuticaudatta 3 2.9; 3.6; 4.6 bandsalso were observedin long-tailedspecies even Aratingaleucophthalmus 2 3; 7.6 Aratingareitrata 3 2.9; 3; 10.5 when other restrictionenzymes were used(data not Aratingasolstitialis shown). Sex-linked bands were absent in known fe- auricapilla 3 4.2; 5.0; 6.0 malesof eight speciesof Amazonaand of Pionusmen- Aratingasolstitialis struus, Deroptyusaccipitrinus, Pionites leucogaster, jandaya 3 4.1; 5.2; 5.8 Pionopsittapileata, egregia, P. frontalis, P. picta, Cyanopsittaspixii 2 3.1; 3.5 and Triclaria malachitacea. We used MboI for members Guarubaguarouba 2 2.8; 5.7 of Amazonabecause this enzyme produced more Nandayusnenday 3 3.9; 4.8; 6.0 polymorphicDNA fingerprint profiles;even when July1997] ShortCommunications and Commentaries 519 we used HaeIII, no sex-linkedfragments were ob- tern of a few intense bands detected in some unsexed served (data not shown). individuals was restricted to females. Discussion.--Untilrecently, only threemethods of Little is known about the phylogeneticrelation- sex identification were available for birds: fecal hor- shipsof psittacines.The availabledata are basedon moneassay, chromosome analysis, and laparoscopy. chromosomal evolution (Valentine 1990, Christidis et Hormone analysesvary with reproductivecondition al. 1991),albumin (Sibley 1960), and the cytochrome- and thus are age and seasondependent. Chromo- b gene (Birt et al. 1992, Leetonet al. 1994).Based on someanalysis is reliablebut time consuming,and in karyotypecorrelations, Valentine (1990) proposed an many occasionsappropriate metaphase cells cannot early separation of Amazonafrom other genera. be found for analysiswithout repeatedattempts. Based on habitat exploration, Mont6n (1977) sug- Birdshave to behandled twice for karyotyping,once gestedthat the short-tailedand the long-tailedbirds for removinga sampleof feathersand a secondtime havebehavioral differences. The presenceof w-chro- for collectingthe growingfeather pulp. Becauselap- mosome-linkedminisatellite sequences in mostlong- aroscopyis a surgicalprocedure, birds are exposed and sharp-tailed psittacines,and their to anesthesiaand to the risksof surgery. absencein the short-tailedspecies, provide support Recently,several DNA techniqueshave been de- for the separateevolution of thesetwo groups.Pre- veloped for sex identification in birds (de Kloet and liminaryanalysis of mitochondrialgene (12S and 16S de Kloet 1992,Griffiths and Tiwari 1993,May et al. rDNAs and cytochrome-b)sequences of nine species 1993). Griffiths and Tiwari (1995) determined the sex of Brazilianparrots also supportsthe separationof of the last Spix'sMacaw (Cyanopsittaspixii) from the long-and short-tailedspecies (Miyaki 1996). shed feathers that were collected in the wild and Acknowledgments.--Thiswork was funded by FA- used as a sourceof DNA for PCR amplification.In PESP,CNPq, and CAPES(Brazil). We thank T. Burke our applicationof DNA fingerprintingin South for a visit to his lab; O. Hanotte and C. E M. Menck Americanparrots, we identified female-specificin- for their constantsupport; P. F. Flechafor invaluable tensebands in somespecies using the human mini- discussion;and L.A. Labruna (ParqueEco16gico do satelliteprobe 33.15 (Miyaki et al. 1992,1993). Other Tiet•, SP), A. L. V. Nunes (SorocabaZoo), N. Kawall, authors also have detected sex-specificbands in L. Maluf, A. Marra, A. Vertematti, C. Isoldi, and M. DNA fingerprintingof birds usingprobe 33.15 (Ra- Silva (aviculturists,Brazil), L. Sanfilippo(Sgo Paulo benoldet al. 1991,Longmire et al. 1992) and probe Zoo), and M. I. Bampi (IBAMA) for blood 33.6 (Graveset al. 1993).Although probe 33.15 could samples.C. Y. M. hasa FundoBUNKA/90 prize.The Jeffreys'probes 33.6 and 33.15are the subjectof pa- not be used to identify the sexof all the specieswe tent No. GBA 2166445and worldwide patents for studied, it was invaluablefor sexingmacaws (Ara, commercialdiagnostic use. We alsothank Associate Anodorhynchus,and Cyanopsitta)and (Aratin- Editor Allan Bakerand two anonymousreviewers ga and Nandayus).These genera belong to the "long, for commentsand editorial suggestions. point-tailed"group and are consideredto be closely related. Our method could not be used to sex the LITERATURE CITED short-tailed parrots in the genusAmazona, and our datasuggest that it alsowas not adequatefor sexing BIRT, t. P., V. L. FRIESEN,J. M. GREEN,W. A. MON- other short-tailed speciessuch as Pionusmenstruus, TEVECCHI,AND W. S. DAVIDSON.1992. Cyto- Pionitesleucogaster, and Pionopsittapileata (which has chrome-b sequencevariation among parrots. sexuallydimorphic ), as well as the "long, Hereditas 117:67-72. wide-tailed" Deroptyusaccipitrinus and Triclariama- BROCK,M. K., AND B. N. WHITE. 1992. Application lachitacea(the latter has sexuallydimorphic plum- of DNA fingerprintingto the recoveryprogram age). The absenceof female-linked bands was not of the endangeredPuerto Rican Parrot. Proceed- due to the factthat a differentrestriction enzyme was ings of the National Academyof SciencesUSA applied. No sex-specificpattern was detectedin the 89:11121-11125. three speciesof Pyrrhurawe studied,each of which BRUFORD,M. W., O. HANOTTE, J. E Y. BROOKFIELD, has a long, sharp-pointedtail. AND t. BURKE.1992. Single locus and multilo- In all caseswhere we performedkaryotyping, we cus DNA fingerprinting.Pages 225-269 in Mo- confirmedthat the intenseband pattern was present leculargenetics analysis of populations.A prac- in all females and absent in males. Because of the dif- tical approach (C. A. R. Hoelzel, Ed.). Oxford ficultiesinherent in chromosomeanalysis (i.e. lack of UniversityPress, New York. growing feathersand failure to obtain appropriate BURKE,T., AND M. K. BRUFORD.1987. DNA finger- metaphasecells), it was impossibleto determinethe printing in birds. Nature 327:149-152. sexof manyof thespecies that we studiedwith probe CHRISTIDIS, L., D. D. SHAW, AND R. SCHODDE. 1991. 33.15. However,the agreementbetween the DNA re- Chromosomal evolution in parrots, 1orikeets suitsand the cytogeneticdata in all speciesfor which and (Aves: Psittaciformes). Hereditas both studieswere performed suggeststhat the pat- 114:47-56. 520 ShortCommunications andCommentaries [Auk,Vol. 114

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