Sex Identification of South American Parrots (Psittacidae, Aves

Sex Identification of South American Parrots (Psittacidae, Aves

516
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Received
3 September
1996,accepted
5 February1997.
Associate
Editor:M. E. Murphy
TheAuk 114(3):516-520,1997
Sex Identification of South American Parrots(Psittacidae,Aves) Using the Human
Minisatellite
Probe 33.15
CRISTINAY. MIYAKI,• J. MAURICIOB. DUARTE,2 RENATOCAPARROZ,
•'2
ADAUTO L. V. NUNES,3 AND ANITA WAJNTALTM
•Departamento
deBiologia,
Universidade
deSaoPaulo,C.P.11,461,CEP05422-970,SaoPaulo,SP,Brazil;
2Departamento
deMelhoramento
Gen•ticoAnimal,FCAVJ,UNESP,
RodoviaCarlosTonanniKm5--Jaboticabal,
CEP 014870-000,St•oPaulo,SP,Brazil;and
3Parque
Zooldgico
deSorocaba,
Sorocaba,
SP,Brazil
Many speciesof SouthAmericanparrotsare endangered,and captivebreeding has becomea standard procedure for speciesconservation.Because
most South American parrots are not sexually dimorphic, an efficientmeansof determining the sex
of individuals is an important tool in establishing
and maintaining a viable breeding population in
captivity.
DNA fingerprinting(Jeffreyset al. 1985)hasbeen
Addresscorrespondence
to this author.E-mail:
[email protected]
appliedin a varietyof wild species,includingbirds
(Burke and Bruford 1987, Wetton et al. 1987). It was
usedto monitorgeneticvariabilityin captivePuerto
Rican Parrots (Amazona vittata; Brock and White
1992)and to establishpaternityin endangeredspecies(Math6 et al. 1993).Recently,we used DNA fin-
gerprintingto identifythe sexof Peach-fronted
(Aratingaaurea)and Golden (Guaruba[Aratinga]guarouba)parakeets(Miyaki et al. 1992)and suggested
that fingerprintingalsocouldbe usedto determine
sex in other psittacines(Miyaki et al. 1993, 1995).
Here, we presentresultson sex determinationusing
the human minisatelliteprobe33.15 (Jeffreyset al.
July1997]
Short
Communications
andCommentaries
517
TABLE
1. Patternof intensebandsin previously
sexedandunsexed
parrots.F andM arenumberof females
and males;N and Nb are number of individuals and numberwith intensebands,respectively.
Sexed
Species
Method
a
Unsexed
F
M
N
Nb
Amazona aestivaaestiva
5
8
k
Bands
b
-
10
0
Total
23
Amazona
aestiva
xanthopteryx
1
3
k
-
0
2
1
7
0
0
0
0
4
6
2
11
Amazona amazonica
Amazona autumnalis diadema
Amazona brasiliensis
2
1
2
2
0
2
k
k
k, 1
Amazona
dufresniana
rhodocorytha
Amazona
farinosa
Amazona
festiva
Amazona
ochrocephala
xantholaema
Amazona
pretrei
0
0
0
1
3
2
0
2
2
1
3
4
k
k
b
d
-
4
3
4
5
4
0
0
0
0
0
4
5
6
7
10
Amazona
xanthops
Anodorhynchus
hyacinthinus
Anodorhynchus
leari
1
3
1
0
5
1
k
k
k, 1
+
+
4
55
1
0
32
1
5
63
3
Ara ararauna
3
2
k, b
+
12
8
17
Arachloroptera
2
5
k, b
+
15
9
7
4
4
4
22
Ara maracana
Ara nobilis
4
2
1
4
k
k
+
+
2
11
0
3
7
17
Aratinga
acuticaudatta
Aratinga
aurea
Aratinga
leucophthalmus
Aratinga
mitrata
Aratinga
solstitialis
auricapilla
Aratinga
solstitialis
jandaya
Cyanopsitta
spixii
Deroptyus
accipitrinus
Guaruba
guarouba
Nandayus
nenday
Pionites
leucogaster
Pionopsitta
pileata
1
2
1
0
0
0
1
2
1
0
0
0
k
k
b
+
+
+
0
32
10
2
8
4
14
5
1
2
4
2
36
12
2
8
4
3
1
3
2
1
1
4
1
3
0
1
1
k
b
k, b
b
k
d
+
+
+
-
0
0
19
21
4
6
9
10
0
0
7
2
25
23
6
8
Pyrrhura
egregia
Pyrrhura
frontalis
Pyrrhura
picta
1
1
1
0
1
1
b
b
b
-
1
2
3
0
0
0
2
4
5
Amazona vinacea
Ara auricollis
Ara macao
Ara manilata
Pionusmenstruus
Triclaria malachitacea
2
2
2
2
1
1
5
1
2
1
k, b
k
k
k
b
d
-
+
+
+
-
-
6
1
7
0
0
1
0
-
12
4
16
10
2
k, karyotypeanalysis;
1,laparoscopy;
b, breeding
behavior;
d, sexualdimorphism.
Female-specific
bands:-, absent;+, present.
or HaelII (for the
1985)in 36 speciesbelongingto 13 generaof South tion enzymeMboI (for Amazona)
othergenera).Fragmentswereseparatedby electroMethods.--Bloodsamples were collected from phoresisthrougha 30-cmlong 1% horizontalagawas stoppedwhen the
birds belongingto aviculturistsand officialestab- rose gel. Electrophoresis
lishments in Brazil. For some individuals, sex was
2-kilobase(kb) markerbandhadmigratedto thebotDNA fragmentswere
determinedby karyotyping,laparoscopy,
or breed- tomof thegel.Thefractionated
ing behavior.Wheneverpossible,growingfeathers transferredontoa nylonmembraneby standardcapblotting(Sambrook
et al. 1989).
(ca.25 daysold) werecollectedfrombirdswhosesex illary Southern
The membranewashybridizedwith minisatellite
wasunknownand processed
for karyotypeanalysis.
et al. 1985),whichwaslabeled
Chromosomepreparationand analysisfollowed 33.15probe(Jeffreys
dCTP or [ct-32P]
dATP.Pre-hybridization
Duarte and Caparroz(1995).The speciesand num- with [ct-32P]
ber of individuals studied are shown in Table 1.
in 0.263M Na2HPO4,1 mM EDTA, 7% SDS,and 1%
The protocolsused to obtain multilocusfinger- BSA at 65øClastedfor 4 h, and the probewas added
printsfollowedBrufordet al. (1992).Foreachbird, 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%
American parrots.
518
ShortCommunications
andCommentaries
A
B
C
D
E
F
G
kbFFMMMFFFFMMFFMFFMFFMMFFMM
H
kb FM
[Auk,Vol. 114
I
F F MMM
23-
4.4-
FIG. 1. ParrotDNA hybridizedwith human minisatelliteprobe33.15.Female-specific
bandsare shown
with arrows. (A) Ara auricollLs,
(B) Am chloroptera,
(C) Ara macao,(D) Ara manilata,(E) Am maracana,
(F) Ara
nobills,(G) Anodorhynchus
hyacinthinus,
(H) Anodorhynchus
leari,(I) Cyanopsitta
spixii.F = female,M = male.
TABLE 2.
Molecular
size of the sex-linked
bands in
SouthAmericanparrots.
Number
Species
Molecular
of bands
size
(kb)
Anodorhynchus
hyacinthinus
Anodorhynchus
leari
3
2
3.1; 5.1; 5.2
3.1; 18.5
Ara ararauna
Ara auricollis
2
4
1.9; 2.5
2.7; 3.7; 3.9; 4.8
Ara chloroptera
4
2.7; 3.7; 4.0; 4.1
Ara
Ara
Ara
Ara
4
3
3
3
2.5;
2.7;
2.7;
2.5;
2
3
2
3
3.7; 7.6
2.9; 3.6; 4.6
3; 7.6
2.9; 3; 10.5
3
4.2; 5.0; 6.0
3
2
2
3
4.1; 5.2; 5.8
3.1; 3.5
2.8; 5.7
3.9; 4.8; 6.0
macao
manilata
maracana
nobilLs
Amtingaaurea
Aratingaacuticaudatta
Aratingaleucophthalmus
Aratingareitrata
Aratingasolstitialis
auricapilla
Aratingasolstitialis
jandaya
Cyanopsitta
spixii
Guarubaguarouba
Nandayusnenday
4.0;
3.5;
3.9;
4.7;
4.1; 4.3
4.0
4.6
11.5
SDS and in IxSSC, 0.1% SDS at 65øC. The filter was
then autoradiographedat -70øC using Kodak RX
film with one or two intensifyingscreens.
Results.--Apatternof two to four intensesex-specific bandswas detectedwith the 33.15 probe in all
femalesstudiedin the generaAra and Aratingaand
alsoin femalesof Anodorhynchus
hyacinthinus,
A. leari,
Cyanopsitta
spixiLGuarubaguarouba,and Nandayus
nenday(see Fig. 1). These intense bands also were
presentin somepreviouslyunsexedindividualsof
Aratingasolstitialis
auricapilla,
A. s.jandaya,
andA. mitrata(Table1). Eachspeciesshoweduniquepatterns
of female-linkedbandsaccordingto their molecular
size(Table2); thesespecies-specific
profilesarea potential tool for speciesidentification.Female-linked
bandsalsowere observedin long-tailedspecieseven
when otherrestrictionenzymeswereused(datanot
shown). Sex-linked bands were absent in known fe-
malesof eight speciesof Amazonaand of Pionusmenstruus, Deroptyusaccipitrinus,Pionites leucogaster,
Pionopsitta
pileata,Pyrrhuraegregia,
P.frontalis,P.picta,
and Triclaria malachitacea. We used MboI for members
of Amazonabecausethis enzyme produced more
polymorphicDNA fingerprint profiles;even when
July1997]
ShortCommunications
andCommentaries
we used HaeIII, no sex-linkedfragmentswere obDiscussion.--Until
recently,only threemethodsof
were available
for birds: fecal hor-
moneassay,chromosome
analysis,and laparoscopy.
Hormoneanalysesvary with reproductivecondition
and thus are age and seasondependent.Chromosomeanalysisis reliablebut time consuming,andin
many occasionsappropriatemetaphasecellscannot
be found for analysiswithout repeatedattempts.
Birdshaveto behandledtwiceforkaryotyping,once
for removinga sampleof feathersand a secondtime
for collectingthe growingfeatherpulp. Because
laparoscopyis a surgicalprocedure,birds are exposed
to anesthesia
and to the risksof surgery.
Recently,severalDNA techniqueshave been developed for sex identification in birds (de Kloet and
de Kloet 1992,Griffithsand Tiwari 1993,May et al.
1993).Griffiths and Tiwari (1995) determined the sex
of the last Spix'sMacaw (Cyanopsitta
spixii) from
shed feathers
that were collected
in the wild
and
used as a sourceof DNA for PCR amplification.In
our applicationof DNA fingerprintingin South
Americanparrots,we identified female-specific
intensebandsin somespeciesusing the human minisatelliteprobe33.15(Miyaki et al. 1992,1993).Other
authors also have detected sex-specificbands in
DNA fingerprintingof birds usingprobe33.15 (Rabenoldet al. 1991,Longmireet al. 1992)and probe
33.6 (Graveset al. 1993).Althoughprobe33.15could
not be used to identify the sexof all the specieswe
studied,it was invaluablefor sexingmacaws(Ara,
Anodorhynchus,
andCyanopsitta)
andconures(Aratinga and Nandayus).
Thesegenerabelongto the "long,
point-tailed"groupand areconsidered
to be closely
related.
Our method
tern of a few intense bands detected in some unsexed
was restricted to females.
individuals
served (data not shown).
sex identification
519
Little is known about the phylogeneticrelationshipsof psittacines.The availabledata are basedon
chromosomal evolution (Valentine 1990, Christidis et
al. 1991),albumin(Sibley1960),andthecytochromeb gene(Birt et al. 1992,Leetonet al. 1994).Basedon
karyotypecorrelations,
Valentine(1990)proposedan
early separationof Amazonafrom other genera.
Based on habitat exploration,Mont6n (1977) suggestedthat the short-tailedand the long-tailedbirds
havebehavioraldifferences.The presenceof w-chromosome-linked
minisatellitesequences
in mostlongand sharp-tailed New World psittacines,and their
absencein the short-tailedspecies,provide support
for the separateevolutionof thesetwo groups.Preliminaryanalysisofmitochondrial
gene(12Sand16S
rDNAs and cytochrome-b)sequences
of nine species
of Brazilianparrotsalsosupportsthe separationof
the long-and short-tailedspecies(Miyaki 1996).
Acknowledgments.--This
work was fundedby FAPESP,CNPq, and CAPES(Brazil). We thank T. Burke
for a visit to his lab; O. Hanotte and C. E M. Menck
for their constantsupport;P.F. Flechafor invaluable
discussion;and L.A. Labruna (ParqueEco16gicodo
Tiet•, SP), A. L. V. Nunes (SorocabaZoo), N. Kawall,
L. Maluf, A. Marra, A. Vertematti, C. Isoldi, and M.
Silva (aviculturists,Brazil), L. Sanfilippo(SgoPaulo
Zoo), and M. I. Bampi (IBAMA) for parrot blood
samples.C. Y. M. hasa FundoBUNKA/90 prize.The
Jeffreys'probes33.6 and 33.15are the subjectof patent No. GBA 2166445and worldwide patentsfor
commercialdiagnosticuse.We alsothank Associate
Editor Allan Bakerand two anonymousreviewers
for commentsand editorialsuggestions.
could not be used to sex the
LITERATURE
short-tailedparrots in the genusAmazona,and our
CITED
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leucogaster,
and Pionopsitta
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sexuallydimorphicplumage),as well as the "long,
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lachitacea
(the latter has sexuallydimorphicplumage). The absenceof female-linked bands was not
dueto the factthata differentrestrictionenzymewas
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