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8,000-yr-old human brain tissuefrom the Windover archaeologicalsite. Nature 323: 803-806.
[Auk,Vol. 105
SIBLEY,C. G., & J. E. AHLQUIST. 1985. The relation-
shipsof someAfrican birds, basedon the genetic
material,DNA. Pp. 115-161 in Proceedingsof the
naturalhistorycollections.LeatherConserv.News
international symposiumon African vertebrates:
2(2): 1-4.
systematics,phylogeny, and evolutionary ecolHIGUCHI, R., B. BOWMAN, g. FREIBERGER,
O. A. RIDER,
ogy (K. L. Schuchmann,Ed.). Bonn,Zool. Forsch.
& A. C. WILSON.1984. DNA sequencesfrom the
Mus. Alexander Koenig.
quagga,an extinct memberof the horsefamily. SOUTHERN,
E. 1975. Detection of specificsequences
Nature 312: 282-284.
amongDNA fragmentsseparatedby gel electroMANIATIS,T., E. F. FRISCH,& J. SAMBROOK.1982. Mophoresis.J. Mol. Biol. 98: 503-517.
lecularcloning:a laboratorymanual.ColdSpring WALLER,
R., & D. E. MCALLISTER.1985. A spot test
Harbor, New York, Cold Spring Harbor Labofor distinguishing formalin from alcohol soluratory.
tions. Proceedingsof the 1985 workshop on the
P•d4BO,
S. 1985. Molecular cloning of ancient Egypcare and maintenanceof natural history collectian mummy DNA. Nature 314: 644-645.
tions (J. Waddington and D. M. Rudkin, Eds.).
RIGBY,P. W. J., M. DIECKMAN, C. RHODES,& P. BERG.
Life SciencesMisc.Publ.,RoyalOntario Museum.
1977. Labeling deoxyribonucleic acid to high
Received20 October1987,accepted18 April 1988.
specificactivity in vitro by nick translationwith
DNA polymeraseI. J. Mol. Biol. 113:237.
HAWKS, C. A., & S. L. WILLIAMS.
1986.
Arsenic
in
Application of Synaptonemal Complex Techniques for Determination of
Diploid Number and Chromosomal Morphology in Birds
DAVID W. HALE, ELIZABETHJ. RYDER,PHILIP D. SUDMAN, AND IRA F. GREENBAUM
Department
of Biology,TexasA&M University,CollegeStation,Texas77843USA
Avian karyologicalstudies are often complicated
by the large numbers of microchromosomes
which
characterizethe chromosomalcomplementsof most
bird species(Shields 1982, 1983). The exact number
of microchromosomes can be difficult to ascertain, as
they are often coveredby the macrochromosomes
or
lost during preparationof mitotic material for cytological analysis.In addition, the small size of the microchromosomes
makesit difficult or impossibleto
determine the position of their centromeres.Consequently, many reports of bird karyotypes present
modal or approximate diploid numbers (e.g. Biederman et al. 1980),or emphasizethe morphologyof
the macrochromosomes
(e.g. Bartlett and Threlfall
1987).We discussa techniquefor accuratedetermination of the diploid number and chromosomalmorphology of birds by electronmicroscopicanalysisof
silver-stainedsynaptonemalcomplexes.
The synaptonemalcomplex(SC)is a ribbonlike proteinaceous
structurewhich formsthe longitudinalaxis
of pachytene bivalents during meiotic prophase I.
Eachmeiotic chromosomeforms an axiswhich, upon
pairing with its homolog,becomesone of the lateral
elementsof the SC. Stainingwith silver differentiates
preferentially the two lateral elementsof the SC from
their associatedchromatin and surrounding nudeoplasm, thereby providing simple linear representations of chromosomal
behavior
and orientation
dur-
ing pachynema.In addition, the positionsof the
centromeres
of the meiotic
chromosomes
are often
discernibleasdark or thickenedregionson the lateral
elementsof the SCsin early pachytenenuclei.
The techniquefor obtainingpachytenenuclei from
the testesof adult male birds (in breeding condition)
was modified
Fig. 1. Giemsa stained somatic karyotype prepared from hypotonically-treatedbone marrow of a
femaleColinus
virginianus.
Thediploidnumberisdearly
82; however, the morphology of the smaller chromosomescannot be determined from this type of
preparation.
from one described
for mammalian
tes-
ticular material (Moses 1977). We used plastic-coated
microscopeslidesto pick up the spermatocytenuclei
after spreadingon the convexsurfaceof a 0.5%(w/v)
NaC1 solution.The slideswere prepared by dipping
them into a Coplin jar that contained a 0.6% (w/v)
solutionof plastic(FalconOptilux petri dish) in chloroform. After air-drying, the edgesof the plasticcoat
October
1988]
Short
Communications
777
Fig. 2. Synaptonemalcomplexkaryotypeconstructedfrom an electronmicrographof a silver-stained
pachyteneoocytefrom Colinus
virginianus.
The autosomalSCsare arrangedby lengthand positionedwith
their darkly stainedcentromericregionson the horizontallines.The partiallypairedZW bivalentis shown
at the upper right. The first row comprisesthe 5 macrochromosome
pairs, excludingthe sexbivalent. The
secondrow includesthe intermediate-sized
autosomes,
and the third and fourth rowscontainthe 27 pairs
of microchromosomes.
Magnification x 3,435.
were sealedwith clear nail polish on the preferred
side of the slide. The 4% paraformaldehydefixative
was prepared without sucroseor DMSO, and adjusted
to pH 8.2-8.5 with 10%formic acid (diluted from 88%
stock)and 1.0N NaOH. Fixationof the surface-spread
spermatocyteswas accomplishedin two steps.First,
the slideswere immersedfor 5 rain in a Coplin jar
containing4%paraformaldehyde
with 0.03%SDS(48.5
ml paraformaldehydesolutionwith 1.5 ml 1.0%SDS).
The slides were
then
immersed
for an additional
5
rain in paraformaldehydewithout SDS in a second
Coplin jar. After the secondfix, the slideswere drained
briefly on end and then swished in 0.4% Photoflo
(Kodak)for 10-15 s. When dry, the testicularmaterial
was stained 6-8 rain with silver nitrate (Howell and
Black 1980).
For electronmicroscopy(EM), the plasticaround
the desiredcellareawasetchedwith a diamond-tipped
scribe,and floatedoff by slowly slipping the slide
into a largedissectingbowl filled with distilled water.
FontaxEM forcepswere usedto place coppergrids
(100 mesh)onto the floatingplastic,which wasthen
lifted off the water surfacewith a strip of Parafilm.
Oncedry, the gridscanbe lifted off the Parafllmwith
forcepsand stored in a dessicatoruntil EM analysis.
The bestelectronmicrographswere obtainedwith a
ZeissEM10Ctransmission
electronmicroscope
at 60
kV with low magnification
setting.Nucleiwere photographed at magnificationsbetween 1,000x and
2,000 x.
Zygoteneandpachyteneoocytes
wereobtainedfrom
femalechickssoonafter hatching.For Northern Bobwhite (Colinusvirginianus),
the largest numbers of
pachytene oocyteswere recovered from the ovaries
of 0-3 day-oldchicksusinga "settle"technique(Mahadevaiahet al. 1984).The techniquesfor preparing
the slides,fixative,andEM samples
wereasdescribed
above for male birds.
The somatickaryotypeof the Northern Bobwhite
(Fig. 1) has been reportedto consistof 82 chromosomes(Benirschke
andHsu 1971).Sixautosomal
pairs
are submetacentric or subtelocentric; the Z chromosome is submetacentric, and the W is acrocentric or
subtelocentric.
The remaining68 chromosomes
ap-
778
ShortCommunications
pear to be acrocentric.Well spread, silver-stained
pachytene nuclei from male and female Northern
Bobwhiteclearlydisplaya full complementof 41 SCs
(Fig. 2). This confirmedthe diploid number previouslydeterminedfrom featherpulp preparations(Benirschke and Hsu 1971). Our SC data generally corroborate the description of the chromosomal
morphologyfrom somaticmetaphases.The SC data
revealed6 pairsof macrochromosomes,
includingthe
sexchromosomes.
The largestautosomalbivalent is
submetacentric,2 macrochromosomepairs are acrocentric,and the remaining2 pairsare subtelocentric.
The Z chromosomeis acrocentric,and approximately
twice as long as the subtelocentricW chromosome.
Of the 8 "intermediate-sized" chromosomepairs, 4
appear subtelocentricand 4 are acrocentric.Two microchromosomepairs are subtelocentric;the remaining 25 pairs of microchromosomes
are clearly acrocentric. The higher number of subtelocentric
autosomesindicated by the SC data reflectsthe less
contractedcondition of the prophaseI chromosomes
and the increasedprecision with which the morphologyof the microchromosomes
canbe resolved.
The surfacespreadingand settletechniquesfor obtaining pachytenemeiocytesand visualizingSCsare
readily applicablefor studiesof avian cytotaxonomy
and cytosystematics.
Thesepreparationsprovide large
numbersof well-spreadpachytenenuclei amenable
to analysisby transmissionelectronmicroscopy.The
silver-stained SCs are easily counted, and provide a
useful and superiormeansof accuratelydetermining
the diploid numberof male and femalebirds.Because
the meiotic chromosomesare completely paired at
pachynema,there is the addedadvantageof analyzing a haploid number of configurations.Moreover,
the centromericregionsarevisibleon the SCsof both
the macrochromosomes
and microchromosomes,
[Auk, Vol. 105
translocations
(Kaelblingand Fechheimer1983)and
pericientric inversions(Kaelbling and Fechheimer
1985)in heterozygousdomesticchickens(Gallusdomesticus).
Synaptonemalcomplex data on chromosomalrearrangements
which occurin naturalpopulationsof birds are not presentlyavailable;however,
numerous examples of intraspecific chromosomal
variationhavebeendocumented(reviewedby Shields
1982). These include polymorphism for pericentric
inversions (Shields 1973, 1976; Hammar and Herlin
1975;Thorneycroft1975;Ansariand Kaul 1979a;Bass
1979)and translocations(Ansari and Kaul 1978, 1979b;
Kaul and Ansari 1979). It is unclear how these types
of polymorphismspersist in avian populations, as
heterozygous
individualsare generallyexpectedto
exhibit reduced reproductive fitness (Shields 1982).
Analysisof SC configurations
couldprovideinsight
into the mechanisms involved
in the maintenance
of
chromosomalpolymorphismsin avian populations,
and yield information relative to the evolutionary
implicationsof chromosomal
rearrangements
in birds.
This researchwassupportedby NIH Grant No. GM27104 and NSF Grant No. BSR-8614569.
We thank W.
F. Krueger and R. Moore, Texas A&M University
Poultry Center, for supplying the Northern Bobwhite.
LITERATURE CITED
ANSARI,H. A., & D. KAUL. 1978. Translocation het-
erozygosity
in thebird,Lonchura
punctulata
(Linn.)
(Ploceidae: Aves). Natl. Acad. Sci. Lett. 1: 83-85.
, & ---.
1979a. Inversion polymorphism
in common Green Pigeon, Treronphoenicoptera
(Latham) (Aves). Jpn. J. Genet. 54: 197-202.
, & --.
1979b. Somaticchromosomes
•f
the Black-headed
Oriole,
Oriolus xanthornus
(Linn.). A probablecaseof translocationheterowhich resolvesthe chromosomalmorphologyto a
zygosity.Experientia35: 739-740.
degreegenerallynot attainableby conventionalkaryS. E., & W. THRELFALL.1987. A comparison
ologicalmethods.The prophaseI configurationsare BARTLETT,
of the karyotypesof six speciesof Charadrimoreelongatethan the correspondingsomaticmetaiformes. Genome 29: 213-220.
phasechromosomes;
hence small chromosomalarms
aremoreeasilydetectedin SCpreparations.Although BASS,R.A. 1979. Chromosomalpolymorphism in
cardinalsCardinaliscardinalis.Can. J.Genet. Cytol.
the SC techniquesare somewhatinvolved, they are
21: 549-553.
substantially
lesstime-consuming
and expensivethan
some of the conventional proceduresfor obtaining BF,NmSCHKE,K., & T. C. HSU, EDS. 1971. Chromosome
atlas:fish,amphibians,reptiles,and birds,vol. 1.
avian chromosomalmaterial (i.e. embryo incubation,
New York, Springer-Verlag.
blood and tissueculturing).
The SCtechniquesalsohaveconsiderable
potential BIEDERMAN,B. M., D. FLORENCE,& C. C. LIN. 1980.
Cytogeneticanalysisof great horned owls (Bubo
in cytogenetic
analysesof known or suspected
chrovirginianus).
Cytogenet.Cell Genet.28: 79-86.
mosomalrearrangementsin birds. As pachyteneSC
configurations
faithfullyreflectthe orientationof the HALE,D. W. 1986. Heterosynapsisand suppression
of chiasmatawithin heterozygouspericentricinpaired meioticchromosomes,
thesetechniquescanbe
versions of the Sitka deer mouse. Chromosoma
employedto investigatethe nature and behaviorof
94: 425-432.
chromosomalrearrangementsin the heterozygous
B., & M. HERLIN.1975. Karyotypesof four
state.Although theseprocedureshave been widely HAMMAR,
bird speciesof the orderPasseriformes.
Hereditas
applied in studyingchromosomalheteromorphisms
in mammals (Mahadevaiah et al. 1984, Hale 1986),
80: 177-184.
similarSCanalysesin birdshavethusfar beenlimited HOWELL,W. M., & D. A. BLACK. 1980. Controlled
silverstainingof nucleolus
organizerregionswith
to examinations
of experimentallyinducedreciprocal
October1988]
ShortCommunications
a protectivecolloidaldeveloper:a 1-stepmethod.
typing in spermatocytesof the Chinese hamster
(Cricetulus
griseus).I. Morphology of the autosomal complementin spread preparations.Chro-
Experientia 36: 1014-1015.
KAELBLING,
M., & N. S. FECHHEIMERß
1983. Synaptonemal complex analysisof chromosomerear-
--,
779
mosoma
60: 99-125.
rangements in domestic fowl, Gallus domesticus. SHIELDS,
G. F. 1973. Chromosomalpolymorphism
Cytogenet. Cell Genet. 36: 567-572.
common to several speciesof Junco(Aves). Can.
& -1985. Synaptonemalcomplex
J. Genet. Cytol. 15: 461-471.
analysis of a pericentric inversion in chromo1976. Meiotic evidence for pericentric in-
some2 of domesticfowl, Gallusdomesticus.
Cy-
version polymorphism in Junco(Aves). Can. J.
togenet. Cell Genet. 39: 82-86.
KAUL,D., & H. g. ANSARI.1979. Chromosomal
polymorphismin a natural population of the Northern Green Barbet, Megalaimazeylanicacaniceps
(Franklin) (Piciformes:Aves). Genetica 49: 1-5.
MAHADEVAIAH,
S., U. MITTWOCH,& M. J.MOSES. 1984.
Pachytenechromosomesin male and female mice
heterozygousfor the Is(7;1)40H insertion.Chromosoma
--.
Genet. Cytol. 18: 747-751.
1982. Comparative avian cytogenetics:a review.
Condor
84: 45-58.
ß 1983. Bird chromosomes.
1: 189-209.
Current
Ornithol.
THORNEYCROFT,
H. B. 1975. A cytogeneticstudy of
the white-throated sparrow, Zonotrichiaalbicollis
(Gmelin). Evolution 29: 611-621.
90: 163-169.
MOSES,
M. J. 1977. Synaptonemalcomplexkaryo-
Received
29 February1988,accepted
25 April 1988.
Influence of Desert Nesting and ForagingDistanceon
Growth Rates in Gray Gulls (Earus modestus)
CARLOSG. GUERRA,• LLOYD C. FITZPATRICK?AND ROBERTOE. AGUILAR•
•Instituto
deInvestigaciones
Oceanologicas,
Universidad
deAntofagasta,
Antofagasta,
Chile,and
2Division
of Environmental
Sciences,
Department
of Biological
Sciences,
University
of NorthTexas,
Denton, Texas 76203 USA
By nesting 35-100 km from the coastin the waterlesspampasof the interior AtacamaDesert of northern Chile, Gray Gull adults limit themselvesto a single daily foraging trip (Howell et al. 1974;Guerra et
al. in press[a, b]; Fitzpatrick et al. in press[a]). Lim-
Negro nestingsiteswere describedby Cikutovic and
Guerra (1983) and Guerra et al. (in press [a]). Each
gull wastaggedwith a numberedmetal band (Model
1242-M, size 10, Nat. Band and Tag Co., Kentucky,
USA). Chicks were weighed with Pesolascales,and
itationson feedingfrequencyand the amountof food wing, culmen, tarsus and total lengths measured as
that adultscan carry (ca. 18 g dry weight; Fitzpatrick describedin Fitzpatrick et al. (in presslb]). Twelve
et al. in press[a];unpubl. data),presumablyaffects gulls were releasedand 11 were taken to the outdoor
growth ratesin young Gray Gulls. General conditions aviary on the Universidad de Antofagastacampus,
of the Atacamaand specificdescriptionsof the Gray located several hundred meters from the beaches
Gull nestingsiteswere reportedby Howell et al. (1974), where free-ranginggulls forage.We attemptedto reGuerraand Cikutovic (1983) and Guerra et al. (in press capture and remeasure the released gulls on six oc[a]). Information on Gray Gulls and their problems casionsbetween 17 Januaryand 10 February 1986.
associatedwith nesting far from food and water in
Eight were recaptured four times; three, three times;
the Atacama,where daily surfaceand air tempera- and one, twice. The free-ranginggulls were alsoused
tures range from 2-61øCand 2.5-38øC,respectively, to studywater-turnoverand feeding energeticsusing
are well documented (Howell et al. 1974; Guerra and
labeled water (MS in prep.).
Fitzpatrick 1987; Guerra et al. 1988, in press [a, b];
Gullsmaintainedin the aviarywere fed oncedaily
Cikutovic et al. in press;Fitzpatrick et al. in press[a,
an ad libitumdiet of fresh or previouslyfrozen sarhi).
dines (Sardinops
sagax)and anchovies(EngraulisrinWe studied growth ratesin free-ranging and cap- gens),the principalfishcomponentof GrayGulls'diet
tive Gray Gull young, and comparedthem to other
(Guerraet al. in presslb]). We weighed and measured
semiprecocialspecieswithin the Laridae.We hypoth- captive gulls every 2-10 days, depending on size,
esized that, because of food and water limitations imuntil the ageof 100 days.Free-ranginggulls beyond
posedby nestingfar from the coast,GrayGullswould 50 dayswere not recapturedeasily and growth data
on them are limited.
have a lower massgrowth-rate than other Laridae.
During early January 1986 we hand-captured 23
We estimatedage of chicks from an empirically
Gray Gull chicks(33-250 g; 1-ca. 45 days old) at a determined wing length-age curve (Ricklefs et al.
nesting site locatedat Cerro Negro, 100 km east of
1980).The curve was basedon wing length-agereAntofagasta(23ø41'S),in the Atacama Desert. Cerro lationshipsfor free-rangingand captivechickswhich