Cooper Ornithological Society
Patterns of Sperm Storage in Relation to Sperm Competition in Passerine Birds
Author(s): James V. Briskie and Robert Montgomerie
Source: The Condor, Vol. 95, No. 2 (May, 1993), pp. 442-454
Published by: University of California Press on behalf of the Cooper Ornithological Society
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The Condor95:442-454
? The CooperOrnithologicalSociety 1993
PATTERNS OF SPERM STORAGE IN RELATIONTO SPERM
COMPETITIONIN PASSERINEBIRDS'
JAMES V. BRISKIE AND ROBERT MONTGOMERIE
Departmentof Biology, Queen'sUniversity,Kingston,OntarioK7L 3N6, Canada
Abstract. Using a techniquefor observingspermstoragetubules(SSTs)in whole-mount
preparationsof oviducttissue,we estimatedthe numberand lengthof SSTsacross20 species
of passerinebirdsin seven subfamilies.The averagenumberof SSTsper femalevariedninefold acrossthe speciesexaminedand was correlatedpositivelywith body mass and relative
egg size, suggestingthat much of the interspecificvariationin the numberof SSTs may be
a consequenceof allometry.However,aftercontrollingfor body mass, the numberof SSTs
per female was also negativelycorrelatedwith the averagelength of SSTs and we suggest
this patterncouldresultfromselectionforgreaterspermlengthwhenaccessto spermstorage
sites is limited by females. SST length varied eight-foldacross the species examined but
unlike the numberof SSTs, SST length was not correlatedwith body mass. Instead, SST
length was stronglyand positively correlatedwith sperm length, suggestinga history of
coevolution betweenmale gametes and sperm storagesites in females that is independent
of body size. Neither mating system nor any of four other morphologicalvariables(testis
length,oviduct length,clutch size, spermstoragecapacity)was consistentlycorrelatedwith
either the number or size of SSTs. Variationbetween species in the potential for sperm
layeringwithin an SST, however, indicatesthat the patternof sperm precedence(i.e., the
patternof paternityrelativeto matingorder)may vary from speciesto species.We propose
that a conflict of interestbetweenthe sexes with respectto sperm storagecould lead to an
arms race between sperm length and SST length and may help explain both the diversity
in spermstoragepatternsamongspeciesand the influenceof spermstorageon the evolution
of matingbehaviorin birds.
Key words: Sperm storage;sperm competition;spermprecedence;sperm;reproductive
strategies;passerines.
INTRODUCTION
Poultry scientists have long known that female
domestic fowl can store spermfor severalweeks
(e.g., Van Drimmelen 1946, Lorenz1950, Lodge
et al. 1971). In poultry, sperm are stored primarilyin specializedspermstoragetubules(SSTs)
located at the junction of the uterusand vagina
(e.g.,Bobret al. 1964,MeroandOgasawara1970)
althoughsome storagemay also occur in the infundibulum (Van Drimmelen 1946). Recent
studies of SSTs in several species of wild birds
(Hatch 1983;BakstandBird 1987;Shugart1988;
Birkhead1987, 1988; Birkheadet al. 1990) suggest that sperm storagemay be a universal feature of avian reproduction.
The significanceof sperm storagefor the evolution of mating patternsin wild birds was recognized by behavioral ecologists only recently
(Hatch 1983, Birkhead1988). One consequence
of sperm storageby females is that sperm from
differentmales are more likelyto co-occurin the
'Received22 July 1992. Accepted1 February1993.
female reproductive tract. Competition between
ejaculates from two or more males over the fertilization of ova of a single female was called
sperm competition by Parker (1970) and subsequent work has shown that this process has
played a large role in the evolution of a variety
of mating behaviors (e.g., copulation frequency,
duration of mate-guarding; Birkhead and Moller
1992a).
Despite a variety of studies of avian SSTs (Mero
and Ogasawara 1970, Shugart 1988, Birkhead
and Hunter 1990, Lessells and Birkhead 1990),
the proximate mechanisms of sperm storage and
their effect on sperm competition are poorly understood. Fertilization in birds typically occurs
in the infundibulum during a brief"fertilization
window" of only 15-30 minutes duration, shortly after ovulation (Howarth 1974). The ovum
then begins its descent down the oviduct where
albumin and shell membranes are deposited.
Unless protected in SSTs, sperm are unlikely to
survive in the oviduct during egg formation and
laying (Lorenz 1966). Thus, specialized sperm
storage sites may have evolved in birds so that
[442]
IN BIRDS
SPERMSTORAGE
inseminations do not have to be synchronized
with the fertilizationwindow to be successfulat
fertilization(Hatch 1983).
Several recent experiments on captive birds
have shown that the last male to copulatebefore
fertilizationenjoys considerablyhigherfertilization success than previous males, even if copulations from the last male are greatly outnumbered by those of the previous male (e.g.,
Compton et al. 1978, Chenget al. 1983, Sims et
al. 1987, Birkhead et al. 1988). For example,
Birkheadet al. (1988) foundthat, in ZebraFinches (Taeniopygiaguttata), the last male to mate
before eggs were fertilized fathered approximately 80% of the subsequent offspring.Such
observationshave suggestedthat sperm may be
stored as stratifiedlayers within SSTs and possibly that the last inseminationis stored nearest
to the oviductallumen, thus giving it the advantage of being the first out during ovulation
(Comptonet al. 1978). On the other hand, Lessells and Birkhead(1990) have used the available
data on poultryto model the sperm storageand
fertilizationprocessbut did not findclearsupport
for this sperm layeringhypothesis. Insteadtheir
analysisshowed that displacementof a previous
male's sperm by that of the last-copulatingmale
could betteraccountfor the observedpatternsof
last-malespermprecedence.Nevertheless,sperm
precedenceexperimentsin birds have been limited to only a few species to date. It may thus be
prematureto generalize sperm storage mechanisms from this small sample, particularlysince
the patternof spermprecedenceis knownto vary
widely from species to species in mammals
(Dewsbury 1984, Birkhead 1988).
AlthoughSSTs seem to be importantin sperm
competition, little is known about variation in
sperm storagecapacity among species and how
such variation may be related to differencesin
mating system or behavior patterns. Shugart
(1988) measuredthe lengthsof SSTs in a dozen
speciesof birdsbut he did not attemptto account
for variationamong species. Recently,Birkhead
and Moiler (1992b) compared the number and
lengthof SSTsacross 11 speciesof domesticbirds
to determine if the duration of sperm storage
could be predicted from SST morphology. Although a weak relationshipwas found, they observeda strongrelationshipbetweenthe number
of SSTs and body mass.
In this paper, we estimate both the number
and lengthof SSTs for a varietyof wild passerine
600
.
443
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200
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5
100
150
200
250
300
Spermlength(rm)
FIGURE 1. Variation in the potential for sperm
stratification among 20 species of passerine birds. For
each species, mean sperm length is plotted against mean
SST length. Lines delineate different SST length: sperm
length ratios. Species (no. of females, males examined)
are: A, Least Flycatcher, Empidonax minimus (3, 3);
B, Tree Swallow, Tachycineta bicolor (4, 4); C, Barn
Swallow, Hirundo rustica (3, 5); D, American Pipit,
Anthus rubescens (3, 3); E, Horned Lark, Eremophila
alpestris(3, 1); F, American Robin, Turdus migratorius
(2, 1); G, Red-eyed Vireo, Vireo olivaceus (2, 1); H,
Yellow Warbler, Dendroica petechia (4, 1); I, American
Redstart, Setophaga ruticilla (2, 1); J, Red-winged
Blackbird, Agelaius phoeniceus (3, 1); K, Yellow-headed Blackbird, Xanthocephalus xanthocephalus (3, 4);
L, Brewer's Blackbird, Euphagus cyanocephalus (3, 2);
M, Brown-headed Cowbird, Molothrus ater (4, 3); N,
Western Meadowlark, Sturnella neglecta (1, I1);O, Lapland Longspur, Calcarius lapponicus (5, 2); P, Smith's
Longspur,C. pictus (3, 4); Q, Snow Bunting,Plectrophenax nivalis(4, 2); R, Common Redpoll, Carduelis
flammea (2, 2); S, Hoary Redpoll, C. hornemanni(1,
2);T, AmericanGoldfinch,C. tristis(4, 2).
species.We then examinehow interspecificvariation in the numberand lengthof SSTsis related
to body size, sperm length, and the degree of
sperm competition expected from the mating
systemof thesespecies.Finally,we describesperm
arrangementpatternswithin SSTsand show that
differencesamongspeciesmay have the potential
to affectsperm precedencepatterns.
METHODS
Oviductsfrom 59 femalesof 20 passerinespecies
representing seven different subfamilies were
collected in Manitoba, Ontario and the Northwest Territories,Canadaduringthe 1988, 1989
and 1990 breedingseasons (see Fig. 1 for list of
species and scientificnames). Within an hour of
collecting each female, we removed the entire
reproductivetract(ovaryto vent). Oviductswere
placedimmediatelyin 10%bufferedformalinin
separatelabelledvials and stored6 to 18 months
444
JAMES V. BRISKIE ANDROBERT MONTGOMERIE
beforeexamination.Only females that were laying or close to laying eggs (as evidenced by advanceddevelopingova) wereused.All birdswere
collected under appropriatefederaland territorial permits.
To prepare an oviduct for examination, all
mesenteries, skin and other obstructingtissues
were carefullyremoved to expose the lower reproductivetract.Oviductlengthwas determined
by tracing the outline of each oviduct with a
measuringthread.If present,developingeggswere
removedand the diameterof the yolk measured.
The oviduct was then straightened,pinned to a
dissectingboard and a single longitudinalincision was made throughthe cloaca, vagina and
uterus.The exposed mucosa was pinned flat and
keep moist with water. Under a dissecting microscope,the uterovaginal(UV)junctionappears
as a distinct band of thin, convoluted folds (see
Bakstand Bird 1987, Birkheadand Hunter 1990,
Birkhead et al. 1990). For each specimen, we
counted the number of folds and measuredthe
approximatewidth of the UV band with a ruler.
To estimate the numberof SSTs, three individual folds (out of a total of 15-20) were removed
in their entirety, beginningfrom the middle of
the vagina and extendingthroughthe entireUV
regionup to and includingabout 0.5 mm of the
uterinefold containingthe shell glands. Any remaining connective tissue was carefullyteased
away and the lamina propria of each fold was
spreadopen onto a clean glass slide so that the
medial surfaceof each half faced up. Each fold
was placed undera cover slip and preparedas a
wet mount. In some instances the preparation
was flattened by gently tapping the cover slip
with a blunt probe.
We countedthe numberof SSTsper fold using
a phasecontrastmicroscope(40 x magnification)
calibratedwith a stage micrometer.Individual
SSTs could be distinguishedand followed along
theirlengthsby fineadjustmentsof the focusand
iris diaphragm(see Fig. 2). SSTs could also be
observedat high magnificationwith a dissecting
microscope and transillumination (see Bakst
1987, Bakst and Bird 1987) but we found the
boundariesof individualSSTsmore distinctand
more easilymeasuredundera phasecontrastmicroscope. Although some SSTs were branched
(< 15%of SSTs per individual, unpubl. data),
spermwere stored primarilyin the distal end of
each tubuleor tubulebranch(pers.observ.,Birk-
head and Hunter 1990, Birkheadet al. 1990). As
we were most interestedin the evolution of the
number of sperm storagesites ratherthan their
configuration,we counted each branchas a separate sperm storagesite for our analyses. Next,
we measuredthe lengthof five haphazardlychosen, clearlyvisible SSTs per fold for a total of 15
per individual bird. BranchedSSTs were measured by following the length of one randomly
chosen branchbeginningfrom its distal end and
continuingto its opening into the lumen of the
oviduct (i.e., the distance sperm travel to enter
or leave an SST). In most preparations,sperm
could be easily discernedwithin tubules by examiningthe tissue in variousfocal planesat high
magnification.Passerinesperm are readily recognizable by their characteristicspiral-shaped
heads (see Fig. 2A). We also attemptedto determine if spermwere presentin SSTs by carefully
openinga few tubules.Photographsof spermand
SST structure(Fig. 2) were taken using Neumarskioptics on a Leitz Dialux 20 microscope.
To calculatethe number of SSTs per female,
we multipliedthe mean numberof SSTsper fold
studied by the total number of folds. Birkhead
and Hunter (1990) and Birkheadet al. (1990)
applied similartechniquesto estimate the number of SSTs in two species of estrildid finches.
When more than one specimen of a species was
available, we calculated a species mean using
mean values from each female.
To determine the relationship between SST
length and sperm length, we collected sperm either by gentle massage of the cloacal protuberance of live birds (see Wolfson 1952) or by salvaging semen from the seminal glomera of
freshly-killed specimens. We also measured
spermcoaxedout ofSSTs in a few cases,avoiding
those that were obviously damaged(e.g., broken
tails) in the process. Sperm collected from tubules did not differ significantlyin length from
that collected directlyfrom males so data from
both sources were combined for analyses. Permanent mounts were made by placing a small
dropletof semen on a cleanglassslide and lightly
smearingit by passing a second glass slide over
the first. The slide was allowed to air dry and
then stored in a dry slide box for later examination. Total sperm length (head plus tail) was
measured under a phase contrast microscope
(100 x magnification;Fig. 2A) by averagingthe
length of 10 haphazardlychosen sperm. As be-
SPERMSTORAGEIN BIRDS
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FIGURE 2. Photomicrographsof sperm (A) and sperm storagetubules(C, E, G), with line tracings(B, D, F,
H) to illustratestructuralfeatures.The line tracingsweremade directlyfrom the photographsbut wereenhanced
to show featuresthat are not readilyvisible in a photographof a singleplane. Spermheads (h) and tails (t) and
the distal ends (d) of SSTs are indicated.In D, F and H the walls of the SSTs are shaded and only one sperm
in each layeris drawn.The scale bar on each drawingis 50 Mmlong. (A-B) Spermof Yellow-headedBlackbird.
(C-D) SSTs of LaplandLongspurshowingtwo distinct layersof sperm fillingthe tubulewhere the heads of the
second layer abut the tails of the first. (E-F) SST of Tree Swallow showing several layers of sperm filling the
tubule-in this case the layersare not discrete.(G-H) SST of AmericanRedstartshowinga singlelayerof sperm
fillingthe tubule.
446
ANDROBERT
MONTGOMERIE
JAMESV. BRISKIE
fore, a single mean was calculatedfor each species fromthe meanvaluesforall individualmales
studied.
We used relative testis length as an index of
sperm production and the intensity of sperm
competition. Interspecificcomparisons of primates (Harcourtet al. 1981), birds(Cartar1985,
Moiller 1991a) and mammals (Kenagy and
Trombulak1986) have shown that non-monogamous animalshave relativelylargertestes than
monogamous species. Large testes in polygamous and promiscuousspeciesare thoughtto be
advantageouswhen the risk of sperm competition is high because they enable a male to produce large quantities of sperm to dilute or displacecompetingejaculates(Harcourtet al. 1981).
We obtaineddata on testis size from freshmaterial collected by us in the field or taken from
labelson museumskins housedin the Royal Ontario Museum, Toronto, the National Museum
of Canada,Ottawa and the Manitoba Museum
of Man and Nature, Winnipeg.When possible,
we used data only from the same geographical
region as the oviduct sample to avoid any bias
due to variation in testis size over the range of
a species(e.g.,Rising 1987).At least 25 measures
of testis size were used for each species. To estimate mean testis lengthat the peakof breeding,
we fitted a second-orderpolynomial regression
betweentestis lengthand date and used the maximum of the regressionfor each species. Body
masses were taken from Dunning (1984).
For all comparative analyses, we used the
methods describedby Harvey and Pagel (1991)
to controlfor phylogeneticeffects.Suchmethods
arerequiredbecausetraitsmay not have evolved
independently among closely related species.
Thus traitsmay be similarin closely relatedspecies simply because of common ancestryrather
than independentevolution and a failureto control for this artificiallyweights sample sizes and
the influenceof some traits on the analysis. We
used Sibley and Ahlquist (1990) to constructa
phylogeny for the species studied. Then, using
Pagel's Evolutionary Covariance Regression
(unpubl.software,see Harvey and Pagel 1991),
we calculated unique linear comparisons or
"contrasts"within each taxon comprisingtwo or
more descendenttaxa. These linearcontrastseffectively reducethe informationat each node in
the phylogenyto a singledatumfor each variable
being studied.
To test for relationsbetween variablesacross
taxa, we determinedwhetherlinearcontrastsfor
one variablewere relatedto those of another.A
variety of statisticalmethods can be used to assess such relations(see Harvey and Pagel 1991)
but we used simple and multiple linear regressions forced through the origin (presented as
"contrastanalysis"). In no case did the use of
rank correlationor binomial tests on the contrasts (see Harvey and Pagel 1991) provide different results.
RESULTS
Sperm storagetubules were observed in all females of the 20 species examined. SSTs were
confined to a 2-5 mm band (oviduct not
stretched)at the junction of the uterus and vagina;no tubuleswere found in the infundibulum
as has been reportedfor domestic chickens(Van
Drimmelen 1946). The estimatedmean number
of SSTs per female varied among species from a
low of 603 in the Tree Swallow (n = 4 individuals) to a high of 5,640 in the Western Meadowlark(n = 1), a more than nine-folddifference
(see Fig. 3). Most (64.8%)of the variationin the
numberof SSTsoccurredat the specieslevel with
lesser amounts explainedby individuals within
species (24.4%) and folds within individuals
(10.9%;nested ANOVA, P < 0.00001 at all levels). The mean lengthofSSTs also variedgreatly
among species (see Fig. 1), with an eight-fold
differencein size from the LeastFlycatcher(71.7
gm)
to the Tree Swallow (581.5 Lim). Most
(60.1%)of the variation in the length of SSTs
occurredat the specieslevel with lesseramounts
explainedby individualswithin species (10.3%),
folds within individuals (2.6%), and samples
within folds (27.0%;nested ANOVA, P < 0.002
at all levels). Sperm(Figs. 2A, B) were visible in
the tubulesof all individualsexamined(Figs.2CH). In all tubules studied, sperm were oriented
with their heads into the blind (distal)end of the
SST (Fig. 2C-H).
VARIATION
IN LENGTHOFSSTs
An analysis of independent contrasts revealed
that SST length was not relatedto female body
mass (b = 0.03, t = 0.12, P = 0.91, n = 16
contrasts,see Table 1 for details).Consequently,
we did not control for body mass in subsequent
analysesof SST length.
As found in a previous analysis (Briskieand
Montgomerie1992),SST lengthwas stronglyand
positively correlatedwith sperm length across
SPERMSTORAGEIN BIRDS
10000
A
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1000
a0.00
R o
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s
H
T
G
K
L
M
No
F
P
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Female mass (g)
0.8
S
Partial b
t
P
n
Spermlength(Lm)
Testis length (mm)
Oviduct length(mm)
1.21
0.48
-0.20
8.80
0.68
0.24
0.001
0.91
0.82
16
16
16
monogamous species. We classifiedmating systems basedon fieldobservationsbut we also used
testis size as an independentmeasureof the genetic matingsystem.However,testis length(controlled for male body mass) was not related to
SST length (Table 1). We did not have enough
independentevolutionaryeventsto compareSST
0.4
S
~
o
-0.4
(3
Test variable
B
H
o
TABLE 1. RelationbetweenSST lengthand various
traitsrelatedto spermstorageand fertilization.Results
presentedhere are from multiple regressionanalyses
(forced through the origin) of independentcontrasts
usedto controlforphylogeny(Harveyand Pagel 1991).
In each case, SST lengthwas regressedagainstthe test
variablewitheithermale mass(fortestissize and sperm
length)or female mass (for oviduct length)held constant. Samplesizes (n) are the numberof independent
contrastsfrom an analysisof 20 species.
*B
10
447
0
0
0.2
0.4
0.6
0.8
S 0.5
Contrastsin female mass
A
FIGURE3. (A) Relationbetweenthe numberof SSTs
and female body mass across 20 species of passerine
birds (species labelled as in Fig. 1) and (B) relation
betweenindependentcontrastsforthesevariables,controllingfor phylogeny(see text for statistics).
species (Table 1, Fig. 4). On average,SST length
was twice that of sperm length so that each SST
could hold about two layersof spermwhen lined
up head to tail. However,it is instructiveto look
at the exceptions to this pattern.In Figure 1 we
have redrawnthe relationshipfrom Briskieand
Montgomerie (1992) but with various SST
length:sperm length ratios indicated. The pattern that emergesis that females in some species
cannot accommodate much more than a single
layerof spermin theirSSTs (e.g.,LeastFlycatcher, Red-eyed Vireo) while three or more layers
of sperm are possible in others (e.g., American
Robin). Indeed, sperm does form layers within
single SSTs (Figs. 2C-F) when tubules are long
enoughto accommodatethem, otherwiseonly a
single layer may form (Figs. 2G, H).
Next, we looked for a correlationbetweenSST
length and mating system because sperm competition is thoughtto differamong avian mating
systems (Birkhead 1988; Birkhead and Meller
1992a), being more intense in polygamousthan
*
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.. . .. .. . . .. ? ... ..• .0 ........
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a -0.5
-0.6
-0.4
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<
0
.5o
-0.2
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0
0.1
0.2
0.3
0.4
0.5
Residual contrasts in sperm length
FIGURE 4. (A) Relation between residual log SST
lengthand residuallog spermlength(residualsfor both
from regressionson female body mass) across 20 species of passerinebirds and (B) relationbetween independent contrastsfor these variables,controllingfor
phylogeny(see Table 1 for statistics).
448
JAMES V. BRISKIE ANDROBERT MONTGOMERIE
A
H
TABLE2. Relationbetweennumberof SSTsandvarious traits relatedto sperm storageand fertilization.
Results presentedhere are from multiple regression
analyses (forced through the origin) of independent
contrastsused to control for phylogeny(Harvey and
Pagel 1991). In each case, the contrastsin numbersof
SSTs were regressedagainstthe test variablewith female body mass controlled.Sample sizes (n) are the
numberof independentcontrastsfrom an analysis of
20 species.
S
S0.4
0.2
*
a0
*+ *4
o
-0.2
-0.4
-0.1
Residuallog egg volume
0.6
B
mb
Test variable
Partial b
t
P
n
Egg volume (mm3)
Oviduct length (mm)
Clutch size
SST length (gm)
SST capacity indexa
Testis length (mm)
2.27
0.99
0.03
-0.59
-0.56
0.08
3.44
1.69
0.28
4.61
1.43
0.15
0.004
0.11
0.78
0.0004
0.18
0.88
15
16
15
16
16
16
0.1
0
0.4
SSST length/sperm length.
as o
kee
*
P
?
-0.2
-0.1
o
o.1
Residualcontrastsin egg volume
FIGURE 5. (A) Relation between the residual log
numberof SSTs (i.e., from regressionof log number
of SSTs on log femalebody mass)and residuallog egg
volume (from regressionof log egg volume on female
body mass)and (B)relationbetweenindependentcontrastsfor these same variables,controllingfor phylogeny (see Table 2 for statistics).
length among mating systems but mean SST
length in polygamous species (306.6 tm, n = 7)
was about 30% longer than mean SST length in
monogamous species (238.5 tm, n = 13). This
suggests that SST length is greater in species with
greater polygamy although further study is needed to determine whether this relationship holds
up when the effects of phylogeny can be controlled statistically.
Oviduct length (controlled for female body
mass) also was not related to SST length (Table
1). A relation between these variables might be
expected if SST size were related to oviduct size
through simple allometry.
VARIATIONIN NUMBER OF SSTs
The numberof SSTsperfemalewas stronglyand
positively related to body mass across species (b
= 0.65, t = 13.7, P = 0.002, n = 16 contrasts;
Fig. 3). Thus, in all subsequent analyses, we con-
trolledfemale body mass statisticallywhen testing for correlationsbetweenthe numberof SSTs
and other variables.
Interspecificvariation in the numberof SSTs
might be related to egg size if relatively larger
eggs require more sperm to ensure fertilization.
For example, Wishart (1987) found that fertility
in chickens decreasedrapidly once the density
of sperm trapped on the egg membrane dropped
below a certainthreshold.If largereggsdilutethe
numberof spermperunit areaof eggmembrane,
moreSSTsmaybe requiredto insurethatenough
sperm are available to maintain this threshold
density and hence maximal fertility. To test this
hypothesis we compared the number of SSTs per
female to egg volume (calculated from measures
given in Harrison [1978]). Ideally, the number
of SSTs should be compared to ovum size at
fertilizationbut data on ovum size are unavailableforthe specieswe studied.Eggsize, however,
is directly correlated with ovum size across species (Ar and Yom-Tov 1978, Sotherland and
Rahn 1987). Among the species we studied, for
example, egg volume was highly correlated with
yolk volume (r = 0.98, P < 0.0001, n = 13). As
predicted, the relative number of SSTs was positively related to relative egg size (both variables
controlledfor female body mass) across species
(Fig. 5, Table 2).
Oviduct length could also influencethe number of SSTs througha dilution effect.Thus, species with relatively large oviducts may require
largersperm stores (and hence more sperm) to
ensure that adequate numbers of sperm reach
IN BIRDS
SPERMSTORAGE
the ova. However, the number of SSTs was not
related to oviduct length across species (Table
2).
Next, we examined the relationshipbetween
number of SSTs and clutch size reasoningthat
species with largerclutchesmight be expectedto
store largerquantitiesof sperm since they have
more eggs to fertilize. The relation between the
number of SSTs and clutch size, however, was
not significant(Table 2).
The numberof SSTs could also be affectedby
SST length, either positively, due to positive allometry,or negatively,as a resultof an energetic
trade-off.In fact,the numberof SSTswas strongly and negatively correlated with SST length
across species (Table 2, Fig. 6). Thus, species
with relativelymany SSTs had small SSTs while
species with relatively few SSTs had SSTs that
were relativelylarge.
If species capable of storing sperm in several
layers can store a greater absolute quantity of
sperm per SST than species which store sperm
in single layers only, then fewer SSTs in total
may be neededto insurean adequatespermstorage capacity.To determineif storagecapacityis
related to the number of SSTs, we calculateda
layeringindex for each species by dividing SST
length by sperm length. Although sperm may
overlapextensivelywithin a singleSST (Figs.2E,
F), SSTs holding several layers of sperm are
probably capable of storing a greater absolute
number of sperm per SST than single layered
SSTs. However, the number of SSTs was not
significantlyrelatedto our sperm capacityindex
(Table 2). Thus, the capacity of single SSTs to
store sperm is not related to their number and
females did not compensate for fewer SSTs by
making largerones that could each potentially
accommodatemore sperm.
Finally, we looked for a relation between the
numberof SSTsand matingsystem.Testislength
(as an index of mating system) was not significantly relatedto the numberof SSTs across species (Table 2). As we had only seven species in
our sample that were not classifiedas monogamous, our sampleof independentevents was too
small to permitstatisticalanalysisof the number
of SSTs against mating system. However, it is
worth noting that the average residual number
of SSTs (i.e., SST numbercontrollingfor female
body mass) in polygamousspecies (-187.1, n =
7) was less than that found in monogamousspecies (101.1, n = 13). These values suggestthat a
449
S0.4
C
0
0.2
6
*
*
0
o0
0
m
0.
C8
S-0.2
-0.4
?
-0.6
-0.4
-0.2
0
0.2
0.4
Residual log SST length
0.4
0
0
o
S.• -0.4
-0.6
0
0.2
0.4
0.6
Residual contrasts in SST length
FIGURE6. (Top)Relationbetweentheresiduallog
numberof SSTsand residuallog SSTlength(bothvariablesregressedon femalebody mass)across20 species
of passerinebirdsand (Bottom)relationbetweenin-
dependentcontrastsfor these same variables,controlling for phylogeny(see Table 2 for statistics).
reduction in the number of SSTs is correlated
with increasingsperm competition.
DISCUSSION
This study provides some of the first estimates
for the numberand lengthof SSTs in free-living
birds, relating differences between species to
variationin behavioraland other morphological
characters.We found that the number of SSTs
variedconsiderablyacrossa sampleof 20 species
of passerinesbut that most of this variationwas
attributableto interspecificdifferencesin female
body mass. Birkheadand Moller (1992b) found
similar results within a smaller sample of domesticated species. These findings are not too
surprisingsince many morphologicaland physiological attributes of organisms are related to
body size (Peters1983).Forexample,largerbirds
may requirea greaternumberof SSTs to ensure
successful fertilizationsimply as compensation
for the higherlikelihood of sperm "gettinglost"
or becoming too diluted in a largerbody.
450
JAMES V. BRISKIE
AND
ROBERT MONTGOMERIE
In a similar manner, selection for increased
egg size (for whateverreason)also may have favored the production of more SSTs to ensure
that fertilitydoes not decline from the dilution
of an otherwiselimited numberof spermspread
over an increasedarea of ovum membrane.Indeed, we found that species with largereggs had
a greaternumberof SSTs even when body mass
was controlled.However,arguingagainstthis dilution hypothesis was the lack of a similar relationshipbetween oviduct length(correctedfor
body mass) and the number of SSTs. Nonetheless,becausespermaretransportedfromthe SSTs
to the infundibulumlargelyvia the contractions
of the uterus(Allenand Grigg1957),it is possible
that few sperm are lost or diluted by increased
oviduct size relative to those losses that occur
when sperm transportis dependentupon sperm
motility (e.g., when swimming up the vagina to
the SSTs or penetratingthe ovum membrane;
Allen and Grigg 1957).
An unexpectedresult of our analysis was the
inverse correlation between the number and
length of SSTs, even when body mass was held
constant statistically. Initially, this suggests a
trade-offbetween the number of SSTs and the
capacity of each SST to store sperm, but there
was no relationshipbetweenthe numberof SSTs
and the amount of sperm each could store (as
measuredby our sperm capacity index). Alternatively,the numberofSSTs mightdecreasewith
increasedSST lengthif it wereenergeticallycostly to maintain largenumbersof large SSTs. Althoughit is not knownhow much femalesinvest
energeticallyin maintaining sperm, such costs
are probably minimal because the number of
spermstoredper femaleis equivalentto only 5%
or less of a singleejaculate.Thus, relativelylittle
sperm is stored despite the fact that most birds
copulaterepeatedlyfor each clutchof eggs(Birkhead 1992).
In a recent study of interspecificvariation in
spermsize, we founda stronginversecorrelation
between the number of SSTs and sperm length
(Briskieand Montgomerie1992). This relationshipis not surprisingsincespermlengthis strongly
correlatedwith SSTlength(seeabove).However,
it does suggest a possible adaptive explanation
for the relationshipbetweenthe numberof SSTs
and SST length that we found in this study. In
mammals, it is known that longer-tailedsperm
can swim faster (Gomendio and Roldan 1991)
and should thereforebe favoredwheneverthere
is intense competition to reach either the ova
(Gomendio and Roldan 1991) or the sites of
sperm storage(Briskieand Montgomerie1992).
In birds, most of the variationamong species in
sperm length is due to differencesin the length
of the sperm's tail (Briskie and Montgomerie
1992); thus, females could conceivably induce
greaterspermcompetitionby reducingthe number of sperm storagesites available,with the resultthatselectionwouldfavorlonger-tailedsperm
that could swim fasterand would reachthe SSTs
more quickly than short-tailed sperm. Since
spermthatarenot held completelywithinan SST
are liable to be swept out by eggs passing down
the oviduct (Lorenz 1966), females,in turn,will
be favored to match the length of their SSTs to
that of the male's sperm. Thus, we suggestthat
sperm competition for limited storagesites will
result in an inverse relationship between the
numberof SSTsand SST lengthvia selectionfor
greatersperm tail-length.
Although the inverse relation between the
length and number of SSTs can be interpreted
as a productof sperm competition, the number
of SSTs was not related to differencesin sperm
competitionas measuredby relativetestis length
as an index of mating system. In species with
moreintensespermcompetition,copulationsare
morefrequentand malesgenerallyproducemore
sperm and their ejaculateshave a higher concentrationof sperm than species with relatively
low sperm competition (Moller 1988, 1991b).
Whethera greaternumber of SSTs is expected
as a consequenceis not clear,since females may
also induce sperm competition by reducingthe
numberof availablespermstoragesites. The absence of a relationshipbetween the number of
SSTs and testis length is also surprisingsince
Birkheadand Moller (1992b) found a positive
correlationacross 11 speciesbetweenthe number
of SSTs and the numberof sperm per ejaculate,
suggestingthat femalesproducemore SSTswhen
males producemore sperm.
SPERMSTORAGE
AND SPERM
PRECEDENCE
In contrastto variation in the number of SSTs,
differencesin SST lengthacrossspecies werenot
correlatedwith body mass. Instead, SST length
was positively correlatedwith sperm length.Although the potential for sperm layering varied
from species to species, a strongrelationshipbetween spermlengthand SST lengthwas obtained
SPERMSTORAGEIN BIRDS
because females in most species had SSTs that
could hold an averageof two layersof sperm. In
our sample of 20 species, SSTs could hold about
two layers of sperm in 15 species, more than
three layers of sperm in two species and only a
single layerin three species (Fig. 1). Why should
the patternof layeringdifferfrom species to species?
One possibility is that sperm long enough to
fill an SST might prevent access to SSTs by the
sperm of other males. In the featherwingbeetles
(Bambariaspp.),spermareparticularlylargeand
the first male to mate fills the female's spermatheca to capacity,therebyexcludingany potential competitorsfor access to her single largeegg
(Dybas and Dybas 1981). If a similar process
operatesin birds, sperm stored by females with
SSTstoo shortto allowspermlayeringmay result
in a paternity pattern biased in favor of firstmating males. However, such a simple mechanism seems unlikely since many SSTs remain
emptyor capableof storingadditionalspermeven
after several inseminations(Vermaand Cherms
1965, Birkheadet al. 1990).
On the other hand, sperm layered within an
SST suggests a proximate mechanism of lastmale sperm precedencein birds. Because SSTs
have only a single opening, it has been hypothesized that the last spermto enteran SST blocks
the exit for spermalreadyinside (Comptonet al.
1978). Duringovulation, such last-storedsperm
will be the first to exit into the oviductal lumen
and make their way to the unfertilizedovum. It
is this advantagethat presumablyfavors the last
male in subsequentfertilizations(Comptonet al.
1978). However, sperm stratificationand last
male precedencecan occur only if SSTs are long
enough to accommodate two or more sperm
lengths.When sperm are stored in a single layer
within short SSTs, all sperm have the opportunity to exit at roughly the same time and the
probabilityof paternityshouldbe determinedby
the proportionof each male's sperm present,all
else being equal. In reality, SST length in most
speciesfallsbetweenone and a few spermlengths
and sperm exhibit varying degrees of stratification and mixing within SSTs. Nevertheless,differencesbetweenspeciesin the proportionof short
and long SSTssuggestthatspermprecedencepatterns may also vary from species to species.
Differencesin the proximate mechanisms of
sperm precedence between species could have
considerableinfluenceon the evolution of male
451
behavior.When most sperm are stored in stratified layers, as in species with many long SSTs
(e.g., AmericanRobin), the last male to copulate
beforefertilizationwill be most likely to fertilize
the next egg laid. In Zebra Finches, SSTs are
about three times as long as sperm (Birkhead
1987) and last male sperm precedencein this
speciesaveragesabout80%(Birkheadet al. 1988).
In this and other species with sperm stratification, males could minimize cuckoldryby insuring they are the last to copulatebeforeeach ovulation.
In contrast,the last male to copulatein species
with a highproportionof shorttubules,and hence
no layering(e.g., Red-eyed Vireo), may achieve
paternityonly in proportionto the numberof its
spermpresentrelativeto total numberof sperm
presentin all SSTs. Thus it may be necessaryfor
extra-pairmales in such species to copulate repeatedlyin orderto outnumberor displacesperm
from previous males. As a result, it is possible
that extra-paircopulationsdo not pose as great
a risk to a pair male's paternityin species with
no sperm layering.Unfortunately,there are currently no data available on levels of sperm precedence in species with small SSTs to test this
hypothesis.
Recently, Lessells and Birkhead (1990) examined several alternative models of sperm
competition to determine whether sperm stratification or sperm displacementbest explained
last-male sperm precedencein domestic chickens. Because last-male precedencedid not decline over time, as would be expected if successive layers of sperm were uncovered as sperm
was depleted in the SSTs, they concluded that
sperm displacementwas the most likely mechanism of spermcompetitionin this species. Our
observationsof SSTanatomyand spermlayering
in a varietyof wild passerinessuggestthat sperm
stratificationmay in fact be an importantmechanism of spermprecedence.Ourresultsalso suggest that the mechanismsof sperm competition
thoughtto occurin chickensmay not necessarily
applyto otherspecies,particularlyif the patterns
of sperm storage(i.e., in single vs. multi-layers)
differ.
AN ARMSRACE?
In correlationalstudiesit is often difficultto separatecausefrom effect.Has SST lengthincreased
in response to changes in sperm length or has
sperm length evolved to accommodatechanges
452
JAMES V. BRISKIE AND ROBERT MONTGOMERIE
tively shortertubules are less likely to exhibit a
last male matingadvantage.Suchan experiment
might also test the effectivenessof "retaliatory
copulations"(Birkheadand Moiler 1992a)across
a rangeof specieswith differentSSTsizes relative
to sperm length. If differencesare found, it may
be possible to relate these patternsto variation
in the mechanisms of sperm storage and vice
versa. Perhapsby labellingsperm (e.g., with radioactive tracers)from specific males, it might
be possible to determine the degree of mixing
and stratificationof sperm from both pair and
extra-paircopulations within SSTs. Such techniques would then be useful for determining
whetherspermdisplacementis more or less likely in species with differentpatternsof layering
within SSTs.
Finally,we needto determinehow storedsperm
areused in the fertilizationprocess.For example,
some sperm probablynever enter the SSTs, but
instead travel directlyto the infundibulum(Howarth 1974). If inseminations are timed to coincidewith ovulation(Birkhead1988),thesenonstored sperm could potentiallyfertilizeeggs and
therebycircumventany selectionimposedby SST
morphology. Indeed, it is possible that sperm
stored in SSTs may seldom be used to fertilize
eggsin species that copulatethroughoutegg-laying (e.g., Smith's Longspur;Briskie 1992), althoughwe could not discernany morphological
differencesin SSTsamongthe speciesin oursample that would suggestsuch a reducedfunction.
Although our study gives only a preliminary
view of the patternsof sperm storage and precedence across species, it is clear that the proximate mechanismsassociatedwith sperm comFURTHER STUDY
petition vary widely from species to species and
Our observationson the number and length of that these differencesmay play an importantrole
SSTs and the arrangementof spermwithin SSTs in shaping the evolution of mating behavior.
suggest several avenues for furtherresearchon Clearly,to understandhow the tremendousvariaspects of avian reproductivebiology not pre- ability in avian mating patternshas evolved, it
viously consideredby behavioralecologists.For will be necessaryto determinehow both females
example,we need to know more about the num- and males manipulatethe processof spermcomber and length of SSTs in a greaterrangeof spe- petition to their own advantage.
cies and matingsystems beforeany generalconclusions can be drawn. Some taxa, such as the
shorebirds(Scolopacidae)or grouse (Phasiani- ACKNOWLEDGMENTS
dae) might be particularlyrewardingfor com- We thankGilles Seutin,SarahKalff,ScottPetrie,
parativeanalysesof sperm storageas they com- Jan Lifjeld,WallyRendelland SariSaundersfor
prise diverse mating systems within closely help collectingoviducts.The ChurchillNorthern
StudiesCentre,the Universityof ManitobaField
relatedgenera.
More experiments on sperm precedenceare Station at Delta Marsh and the Queen's Unialso needed to determine if species with rela- versity BiologicalStation provided logistic sup-
in SST length?From the female's perspective,
changing SST length relative to sperm length
could give her control over sperm precedence.
On the one hand, by having long SSTs, females
could control which male fertilizes each egg by
makingsurehe is the last to copulatebeforeovulation. This strategymight be important if she
wishes either to devalue a forcedextra-paircopulation or to be fertilizedby a male other than
her own mate. Alternatively, if greater sperm
mixing occurs in short SSTs, this process could
induce greater male-male competition and it
might be a strategyto insure that a male with
the mostviableandfastestspermorhighestsperm
productionfertilizesmost of her clutch (Knowlton and Greenwell 1984).
From the male's perspective, sperm stratification resultingin last male precedenceallows
males to devalue extra-pair inseminations by
simply copulating again with his mate before
ovulation. Alternativelywhen sperm are stored
in single layers, extra-paircopulations may be
swampedby resident male sperm. When males
and females are under differentselection pressures with respectto extra-paircopulations,the
net resultmay be an "armsrace"(Dawkinsand
Krebs1979)betweenspermlengthandSSTlength
for control over sperm precedencepatterns.Because the ratio between sperm length and SST
length varies from species to species, independent of body mass and phylogeny, we suggest
thatthe evolution of both of these structuresmay
be the product of escalating adaptations and
counter-adaptationsbetweenthe sexes to control
the proximatemechanismof sperm precedence.
SPERMSTORAGE
IN BIRDS
port and accommodation during the course of
this study. Fundingwas providedby Indian and
Northern Affairs Canada (Northern Scientific
TrainingGrant),a NaturalSciencesand Engineering Research Council (NSERC) Scholarshipto
J.V.B. and grantsfrom NSERC and the Queen's
University Advisory Research Committee to
R.M.D. Promislow provided sage advice on interpretingthe comparative analyses and T. R.
Birkheadand two anonymous reviewers made
many useful comments on the manuscript.
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