Predicting variation in sperm precedence
P E N N Y A. C O O K*, I A N F. H A R V E Y G E O F F R E Y A. P A R K E R
Population Biolog Research Group, School of Biological Sciences, Uniersit of Lierpool, PO Box 147,
Lierpool L69 3BX, UK
SUMMARY
Sperm competition theory predicts that males are adapted for success in sperm competition by the
production of large numbers of sperm. This is supported by both inter- and intraspecific studies showing
that males mating under high sperm competition risk increase investment in sperm production. Such an
increase in sperm production is an advantage if sperm mix randomly or if sperm displacement occurs.
When two males mate with the same female, the measurement of the proportion of eggs fertilized by the
second male to mate (termed P ) has been used to help elucidate sperm competition mechanisms. P is
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usually quoted as a mean value, with little attention being paid to its variance, although P estimates are
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notoriously variable. By predicting an expected variance for P , additional information on sperm
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competition mechanisms may be obtained. Here we present a technique for analysing the variance in P
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when a given mechanism of P is assumed. We apply this technique to P data collected from Plodia
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interpunctella (Lepidoptera, Pyralidae), assuming a ‘ fair raffle ’ mechanism of sperm competition. We
compare observed distributions of P with theoretical distributions generated assuming random mixing of
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two ejaculates drawn randomly from a population of known mean and variance in sperm numbers.
Ejaculates of known size were obtained by counting the number of sperm ejaculated by males mating for
the first (large ejaculate) or second (small ejaculate) time. Females either received two small, or one small
and one large ejaculate, and the distribution of P (estimated using the sterile male technique) was
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compared with our theoretical predictions. The observed variance in P was greater than our model
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prediction, thus we conclude that sperm from P. interpunctella do not mix randomly before fertilization.
pattern is known. For example, in the odonate species
Calopterx maculata the mating male removes virtually
all of the previous sperm, causing a P value of nearly
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one (Waage 1979) and in the dungfly Scatophaga
stercoraria the mating male appears to displace a
proportion of the previous sperm with his own ejaculate
leading to a mean P of 0.8 (Parker 1970 b ; Parker &
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Simmons 1991 ; Simmons & Parker 1992). However, in
many species P values range from zero to one (see
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reviews in Lewis & Austad 1990 ; Simmons & SivaJothy 1997), and few studies have examined this
intraspecific variation. In some studies that have done
so, male body size has been an important factor, with
larger males having increased success in sperm competition (Lewis & Austad 1990 ; LaMunyon & Eisner
1993 a ; C. Bissoondath & C. Wiklund, unpublished
data) ; although this was not the case in studies by Sva$ rd
& McNeil (1994), Conner (1995) and Radwan (1996).
Large males may have increased fertilization success by
producing more sperm or larger spermatophores
(LaMunyon & Eisner 1993 b), or by producing sperm
at a faster rate (Simmons & Parker 1992). Duration of
copula may also effect fertilization success, for example,
if sperm are transferred continuously during mating.
Males that copulate for longer have higher fertilization
success in some species (Dickinson 1986 ; Rubenstein
1989 ; Parker & Simmons 1991 ; Simmons & Parker
1992).
It is usually assumed implicitly in P studies that
#
when two males mate with the same female they both
ejaculate the same number of sperm. However, this
1. I N T R O D U C T I O N
Sperm competition, which occurs when sperm from
more than one male compete for a given set of eggs
(Parker 1970 a), is now recognized as being important in
the evolution of male characteristics (e.g. Smith 1984 ;
Birkhead & Møller 1992 ; Baker & Bellis 1995). For
example, comparative studies have shown that males of
species with high levels of sperm competition have
relatively larger testes (Short 1979 ; Møller 1988 a, b ;
Jennions & Passmore 1993 ; Gage 1994), and produce
larger ejaculates (Sva$ rd & Wiklund 1989 ; Wedell
1993) and more sperm (Møller 1988 a). Theory predicts
that the production of large numbers of sperm is an
advantage in sperm competition (Parker 1982, 1984,
1993) if, for example, sperm mix randomly within the
female (e.g. Martin et al. 1974 ; Simmons 1987 ; Wedell
1991) or displace previous males’ ejaculates (Parker &
Simmons 1991 ; Simmons & Parker 1992).
A male’s fertilization success will generally increase
with the number of his sperm relative to those of other
males, depending on the underlying mechanism of
sperm competition (Parker et al. 1990). The outcome of
sperm competition is measured as the proportion of the
eggs fertilized by the second of the two males to mate
and is termed P (Boorman & Parker 1976). Typically,
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a mean P value for a species is quoted, and for a few
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species the mechanism behind the sperm precedence
* Current address and author for correspondence : Department of
Zoology, University of Stockholm, S-106 91 Stockholm, Sweden.
Phil. Trans. R. Soc. Lond. B (1997) 352, 771–780
Printed in Great Britain
771
# 1997 The Royal Society
P. A. Cook and others Variation in sperm precedence
Phil. Trans. R. Soc. Lond. B (1997)
2. T H E O R E T I C A L B A C K G R O U N D
We first derive probability distributions of the P
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values that would arise if the sperm from two ejaculates
drawn from populations of known mean and variance
in sperm number mixed randomly and if each sperm
has an equal chance of being selected for fertilization.
We competed ‘ large ’ and ‘ small ’ ejaculates of
known mean and variance in sperm numbers (figure
1 a ; for methods see § 3 b). Enhancing the variation in
25
eupyrene sperm number (Χ1000)
may not necessarily be the case. Several factors may
influence how many sperm a male delivers, for
example, in the moth Plodia interpunctella sperm numbers
are reduced when males are reared on a restricted diet
(Gage & Cook 1994). Additionally, males may vary
sperm number according to mating history : in P.
interpunctella males become depleted, producing fewer
sperm on second and third matings compared with
virgin matings (Gage & Cook 1994 ; Cook & Gage
1995). Sexually exhausted males of the bruchid beetle
Callosobruchus maculatus achieve lower fertilization success than those on their first mating (Eady 1995). In
contrast, male Pieris rapae butterflies increase sperm
number from their first to second matings, possibly due
to higher risk of sperm competition later in the flight
season (Cook & Wedell 1996). Studies on a variety of
species show that the number of sperm ejaculated by a
male increases when sperm competition risk is high
(Baker & Bellis 1989, 1993 ; Bellis et al. 1990 ; Gage
1991 ; Gage & Baker 1991 ; Simmons et al. 1993 ; Cook
& Gage 1995). Males may increase the number of
sperm they inseminate to increase the representation of
their sperm in the fertilization set. Such an increase in
sperm number as a result of mating with a non-virgin
female could contribute towards the observed high
values of P seen in many species.
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It is also clear that a large amount of unexplained
variation in P occurs within the majority of insect
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species (Lewis & Austad 1990 ; Simmons & Siva-Jothy
1997). We suggest the following approach for analysing
intraspecific variation in P : (i) assume a given
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mechanism of sperm competition ; (ii) examine the
variation in sperm numbers delivered during mating
by the population of males used for the sperm
competition study ; (iii) given (i), use (ii) to predict the
variation in P ; (iv) compare the observed and
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predicted P distributions : if there is a fit, we have
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circumstantial evidence for the causes of variation in P
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(although we may not know why number of sperm
transferred varies within the sample of males used in
the experiment). If observed and predicted distributions differ, we can claim that the mechanism of sperm
competition is unlikely to be the one we have assumed
under (i).
In the present paper we demonstrate this method by
applying it to data collected on the pyralid moth Plodia
interpunctella (Hu$ bner). P in P. interpunctella is 0.68
#
(Gwynne 1984, using data from Brower 1975) ; a value
that superficially indicates high sperm mixing. Sperm
numbers in P. interpunctella are quite variable (Gage &
Cook 1994 ; Cook & Gage 1995), and males mating
with already mated females increase the number of
sperm they ejaculate compared to those copulating
with virgin females (Cook & Gage 1995). From a
knowledge of the mean and variance of sperm numbers
in each ejaculate we predict what the distribution of P
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values would be if the sperm mix randomly within the
female storage organ, and we test this hypothesis
against observed distributions of P .
#
(a)
20
female status
non-virgin
15
virgin
10
5
10
10
7
10
0
first mating
second mating
male status
small
(b)
large
probability
772
0
10000
20000
30000
sperm number
Figure 1. (a) The mean number of eupyrene sperm (bars are
standard errors ; numbers at bases of bars are sample sizes)
produced by males on their first (‘ large ’ ejaculate) and
second (‘ small ’ ejaculate) matings when mating with females
that were either non-virgin (having previously received a
‘ small’ ejaculate) or virgin. Apyrene numbers are not shown
as they are not used in the model, however the pattern is the
same. (b) As males provide both virgin and non-virgin
females with similar numbers of eupyrene sperm, normal
plots for ‘ large ’ (males’ first matings ; XF ¯ 17 075, s.d. ¯
5006) and ‘ small ’ (males’ second matings ; XF ¯ 5873, s.d. ¯
3095) ejaculates are fitted from the combined data ; these
sperm number distributions are then used in the model (§ 2).
Variation in sperm precedence
sperm numbers between the two competing males
increases the power of the test for fit between predicted
and observed distributions. From fitted normal probability plots (figure 1 b), the probability (q) of male 1
producing an ejaculate of size n , and the probability
"
(r) of male 2 producing an ejaculate of size n can be
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calculated. The probability of these two ejaculates
meeting each other in the female tract is therefore
qn rn . If the sperm then mix randomly and each sperm
" #
has an equal chance of being picked for fertilization,
the proportion of the eggs to be fertilized by the second
male is the same as the proportion of his sperm in the
female tract, that is :
n
(1)
P¯ #
# n n
" #
i.e. the ‘ fair raffle ’ principle of Parker et al. (1990).
Computer simulations (using Minitab, Release 9)
were carried out to generate P distributions that would
#
occur as a result of sperm number variation : (i) A
matrix was constructed to calculate the probabilities of
all the possible combinations of pairs of ejaculates from
males 1 and 2. The simulations used a range of
1000–12 000 sperm (increment 1000 sperm) for ‘ small ’
ejaculates, and a range of 2000–24 000 sperm (increment 2000 sperm) for ‘ large ’ ejaculates (these
sperm number ranges were selected to cover those
shown in figure 1 b). (ii) A corresponding matrix was
constructed to calculate the P values that would occur
#
if the two ejaculates met (from equation (1)). (iii) Each
P value therefore has an associated probability in the
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corresponding matrix. P values were assigned into the
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categories : 0–0.05, 0.05–0.1, … 0.95–1.0. The probabilities from the corresponding matrix were summed
for each category of P . This generates theoretical P
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distributions that occur if the proportion of eggs
fertilized by the second male was purely a result of
random sperm mixing (see line on figure 2).
3. M A T E R I A L S A N D M E T H O D S
(a) Rearing technique
Plodia interpunctella was cultured in constant condition
rooms at 25 (³2) °C with a 16L : 8D photoperiod.
Larvae were reared on a diet of bran midlings, glycerol
and yeast in a 10 : 1 : 1 ratio and at density of about 100
eggs per 400 ml of medium. Virgin animals were
obtained by isolating individual 5th instar larvae from
the stock culture in 30 ml vials with a small amount of
food medium.
(b) Sperm counts
Lepidoptera produce two types of sperm, a fertile
‘ eupyrene ’ form and a non-fertile, anucleate ‘ apyrene ’
form. The function of the apyrene sperm type is
unknown (although they may influence female sexual
receptivity : Silberglied et al. 1984 ; Cook & Gage
1995), therefore their numbers are not incorporated in
the model.
We manipulated ejaculate size by allowing given
males to mate for the first (‘ large ’ ejaculate) or second
Phil. Trans. R. Soc. Lond. B (1997)
P. A. Cook and others 773
time (‘ small ’ ejaculate) ; male P. interpunctella predictably produce ejaculates with decreasing numbers
of sperm over successive matings (Gage & Cook 1994 ;
Cook & Gage 1995). Male P. interpunctella have been
shown to vary the size of their ejaculate depending on
whether females are virgin or non-virgin (Cook &
Gage 1995), therefore the number of sperm produced
by males on their first and second matings to both
virgin and non-virgin females was ascertained. Females,
3–4 days old, were mated either once or twice to 3–4day-old males. Singly-mated females received either a
small or a large ejaculate, and the number of sperm in
the spermatophore were counted using established
protocols (Gage & Cook 1994 ; Cook & Gage 1995).
Doubly-mated females all received a small ejaculate at
the first mating. After 36 h, females were mated to a
male on either his first or second mating, and the
number of sperm transferred were counted. P. interpunctella are more active during the dark cycle.
However, virgin females mate readily in the light cycle.
All first matings were therefore carried out in the light
cycle. Non-virgin females are generally more reluctant
to mate during the light cycle, so for second matings,
pairs were observed throughout the dark cycle. About
half of the females remated under these conditions,
therefore twice as many doubly-mated as singly-mated
treatments were set up.
In contrast to previous results (Cook & Gage 1995 :
see § 5 a), female mating status (virgin or not) had no
effect on the number of sperm transferred by males
(figure 1 a ; first mating : eupyrene, F , ¯ 0.43 ; n.s. ;
" ")
apyrene, F , ¯ 0.05, n.s ; second mating : eupyrene,
" ")
F , ¯ 0.25, n.s. ; apyrene, F , ¯ 0.35, n.s). The mean
" "&
" "&
and variance in the numbers of sperm in a ‘ small ’
(male on his second mating) and a ‘ large ’ (male on his
first mating) ejaculate were calculated. As there were
no significant differences in the number of sperm
produced by males mating with virgin or non-virgin
females, the data for each size of ejaculate were
combined to estimate the parameters. The distributions
are not significantly different from normal (the data
were highly correlated with their normal scores,
equivalent to a Shapiro–Wilk test (Minitab Reference
Manual 1991) ; large ejaculates, r ¯ 0.978, p " 0.1,
n ¯ 17 ; small ejaculates, r ¯ 0.992, p " 0.1, n ¯ 20).
To check that irradiated and normal males did not
differ in the number of sperm they transfer, virgin 1- or
2-day-old irradiated and normal males were mated
and the number of sperm they ejaculated was counted.
The irradiated and normal males did not differ in the
number of eupyrene sperm ejaculated (normal : XF ³s.e.
¯ 11 676³1058, n ¯ 18 ; sterile : XF ¯ 12 996³1031,
n ¯ 17 ; F , ¯ 0.80, n.s.), but irradiated males ejacu" $$
lated more apyrene sperm than normal males (normal :
XF ¯ 117 207³10 776, n ¯ 18 ; sterile : XF ¯ 155 654³
11 088, n ¯ 17 ; F , ¯ 6.18, p ¯ 0.018).
" $$
(c) P2 experiment
To estimate P , two males were mated with the same
#
female and the number of offspring sired by each of the
males was counted. We used the sterile male technique :
males were exposed to a sublethal doses of irradiation
774
P. A. Cook and others Variation in sperm precedence
so that although they still produced sperm that were
capable of fertilizing the egg, the resulting embryos
suffered lethal mutations in the early stages of
development. In a double mating, eggs that failed to
hatch were therefore attributed to the sterile male and
those that hatched to the normal male. There are two
disadvantages with this technique : first, there may be
differences in the competitiveness of normal and sterile
male’s sperm and to control for this the mating order
was reversed (Parker 1970 a) ; second, paternity assignment can be ambiguous, as a proportion of eggs
that are fertilized by a normal male usually fail to
hatch and some of the progeny from a sterile male may
develop. The proportion of hatching eggs attributed to
each male from the double matings was therefore
corrected using the mean fertility of control females
mating with either two normal or two sterile males.
Two equations have been published to incorporate the
correction factors (Boorman & Parker 1976 ; Sille! nTullberg 1981). However, these are algebraically
equivalent (Appendix 1) and we used the following
form to calculate PN, the proportion of eggs fertilized
by the normal (i.e. non-irradiated) male :
PN ¯
(x®)
(p®)
(2)
where x is the proportion of eggs that hatch from the
sterile–normal and normal–sterile matings, p is the
proportion hatching after two normal matings and is
the proportion hatching after two sterile matings.
Females each received two ejaculates, with all
females first receiving a small ejaculate, as such females
remate more readily. For their second mating, females
received either a large ejaculate or another small
ejaculate. Experimental females were mated alternately to sterile and to normal males, and vice versa,
and controls were mated to two sterile or two normal
males, giving rise to eight categories.
All the adults eclosing over a 2-day period were
collected and sexed. Half the males were irradiated on
the same day using 350 Gy (at a rate of 4.5 Gy min−")
from a gamma radiation source at the John Moores
University, Liverpool (a dose sufficient to induce 98 %
sterility in the small–small and 95 % sterility in the
small–large sterile controls). Males were randomly
assigned first or second male roles. Females were either
assigned to experimental or to control categories, or
they were used to deplete the males.
Males (both sterile and normal) that were to be in
the first male role were mated for the first time one day
prior to the experiment in order to deplete them. For
females’ first matings, females were placed with ‘ first
males ’ (now on their second mating) and after
copulation, males were removed. The following day
any female that had laid more than about 20 eggs was
discarded from the experiment. Females were then
given the opportunity to mate for the second time (and
were later dissected upon death to ascertain the
number of matings from spermatophore counts ;
Drummond 1984), with a male transferring either a
‘ small ’ ejaculate (i.e. already mated on the previous
day) or a ‘ large ’ ejaculate (i.e. virgin). For second
matings, pairs were housed together for 24 h (to allow
Phil. Trans. R. Soc. Lond. B (1997)
maximum opportunity for remating), and the second
male was removed the following morning. Females
that had mated only once or (as occurred very rarely)
more than twice were removed from the analysis.
Males were placed in the freezer after their mating
opportunity and after the experiment male size was
estimated (see § 3 d). Males and females were all 3–4
days old on females’ first matings and 4–5 days old on
females’ second matings.
After females were separated from their ‘ second
males ’, they were isolated in 30 ml plastic tubes and
eggs were collected every 2 days for the duration of the
female lifespan. P. interpunctella do not require a
substrate on which to oviposit ; eggs were either found
loose in the tube or adhered to the walls. Up to 70 eggs
(depending on how many had been laid) were sampled
randomly and sprinkled in a small Petri dish on sticky
paper, allowing larvae to hatch and preventing them
from escaping, eating the egg cases or cannibalizing
siblings. After 5 days, the numbers of unhatched and
empty egg cases were counted using a binocular
microscope and the proportion of eggs to hatch was
calculated.
The data for the proportion of hatching eggs were
divided into ‘ early ’ (those laid in the first 2 days) and
‘ late ’ (laid in the remainder of the female’s life). The
corrected proportion of eggs fertilized by the normal
male (PN ; equation (2)) was calculated for early and
late egg collections separately. The mean proportions
of viable eggs from normal–normal controls (p) were
0.94 (early eggs) and 0.75 (late eggs) for ‘ small–small ’
treatments, and 0.94 (early eggs) and 0.68 (late eggs)
for ‘ small–large ’ treatments. The mean proportions of
viable eggs from the sterile–sterile controls () were
0.02 (early eggs) and 0.01 (late eggs) for ‘ small–small ’
treatments, and 0.05 (early eggs) and 0 (late eggs) for
‘ small–large ’ treatments. In the case of sterile–normal
matings, PN is the P value. For normal–sterile matings,
#
values of PN are P values (the proportion eggs fertilized
"
by the first male) and were therefore converted to P
#
values by subtracting them from one.
The resulting P dataset had some values of less than
#
zero and some of greater than one. The values less than
zero were caused by some of the sterile–sterile controls
being more fertile than the experimentals (i.e. when
" x). Values greater than one were caused by some
of the normal–normal controls being less fertile than the
experimentals (i.e. when p ! x). The datasets were
therefore transformed so that the data lay in the range
0 to 1 as follows :
(P i®P min)
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#
Corrected P i ¯
(3)
#
(P max®P min)
#
#
where P i are individual P values, P min is the lowest
#
#
#
and P max the highest P value in the dataset.
#
#
(d) Measurement of male size
Male size was estimated by measuring the length of
the forewing. Accurate measurements were obtained
by removing the scales from wings (following the
Variation in sperm precedence
technique given in Reid 1976) and mounting the wings
on glass slides. The length between the wing margin}
vein one junction and the point of wing insertion for
both left and right wings was measured using an eye
piece graticule at ¬15 magnification. The mean of the
two measurements was taken.
4. R E S U L T S
Mean P values vary from 0.34 to 0.88 (table 1)
#
suggesting some sperm mixing (cf. Brower 1975).
However, expressing the data as means gives little
indication of the true pattern of sperm precedence ; the
frequency distributions of P are shown in figure 2.
#
Although the distributions in figure 2 show the two
mating orders combined for simplicity, Kolmogorov–
Smirnov goodness-of-fit tests were carried out on the
mating orders separately. The expected and observed
distributions differ significantly when a small ejaculate
is followed by a large ejaculate, for both early
(normal–sterile : D . ¯ 0.55, n ¯ 17, p ! 0.01 ; sterile–
!&
normal : D . ¯ 0.71, n ¯ 19, p ! 0.01) and late egg
!&
collections (normal–sterile : D . ¯ 0.46, n ¯ 19, p !
!&
Table 1. Mean P alues, standard deiations and sample
#
sies for all treatments in both earl (the first 2 das of egg
laing) and late (eggs laid subsequentl) egg collections
(The range of P values was 0 to 1 in all cases. Females each
#
received two ejaculates : ø is a small ejaculate from an
irradiated male, o is small ejaculate from a normal male, Ø is
large ejaculate from an irradiated male and O is large
ejaculate from a normal male.)
treatment
early
late
first
second
XF
s.d
n
XF
s.d.
n
o
ø
o
ø
Ø
O
ø
o
0.78
0.88
0.34
0.80
0.37
0.22
0.37
0.32
17
19
12
18
0.78
0.75
0.78
0.46
0.33
0.27
0.38
0.38
19
23
15
16
Phil. Trans. R. Soc. Lond. B (1997)
5
0
0.25
0.5
0.75
1
0.75
1
P2
10
(b)
7.5
frequency
To test whether the observed distribution of P
#
values was the same as the predicted P distributions
#
shown in figure 2, the data for the four treatments were
first converted to the same categories of P values as the
#
theoretical P distributions (0–0.05, 0.05–0.1, …
#
0.95–1.0). The cumulative observed distributions were
then compared with the cumulative theoretical P
#
probability distributions using Kolmogorov–Smirnov
goodness-of-fit tests. Two-sample Kolmogorov–
Smirnov tests were used to test for differences between
pairs of observed distributions. These non-parametric
tests have the null hypothesis that the observed and
expected (goodness-of-fit test) or the two observed
(two-sample test) distributions have identical shapes,
and are sensitive to differences in location, dispersion
and skewness (Sokal & Rohlf 1995).
(a)
10
frequency
(e) Analysis
15
P. A. Cook and others 775
5
2.5
0
0.25
0.5
P2
Figure 2. Expected P if ejaculates of known mean and
#
variance in sperm number (from figure 1 b) mix randomly
(line) compared with the observed data (bars) from eggs laid
early in the female’s life (for both mating orders combined).
(a) Sperm from a large ejaculate competing with sperm from
an initial small ejaculate, and (b) sperm from a small
ejaculate competing with that of an initial small ejaculate.
0.01 ; sterile–normal : D . ¯ 0.37, n ¯ 23, p ! 0.01).
!&
Similarly, expected and observed distributions differ
significantly when a small ejaculate is followed by
another small ejaculate, in both early (normal–sterile :
D . ¯ 0.56, n ¯ 12, p ! 0.01 ; sterile–normal : D . ¯
!&
!&
0.7, n ¯ 18, p ! 0.01) and late egg collections
(normal–sterile D . ¯ 0.7, n ¯ 15, p! 0.01 ; sterile–
!&
normal : D . ¯ 0.39, n ¯ 16, p ! 0.01).
!&
Two-sample Kolmogorov–Smirnov tests between
pairs of observed distributions were also carried out.
Mating order (whether the irradiated male mated first
or second) significantly affects the distribution of P
#
values when two small ejaculates compete (in both
early and late clutches), but not when a small ejaculate
is followed by a large one (table 2). The effect of
ejaculate size on P is ambiguous. Although it appears
#
776
P. A. Cook and others Variation in sperm precedence
Table 2. Kolmogoro-Smirno tWo-sample tests to compare the
distributions of P betWeen the different mating orders and
#
ejaculate sies
(Females each receive two ejaculates : ø is a small ejaculate
from an irradiated male ; o is small, normal ; Ø is large,
irradiated and O is large, normal. Early eggs were those
collected during the first 2 days of oviposition and late eggs
were all those collected subsequently. For sample sizes, see
table 1.)
early
mating order
effects
o Ø and ø O
o ø and ø o
ejaculate size
effects
o Ø and o ø
ø O and ø o
late
Dmax
p
Dmax
p
0.42
0.64
n.s.
! 0.001*
0.37
0.55
n.s.
0.012*
0.57
0.28
0.013*
N.S.
0.21
0.51
N.S.
0.01*
* Remain significant at an experiment-wise error rate of
0.05 following the sequential Bonferroni technique (Rice
1989).
Table 3. Spearman’s rank correlations betWeen relatie male
sie (male 2}male 1) and P
#
(Females each receive two ejaculates (treatment) : ø is a small
ejaculate from an irradiated male ; o is small, normal ; Ø is
large, irradiated and O is large, normal. Early eggs were
those collected during the first 2 days of oviposition and late
eggs were all those collected subsequently.)
treatment
early
late
first
second
n
rs
p
n
rs
p
o
ø
o
ø
Ø
O
ø
o
16
17
11
17
0.10
0.23
®0.48
0.06
n.s.
n.s.
n.s.
n.s.
14
20
11
15
0.13
®0.12
®0.43
®0.13
n.s.
n.s.
n.s.
n.s.
from figure 2 that large ejaculates are more successful,
when the data are analysed for mating order separately, no clear pattern emerges. Instead, the distributions seem more strongly affected by mating order and
whether eggs are laid early or late in the female lifespan
(table 2). Fertilization success in this species does not
appear to be related to male size (table 3).
5. D I S C U S S I O N
(a) Experimental
The observed distributions of sperm numbers (figure
1) show a large amount of variation and, if the sperm
were competing as in the fair raffle model (Parker et al.
1990), our model predicts a corresponding large
variation in the P (figure 2). For example, in the case
#
of an initial small ejaculate competing with a later
Phil. Trans. R. Soc. Lond. B (1997)
large ejaculate, P would be expected to vary from 0.5
#
to 1 (figure 2 a). The observed distributions of P values
#
vary from 0 to 1. However, the shapes of the
distributions do not fit with the model based on the
‘ fair raffle ’ hypothesis (Parker et al. 1990).
Here the number of sperm transferred by each male
was known ; however, several factors make it difficult to
assess accurately whether these sperm are mixing
randomly in the female storage organs. Fertilization
success may become biased towards the second male to
mate if the female oviposits between the two matings.
This was controlled by discarding females that laid
more than 20 eggs. Sperm from the first mating may
also be passively lost from storage (Tsubaki &
Yamagishi 1991 ; Eady 1994). Although the time
between the two matings was as short as possible,
sufficient time had to be allowed for the sperm to travel
to the storage organs allowing (i) enough room in the
bursa for another spermatophore, and (ii) the female to
become receptive (the presence of a full spermatophore
in the bursa delays female remating ; Sugawara 1979).
In other invertebrate studies that examine intraspecific variation in P , male size has been important in
#
determining fertilization success. In two lepidopteran
species, large males transfer larger spermatophores and
have greater fertilization success (LaMunyon & Eisner
1993 b ; C. Bissoondath & C. Wiklund, unpublished
data). Larger males of the dungfly Scatophaga stercoraria
transfer sperm at a faster rate but copulate for a
compensatory shorter time ; thus their P is the same as
#
that of small males (Simmons & Parker 1992).
However, in P. interpunctella, large males do not transfer
more sperm (unpublished data) and, in common with
another lepidopteran (Sva$ rd & McNeil 1994), male
size does not correlate with P . Interestingly, in the
#
mite Rhioglphus robini, fertilization success correlates
only with sperm size and not with sperm numbers or
male size (Radwan 1996).
Several explanations have been put forward to
explain observed P patterns in Lepidoptera. In species
#
where females remate very rapidly it has been suggested
that the second male’s spermatophore may displace
that of the first so that few of the first male’s sperm
reach the spermatheca (Drummond 1984). This is
unlikely to be the case here, as females were allowed
adequate time (24 h) between each mating for sperm
to be transported to the storage organs. Another
suggestion is that the pattern of second male precedence could be caused by the tubular shape of the
spermatheca (Walker 1980). The second male may
have an advantage because the sperm from the
previous males is displaced backwards. If sperm
displacement occurs, larger ejaculates may displace
more of the previous sperm. This could account for the
more consistent second male advantage when the male
produces a large ejaculate rather than a small one
(figure 2 a compared with 2 b).
It is hard to explain the occurrence of occasional low
P values in our data. For example, three females out of
#
37 in the small–large treatment apparently used sperm
from the first male (the small ejaculate) to fertilize all
their eggs (figure 2 a). In these cases it is possible that
(i) the female was sterile (and her eggs therefore
Variation in sperm precedence
Phil. Trans. R. Soc. Lond. B (1997)
µ2 is twice µ1;
CV for both males is 30
(a)
µ2 is twice µ1;
CV for both males is 15
probability
µ2 is ten times µ1;
CV for both males is 30
0
1
0.5
P2
(b)
CV1 = 15; CV2 = 30
CV1 = 30; CV2 = 15
CV1 = CV2 = 30
probability
attributed to the sterile male), but this is unlikely to
account for many low P values, as complete infertility
#
in normal matings is rare (of 197 females double-mated
to normal males, only one female was completely
infertile ; unpublished data) ; or (ii) transfer of sperm
from the second spermatophore failed. Although, upon
post mortem, data from females whose spermatophore
had obviously not drained were omitted, it is possible
that we were not always able to detect failures of sperm
transport. An alternative explanation for such ‘ all or
none ’ P patterns (that are more apparent in some
#
other species : Retnakaran 1974 ; LaMunyon & Eisner
1993 a ; Sva$ rd & McNeil 1994) is that female
Lepidoptera exert choice over ejaculates (LaMunyon
& Eisner 1993 a, b). As females appear to control the
movement of sperm from the bursa to the spermatheca
(Tschudi-Rein & Benz 1990 ; LaMunyon & Eisner
1993 a), they may also have the potential to select an
individual male’s sperm.
Sterilization treatment has a significant effect on P
#
when two small ejaculates compete (table 2). This
difference in sperm competitiveness does not seem to
occur when males are on their first mating (producing
a large ejaculate) as second males transferring large
ejaculates do equally well (table 2). However, on a
male’s second mating (small ejaculate), irradiation
seems to cause the sperm to be considerably less
competitive than that of the normal male (tables 1 and
2). Sterilization may have a greater effect on the sperm
used in the second ejaculate if these sperm had been
still developing at the time of the irradiation dose.
However, this is not consistent with the apparent
reversal of the sperm precedence pattern later in the
female’s life, with sperm from the sterile male being
used (table 1).
Another effect of the sterilization treatment was the
production of significantly more apyrene (non-fertile),
but not eupyrene (fertile), sperm by sterile males than
normal males in first ejaculates (§ 3 b). Male P.
interpunctella appear to have a mechanism whereby they
change the number of apyrene sperm relative to
eupyrene sperm depending on female quality (Cook &
Gage 1995). It is possible that if the irradiation
treatment damaged this mechanism there would be a
resulting change in apyrene sperm numbers.
In this study, variation in P cannot be explained by
#
an increase in sperm numbers ejaculated by the second
male to mate in response to sperm competition risk
(figure 1 a). In contrast, Cook and Gage (1995) found
that the number of eupyrene sperm ejaculated by male
P. interpunctella depended on sperm competition risk :
males increased the number of sperm when mating
with non-virgin females. There are two possible reasons
why we find no such effect in the present study : (i) in
the previous study males used both female age and
mating status as cues to determine how many sperm to
ejaculate, whereas here we use females of an intermediate age ; and (ii) previously males were found
to ejaculate larger numbers of sperm to females that
already contained a ‘ large ’ ejaculate compared to
those that contained ‘ small ’ ejaculates, whereas in the
present study, females’ first matings were always to
males producing a ‘ small ’ ejaculate.
P. A. Cook and others 777
0
0.5
1
P2
Figure 3. The effect of altering the mean and variance of
sperm numbers on the predicted P . (a) The coefficient of
#
variation (CV) is the same for both males. Increasing the
mean size of a second male’s ejaculate (thin line) or reducing
the variation in both males’ ejaculates (broken line) decreases
the predicted variation in P . µ and µ are the mean sperm
# "
#
numbers for the first and second male respectively. (b) When
the mean numbers of sperm ejaculated by both males are
kept constant (here the second male’s ejaculate is twice that
of the first male’s), reducing the CV of either male (thin line
and broken line) decreases the predicted variation in P . CV
#
"
and CV are the coefficients of variation for sperm number
#
for the first and second male respectively.
(b) Theoretical
In principle it seems that the technique we have
applied could be used in all sperm competition studies
to provide evidence for various mechanisms of sperm
competition. Our technique relies on estimating the
population variance in sperm numbers. However,
caution is needed as variation due to sampling occurs
at two levels. There is for most studies (i) an error
associated with subsampling sperm to deduce an
individual male’s sperm count, and (ii) an error
associated with sampling males to deduce the population variance. The present study assumes no error in
778
P. A. Cook and others Variation in sperm precedence
measuring an individual’s sperm count ; this is a
reasonable assumption in this case because the total
number of eupyrene sperm is counted directly (see
Gage & Cook 1994). However, most sperm counts rely
on subsampling and under such circumstances appropriate techniques should be used to estimate
the population (inter-individual) variance in sperm
numbers.
It is useful to obtain some feel for the behaviour of
our model under random sperm mixing. Figure 3
shows some predicted P distributions when means and
#
coefficients of variation of sperm numbers of two
competing ejaculates are varied. The second male
always has the larger ejaculate. The main effects are (i)
reducing the coefficient of variation at a given sperm
number reduces the predicted variation in P (figure
#
3 a) ; (ii) increasing the difference in mean sperm
number at a given coefficient of variation reduces the
predicted variation as well as increasing P (figure 3 a) ;
#
and (iii) reducing the coefficient of variation of either
of the males reduces the predicted variation in P ,
#
although this effect is small relative to the effect of
changing sperm numbers (figure 3 b).
(c) Conclusion
Our study demonstrates convincingly that sperm
competition does not occur by random mixing,
following a ‘ fair raffle ’ (Parker et al. 1990) in P.
interpunctella. We could investigate further possible
mechanisms of sperm competition (e.g. sperm displacement or stratification) using the same technique.
To do this, we would simply insert the appropriate
alternative form into equation (1) : the rest of the
method for generating the predicted distributions
would then proceed in an identical fashion. The
equation for sperm displacement with instant random
mixing is given in Parker and Simmons (1991, equation
(11)). However, the strong bimodal distribution of P is
#
suggestive of female choice of ejaculates. In principle,
female preferential use of sperm could also be modelled
by our technique, as P is likely still to depend
#
(although less strongly) on sperm numbers. To do this
we would need to quantify the bias algebraically and
substitute this definition into equation (1).
This paper is, in essence, a starting point for further
analyses of P data. We suggest that variance in sperm
#
numbers can be used routinely to predict P distribu#
tions ; a test against the observed distribution then gives
a more sensitive test than using simply the mean P .
#
We are extremely grateful to Eric Corbett for irradiating the
moths and Douglas Reed for computer expertise. We also
thank Matt Gage, Steve Sait and Leigh Simmons for advice
and discussion, and two anonymous referees for providing
constructive comments. P. A. C. was supported by a BBSRC
studentship and G. A. P. was supported by NERC grant
GR3}9264.
REFERENCES
Baker, R. R. & Bellis, M. A. 1989 Number of sperm in
human ejaculates varies in accordance with sperm competition theory. Anim. Beha. 37, 867–869.
Baker, R. R. & Bellis, M. A. 1993 Human sperm comPhil. Trans. R. Soc. Lond. B (1997)
petition : ejaculate adjustment by males and the function of
masturbation. Anim. Beha. 46, 861–885.
Baker, R. R. & Bellis, M. A. 1995 Human sperm competition :
copulation, masturbation and infidelit. London : Chapman &
Hall.
Bellis, M. A., Baker, R. R. & Gage, M. J. G. 1990 Variation
in rat ejaculates consistent with the kamikaze-sperm
hypothesis. J. Mamm. 71, 479–480.
Birkhead, T. R. & Møller, A. P. 1992 . Sperm competition in
birds : eolutionar causes and consequences. London : Academic
Press.
Boorman, E. & Parker, G. A. 1976 Sperm (ejaculate)
competition in Drosophila melanogaster, and the reproductive
value of females to males in relation to female age and
mating status. Ecol. Entomol. 1, 145–155.
Brower, J. H. 1975 Sperm precedence in the Indian meal
moth Plodia interpunctella. Ann. Ent. Soc. Am. 68, 78–80.
Conner, J. K. 1995 Extreme variability in sperm precedence
in the fungus beetle, Boliotherus cornutus (Coleoptera
Tenebrionidae). Ethol. Ecol. Eol. 7, 277–280.
Cook, P. A. & Gage, M. J. G. 1995 Effects of risks of sperm
competition on the numbers of eupyrene and apyrene
sperm ejaculated by the moth Plodia interpunctella
(Lepidoptera : Pyralidae). Beha. Ecol. Sociobiol. 36,
261–268.
Cook, P. A. & Wedell, N. 1996 Ejaculate dynamics in
butterflies : a strategy for maximizing fertilization success ?
Proc. R. Soc. Lond. B 263, 1047–1051.
Dickinson, J. L. 1986 Prolonged mating in the milkweed
leaf beetle Labidomera cliicollis cliicollis (Coleoptera :
Chrysomelidae) : at test of the ‘ sperm-loading ’ hypothesis.
Beha. Ecol. Sociobiol. 18, 331–338.
Drummond, B. A. 1984 Multiple mating and sperm
competition in the Lepidoptera. Sperm competition and the
eolution of animal mating sstems (ed. R. L. Smith), pp.
291–370. London : Academic Press.
Eady, P. 1994 Sperm transfer and storage in relation to
sperm competition in Callosobruchus maculatus. Beha. Ecol.
Sociobiol. 35, 123–129.
Eady, P. E. 1995 Why do male Callosobruchus maculatus
beetles inseminate so many sperm ? Beha. Ecol. Sociobiol.
36, 25–32.
Gage, M. J. G. 1991 Risk of sperm competition directly
affects ejaculate size in the Mediterranean fruit fly. Anim.
Beha. 42, 1036–1037.
Gage, M. J. G. 1994 Associations between body size, mating
pattern, testes size and sperm lengths across butterflies.
Proc. R. Soc. Lond. B 258, 247–254.
Gage, M. J. G. & Baker, R. R. 1991 Ejaculate size varies
with sociosexual situation in an insect. Ecol. Ent. 16,
331–337.
Gage, M. J. G. & Cook, P. A. 1994 Sperm size or numbers ?
Effects of nutritional stress upon eupyrene and apyrene
sperm production strategies in the moth Plodia interpunctella
(Lepidoptera : Pyralidae) Funct. Ecol. 8, 594–599.
Gwynne, D. T. 1984 Male mating effort, confidence of
paternity, and insect sperm competition. Sperm competition
and the eolution of animal mating sstems (ed. R. L. Smith),
pp. 117–151. London : Academic Press.
Jennions, M. D. & Passmore, N. I. 1993 Sperm competition
in frogs : testis size and a ‘ sterile male ’ experiment on
Chiromantis xerampelina (Rhacophoridae). Biol. J. Linn. Soc.
50, 211–220.
LaMunyon, C. W. & Eisner, T. 1993 a Postcopulatory
sexual selection in an arctiid moth (Utetheisa ornatrix). Proc.
natn. Acad. Sci. U.S.A. 90, 4689–4692.
LaMunyon, C. W. & Eisner, T. 1993 b Spermatophore size
as determinant of paternity in an arctiid moth (Utetheisa
ornatrix). Proc. Natn. Acad. Sci. U.S.A. 91, 7081–7084.
Variation in sperm precedence
Lewis, S. M. & Austad, S. N. 1990 Sources of intraspecific
variation in sperm precedence in red flour beetles. Am. Nat.
135, 351–359.
Martin, P. A., Reimers, T. J., Lodge, J. R. & Dziuk, P. J.
1974 The effect of ratios and numbers of spermatozoa
mixed from two males on proportions of offspring. J.
Reprod. Fert. 39, 251–258.
Minitab Reference Manual 1993 Release 9. State College,
Pennsylvania : Minitab Inc.
Møller, A. P. 1988 a Ejaculate quality, testes quality and
sperm competition in primates. J. Human Eol. 17,
489–502.
Møller, A. P. 1988 b Testes size, ejaculate quality and sperm
competition in birds. Biol. J. Linn. Soc. 33, 273–283.
Parker, G. A. 1970 a Sperm competition and its evolutionary
consequences in the insects. Biol. Re. 45, 525–567.
Parker, G. A. 1970 b Sperm competition and its evolutionary
effect on copula duration in the fly Scatophaga stercoraria. J.
Insect Phsiol. 16, 1301–1328.
Parker, G. A. 1982 Why are there so many tiny sperm ?
Sperm competition and the maintenance of two sexes. J.
theor. Biol. 96, 281–294.
Parker, G. A. 1984 Sperm competition and the evolution of
animal mating strategies. In Sperm competition and the
eolution of animal mating sstems (ed. R. L. Smith), pp. 1–60.
London : Academic Press.
Parker, G. A. 1993 Sperm competition games : sperm size
and sperm number under adult control. Proc. R. Soc. Lond.
B 253, 245–254.
Parker, G. A. & Simmons, L. W. 1991 A model of constant
random sperm displacement during mating : evidence
from Scatophaga. Proc. R. Soc. Lond. B 246, 107–115.
Parker, G. A., Simmons, L. W. & Kirk, H. 1990 Analysing
sperm competition data : simple models for predicting
mechanisms. Beha. Ecol. Sociobiol. 27, 55–65.
Reid, J. 1976 Techniques. In The moths and butterflies of Great
Britain and Ireland, vol. 1 (ed. J. Heath), pp. 117–132.
London : Curwen Press.
Radwan, J. 1996 Intraspecific variation in sperm competition success in the bulb mite : a role for sperm size. Proc.
R. Soc. Lond. B 263, 855–859.
Retnakaran, A. 1974 The mechanism of sperm precedence
in the spruce budworm, Choristoneura fumiferana
(Lepidoptera : Tortricidae). Can. Ent. 106, 1189–1194.
Rice, W. R. 1989 Analyzing tables of statistical tests.
Eolution 43, 223–225.
Rubenstein, D. I. 1989 Sperm competition in the water
strider, Gerris remigis. Anim. Beha. 38, 631–636.
Short, R. V. 1979 Sexual selection and its component parts,
somatic and genital selection as illustrated by man and the
great apes. Ad. Stud Beha. 9, 131–158.
Silberglied, R. L., Shepherd, J. G. & Dickinson, J. L. 1984
Eunuchs : the role of apyrene sperm in Lepidoptera ? Am.
Nat. 123, 255–265.
Sille! n-Tullberg, B. 1981 Prolonged copulation : A male
‘ postcopulatory ’ strategy in a promiscuous species, Lgaeus
equestris (Heteroptera : Lygaeidae). Beha. Ecol. Sociobiol. 9,
283–289.
Simmons, L. W. 1987 Sperm competition as a mechanism
of female choice in the field cricket, Grllus bimaculatus.
Beha. Ecol. Sociobiol. 21, 197–202.
Simmons, L. W., Craig, M., Llorens, T., Schinzig, M. &
Hosken, D. 1993 Bushcricket spermatophores vary in
accord with sperm competition and parental investment
theory. Proc. R. Soc. Lond. B 251, 183–186.
Simmons, L. W. & Parker, G. A. 1992 Individual variation
in sperm competition success of yellow dungflies, Scatophaga
stercoraria. Eolution 46, 366–375.
Simmons, L. W. & Siva-Jothy, M. 1997 Sperm competition
Phil. Trans. R. Soc. Lond. B (1997)
P. A. Cook and others 779
in insects : mechanisms and the potential for selection. In
Sperm competition and sexual selection (ed. T. R. Birkhead &
A. P. Møller), London : Academic Press. (In the press.)
Smith, R. L. 1984 Sperm competition and the eolution of animal
mating sstems. London : Academic Press.
Sokal, R. R. & Rohlf, F. J. 1995 Biometr. New York :
Freeman.
Sugawara, T. 1979 Stretch reception in the bursa copulatrix
of the butterfly, Pieris rapae cruciora, and its role in
behaviour. J. comp. Phsiol. 130, 191–199.
Sva$ rd, L. & McNeil, J. N. 1994 Female benefit, male risk :
Polyandry in the true armyworm Pseudaletia unipuncta.
Beha. Ecol. Sociobiol. 35, 319–326.
Sva$ rd, L. & Wiklund, C. 1989 Mass and production rate of
ejaculates in relation to monandry}polyandry in butterflies. Beha. Ecol. Sociobiol. 24, 395–402.
Tschudi-Rein, K. & Benz, G. 1990 Mechanisms of sperm
transfer in female Pieris brassicae (Lepidoptera : Pieridae).
Ann. ent. Soc. Am. 83, 1158–1164.
Tsubaki, Y. & Yamagishi, M. 1991 ‘ Longevity ’ of sperm
within the female of the melon fly, Dacus cucurbitae
(Diptera : Tephriditae), and its relevance to sperm
competition. J. Insect Beha. 4, 243–250.
Waage, J. K. 1979 Dual function of the damsel fly penis :
sperm removal and transfer. Science 203, 916–918.
Walker, W. F. 1980 Sperm utilization strategies in nonsocial
insects. Am. Nat. 115, 780–799.
Wedell, N. 1991 Sperm competition selects for nuptial
feeding in a bushcricket. Eolution 45, 1975–1978.
Wedell, N. 1993 Spermatophore size in bushcrickets :
Comparative evidence for nuptial gifts as sperm protection
device. Eolution 47, 1203–1212.
Receied 22 October 1996 ; accepted 11 December 1996
APPENDIX 1
Two equations have been published to incorporate
the correction factors when using the sterile male
technique to calculate P (Boorman & Parker 1976 ;
#
Sille! n-Tullberg 1981). Boorman and Parker (1976)
showed that from the sterile–normal and normal–
sterile matings, the proportion of eggs that hatch (x) is
adjusted using the fertility after two normal matings
(p) and two sterile matings () to give the proportion of
eggs fertilized by the irradiated male (PR) :
E
01
01
x
p
n
p
1®
p
G
1®
0 1
x
PR ¯ 1®
p
F
.
(A 1)
H
Sille! n-Tullberg (1981) published an equation where
the mean proportion of eggs fertilized by the normal
male (PN) is calculated by :
PN ¯
(x®)
.
( p®)
(A 2)
The eggs not fertilized by the normal male are
attributed to the sterile male, so PR (the proportion of
eggs fertilized by the sterile male) therefore will be
(from equation (A 2)) :
(x®)
.
( p®)
PR ¯ 1®
(A 3)
780
P. A. Cook and others Variation in sperm precedence
It can be shown that equations (A 1) and (A 3) are
algebraically equivalent. By substituting the terms x}p
and }p from (A 1) for e and f respectively, equation
(A 1) becomes :
(1®e)
PR ¯ (1®e)f
(1®f )
(A 4)
and simplifies to :
(1®e)
PR ¯
(1®f )
Phil. Trans. R. Soc. Lond. B (1997)
0Pp 1 ¯ 1®xp
PR®
R
(A 5)
(A 8)
This is the same as :
PR ( p®) ¯ ( p®)®(x®)
(A 6)
(A 7)
which, when multiplied by p becomes :
PR ( p®) ¯ p®x
and so :
PR (1®f ) ¯ 1®e.
Substituting e and f back to x}p and }p it can be
shown that :
which rearranges to form equation (A 3).
(A 9)