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Sperm number trumps sperm size
in mammalian ejaculate evolution
Stefan Lüpold† and John L. Fitzpatrick‡
Research
Cite this article: Lüpold S, Fitzpatrick JL.
2015 Sperm number trumps sperm size
in mammalian ejaculate evolution. Proc. R. Soc.
B 282: 20152122.
http://dx.doi.org/10.1098/rspb.2015.2122
Received: 3 September 2015
Accepted: 23 October 2015
Subject Areas:
evolution
Keywords:
postcopulatory sexual selection, ejaculate
investment, sperm dilution hypothesis,
metabolic constraint hypothesis,
body size, meta-analysis
Author for correspondence:
Stefan Lüpold
e-mail: [email protected]
†
Present address: Department of Evolutionary
Biology and Environmental Sciences, University
of Zurich-Irchel, Winterthurerstrasse 190,
8057 Zurich, Switzerland.
‡
Present address: Department of Zoology/
Ethology, Stockholm University, Svante
Arrhenius väg 18B, Stockholm 10691, Sweden.
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2015.2122 or
via http://rspb.royalsocietypublishing.org.
Computational and Evolutionary Biology, Faculty of Life Sciences, University of Manchester,
Manchester M13 9PT, UK
SL, 0000-0002-5069-1992
Postcopulatory sexual selection is widely accepted to underlie the extraordinary diversification of sperm morphology. However, why does it favour
longer sperm in some taxa but shorter in others? Two recent hypotheses
addressing this discrepancy offered contradictory explanations. Under the
sperm dilution hypothesis, selection via sperm density in the female reproductive tract favours more but smaller sperm in large, but the reverse in small,
species. Conversely, the metabolic constraint hypothesis maintains that ejaculates respond positively to selection in small endothermic animals with high
metabolic rates, whereas low metabolic rates constrain their evolution in
large species. Here, we resolve this debate by capitalizing on the substantial
variation in mammalian body size and reproductive physiology. Evolutionary
responses shifted from sperm length to number with increasing mammalian
body size, thus supporting the sperm dilution hypothesis. Our findings
demonstrate that body-size-mediated trade-offs between sperm size and
number can explain the extreme diversification in sperm phenotypes.
1. Introduction
Female promiscuity can cause sperm from different males to compete to fertilize a
female’s limited supply of eggs [1]. The resulting selection on ejaculates to maximize fertilization success is viewed as the leading factor driving the dramatic
diversification of sperm size and shape, as well as ejaculate traits more broadly
(reviewed in [2]). Sperm competition theory initially focused on sperm numbers,
proposing that males transferring more sperm gained a numerical competitive
advantage analogous to buying more raffle tickets [3–5]. Within finite resources
allocated to sperm production, however, investing in greater sperm numbers can
be achieved only by reducing sperm size. This trade-off between sperm size and
number soon became integral to sperm competition models, including those
explaining the evolution of numerous tiny sperm relative to the fewer and usually
much larger ova [3,6], which is ultimately also an important contributor to the
evolution of mating systems [7–9].
Although sperm competition was predicted to favour smaller but more
numerous spermatozoa, there is accumulating evidence that longer sperm may
also result in fitness benefits and thus be under directional selection, resulting
in longer sperm in species experiencing relatively high levels of sperm competition (e.g. [10–16]). To address the inconsistent directions of sperm evolution
in response to sperm competition among taxa (see [17–20] for reviews), two
hypotheses were recently proposed. First, the ‘sperm dilution hypothesis’ states
that although males under intense sperm competition should always invest
more in gametes, the strength of selection on sperm size depends on the sperm
density prior to fertilization [21,22]. As the limits of sperm production are
approached at high levels of sperm competition (e.g. [23,24]), trade-offs between
sperm size and number are expected to generate stronger selection on sperm size
when dilution effects are low and sperm actively interact with one another [21,22],
such as in small organisms where the high sperm density within a relatively small
reproductive tract often results in sperm displacement [25–27]. However, tradeoffs result in stronger selection on sperm number when dilution effects are high
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
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2. Material and methods
(a) Data collection and study taxa
We compiled species-specific values of sperm length, total
number of sperm in ejaculates, combined testes mass, and male
and female body masses of 100 mammalian species from the
literature (electronic supplementary material, table S1). We
used only sperm counts from samples that were obtained by electroejaculation or as natural ejaculates (for details see [37]) and
omitted any samples collected from the epididymes as these
are less likely to reflect natural ejaculates. In some cases, the
total sperm number was not provided but it could be calculated
as the product of ejaculate volume and sperm concentration.
(b) Statistical analyses
We conducted all analyses using the statistical package R v. 3.2
(R Core Team 2015) and transformed all data by logarithmic
transformations to meet the parametric requirements of the
2
Proc. R. Soc. B 282: 20152122
m*/s* ratio at a higher rate with greater body size and thus
strengthens its negative relationship with the measure of
sperm competition [21]. Under the metabolic constraint
hypothesis, however, we predicted that the strength of the
relationship between m*/s* and sperm competition should
not change systematically with body size. This is because of
the assumption underlying this hypothesis that the high
resource turnover and efficient sperm production allows
small species to increase both sperm size and number in
response to selection, and both traits are increasingly constrained in their response to selection as body size increases
[24,28]. Thus, although selection may consistently favour one
trait over the other, leading to a bias towards either m* or s*
within any body-size category, the m*/s* ratio itself is not predicted to covary more tightly with sperm competition levels
among large-bodied compared to smaller bodied species.
Using these contrasting predictions between the two
hypotheses, we evaluated the evolution of sperm size and
number in mammals and tested whether the observed macroevolutionary patterns supported the sperm dilution or the
metabolic constraint hypothesis. The extraordinary variation
in body size in this taxon, spanning six orders of magnitude
in our study, makes mammals a powerful system to test
whether the response of ejaculates to sperm competition
changes with female body size, which we use as a proxy of
the female reproductive tract, given that the different parts
of the female reproductive tract strongly covary with body
size [33,34] (also see §2b). Additionally, the mammalian
phylogeny is well resolved (e.g. [35,36]) and their reproductive physiology and ejaculate traits are well characterized
[14,37]. Finally, there has been a long-standing debate about
the variation in sperm size in response to sperm competition
among mammals, with conflicting results between studies
depending on the combination of species examined
[14,38–40]. This ambiguity in evolutionary responses to
sperm competition may in part be due to unresolved impacts
of body-size-dependent relationships between gamete
investment and sperm competition levels.
Across the large sample of mammalian species examined,
our results support the sperm dilution, not metabolic constraint hypothesis, and demonstrate that body size-mediated
trade-offs between sperm size and number can explain the
extreme diversification in sperm phenotypes.
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(i.e. in large organisms where sperm are diluted in the female’s
reproductive tract and thus at relatively low densities [21,22]).
Alternatively, the ‘metabolic constraint hypothesis’ argues that
sperm size, and gamete investment generally, responds to
selection more intensely in species with higher mass-specific
and cellular metabolic rates, based on the assumption that in
these species resource processing of fast-dividing cells like
germ cells is more efficient, resulting in enough resources to
increase sperm size and/or number when under selection
[24,28,29]. Following this hypothesis, the metabolic constraints
on evolving longer and/or more abundant sperm should be
relatively low in small-bodied endothermic animals with
high metabolic rates, but high in large-bodied species with relatively low metabolic rates [24,28]. If so, both sperm size and
number would be predicted to covary strongly positively
with the level of sperm competition in small-bodied species
but to be independent of, or only weakly associated with,
sperm competition in larger bodied species owing to the stronger constraint on their evolution. Consequently, whereas both
hypotheses suggest that the strength of selection acting on
sperm size is ultimately dependent on body size, their predictions regarding the effect of body size on gamete investment
are fundamentally different.
Here, we aimed to resolve the debate between the sperm
dilution and the metabolic constraint hypotheses and to
assess their applicability in explaining the macroevolutionary
variation in sperm size by assessing it jointly with variation
in sperm number. Using Parker et al.’s [21] terminology, we
examined the relationship between sperm competition and
both total gamete investment (m*s*; i.e. product of sperm
size m* and sperm number s*) and relative investment in
sperm size and number (m*/s*), respectively. Examining the
response of these combined traits to sperm competition along
the body-size spectrum targets the contrasting predictions
between the two hypotheses to address our goal.
The sperm dilution hypothesis, under which m*s* is
driven almost exclusively by variation in sperm number
[21,22], predicts a positive relationship between m*s* and
relative testes size (as a proxy of sperm competition [30,31])
for all body sizes, but this relationship should become stronger with increasing body size because sperm number is
under intense selection by both sperm competition and a
growing risk of sperm loss or dilution in the longer and
more voluminous female reproductive tract of larger species
[32]. By contrast, to support the metabolic constraint hypothesis, we predicted that the relationship between m*s* and
sperm competition should be strong in small species due to
their high propensity to increase both ejaculate traits in
response to sperm competition and low metabolic constraints,
but it should become weaker in larger species where lower
metabolic rates and less efficient sperm production are
thought to constrain their ability to increase sperm size and
number [24,28].
For the relative investment in sperm size and number,
expressed by the m*/s* ratio, the sperm dilution hypothesis
predicts that selection acting primarily on sperm number
(i.e. raffle-like sperm competition) should result in a negative
relationship between m*/s* and sperm competition risk, and
this relationship should gradually become more strongly
negative with any incremental increase in body size. This is
due to the intense selection on sperm number by both
sperm competition and the risk of sperm loss, which accelerates the interspecific variance in sperm number, s*, lowers the
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Table 1. Phylogenetically controlled associations between different measures of gamete investment and relative testes mass across 100 mammalian species.
(The partial correlation coefficients r are presented with their lower (LCL) and upper (UCL) non-central 95% confidence limits.)
partial r (LCL, UCL)
t
p
total sperm number
testes mass
body mass
0.61 (0.47, 0.70)
– 0.08 ( – 0.27, 0.12)
7.52
– 0.80
,0.0001
0.42
total sperm length
testes mass
body mass
0.04 ( – 0.15, 0.23)
– 0.17 ( – 0.35, 0.02)
0.42
– 1.74
0.68
0.09
0.900.003,0.17
total gamete investment (m*s*)
testes mass
0.61 (0.48, 0.71)
7.62
,0.0001
,0.0010.80,,0.001
relative investment (m*/s*)
body mass
testes mass
– 0.15 ( – 0.33, 0.06)
– 0.59 ( – 0.69, – 0.44)
– 1.44
– 7.11
0.15
,0.0001
0.050.57,,0.001
body mass
0.01 ( – 0.19, 0.20)
statistical models. These transformations also helped to avoid the
problematic use of ratios in phylogenetic models in the case of
the ratio between sperm size and number, because ln(sperm
size : sperm number) is equivalent to ln(sperm size) – ln(sperm
number). To statistically account for shared ancestry among
species, we conducted phylogenetic generalized least-squared
(PGLS) regressions [41,42], based on the same molecular phylogenies as in previous large-scale studies on the traits and taxa
used [14,37] (electronic supplementary material, figure S1).
PGLS models estimate the phylogenetic scaling parameter l to
evaluate the phylogenetic relationship of the covariance in the
residuals [41]. Using likelihood ratio tests, we established whether
the models with the maximum-likelihood value of l differed from
models with values of l set to 0 or 1, respectively. Values of l close
to 0 indicate phylogenetic independence and those close to 1
suggest a strong phylogenetic association of the traits [41].
Throughout this paper, we present l values with their associated
p-values of these likelihood ratio tests in superscripts (first against
l ¼ 0, second against l ¼ 1).
To examine the relationship between ejaculate traits and
sperm competition levels, we conducted phylogenetic multiple
regressions as described above, using combined testes mass as
the predictor and male body size as a covariate to account for
allometric effects [43,44]. Relative testes mass is a widely used
proxy of sperm competition levels as it covaries positively with
indices of multiple paternity in various taxa, including mammals
[30]. To better illustrate shifts in m*s* or m*/s* in relation to
female body mass (as a proxy of the size of the reproductive
tract; see below), we used a sliding-window approach [45]. In
brief, we fitted the above multiple PGLS models within a sliding
window that was four units wide on the scale of ln(female body
mass) and then slid the window along this scale from the smallest to the largest female body masses at increments of 1.5 units.
For each such window, we calculated the partial correlation coefficient between combined testes mass and either m*s* or m*/s*
and transformed it to Zr using Fisher’s transformation, weighted
by sample size [46]. We then plotted these Zr-values against the
mean female body mass calculated for each sliding window.
Finally, to confirm the consistency and robustness of these
analyses, we repeated them using different window sizes and
starting points (electronic supplementary material, figure S2).
We used female body mass as a proxy of the size of the reproductive tract because not enough data were available to use the
reproductive tract size directly. However, restricting Anderson
et al.’s [34] data on oviduct length and female body mass to the
taxa used in our study (i.e. excluding Marsupialia and Chiroptera)
revealed a highly significant positive relationship between the two
traits (n ¼ 39; r ¼ 0.87, p , 0.0001; l , 0.00011.0,,0.001), with an
allometric slope between oviduct length and the body mass
(cube-root-transformed for equal dimensionality) of 0.79 (95%
0.10
0.050.64,,0.001
0.92
confidence interval: 0.66– 0.94). Whereas female body mass covaries with oviduct length and other components of the female
reproductive tract (also see [33,34]), it is also highly correlated
with the mass-specific metabolic rate [47], thereby providing a
good proxy for the assumptions underlying both the sperm
dilution and metabolic constraint hypotheses.
3. Results
First, we examined the relationship of both sperm length
and sperm number with sperm competition risk, using body
size-corrected testes mass as a proxy measure [17,30,31], in separate phylogenetic multiple regressions across all 100 species.
These analyses revealed that sperm number, but not sperm
length, increased significantly with relative testes mass
(table 1). Additionally, as predicted theoretically [21], across
all 100 mammalian species examined the association between
relative testes mass and the total investment in the cellular component of ejaculates (m*s*) was strongly positive (table 1 and
figure 1a), whereas that with relative investments (m*/s*) was
strongly negative (table 1 and figure 1b). The same patterns
were also apparent within each of the four most speciose
taxa in our dataset (Artiodactyla, Carnivora and Primates,
Rodentia), albeit not significantly so in the Rodentia, which
was limited to only nine species (compared to N 20 in the
other three taxa; electronic supplementary material, table S2).
Next, we determined if the strength of the association
between sperm competition risk and total and relative
gamete investment varied across different body-size categories. The response to sperm competition can be expressed
as the effect size, r, of the relationship between measures of
gamete investment (which dilution effects and metabolism
could constrain) and relative testes mass as a proxy measure
of sperm competition [17,30,31]. Large effect sizes indicate
that gamete investment varies tightly with different degrees
of selection, whereas small effect sizes would suggest a limited
response to variation in sperm competition levels. However, as
effect sizes are influenced strongly by sample size, we converted r values to Fisher’s sample-size-corrected Zr (i.e. using
weighted Fisher’s transformations of r [46]) to facilitate comparisons of responses of total and relative gamete investment
in response to sperm competition risk across the broad range
of mammalian body sizes.
Using standardized Zr values of the relationships
between either m*s* or m*/s* and relative testes mass, we
Proc. R. Soc. B 282: 20152122
predictor
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l
response
3
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(a)
(a)
3
4
40
55
0
–1
–2
–3
49
20
30
16
10
11
0
(b) 4
(b)
3
weighted effect size Zr (m*/s*)
–4
2
residual m*/s*
30
Proc. R. Soc. B 282: 20152122
weighted effect size Zr (m*s*)
residual m*s*
1
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61
2
1
0
–1
–2
0
–5
–10
16
11
30
–15
–20
49
–25
–30
61
55
–3
–2
–1
0
1
residual testes mass
2
Figure 1. Partial regression plots showing the relationships across 100 mammalian species of (a) the total gametic investment ( product of sperm length
and number) and (b) the relative gametic investment (sperm length/sperm
number) with testes mass after removing variation in body size from both
variables.
employed a sliding-window approach [45] to determine how
the strength of these associations varied across different
body-size categories, using female body mass as a proxy of
female reproductive tract size (for details see §2b). Comparing the calculated Zr-values between different body size
categories allowed us to estimate size-dependent responses
to selection in order to test specific predictions that can distinguish between the two major hypotheses (§1). These
analyses revealed that the relationships between relative
testes mass and either m*s* or m*/s* became stronger, albeit
in opposing directions, with increases in female body mass
(figure 2). These results highlight that the total ejaculate
investment increases with sperm competition risk and body
size, but that the relative investment in sperm number becomes
stronger compared to that in sperm length as species increase
in size, consistent with the predictions from the sperm dilution
hypothesis [21,22]. Since the unequal distribution of species
along the body-size spectrum rendered these analyses sensitive
to changes in both the width of the sliding window and the
position of the first window (which then determined the cutoffs for each of the following windows), we repeated these analyses with varying window sizes and starting points and found
largely consistent patterns regardless of the parameter settings
(electronic supplementary material, figure S2).
To test whether the size-dependent variation in the
relationship of m*s* or m*/s* with relative testes size was
5
6
7
8
9
10
ln mean female body mass
11
Figure 2. Sliding-window analysis expressing, for each window, ln(mean
female body mass) and the weighted Zr-value as the sample size-controlled
effect size of a relationship between relative testes mass and (a) the total
ejaculate investment (m*s*), or (b) the relative investment between sperm
size and number (m*/s*). These Zr-values reflect the strength of the examined relationship within each window (the farther away from zero, the
stronger the relationship). For each window, we restricted the dataset to a
range of body sizes spanning four units along the x-axis and moved the
window along this axis at intervals of 1.5 units. The first window had its
midpoint at 4.3, the last at 11.8. Numbers above points indicate the
number of species falling into each window. All p-values of the relationships
underlying each data point were p , 0.001 except for the one labelled with
11 in (a) ( p ¼ 0.015) and the one labelled with 16 in (b) ( p ¼ 0.056).
driven solely by sperm number, given that sperm length was
not associated with size-controlled testes mass across all
100 species (table 1), we also applied the sliding-window
approach to both ejaculate traits separately. We found sperm
number to covary positively with relative testes mass within
each body-size category (all p 0.01), but more tightly so
among larger bodied species (electronic supplementary
material, figure S3a). The corresponding associations with
sperm length were statistically significant only within the
second-smallest size category ( p ¼ 0.02), and a negative
trend between the weighted Zr values and mean female
body mass was apparent (electronic supplementary material,
figure S3b). We confirmed this trend across the much
larger dataset of sperm length in Tourmente et al.’s [14] study
(n ¼ 226 eutherian mammal species; electronic supplementary
material, figure S4). These separate results combined suggest
that the size-dependent shifts in m*s* or m*/s* reported
above are not the result of increasing variation in sperm
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4. Discussion
Data accessibility. All data used in this paper have been archived in the
Dryad repository (http://dx.doi.org/10.5061/dryad.h0246).
Authors’ contributions. Both authors conceived the study, S.L. collected
and analysed the data, and both authors wrote the manuscript.
Competing interests. All authors declare that they have no competing
financial interest.
Funding. We received no funding for this study.
5
Proc. R. Soc. B 282: 20152122
Across our sample of eutherian mammals, we found greater
total investment in the gametic fraction of ejaculates in
response to increasing sperm competition risk. However,
increases in sperm number in response to sperm competition
were stronger than increases in sperm size. Furthermore, the
strength of these effects increased with female body size, and
thus likely with the size of the female reproductive tract
[33,34]. These results offer robust support for the sperm
dilution hypothesis [21,22,32], suggesting that in large species
the marginal benefits of transferring greater sperm numbers
outweigh those of transferring longer sperm to a greater
extent than in smaller species.
In their comparison of passerine birds and fruit flies,
Immler et al. [22] illustrated that high sperm density within
the female reproductive tract of the small-bodied flies increases
the benefits of sperm size due to direct competition for storage
and sperm displacing one another [25–27], whereas selection is
stronger on sperm number than on sperm size in the birds, in
which the mechanism of sperm competition is more rafflelike [22,48] (also see [49] for a similar pattern in mammals).
Our meta-analysis corroborates and expands these findings by showing continuous size-dependent variation in the
importance of sperm number over that of sperm size. With
increasing mammalian body size, incremental increases in
sperm competition risk were tightly associated with a shift
towards investing more in sperm number rather than sperm
length. Thus, among mammals, selection is stronger on
sperm number in large-bodied compared to small-bodied
species. Interestingly, it is among these smaller species where
more complex sperm morphology is observed, including
apical hooks or other processes protruding from the sperm
heads in various murid rodents, for example, which are
thought to facilitate sperm cooperation and sperm transport
[50,51]. It is thus plausible that the smaller body size increases
the density of sperm relative to the female reproductive tract
and lowers the risk of sperm loss, thereby creating an environment where variation in sperm morphology (or sperm quality
in general) can explain a relatively greater proportion of overall
ejaculate competitiveness. Consistently, several sperm-quality
traits that covaried positively with the level of sperm competition also decreased with body size, whereas sperm number
tended to increase, albeit not significantly so, in a previous
study across a broad range of mammalian species [37].
Additionally, in our separate analyses of sperm length and
number, the association with the level of sperm competition
across different body-size categories decreased for sperm
length but increased for sperm number, indicating that selection on sperm length is likely to become weaker as that on
sperm number increases. These findings corroborate the
general trends for the total and relative gametic investment
(above) and highlight the increasing importance of sperm
number in response to counteract the risk of sperm dilution
or loss as body size increases.
Our results do not dismiss the importance of sperm morphology or sperm quality, even for relatively large mammals
where sperm quality has been shown to influence male fertility
(e.g. Iberian red deer Cervus elaphus hispanicus [52]). However,
sperm-quality effects are likely to be more subtle and
manifested mainly after controlling for sperm numbers (also
see [52]). Superior quality may help sperm traverse the challenging environment of the female reproductive tract faster and
thus in greater numbers [53,54], and it may be critical once
sperm compete for fertilization near the ovum. Yet, sperm
dilution and sperm loss are likely to be chance effects, thereby
raising the importance of sperm number where greater distance has to be covered. For example, in humans, a mere
0.004% (approx. 250 sperm) of all motile sperm inseminated
make it past the uterus [55], and it seems likely that the efficiency of sperm transport would vary with the size of the
female reproductive tract.
In contrast to the sperm dilution hypothesis, our joint
examination of sperm size and number variation did not support the metabolic constraint hypothesis. It seems plausible
that the lack of a link between sperm size and sperm competition in large species, which was attributed to a metabolic
constraint [28,29], may simply be the result of relaxed selection
on sperm size due to the comparatively much greater advantage of increasing sperm number. Effects of the metabolic
rate and of body size per se are inherently difficult to separate.
In fact, the mass-specific metabolic rate (basal metabolic rate
divided by body mass), as used in the tests of the metabolic
constraint hypothesis [24,28,29], does not remove the effect of
body size from the metabolic rate (e.g. [56]). Across mammals,
approximately 90% of the mass-specific metabolic rate is
explained by body size variation alone [47], so its relationship
with sperm length largely reflects an inverted association of
sperm length with body mass. Consequently, it is possible
that the metabolic rate and the intensity of selection on
sperm size independently vary with body size.
In conclusion, we document size-dependent and gradual
shifts in ejaculate allocation, with large species exhibiting
stronger selection on sperm number than on sperm size compared to smaller species. This gradual change in the relative
investment in ejaculate traits supports the sperm dilution
hypothesis, but is not consistent with the metabolic constraint
hypothesis, thereby resolving the debate between these two
competing explanations of how body size affects patterns of
sperm evolution. Additionally, Parker [57] recently proposed
an extensive theoretical framework explaining how changes
in the selective environment, associated with stepwise
transitions from simple sperm-broadcasting life forms to complex and mobile organisms with internal fertilization, have
shaped the evolutionary dynamics of, and selection on,
gamete investment throughout animal history. In this context,
our findings add an important piece to Parker’s [57] predictions and have broad implications for our understanding of
ejaculate evolution by showing how variation in body size
affects the selective environment in internal fertilizers and
thus the trajectory of sperm size evolution.
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number alone, with the effect of sperm competition on sperm
length being invariably weak across all body size categories.
Rather, the strength of the relationship between sperm length
and relative testes mass tended to decline gradually with
increasing body size while it became stronger for sperm
number, as predicted by the sperm dilution hypothesis [21,22].
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Acknowledgements. We thank O. Garcia Alvarez, R. Martins Crivelaro,
B. Pukazhenthi and A. Schulte-Hostedde for providing unpublished
data, and S. Immler, R. Montgomerie and G. Parker for helpful
comments on the manuscript.
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