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ORIGINAL PAPER
Variation in sperm morphometry and sperm competition
among barn swallow (Hirundo rustica) populations
Terje Laskemoen &Tomas Albrecht &Andrea Bonisoli-Alquati &
Jaroslav Cepak &Florentino de Lope &Ignacio G. Hermosell &
Lars Erik Johannessen &Oddmund Kleven &Alfonso Marzal &
Timothy A. Mousseau &Anders P. Møller &
Raleigh J. Robertson &Geir Rudolfsen &
Nicola Saino &Yoni Vortman &Jan T. Lifjeld
Received: 7 September 2012 /Revised: 2 November 2012 / Accepted: 7 November 2012 / Published online: 18 November 2012
#Springer-Verlag Berlin Heidelberg 2012
Abstract Spermatozoa vary greatly in size and shape among
species across the animal kingdom. Postcopulatory sexual
selection is thought to be the major evolutionary force driving
this diversity. In contrast, less is known about how sperm size
varies among populations of the same species. Here, we
investigate geographic variation in sperm size in barn swal-
lows Hirundo rustica, a socially monogamous passerine with
a wide Holarctic breeding distribution. We included samples
Communicated by S. Pruett-Jones
Electronic supplementary material The online version of this article
(doi:10.1007/s00265-012-1450-0) contains supplementary material,
which is available to authorized users.
T. Laskemoen (*):L. E. Johannessen :O. Kleven :J. T. Lifjeld
Natural History Museum, University of Oslo,
P.O. Box 1172(Blindern, 0318 Oslo, Norway
e-mail: terje.laskemoen@nhm.uio.no
T. Albrecht
Institute of Vertebrate Biology, v.v.i., Academy of Sciences of the
Czech Republic, Brno, Czech Republic
T. Albrecht
Department of Zoology, Faculty of Science, Charles University in
Prague, Prague, Czech Republic
A. Bonisoli-Alquati :T. A. Mousseau
Department of Biological Sciences, University of South Carolina,
Columbia, SC, 29208, USA
J. Cepak
Bird-ringing Station, National Museum, Prague,
Czech Republic
F. de Lope :I. G. Hermosell :A. Marzal
Departamento de Anatomia, Biologia Celular y Zoologia,
Universidad de Extremadura, Badajoz, Spain
O. Kleven
Norwegian Institute for Nature Research-NINA,
P. O. Box 5685(Sluppen, 7485 Trondheim, Norway
A. P. Møller
Laboratoire d’Ecologie, Systématique et Evolution, CNRS UMR
8079, Université Paris-Sud,
Orsay Cedex, France
R. J. Robertson
Department of Biology, Queen’s University, Kingston,
Ontario, K7L 3N6, Canada
G. Rudolfsen
Department for Environmental Radioactivity, Norwegian
Radiation Protection Authority, Fram Center,
9296 Tromsø, Norway
N. Saino
Dipartimento di Biologia, Universit’a degli Studi di Milano,
via Celoria 26,
20133 Milano, Italy
Y. Vortman
Department of Zoology, Faculty of Life Sciences,
Tel-Aviv University,
Ramat Aviv, Tel-Aviv, 69978, Israel
Behav Ecol Sociobiol (2013) 67:301–309
DOI 10.1007/s00265-012-1450-0
from seven populations and three subspecies: five populations
of ssp. rustica in Europe (Czech, Italy, Norway, Spain, and
Ukraine), one population of ssp. transitiva in Israel, and one
population of ssp. erythrogaster in Canada. All sperm traits
(head length, midpiece length, tail length, and total length)
varied significantly among populations. The variation among
the European rustica populations was much lower than the
differences among subspecies, indicating that sperm traits
reflect phylogenetic distance. We also performed a test of
the relationship between the coefficient of between-male var-
iation in total sperm length and extrapair paternity levels
across different populations within a species. Recent studies
have found a strong negative relationship between sperm size
variation and extrapair paternity among species. Here, we
show a similar negative relationship among six barn swallow
populations, which suggests that the variance in male sperm
length in a population is shaped by the strength of stabilizing
postcopulatory sexual selection.
Keywords Barn swallow .Extrapair paternity .Hirundo
rustica .Sperm competition .Sperm size
Introduction
Spermatozoa show enormous variation in size and shape
across animal taxa (e.g., Cohen 1977). The evolutionary
forces shaping this variation have received increasing atten-
tion but are not fully understood (Birkhead et al. 2009).
Polyandry, i.e., females mating with more than one male,
is common across the animal kingdom, and this constitutes a
potentially powerful source of postcopulatory sexual selec-
tion on sperm traits, either through sperm competition
(Parker 1970) or cryptic female choice (Eberhard 1996).
It is well documented that spermatozoa vary considerably
in size both within (e.g., Ward 1998; Morrow and Gage
2001b; Laskemoen et al. 2007) and among species (e.g.,
Gage 1998; Calhim et al. 2007; Kleven et al. 2008; Lüpold
et al. 2009). Although geographical variation in sperm size
within species has been studied in a range of species, the
patterns are complex and not clear. For example, in guppies
(Poecilia reticulata), Elgee et al. (2010) found that males
from populations with high risk of predation had faster
swimming sperm and sperm with longer midpieces than
males from populations with lower risk of predation.
However, total sperm length did not differ between guppy
populations (Elgee et al. 2010). Studies of several droso-
philid flies have found significant variation in sperm size
among populations (Snook 2001; Pitnick et al. 2003; Joly et
al. 2004). Yellow dung flies (Scatophaga stercoraria) have
been shown to have longer sperm at high temperature when
raised experimentally in laboratory conditions (Blanckenhorn
and Hellriegel 2002). However, another study of the same
species did not find differences in sperm length in three natural
populations (Hosken et al. 2003). A comparative study of
sperm morphology of rhacophorid frogs included samples of
the species (Chirixalus eiffingeri) from both Japan and Taiwan
and documented significant differences in sperm size between
these two populations (Kuramoto 1996). A study of quacking
frogs (Crinia georgiana) documented significant variation in
sperm size and relative number of sperm among four popula-
tions (Hettyey and Roberts 2006). The sea urchin
(Strongylocentrotus droebachiensis) was found to have high
diversity in sperm traits, except total sperm length, among
three different populations (Manier and Palumbi 2008).
Minoretti and Baur (2006) documented significant differences
in sperm size among four populations of the land snail
(Arianta arbustorum). In birds, two recent studies have inves-
tigated geographic variation in sperm size, both documenting
significant differences among populations (Lüpold et al. 2011;
Schmoll and Kleven 2011 ). In red-winged blackbirds
(Agelaius phoeniceus), sperm morphology varied significant-
ly among the 17 study sites in continental United States
(Lüpold et al. 2011). Lüpold et al. (2011) also found a gradual
increase in sperm size from southwest to northeast of the
breeding range, and further, a negative relationship between
sperm length and body size. Schmoll and Kleven (2011)
investigated variation in sperm size within and between two
populations of coal tits (Periparus ater) and documented
significant variation in total sperm length and sperm head
length between the two populations. These two species are
characterized by little or moderate gene flow, which could
help explain geographical differences in sperm traits with high
heritability. A call is thus made for investigation of variation in
sperm size among populations in species with a higher degree
of gene flow among populations.
Recently, comparative studies of passerine birds have
shown that the intraspecific variation in total sperm length
and other sperm components are negatively associated with
the risk of sperm competition (Calhim et al. 2007; Immler et
al. 2008; Kleven et al. 2008; Lifjeld et al. 2010). This can be
interpreted as evidence of stronger stabilizing selection on
males producing an optimal sperm type when the risk of
sperm competition increases. However, this relationship has
not been investigated among populations of the same spe-
cies. It is well known that levels of extrapair paternity vary
among populations (Petrie and Kempenaers 1998), and in
this study, we ask if this variation amongst populations in
sperm competition could also be reflected in between-male
variation in sperm size.
The barn swallow (Hirundo rustica) is a socially monog-
amous passerine with a broad Holarctic breeding distribu-
tion (Møller 1994b; Turner 2006). The barn swallow has
been thoroughly studied over the last decades and is con-
sidered a model species for studies on sexual selection, mate
choice, and sperm competition (e.g., Møller 1988; Møller
302 Behav Ecol Sociobiol (2013) 67:301–309
1994a; Møller 1994b; Saino et al. 1997; Safran et al. 2005;
Kleven et al. 2006; Lifjeld et al. 2011). Currently, six dif-
ferent subspecies of barn swallows are recognized (Turner
2006; Dor et al. 2010). However, some of these subspecies
are geographically isolated, preventing gene flow (Turner
2006). A recent phylogeny of barn swallows and other
Hirundo species found that the European H. rustica rustica
and East-Mediterranean barn swallows H.rustica transitiva
cluster together, as do the Asian and American barn swal-
lows (Dor et al. 2010). Dor et al. (2010) also reported
pairwise molecular distances within and between these
clades, showing low distance within the European-
Mediterranean clade and substantially greater distance be-
tween the Asian-American and the European-Mediterranean
clades. Furthermore, there is evidence from barcoding of the
mitochondrial cytochrome c oxidase I (CO1) gene showing
that the North American barn swallow H. rustica eryth-
rogaster differs almost 2 % in genetic distance from the
European barn swallow (Johnsen et al. 2010).
Here, we investigate geographic variation in sperm mor-
phometry in barn swallows. We examined sperm morphome-
try in seven different barn swallow populations representing
three subspecies: five populations of European (Czech, Italy,
Norway, Spain and Ukraine), one population of East-
Mediterranean (Israel), and one North American population
(Canada). Based on the prior knowledge of genetic variation
(Dor et al. 2010; Johnsen et al. 2010), we predicted that
European and Mediterranean barn swallows would exhibit
more similar sperm morphometry when compared to North
American barn swallows. We also tested the hypothesis that
sperm size variation is negatively related to the risk of sperm
competition across populations.
Methods
Field procedures
We captured and sampled male barn swallows from seven
different populations; Czech Republic (2009), Italy (2011),
Norway (2008 and 2009), Spain (2010 and 2011), Ukraine
(2010), Israel (2010 and 2011), and Canada (2006). See
supplementary Table S1 for detailed sampling location infor-
mation. Notably, European barnswallows migrate through the
breeding areas of East-Mediterranean barn swallows in Israel,
which could potentially cause sampling errors in this area.
However, all birds sampled in Israel were earlier banded and
belonged to a monitored population of East-Mediterranean
barn swallows. To prevent pseudo-replication of already sam-
pled birds, all unbanded birds were banded with unique alu-
minium bands.We obtained ejaculate samples either by gently
massaging the males' cloacal protuberance following a mod-
ified method from Wolfson (1952) or from fecal samples
following Immler and Birkhead (2005). The ejaculate or fecal
sample was immediately fixed in a 5 % formalin solution and
stored until slide preparation. After sampling, all birds were
released in the immediate vicinity of their respective colonies.
Sperm morphometry
For each sperm sample, a small aliquot of approximately
15 μl was applied on a microscope slide, allowed to air-dry,
and subsequently gently rinsed with distilled water and air-
dried again. We measured the head, midpiece, and tail
(±0.1 μm) of ten intact spermatozoa per male. Measuring
ten sperms per male has been shown to give representative
estimations of an individual's mean sperm length
(Laskemoen et al. 2007). Total length is the sum of all three
separate sperm traits. For one individual, we measured the
same ten sperms twice to establish the repeatability of our
measurements, following Lessells and Boag (1987). The
measurements were highly repeatable (head: F
9,10
06.83,
P00.003, r00.74; midpiece: F
9,10
0114.0, P<0.001, r0
0.98; tail: F
9,10
0105.8, P<0.001, r00.98). For the samples
of North American barn swallows, we used a Zeiss
AxioCam HRc camera mounted on a Zeiss Axioplan 2 light
microscope to obtain digital images of spermatozoa at a
magnification of 200×. Further, the morphometric measure-
ments were conducted using the software Zeiss AxioVision
4.1 (© Carl Zeiss Vision GmbH, Germany). For all other
samples, we used a Leica DFC420 camera mounted on a
Leica DM6000 B digital light microscope to obtain digital
images at magnifications of 200×. The morphometric meas-
urements were conducted using Leica Application Suite
(version 2.6.0 R1). The use of two different microscope
setups could potentially cause artificial differences. In order
to establish whether the different microscope systems gave
different results, we measured ten randomly chosen individ-
uals of the North American barn swallows on the Leica
microscope system as well. No significant differences were
observed (average head length, 14.3 vs. 14.3 μm; average
midpiece length, 59.8 vs. 59.9 μm; average tail length, 14.4
vs. 14.2 μm, Zeiss vs. Leica systems, respectively; paired t
tests: all t<0.57, all P>0.58). To avoid observer effects, one
person (TL) conducted all morphometric measurements. In
an earlier study of sperm characteristics of Ukrainian barn
swallows, Møller et al. (2008) found differences in both sperm
size and proportion of abnormal sperm between birds sampled
near the Chernobyl area and those sampled at control areas
southwest of Chernobyl. Therefore, we tested for differences
in sperm traits between the four Ukrainian localities in the
present dataset. We found no significant differences in any of
the measured sperm traits (ANOVA, all F
3,21
<1.46, all P>
0.25). Hence, we pooled all individuals from Ukraine as one
population. Notably, the Ukrainian samples included in the
present study are not the same as in Møller et al. (2008).
Behav Ecol Sociobiol (2013) 67:301–309 303
Extrapair paternity data
We extracted data on extrapair paternity from five populations
from the literature (Canada, Italy, Israel, Spain, and Ukraine).
In addition, we obtained unpublished data on extrapair pater-
nity from the Czech population (T. Albrecht, J. Kreisinger, and
R. Michalkova unpublished data). Notably, all extrapair pa-
ternity data originate from the same populations from which
we obtained sperm samples. A summary of paternity data and
sample sizes are presented in Table 1.
Statistical analyses
We included all sperm measurements, i.e., ten sperms per
male, and used General Linear Mixed Models (GLMM) with
population as fixed factor and individual as random factor in
our tests for differences in sperm traits among populations.
For the traits that showed significant variation among popu-
lations, we estimated marginal means and conducted all pos-
sible pairwise comparisons, Bonferroni-adjusted for multiple
comparisons. We calculated the coefficient of variation in
sperm length between males (CV
bm
) for all populations using
the formula: CV
bm
0SD/mean×100. Furthermore, as the co-
efficient of variation for small sample sizes tends to be under-
estimated, weapplied the formula recommended by Sokal and
Rohlf (1995): CV
adj
0CV×(1 + (1/4n)). Hence, all CV
bm
val-
ues reported here are adjusted for sample size. Statistical tests
were conducted using Statistica v7.1 (StatSoft Inc), SPSS
v19.0.0 (SPSS Inc.), and figures were drawn using Origin
v7.0300 (OriginLab Corporation).
Results
Sperm morphometry
Sperm head length, midpiece length, and total length dif-
fered significantly among populations (GLMMs: head
length: F
6, 183
017.4, P<0.001; midpiece length: F
6, 183
0
15.9, P<0.001; total length: F
6, 183
010.3, P<0.001). Thus,
for these traits, we estimated marginal means and pair-
wise comparisons to reveal which populations differed
(Table S2–S4). Descriptive statistics of sperm morphom-
etry from all seven populations are presented in Table 2.
Sperm tail length did not differ significantly among
populations (F
6, 183
02.0, P00.07 ). Generally, the
North American barn swallows differed most from the
other populations in sperm morphometry, with longer
sperm heads than individuals from all the other popula-
tions and shorter midpieces than all but the Italian and
East-Mediterranean populations (Fig. 1a, b;TableS2,
S3). Considering total sperm length, North American barn
swallows generally had shorter sperm than the European
populations but did not differ from the population of East-
Mediterranean barn swallows (Fig. 1c; Table S4).
Two of our populations (Norway and Spain) included
samples from two different seasons, and thus we tested for
potential year-effects on sperm size. However, we found no
significant effect of sample year on any of the sperm traits
measured (Norway: all F
1,24
<0.69, P>0.42; Spain: all F
1,11
<2.48, all P>0.14).
Among-male variation in sperm size and extrapair paternity
levels
The populations showed somewhat different values of
among male variation in sperm size (CV
bm
)(Table2).
When examining the relationship between CV
bm
and
percentage broods containing one or more extrapair
young (EPB), we found a significant negative relation-
ship (Pearson's: r0−0.96, N06, P00.003) (Fig. 2a). The
relationship between CV
bm
and percentage extrapair
young (EPY) also went in the predicted negative direc-
tion, although this was not statistically significant
(Pearson's: r0−0.62, N06, P00.19) (Fig. 2b). Hence, both
tests were in the predicted direction with decreasing CV
bm
Table 1 Summary of extrapair paternity data from six populations of barn swallows Hirundo rustica used in the present study
Country EPY (95 % CI)
a
EPB
b
Nyoung/Nbroods Source
Czech 23.1 (15.2–31.1) 60.0 108/25 (T. Albrecht, J. Kreisinger
and R. Michalkova
unpublished data)
Italy 29.0 (22.9–35.1) 52.0 214/52 (Saino et al. 1999)
Spain 17.8 (14.9–20.7) 32.4 674/170 (Møller et al. 2003)
Ukraine 30.0 (20.0–40.0) 50.0 80/18 (Ellegren et al. 1997)
Israel 15.3 (9.7–20.9) 43.9 161/41 (Vortman et al. 2011)
Canada 28.8 (25.9–31.7) 48.1 917/210 (Kleven et al. 2005)
a
Percentage extrapair young with the 95 % lower and upper confidence limits of the estimate indicated
b
Percentage broods containing one or more extrapair young
304 Behav Ecol Sociobiol (2013) 67:301–309
being associated with increasing level of extrapair paternity
(Lifjeld et al. 2010).
Discussion
Our analysis revealed significant differences in sperm mor-
phometry among barn swallow populations and subspecies.
North American barn swallows from Canada had signifi-
cantly shorter sperm than European barn swallows from
Czech, Norway, Spain, and Ukraine but did not differ sig-
nificantly from European barn swallows from Italy and
East-Mediterranean barn swallows from Israel. When exam-
ining the three different sperm traits included in our analyses
(head length, midpiece length, and tail length), North
American barn swallows were characterized by longer
sperm heads than all other populations and shorter midpie-
ces than all other populations except the East-Mediterranean
one. Further, we found that between male variation in sperm
length (CV
bm
) was negatively related to levels of extrapair
paternity, both expressed as percentage broods contain-
ing extrapair young (EPB) and percentage extrapair
young (EPY).
Based on the phylogenetic relationships between the
three subspecies of barn swallows included in our analyses
(Dor et al. 2010), we predicted that European and East-
Mediterranean barn swallows should be more similar to
each other in sperm morphometry compared to North
American barn swallows. Although our findings were not
clear, some of the sperm traits coincided with this predic-
tion, e.g., spermatozoa of North American barn swallows
had longer heads and shorter midpieces than all other pop-
ulations. Total sperm length showed a different pattern, with
European barn swallows having significantly longer sperm
than both East-Mediterranean and North American barn
swallows, whereas the two latter did not differ significantly
for this trait. Interestingly, sperm head size seems to be a
trait that often differs among populations. It was the most
variable sperm trait in the coal tit study (Schmoll and
Kleven 2011). Also, in a recent comparison of wild and
domesticated zebra finches (Taeniopygia guttata), sperm
head length showed the greatest difference between the
two groups (Immler et al. 2012). We do not know if genetic
drift or selection is causing the differentiation in sperm
morphometry among populations. Both factors can however
be important for differentiation in sperm morphometry and
are not mutually exclusive. Whereas the studies of coal tits
and red-winged blackbirds documented significant differen-
tiation in sperm size between relatively close populations
(Lüpold et al. 2011; Schmoll and Kleven 2011), this study
documented little differentiation in sperm size across the
five European barn swallow populations. Another study on
pied flycatchers (Ficedula hypoleuca) found a similar pat-
tern as the present study, with no difference in sperm size
between one German and two Norwegian populations
(Lifjeld et al. 2012). The discrepancy between the coal tit
and red-winged blackbird on one hand and the pied fly-
catcher and barn swallow studies on the other, might be
explained by differences in gene flow among populations.
Both coal tits and red-winged blackbirds are resident or
short distance migratory birds, whereas pied flycatchers
and barn swallows are long distance migrants with clear
differences in dispersal distance (Paradis et al. 1999).
Notably, the East-Mediterranean barn swallow is sedentary
but still is subject to high levels of gene flow supposedly
from migrating European barn swallows (Dor et al. 2012).
Thus, gene flow could be more prominent in the two latter
species.
Notably, it has been shown in Gouldian finches
(Erythrura gouldiae) that males can exhibit plasticity in
Table 2 Descriptive statistics of sperm morphometry in seven populations of barn swallows Hirundo rustica (see supplementary Table S2–S4 for
pairwise comparisons between all populations)
Head length (μm) Midpiece length (μm) Tail length (μm) Total length (μm) CV
bm
a
CV
wm
b
Mean± SD Range Mean± SD Range Mean± SD Range Mean± SD Range
Czech (n020) 13.1±0.5 11.7–13.9 62.2 ±2.8 57.3–66.9 14.7± 2.2 9.7–18.7 89.9 ± 2.1 86.4–94.2 2.34 1.52
Italy (n08) 13.0± 0.3 12.6–13.4 64.4 ± 1.3 62.9–66.4 13.0±2.6 10.2–18.7 90.3 ± 2.2 88.0–94.8 2.54 1.65
Norway (n026) 13.2± 0.5 12.1–14.2 61.6 ± 3.1 54.2–65.9 15.8±3.4 11.6–25.6 90.6 ± 2.4 86.4–94.6 2.63 1.67
Spain (n013) 13.4± 0.5 12.8–14.6 62.5 ±2.5 58.3–66.0 15.0± 2.2 11.5–20.3 91.0±2.9 85.3–96.9 3.29 1.66
Ukraine (n025) 13.2± 0.4 12.2–14.2 62.6 ± 2.3 57.7–66.6 15.4±2.9 11.0–24.5 91.2 ± 2.5 86.2–96.8 2.76 1.92
Israel (n08) 13.2± 0.6 12.4–14.0 61.7 ± 2.9 57.5–66.0 13.6±1.2 11.5–15.5 88.5 ± 2.8 84.3–92.2 3.13 2.24
Canada (n090) 14.0± 0.7 12.5–15.5 59.0 ± 2.1 52.3–63.3 14.8±2.1 10.2–20.1 87.9 ± 2.5 81.4–93.0 2.82 1.74
a
Coefficient of variation of total sperm length calculated as SD/mean*100 and adjusted for sample size following the formula (CV*(1 +(1/4n)))
(Sokal and Rohlf 1995)
b
Average coefficient of variation of total sperm length within males, based on ten sperms per male
Behav Ecol Sociobiol (2013) 67:301–309 305
sperm morphometry within the same breeding season when
the social environment is altered (Immler et al. 2010). Males
were found to increase the relative size of the sperm mid-
piece when placed in intermediate to high competitive envi-
ronments and increase the size of the sperm tail when facing
Fig 1 Sperm ahead, bmidpiece, and ctotal length, in seven popula-
tions of barn swallows Hirundo rustica. Average values are indicated
as small squares,boxes indicate ±SE, and whiskers indicate ±95 %
confidence intervals. Sample sizes are; Czech n020 males, Italy n0
8males,Norwayn026 males, Spain n013 males, Ukraine n025
males, Israel n08 males, and Canada n090 males. See “Results”
section for test statistics
Fig 2 Relationship between apercentage broods containing extrapair
young and coefficient of variation (adjusted for sample size) (CV
bm
) in
sperm length between males and bpercentage extrapair young and
CV
bm
, in six populations of barn swallows. Numbers represent the
following populations; (1) Czech, (2) Italy, (3) Ukraine, (4) Canada, (5)
Israel, and (6) Spain. See “Results”section for test statistics
306 Behav Ecol Sociobiol (2013) 67:301–309
low to intermediate competitive environments (Immler et al.
2010). This could imply that the relative size of the sperm
midpiece and tail could change across the breeding season
in barn swallows as well. Similarly, a recent study of sperm
size variation in house wrens (Troglodytes aedon) found that
total sperm length was highly consistent across the breeding
season, whereas the flagellum:head length ratio increased
during the season, indicating phenotypic plasticity or adjust-
ment in the relative size of sperm components (Cramer et al.
2012). However, repeated samples across the breeding sea-
son of North American and Czech barn swallows have
shown very high repeatability of all sperm traits (own un-
published data).
Sperm head, midpiece, and flagellum length have been
shown to be heritable in the zebra finch (Birkhead et al.
2005) and in several other taxa (e.g. Ward 2000; Morrow
and Gage 2001a; Baer et al. 2006). Thus, plasticity in sperm
morphometry within the same breeding season might be
rather marginal and not biasing the population differences
reported here.
To the best of our knowledge, this is the first study
examining the relationship between sperm size variation
and extrapair paternity levels across populations within the
same species. A few studies have previously demonstrated
that this relationship is significantly negative in passerine
birds (Calhim et al. 2007; Kleven et al. 2008; Lifjeld et al.
2010). Recently, a similar negative relationship between
sperm size variation and polyandry has been demonstrated
in social insects (Fitzpatrick and Baer 2011). As extrapair
paternity levels are known to vary among populations of the
same species (e.g., Griffith et al. 2002), with barn swallows
being one of the species showing considerable variation
(Saino et al. 1999; Møller et al. 2003; Kleven et al. 2005;
Vortman et al. 2011), we tested associations between CV
bm
and levels of extrapair paternity data across populations of this
species. Both EPB and EPY were negatively related to CV
bm
,
as predicted from the aforementioned interspecific studies.
The relationship between CV
bm
and EPY was, however, not
statistically significant. Although our sample size in the
present dataset is small, we argue that this indicates that
CV
bm
and risk of sperm competition are negatively related
also within species, further supporting the notion that risk of
sperm competition induces stabilizing selection on sperm size.
The present study shows little variation in sperm size/
morphometry among European and East-Mediterranean
barn swallows, and somewhat larger variation between
European-East-Mediterranean and North American barn
swallows. These results make sense in the light of the
genetic difference and the large geographic distance be-
tween populations. The subspecies rustica and transitiva
are genetically closely related (Dor et al. 2010), and they
do not differ much in sperm morphometry. The subspecies
erythrogaster is genetically quite distant from both rustica
and transitiva (Dor et al. 2010; Johnsen et al. 2010), and this
is also reflected in the observed sperm morphometry. In
conclusion, sperm morphometry might give an indication
of genetic distance, not only between species, but also
between phylogenetic lines within species. Further, we
found negative relationships between CV
bm
and two meas-
ures of extrapair paternity, EPB and EPY, indicating that the
same pattern as have been found among species (Calhim et
al. 2007; Kleven et al. 2008; Lifjeld et al. 2010; Fitzpatrick
and Baer 2011), also is present intraspecifically, and which
strengthens the explanation that sperm competition exerts a
stabilizing selection pressure on sperm size variation.
Acknowledgments We are grateful to all people that assisted with
field work, especially Frode Fossøy in Canada, Bjørn Aksel Bjerke in
Norway, and Luz Garcia-Longoria in Spain. A special thanks to Gustav
Thorsø Mohr for allowing us to trap inside the barn at Thorsø
Herregård. We thank two anonymous reviewers for helpful comments
on an earlier draft of the manuscript. This study was supported by
funding from the Czech Science Foundation (to TA, project no.
P506/12/2472), a Fondazione Cariplo grant (to NS, grant no. 2009–
3496), the Ministry of Culture of the Czech Republic (to JC, grant no.
DKRVO 00023272), the Natural Sciences and Engineering Research
Council of Canada (to RJR), the Norwegian Research Council (to JTL,
OK, LEJ and TL), the Samuel Freeman Charitable Trust (to TAM), and
the Spanish Ministry of Economy and Competitivness (to IGH, FdL and
AM, grant no. CGL 2012–36665).
Ethical standards All authors declare that the present study com-
plies with the current laws and ethical standards of animal research in
Canada, Czech Republic, Israel, Italy, Norway, Spain, and Ukraine.
Conflict of interest The authors declare that they have no conflict of
interest
References
Baer B, de Jong G, Schmid-Hempel R, Schmid-Hempel P, Høeg JT,
Boomsma JJ (2006) Heritability of sperm length in the bumblebee
Bombus terrestris. Genetica 127:11–23
Birkhead TR, Pellatt EJ, Brekke P, Yeates R, Castillo-Juarez H (2005)
Genetic effects on sperm design in the zebra finch. Nature
434:383–387
Birkhead TR, Hosken DJ, Pitnick S (2009) Sperm biology: an evolu-
tionary perspective. Academic Press, Oxford
Blanckenhorn WU, Hellriegel B (2002) Against Bergmann's rule: fly
sperm size increases with temperature. Ecol Lett 5:7–10
Calhim S, Immler S, Birkhead TR (2007) Postcopulatory sexual selec-
tion is associated with reduced variation in sperm morphology.
PLoS One 2:e413
Cohen J (1977) Reproduction. Butterworths, London
Cramer ERA, Laskemoen T, Kleven O, Lifjeld JT (2012) Sperm
length variation in House Wrens Troglodytes aedon. J Ornithol
DOI: 10.1007/s10336–012-0878-3
Dor R, Safran RJ, Sheldon FH, Winkler DW, Lovette IJ (2010) Phy-
logeny of the genus Hirundo and the barn swallow subspecies
complex. Mol Phyl Evol 56:409–418
Dor R, Safran RJ, Vortman Y, Lotem A, McGowan A, Evans MR,
Lovette IJ (2012) Population genetics and morphological
Behav Ecol Sociobiol (2013) 67:301–309 307
comparisons of migratory European (Hirundo rustica rustica) and
sedentary East-Mediterranean (Hirundo rustica transitiva) barn
swallows. J Hered 103:55–63
Eberhard WG (1996) Female control: sexual selection by cryptic
female choice. Princeton University Press, Princeton, NJ
Elgee KE, Evans JP, Ramnarine IW, Rush SA, Pitcher TE (2010)
Geographic variation in sperm traits reflects predation risk and
natural rates of multiple paternity in the guppy. J Evol Biol
23:1331–1338
Ellegren H, Lindgren G, Primmer CR, Møller AP (1997) Fitness loss
and germline mutations in barn swallows breeding in Chernobyl.
Nature 389:593–596
Fitzpatrick JL, Baer B (2011) Polyandry reduces sperm length varia-
tion in social insects. Evolution 65:3006–3012
Gage MJG (1998) Mammalian sperm morphometry. Proc R Soc Lond
B 265:97–103
Griffith SC, Owens IPF, Thuman KA (2002) Extra pair paternity in
birds: a review of interspecific variation and adaptive function.
Mol Ecol 11:2195–2212
Hettyey A, Roberts JD (2006) Sperm traits of the quacking frog, Crinia
georgiana: intra- and interpopulation variation in a species with a
high risk of sperm competition. Behav Ecol Sociobiol 59:389–396
Hosken DJ, Garner TWJ, Blanckenhorn WU (2003) Asymmetry, testis
and sperm size in yellow dung flies. Funct Ecol 17:231–236
Immler S, Birkhead TR (2005) A non-invasive method for obtaining
spermatozoa from birds. Ibis 147:827–830
Immler S, Calhim S, Birkhead TR (2008) Increased postcopulatory
sexual selection reduces the intramale variation in sperm design.
Evolution 62:1538–1543
Immler S, Pryke SR, Birkhead TR, Griffith SC (2010) Pronounced
within-individual plasticity in sperm morphometry across social
environments. Evolution 64:1634–1643
Immler S, Griffith SC, Zann R, Birkhead TR (2012) Intra-specific
variance in sperm morphometry: a comparison between wild
and domesticated Zebra Finches Taeniopygia guttata.Ibis
154:480–487
Johnsen A, Rindal E, Ericson PGP, Zuccon D, Kerr KCR, Stoeckle
MY, Lifjeld JT (2010) DNA barcoding of Scandinavian birds
reveals divergent lineages in trans-Atlantic species. J Ornithol
151:565–578
Joly D, Korol A, Nevo E (2004) Sperm size evolution in Drosophila:
inter- and intraspecific analysis. Genetica 120:233–244
Kleven O, Jacobsen F, Robertson RJ, Lifjeld JT (2005) Extrapair
mating between relatives in the barn swallow: a role for kin
selection? Biol Lett 1:389–392
Kleven O, Jacobsen F, Izadnegahdar R, Robertson RJ, Lifjeld JT
(2006) Male tail streamer length predicts fertilization success in
the North American barn swallow (Hirundo rustica eryth-
rogaster). Behav Ecol Sociobiol 59:412–418
Kleven O, Laskemoen T, Fossøy F, Robertson RJ, Lifjeld JT (2008)
Intraspecific variation in sperm length is negatively related to
sperm competition in passerine birds. Evolution 62:494–499
Kuramoto M (1996) Generic differentiation of sperm morphology in
treefrogs from Japan and Taiwan. J Herpetol 30:437–443
Laskemoen T, Kleven O, Fossøy F, Lifjeld JT (2007) Intraspecific
variation in sperm length in two passerine species, the bluethroat
Luscinia svecica and the willow warbler Phylloscopus trochilus.
Ornis Fenn 84:131–139
Lessells CM, Boag PT (1987) Unrepeatable repeatabilities: a common
mistake. Auk 104:116–121
Lifjeld JT, Laskemoen T, Kleven O, Albrecht T, Robertson RJ (2010)
Sperm length variation as a predictor of extrapair paternity in
passerine birds. PLoS One 5:e13456
Lifjeld JT, Kleven O, Jacobsen F, McGraw KJ, Safran RJ, Robertson
RJ (2011) Age before beauty? Relationships between fertilization
success and age-dependent ornaments in barn swallows. Behav
Ecol Sociobiol 65:1687–1697
Lifjeld JT, Laskemoen T, Kleven O, Pedersen ATM, Lampe HM, Rudolf-
sen G, Schmoll T, Slagsvold T (2012) No evidence for pre-
copulatory sexual selection in a passerine bird. PLoS One 7:e32611
Lüpold S, Linz GM, Birkhead TR (2009) Sperm design and variation
in the New World blackbirds (Icteridae). Behav Ecol Sociobiol
63:899–909
Lüpold S, Westneat DF, Birkhead TR (2011) Geographical variation in
sperm morphology in the red-winged blackbird (Agelaius phoe-
niceus). Evol Ecol 25:373–390
Manier MK, Palumbi SR (2008) Intraspecific divergence in sperm
morphology of the green sea urchin, Strongylocentrotus droeba-
chiensis: implications for selection in broadcast spawners. BMC
Evol Biol 8:283
Minoretti N, Baur B (2006) Among- and within-population variation in
sperm quality in the simultaneously hermaphroditic land snail
Arianta arbustorum. Behav Ecol Sociobiol 60:270–280
Møller AP (1988) Female choice selects for male sexual tail ornaments
in the monogamous swallow. Nature 332:640–642
Møller AP (1994a) Repeatability of female choice in a monogamous
swallow. Anim Behav 47:643–648
Møller AP (1994b) Sexual selection and the barn swallow. Oxford
University Press, Oxford
Møller AP, Brohede J, Cuervo JJ, de Lo pe F, Primmer C (2003)
Extrapair paternity in relation to sexual ornamentation, arrival
date, and condition in a migratory bird. Behav Ecol 14:707–712
Møller AP, Mousseau TA, Lynn C, Ostermiller S, Rudolfsen G (2008)
Impaired swimming behaviour and morphology of sperm from
barn swallows Hirundo rustica in Chernobyl. Mutat Res Genet
Tox Environ Mutagen 650:210–216
Morrow EH, Gage MJG (2001a) Artificial selection and heritability of
sperm length in Gryllus bimaculatus. Heredity 87:356–362
Morrow EH, Gage MJG (2001b) Consistent significant variation be-
tween individual males in spermatozoal morphometry. J Zool
Lond 254:147–153
Paradis E, Baillie SR, Sutherland WJ, Gregory RD (1999) Dispersal
and spatial scale affect synchrony in spatial population dynamics.
Ecol Lett 2:114–120
Parker GA (1970) Sperm competition and its evolutionary consequen-
ces in the insects. Biol Rev 45:525–567
Petrie M, Kempenaers B (1998) Extra-pair paternity in birds: explain-
ing variation between species and populations. Trends Ecol Evol
13:52–58
Pitnick S, Miller GT, Schneider B, Markow TA (2003) Ejaculate-
female coevolution in Drosophila mojavensis. Proc R Soc Lond
B 270:1507–1512
Safran RJ, Neumann CR, McGraw KJ, Lovette IJ (2005) Dynamic
paternity allocation as a function of male plumage color in barn
swallows. Science 309:2210–2212
Saino N, Primmer CR, Ellegren H, Møller AP (1997) An experimental
study of paternity and tail ornamentation in the barn swallow
(Hirundo rustica). Evolution 51:562–570
Saino N, Primmer CR, Ellegren H, Møller AP (1999) Breeding syn-
chrony and paternity in the barn swallow (Hirundo rustica).
Behav Ecol Sociobiol 45:211–218
Schmoll T, Kleven O (2011) Sperm dimensions differ between two
coal tit Periparus ater populations. J Ornithol 152:515–520
Snook RR (2001) Absence of latitudinal clines in sperm characters in
North American populations of Drosophila subobscura (Diptera:
Drosophilidae). Pan-Pac Entomol 77:261–271
308 Behav Ecol Sociobiol (2013) 67:301–309
Sokal RR, Rohlf FJ (1995) Biometry: the principles and practice of
statistics in biological research. Freeman, New York
Turner AK (2006) The barn swallow. T. & A. D, Poyser, London
Vortman Y, Lotem A, Dor R, Lovette IJ, Safran RJ (2011) The sexual
signals of the East-Mediterranean barn swallow: a different swal-
low tale. Behav Ecol 22:1344–1352
Ward PI (1998) Intraspecific variation in sperm size characters. Hered-
ity 80:655–659
Ward PI (2000) Sperm length is heritable and sex-linked in the yellow
dung fly (Scathophaga stercoraria). J Zool Lond 251:349–353
Wolfson A (1952) The cloacal protuberance: a means for determining
breeding condition in live male passerines. Bird Band 23:159–165
Behav Ecol Sociobiol (2013) 67:301–309 309
