Sperm competitiveness differs between two frog populations with different breeding systems

Abstract

In species with simultaneous polyandry, male-biased operational sex ratio is expected to increase the risk of sperm competition and thus sperm traits affecting siring success can differ among populations. Here, we test the hypothesis that high male–female ratios will enhance sperm competitiveness of Rana temporaria males. In this species, local populations can show either prolonged or explosive breeding. In a context of sperm competition and in controlled laboratory conditions, prolonged-breeding males sired a higher proportion of eggs than explosive-breeding males, regardless of female origin. This study demonstrates intrapopulation variation in siring success under a situation of sperm competition, consistent with the prolonged-explosive dichotomy of breeding strategies.

Full text

Sperm competitiveness differs between two frog
populations with different breeding systems
D. Álvarez1,2, L. Viesca1,2 & A. G. Nicieza1,2
1 Ecology Unit, Department of Organisms and Systems Biology, University of Oviedo, Oviedo, Spain
2 Research Unit of Biodiversity (UO-CSIC-PA), Mieres, Spain
Keywords
sperm competition; sex ratio, mating
system; paternity; amphibian.
Correspondence
David Álvarez, C/Catedrático Rodrigo Uría
s/n, Oviedo, Asturias 33071, Spain. Tel:
+34-985223794; Fax: +34-985104866
E-mail: dalvarezf@gmail.com
Editor: Mark-Oliver Rödel
Received 17 July 2013; revised 29 October
2013; accepted 29 October 2013
doi:10.1111/jzo.12093
Abstract
In species with simultaneous polyandry, male-biased operational sex ratio is
expected to increase the risk of sperm competition and thus sperm traits affect-
ing siring success can differ among populations. Here, we test the hypothesis
that high male–female ratios will enhance sperm competitiveness of Rana
temporaria males. In this species, local populations can show either prolonged or
explosive breeding. In a context of sperm competition and in controlled labora-
tory conditions, prolonged-breeding males sired a higher proportion of eggs
than explosive-breeding males, regardless of female origin. This study demon-
strates intrapopulation variation in siring success under a situation of sperm
competition, consistent with the prolonged-explosive dichotomy of breeding
strategies.
Introduction
Sexual selection theory predicts that male mating success
should be affected by the operational sex ratio (OSR;
Andersson, 1994), the intensity of intrasexual selection
increases the more unbalanced the OSR is towards males
(Kvarnemo & Ahnesjö, 1996). Selection can act via female
monopolization, or through sperm competition, or both.
Therefore, the evolution of sperm competition can be associ-
ated with the long-term intensity of male–male competition.
Differences in siring success under sperm competition can be
mediated by variation in sperm number (Parker & Ball, 2005)
or sperm characteristics (Snook, 2005; Hettyey & Roberts,
2006; Dziminski et al., 2009), but sperm quality can be more
important in determining fertilization success under competi-
tive situations (Birkhead et al., 1999; García-González &
Simmons, 2005; Firman & Simmons, 2008).
Theoretically, geographic variation in mating systems and
OSRs can promote local adaptation in sperm traits (Leach &
Montgomerie, 2000). Despite sperm competition being taxo-
nomically widespread (Birkhead, 1995) and OSR being highly
variable, how spatial variation in OSRs can affect geographic
variation in sperm competition is unknown. Anuran amphib-
ians are ideal models to test hypotheses about the evolution of
sperm competition in spatially structured species; most species
have external aquatic fertilization, and multiple mating is
common (Laurila & Seppa, 1998; Roberts et al., 1999; Lodé &
Lesbarrères, 2004; Vieites et al., 2004), and they can be clas-
sified as either explosive or prolonged breeders (Wells, 2007).
The reproduction of prolonged breeders extends over months,
and males stay at the breeding sites for weeks, whereas females
usually arrive at the ponds just a few hours before spawning;
this results in male-biased OSRs. In contrast, in explosive
systems, the highly synchronous arrival of females at the
breeding sites lead to more balanced OSR, which is expected
to relax the intensity of male–male competition (Andersson,
1994).
Here, we sought to explore whether long-term OSR can
affect sperm competition and thus result in geographic vari-
ation in sperm competitiveness. However, it should be noted
that, to be a selective force, differences in operational sex ratio
must lead to differences in the average number of males
mating with each female. Here we have assumed that relation-
ship, but that link remains unexplored. The rationale is to
determine whether males from explosive and prolonged
systems differ in sperm ‘quality’ traits, with the hypothesis
that prolonged systems have more competitive sperm. Insight
on local-scale differences in the fertilization potential of males
is important because it can affect the pattern of gene flow in
subdivided populations (i.e. either boosting or minimizing the
genetic impact of migrants on the receptor populations), and
therefore, it has an obvious interest for conservation genetics.
Until now, only one study (see Dziminski et al., 2010) had
examined the hypothesis that spatial variation in OSR can
promote geographic variation in male reproductive ability.
Our model species was the common frog (Rana temporaria),
an anuran species that mates by amplexus and for which
simultaneous polyandry and clutch piracy has been reported
bs_bs_banner
Journal of Zoology
Journal of Zoology. Print ISSN 0952-8369
202 Journal of Zoology 292 (2014) 202–205 © 2013 The Zoological Society of London

(Laurila & Seppa, 1998; Vieites et al., 2004). R. temporaria is
considered an explosive breeder, but prolonged breeding is
common in some areas. In the Cantabrian region (south-
western end of the species range), mountain populations are
explosive breeders that usually reproduce before complete
melting of ice cover (winter–spring breeders). In contrast, at
low altitudes, the reproductive period can last up to 6 months
(September–February), with multiple peaks of activity
associated with rainy nights. This is an exceptional system
because the explosive-prolonged dichotomy of breeding
occurs within a single species. In this study, we used an
experimental approach to compare the fertilization rates of
prolonged versus explosive R. temporaria males after control-
ling for sperm concentration. By doing so, we focus on sperm
traits instead of sperm production, which can be more depend-
ent on temporal variation. If the intensity of intrasexual
competition is positively correlated with the operative
male : female ratio, we should expect that males from low-
altitude (prolonged breeding) populations show a higher
sperm competitive ability than males from high-altitude
(explosive breeding) populations.
Material and methods
The experimental animals were adult R. temporaria from two
local populations: Áliva (43.17884°N, 4.76479°W; 1420 m
elevation) and Color (43.29484°N, 5.27711°W; 380 m eleva-
tion). These populations belong to different genetic units, but
the same mtDNA lineage (authors, in prep.). Color frogs are
prolonged breeders (late August to February) and OSR is
highly biased towards males [mean (±sd) OSR across 2008–
2009 season: 11.7 ±6.2]. Color frogs were captured on 17
January 2010. In contrast, the Áliva population is an example
of explosive breeding, within 1–2 weeks in March. Although,
because of the short breeding period (less than 3 weeks), we
lack exhaustive data on Áliva sex ratio, single-day counts for
six nearby populations with similar reproductive timing
(explosive breeding) during the 2009 season revealed a much
lower male-biased OSR (mean ±sd: 4.3 ±2.4).
Áliva frogs were captured on 25 October 2009, just before
hibernation, when they have already acquired all the energy
for overwintering and breeding at the start of ice melting. The
hibernating period starts by late October/early November due
to snow cover and low temperatures. Frogs from Áliva popu-
lation were kept in dark small outdoor enclosures exposed to
the same weather conditions (temperature, precipitation, etc.)
experienced by the frogs from the low-altitude population
until the beginning of the experiment.
The fertilization experiment was conducted on 19 January
2010. We used six males and three females from each popula-
tion. All individuals were administered three doses (−24 h,
−12 h, −2 h) of Luteinizing-Hormone-Releasing-Hormone-
analogue (LHRHa; Sigma-Aldrich St. Louis, MO, USA) to
induce spermiation and ovulation (Browne et al., 2006).
After 24 h from the first dose, we stripped females by press-
ing their abdomen and gently pushed the male abdomen to
obtain eggs and sperm. To produce equivalent sperm doses,
milt samples were centrifuged in a capillary tube for 195 s at
12 145 g using a Biocem 20 Centrifuge (Orto Alresa, Madrid,
Spain). We calculated the spermatocrit value and delivered
fixed volumes of sperm to control for variation in sperm con-
centration. Sperm doses from two males were placed as sepa-
rate droplets in a Petri dish, and 50 mL of water were added to
ensure a complete homogenization. A sample of eggs from
each female clutch (range 60–129 eggs, see Table 1) was depos-
ited in the center of a Petri dish. Finally, the mixed-sperm dose
was added to the Petri dish containing the eggs of a single
female. Each female was crossed with two males of different
origin (Aliva vs. Color) to produce a total of six mixed
clutches. After 10 min, egg masses were washed, placed in
500-mL plastic vessels and incubated at 15°C during 20 d.
All the hatched tadpoles and a toe clip from the female and
both males of each family were preserved in absolute alcohol
for further genotyping. To assign the paternities, we used four
microsatellites: BFG072, BFG093, BFG183 and BFG241
(Matsuba & Merilä, 2009).
Extraction of genomic DNA and polymerase
chain reaction (PCR) reactions
Whole genomic DNA was isolated from tissues with a stand-
ard Chelex extraction consisting of 500 μL of a 10% Chelex
solution (Chelex-100, Bio-Rad, Hercules, CA, USA) incubate
with 7 μg proteinase K as 55°C for 60 min and 100°C for
20 min. Parental frogs were genotyped using a PCR multiplex,
analysing the four microsatelites at the same time. In this case,
we used 6–20 ng of template DNA, 0.3–0.7 μM of primer and
5μL of Qiagen Multiplex PCR Master Mix (Qiagen GmbH,
Hilden, Germany). The PCR started from a Taq polymerase
activation step at 95°C for 15 min, followed by 38 cycles of a
30-s denaturation step at 94°C, a 30-s annealing step at 55°C
and a 30-s extension step at 72°C, and then a final extension
step at 60°C for 30 min. After genotyping, all the parental
frogs for the four selected microsatellites, we confirmed that
locus BFG093 and BFG183 had different alleles in Áliva and
Color populations, and therefore, we genotyped all hatched
larvae for those microsatellites. These two microsatellites were
amplified independently using 6–20 ng of template DNA, 0.3–
0.7 μM of primer, 250 μM of dNTPs (Promega, Madison, WI
Table 1 Sample of eggs used in each of the crosses, number of
fertilized eggs (percentage in brackets), and number of hatched larvae
assigned to Color and Áliva males (percentage in brackets) Mean (±SD)
percentage of fertilized eggs in each population are shown in bold.
Dam Eggs Fertilized (%)
Sire
Color (%) Áliva (%)
Áliva 1 129 35 (27.1) 27 (77.1) 8 (22.9)
Áliva 2 60 47 (78.3) 32 (68.1) 15 (31.9)
Áliva 3 109 51 (46.8) 39 (76.5) 15 (23.5)
Mean ±SD 50.7 ±25.8 73.9 ±5.0 26.1 ±5.0
Color 1 100 0 (0) ––
Color 2 67 61 (91.0) 54 (88.5) 7 (11.5)
Color 3 64 21 (32.8) 20 (95.2) 1 (4.8)
Mean ±SD 61.9 ±41.1 91.8 ±4.7 8.1 ±4.7
D. Álvarez, L. Viesca and A. G. Nicieza Sex ratio and sperm competition in frogs
Journal of Zoology 292 (2014) 202–205 © 2013 The Zoological Society of London 203

USA), 0.5 U of Go Taq® Flexi DNA Polymerase (Promega),
2.0–2.5 mM of Mg2+,2μL of 5x colorless Go Taq Flexi Buffer
and 2 μL of 5x Green Go Taq Flexi Buffer. PCR cycles were
starting with 5 min at 94°C following by 40 cycles consisted in
30 s denaturation at 94°C, 30 s annealing at 56°C and extend-
ing 30 s at 72°C. After 40 cycles, 20 additionally minutes at
72°C were left for elongation. PCR reactions were performed
on Applied Biosystems 2720 Thermal (Applied Biosystems,
Foster City, CA, USA). PCR products were separated and
detected by capillary electrophoresis on an ABI PRISM®
3130xl Genetic Analyzer (Applied Biosystems).
Statistical analyses
To examine effects of male origin on fertilization rate, we used
a generalized estimation equation (GEE) with repeated meas-
urements, a Poisson error distribution and log link function.
The number of tadpoles assigned to each male was the
dependent variable, and the origin population of males and
females were categorical variables. Since each female was
crossed with two males, ‘Female’ was treated as a repeated
variable. All the analyses were performed with the SPSS v.19
statistical package (SPSS Inc., Chicago, IL, USA).
The work met the Spanish legal requirements about animal
welfare supervised by the University of Oviedo (license
number 8-INV-2012). Field work and permits of capture was
supervised and approved by Principado de Asturias (permit
number 2010/000371) and Picos de Europa National Park
(permit numbers CO/09/0571/2009).
Results
Sperm cell concentration was quite variable among males
(percentage of total sperm volume, mean ±1se: 6.92 ±
1.75%), but there were no significant differences between
populations (Mann–Whitney two-tailed U-test: U=13.0;
P=0.48). All the eggs from one clutch (Color female 1)
remained unfertilized. For the other five females, fertilization
rate ranged from 27.1% and 91.0%, and we assigned the pater-
nity of all the larvae unequivocally (Table 1).
Regardless of female origin, Color males consistently
fertilized a larger proportion of eggs than their Aliva counter-
parts (Fig. 1). In fact, GEE analysis confirmed the existence
of a significant male effect on fertilization success (Wald
χ2=85.37; d.f. =1; P<0.0001). However, there was no sig-
nificant female effect (Wald χ2=0.036; d.f. =1; P=0.85). In
addition, the differences between Áliva and Color males in
fertilization rate were greater for the clutches produced by
Color females (Table 1).
Discussion
Our data showed interpopulation variation in the fertilization
ability of male common frogs under a situation of sperm
competition. Although geographic variation in OSR and male
reproductive ability was studied previously (Dziminski et al.,
2010), this is the first time that differences in sperm competi-
tiveness have been observed between males from different
populations with different breeding systems of the same
species. Males from Color showed a consistent fertilization
advantage over Áliva males, thus suggesting that these differ-
ences could be associated with differences in the mating
systems and the resulting OSRs. However, because of the
differences in phenology between Aliva and Color popula-
tions and although all individuals were subjected to the same
conditions, results should be interpreted with caution.
Males can deal with competition for females in two ways.
First, female monopolization can be achieved by fighting.
Alternatively, females can mate simultaneously with multiple
males, and the sperm from these compete to fertilize the eggs
(simultaneous polyandry). In populations with prolonged
breeding, we expect a sequential entry of females to promote
competition among males, resulting in either female monopo-
lization or sperm competition. In contrast, if reproduction is
highly synchronized (explosive), the incidence of multiple-
male mating is expected to be lower, and therefore, selection
for sperm traits involved in rapid fertilization should be also
lower. In R. temporaria, simultaneous polyandry (Laurila &
Seppa, 1998) can be associated with multiple-male mating and
post-mating clutch piracy (Vieites et al., 2004). We thus
hypothesize that these effects would be greater in populations
with prolonged breeding and male-biased OSR, therefore
increasing the risk of sperm competition in relation to explo-
sive systems.
In a context of simultaneous polyandry, sperm motility
rather than sperm number can be the main determining factor
(Snook, 2005), and there are evidences that sperm competition
selects for increased sperm length and slow sperm velocity in
frogs (Byrne, Simmons & Roberts, 2003; Dziminski et al.,
2009). Unfortunately, we could not perform a test of sperm
quality (live/dead sperm, morphology, etc.) during normal
breeding season for each population since sperm quality may
change with the season. Therefore, our finding that Color
males consistently outperformed Áliva males fits well with the
Figure 1 Siring success of pairs of males from Color and Áliva popu-
lations crossed with single females from Color (○) or Áliva (●). Dashed
and solid lines represent the differences between the two males mated
to a given female from Color or Áliva, respectively.
Sex ratio and sperm competition in frogs D. Álvarez, L. Viesca and A. G. Nicieza
204 Journal of Zoology 292 (2014) 202–205 © 2013 The Zoological Society of London

idea that more male-biased OSRs results in more risk of sperm
competition, which in turn would select for a higher sperm
performance. On the other hand, recent work has stressed the
importance of the female–male genetic similarity (Tregenza &
Wedell, 2000; Sherman et al., 2008). At the within-population
level, siring success of male tree frog (Litoria peronii) was
related to the genetic similarity with females (Sherman et al.,
2008). However, as in the present study, after controlling for
sperm concentration, male–male difference in siring success
was consistent across females, suggesting that in L. peronii,
male siring success is primarily determined by among-male
variation with less influence of male–female relatedness
(Sherman, Wapstra & Olsson, 2009). Our finding that the
advantage of Color males was much greater for Color than for
Áliva females also suggests some effect of genetic compatibil-
ity. This is interesting because, most likely, the processes dic-
tated by genetic compatibility and sperm ‘quality’ could
operate simultaneously (Sherman et al., 2009). Altogether,
our results indicate that, in a scenario of population interac-
tion, differences between populations in sperm traits could
accelerate genetic introgression, but the magnitude of this
effect will be dependent on the genetic compatibility of indi-
viduals from different populations.
Acknowledgements
Financial support was provided by the Ministerio de Ciencia
e Innovación and FEDER funds (MICINN, project
#CGL2009–12767-C02-01), Ministerio de Medio Ambiente,
Medio Rural y Marino (project # MMAMRM-08-38/2008)
and FICYT (#COF09-04). Jon Loman and an anonymous
reviewer provided valuable comments on an earlier draft of
the manuscript.
References
Andersson, M. (1994). Sexual selection. Princeton: Princeton
University Press.
Birkhead, T.R. (1995). Sperm competition: evolutionary
causes and consequences. Reprod. Fertil. Dev. 7, 755–775.
Birkhead, T.R., Martinez, J.G., Burke, T. & Froman, D.P.
(1999). Sperm mobility determines the outcome of sperm
competition in the domestic fowl. Proc. R. Soc. Lond. B
266, 1759–1764.
Browne, R.K., Seratt, J., Vance, C. & Kouba, A. (2006). Hor-
monal priming, induction of ovulation and in-vitro fertili-
zation of the endangered Wyoming toad (Bufo baxteri).
Reprod. Biol. Endocrinol. 4, 34.
Byrne, P.G., Simmons, L.W. & Roberts, J.D. (2003). Sperm
competition and the evolution of gamete morphology in
frogs. Proc. R. Soc. Lond. B 270, 2079–2086.
Dziminski, M.A., Roberts, J.D., Beveridge, M. & Simmons,
L.W. (2009). Sperm competitiveness in frogs: slow
and steady wins the race. Proc. R. Soc. B 276, 3955–3961.
Dziminski, M.A., Roberts, J.D., Beveridge, M. & Simmons,
L.W. (2010). Among-population covariation between sperm
competition and ejaculate expenditure in frogs. Behav. Ecol.
21, 322–328.
Firman, R.C. & Simmons, L.W. (2008). Polyandry facilitates
postcopulatory inbreeding avoidance in house mice. Evolu-
tion 62, 603–611.
García-González, F. & Simmons, L.W. (2005). Sperm viabil-
ity matters in insect sperm competition. Curr. Biol. 15, 271–
275.
Hettyey, A. & Roberts, J.D. (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.
Kvarnemo, C. & Ahnesjö, I. (1996). The dynamics of opera-
tional sex ratios and competition for mates. Trends Ecol.
Evol. 11, 404–408.
Laurila, A. & Seppa, P. (1998). Multiple paternity in the
common frog (Rana temporaria): genetic evidence from
tadpole kin groups. Biol. J. Linn. Soc. 63, 221–232.
Leach, B. & Montgomerie, R. (2000). Sperm characteristics
associated with different male reproductive tactics in
bluegills (Lepomis macrochirus). Behav. Ecol. Sociobiol. 41,
31–37.
Lodé, T. & Lesbarrères, D. (2004). Multiple paternity in Rana
dalmatina, a monogamous territorial breeding anuran.
Naturwissenschaften 91, 44–47.
Matsuba, C. & Merilä, J. (2009). Isolation and characteriza-
tion of 145 polymorphic microsatellite loci for the common
frog (Rana temporaria). Mol. Ecol. Resour. 9, 555–562.
Parker, G.A. & Ball, M.A. (2005). Sperm competition, mating
rate and the evolution of testes and ejaculate sizes: a popu-
lation model. Biol. Lett. 1, 235–238.
Roberts, J.D., Standish, R.J., Byrne, P.G. & Doughty, P.
(1999). Synchronous polyandry and multiple paternity in
the frog Crinia georgiana (Anura: Myobatrachidae). Anim.
Behav. 57, 721–726.
Sherman, C.D.H., Wapstra, E., Uller, T. & Olsson, M. (2008).
Males with high genetic similarity to females sire more off-
spring in sperm competition in Peron’s tree frog Litoria
peronii.Proc. R. Soc. B 275, 971–978.
Sherman, C.D.H., Wapstra, E. & Olsson, M. (2009). Consist-
ent male-male paternity differences across female geno-
types. Biol. Lett. 5, 232–234.
Snook, R.R. (2005). Sperm in competition: not playing by the
numbers. Trends Ecol. Evol. 20, 46–53.
Tregenza, T. & Wedell, N. (2000). Genetic compatibility, mate
choice and patterns of parentage: invited review. Mol. Ecol.
9, 1013–1027.
Vieites, D.R., Nieto-Román, S., Barluenga, M., Palanca, A.,
Vences, M. & Meyer, A. (2004). Post-mating clutch piracy
in an amphibian. Nature 431, 305–308.
Wells, K.D. (2007). The ecology and behaviour of amphibians.
Chicago: University of Chicago Press.
D. Álvarez, L. Viesca and A. G. Nicieza Sex ratio and sperm competition in frogs
Journal of Zoology 292 (2014) 202–205 © 2013 The Zoological Society of London 205